Clinical Studies and Therapies in Parkinson's Disease: Translations from Preclinical Models 0128221208, 9780128221204

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CLINICAL STUDIES AND THERAPIES IN PARKINSON’S DISEASE

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CLINICAL STUDIES AND THERAPIES IN PARKINSON’S DISEASE Translations from Preclinical Models

JUAN SEGURA-AGUILAR University of Chile, Santiago, Chile

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2021 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-822120-4 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals Publisher: Nikki Levy Acquisitions Editor: Joslyn Chaiprasert-Paguio Editorial Project Manager: Devlin Person Production Project Manager: Swapna Srinivasan Cover Designer: Greg Harris

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I would like to dedicate this book to Maria Ester Munoz Herrera, my wife and life partner during the difficult times I have lived, including my clandestine fight against the Pinochet dictatorship in Chile, kidnapping and torture by Pinochet’s covert intelligence service (DINA), disappearance to the Villa Grimaldi torture center for 3 months, detention in the Tres Alamos concentration camp for 9 months, and eventual exile in Sweden. Maria Ester was a fundamental support who enabled me to study and pursue a doctorate at the University of Stockholm; she cared for my three children so that I could dedicate myself to study. She accompanied me on my return to Chile, where I joined the Faculty of Medicine of the University of Chile. I want to thank Maria EsterdI have been so fortunate to have received all her love and support for nearly 50 years.

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Contents

1. Parkinson’s disease A. Epidemiology References B. Parkinson’s disease classification C. Parkinsonism C.1. Drug-induced parkinsonism References C.2. Paraquat-induced parkinsonism References C.3. Copper-induced parkinsonism References C.4. Manganese-induced parkinsonism References C.5. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridineeinduced parkinsonism References D. Idiopathic Parkinson’s disease References D.1. Nonmotor symptoms References D.1.1. Olfactory dysfunction References D.1.2. Rapid eye movement sleep behavior disorder References D.1.3. Depression References D.1.4. Constipation References D.1.5. Excessive daytime somnolence References D.1.6. Insomnia References D.1.7. Anxiety References D.1.8. Cognitive decline References D.1.9. Parkinson’s disease dementia and dementia with Lewy bodies References

vii

1 2 2 2 2 6 7 8 10 12 13 16 18 22 23 24 25 25 26 29 31 36 38 43 46 49 51 53 54 55 56 59 60 65 67 74

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D.1.10. Orthostatic hypotension 75 References 77 D.1.11. Visual disturbances 77 References 79 D.2. Motor symptoms 80 D.2.1. Alpha-synuclein role in degeneration of nigrostriatal neurons 80 References 88 References 110 D.2.2. Mitochondrial dysfunction 116 References 123 D.2.3. Protein degradation dysfunction role in degeneration of nigrostriatal neurons 125 References 131 D.2.4. Role of oxidative stress in degeneration of nigrostriatal neurons 134 References 140 D.2.5. Neuroinflammation’s role in degeneration of nigrostriatal neurons 142 References 147 D.2.6. Role of endoplasmic reticulum stress in degeneration of nigrostriatal neurons 150 References 154 D.3. Diagnosis of idiopathic Parkinson’s disease 156 Reference 157 E. Genetic Parkinson’s disease 157 E.1. Autosomal-dominant mutations 157 E.2. Autosomal-recessive mutations 160 References 168

2. Parkinson’s pharmacological therapy Dopaminergic drugs Anticholinergic drugs Other pharmacological treatments New targets and disease-modifying drugs for Parkinson’s disease treatment in phase 3 Drugs in clinical trials References

173 176 177 177 181 182

3. Dopamine synthesis Tyrosine hydroxylase Aromatic amino acid decarboxylase References

187 189 192

4. Dopamine storage and release References

200

5. Dopamine oxidative deamination Monoamine oxidases

203

Contents

Monoamine oxidase-B References

ix 204 206

6. Dopamine methylation Catechol ortho-methyltransferase References

209 212

7. Dopamine oxidation to neuromelanin and neurotoxic metabolites Dopamine ortho-quinone Aminochrome 5,6-Indolequinone Dopaminochrome Neuromelanin References

213 215 218 218 221 223

8. Neuroprotective mechanisms against dopamine oxidation-dependent neurotoxicity Vesicular monoamine transporter-2 DT-diaphorase Glutathione transferase-M2-2 Astrocytes neuroprotection against aminochrome neurotoxicity References

229 230 232 234 237

9. Exogenous neurotoxins as a preclinical model for Parkinson’s disease 6-Hydroxydopamine 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine Rotenone Exogenous neurotoxin preclinical models for Parkinson’s disease References

241 243 245 246 249

10. Preclinical models based on genetic mutations associated with the familial form of Parkinson’s disease References

259

11. Preclinical models based on endogenous neurotoxins Alpha-synuclein

264

x 3,4-Dihydroxyphenylacetaldehyde Aminochrome References

Contents

267 269 277

12. Conclusions Index

287

C H A P T E R

1 Parkinson’s disease A. Epidemiology Parkinson’s disease is the second-most prevalent neurodegenerative disease after Alzheimer disease. The prevalence in developed countries is 1% in populations over 60 years old [1,2], but prevalence increases with age to 4% for those over 80 years of age, suggesting that age is a risk factor in Parkinson’s disease. A prevalence of 3.5% has been estimated for Parkinson’s disease at 85e89 years of age in Europe [3]. The incidence of Parkinson’s disease in developed countries is 8 to 18 persons per 100,000 [1]. A meta-analysis of 13 publications showed that the prevalence of Parkinson’s disease in China was lower than in developed countries, but the incidence of Parkinson’s disease was higher. The prevalence of Parkinson’s disease also increases with age in China, and a higher prevalence of Parkinson’s disease was found in men than in women in China [4]. Sex differences in Parkinson’s disease prevalence in developed countries is controversial due to some reports suggest a higher prevalence of Parkinson’s disease in men than in women, whereas other reports did not find significant differences in Parkinson’s disease prevalence between gender [1]. The prevalence of Parkinson’s disease in South Africa was 2.8 times higher in white compared with black patients [5]. A global epidemiological study revealed a marked increase in the prevalence of Parkinson’s disease by comparing data from 1990 to 2016. In 1990, 2.5 million individuals had Parkinson’s disease globally compared with 6.1 million in 2016. This study also revealed that Parkinson’s disease caused 211,000 deaths in 2016 [6]. These results can be explained by the fact that age is a risk factor for the disease and the increase in life expectancy implies an increase in the number of elderly people who are living longer. However, in this study the increase in prevalence is also observed in most regions when the results are compared by age. This

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global study also pointed to higher Parkinson’s disease prevalence in men than in women and also that industrialized countries experienced the highest increases in mortality and prevalence associated with Parkinson’s disease [6].

References [1] de Lau LM, Breteler MM. Epidemiology of Parkinson’s disease. Lancet Neurol 2006;5: 525e35. [2] Tysnes OB, Storstein A. Epidemiology of Parkinson’s disease. J Neural Transm 2017;124: 901e5. [3] Clarke CE, Moore AP. Parkinson’s disease. Am Fam Physician 2007;75:1045e8. [4] Ma CL, Su L, Xie JJ, Long JX, Wu P, Gu L. The prevalence and incidence of Parkinson’s disease in China: a systematic review and meta-analysis. J Neural Transm 2014;121: 123e34. [5] Amod FH, Bhigjee AI. Clinical series of Parkinson’s disease in KwaZulu-Natal, South Africa: retrospective chart review. J Neurol Sci 2019;401:62e5. [6] GBD 2016 Parkinson’s Disease Collaborators. Global, regional, and national burden of Parkinson’s disease, 1990e2016: a systematic analysis for the global burden of disease study 2016. Lancet Neurol 2018;17:939e53.

B. Parkinson’s disease classification The concept of Parkinson’s disease is used indiscriminately without considering the origin of symptoms. Idiopathic Parkinson’s disease is the largest of the parkinsonian groups (70%), and the cause of this disease is still unknown. The second-most important parkinsonian group is those who have a defined reason for motor symptoms, and this group represents about 20% of parkinsonian cases. The third-most important group by number of cases is the familial form of Parkinson’s disease, in which the disease is associated with a specific mutation that is responsible for 5% to 10% of total parkinsonian Fig. 1.1.

C. Parkinsonism C.1. Drug-induced parkinsonism The second cause of parkinsonism is drug-induced parkinsonism in which parkinsonism is induced by a known agent [1]. The existence of drug-induced parkinsonism is an important factor in erroneous diagnoses in Parkinson’s disease. Clinical insecurity in Parkinson’s disease diagnosis is usual when patients are prescribed dopamine-blocking medications; it has been suggested that DAT-SPECT imaging can improve diagnostic certainty [2]. DAT-SPECT imaging is a useful tool in the early diagnosis of Parkinson’s disease and allow for other

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FIGURE 1.1 Parkinson’s disease classification. The largest parkinsonian group is classified as idiopathic Parkinson’s disease. The second-largest parkinsonian syndrome group is classified as patients with parkinsonism. Finally, the smallest parkinsonian syndrome group is composed of genetic Parkinson’s disease.

nondegenerative parkinsonian disorders such drug-induced parkinsonism, dystonic tremor, psychogenic parkinsonism and essential tremor to be disregarded because this technique can truthfully detect dopaminergic presynaptic deficit [3,4]. Transcranial sonography of the substantia nigra has been extensively used to diagnose Parkinson’s disease, and it can also be used to diagnose

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drug-induced parkinsonism. Transcranial sonographic substantia nigra echogenicity of drug-induced parkinsonism patients and control are the same, contrasting with a significant increase in echogenicity in Parkinson’s disease patients [5]. It has been suggested that nonmotor symptoms, principally excessive daytime, urinary symptoms, restless leg syndrome, sleepiness, hyposmia and attention deficit may be useful to discriminate between Parkinson’s disease and drug-induced parkinsonism in the early stages [6]. Drugs used as antipsychotic agents, antidepressants, cholinomimetics, antiemetics, antiepileptic drugs, antivertigo medications, calcium channel antagonists, and antiarrhythmics have been associated with druginduced parkinsonism [7]. The symptoms of drug-induced parkinsonism induced by drug side effects will disappear when the offending drug is withdrawn. However, in some patients, symptoms persist or may worsen over time when neuroleptics have been withdrawn, suggesting that a degenerative process has probably been initiated in the patient before the use of neuroleptics that hastened this process. We must remember that motor symptoms appear after the loss of 60% to 70% of dopaminergic neurons that contain neuromelanin. A causal relationship has been suggested between neuroleptic exposure and Parkinson’s disease based on the unexpectedly high prevalence of Parkinson’s disease following neuroleptic exposure [8]. Trimetazidine, an antianginal drug used in European countries and Asia, was associated with parkinsonism in trimetazidine users [9]. Tacrolimus treatment prescribed for immunosuppression after orthotopic liver transplant induced severe, symmetric parkinsonism, which included resting tremor, rigidity, bradykinesia, and postural instability [10]. Lenalidomide has been reported to induce reversible parkinsonism [11]. This parkinsonism is induced by a blockade of postsynaptic receptors for dopamine that is generally reversible and is directly related to the dose. Drug-induced parkinsonism symptoms in general disappear within 6 months of withdrawal of drug treatment. In a study done in Olmsted County, Minnesota, with 364 incident cases of parkinsonism, 20% of the cases were drug-induced parkinsonism [12]. A 3-decade-long study on drug-induced parkinsonism was performed in a geographically demarcated American population (Olmsted County, Minnesota, from 1976 through 2005). Of 906 cases of parkinsonism from 1976 to 2005, 11.5% of the total, or 108 individuals, presented drug-induced parkinsonism [13]. A recent publication estimates the prevalence of druginduced parkinsonism in users of antipsychotics at 20 to 35% [14]. A study done in France with stabilized schizophrenia patients revealed that the prevalence of group-induced parkinsonism was 13% [15]. It was reported that 12% of a population aged 75 years or more exhibited drug-induced parkinsonism in Brazil [16]. In Korea, the annual

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FIGURE 1.2 Drugs used for therapeutic treatment that induce parkinsonism. Typical antipsychotic drugs that induce parkinsonism include haloperidol, amisulpride, sulpiride, levomepromazine, and promazine, calcium channel antagonists (L-channel) such as diltiazem, antidepressants such as fluoxetine and sertraline, antiarrhythmics such as amiodarone, and antiemetic and gastric mobility agents such as itopride [17].

prevalence of drug-induced parkinsonism was reported to have increases from 0.000041% to 0.00007% from 2009 to 2015, and the common drug that induced drug-induced parkinsonism comprised derivatives of benzamide [17]. It is generally believed that drug-induced parkinsonism is

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described by symmetry of symptoms. A study of 11 patients with a diagnosis of drug-induced parkinsonism and asymmetric symptoms with SPECT-DaTSCAN did not confirm asymmetry of drug-induced parkinsonism [18]. A study of 21 cases of drug-induced parkinsonism revealed that the age at onset was between 40 and 87 years with a Hoehn and Yahr Scale score of 4%, and 70% of the cases were induced by sulpiride. Interestingly, the drugs responsible for drug-induced parkinsonism were not prescribed by neurological or psychiatric departments [19] Fig. 1.2.

References [1] Brigo F, Erro R, Marangi A, Bhatia K, Tinazzi M. Differentiating drug-induced parkinsonism from Parkinson’s disease: an update on non-motor symptoms and investigations. Park Relat Disord 2014;20:808e14. [2] Yomtoob J, Koloms K. Bega D DAT-SPECT imaging in cases of drug-induced parkinsonism in a specialty movement disorders practice. Park Relat Disord 2018;53:37e41. [3] Ba F, Martin WR. Dopamine transporter imaging as a diagnostic tool for parkinsonism and related disorders in clinical practice. Park Relat Disord 2015;21:87e94. [4] Rodriguez-Porcel F, Jamali S, Duker AP, Espay AJ. Dopamine transporter scanning in the evaluation of patients with suspected Parkinsonism: a case-based user’s guide. Expert Rev Neurother 2016;16:23e9. [5] Oh YS, Kwon DY, Kim JS, Park MH, Berg D. Transcranial sonographic findings may predict prognosis of gastroprokinetic drug-induced parkinsonism. Park Relat Disord 2018;46:36e40. [6] Kim JS, Youn J, Shin H, Cho JW. Nonmotor symptoms in drug-induced parkinsonism and drug-naı¨ve Parkinson disease. Can J Neurol Sci 2013;40:36e41. [7] Mena MA, de Yebenes JG. Drug-induced parkinsonism. Expet Opin Drug Saf 2006;5: 759e71. [8] Erro R, Bhatia KP, Tinazzi M. Parkinsonism following neuroleptic exposure: a doublehit hypothesis? Mov Disord 2015;30:780e5. [9] Kwon J, Yu YM, Kim S, Jeong KH, Lee E. Association between trimetazidine and parkinsonism: a population-based study. Neuroepidemiology 2019;52:220e6.  [10] Gmitterova´ K, Mina´r M, Zigrai M, Kosutzka´ Z, Kusnı´rova´ A, Valkovic P. Tacrolimusinduced parkinsonism in a patient after liver transplantation - case report. BMC Neurol 2018;18:44. [11] Argente-Escrig H, Martinez JC, Go´mez E, Balaguer A, Sevilla T, Bataller L. Lenalidomide induced reversible parkinsonism, dystonia, and dementia in subclinical Creutzfeldt-Jakob disease. J Neurol Sci 2018;393:140e1. [12] Bower JH, Maraganore DM, McDonnell SK, Rocca WA. Incidence and distribution of parkinsonism in Olmsted County, Minnesota, 1976e1990. Neurology 1999;52: 1214e20. [13] Savica R, Grossardt BR, Bower JH, Ahlskog JE, Mielke MM, Rocca WA. Incidence and time trends of drug-induced parkinsonism: a 30-year population-based study. Mov Disord 2017;32:227e34. [14] Ward KM, Citrome L. Antipsychotic-related movement disorders: drug-induced parkinsonism vs. Tardive dyskinesia-key differences in pathophysiology and clinical management. Neurol Ther 2018;7:233e48. [15] Misdrahi D, Tessier A, Daubigney A, et al. Prevalence of and risk factors for extrapyramidal side effects of antipsychotics: results from the national FACE-SZ cohort. J Clin Psychiatr 2019;80. pii: 18m12246.

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[16] Vale TC, Barbosa MT, Resende EPF, et al. Parkinsonism in a population-based study of individuals aged 75þ years: the Pieta` study. Park Relat Disord 2018;56:76e81. [17] Byun JH, Cho H, Kim YJ, Kim JS, Baik JS, Jang S, Ma HI. Trends in the prevalence of drug-induced parkinsonism in Korea. Yonsei Med J 2019;60:760e7. [18] Gajos A, Dąbrowski J, Bie nkiewicz M, Płachci nska A, Kusmierek J, Bogucki A. The symptoms asymmetry of drug-induced parkinsonism is not related to nigrostriatal cell degeneration: a SPECT-DaTSCAN study. Neurol Neurochir Pol 2019;53:311e4. [19] Shiraiwa N, Tamaoka A, Ohkoshi N. Clinical features of drug-induced Parkinsonism. Neurol Int 2018;10:7877.

C.2. Paraquat-induced parkinsonism Drug-induced parkinsonism is not only related to the use of drugs prescribed in disease treatment in which the drugs block postsynaptic receptors for dopamine. However, human beings are exposed to a large number of chemicals used in agriculture, domestic activities, and drug abuse. Exposure to glyphosate in the absence of protective equipment for over 3 h daily over a week induced parkinsonism symptoms such as bradykinesia, rigidity, reduced arm swing, mask face, and rest tremors [1]. Paraquat (1,10 -dimethyl-4e40 -bipyridinium dichloride) is a nonselective quaternary ammonium herbicide used in agriculture that is forbidden in many countries. The herbicide paraquat is used in agriculture and is sold in 90 countries, including the USA, Canada, Australia, Japan, New Zealand, Chile, China, and others [2]. Paraquat exposure has been associated with parkinsonism [3e8]. Paraquat can cross the bloodebrain barrier because systemic administration of paraquat results in regional distribution of the herbicide within the brain, mostly in the hypothalamus and prefrontal cortex [9]. Paraquat transport into dopaminergic neurons is mediated by dopamine transporter, but the divalent cation paraquat2þ must be converted to its monovalent cation paraquatþ to be the substrate for dopamine transporters. Microglial NADPH oxidase has been proposed to catalyze paraquat2þ reduction to paraquatþ. The organic cation transporter-3 also transports paraquat and is amply expressed in nondopaminergic neurons in the nigrostriatal region [10]. Mitochondrial quality control and dynamics play an essential role in the maintenance of mitochondrial function and homeostasis. Paraquat induces mitochondrial dysfunction by disturbing mitochondrial dynamics both in vitro and in vivo [11]. Paraquat induces mitochondrial damage by (1) decreasing mitochondrial cristae; (2) increasing autophagy vesicles and vacuole area; and (3) impairment of mitochondrial membrane potential and decrease ATP level [12]. Paraquat induces oxidative stress, and superoxide dismutase has been demonstrated to reduce motor slowing and mitochondrial damage [13]. Paraquat induces glutamate efflux-starting excitotoxicity mediated by reactive nitrogen species [14].

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Paraquat induces an increase in glucose uptake y that increases the levels of Naþ-glucose transporters isoform-1 proteins and glucose transporter type-4. Overexpression of alpha-synuclein triggers glucose accumulation and paraquat toxicity, which is reduced by inhibiting the pentose phosphate pathway and glucose metabolism/transport [15]. Maneb, a fungicide, has been reported to be associated with parkinsonism [16]. It has been reported that paraquat and maneb together exert a synergistic effect [17,18]. Paraquat and maneb induce activation of NADPH oxidase and ferroptosis in SH-SY5Y dopaminergic cells. NFE2L2 and PPARGC1a genes encode transcription factor participation in the regulation of antioxidant enzyme expression. NFE2L2 and PPARGC1a are involved in paraquat/maneb-induced parkinsonism [19]. Paraquat/maneb exposure induces toxicity in the hippocampus in the early life of rats that may impair memory, learning, and cognition [20]. In vivo, NADPH oxidase involvement in ferroptosis resulted in neuroinflammation, lipid peroxidation, reduced iron content, and neurodegeneration of dopaminergic neurons [21]. In vivo, paraquat induces memory impairment, motor deficit, oxidative stress, and neuroinflammation [22,23]. Paraquat induces severe lung damage resulting in pulmonary fibrosis, but the mechanism is unknown. Paraquat induces endoplasmic reticulum stress-dependent cell death in human lung epithelial A549 cells by disrupting the expression levels of unfolded protein response-related molecules [24]. Paraquat induces activation of autophagy in response to early endoplasmic reticulum stress, which is speeded in cells that overexpress wild-type apoptosis signal-regulating kinase 1 [25]. Paraquat also induces dysfunction of protein both proteasomal and lysosomal degradation systems. Paraquat impairs proteasomal activity, which is a late event in cell death progression and autophagy flux [26]. Paraquat induces cardiac contractile dysfunction, and ablation of the innate proinflammatory mediator toll-like receptor-4 improved paraquatdependent myocardial contractile dysfunction, probably through a reduction in inflammation, endoplasmic reticulum stress, and apoptosis [27]. Paraquat induces changes in miRNA expression such as downregulation of miR-17-5p expression, which suggests that miRNA involvement in the progression of neurodegeneration in paraquatinduced parkinsonism [18].

References [1] Zheng Q, Yin J, Zhu L, Jiao L, Xu Z. Reversible Parkinsonism induced by acute exposure glyphosate. Park Relat Disord 2018;50:121. [2] Bastı´as-Candia S, Zolezzi JM, Inestrosa NC. Revisiting the paraquat-induced sporadic Parkinson’s disease-like model. Mol Neurobiol 2019;56:1044e55.

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[3] Tanner CM, Kamel F, Ross GW, et al. Rotenone, paraquat, and Parkinson’s disease. Environ Health Perspect 2011;119:866e72. [4] Leo´n-Verastegui AG. Parkinson’s disease due to laboral exposition to paraquat. Rev Med Inst Mex Seguro Soc 2012;50:665e72. [5] Costello S, Cockburn M, Bronstein J, Zhang X, Ritz B. Parkinson’s disease and residential exposure to maneb and paraquat from agricultural applications in the central valley of California. Am J Epidemiol 2009;169:919e26. [6] Kamel F, Tanner C, Umbach D, Hoppin J, Alavanja M, Blair A, Comyns K, Goldman S, Korell M, Langston J. Pesticide exposure and self-reported Parkinson’s disease in the agricultural health study. Am J Epidemiol. 2007;165:364e74. [7] Le Couteur DG, Mc Lean AJ, Taylor MC, Woodham BL, Board PG. Pesticides and Parkinson’s disease. Biomed Pharmacother 1999;53:122e30. [8] Dick FD, De Palma G, Ahmadi A, Scott NW, Prescott GJ, Bennett J, et al. Environmental risk factors for Parkinson’s disease and parkinsonism: the Geoparkinson study. Occup Environ Med 2007;64:666e72. [9] Corasaniti MT, Strongoli MC, Pisanelli A, Bruno P, Rotiroti D, Nappi G, Nistico` G. Distribution of paraquat into the brain after its systemic injection in rats. Funct Neurol 1992;7:51e6. [10] Rappold PM, Cui M, Chesser AS, Tibbett J, Grima JC, Duan L, Sen N, Javitch JA, Tieu K. Paraquat neurotoxicity is mediated by the dopamine transporter and organic cation transporter-3. Proc Natl Acad Sci U S A 2011;108:20766e71. [11] Zhao F, Wang W, Wang C, Siedlak SL, Fujioka H, Tang B, Zhu X. Mfn2 protects dopaminergic neurons exposed to paraquat both in vitro and in vivo: implications for idiopathic Parkinson’s disease. Biochim Biophys Acta Mol Basis Dis 2017;1863:1359e70. [12] Wu S, Lei L, Song Y, Liu M, Lu S, Lou D, Shi Y, Wang Z, He D. Mutation of hop-1 and pink-1 attenuates vulnerability of neurotoxicity in C. elegans: the role of mitochondriaassociated membrane proteins in Parkinsonism. Exp Neurol 2018;309:67e78. [13] Filograna R, Godena VK, Sanchez-Martinez A, Ferrari E, Casella L, Beltramini M, Bubacco L, Whitworth AJ, Bisaglia M. Superoxide dismutase (SOD)-mimetic M40403 is protective in cell and fly models of paraquat toxicity. J. Biol. Chem. 2016;291:9257e67. [14] Shimuzu K, Matsubara K, Ohtaki K, Fujimaru S, Saito O, Shiono H. Paraquat induces long-lasting dopamine overflow through the excitotoxic pathway in the striatum of freely moving rats. Brain Res 2003;976:243e52. [15] Anandhan A, Lei S, Levytskyy R, et al. Glucose metabolism and AMPK signaling regulate dopaminergic cell death induced by gene (a-Synuclein)-environment (paraquat) interactions. Mol Neurobiol 2017;54:3825e42. [16] Ferraz HB, Bertolucci PH, Pereira JS, Lima JG, Andrade LA. Chronic exposure to the fungicide maneb may produce symptoms and signs of CNS manganese intoxication. Neurology 1988;38:550e3. [17] Thiruchelvam M, Richfield EK, Baggs RB, Tank AW, Cory-Slechta DA. The nigrostriatal dopaminergic system as a preferential target of repeated exposures to combined paraquat and maneb: implications for Parkinson’s disease. J. Neurosci. 2000;20:9207e14. [18] Wang Q, Zhan Y, Ren N, Wang Z, Zhang Q, Wu S, Li H. Paraquat and MPTP alter microRNA expression profiles, and downregulated expression of miR-17-5p contributes to PQ-induced dopaminergic neurodegeneration. J Appl Toxicol 2018;38:665e77. [19] Paul KC, Sinsheimer JS, Cockburn M, Bronstein JM, Bordelon Y, Ritz B. NFE2L2, PPARGC1a, and pesticides and Parkinson’s disease risk and progression. Mech Ageing Dev 2018;173:1e8. [20] Li B, He X, Sun Y, Li B. Developmental exposure to paraquat and maneb can impair cognition, learning and memory in Sprague-Dawley rats. Mol Biosyst 2016;12:3088e97. [21] Hou L, Huang R, Sun F, Zhang L, Wang Q. NADPH oxidase regulates paraquat and maneb-induced dopaminergic neurodegeneration through ferroptosis. Toxicology 2019;417:64e73.

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[22] Ishola IO, Akinyede AA, Adeluwa TP, Micah C. Novel action of vinpocetine in the prevention of paraquat-induced parkinsonism in mice: involvement of oxidative stress and neuroinflammation. Metab Brain Dis 2018;33:1493e500. [23] Ishola IO, Akataobi OE, Alade AA, Adeyemi OO. Glimepiride prevents paraquatinduced Parkinsonism in mice: involvement of oxidative stress and neuroinflammation. Fundam Clin Pharmacol 2019;33:277e85. [24] Omura T, Asari M, Yamamoto J, et al. Sodium tauroursodeoxycholate prevents paraquat-induced cell death by suppressing endoplasmic reticulum stress responses in human lung epithelial A549 cells. Biochem Biophys Res Commun 2013;432:689e94. [25] Niso-Santano M, Bravo-San Pedro JM, Go´mez-Sa´nchez R, Climent V, Soler G, Fuentes JM, Gonza´lez-Polo RA. ASK1 overexpression accelerates paraquat-induced autophagy via endoplasmic reticulum stress. Toxicol Sci 2011;119:156e68. [26] Navarro-Yepes J, Anandhan A, Bradley E, et al. Inhibition of protein ubiquitination by paraquat and 1-methyl-4-phenylpyridinium impairs ubiquitin-dependent protein degradation pathways. Mol Neurobiol 2016;53:5229e51. [27] Lei Y, Li X, Yuan F, Liu L, Zhang J, Yang Y, Zhao J, Han Y, Ren J, Fu X. Toll-like receptor 4 ablation rescues against paraquat-triggered myocardial dysfunction: role of ER stress and apoptosis. Environ Toxicol 2017;32:656e68.

C.3. Copper-induced parkinsonism Young workers exposed to high concentrations of copper in mineralprocessing refineries in Chile developed parkinsonism [1]. One of these workers reported that due to the high temperatures at which the copper melts, protective masks are removed, and therefore, workers inhale high concentrations of copper. He also mentioned that even the lunchroom was highly contaminated with copper. Wilson’s disease is an autosomalrecessive disease caused by mutations in a transmembrane coppertransporter ATPase (ATP7B gene) that transports copper excess from the liver to the bile for excretion from the body [2e4]. This inability to remove excess copper results in copper accumulation in multiple organs in the body including the brain. The typical symptoms of Wilson’s disease include cirrhosis, the ocular finding of KayserFleischer rings, and neurological manifestations [4]. A study of 281 Wilson’s disease patients evaluated over 3 decades revealed that the major symptoms of Wilson’s disease were neurological (69%). Interestingly, 62% of neurologic symptoms were caused by parkinsonism. This means that 43% of all patients in this study presented parkinsonism, and only 15% of total symptoms were hepatic [5]. Wilson’s disease is treated with copper chelators and zinc salts. Early genetic diagnosis of the disease is important, and new therapies are under development to circumvent ATP7B-deficiency [3]. In difficult cases, liver transplantation has been used, and tetrathiomolybdate salts are under clinical trial [6]. The ATP7B gene encodes an ATPase located in the liver transmembrane whose function is to transport excess copper from the liver to the bile for excretion. A mutation of the ATP7B gene implies an increase in

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FIGURE 1.3 Possible mechanism for copper-induced parkinsonism. Possible mechanism for copper-induced parkinsonism. The positive charges of copper (II) bind to the negative charges of hydroxyl groups when they are dissociated. This copperedopamine complex has affinity for the dopamine transporter that transports the complex into the cytosol of dopaminergic neurons. The copper (II) in the complex is reduced by oxidizing dopamine to aminochrome, releasing free copper (I). Aminochrome has been shown to be able to induce alpha-synuclein aggregation to neurotoxic oligomers, mitochondrial dysfunction, oxidative stress, autophagy dysfunction, endoplasmic reticulum stress, proteosomal dysfunction, and neuroinflammation.

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the concentration of copper in the liver, which goes into the bloodstream and accumulates in different tissues. The brain is one of the tissues where copper accumulates (Cu2þ). Under normal conditions, dopamine is released from dopaminergic neurons under neurotransmission and binds to postsynaptic dopaminergic receptors. Subsequently, reuptake of free dopamine from the interneuronal space is mediated by dopamine transporter localized in the membranes of presynaptic neurons. Under high levels of brain copper (Cu2þ), free dopamine in the intersynaptic space can form a complex with Cu2þ composed of a copper (Cu2þ) molecule and a dopamine molecule [7]. This dopamine-Cu2þ complex has affinity for the dopamine transporter that transports this complex into cytosolic dopaminergic neurons. In the cytosol of dopaminergic neurons, the dopamine-Cu2þ complex Cu2þ is reduced to Cuþ by oxidation of dopamine to aminochrome. In this oxidoreduction reaction, Cuþ and aminochrome are released from the complex. Aminochrome can be neurotoxic in dopaminergic neurons, but the enzyme DT-diaphorase prevents aminochrome neurotoxicity by catalyzing its two-electron reduction. However, when the aminochrome concentration is too high, the protective capacity of the DT-diaphorase enzyme is suppressed. Experiments with high concentrations of copper suppressed DT-diaphorase protective capacity inducing neurotoxicity and loss of tyrosine hydroxylase-positive neurons [8]. The ability of copper (Cu2þ) to complex with dopamine, its specific transport by neurons that express dopamine transporter, and the catalysis of dopamine oxidation to aminochrome in the copperedopamine complex explains the high incidence of parkinsonism in Wilson’s disease. Aminochrome is neurotoxic by inducing the aggregation of alpha-synuclein to neurotoxic oligomers, mitochondrial dysfunction, oxidative stress, autophagy dysfunction, endoplasmic reticulum stress, proteasomal system dysfunction, and neuroinflammation (Fig. 1.3).

References [1] Caviedes P, Segura-Aguilar J. The price of development in Chile. Neuroreport 2001;12: A25. [2] Bandmann O, Weiss KH, Kaler SG. Wilson’s disease and other neurological copper disorders. Lancet Neurol 2015;14:103e13. [3] Ranucci G, Polishchuck R, Iorio R. Wilson’s disease: prospective developments towards new therapies. World J Gastroenterol 2017;23:5451e6. [4] Capone K, Azzam RK. Wilson’s disease: a review for the general pediatrician. Pediatr Ann 2018;47:e440e4. [5] Taly AB, Meenakshi-Sundaram S, Sinha S, Swamy HS, Arunodaya GR. Wilson disease: description of 282 patients evaluated over 3 decades. Medicine 2007;86:112e21. [6] Członkowska A, Litwin T, Dusek P, Ferenci P, Lutsenko S, Medici V, Rybakowski JK, Weiss KH, Schilsky ML. Wilson disease. Nat Rev Dis Primers 2018;4:21.

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[7] Paris I, Dagnino-Subiabre A, Marcelain K, et al. Copper neurotoxicity is dependent on dopamine-mediated copper uptake and one-electron reduction of aminochrome in a rat substantia nigra neuronal cell line. J Neurochem 2001;77:519e29. [8] Dı´az-Ve´liz G, Paris I, Mora S, Raisman-Vozari R, Segura-Aguilar J. Copper neurotoxicity in rat substantia nigra and striatum is dependent on DT-diaphorase inhibition. Chem Res Toxicol 2008;21:1180e5.

C.4. Manganese-induced parkinsonism Prolonged and excessive manganese inhalation in occupational activities, such as mining, welding, and other industries, induces manganese accumulation in certain brain regions that causes extrapyramidal motor disorder and central nervous system dysfunctions [1e3]. High exposure to manganese results in an accumulation in the brain related to the control of motor and nonmotor functions, and exposure also induces progressive neuronal degeneration, specifically in the striatum, substantia nigra, subthalamic nucleus, and globus pallidus [4e8]. A welder exposed for 30 years presented rigidity, bradykinesia, writing clumsiness, masked face, and postural instability. Magnetic resonance imaging of the brain showed hyperintensity lesions in the bilateral globus pallidus, pontine tegmentum, dentate nucleus, midbrain, and cerebral white matter. The patient had high manganese levels of urine and serum [9]. A man occupationally exposed to manganese presented symptoms that included resting tremor, bradykinesia, and masked face. The patient was not responsive to levodopa treatment, and his fluorodopa PET scan was normal [10]. A man who received parenteral nutrition for intestinal failure presented a significantly increased manganese concentration and had developed resting tremor and extrapyramidal dyskinesia. This man did not have a history of parkinsonism, essential tremor, or other neurological symptoms [11]. Magnetic resonance imaging brain scanning of a welder showed bilateral hyperdensity of the globus pallidus and a high manganese serum level, and he presented with a paranoid psychotic state [12]. The fungicide maneb (manganese ethylene-bis-dithiocarbamate) induced parkinsonism in agricultural workers, who presented rigidity, bradykinesia, postural tremor, cerebellar signs, and nervousness, memory complaints, headache, fatigue, and sleepiness [13,14]. Six workers with high exposures to manganese at a ferromanganese factory had increased manganese concentrations in the blood and presented with bradykinesia and rigidity [15]. A postmortem study of a man with chronic manganese exposure revealed neurodegeneration in the basal ganglia, in which the pallidum

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was the most damaged, while the substantia nigra was not affected. In addition, this study did not find any differences in manganese distribution in the brain in comparison with control and Parkinson’s disease brain (Yamada, 1986). A patient with manganese toxicity secondary to endstage liver disease showed reduced striatal uptake of FDOPA, suggesting a dysfunction of the nigrostriatal system [16]. A young welder exposed to welding fumes presented rigidity, bradykinesia, and tremor, and 2 years later also presented anxiety, depression with psychotic features, and emotional dysfunction with irritability [17]. A young welder presented rigidity, tremors, progressive cognitive slowing, bradykinesia, and gait instability without effect of levodopa. Magnetic resonance image showed an increased signal intensity on T1-weighted in the bilateral basal ganglia and elevated level of manganese in the serum and urine [18]. Manganese induces motor and sensory disorders and cognitive and neuropsychiatric deficits [19,20]. In idiopathic Parkinson’s disease, the nigrostriatal neuronal system is affected, and manganese parkinsonism degeneration predominantly occurs with the globus pallidus [10]. The major pathological findings occur in the pallidum and striatum [21]. The correlation between dysregulation of dopamine neurotransmission and high manganese levels in the brain are controversial. Manganese exposure induce dopamine depletion has been reported [22,23], whereas other reports have not found differences [24]. A positron-emission tomography study showed remarkable damage in the striatum of manganese-exposed animals when they measured amphetamine-induced dopamine release [25]. It has been reported that degeneration of midbrain dopamine neurons is not involved in manganese-induced parkinsonism, and levodopa therapy does not affect this parkinsonism [26]. Glutamatergic neurotransmission has been reportedly altered by chronic manganese exposure by activating ionotropic glutamate receptors, impairing synaptic homeostasis and ionic concentration, inducing mitochondrial dysfunction, and impairing cellular metabolism [27,28]. Manganese is a cofactor for the enzyme glutamine synthetase and is expressed mainly in astrocytes. Glutamine synthetase catalyzes the synthesis of glutamine from glutamate and ammonium to mobilize glutamate from the brain to the liver and/or kidney for amino group elimination. Reduction in glutamine synthetase increases glutamate signaling and resulting in excitotoxicity [29]. Manganese accumulation in the basal ganglia exhibited enhanced sensitivity of postsynaptic glutamate receptors through stimulation of glutamate receptors [30]. Manganese depositions are mainly found in gamma-aminobutyric acidergic neurons of the globus pallidus of basal

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ganglia. The possible role of manganese on gamma-aminobutyric acidergic neurotransmission is controversial based on manganese exposure. Weekly administration of doses of manganese for 7e59 weeks to cynomolgus macaque monkeys revealed a reduction in glutamine synthetase expression in the globus pallidus with a concomitant increase in amyloid beta precursor-like protein-1 gene expression, but this study did not show differences in glutamatergic and gamma-aminobutyric acidergic neurotransmission compared with that of control animals [31]. Dopaminergic neurons are vulnerable to manganese exposure because the postsynaptic structures containing D1 receptors are affected, whereas manganese do not induced changes in gamma-aminobutyric acid receptors or muscarinic acetylcholine receptors [32]. Chronic manganese administration induces an increased in gamma-aminobutyric acid content in caudate nucleus of rats [33]. Manganese exposure increases this metal concentration in the brain and increase in gamma-aminobutyric acid and the ratio between gamma-aminobutyric acid to glutamate, suggesting an enhanced inhibitory transmission in the brain [34]. A decrease in brain GABA levels and uptake in synaptosomes was observed after manganese exposure [35]. It has been reported that the formation of alpha-synuclein oligomers is one of the major reasons of manganese neurotoxicity [36]. Manganese redox ability is probably involved in manganese neurotoxicity and atypical parkinsonism. Manganese (II) can oxidize to manganese (III) that is a potent oxidizing agent. Manganese (III) has been reported to oxidize dopamine to aminochrome both in the presence or absence of oxygen. Aminochrome has been reported to be neurotoxic when it is reduced by one electron to leukoaminochrome o-semiquinone radical, which autoxidize in the presence of oxygen generating reactive oxygen species [37,38]. In addition, aminochrome has been reported to induce mitochondrial dysfunction, neuroinflammation, protein degradation dysfunction of both proteasomal and lysosomal systems, endoplasmic reticulum stress and alpha-synuclein neurotoxic oligomers [39]. It has been reported that the formation of alpha-synuclein oligomers is one of the major reasons of manganese neurotoxicity [36]. Aminochrome generated during dopamine oxidation to neuromelanin in dopaminergic neurons containing neuromelanin has been reported to induce the formation neurotoxic oligomers [40]. Thus, it seems to be plausible that one-way manganese induces neurotoxicity is through oxidizing dopamine to aminochrome when manganese in the oxidized state (III) catalyzes dopamine oxidation to aminochrome in neurons where dopamine exists. Aminochrome is neurotoxic through inducing mitochondrial dysfunction, the aggregation of alpha-synuclein to neurotoxic oligomers, the dysfunction of both lysosomal and proteasomal protein degradation systems, endoplasmic reticulum stress, neuroinflammation, and oxidative stress [39], Fig. 1.4.

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FIGURE 1.4 Possible mechanism of manganese-induced parkinsonism. Manganese (II) is transported into dopaminergic neurons where manganese (II) is oxidized to manganese (III) that it is a potent oxidizing agent. Manganese (III) can oxidize dopamine to aminochrome in the presence or absence of oxygen. In dopaminergic neurons, the flavoenzyme DT-diaphorase reduces aminochrome with two electrons to leukoaminochrome, thus preventing aminochrome-induced neurotoxicity. The inhibition of DT-diaphorase allows aminochrome to induce alpha-synuclein aggregation to neurotoxic oligomers, mitochondrial dysfunction, protein degradation dysfunction of both lysosomal (autophagy) and proteasomal systems, endoplasmic reticulum stress, neuroinflammation, and oxidative stress. The neurotoxic metabolism of aminochrome induces neurodegeneration of dopaminergic neurons, and finally, manganese-induced parkinsonism.

References [1] Perl DP, Olanow CW. The neuropathology of manganese-induced parkinsonism. J Neuropathol Exp Neurol. 2007;66:675e82. [2] Dobson AW, Erikson KM, Aschner M. Manganese neurotoxicity. Ann N Y Acad Sci 2004;1012:115e28. [3] Kwakye GF, Paoliello MM, Mukhopadhyay S, Bowman AB, Aschner M. Manganeseinduced parkinsonism and Parkinson’s disease: shared and distinguishable features. Int J Environ Res Publ Health 2015;12:7519e40. [4] Klos K, Chandler M, Kumar N, Ahlskog J, Josephs K. Neuropsychological profiles of manganese neurotoxicity. Eur. J. Neurol. 2006;13:1139e41.

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[5] Bouabid S, Delaville C, De Deurwaerde`re P, Lakhdar-Ghazal N, Benazzouz A. Manganese-induced atypical parkinsonism is associated with altered Basal Ganglia activity and changes in tissue levels of monoamines in the rat. PLoS One 2014;9:e98952. [6] Uchino A, Noguchi T, Nomiyama K, Takase Y, Nakazono T, Nojiri J, Kudo S. Manganese accumulation in the brain: MR imaging. Neuroradiology 2007;49:715e20. [7] Peres TV, Schettinger MR, Chen P, et al. Manganese-induced neurotoxicity: a review of its behavioral consequences and neuroprotective strategies. BMC Pharmacol Toxicol 2016;17:57. [8] Josephs KA, Ahlskog JE, Klos KJ, Kumar N, Fealey RD, Trenerry MR, Cowl CT. Neurologic manifestations in welders with pallidal MRI T1 hyperintensity. Neurology 2005; 64:2033e9. [9] Sato K, Ueyama H, Arakawa R, Kumamoto T, Tsuda T. A case of welder presenting with parkinsonism after chronic manganese exposure. Rinsho Shinkeigaku 2000;40: 1110e5. [10] Kim JW, Kim Y, Cheong HK, Ito K. Manganese induced parkinsonism: a case report. J Kor Med Sci 1998;13:437e9. [11] Walter E, Alsaffar S, Livingstone C, Ashley SL. Manganese toxicity in critical care: case report, literature review and recommendations for practice. J Intensive Care Soc 2016; 17:252e7. [12] Verhoeven WM, Egger JI, Kuijpers HJ. Manganese and acute paranoid psychosis: a case report. J Med Case Rep 2011;12(5):146. [13] Ferraz HB, Bertolucci PHF, Pereira JS, Lima JGC, Andrade LAF. Chronic exposure to the fungicide maneb may produce symptoms and signs of CNS manganese intoxication. Neurology 1988;38:550e3. [14] Meco G, Bonifati V, Vanacore N, Fabrizio E. Parkinsonism after chronic exposure to the fungicide maneb (manganese ethylene-bis-dithiocarbamate). Scand J Work Environ Health 1994;20:301e5. [15] Huang CC1, Chu NS, Lu CS, Wang JD, Tsai JL, Tzeng JL, Wolters EC, Calne DB. Chronic manganese intoxication. Arch Neurol 1989;46:1104e6. [16] Criswell SR, Perlmutter JS, Crippin JS, et al. Reduced uptake of FDOPA PET in endstage liver disease with elevated manganese levels. Arch Neurol 2012;69:394e7. [17] Bowler RM, Koller W, Schulz PE. Parkinsonism due to manganism in a welder: neurological and neuropsychological sequelae. Neurotoxicology 2006;27:327e32. [18] Sadek AH, Rauch R, Schulz PE. Parkinsonism due to manganism in a welder. Int J Toxicol 2003;22:393e401. [19] Bakthavatsalam S, Das Sharma S, Sonawane M, Thirumalai V, Datta A. A zebrafish model of manganism reveals reversible and treatable symptoms that are independent of neurotoxicity. Dis Model Mech 2014;7:1239e51. [20] Scholten JM. On manganese encephalopathy; description of a case. Folia Psychiatr Neurol Neurochir Neerl 1953;56:878e84. [21] Kim Y, Kim JM, Kim JW, Yoo CI, Lee CR, Lee JH, Kim HK, Yang SO, Chung HK, Lee DS, Jeon B. Dopamine transporter density is decreased in parkinsonian patients with a history of manganese exposure: what does it mean? Mov Disord 2002;17:568e75. [22] Madison JL, Wegrzynowicz M, Aschner M, Bowman AB. Disease-toxicant interactions in manganese exposed huntington disease mice: early changes in striatal neuron morphology and dopamine metabolism. PLoS One 2012;7:e31024. [23] Seth PK, Chandra SV. Neurotransmitters and neurotransmitter receptors in developing and adult rats during manganese poisoning. Neurotoxicology 1984;5:67e76. [24] Calabresi P, Ammassari-Teule M, et al. A synaptic mechanism underlying the behavioral abnormalities induced by manganese intoxication. Neurobiol. Dis. 2001;8:419e32. [25] Guilarte TR, Burton NC, McGlothan JL, et al. Impairment of nigrostriatal dopamine neurotransmission by manganese is mediated by pre-synaptic mechanism(s): implications to manganese-induced parkinsonism. J Neurochem 2008;107:1236e47.

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[26] Guilarte TR. Manganese and Parkinson’s disease: a critical review and new findings. Environ. Health Perspect. 2010;118:1071e80. [27] Gavin CE, Gunter KK, Gunter TE. Manganese and calcium transport in mitochondria: implications for manganese toxicity. Neurotoxicology 1999;20:445e53. [28] Castilho RF, Ward MW, Nicholls DG. Oxidative stress, mitochondrial function, and acute glutamate excitotoxicity in cultured cerebellar granule cells. J Neurochem 1999; 72:1394e401. [29] Maciejewski PK, Rothman DL. Proposed cycles for functional glutamate trafficking in synaptic neurotransmission. Neurochem Int 2008;52:809e25. [30] Spadoni F, Stefani A, Morello M, Lavaroni F, Giacomini P, Sancesario G. Selective vulnerability of pallidal neurons in the early phases of manganese intoxication. Exp Brain Res 2000;135:544e51. [31] Burton NC, Guilarte TR. Manganese neurotoxicity: lessons learned from longitudinal studies in nonhuman primates. Environ Health Perspect 2009;117:325e32. [32] Eriksson H, Gillberg PG, Aquilonius SM, Hedstro¨m KG, Heilbronn E. Receptor alterations in manganese intoxicated monkeys. Arch Toxicol 1992;66:359e64. [33] Bonilla E. Increased GABA content in caudate nucleus of rats after chronic manganese chloride administration. J Neurochem 1978;31:551e2. [34] Garcia SJ, Gellein K, Syversen T, Aschner M. A manganese-enhanced diet alters brain metals and transporters in the developing rat. Toxicol Sci 2006;92:516e25. [35] Anderson JG, Fordahl SC, Cooney PT, Weaver TL, Colyer CL, Erikson KM. Manganese exposure alters extracellular GABA, GABA receptor and transporter protein and mRNA levels in the developing rat brain. Neurotoxicology 2008;29:1044e53. [36] Yan DY, Liu C, Tan X, et al. Mn-induced neurocytes injury and autophagy dysfunction in alpha-synuclein wild-type and knock-out mice: highlighting the role of alphasynuclein. Neurotox Res 2019;36:66e80. [37] Segura-Aguilar J, Lind C. On the mechanism of the Mn3(þ)-induced neurotoxicity of dopamine: prevention of quinone-derived oxygen toxicity by DT diaphorase and superoxide dismutase. Chem Biol Interact 1989;72:309e24. [38] Arriagada C, Paris I, Sanchez de las Matas MJ, et al. On the neurotoxicity mechanism of leukoaminochrome o-semiquinone radical derived from dopamine oxidation: mitochondria damage, necrosis, and hydroxyl radical formation. Neurobiol Dis 2004;16: 468e77. [39] Herrera A, Mun˜oz P, Steinbusch HWM, Segura-Aguilar J. Are dopamine oxidation metabolites involved in the loss of dopaminergic neurons in the nigrostriatal system in Parkinson’s disease? ACS Chem Neurosci 2017;8:702e11. [40] Mun˜oz P, Cardenas S, Huenchuguala S, et al. DT-diaphorase prevents aminochromeinduced alpha-synuclein oligomer formation and neurotoxicity. Toxicol Sci 2015;145: 37e47.

C.5. 1-Methyl-4-phenyl-1,2,3,6tetrahydropyridineeinduced parkinsonism Drug addicts using a “synthetic heroin” obtained on the streets of Northern California developed severe parkinsonism with typical symptoms of Parkinson’s disease such as tremor and asymmetry. They also developed nonmotor symptoms that included deficits in executive function and facial seborrhea. The most interesting observation was that the symptoms appeared only 3 days after the consumption of “synthetic

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heroin” [1]. These patients were responsive to L-dopa treatment, but some developed L-dopa-induced dyskinesias like those seen in Parkinson’s disease. One patient developed L-dopa-induced dyskinesias after 6 weeks of treatment with L-dopa. The analysis of this “synthetic heroin” revealed that it was almost pure 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a coproduct of 1-methyl-4-phenyl-4-propionoxypiperidine synthesis [2]. This discovery was published in 1983 and suggested that 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine induces selective degeneration in the substantia nigra [3]. This discovery provided a new direction for Parkinson’s disease research, because for the first time, a drug had induced nearly identical symptoms of the disease in humans, and 1-methyl-4-phenyl-4propionoxypiperidine was used as the model neurotoxin in the animal experimental model of Parkinson’s disease. Biochemical and histological studies performed in primates treated with 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine revealed that 80% of neurons were lost in the substantia nigra accompanied with a significant decrease in dopamine-2 receptor density [4]. Treatment of cats with 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine induces denervation of dopaminergic neurons and loss of dopamine uptake in the substantia nigra pars compacta, putamen, nucleus accumbens, and caudate nucleus. The animals presented Parkinson’s disease-like symptoms such as bradykinesia, rigidity, akinesia, and postural instability [5]. Treatment of marmosets with 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine induced partial cell loss in the ventral tegmental area but near total deletion of cells in the substantia nigra pars compacta. A severe loss of dopamine uptake was observed in the substantia nigra pars compacta, putamen, and caudate. The postsynaptic dopamine receptor D1 was increased after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment in the caudate and putamen [6]. Monkeys with 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine-induced parkinsonism treated chronic administration of L-dopa exhibited motor fluctuations, and dyskinesias. A significant decrease in presynaptic dopamine uptake was observed in putamen and caudate without an increase in striatal dopamine levels [7]. The selective neurotoxic action of 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine is not self-induced, because it requires biotransformation into a specific dopaminergic neurotoxin (1-methyl-4phenylpyridinium). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine is hydrophobic and volatile, and it can cross over the bloodebrain barrier, where it is converted to 1-methyl-4-phenylpyridinium dopaminergic neurotoxin in the astrocytes. Astrocyte cultures convert 1-methyl-4phenyl-1,2,3,6- tetrahydropyridine to the 1-methyl-4-phenylpyridinium ion. The monoamine oxidase inhibitor pargyline reduced the biotransformation of 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine. Monoamine oxidase B is responsible for the conversion of the 1-methyl-4-phenyl-

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1,2,3,6-tetrahydropyridine to 1-methyl-4-phenylpyridinium-selective dopaminergic neurotoxin [8e10]. 1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine induces loss in the dopamine nigrostriatal system accompanied with a decrease in dopamine metabolites dihydroxyphenylacetic acid and homovanillic acid and inhibition of dopamine uptake. No significant loss of dopamine in other brain areas was observed in 1-methyl-4-phenyl-1,2,5,6tetrahydropyridine-treated animals. The monoamine oxidase B inhibitor deprenyl, but not the monoamine oxidase-A inhibitor clorgiline, prevents 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine-induced degeneration [9]. The selectivity of nigrostriatal degeneration induced by the 1-methyl-4phenylpyridinium ion depends on its affinity with the dopamine transporter. The uptake of 1-methyl-4-phenylpyridinium ion into the striatum was inhibited by dopamine transporter inhibitor nomifensine and dopamine [11]. 1-Methyl-4-phenylpyridinium ion accumulates in mitochondria of dopaminergic neurons, inhibiting NAD þ reduction to the NADH required to drive mitochondrial electron transport. The lack of NADH inhibits both the mitochondrial electron transport and the coupled oxidative phosphorylation to produce ATP, inducing mitochondrial dysfunction [12]. The methyl-4-phenylpyridine ion inhibits mitochondrial complex I, as complex II is unaffected because succinate oxidation was not inhibited [13]. The ATP levels in the striatum, cerebellum, and ventral mesencephalon decreased in animals treated with 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine [14]. These results suggest that 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine-induced neurodegeneration involves mitochondrial dysfunction. Animals treated with 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine exhibit decreased VMAT-2 expression and activity [15]. The 1-methyl-4phenylpyridinium ion was taken up into synaptic vesicles from the mouse striatum, and this uptake was dependent on temperature and Mg2þ/ATP. This uptake was dopamine-, tetrabenazine-, and amphetamine-sensitive, suggesting that dopamine and 1-methyl-4phenylpyridinium ion uptake use the same vesicular transporter [16]. Dopamine uptake from cytosol to synaptic vesicles is mediated by vesicular monoamine transporter-2, and the presence of 1-methyl-4phenylpyridinium ion in dopaminergic neurons of the nigrostriatal system will compete with dopamine, resulting in the presence of cytosolic dopamine that autoxidizes to neuromelanin with the formation of neurotoxic orthoquinones. Incubation of the 1-methyl-4phenylpyridinium ion in a catecholaminergic cell line in the presence of dicoumarol, an inhibitor of DT-diaphorase that it is a protective enzyme against orthoquinone neurotoxicity, increases its neurotoxicity fourfold accompanied with DNA fragmentation and calpain activation. These results suggest that dopamine oxidation is involved in 1-methyl-4phenylpyridinium ion neurotoxicity [17] (Fig. 1.5).

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FIGURE 1.5 1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP)-induced neurotoxicity. 1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine in the brain is converted to the 1-methyl-4phenylpyridinium ion by monoamine oxidase B (MAO-B) in the astrocytes. 1-Methyl-4phenylpyridinium ion has very high affinity to dopamine transporter localized in the outer membrane of dopaminergic neurons. The dopaminergic neuron 1-methyl-4-phenylpyridinium ion accumulates inside mitochondria, inhibiting the complex I mitochondrial electron transport chain, which results in mitochondrial dysfunction. In addition, 1-methyl-4phenylpyridinium ion has high affinity to vesicular monoamine transport-2 (VMAT-2), thus competing with dopamine. Free cytosolic dopamine autoxidizes to form aminochrome, which can be neurotoxic.

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References [1] Williams A. MPTP toxicity: clinical features. J Neural Transm Suppl 1986;20:5e9. [2] Langston JW. The MPTP story. J Parkinsons Dis 2017;7:S11e9. [3] Langston JW, Ballard P, Tetrud JW, Irwin I. Chronic Parkinsonism in humans due to a product of meperidine-analog synthesis. Science 1983;219:979e80. [4] Hantraye P, Loc’h C, Tacke U, et al. "In vivo" visualization by positron emission tomography of the progressive striatal dopamine receptor damage occurring in MPTPintoxicated non-human primates. Life Sci 1986;39:1375e82. [5] Frohna PA, Rothblat DS, Joyce JN, Schneider JS. Alterations in dopamine uptake sites and D1 and D2 receptors in cats symptomatic for and recovered from experimental parkinsonism. Synapse 1995;19:46e55. [6] Gnanalingham KK, Smith LA, Hunter AJ, Jenner P, Marsden CD. Alterations in striatal and extrastriatal D-1 and D-2 dopamine receptors in the MPTP-treated common marmoset: an autoradiographic study. Synapse 1993;14:184e94. [7] Rioux L, Frohna PA, Joyce JN, Schneider JS. The effects of chronic levodopa treatment on pre- and postsynaptic markers of dopaminergic function in striatum of parkinsonian monkeys. Mov Disord 1997;12:148e58. [8] Langston JW, Irwin I, Langston EB, Forno LS. 1-Methyl-4-phenylpyridinium ion (MPPþ): identification of a metabolite of MPTP, a toxin selective to the substantia nigra. Neurosci Lett 1984;48:87e92. [9] Heikkila RE, Manzino L, Cabbat FS, Duvoisin RC. Protection against the dopaminergic neurotoxicity of 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine by monoamine oxidase inhibitors. Nature 1984;311:467e9. [10] Ransom BR, Kunis DM, Irwin I, Langston JW. Astrocytes convert the parkinsonism inducing neurotoxin, MPTP, to its active metabolite, MPPþ. Neurosci Lett. 1987;75: 323e8. [11] Shen RS, Abell CW, Gessner W, Brossi A. Serotonergic conversion of MPTP and dopaminergic accumulation of MPPþ. FEBS Lett 1985;189:225e30. [12] Ramsay RR, Dadgar J, Trevor A, Singer TP. Energy-driven uptake of N-methyl-4phenylpyridine by brain mitochondria mediates the neurotoxicity of MPTP. Life Sci 1986;39:581e8. [13] Ramsay RR, Salach JI, Singer TP. Uptake of the neurotoxin 1-methyl-4-phenylpyridine (MPPþ) by mitochondria and its relation to the inhibition of the mitochondrial oxidation of NADþ-linked substrates by MPPþ. Biochem Biophys Res Commun 1986;134: 743e8. [14] Chan P, DeLanney LE, Irwin I, Langston JW, Di Monte D. Rapid ATP loss caused by 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mouse brain. J Neurochem 1991;57: 348e51. [15] Singh S, Singh K, Patel S, et al. Nicotine and caffeine-mediated modulation in the expression of toxicant responsive genes and vesicular monoamine transporter-2 in 1-methyl 4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson’s disease phenotype in mouse. Brain Res 2008;1207:193e206. [16] Del Zompo M, Piccardi MP, Ruiu S, Quartu M, Gessa GL, Vaccari A. Selective MPPþ uptake into synaptic dopamine vesicles: possible involvement in MPTP neurotoxicity. Br J Pharmacol 1993;109:411e4. [17] Aguilar Herna´ndez R, Sa´nchez De Las Matas MJ, Arriagada C, et al. MPP(þ)-induced degeneration is potentiated by dicoumarol in cultures of the RCSN-3 dopaminergic cell line. Implications of neuromelanin in oxidative metabolism of dopamine neurotoxicity. Neurotox Res 2003;5:407e10.

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D. Idiopathic Parkinson’s disease For a long time, Parkinson’s disease was considered a movement disorder because its diagnosis was based on motor symptoms. However, patients presented several symptoms not related to motor impairment. The proposal of Parkinson’s disease stages, where nonmotor symptoms appear many years before motor symptoms provided an important input for research on nonmotor symptoms. According to Braak Parkinson’s disease stages, Lewy bodies and Lewy neurites appear years before motor symptoms become evident [1,2]. Alpha-synuclein is the major component of these abnormal proteinaceous bodies, for which the alpha-synuclein monomer aggregates to fibrils that are deposited in Lewy bodies [3,4]. The inclusion-body pathology appears first in the olfactory bulb/ anterior, olfactory nucleus, and medulla oblongata/pontine tegmentum and is denominated by stages 1e2, which are presymptomatic stages. Disease progression to stage 2 has not induced neurodegeneration in the substantia nigra. The ascendant progressions of the disease occur in the basal part of the midbrain and forebrain (stage 3) where Lewy neurites and Lewy bodies begin to form, initiating a very slow degeneration of dopaminergic neurons containing neuromelanin. Stages 3e4 affect the substantia nigra and other nuclear grays of the forebrain and midbrain, thus inducing pathological changes that result in the apparition of motor symptoms. In stages 5e6, the disease progresses to the mature neocortex, where the neurodegenerative process is in a very advanced stage, and the dark substantia nigra are very pale. All disease symptoms are fully developed after years of disease progression [1]. A timeline for the disease before the apparition of motor symptoms has been proposed. According to published works, the prodromal stage takes 2e5 years, the early motor stage takes 3e6 years, and the early to middle stages take 4e12 years. Between the third and ninth year of the early to middle stages, the disease is diagnosed when motor symptoms become evident. This implies that disease development is ongoing for 8 to 20 years before the onset of motor symptoms. This is important to consider when choosing preclinical models to represent what occurs in the disease, because the disease’s development process is extremely slow [5] Fig. 1.6.

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FIGURE 1.6 The development of Parkinson’s disease over time. Development of Parkinson’s disease spans many years. The disease has always been recognized by its motor symptoms, but nonmotor symptoms appear many years before motor symptoms do.

References [1] Braak H, Ghebremedhin E, Ru¨b U, Bratzke H, Del Tredici K. Stages in the development of Parkinson’s disease-related pathology. Cell Tissue Res 2004;318:121e34. [2] Braak H, Del Tredici K, Ru¨b U, de Vos RA, Jansen Steur EN, Braak E. Staging of brain pathology related to sporadic Parkinson’s disease. Neurobiol Aging 2003;24: 197e211.

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[3] Wakabayashi K, Takahashi H, Obata K, Ikuta F. Immunocytochemical localization of synaptic vesicle-specific protein in Lewy body-containing neurons in Parkinson’s disease. Neurosci Lett 1992;138:237e40. [4] Spillantini MG, Schmidt ML, Lee VMY, Trojanowski JQ, Jakes R, Goedert M. a-Synuclein in Lewy bodies. Nature 1997;388:839e40. [5] Schapira AHV, Chaudhuri KR, Jenner P. Non-motor features of Parkinson disease. Nat Rev Neurosci 2017;18:435e50.

D.1. Nonmotor symptoms A study performed in the United Kingdom using the Health Improvement Network’s primary care database included 8166 Parkinson’s disease patients and 46,775 individuals without the disease as control. The information extracted from this database includes early or possible prediagnostic symptoms that can appear years before other diagnoses such as motor symptoms (rigidity, tremor, neck stiffness, balance deficiencies, and shoulder stiffness), neuropsychiatric disorders (cognitive decay, memory deficiency, depression, anxiety, and apathy), autonomic symptoms (hypotension, constipation, urinary dysfunction, erectile dysfunction, and dizziness) and other symptoms (insomnia, hypersalivation, fatigue, anosmia, and rapid eye movement sleep behavior disorder). Based on symptoms reported, less than 1% were excluded from more evaluation; these included hypersalivation, anosmia, apathy, rapid eye movement sleep behavior disorder, and cognitive decay. Individuals who had been diagnosed with Parkinson’s disease 5 years before had a greater occurrence of tremor, balance impairment, constipation, hypotension, erectile dysfunction, urinary dysfunction, dizziness, fatigue, depression, and anxiety than individuals who did not develop the disease. Individuals who had been diagnosed with Parkinson’s disease 10 years before had a higher occurrence of tremor and constipation than individuals who did not develop Parkinson’s disease [1]. Nonmotor symptoms precede motor symptoms and can be a tool to detect individuals long before they develop motor symptoms. One of the problems of nonmotor symptom diagnosis is that the patient has not been in contact with a neurologist at an early stage before motor symptoms are present. A guideline for diagnosis, assessment, and management of nonmotor symptoms would improve early diagnosis and treatment [2].

References [1] Schrag A, Horsfall L, Walters K, Noyce A, Petersen I. Prediagnostic presentations of Parkinson’s disease in primary care: a case-control study. Lancet Neurol 2015;14:57e64. [2] De Rui M, Inelmen EM, Trevisan C, Pigozzo S, Manzato E, Sergi G. Parkinson’s disease and the non-motor symptoms: hyposmia, weight loss, osteosarcopenia. Aging Clin Exp Res 2020;32:1211e8.

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D.1.1. Olfactory dysfunction According Braak, stage one suggests that anterior olfactory nucleus and olfactory bulb degeneration result in nonmotor symptoms such as olfactory dysfunction. The frequency of olfactory dysfunction in Parkinson’s disease is controversial because some studies report that up to 90% of patients with the idiopathic form of the disease have hyposmia [1]. In a study performed in southern China of 110 patients with Parkinson’s disease, 40% had no olfactory dysfunction [2]. Another study reported that 30% of Parkinson’s disease patients with motor symptoms had no olfactory dysfunction in a study performed with 169 patients who had no differences in their responses to levodopa or motor scale [3]. A study with the aim to identify bromines for use in identifying olfactory dysfunction and explore the association between cognitive function and hyposmia in early, drug-naive, and nondemented Parkinson’s disease patients in China revealed that isovaleric acid and b-phenylethyl alcohol were the best compounds to detect hyposmia in early nondemented Parkinson’s disease patients. In addition, hyposmia was usual in nondemented, early, and drug-naive Parkinson’s disease patients [4]. A study on the effect of olfactory training was performed with Parkinson’s disease patients who also had hyposmia and demonstrated significant improvement in the identification of odors in an olfactory test after sameday or long-term training sessions [5]. A diet low in thiamin and folate before Parkinson’s disease diagnosis was found to be associated with olfactory dysfunction [6]. The olfactory function of Parkinson’s disease patients was determined using a smell identification test, showing that hyposmia was correlated with motor and nonmotor symptoms such as sleep disturbances, depression, anxiety, and autonomic dysfunction [7]. Evaluation of odoridentification capacity on both sides of the nose in 20 early-stage Parkinson’s disease cases without treatment was performed. All patients presented bilateral olfactory dysfunction, and no relationship was observed between the degree of bradykinesia, rigidity, tremor, or gait disturbance and olfactory test scores [8]. A prospective study of relatives of Parkinson’s disease patients was performed by testing olfactory identification, detection, and discrimination capacity and the nigrostriatal dopaminergic function by determining dopamine transporter activity using single-photon-emission computed tomography [123I] beta-CIT at baseline and 2 years from baseline. Ten percent of those with idiopathic hyposmia, who also had reduced nigrostriatal dopaminergic function at baseline, developed Parkinson’s disease [9]. A study of olfactory function performed in HonolulueAsia

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with a population over 70 years old and without Parkinson’s disease suggested that reduced olfaction can be detected 4 years before the onset of clinical Parkinson’s disease [10]. The raphe nuclei, the nucleus basalis of Meynert, and the locus coeruleus have been reported to be associated with olfactory dysfunction in Parkinson’s disease. In addition, the association of the autonomic nervous system with Parkinson’s disease symptoms indicates that insufficiencies in noradrenergic, serotonergic, and cholinergic function may contribute to olfactory dysfunction [11]. It has been reported that olfactory bulb volume is not changed in tremor-dominant Parkinson’s disease in comparison with controls, but in nontremor-dominant Parkinson’s disease, a decrease in olfactory bulb volume was observed. The same study did not observe a variation in olfactory bulb volume when comparing all Parkinson’s disease patients by Unified Parkinson’s Disease Rating Scale motor scores, duration of the disease, patient age, and age at onset of the disease [12]. The role of olfactory dysfunction in delirium after surgery under general anesthesia was studied in 34 Parkinson’s disease cases without or with postoperative delirium. This study revealed that olfactory dysfunction is associated with postoperative delirium in Parkinson’s disease patients [13]. Short-latency afferent inhibition response partly reveals central cholinergic dysfunction and a relationship with the severity of olfactory dysfunction in Parkinson’s disease [14]. It has been proposed that severe olfactory impairment and shortlatency afferent inhibition alterations support the hypothesis of cholinergic dysfunction in some Parkinson’s disease patients who will possibly develop a dementia [15]. It has been suggested that olfactory dysfunction can be used as a predictor of cognitive decline because olfactory dysfunction is correlated with other nonmotor symptoms of Parkinson’s disease [16]. A study performed with patients newly diagnosed with Parkinson’s disease with hyposmia revealed that 42% had developed dementia after 10 years, suggesting that olfactory dysfunction increases the risk of dementia [1]. A study with the aim to determine the association between cognitive function and olfactory dysfunction with Parkinson’s disease patients at the time of diagnosis showed that olfactory dysfunction was more dominant in Parkinson’s disease patients with mild cognitive impairment than in patients with normal cognition [17]. Parkinson’s disease patients presenting severe hyposmia have increased risk of developing dementia and cerebral metabolic weakening [18]. Hyposmia has been proposed as preceding clinical motor symptoms in Parkinson’s disease by several years because of neuropathological changes in the olfactory-related brain regions, and severe olfactory

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dysfunction is probably associated with Parkinson’s disease dementia [19]. However, a study performed with 63 Parkinson’s disease patients with cognitive impairment and olfactory dysfunction revealed that these nonmotor signs were negatively correlated [20]. The olfactory bulb volume is correlated with olfactory function, and a study revealed that olfactory bulb volume is minor in idiopathic Parkinson’s disease [21]. Olfactory dysfunction was determined to be closely associated with atrophy of brain regions such as the amygdala and other limbic structures via volumetric magnetic resonance imaging [18]. However, another study using high-resolution magnetic resonance imaging did not detect a volume loss of the olfactory bulb in Parkinson’s disease patients, suggesting that this technique is not suitable for detecting idiopathic Parkinson’s disease [22]. Quantitative analysis of the entire olfactory bulb revealed ventral glomerular insufficiency in Parkinson’s disease, and alpha-synuclein immunopositive voxels are correlated with lower global glomerular voxel volume [23]. A study on how olfactory dysfunction affects the white matter microstructure in untreated or newly diagnosed Parkinson’s disease patients showed that patients with hyposmia presented a significant reduction in local and global efficiency and a disrupted connection between the left rectus and right medial orbitofrontal cortex and had inferior frontal-related cognitive function. They propose that disconnectivity between the bilateral olfactory circuitry can be used as a marker for olfactory dysfunction in Parkinson’s disease [24]. A study focused on the relationship of olfactory dysfunction and gray matter volume in brain regions subtending primary and secondary olfactory handling revealed that Parkinson’s disease patients have greater gray matter volume loss confined to orbitofrontal cortex and bilateral piriform cortex [25]. A comparison of the reduction in olfactory perception between Parkinson’s disease patients and hyposmia patients with other causes revealed that Parkinson’s disease patients have a marked difference in late electroencephalographic components, thus indicating a decline in central brain networks as a fundamental factor for olfactory loss in Parkinson’s disease. They observed that the cause of olfactory dysfunction in Parkinson’s disease does not depend on damage to the peripheral olfactory system but rather on a more central deficit [26]. The correlation between olfactory dysfunction and a possible decrease in dopamine uptake mediated by dopamine transporter was determined in Parkinson’s disease patients with hyposmia using positron-emission tomography and magnetic resonance imaging. The left posterior and anterior putamen and the bilateral caudates of the hyposmia Parkinson’s disease patients exhibited significantly reduced dopamine transporter uptake [27].

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A study performed with 166 Parkinson’s disease without dementia demonstrated that 82% had olfactory dysfunction that was associated with disease severity determined by evaluating motor symptoms with the Unified Parkinson’s Disease Rating Scale III, Hoehn and Yahr, levodopa equivalent dose, and freezing of gait questionnaire [28]. The progression of olfactory dysfunction was studied in 25 Parkinson’s disease patients for 4 years using the University of Pennsylvania Smell Identification Test, and structural magnetic resonance imaging. They found that the olfactory test was positively correlated with volume change in the right thalamus, right caudate nucleus, and left putamen [29]. The possible correlation among motor and nonmotor symptoms such as cognitive impairment, olfactory function, fatigue and apathy was studied in 96 Parkinson’s disease patients. This study concluded that both motor symptoms and apathy were associated with olfactory dysfunction, suggesting that the development of olfactory dysfunction is associated with motor symptoms and nonmotor apathy symptoms [30]. A study on the relationship between lateralized microstructural changes in anterior olfactory structures (the anterior end of the olfactory tract and bulbs) and lateralized motor impairment was performed. Interestingly, no relationship was observed between lateralized microstructural changes in the anterior olfactory structures and lateralized motor impairment, suggesting that asymmetries in the anterior olfactory structures microstructure are not related to the laterality of Parkinson’s disease motor symptoms [31]. The possible role of comorbid depression on olfactory dysfunction was studied by comparing Parkinson’s 30 patients without depression and 30 with depression. This study demonstrated that depression does not contribute to olfactory impairment in Parkinson’s disease [32]. The level of serum vitamin D was found to be associated with olfactory dysfunction in Parkinson’s disease, although the mechanism is complete unknown and more research is required. [33, 34].

References [1] Domello¨f ME, Lundin KF, Edstro¨m M, Forsgren L. Olfactory dysfunction and dementia in newly diagnosed patients with Parkinson’s disease. Park Relat Disord 2017;38:41e7. [2] Wang XY, Han YY, Li G, Zhang B. Association between autonomic dysfunction and olfactory dysfunction in Parkinson’s disease in southern Chinese. BMC Neurol 2019;19:17. [3] Rossi M, Escobar AM, Bril A, et al. Motor features in Parkinson’s disease with normal olfactory function. Mov Disord 2016;31:1414e7. [4] Mao CJ, Wang F, Chen JP, Yang YP, Chen J, Huang JY, Liu CF. Odor selectivity of hyposmia and cognitive impairment in patients with Parkinson’s disease. Clin Interv Aging 2017;12:1637e44. [5] Knudsen K, Flensborg Damholdt M, Mouridsen K, Borghammer P. Olfactory function in Parkinson’s disease - effects of training. Acta Neurol Scand 2015;132:395e400. [6] Ha˚glin L1, Johansson I2, Forsgren L3, Ba¨ckman L1. Intake of vitamin B before onset of Parkinson’s disease and atypical parkinsonism and olfactory function at the time of diagnosis. Eur J Clin Nutr 2017;71:97e102.

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[7] Roos DS, Twisk JWR, Raijmakers PGHM, Doty RL, Berendse HW. Hyposmia as a marker of (non-) motor disease severity in Parkinson’s disease. J Neural Transm 2019;126:1471e8. [8] Doty RL, Stern MB, Pfeiffer C, Gollomp SM, Hurtig HI. Bilateral olfactory dysfunction in early stage treated and untreated idiopathic Parkinson’s disease. J Neurol Neurosurg Psychiatry 1992;55:138e42. [9] Ponsen MM, Stoffers D, Booij J, van Eck-Smit BL, Wolters ECh, Berendse HW. Idiopathic hyposmia as a preclinical sign of Parkinson’s disease. Ann Neurol 2004;56:173e81. [10] Ross GW, Petrovitch H, Abbott RD, Tanner CM, Popper J, Masaki K, Launer L, White LR. Association of olfactory dysfunction with risk for future Parkinson’s disease. Ann Neurol 2008;63:167e73. [11] Doty RL. Olfactory dysfunction in Parkinson disease. Nat Rev Neurol 2012;8:329e39. [12] Altinayar S, Oner S, Can S, Kizilay A, Kamisli S, Sarac K. Olfactory disfunction and its relation olfactory bulb volume in Parkinson’s disease. Eur Rev Med Pharmacol Sci 2014;18:3659e64. [13] Kim MS, Yoon JH, Kim HJ, Yong SW, Hong JM. Olfactory dysfunction is related to postoperative delirium in Parkinson’s disease. J Neural Transm 2016;123:589e94. [14] Oh E, Park J, Youn J, Kim JS, Park S, Jang W6. Olfactory dysfunction in early Parkinson’s disease is associated with short latency afferent inhibition reflecting central cholinergic dysfunction. Clin Neurophysiol 2017;128:1061e8. [15] Versace V, Langthaler PB, Sebastianelli L, Ho¨ller Y, Brigo F, Orioli A, Saltuari L, Nardone R. Impaired cholinergic transmission in patients with Parkinson’s disease and olfactory dysfunction. J Neurol Sci 2017;377:55e61. [16] Fullard ME, Morley JF, Duda JE. Olfactory dysfunction as an early biomarker in Parkinson’s disease. Neurosci Bull 2017;33:515e25. [17] Park JW, Kwon DY, Choi JH, Park MH, Yoon HK. Olfactory dysfunctions in drug-naı¨ve Parkinson’s disease with mild cognitive impairment. Park Relat Disord 2018;46:69e73. [18] Takeda A, Baba T, Kikuchi A, Hasegawa T, Sugeno N, Konno M, Miura E, Mori E. Olfactory dysfunction and dementia in Parkinson’s disease. J Parkinsons Dis 2014;4: 181e7. [19] Taniguchi S, Takeda A. Olfactory dysfunction. Nihon Rinsho 2017;75:119e23. [20] Guo S, Li L, Dai Y, Tang Q, Chen Y, Li S, Chen J, Mao C, Li J, Liu C. Correlations between olfactory and cognitive functions in early stage Parkinson’s disease. Zhonghua Yi Xue Za Zhi 2015;95:489e92. [21] Hang W, Liu G, Han T, Zhang P. Zhang J Olfactory function in patients with idiopathic Parkinson’s disease Zhonghua Er Bi. Yan Hou Tou Jing Wai Ke Za Zhi 2015;50:20e4. [22] Paschen L, Schmidt N, Wolff S, Cnyrim C, van Eimeren T, Zeuner KE, Deuschl G, Witt K. The olfactory bulb volume in patients with idiopathic Parkinson’s disease. Eur J Neurol 2015;22:1068e73. [23] Zapiec B, Dieriks BV, Tan S, Faull RLM, Mombaerts P, Curtis MA. A ventral glomerular deficit in Parkinson’s disease revealed by whole olfactory bulb reconstruction. Brain 2017;140:2722e36. [24] Wen MC, Xu Z, Lu Z, Chan LL, Tan EK, Tan LCS. Microstructural network alterations of olfactory dysfunction in newly diagnosed Parkinson’s disease. Sci Rep 2017;7:12559. [25] Lee EY, Eslinger PJ, Du G, Kong L, Lewis MM, Huang X. Olfactory-related cortical atrophy is associated with olfactory dysfunction in Parkinson’s disease. Mov Disord 2014;29:1205e8. [26] Iannilli E, Stephan L, Hummel T, Reichmann H, Haehner A. Olfactory impairment in Parkinson’s disease is a consequence of central nervous system decline. J Neurol 2017;264:1236e46. [27] Oh YS, Kim JS, Hwang EJ, Lyoo CH. Striatal dopamine uptake and olfactory dysfunction in patients with early Parkinson’s disease. Park Relat Disord 2018;56:47e51.

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[28] Cavaco S, Gonc¸alves A, Mendes A, et al. Abnormal olfaction in Parkinson’s disease is related to faster disease progression. Behav Neurol 2015;2015:976589. [29] Campabadal A, Uribe C, Segura B, et al. Brain correlates of progressive olfactory loss in Parkinson’s disease. Park Relat Disord 2017;41:44e50. [30] Masala C, Solla P, Liscia A, Defazio G, Saba L, Cannas A, Cavazzana A, Hummel T, Haehner A. Correlation among olfactory function, motors’ symptoms, cognitive impairment, apathy, and fatigue in patients with Parkinson’s disease. J Neurol 2018; 265:1764e71. [31] Joshi N, Rolheiser TM, Fisk JD, et al. Lateralized microstructural changes in early-stage Parkinson’s disease in anterior olfactory structures, but not in substantia nigra. J Neurol 2017;264:1497e505. [32] Rossi M, Perez-Lloret S, Millar Vernetti P, Drucaroff L, Costanzo E, Ballesteros D, Bril A, Cerquetti D, Guinjoan S, Merello M. Olfactory dysfunction evaluation is not affected by comorbid depression in Parkinson’s disease. Mov Disord 2015;30:1275e9. [33] Fullard ME, Duda JE. A Review of the Relationship Between Vitamin D and Parkinson Disease Symptoms. Front Neurol. 2020;11:454. [34] Kim JE, Oh E, Park J, Youn J, Kim JS, Jang W. Serum 25-hydroxyvitamin D3 level may be associated with olfactory dysfunction in de novo Parkinson’s disease. J Clin Neurosci. 2018;57:131e5.

D.1.2. Rapid eye movement sleep behavior disorder Rapid eye movement sleep behavior disorder is another nonmotor symptom in Parkinson’s disease, but this disorder also affects other neurodegenerative pathologies such as dementia with Lewy bodies and multiple-system atrophy. Under normal and healthy conditions, the skeletal muscles are in a state of motor paralysis during rapid eye movement sleep. Rapid eye movement sleep behavior disorder is characterized by the absence of motor paralysis, called atonia, during sleep, allowing active behavior during dreaming such as exaggerated and violent movements [1,2]. Rapid eye movement sleep behavior disorder is one of the premotor symptoms of Parkinson’s disease. However, a study reported that the prevalence of this premotor symptom is more prevalent in men than in women and also observed after the onset of motor symptoms. Less than 1% of the adult population is affected with rapid eye movement sleep behavior disorder, but 25% to 50% of patients with dementia with Lewy bodies, Parkinson’s disease, and multisystem atrophy are affected with this disorder [3]. Thirty-three to forty-six percent of Parkinson’s disease patients are affected by rapid eye movement sleep behavior disorder [4]. A metaand meta-regression analysis based on nearly 7000 patients concluded that the prevalence of rapid eye movement sleep behavior disorder was 42.3% [5]. A new meta-analysis reported that the risk for rapid eye movement sleep behavior disorder developing into neurodegeneration was 34%, 82%, and 97% after 5, 10, and 14 years, respectively [6]. Several hypotheses about the rapid eye movement sleep function have been proposed such as (1) a role for brain development [7,8]; (2) memory

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formation and consolidation [9e12]; and (3) restoration of aminergic neurons/receptor function. However, the reports supporting these functions are controversial, and therefore, rapid eye movement’s sleep function remains unknown [13]. It remains unclear whether rapid eye movement sleep behavior disorder is a secondary effect of neurodegeneration or directly influences neurodegenerative processes [14]. Rapid eye movement sleep behavior disorder is characterized by alpha-synuclein aggregations in the brainstem and sometimes violent and aggressive behavior during sleep with fleeing and fighting and actions [15e17]. In most cases, rapid eye movement sleep behavior disorder occurs before the existence of the synucleinopathies that cause degeneration in structures such as the caudal brainstem, where the circuits of rapid eye movement sleep are situated [1]. Rapid eye movement sleep behavior disorder in the absence of neurologic pathology is considered an idiopathic form of this sleep disorder. However, neuropsychological and neurophysiological function studies of idiopathic rapid eye movement sleep behavior disorder have reported indications of central nerve system dysfunction during both sleep and wakefulness in these patients, questioning the definition of the idiopathic form of this sleep disease in the absence of neurological disorders [18]. Rapid eye movement sleep behavior disorder is related to neurodegeneration, and some idiopathic and secondary forms of rapid eye movement sleep behavior disorder are associated with or related to narcolepsy, autoimmune disorders, focal brainstem damage, and drugs [3]. A study of 53 Parkinson’s disease patients with rapid eye movement sleep behavior disorder reported inferior and clinically reduced performance on several cognitive tests in comparison with healthy individuals and patients without this disorder. The authors suggested that rapid eye movement sleep behavior disorder is associated with higher mild cognitive impairment diagnosis frequency and a more damaged cognitive profile [4]. A study of the possible role of gender in rapid eye movement sleep behavior disorder using video polysomnography to determine muscular activity, sleep structure, and motor events during rapid eye movement sleep revealed that men have more motor events and muscular phasic activity [19]. A prospective study done with Parkinson’s disease patients using dopamine transporter positron-emission tomography revealed that patients with clinically probable rapid eye movement sleep behavior disorder have greater initial motor deficits and dopamine transporter activity in the putamen [20]. It has been suggested that dopamine transporter deficit will be useful in predicting synucleinopathy in idiopathic rapid eye movement sleep behavior disorder. Dopamine transporter activity was determined using dopamine transporter single-photon emission computed tomography in

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which the patients received a single injection of ligand 123-I-2b-carbomethoxy-3b-(4-iodophenyl)-N-(3-fluoropropyl) nortropane after thyroid blockade. A reduction in I-2b-carbomethoxy-3b-(4-iodophenyl)-N-(3fluoropropyl) nortropane putamen uptake of more than 25% is expected to develop synucleinopathy 3 years after examination [21]. A study on the correlation between rapid eye movement sleep behavior disorder and striatal dopamine transporter dysfunction was performed using three groups of patients (healthy controls, Parkinson’s disease, and rapid eye movement sleep behavior disorder). The rapid eye movement sleep behavior disorders were divided into two subgroups, abnormal or relatively normal striatal dopamine transporter binding. They reported modest correlations and suggested that nigrostriatal dopaminergic denervation cannot explain differences in network activity [22]. It has been proposed that increased submentalis rapid eye movement sleep in the absence of atonia is a possible biomarker for supposed synucleinopathy etiologies in parkinsonism because polysomnography curves from tauopathies are different from those of synucleinopathies [23]. The use of magnetic resonance imaging has been proposed to determine substantia nigra damage in idiopathic rapid eye movement sleep behavior disorder by determining neuromelanin levels. The motor symptoms in Parkinson’s disease appear when around 60% to 70% of dopaminergic neurons containing neuromelanin are lost, and therefore neuromelanin was considered a good marker for degeneration of the dopaminergic nigrostriatal system. Neuromelanin imaging in idiopathic rapid eye movement sleep behavior disorder revealed a decrease in neuromelanin volume, suggesting that this technique can be used to determine the level of neurodegeneration of nigrostriatal neurons [24]. The existence of common neurobiological pathways leading to development of rapid eye movement sleep behavior disorder, visual hallucinations, and cognitive impairment have been reported based on electrophysiology, neuroimaging, neuropsychological, clinical, and epidemiological studies. In addition, cholinergic dysfunction and gray matter atrophy of the parahippocampal cortices and hippocampus has been reported in Parkinson’s disease patients with rapid eye movement sleep behavior disorder, cognitive impairment, and visual hallucinations [25]. Parkinson’s disease patients with early onset of rapid eye movement sleep behavior disorder have high night urination frequency before motor symptoms occur [26]. The possible role of rapid eye movement sleep behavior disorder on obstructive sleep apnea was studied in 174 Parkinson’s disease patients. They found that rapid eye movement sleep behavior disorder decreases obstructive sleep apnea, and cognitive impairment was increased in Parkinson’s disease patients who had both

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rapid eye movement sleep behavior disorder and obstructive sleep apnea [27]. Rapid eye movement sleep behavior disorder in women with Parkinson’s disease was studied in 156 patients in which 27% developed rapid eye movement sleep behavior disorder. The most common features of rapid eye movement sleep behavior disorder in women are its onset after Parkinson’s symptoms, older age, reduced tremor score, and increased axial signs score [28]. It has been reported that alterations in gait and coupling of posture like those observed in Parkinson’s disease can also be detected in rapid eye movement sleep behavior disorder patients before findings of a neurodegenerative disorder. These results suggest that the pathogenesis of freezing of gait is associated with abnormal regulation of muscle tone in rapid eye movement sleep [29]. A study on balance and gait impairments in idiopathic rapid eye movement sleep behavior disorder patients revealed small impairments in these patients under fast-paced dual-task gait conditions, suggesting a premature progressive degeneration in the brainstem that regulates both gait coordination and rapid eye movement sleep [30]. Parkinson’s disease patients with rapid eye movement sleep behavior disorder exhibited a reduction in gray matter volume of the hippocampus and left posterior cingulate. In addition, gray matter was reduced in the left medial frontal gyrus, postcentral gyrus, precuneus, cuneus, and both inferior parietal lobules. No significant changes in white matter were observed [31]. The relationship between sleep-disordered breathing and rapid eye movement sleep behavior disorder was studied in Parkinson’s disease patients and showed that sleep-disordered breathing is more severe and frequent in Parkinson’s disease patients with rapid eye movement sleep behavior disorder and led to increased hypoxia risk during sleep [32]. It has been reported that rapid eye movement sleep behavior disorder is also present during childhood in neurodevelopmental disabilities associated with Moebius syndrome, autism, and SmitheMagenis syndrome, structural brainstem lesions associated with Chiari malformation type and Pontine glioma, primary hypersomnia/degeneration associated with narcolepsy/narcolepsyecataplexy, movement disorders associated with juvenile Tourette syndrome and Parkinson’s disease, and medicationinduced sleep disorder associated with selective serotonin reuptake inhibitors such as venlafaxine, clomipramine, and fluoxetine as well as tricyclic agents [33]. A study based on a rapid eye movement sleep behavior disorder screening questionnaire performed in adults over 50 years of age concluded that the presence of olfactory disturbance, imbalance, head injury, hyperlipidemia, lower education, constipation, atrial fibrillation, and the use of selective serotonin reuptake inhibitors, benzodiazepine, and alcohol are risk factors for developing rapid eye movement sleep

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behavior disorder [34]. In a study of idiopathic rapid eye movement sleep behavior disorder in those with no signs of Parkinson’s disease or dementia, 20 patients were examined using resting-state electroencephalography and exhibited a decrease in delta-band functional connectivity [35]. The possible relationship between idiopathic rapid eye movement sleep behavior disorder and neuroinflammation has been proposed, but a study performed with patients without dementia or parkinsonism exhibited no statistically significant difference in inflammation markers such as tumor necrosis factor-a, interleukin-2, interleukin-1b, interleukin6, and interleukin-10 [36]. It has been suggested that an increase in polysomnographic rapid eye movement sleep without atonia is probably a biomarker for diagnosis of mild cognitive impairment or parkinsonism [37,38]. A study with 45 newly diagnosed Parkinson’s disease patients suggested that rapid eye movement sleep behavior disorder in an initial stage of the disease is correlated with higher risk of functional dependency [36]. One of the features of synucleinopathies is a visual dysfunction also reported to be present in patients with idiopathic rapid eye movement sleep behavior disorder and Parkinson’s disease but in a lower severity. Visual dysfunction is also associated with age, motor symptoms, and cognitive decrease [37]. A study with the aim to understand the factors involved in excessive daytime sleepiness in Parkinson’s disease patients revealed that excessive daytime sleepiness seems to be associated with rapid eye movement sleep, age, cognitive impairment, male sex, and worsened quality of life [38]. A group of Parkinson’s disease patients evaluated with the Unified Parkinson’s Disease Rating Scale, video polysomnography to determine rapid eye movement sleep behavior disorder, the Starkstein apathy test, the Epworth sleepiness test, the mini-mental state examination, and Hamilton depression scales revealed that rapid eye movement sleep behavior disorder is associated with apathy and depressive symptoms [39]. A study performed with nearly 12,000 people who did not have Parkinson’s disease, dementia, stroke, head injury, or cancer concluded that people who consume alcohol have a higher probability of developing rapid eye movement sleep behavior disorder [40]. A study with the aim to investigate the sleep and circadian phenotypes in Parkinson’s disease patients reported that these patients showed reduced rapid eye movement sleep, reduced sleep efficiency, increased sleep latency, decreased circulating melatonin levels, increased cortisol levels, and changed Bmal1 expression [41,42]. A randomized, doubleblind, placebo-controlled clinical study to test the ability of melatonin to reduce rapid eye movement sleep behavior disorder in 30 Parkinson’s disease patients with prolonged-release melatonin during 8 weeks and 4 weeks of observation pre- and posttreatment did not show a difference

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from the control group [43]. The main nonpsychotropic element of the Cannabis sativa plant cannabidiol reduces rapid eye movement sleep behavior disorder [2].

References [1] McKenna D, Peever J. Degeneration of rapid eye movement sleep circuitry underlies rapid eye movement sleep behavior disorder. Mov Disord 2017;32:636e44. [2] Chagas MH, Eckeli AL, Zuardi AW, et al. Cannabidiol can improve complex sleeprelated behaviours associated with rapid eye movement sleep behaviour disorder in Parkinson’s disease patients: a case series. J Clin Pharm Therapeut 2014t;39:564e6. [3] Bassetti CL, Bargiotas P. REM sleep behavior disorder. Front Neurol Neurosci 2018;41: 104e16. [4] Jozwiak N, Postuma RB, Montplaisir J, et al. REM sleep behavior disorder and cognitive impairment in Parkinson’s disease. Sleep 2017;40:zsx101. [5] Zhang X, Sun X, Wang J, Tang L, Xie A. Prevalence of rapid eye movement sleep behavior disorder (RBD) in Parkinson’s disease: a meta and meta-regression analysis. Neurol Sci 2017;38:163e70. [6] Galbiati A, Verga L, Giora E, Zucconi M, Ferini-Strambi L. The risk of neurodegeneration in REM sleep behavior disorder: a systematic review and meta-analysis of longitudinal studies. Sleep Med Rev 2019;43:37e46. [7] Lyamin OI, Lapierre JL, Mukhametov LM. Sleep in aquatic species. In: Kushida C, editor. The encyclopedia of sleep, vol. 1. Waltham, MA: Academic Press; 2013. p. 57e62. [8] Roffwarg HP, Muzio JN, Dement WC. Ontogenetic development of the human sleepdream cycle. Science 1966;152:604e19. [9] Frank MG. Sleep and plasticity in the visual cortex: more than meets the eye. Curr Opin Neurobiol 2017;44(8):12e71. [10] Dumoulin Bridi MC, Aton SJ, Seibt J, Renouard L, Coleman T, Frank MG. Rapid eye movement sleep promotes cortical plasticity in the developing brain. Sci Adv 2015;1: e1500105. [11] Poe GR, Walsh CM, Bjorness TE. Cognitive neuroscience of sleep. Prog Brain Res 2010; 185:1e19. [12] Diekelmann S, Born J. The memory function of sleep. Nat Rev Neurosci 2010;11:114e26. [13] Peever J, Fuller PM. The biology of REM sleep. Curr Biol 2017;27:R1237e48. [14] Pillai JA, Leverenz JB. Sleep and neurodegeneration: a critical appraisal. Chest 2017; 151:1375e86. [15] Barone DA, Henchcliffe C. Rapid eye movement sleep behavior disorder and the link to alpha-synucleinopathies. Clin Neurophysiol 2018;129:1551e64. [16] Howell MJ, Schenck CH. Rapid eye movement sleep behavior disorder and neurodegenerative disease. JAMA Neurol 2015;72:707e12. [17] Reichmann H. Premotor diagnosis of Parkinson’s disease. Neurosci Bull 2017;33: 526e34. [18] Ferini-Strambi L, Marelli S, Galbiati A, Rinaldi F, Giora E. REM sleep behavior disorder (RBD) as a marker of neurodegenerative disorders. Arch Ital Biol 2014;152:129e46. [19] Bugalho P, Salavisa M. Factors influencing the presentation of REM sleep behavior disorder: the relative importance of sex, associated neurological disorder, and context of referral to polysomnography. J Clin Sleep Med 2019. pii: jc-18-00773. [20] Chung SJ, Lee Y, Lee JJ, Lee PH, Sohn YH. Rapid eye movement sleep behaviour disorder and striatal dopamine depletion in patients with Parkinson’s disease. Eur J Neurol 2017;24:1314e9.

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[21] Iranzo A, Santamarı´a J, Valldeoriola F, et al. Dopamine transporter imaging deficit predicts early transition to synucleinopathy in idiopathic rapid eye movement sleep behavior disorder. Ann Neurol 2017;82:419e28. [22] Huang Z, Jiang C, Li L, et al. Correlations between dopaminergic dysfunction and abnormal metabolic network activity in REM sleep behavior disorder. J Cerebr Blood Flow Metabol 2020;40:552e62. [23] McCarter SJ, Feemster JC, Tabatabai GM, et al. Submentalis rapid eye movement sleep muscle activity: a potential biomarker for synucleinopathy. Ann Neurol 2019;86: 969e74. [24] Pyatigorskaya N, Gaurav R, Arnaldi D, et al. Magnetic resonance imaging biomarkers to assess substantia nigra damage in idiopathic rapid eye movement sleep behavior disorder. Sleep 2017;40. https://doi.org/10.1093/sleep/zsx149. [25] Lenka A, Hegde S, Jhunjhunwala KR, Pal PK. Interactions of visual hallucinations, rapid eye movement sleep behavior disorder and cognitive impairment in Parkinson’s disease: a review. Park Relat Disord 2016;22:1e8. [26] Nodel MR, Ukraintseva YV, Yakhno NN. Syndrome of rapid eye movement sleep behavior disorder and nocturia in Parkinson’s disease. Zh Nevrol Psikhiatr Im S S Korsakova 2017;117:15e20. [27] Huang JY, Zhang JR, Shen Y, et al. Effect of rapid eye movement sleep behavior disorder on obstructive sleep apnea severity and cognition of Parkinson’s disease patients. Chin Med J 2018;131:899e906. [28] Mahale RR, Yadav R, Pal PK. Rapid eye movement sleep behaviour disorder in women with Parkinson’s disease is an underdiagnosed entity. J Clin Neurosci 2016;28:43e6. [29] Alibiglou L, Videnovic A, Planetta PJ, Vaillancourt DE, MacKinnon CD. Subliminal gait initiation deficits in rapid eye movement sleep behavior disorder: a harbinger of freezing of gait? Mov Disord 2016;31:1711e9. [30] Ehgoetz Martens KA, Matar E, Hall JM, Phillips J, Szeto JYY, Gouelle A, Grunstein RR, Halliday GM, Lewis SJG. Subtle gait and balance impairments occur in idiopathic rapid eye movement sleep behavior disorder. Mov Disord 2019;34:1374e80. [31] Lim JS, Shin SA, Lee JY, Nam H, Lee JY, Kim YK. Neural substrates of rapid eye movement sleep behavior disorder in Parkinson’s disease. Park Relat Disord 2016;23:31e6. [32] Zhang LY, Liu WY, Kang WY, Yang Q, Wang XY, Ding JQ, Chen SD, Liu J. Association of rapid eye movement sleep behavior disorder with sleep-disordered breathing in Parkinson’s disease. Sleep Med 2016;20:110e5. [33] Kotagal S. Rapid eye movement sleep behavior disorder during childhood. Sleep Med Clin 2015;10:163e7. [34] Ma C, Pavlova M, Li J, Liu Y, Sun Y, Huang Z, Wu S, Gao X. Alcohol consumption and probable rapid eye movement sleep behavior disorder. Ann Clin Transl Neurol 2018;5: 1176e83. [35] Sunwoo JS, Lee S, Kim JH, et al. Altered functional connectivity in idiopathic rapid eye movement sleep behavior disorder: a resting-state EEG study. Sleep 2017;40. https:// doi.org/10.1093/sleep/zsx058. [36] Kim R, Yoo D, Im JH, Kim HJ, Jeon B. REM sleep behavior disorder predicts functional dependency in early Parkinson’s disease. Park Relat Disord 2019;66:138e42. [37] (a) Li Y, Zhang H, Mao W, Liu X, Hao S, Zhou Y, Ma J, Gu Z, Chan P. Visual dysfunction in patients with idiopathic rapid eye movement sleep behavior disorder. Neurosci Lett 2019;709:134360. (b) McCarter SJ, Sandness DJ, McCarter AR, Feemster JC, Teigen LN, Timm PC, Boeve BF, Silber MH, St Louis EK. REM sleep muscle activity in idiopathic REM sleep behavior disorder predicts phenoconversion. Neurology 2019;93:e1171e9. ¨ , Benbir Senel G, Karadeniz D. Rapid eye movement sleep without atonia [38] (a) Dede HO constitutes increased risk for neurodegenerative disorders. Acta Neurol Scand 2019; 140:399e404. (b) Xiang YQ, Xu Q, Sun QY, Wang ZQ, Tian Y, Fang LJ, Yang Y,

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Tan JQ, Yan XX, Tang BS, Guo JF. Clinical features and correlates of excessive daytime sleepiness in Parkinson’s disease. Front Neurol 2019;10:121. Bargiotas P, Ntafouli M, Lachenmayer ML, Krack P, Schu¨pbach WMM, Bassetti CLA. Apathy in Parkinson’s disease with REM sleep behavior disorder. J Neurol Sci 2019; 399:194e8. Ma JF1, Qiao Y, Gao X, Liang L, Liu XL, Li DH, Tang HD, Chen SD. A community-based study of risk factors for probable rapid eye movement sleep behavior disorder. Sleep Med 2017;30:71e6. Breen DP, Vuono R, Nawarathna U, Fisher K, Shneerson JM, Reddy AB, Barker RA. Sleep and circadian rhythm regulation in early Parkinson disease. JAMA Neurol 2014;71:589e95. Mantovani S, Smith SS, Gordon R, O’Sullivan JD. An overview of sleep and circadian dysfunction in Parkinson’s disease. J Sleep Res 2018;27:e12673. Gilat M, Coeytaux Jackson A, Marshall NS, et al. Melatonin for rapid eye movement sleep behavior disorder in Parkinson’s disease: a randomised controlled trial. Mov Disord 2020;35:344e9.

D.1.3. Depression Depression is described by anhedonia, a reduced positive vision of life, depressed mood, and an increased negative view of life. Depressive disorder is a common nonmotor neuropsychiatric symptom of Parkinson’s disease. In Parkinson’s disease, we can observe three types of depressive disorders: dysthymia (also called persistent depressive), minor depression, and major depression. Depression is one of the most prevalent neuropsychiatric disturbances in Parkinson’s disease (around 40%e50%) [1], but there are different prevalence values, with reported ranges from 2.7% to 90% [2]. Depressive disorders are diagnosed using diagnostic and statistical manual of mental disorders criteria that include the presence of the following symptoms for 2 consecutive weeks and implicate a variation from baseline: decreased interest or pleasure in most activities most of the day; depressed mood most of the day; Insomnia or hypersomnia; significant weight change (5%) or change in appetite; psychomotor agitation or retardation; feelings of worthlessness or excessive or inappropriate guilt; fatigue or loss of energy; recurrent thoughts of death or suicide or a suicide plan; and a diminished ability to think or concentrate, or indecisiveness. Major depression diagnosis requires the presence of at least five symptoms, whereas at least two symptoms are required to be present for diagnosis of minor depression [3,4]. A study reported that the most important feature in women with Parkinson’s disease with depression was melancholy, whereas in men, it was loss of libido as well as increased apathy [5]. The association between striatal signaling and observed impairment in reversal learning from reward versus punishment was dependent on the presence of depression symptoms in Parkinson’s disease [2,3]. The relationship between body

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posture and depression in Parkinson’s disease was studied using Beck Depression Inventory scores. Parkinson’s disease patients with depression in the standing position exhibited increased distance between the head and pelvis and anterior tilting of the head from the pelvis. The degree of flexion was correlated with the severity of depression symptoms [6]. The molecular mechanisms of depression in Parkinson’s disease remain unknown, but it has been proposed that changes in serotonergic, dopaminergic, and noradrenergic systems, brain structure, and levels of inflammatory factors are involved in depression development [7]. The association of depression to a mutation in serotonin transporter promotor polymorphism is controversial because a meta-analysis of the correlation between the development of depression and stress and serotonin transporter promotor polymorphism showed a positive association. However, other studies do not report any significant association between depression, stress, and serotonin transporter promotor polymorphism [8]. It has been proposed that stress is correlated with increased depressive symptoms, and these symptoms will increase in patients carrying one short allele of the serotonin transporter gene promoter [9]. Stress seems to play an important role in the development of depression by altering neuronal pathways and their neurotransmitters signals such as dopamine, noradrenaline, and serotonin, resulting in dysfunction of the hypothalamicepituitaryeadrenal axis. The dysfunction of this hypothalamicepituitaryeadrenal axis probably induces behavioral deficits and/or mood disorders and cause hormonal imbalances [10,11]. Hypothalamusepituitaryeadrenal axis activity is induced by stress that results in the generation of glucocorticoids by the adrenals. In the hypothalamicepituitaryeadrenal axis, the hypothalamus releases a corticotropin-releasing hormone that it is an important regulator of stress response. Corticotropin-releasing hormone binds corticotropin-releasing hormone-receptor in the pituitary, inducing the release of adrenocorticotropic hormone, which triggers the synthesis of glucocorticoids. The action of corticotropin-releasing hormone is regulated by corticotropinreleasing hormone-binding protein, which has higher affinity to corticotropin-releasing hormone than to the corticotropin-releasing hormone-receptor in the pituitary. Corticotropin-releasing hormonebinding protein regulates stress response [12]. Glucocorticoid receptors are expressed in the brain, and glucocorticoids can act as transcription factors regulating gene expression in different regions of the brain. Animal studies have shown that glucocorticoid secretion increases when a pregnant female is exposed to stress. Glucocorticoids in the placenta affect the fetus by increasing hypothalamusepituitaryeadrenal axis activity and change brain development [13,14]. Glucocorticoids inhibit neurogenesis, retard the

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maturation of glia, neurons and myelin formation, and disturb neuronal structure and synapse formation [15]. Normal brain maturation depends on glucocorticoids because they induce terminal maturation, remodel dendrites and axons, and disturb cell survival. Glucocorticoids decrease glycerol-3-phosphate dehydrogenase, thus affecting myelin lipid biosynthesis [16]. Retrospective studies on children whose mothers were exposed to synthetic glucocorticoids show an association with attention-deficit/hyperactivity disorder (ADHD)-like symptoms [17]. A study performed of women around 45 years of age showed that those who had experienced childhood adversity such as sexual abuse or emotional neglect had an increase in glucocorticoid receptor expression [18]. A pregnancy exposed to stress, depression, or anxiety increase the risk that children will develop impaired cognitive development or ADHD [19] (Fig. 1.7). Three monoaminergic hypotheses have been proposed to explain the molecular mechanisms involved in depression: (1) The norepinephrine hypothesis of depression. The neurotransmitter norepinephrine is related to motivation, mood, and cognitive and emotional functions [20,21]. The major norepinephrine released in the brain is done through the locus coeruleus [22], and norepinephrine deficiency affects brain regions associated with depression such as the hippocampus, prefrontal cortex, and hypothalamus [23]. Selective norepinephrine reuptake inhibitors have been used to treat depression by increasing norepinephrine levels [23]. The increase in norepinephrine levels using selective norepinephrine reuptake inhibitors has been used in depression treatment [23]. Norepinephrine depletion does not cause depression in healthy individuals nor does it worsen the symptoms of a patient with major depression [24]; (2) Serotonin hypothesis of depression. The neurotransmitter serotonin is primarily generated by serotonergic neurons localized in the dorsal raphe nucleus of the brainstem and innervates into the amygdala, striatum, and prefrontal cortex [25]. Serotonergic neurons release serotonin to the synaptic cleft under neurotransmission, and serotonin transporters take up serotonin from the synaptic cleft into serotoninergic neurons to modulate several brain functions such as emotion and mood [25]. Abnormal serotonin levels in the brain have been associated with depression because low serotonin levels under tryptophan depletion produce acute symptomatic relapse in stabilized patients with depression [26]. Animals exposed to early-life stress by maternal separation induced an increase in the number of serotonin neurons in the dorsal raphe [27]. Selective serotonin reuptake inhibitors are used to treat maternal affective disorders, and the question is what happens during infant development that requires a serotonergic environment. A study on the effect of the selective serotonin reuptake inhibitor fluoxetine on

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FIGURE 1.7 The effect of stress on hypothalamusepituitaryeadrenal axis activity. Under stress conditions, the hypothalamus secretes corticotropin-releasing hormone (CRH) that binds to the corticotropin-releasing hormone receptor modulated by the corticotropinreleasing hormone-binding protein, which has much more affinity for the receptor than for corticotropin-releasing hormone. The corticotropin-releasing hormone-binding protein can inhibit the release of adrenocorticotropic hormone (ACTH) from pituitary. The adrenocorticotropic hormone binds adrenocorticotropic hormone-receptor, also called melanocortin-2 receptor (MC2R), which is a G protein-coupled receptor that acts by activating cAMP as a second messenger. The production of cAMP under the action of adrenocorticotropic hormone is stimulated by melanocortin-2 receptor accessory protein 1 (MRAP1). The adrenal cortex releases cortisol/corticosterone that induces changes in brain development, brain plasticity, and mood disorders.

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preadolescent animals showed a reduction in glucocorticoid receptor density, synaptophysin, and postsynaptic density protein 95 in the cingulate cortex [28]. Early-life stress induces DNA methylation of the neuropeptide corticotropin-releasing factor gene promoter in the central amygdala of adult male rats [29]. Serotonin transporter deletion induces anxiety and depression associated with reduced neuronal plasticity and a decrease in brain-derived neurotrophic factor expression. The deletion of serotonin transporter probably increases stress vulnerability during brain maturation [30]. A study on the effect of mild adversity in rat pups that were temporarily denied contact with their mother induced depression in adulthood. The depressive-like and anxious behavior was concomitant with alterations in the serotonergic system, such as increased levels of serotonin receptor in the hippocampus, decreased serotonin levels, and increased serotonin turnover [31]. Selective serotonin reuptake inhibitors such as fluoxetine have been used to treat depression for a long time. A recent study reported that fluoxetine induces neuronal plasticity in the hippocampus in a rat model of depression, and the authors concluded that fluoxetine-dependent neurite formation depends on an increase in GAP-43 that may be a relevant mechanism by which fluoxetine enhances hippocampal neuroplasticity and may explain its antidepressant action [32]; (3) Dopamine hypothesis of depression. Stress affects dopaminergic projections in both the mesolimbic and the mesocortical neuronal systems [10,33]. Dopaminergic neurons in the mesolimbic system in the brain’s reward system facilitate vulnerability to antidepressant action and social defeat [33]. It has been proposed that stress is involved in the development of nonmotor symptoms in depression and Parkinson’s disease because stress may induce depression and later exacerbate motor symptoms [34]. A healthy thirty-eight-year-old woman without neurological diseases developed tremor at rest after a week of exposure to a severe stress situation. All analysis such as electromyography and positronemission tomography scanning supported a diagnosis of early-stage Parkinson’s disease. In addition, the tremors were relieved by the use of antiparkinsonian drugs [35]. An experiment in which animals were treated with either corticosterone supplementation with adrenalectomy or restraint stress revealed that under both conditions, glucocorticoid receptor density increased in the substantia nigra, ventral tegmental area, motor cortex, and striatum in the nonlesioned side of animals. Both treatments increased mRNA expression of calcyon, a protein related to D1 and D5 receptors, in the nonlesioned side of animals [36]. Stress impacts behaviors mediated by the dopaminergic system such as cross-sensitization with drugs of abuse, locomotor activity, appetite, and sexual activity. Early-life stress

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events may contribute to depression by involving central dopamine dysfunction [37]. An increase in dopamine metabolism was observed in animals exposed to conditioned fear stress in brain regions related to dopamine systems except the striatum, while serotonin metabolism increased in the amygdala, medial prefrontal cortex and nucleus accumbens. These results suggest that regional activation of dopamine and serotonin metabolism depends on stress intensity and duration [38]. Anhedonia is linked with dopamine system dysfunction. The acute use of subanesthetic doses of ketamine induces a rapid decrease in depression symptoms in patients with a major depressive episode, thus reducing suicidal symptoms [39,40]. Ketamine induces changes in dopamine, serotonin, and other monoamine systems [41,42]. It has been proposed that the dopamine system has an important role in anhedonia symptoms in depression because of dopamine system dysregulation where the regulatory afferent circuits are affected [43,44]. Postlesion stress in animals treated with 6hydroxydopamine showed a decrease in neurotrophic factor levels with a concomitant increase in corticosterone concentration in stressed rats [45]. The effect of yoga and resistance training exercise on idiopathic Parkinson’s disease patients who walk with or without an assistive device and were able to stand unaided was performed, revealing that yoga and resistance training exercise were equally effective at improving mobility and motor dysfunction among patients with mild to moderate Parkinson’s disease. However, the yoga program also reduced depression, anxiety, and symptoms [46].

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methylation of the corticotropin-releasing factor gene promoter region in the adult rat brain. Dev Psychopathol 2015;27:123e35. Calabrese F, Guidotti G, Middelman A, Racagni G, Homberg J, Riva MA. Lack of serotonin transporter alters BDNF expression in the rat brain during early postnatal development. Mol Neurobiol 2013;48:244e56. Diamantopoulou A, Kalpachidou T, Aspiotis G, Gampierakis I, Stylianopoulou F, Stamatakis A. An early experience of mild adversity involving temporary denial of maternal contact affects the serotonergic system of adult male rats and leads to a depressive-like phenotype and inability to adapt to a chronic social stress. Physiol Behav 2018;184:46e54. Zavvari F, Nahavandi A, Goudarzi M. Fluoxetine attenuates stress-induced depressivelike behavior through modulation of hippocampal GAP43 and neurogenesis in male rats. J Chem Neuroanat 2019:101711. Cao JL, Covington 3rd HE, Friedman AK, Wilkinson MB, Walsh JJ, Cooper DC, Nestler EJ, Han MH. Mesolimbic dopamine neurons in the brain reward circuit mediate susceptibility to social defeat and antidepressant action. J Neurosci 2010; 30:16453e8. Hemmerle AM, Herman JP, Seroogy KB. Stress, depression and Parkinson’s disease. Exp Neurol 2012;233:79e86. Zou K, Guo W, Tang G, Zheng B, Zheng Z. A case of early onset Parkinson’s disease after major stress. Neuropsychiatric Dis Treat 2013;9:1067e9. Hao Y, Shabanpoor A, Metz GA. Stress and corticosterone alter synaptic plasticity in a rat model of Parkinson’s disease. Neurosci Lett 2017;651:79e87. Pani L, Porcella A, Gessa GL. The role of stress in the pathophysiology of the dopaminergic system. Mol Psychiatr 2000;5:14e21. Inoue T, Tsuchiya K, Koyama T. Regional changes in dopamine and serotonin activation with various intensity of physical and psychological stress in the rat brain. Pharmacol Biochem Behav 1994;49:911e20. Rinco´n-Corte´s M, Grace AA. Antidepressant effects of ketamine on depression-related phenotypes and dopamine dysfunction in rodent models of stress. Behav Brain Res 2020;379:112367. Andrade C. Ketamine for depression, 2: diagnostic and contextual indications. J Clin Psychiatr 2017;78:e555e8. Kapur S, Seeman P. NMDA receptor antagonists ketamine and PCP have direct effects on the dopamine D(2) and serotonin 5-HT(2)receptors-implications for models of schizophrenia. Mol Psychiatr 2002;7:837e44. Nishimura MDM, Sato MDPK, Okada MDT, Yoshiya MDPI, Schloss PP, Shimada MDPS, Tohyama MDPM. Ketamine inhibits monoamine transporters expressed in human embryonic kidney 293 cells. Anesthesiology 1998;88:768e74. Belujon P, Grace AA. Dopamine system dysregulation in major depressive disorders. Int J Neuropsychopharmacol 2017;20:1036e46. Szczypi nski JJ, Gola M. Dopamine dysregulation hypothesis: the common basis for motivational anhedonia in major depressive disorder and schizophrenia? Rev Neurosci 2018;29:727e44. Ngema PN, Mabandla MV. Post 6-OHDA lesion exposure to stress affects neurotrophic factor expression and aggravates motor impairment. Metab Brain Dis. 2017;32: 1061e7. Kwok JYY, Kwan JCY, Auyeung M3, Mok VCT4, Lau CKY5, Choi KC6, Chan HYL6. Effects of mindfulness yoga vs stretching and resistance training exercises on anxiety and depression for people with Parkinson disease: a randomized clinical trial. JAMA Neurol 2019;76:755e63.

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D.1.4. Constipation Constipation is considered one of the nonmotor symptoms of Parkinson’s disease that develops during the initial phase of the disease [1,2]. Lewy bodies containing alpha-synuclein aggregations have been observed in the neurons of the gastrointestinal tract of Parkinson’s disease patients 20 years before clinical diagnosis [3e5]. The autonomic nervous system comprises parasympathetic, sympathetic, and enteric systems, but the enteric nervous system is the largest, and it is vital for life. This system is complicated, and it is capable of arranging gastrointestinal conduct autonomously of the central nervous system input [6]. The digestive system relates to the enteric nervous system localized inside of gastrointestinal tract membranes and innervation from the central nervous system. The neuronal cell bodies are localized in the enteric nervous system, and their axons relate to the pancreas, sympathetic ganglia, gallbladder, trachea, and brainstem and spinal cord [7,8]. In general, the enteric nervous system works in conjunction with the central nervous system whereby the central nervous system stimulates enteric conduct and the intestine to send signals to the brain. The majority of vagal fibers from the gut to the brain send information to the brain, such as sensations of satiety, bloating, or nausea, suggesting that in gutebrain communication, the gut is more a transmitter than a receiver of signals. There is also a flow of information between sympathetic prevertebral ganglia and the enteric nervous system [7e9]. It seems that the vagus can differentiate between pathogenic and nonpathogenic bacteria. It has been proposed that the vagus sends signals to the brain to induce anxiolytic and anxiogenic effects. Vagal signals from the gut to the brain can induce an antiinflammatory response that includes acetylcholine release [10]. A clinical study of patients with major depressive episode with stimulation of the vagus nerve performed by neurocybernetic prosthesis concluded that vagus nerve stimulation has antidepressive effects [11]. A 5-year prospective study concluded that adjunctive vagus nerve stimulation in patients with treatment-resistant depression has antidepressant effects [12]. Vagus nerve stimulation has also had positive results in treating patients with refractory epilepsy; the positive effect is not immediate but increases over time. It has been proposed that the possible mechanism of action of vagus nerve stimulation is related to modulation of the thalamus, locus coeruleus, and limbic serotonergic and noradrenergic projections [13]. A study of refractory epilepsy patients treated with vagus nerve stimulation demonstrated an improvement in working memory [14]. The American Food and Drug Administration has accepted vagus nerve stimulation for the treatment of patients with depression,

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epilepsy, and headache. In animals, vagus nerve stimulation decreases neurological deficits and infarct volume, and its increases cognition and memory in rats with stroke lesions. Therefore, it has been proposed that vagus nerve stimulation can be useful in treating ischemic stroke [15]. Gutebrain communication allows signals related to intestinal function to be sent to the brain, but this gutebrain communication can also be used to transfer varicella-zoster virus to the brain [6]. Several lines of evidence suggest the presence of the dopamine system in sympathetic neurons where transcripts encoding dopamine transporter and tyrosine hydroxylase were present in murine intestine. Immunopositive tyrosine hydroxylase was demonstrated in the colon, small intestine, and stomach. Surgical removal of sympathetic nerves does not change tyrosine hydroxylase immunopositive results and mRNA expression of dopamine transporter and tyrosine hydroxylase [16]. A study performed with germ-free mice demonstrated that gut microbiota changes behavior and brain development by reducing anxiety and increasing motor activity. The authors suggested that the microbiota presence affected neuronal circuits related to anxiety behavior and motor control [17]. Gut microbiota can generate and use neurotransmitters such as serotonin, dopamine, norepinephrine, or gamma-aminobutyric acid to alter human neurotransmitter levels [18]. Anxiety induced by alcohol withdrawal was demonstrated by transplanting enteric microbiota from alcohol-drinking mice to control animal drinking water. In addition, this transplantation of the microbiota of alcohol-drinking mice induced the expression of genes associated with alcohol addiction [19]. A study performed with the MitoPark mouse model in which the mitochondrial transcription factor A of the cre/lox system was selectively removed from dopaminergic neurons showed that a decrease in gastrointestinal motility was the first symptom observed in the mouse model for Parkinson’s disease after 8 weeks. After 24 weeks, signs of constipation were observed such as decreased fecal water content and longer colon transit time. Other symptoms observed were activation of enteric glial cells, intestinal inflammation, and loss of tyrosine hydroxylaseimmunoreactive neurons in the gut and midbrain region [20]. Gram-negative bacteria with Pink1 gene deleted were used to induce an intestinal infection that induced the formation of mitochondria-specific CD8þ T cells, suggesting that Pink1 is an inhibitor of the immune system. Interestingly, a decrease in dopaminergic neuron terminals in the striatum was observed with concomitant motor dysfunction in which treatment with L-dopa recovered motor dysfunction [21]. Colon biopsies obtained from Parkinson’s disease patients showed increased proinflammatory gene profiles such as cytokine, the bacterial endotoxin-specific ligand TLR4, and CD3þ T cells in comparison with control samples. They found

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an increase in the expression of the bacterial endotoxin-specific ligand TLR4, CD3þ T cells, suggesting an important role of TLR4 in gutebrain axis inflammation [22]. Enteric neuronal system is normally targeted by nutrients but also by the release of microbiota inflammatory factors. Intestine inflammation seems to play an important role in the induction of several pathologies such as Parkinson’s disease, ulcerative colitis, Crohn disease and diabetes type 2 [23]. A study performed in India revealed that 45% of Parkinson’s disease patients with malnutrition or at risk of malnourishment present constipation, sialorrhea and dysphagia in comparison with patients with normal nutrition [24]. Changes in the microbiota seem to play a role in gutebrain axis-generated inflammation that may be involved in initial symptoms of Parkinson’s disease. It has been reported that Helicobacter pylori bacteria play a role in Parkinson’s disease because Parkinson’s disease patients infected with H. pylori develop worse motor dysfunction than patients not infected with this bacterium. The abolition of H. pylori in patients with Parkinson’s disease improves motor dysfunction in comparison with patients infected with this bacterium. H. pylori bacteria probably metabolize levodopa by oxidizing its catechol structure to levodopa orthoquinone, which rearranges and polymerizes to form melanin. This is an irreversible reaction and will reduce the levodopa concentration, which explains levodopa’s reduced absorption in patients infected with this bacterium. Interestingly, Parkinson’s disease patients are more prone to be infected with H. pylori bacteria than normal individuals are [25]. A study of nearly 200 Parkinson’s disease patients to study microbiota composition by sequencing the 16S rRNA gene of DNA extracted from stool revealed a significant alteration of microbiota including the Lactobacillaceae, Lachnospiraceae, Christensenellaceae, [Tissierellaceae], Pasteurellaceae, Verrucomicrobiaceae, and Bifidobacteriaceae families. The change in microbiota resulted in changes in the metabolism of xenobiotics including therapeutic drugs and plant-derived compounds [26]. A study performed in stool samples of Parkinson’s disease patients to identify possible early-stage markers of intestinal inflammation revealed increased levels of CXCL8, interleukin-1a, C-reactive protein, and interleukin-1b, thus suggesting that intestinal inflammation occurs in Parkinson’s disease patients [27]. Based on the possible relationship between Parkinson’s disease and increased intestinal permeability and intestinal inflammation, a study done with Parkinson’s disease patients focused on searching for markers of intestine inflammation (lactoferrin and calprotectin) and intestinal permeability (zonulin and alpha-1-antitrypsin). This study revealed that zonulin, alpha-1-antitrypsin, and calprotectin were significantly increased in Parkinson’s disease patient samples [28,29]. The use of

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probiotic such as of Bifidobacterium and Lactobacillus to treat constipation in Parkinson’s disease patients has been effective, but the effect of probiotics on motor symptoms and disease progression is completely unclear [30]. A study of pediatric patients reported that expression of alphasynuclein in enteric neurites of the gastrointestinal tract was positively associated with the level of chronic and acute inflammation in the intestinal wall. In addition, the monomeric and oligomeric forms of alphasynuclein induce dendritic cell maturation and monocyte and neutrophil migration dependent on the integrin subunit CD11b [31]. A study done with fecal samples and sigmoid mucosal biopsies collected from Parkinson’s disease patients reported that butyrate-producing bacteria considered antiinflammatory and from Blautia, Roseburia, and Coprococcus were significantly decreased in Parkinson’s disease samples compared with controls. On the other hand, mucosa samples from Parkinson’s disease patients have increased levels of Proteobacteria of the genus Ralstonia, which are considered proinflammatory [32]. Glia cells are the major constituent of the enteric nervous system and play several important roles such as (1) preserving the integrity of the intestinal epithelium barrier, which is important for digestive diseases, inflammatory bowel diseases, postoperative ileus, obesity, or Parkinson’s disease; (2) regulating gut homeostasis and its involvement in extradigestive and digestive diseases; (3) exerting neuroprotective roles; (4) regulating neuromediator expression; and (5) working as a neuronal glial as well as neuronal progenitor in the enteric nervous system [33]. It has been proposed that enteric glial cells play a role in chronic constipation and other gastrointestinal disorders associated with intestine inflammation such as Crohn’s disease and ulcerative colitis in Parkinson’s disease and reported enteric glial cell dysfunction [34]. Glia cells from the enteric and central nervous systems express glial fibrillary acidic protein, and a study done with Parkinson’s disease, multiple-system atrophy, and progressive supranuclear palsy patients reported that only Parkinson’s disease patients showed a significant increase in glial fibrillary acidic protein levels in colonic biopsies. Parkinson’s disease patients presented a lower phosphorylation level of expression of glial fibrillary acidic protein at serine 13 in comparison with control individuals [35].

References [1] Postuma RB, Berg D, Stern M, Poewe W, Olanow CW, Oertel W6, Obeso J, Marek K8, Litvan I, Lang AE1, Halliday G, Goetz CG, Gasser T, Dubois B, Chan P, Bloem BR, Adler CH, Deuschl G. MDS clinical diagnostic criteria for Parkinson’s disease. Mov Disord 2015;30:1591e601. [2] Knudsen K, Krogh K, Østergaard K, Borghammer P. Constipation in Parkinson’s disease: subjective symptoms, objective markers, and new perspectives. Mov Disord 2017;32:94e105.

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[3] Beach TG, Adler CH, Sue LI, et al. Multi-organ distribution of phosphorylatedasynuclein histopathology in subjects with Lewybody disorders. Acta Neuropathol 2010;119:689e702. [4] Gelpi E, Navarro-Otano J, Tolosa E, et al. Multiple organ involvement by alphasynuclein pathology in Lewy body disorders. Mov Disord 2014;29:1010e1018.9. [5] Stokholm MG, Danielsen EH, Hamilton-Dutoit SJ, Borghammer P. Pathological alphasynuclein in gastrointestinal tissues from prodromal Parkinson disease patients. Ann Neurol 2016;79:940e9. [6] Rao M, Gershon MD. The bowel and beyond: the enteric nervous system in neurological disorders. Nat Rev Gastroenterol Hepatol 2016;13:517e28. [7] Furness JB, Callaghan BP, Rivera LR, Cho HJ. The enteric nervous system and gastrointestinal innervation: integrated local and central control. Adv Exp Med Biol 2014;817: 39e71. [8] a Furness JB, Callaghan BP, Rivera LR, Cho HJ. The enteric nervous system and gastrointestinal innervation: integrated local and central control. Adv Exp Med Biol 2014;817: 39e71.b Furness JB. The enteric nervous system and neurogastroenterology. Nat Rev Gastroenterol Hepatol 2012;9:286e94. [9] Furness JB. Integrated neural and endocrine control of gastrointestinal function. Adv Exp Med Biol 2016;891:159e73. [10] Forsythe P, Bienenstock J, Kunze WA. Vagal pathways for microbiomeebrainegut axis communication. Adv Exp Med Biol 2014;817:115e33. [11] Rush AJ, George MS, Sackeim HA, Marangell LB, Husain MM, Giller C, Nahas Z, Haines S, Simpson Jr RK, Goodman R. Vagus nerve stimulation (VNS) for treatmentresistant depressions: a multicenter study. Biol Psychiatr 2000;47:276e86. [12] Aaronson ST, Sears P, Ruvuna F, Bunker M, Conway CR, Dougherty DD, Reimherr FW, Schwartz TL, Zajecka JM. A 5-year observational study of patients with treatmentresistant depression treated with vagus nerve stimulation or treatment as usual: comparison of response, remission, and suicidality. Am J Psychiatr 2017;174:640e8. [13] Oliveira TVHF, Francisco AN, Demartini Junior Z, Stebel SL. The role of vagus nerve stimulation in refractory epilepsy. Arq Neuropsiquiatr 2017;75:657e66. [14] Sun L, Pera¨kyla¨ J, Holm K, Haapasalo J, Lehtima¨ki K, Ogawa KH, Peltola J, Hartikainen KM. Vagus nerve stimulation improves working memory performance. J Clin Exp Neuropsychol 2017;39:954e64. [15] Ma J, Qiao P, Li Q, Wang Y, Zhang L, Yan LJ, Cai Z. Vagus nerve stimulation as a promising adjunctive treatment for ischemic stroke. Neurochem Int 2019;131:104539. [16] Li ZS, Pham TD, Tamir H, Chen JJ, Gershon MD. Enteric dopaminergic neurons: definition, developmental lineage, and effects of extrinsic denervation. J Neurosci 2004;24: 1330e9. [17] Diaz Heijtz R, Wang S, Anuar F, Qian Y, Bjo¨rkholm B, Samuelsson A, Hibberd ML, Forssberg H, Pettersson S. Normal gut microbiota modulates brain development and behavior. Proc Natl Acad Sci U S A. 2011;108:3047e52. [18] Strandwitz P. Neurotransmitter modulation by the gut microbiota. Brain Res 2018;1693: 128e33. [19] Xiao HW1, Ge C1, Feng GX1, Li Y1, Luo D1, Dong JL1, Li H1, Wang H2, Cui M3, Fan SJ4. Gut microbiota modulates alcohol withdrawal-induced anxiety in mice. Toxicol Lett 2018;287:23e30. [20] Ghaisas S, Langley MR, Palanisamy BN, Dutta S, Narayanaswamy K, Plummer PJ, Sarkar S, Ay M, Jin H, Anantharam V, Kanthasamy A, Kanthasamy AG. MitoPark transgenic mouse model recapitulates the gastrointestinal dysfunction and gut-microbiome changes of Parkinson’s disease. Neurotoxicology 2019;75:186e99. [21] Matheoud D, Cannon T, Voisin A, Penttinen AM, Ramet L, Fahmy AM, Ducrot C, Laplante A, Bourque MJ, Zhu L, Cayrol R, Le Campion A, McBride HM, Gruenheid S, Trudeau LE, Desjardins M. Intestinal infection triggers Parkinson’s disease-like symptoms in Pink1-/- mice. Nature 2019;571:565e9.

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[22] Perez-Pardo P, Dodiya HB, Engen PA, Forsyth CB, Huschens AM, Shaikh M, Voigt RM, Naqib A, Green SJ, Kordower JH, Shannon KM, Garssen J, Kraneveld AD, Keshavarzian A. Role of TLR4 in the gut-brain axis in Parkinson’s disease: a translational study from men to mice. Gut 2019;68:829e43. [23] Bessac A, Cani PD, Meunier E, Dietrich G, Knauf C. Inflammation and gut-brain Axis during type 2 diabetes: focus on the crosstalk between intestinal immune cells and enteric nervous system. Front Neurosci 2018;12:725. [24] Paul BS, Singh T, Paul G, Jain D, Singh G, Kaushal S, Chhina RS. Prevalence of malnutrition in Parkinson’s disease and correlation with gastrointestinal symptoms. Ann Indian Acad Neurol 2019;22:447e52. [25] McGee DJ, Lu XH, Disbrow EA. Stomaching the possibility of a pathogenic role for Helicobacter pylori in Parkinson’s disease. J Parkinsons Dis 2018;8:367e74. [26] Hill-Burns EM, Debelius JW, Morton JT, Wissemann WT, Lewis MR, Wallen ZD, Peddada SD, Factor SA, Molho E, Zabetian CP, Knight R, Payami H. Parkinson’s disease and Parkinson’s disease medications have distinct signatures of the gut microbiome. Mov Disord 2017;32:739e49. [27] Houser MC, Chang J, Factor SA, Molho ES, Zabetian CP, Hill-Burns EM, Payami H, Hertzberg VS, Tansey MG. Stool immune profiles evince gastrointestinal inflammation in Parkinson’s disease. Mov Disord 2018;33:793e804. [28] Schwiertz A, Spiegel J, Dillmann U, Grundmann D, Bu¨rmann J2, Faßbender K2, Scha¨fer KH3, Unger MM4. Fecal markers of intestinal inflammation and intestinal permeability are elevated in Parkinson’s disease. Park Relat Disord 2018;50:104e7. [29] Mulak A, Koszewicz M, Panek-Jeziorna M, Koziorowska-Gawron E, Budrewicz S. Fecal calprotectin as a marker of the gut immune system Activation is elevated in Parkinson’s disease. Front Neurosci 2019;13:992. [30] Van Laar T, Boertien JM, Herranz AH. Faecal transplantation, pro- and prebiotics in Parkinson’s disease; hope or hype? J Parkinsons Dis 2019;9:S371e9. [31] Stolzenberg E1, Berry D, Yang, Lee EY, Kroemer A, Kaufman S, Wong GCL, Oppenheim JJ, Sen S, Fishbein T, Bax A, Harris B, Barbut D, Zasloff MA. A role for neuronal alpha-synuclein in gastrointestinal immunity. J Innate Immun 2017;9:456e63. [32] Keshavarzian A, Green SJ, Engen PA, Voigt RM, Naqib A, Forsyth CB, Mutlu E, Shannon KM. Colonic bacterial composition in Parkinson’s disease. Mov Disord 2015;30:1351e60. [33] Neunlist M, Rolli-Derkinderen M, Latorre R, Van Landeghem L, Coron E, Derkinderen P, De Giorgio R. Enteric glial cells: recent developments and future directions. Gastroenterology 2014;147:1230e7. [34] Clairembault T, Leclair-Visonneau L, Neunlist M, Derkinderen P. Enteric glial cells: new players in Parkinson’s disease? Mov Disord 2015;30:494e8. [35] Clairembault T, Kamphuis W, Leclair-Visonneau L, Rolli-Derkinderen M, Coron E, Neunlist M, Hol EM, Derkinderen P. Enteric GFAP expression and phosphorylation in Parkinson’s disease. J Neurochem 2014;130:805e15.

D.1.5. Excessive daytime somnolence Various forms of sleep disturbance are observed in Parkinson’s disease such as rapid eye movement sleep behavior disorders, insomnia, excessive daytime sleepiness, and restless legs syndrome [1]. Excessive daytime sleepiness is a nonmotor symptom observed in Parkinson’s disease patients that is different from those of nocturnal sleep disturbances, because improvement in nocturnal sleep after administration of

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rotigotine did not improve daytime sleepiness [2]. The possible relationship between excessive daytime sleepiness and other nonmotor symptoms in Parkinson’s disease have been studied, but no relationship has been found [3]. Excessive daytime sleepiness is present in other neurological disorders such as multiple sclerosis, epilepsy, myotonic dystrophies, and dementia [4]. Around 12% of Parkinson’s disease patients present excessive daytime sleepiness before the start of treatment that increases constantly over time. In more advanced stages of the disease, excessive daytime sleepiness can relate to dementia, but to date, it is unclear whether excessive daytime sleepiness has an impact on Parkinson’s disease progression [5]. Another study on excessive daytime sleepiness prevalence reported 22.5% prevalence in Parkinson’s disease patients [6]. A study of near 3000 individuals without diagnosed Parkinson’s disease was performed by 11 years of monitoring to determine the possible role of excessive daytime sleepiness or objective napping preceding Parkinson’s disease. They concluded that only objective long napping was related to a higher risk of Parkinson’s disease development in older men [7]. A study performed with 12 Parkinson’s disease patients and 6 healthy controls using [11C] rolipram positron-emission tomography and a multimodal magnetic resonance imaging scan revealed that patients with excessive daytime sleepiness exhibited a significant increase in phosphodiesterase 4 expression in the hippocampus, limbic striatum, hypothalamus, and caudate [8]. A study to determine a possible correlation between dopaminergic impairment and the development of excessive daytime sleepiness was performed in Parkinson’s disease patients using dopaminergic specific single-photon-emission computed tomography molecular imaging of dopamine transporter. Patients with excessive daytime sleepiness presented inferior motor and nonmotor Unified Parkinson’s Disease Rating Scale scores, worsened cognitive and autonomic function, depression, and decreased dopamine transporter in caudate. However, the authors suggest that dopamine reduction in the caudate may not be specific to dopaminergic degeneration [9]. Excessive daytime sleepiness affects the social life of Parkinson’s disease patients, and treatment includes pharmacological drugs such as methylphenidate, atomoxetine, caffeine, modafinil, istradefylline, and sodium oxybate and nonpharmacologic treatments such as light therapy, repetitive transcranial magnetic stimulation, and cognitive behavioral therapy [10]. A clinical study to test the possible use of sodium oxybate, a drug used in narcolepsy treatment, to treat excessive daytime sleepiness showed that sodium oxybate was well tolerated and improved excessive daytime sleepiness symptoms [11]. A clinical study of Parkinson’s disease patients with excessive daytime sleepiness to test the efficacy and safety of light therapy revealed that

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bright light therapy resulted in important progress in excessive daytime sleepiness as determined by the Epworth Sleepiness Scale score. The use of the Pittsburg Sleep Quality Index to determine improvements in excessive daytime sleepiness showed that both dim-red and bright light therapy demonstrated improvement [12]. The level of melatonin was determined throughout the day in Parkinson’s disease patients treated with dopaminergic therapy. Patients with excessive daytime sleepiness have a significantly lower amplitude of the melatonin rhythm, suggesting that these patients have circadian dysfunction [13]. A study of 48 patients with Parkinson’s disease was performed using questionnaires such as the Pittsburgh Sleep Quality Index, Hamilton Anxiety Rating Scale, Beck Depression Inventory, Epworth Sleepiness Scale, and Parkinson’s Disease Questionnaire and revealed that excessive daytime sleepiness is associated with depression and anxiety [14]. A study explored the role of brain white matter microstructure changes in Parkinson’s disease patients using diffusion magnetic resonance imaging connectometry in patients with excessive daytime sleepiness and reported microstructural alterations with respect to sleep-related circuits in comparison with patients without excessive daytime sleepiness [15]. A comparison of several studies based on Parkinson’s disease patients with excessive daytime sleepiness showed decreased functional features such as cerebral metabolism, white matter integrity and brain volume, and neural structures. In addition, a correlation between depression and excessive daytime sleepiness was observed, while the correlation between anxiety and excessive daytime sleepiness was small [16]. Parkinson’s disease with excessive daytime sleepiness was correlated with depression, nontremor symptoms, autonomic dysfunction, probable behavioral disorder, and anxiety but not motor severity or cognitive dysfunction. Patients with idiopathic rapid eye movement sleep behavior disorder have higher risk of developing Parkinson’s disease when they also present with excessive daytime sleepiness symptoms [17].

References [1] Falup-Pecurariu C, Diaconu S¸. Sleep dysfunction in Parkinson’s disease. Int Rev Neurobiol 2017;133:719e42. [2] Liguori C, Mercuri NB, Albanese M, Olivola E, Stefani A, Pierantozzi M. Daytime sleepiness may be an independent symptom unrelated to sleep quality in Parkinson’s disease. J Neurol 2019;266:636e41. [3] Ho¨glund A, Broman JE, Pa˚lhagen S, Fredrikson S, Hagell P. Is excessive daytime sleepiness a separate manifestation in Parkinson’s disease? Acta Neurol Scand 2015;132:97e104. [4] Maestri M, Romigi A, Schirru A, et al. Excessive daytime sleepiness and fatigue in neurological disorders. Sleep Breath 2020;24:413e24. [5] Gjerstad MD, Alves G, Maple-Grødem J. Excessive daytime sleepiness and REM sleep behavior disorders in Parkinson’s disease: a narrative review on early intervention with implications to neuroprotection. Front Neurol 2018;9:961.

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[6] Phattanarudee S, Sangthong S, Bhidayasiri R. Association between sleep disturbances and daytime somnolence in Parkinson’s disease. Eur Neurol 2018;80:268e76. [7] Leng Y, Goldman SM, Cawthon PM, Stone KL, Ancoli-Israel S, Yaffe K. Excessive daytime sleepiness, objective napping and 11-year risk of Parkinson’s disease in older men. Int J Epidemiol 2018;47:1679e86. [8] Wilson H, Pagano G, Niccolini F, Muhlert N, Mehta MA, Searle G, Gunn RN, Rabiner EA, Foltynie T, Politis M. The role of phosphodiesterase 4 in excessive daytime sleepiness in Parkinson’s disease. Park Relat Disord 2019;(19):30073-2. pii: S1353-8020. [9] Yousaf T, Pagano G, Niccolini F, Politis M. Excessive daytime sleepiness may be associated with caudate denervation in Parkinson disease. J Neurol Sci 2018;387:220e7. [10] Shen Y, Huang JY, Li J, Liu CF. Excessive daytime sleepiness in Parkinson’s disease: clinical implications and management. Chin Med J 2018;131:974e81. [11] Bu¨chele F, Hackius M, Schreglmann SR, Omlor W, Werth E, Maric A, Imbach LL, Ha¨gele-Link S, Waldvogel D, Baumann CR. Sodium oxybate for excessive daytime sleepiness and sleep disturbance in Parkinson disease: a randomized clinical trial. JAMA Neurol 2018;75:114e8. [12] Videnovic A, Klerman EB, Wang W, Marconi A, Kuhta T, Zee PC. Timed light therapy for sleep and daytime sleepiness associated with Parkinson disease: a randomized clinical trial. JAMA Neurol 2017;74:411e8. [13] Videnovic A, Noble C, Reid KJ, Peng J, Turek FW, Marconi A, Rademaker AW, Simuni T, Zadikoff C, Zee PC. Circadian melatonin rhythm and excessive daytime sleepiness in Parkinson disease. JAMA Neurol 2014;71:463e639. [14] Palmeri R, Lo Buono V, Bonanno L, et al. Potential predictors of quality of life in Parkinson’s Disease: sleep and mood disorders. J Clin Neurosci 2019;70:113e7. [15] Ashraf-Ganjouei A, Kheiri G, Masoudi M, Mohajer B, Mojtahed Zadeh M, Saberi P, Shirin Shandiz M, Aarabi MH. White matter tract alterations in drug-naı¨ve Parkinson’s disease patients with excessive daytime sleepiness. Front Neurol 2019;10:378. [16] Wen MC, Chan LL, Tan LCS, Tan EK. Mood and neural correlates of excessive daytime sleepiness in Parkinson’s disease. Acta Neurol Scand 2017;136:84e96. [17] Zhou J, Zhang J, Lam SP, et al. Excessive daytime sleepiness predicts neurodegeneration in idiopathic REM sleep behavior disorder. Sleep 2017;40. https://doi.org/ 10.1093/sleep/zsx041.

D.1.6. Insomnia Sleep disorders are a problem that affects the quality of life of Parkinson’s disease patients. The most common insomnia symptoms in Parkinson’s disease are early morning awakening and sleep disruption [1]. Insomnia seems to be associated with nonmotor symptoms of Parkinson’s disease [2]. A primary diagnosis of insomnia is based on validated questionnaires and medical history. To evaluate the circadian sleepewake rhythm, actigraphy and polysomnography are used to determine sleep duration in differential diagnoses [3]. A study of 412 patients showed that at the initial point of the study, only 33% of patients had insomnia, but this increased to 50% within the study period. Treatment of Parkinson’s disease with high doses of dopamine agonists as well as motor fluctuations and depressive symptoms have been associated with severe insomnia [4]. A study to identify

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possible factors associated with chronic insomnia in patients with Parkinson’s disease revealed that large rapid eye movement sleep disorder, a routine of staying in bed without sleeping, the absence of obstructive sleep apnea, and high Pittsburgh Sleep Quality Index scores are associated with chronic insomnia [5]. It has been proposed that mood disorder symptoms such as depression and anxiety are a risk factor for insomnia in Parkinson’s disease. In addition, they propose that the correlation between anxiety and insomnia is bidirectional, suggesting that both sleep syndromes and anxiety can initiate a negative spiral in Parkinson’s disease patients by potentiating each other [6]. A study performed with a population of predominantly Hispanic males with moderate Parkinson’s disease revealed that insomnia symptoms were present in 46% of study participants [7]. It has been proposed that nutritional supplementation and dietary modification may improve insomnia and other symptoms such as constipation and depression, because gastrointestinal activity is impaired during early stages of the disease [8]. A study of 68 Parkinson’s disease patients revealed that 56% self-reported insomnia associated with presleep cognitive arousal, and cognitive behavioral therapy was proposed to treat chronic insomnia [9]. A clinical trial (ACTRN12617001103358) is underway to test the effect of melatonin in 44 patients with Parkinson’s disease [10]. Exposure to polychromatic light was found to significantly improve insomnia and suggests that light therapy can be used by Parkinson’s disease patients [11]. Fourteen randomized controlled trials were performed to study the efficacy of eszopiclone in comparison with active control or placebo and concluded that Eszopiclone appears to be an efficient drug for insomnia [12]. A meta-analysis that included six randomized trials with 2809 patients with primary insomnia concluded that eszopiclone is a safe and effective drug for the treatment of primary insomnia, particularly in elderly patients [13].

References [1] Ylikoski A, Martikainen K, Sieminski M, Partinen M. Parkinson’s disease and insomnia. Neurol Sci 2015;36:2003e10. [2] Chung S, Bohnen NI, Albin RL, Frey KA, Mu¨ller ML, Chervin RD. Insomnia and sleepiness in Parkinson disease: associations with symptoms and comorbidities. J Clin Sleep Med 2013;9:1131e7. [3] Mayer G, Jennum P, Riemann D, Dauvilliers Y. Insomnia in central neurologic diseaseseoccurrence and management. Sleep Med Rev 2011;15:369e78. [4] Zhu K, van Hilten JJ, Marinus J. The course of insomnia in Parkinson’s disease. Park Relat Disord 2016;33:51e7. [5] Sobreira-Neto MA, Pena-Pereira MA, Sobreira EST, et al. Chronic insomnia in patients with Parkinson disease: which associated factors are relevant? J Geriatr Psychiatr Neurol 2020;33:22e7.

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[6] Rutten S, Vriend C, van der Werf YD, Berendse HW, Weintraub D, van den Heuvel OA. The bidirectional longitudinal relationship between insomnia, depression and anxiety in patients with early-stage, medication-naı¨ve Parkinson’s disease. Park Relat Disord 2017;39:31e6. [7] Shafazand S, Wallace DM, Arheart KL, Vargas S, Luca CC, Moore H, Katzen H, Levin B, Singer C. Insomnia, sleep quality, and quality of life in mild to moderate Parkinson’s disease. Ann Am Thorac Soc 2017;14:412e9. [8] Mischley LK. Nutrition and nonmotor symptoms of Parkinson’s disease. Int Rev Neurobiol 2017;134:1143e61. [9] Lebrun C, Ge´ly-Nargeot MC, Maudarbocus KH, Rossignol A, Geny C, Bayard S. Presleep cognitive arousal and insomnia comorbid to Parkinson disease: evidence for a serial mediation model of sleep-related safety behaviors and dysfunctional beliefs about sleep. J Clin Sleep Med 2019;15:1217e24. [10] Nikles J, O’Sullivan JD, Mitchell GK, Smith SS, McGree JM, Senior H, Dissanyaka N, Ritchie A. Protocol: using N-of-1 tests to identify responders to melatonin for sleep disturbance in Parkinson’s disease. Contemp Clin Trials Commun 2019;15:100397. [11] Martino JK1, Freelance CB2, Willis GL3. The effect of light exposure on insomnia and nocturnal movement in Parkinson’s disease: an open label, retrospective, longitudinal study. Sleep Med 2018;44:24e31. [12] Ro¨sner S, Englbrecht C, Wehrle R, Hajak G, Soyka M. Eszopiclone for insomnia. Cochrane Database Syst Rev 2018;10:CD010703. [13] Liang L, Huang Y, Xu R, Wei Y, Xiao L, Wang G. Eszopiclone for the treatment of primary insomnia: a systematic review and meta-analysis of double-blind, randomized, placebo-controlled trials. Sleep Med 2019;62:6e13.

D.1.7. Anxiety Anxiety is an important influence on the quality of life of Parkinson’s disease patients, and a study of 400 Parkinson’s disease patients revealed that anxiety is associated with insomnia, female gender, excessive daytime sleepiness, autonomic dysfunction, and cognitive impairment [1]. There are six validated tests for anxiety diagnosis: the Geriatric Anxiety Inventory, Hospital Anxiety and Depression Scale, Hamilton Anxiety Rating Scale, Beck Anxiety Inventory, Mini-Social Phobia Inventory, and Parkinson’s Anxiety Scale. The Geriatric Anxiety Inventory is easy to use with acceptable specificity and sensitivity. The Parkinson Anxiety Scale was developed for Parkinson’s disease, but its sensitivity is low [2]. Another study analyzed 49 articles found in the literature and concluded that anxiety in Parkinson’s disease patients is associated with treatment-dependent side effects (dyskinesia, on/off fluctuations), motor symptoms (symptom severity, dystonia, tremor, freezing of gait, and bradykinesia), demographic and clinical characteristics (disease stage, duration, progression, gender, and age), and nonmotor symptoms (depression, cognitive impairment, sleep abnormalities, and fatigue) [3]. A study of 90 Parkinson’s disease patients without dementia in order to characterize the most common anxiety symptoms reported in the literature revealed that the most frequent anxiety symptoms were fear, agitation, distress, embarrassment, worry, and social withdrawal as a consequence of pharmacological therapy side effects and motor symptoms [4].

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Anxiety seems to be a psychological response to stress induced by the situation involved in having Parkinson’s disease or may be associated with neurochemical changes induced by the disease. The drugs used in Parkinson’s disease treatment may also play a role in anxiety development. Anxiety in Parkinson’s disease patients seems to be grouped in the phobic, panic, and generalized anxiety disorder zones. The level of coexistence between depression and anxiety is high in Parkinson’s disease patients [5]. A study performed to determine clinical and sociodemographic features in the prediction of anxiety revealed that severe anxiety was correlated with depressive symptoms and compulsive behavior. Advanced age, rapid eye movement sleep behavior disorder, and inferior cognitive function are predictors of increased anxiety [6]. The prevalence of anxiety in Parkinson’s disease patients varies by the countries in which the studies are carried out. A study performed in Hungary with 190 patients with Parkinson’s disease showed that the prevalence of anxiety was 35.8% [7]. A study performed in China with 400 Parkinson’s disease patients showed that 26% of patients had anxiety, and the risk factors to develop Parkinson’s disease with depression were anxiety, severe motor function, living alone without a partner, poor sleep quality, tumor diseases, and dyskinesia [8]. In another study, half the Parkinson’s disease patients had anxiety symptoms at a much higher prevalence than in the normal population. They found also a negative correlation between the volume of the left amygdala and a subscale of the Beck Anxiety Inventory, but not for total test, indicating that it was independent of dopaminergic or anxiolytic medication status, the severity of motor symptoms, and autonomic dysfunction. It remains unclear whether a decrease in left amygdala volume is a consequence of chronic anxiety symptoms, depends on a Parkinson’s diseaseeassociated neurobiological vulnerability to anxiety, or is the result of a premorbid trait. The authors of this study propose that the development of anxiety symptoms and a concomitant reduction in amygdala volume depend on Parkinson’s disease pathology [9]. Beck Anxiety Inventory motor aspects were strongly associated with motor function (Unified Parkinson’s Disease Rating Scale), tremor severity, depression, and quality of life. The total score for the Beck Anxiety Inventory was found to be correlated with inferior quality of life [10]. The relationship between anxiety and dopaminergic neurons has been controversial. Nonmotor symptoms such as insomnia, dysautonomia cognitive impairment, and daytime somnolence not responsive to dopaminergic therapy are risk factors for anxiety, thus suggesting that it is dopaminergic-independent. However, dopaminergic neurons localized in the substantia nigra project their axons to nondopaminergic brain regions, thus modulating their activity. The loss of dopamine in the caudate

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nucleus, anterior cingulate, frontal cortical areas, amygdala, nucleus accumbens and other limbic areas is associated with anxiety in Parkinson’s disease [11]. The analysis of eight functional imaging studies on anxiety in Parkinson’s disease patients performed using single-photon emission computed tomography or position-emission tomography techniques revealed an inverse correlation between the severity of anxiety symptoms and dopaminergic density in the putamen and caudate [12]. The study of cortical thickness performed using corticometry by magnetic resonance imaging revealed that the cortical thinning in the rectus, cingulum, and right frontal Orb were pathological causes of clinical signs such as anxious mood, depressed mood, psychosis, cognitive impairment, apathy, hallucinations, sleep problems, short-term memory stores, central execution, and nighttime or daytime sleepiness in midstage Parkinson’s disease patients [13]. The dorsal anterior cingulate cortex is involved in social behaviors, motivational processes, conflict monitoring, reward encoding, and response initiation. An impairment of the dorsal anterior cingulate cortex has been reported in Parkinson’s disease patients with anxiety and depression [14]. Glucose metabolism was studied in Parkinson’s disease patients with anxiety using the resting-state F-18 fluorodeoxyglucose positron-emission tomography scan to determine cerebral metabolic activity. This study revealed that metabolic reductions of the striatal areas and prefrontal cortex were associated with anxiety in Parkinson’s disease patients, suggesting that this deficit might impact anxiety in Parkinson’s disease [15]. The possible relationship between cognitive dysfunction and anxiety was studied in 77 patients without dementia using the Beck Anxiety Inventory and neurocognitive function by determining attention/working memory, categorical fluency, and executive function (phonemic fluency and set-shifting). They concluded that anxiety is associated with set-shifting and a specific domain of executive function in Parkinson’s disease without dementia [16]. The possible relationship between anxiety and cognitive dysfunction in Parkinson’s disease was studied in 185 newly diagnosed patients. Anxiety was determined using the Unified Parkinson’s Disease Rating Scale clinician-rated anxiety item. Cognitive dysfunction was determined using different neuropsychological tests such as the Montreal Cognitive Assessment, global cognitive function was assessed with the Mini-Mental State Exam, the Digit Vigilance Accuracy Test from cognitive drug research, and the Power of Attention battery. Memory was determined using spatial recognition memory, pattern recognition memory, and paired associates learning. This study concluded that anxiety symptoms are associated with cognitive dysfunction, especially memory dysfunction [17].

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The relationship between balance and gait disturbance was studied in 212 Parkinson’s disease patients using standardized scales such as the Montreal Cognitive Assessment, MontgomeryeAsberg Depression Rating Scale, and Hamilton Anxiety Scale to determine the mood and cognitive status correlated with patient motor symptoms such as balance and gait disturbance, rigidity, tremor, and bradykinesia. Interestingly, only balance and gait disturbance were found to be associated with a high level of anxiety. Those patients affected by balance and gait disturbance and anxiety exhibited higher levels of depression [18]. Another study on the role of anxiety on balance in Parkinson’s disease patients was performed with 42 participants comprising patients and controls with high and low anxiety who participated in 10 quiet standing trials on a force platform under two different conditions, on a platform located on the ground and on an elevated platform, before the anxiety of participants was measured. The authors concluded that anxiety affects balance control in Parkinson’s disease, particularly of those who are highly anxious. Parkinson’s disease patients were also tested in the absence and presence of dopaminergic medication, and anxiety seems to be modulated by dopamine [19]. The presence of restless legs syndrome increases the severity and prevalence of anxiety and depression in Parkinson’s disease patients, as revealed by a study of patients with restless legs syndrome and Parkinson’s disease [20].

References [1] Zhu K, van Hilten JJ, Marinus J. Onset and evolution of anxiety in Parkinson’s disease. Eur J Neurol 2017;24:404e11. [2] Mele B, Holroyd-Leduc J, Smith EE, Pringsheim T, Ismail Z, Goodarzi Z. Detecting anxiety in individuals with Parkinson disease: a systematic review. Neurology 2018;90:e39e47. [3] Lutz SG, Holmes JD, Ready EA, Jenkins ME, Johnson AM. Clinical presentation of anxiety in Parkinson’s disease: a scoping review. OTJR (Thorofare N J) 2016;36:134e47. [4] Dissanayaka NN, O’Sullivan JD, Pachana NA, Marsh R, Silburn PA, White EX, Torbey E, Mellick GD, Copland DA, Byrne GJ. Disease-specific anxiety symptomatology in Parkinson’s disease. Int Psychogeriatr 2016;28:1153e63. [5] Walsh K, Bennett G. Parkinson’s disease and anxiety. Postgrad Med J 2001;77:89e93. [6] Rutten S, van der Ven PM, Weintraub D, Pontone GM, Leentjens AFG, Berendse HW, van der Werf YD, van den Heuvel OA. Predictors of anxiety in early-stage Parkinson’s disease - results from the first two years of a prospective cohort study. Park Relat Disord 2017;43:49e55. [7] Kova´cs M, Makkos A, Weintraut R, Kara´di K, Janszky J, Kova´cs N. Prevalence of Anxiety among Hungarian Subjects with Parkinson’s Disease. Behav Neurol. 2017;2017: 1470149. [8] Cui SS, Du JJ, Fu R, Lin YQ, Huang P, He YC, Gao C, Wang HL, Chen SD. Prevalence and risk factors for depression and anxiety in Chinese patients with Parkinson disease. BMC Geriatr 2017;17:270. [9] Vriend C, Boedhoe PS, Rutten S, Berendse HW, van der Werf YD, van den Heuvel OA. A smaller amygdala is associated with anxiety in Parkinson’s disease: a combined FreeSurfer-VBM study. J Neurol Neurosurg Psychiatry 2016;87:493e500.

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[10] Salazar RD, Le AM, Neargarder S, Cronin-Golomb A. The impact of motor symptoms on self-reported anxiety in Parkinson’s disease. Park Relat Disord 2017;38:26e30. [11] Pontone GM. Anxiety in Parkinson’s: a complex syndrome of non-dopaminergic and dopaminergic etiology. Eur J Neurol 2017;24:541e2. [12] Wen MC, Chan LL, Tan LC, Tan EK. Depression, anxiety, and apathy in Parkinson’s disease: insights from neuroimaging studies. Eur J Neurol 2016;23:1001e19. [13] Deng X, Tang CY, Zhang J, Zhu L, Xie ZC, Gong HH, Xiao XZ, Xu RS. The cortical thickness correlates of clinical manifestations in the mid-stage sporadic Parkinson’s disease. Neurosci Lett 2016;633:279e89. [14] Thobois S, Prange S, Sgambato-Faure V, Tremblay L, Broussolle E. Imaging the etiology of apathy, anxiety, and depression in Parkinson’s disease: implication for treatment. Curr Neurol Neurosci Rep 2017;17:76. [15] Wang X, Zhang J, Yuan Y, Li T, Zhang L, Ding J, Jiang S, Li J, Zhu L, Zhang K. Cerebral metabolic change in Parkinson’s disease patients with anxiety: a FDG-PET study. Neurosci Lett 2017;653:202e7. [16] Reynolds GO, Hanna KK, Neargarder S, Cronin-Golomb A. The relation of anxiety and cognition in Parkinson’s disease. Neuropsychology 2017;31:596e604. [17] Dissanayaka NNW, Lawson RA, Yarnall AJ, Duncan GW, Breen DP, Khoo TK, Barker RA, Burn DJ, ICICLE-PD study group. Anxiety is associated with cognitive impairment in newly-diagnosed Parkinson’s disease. Park Relat Disord 2017;36:63e8.  [18] Sumec R, Rektorova´ I, Jech R, Mens´ıkova´ K, et al. Motion and emotion: anxiety-axial connections in Parkinson’s disease. J Neural Transm 2017;124:369e77. [19] Ehgoetz Martens KA, Lefaivre SC, Beck EN, Chow R, Pieruccini-Faria F, Ellard CG, Almeida QJ. Anxiety provokes balance deficits that are selectively dopa-responsive in Parkinson’s disease. Neuroscience 2017;340:436e44. [20] Rana AQ, Mosabbir AA, Qureshi AR, Abbas M, Rana MA. Restless leg syndrome: a risk factor of higher prevalence of anxiety and depression in Parkinson’s disease patients. Neurol Res 2016;38:309e12.

D.1.8. Cognitive decline Cognitive decline is an important as well as the most common nonmotor symptom in Parkinson’s disease. Parkinson’s disease patients present with rapid cognitive decline in memory and executive, visuospatial, and attentional cognitive domains in comparison with agematched healthy individuals. A wide spectrum of cognitive disabilities can be observed in Parkinson’s disease patients including normal cognition, mild cognitive impairment, and moderate and severe dementia. Parkinson’s disease patients are at greater risk of developing dementia than the population without the disease, and it has been suggested that Parkinson’s disease patients who live for more than 10 years after diagnosis will develop dementia [1]. Mild cognitive impairment is characterized by the appearance of memory, thinking, language, and judgment problems that are more evident than those observed under normal aging and is a stage before the development of dementia. Mild cognitive impairment in Parkinson’s disease could be a dementia prestage in which the patient begins to walk irreversibly toward dementia. However, these possible biomarkers must

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be validated. A study conducted in Norway of 182 patients with Parkinson’s disease with a follow-up over 3 years revealed that 21% of patients with mild cognitive impairment status showed reversal to a normal cognitive stage. Twenty percent of recently diagnosed Parkinson’s disease patients reversed their mild cognitive impairment status to a normal state of cognition within 1 year [2]. The possible role of computerized working memory training intervention in cognitively unimpaired Parkinson’s disease patients was performed in 76 patients and showed an improvement after training that seemed to have a stabilizing effect [3]. The positive effect of computer-based cognitive training has been reviewed, and a possible limitation is heterogeneity among studies because of differences in software, patient disease stage, and the duration and number of training sessions [4]. Exercise training has also been demonstrated to improve motor and nonmotor symptoms of Parkinson’s disease such as cognitive impairment [5]. The possible dual-task role in cognition in Parkinson’s disease was studied using walking and cycling as motor tasks. The cognitive-cycling dual-task has a facilitative effect on cognition, suggesting that cognitivecycling dual-task training may improve motor and cognitive functions in Parkinson’s disease [6]. The identification of biomarkers for mild cognitive impairment will allow early detection of patients at potential risk of developing dementia. Features that possibly indicate various dementia risks have been reported, such as amyloid beta in cerebrospinal fluid, decreased cerebral cholinergic metabolism, and innervation determined by positronemission tomography principally in posterior areas, hippocampal atrophy in magnetic resonance imaging, low values of uric acid, and insulin-like and epidermal growth factors in plasma [7]. The diagnosis of mild cognitive impairment in Parkinson’s disease patients depends on the criteria used by the neurologist. The Movement Disorder Society published guidelines for diagnosis of Parkinson’s disease with mild cognitive impairment, but depending on the criteria used, different results may be obtained for the same study. The 60% of Parkinson’s disease patients diagnosed with the Society’s criteria II level presented with mild cognitive impairment, whereas only 23% of patients were diagnosed with mild cognitive impairment when the Society’s level I criteria were used. In addition, this study confirms that mild cognitive impairment is a risk factor for developing dementia and shows that memory impairment, neurological impairment, and educational level are predictors of developing cognitive impairment [8]. A study performed with Chinese Parkinson’s disease patients suggests that the most useful predictive model to determine mild cognitive dysfunction is a higher Mini-Mental State Examination score and no test decline in the visuospatial index and delayed recall scores of the

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twelve-item Word Recall Test [9]. Self-rating is probably more sensitive to the impact of cognitive changes on instrumental activities of daily living function in the early steps of cognitive decline in Parkinson’s disease. Changes in processing speed, learning, and executive function may suggest a higher likelihood of impairment in instrumental activities of daily living [10]. A study of nondemented Parkinson’s disease patients revealed that the bilateral hippocampal volume decreased and was significantly correlated with the Montreal Cognitive Assessment score, suggesting hippocampus vulnerability during the progression of Parkinson’s disease [11]. The prevalence of mild cognitive impairment in Parkinson’s disease varies depending on the study performed and its population. Some publications have reported that 25% to 30% of patients with Parkinson’s disease have mild cognitive impairment [1]. The prevalence in an Australian study of 70 Parkinson’s disease patients was 45% [12]. Other reports have indicated that around 30% of Parkinson’s disease patients have presented with mild cognitive impairment [13]. The molecular mechanism responsible for the development of Parkinson’s disease dementia is unknown, but it is known that a dysfunction of the metabolism of alpha-synuclein and amyloid protein and a cholinergic deficit contribute to cognitive impairment in Parkinson’s disease [14]. A study to investigate the existence of possible predictors of dementia was performed using a longitudinal approach with a 5-year follow-up. They reported the existence of three clinical dementia predictors in Parkinson’s diseasedspecifically, poor semantic fluency, older age, and inability to precisely copy an intersecting-pentagons figure. The possible influence of the microtubule-associated protein TAU gene (H1/H1) on cognitive dysfunction and dementia in Parkinson’s disease was also studied by genotyping brain tissue from the frontal cortex of patients with idiopathic Parkinson’s disease or dementia with Lewy bodies. Microtubule-associated protein TAU H1/H1 was found to be an independent predictor of dementia risk. No effect of catechol-o-methyltransferase (Val(158)Met) mutation on dementia was observed in this study [15]. A study was performed to determine the possible effect of metabolic syndrome on cognitive impairment in Parkinson’s disease with patients older than 60 years of age and revealed that metabolic syndrome increases the risk of cognitive impairment. In addition, the severity of cognitive impairment is correlated with metabolic syndrome [16]. The cognitive profiles of Parkinson’s disease patients with motor symptoms were compared and correlated with cognitive function. Akinetic-rigid Parkinson’s disease patients have more deficient results in attention, working memory, visuospatial abilities, card sorting, and formalelexical word fluency. These results suggest that cognitive deficits

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are more severe in akinetic-rigid Parkinson’s disease patients in comparison with patients with tremor dominant motor symptoms [17]. The association between nonmotor symptoms in Parkinson’s disease and white matter lesions was studied in 15 patients and revealed a relationship between white matter lesions and nonmotor symptoms such as depression, anxiety, and fatigue [18]. Decreased connectivity was observed in the subnetworks of white matter structural organizations in Parkinson’s disease patients with mild cognitive impairment [19]. Low performance by Parkinson’s disease patients with mild cognitive impairment on the FarnswortheMunsell 100 hue test is correlated with white matter alterations observed using high-definition neuroanatomical magnetic resonance imaging [20]. White matter lesions are seen more frequently in patients with Parkinson’s disease with mild cognitive impairment, where there are important relationships among memory performance, language, and executive function as well as an increased number of white matter injuries [21]. Important modifications in the spatial distribution of white matter hyperintensities were observed in Parkinson’s disease patients with mild cognitive impairment, especially in periventricular regions determined using voxel-wise lesion probability maps analyses [22]. The impairment of transfer information through callosal-cortical and interhemispheric projections because of microstructural white matter anomalies of the corpus callosum may be involved in cognitive impairment in Parkinson’s disease [23]. It has been proposed that an increase in central white matter diffusivity in specific regions suggests early axonal damage. However, these observations are not related to gray matter volume damage [24]. Parkinson’s disease patients with mild cognitive impairment have different functional connectivity in the bilateral parietal and right frontal areas as determined by seed-based resting-state analysis. White matter hyperintensities are considered markers of small-vessel disease associated with cognitive decline in Alzheimer disease, but its role in Parkinson’s disease is unclear. A study performed with de novo Parkinson’s disease patients revealed that patients with increased baseline white matter hyperintensities presented significantly greater cognitive impairment. The authors of this study proposed that the presence of white matter hyperintensities is a warning sign for future development of cortical atrophy and cognitive decline in de novo Parkinson’s disease patients [25]. A study that performed diffusion tensor magnetic resonance imaging scans with the aim to investigate white and gray matter damage in relation to cognitive impairment in Parkinson’s disease revealed that Parkinson’s disease patients with mild cognitive impairment presented white matter damage in the body of the corpus callosum and superior longitudinal fasciculi, uncinate, and anterior inferior fronto-occipital. The

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authors concluded that subtle cognitive deterioration in Parkinson’s disease is correlated with aberrations of interhemispheric and frontal white matter connections but not with gray matter atrophy [26]. Voxel-based morphometry techniques used to study changes in the brain structure in Parkinson’s disease patients with mild cognitive impairment revealed that Parkinson’s disease with mild cognitive impairment has gray matter atrophy localized in the basal ganglia, limbic lobe, frontal lobe, and cerebellum. In addition, Parkinson’s disease patients with normal cognition presented gray matter atrophy essentially in the limbic lobe, prefrontal lobe, and left temporal gyrus [27]. The possible cognitive-associated changes in gray matter structure and function in Parkinson’s disease patients were studied using resting-state functional connectivity and voxel-based morphometry. They found that subtle cognitive deterioration in Parkinson’s disease is correlated with functional and structural impairment of the prefrontal cortex and temporal lobe [28]. A possible cholinergic deficiency in the frontal brain areas is probably important for the rapid progression of cognitive deterioration in Parkinson’s disease because a reduced seed-based restingstate functional connectivity it was observed between the frontal area and substantia innominata that can be correlated with early-onset mild cognitive impairment [29]. A study about the structural brain connectome in Parkinson’s disease patients with mild cognitive impairment revealed that these patients presented global network alterations characterized by a large frontoparietal network and basal ganglia [30]. A study performed with two groups of Parkinson’s disease patients with normal and mild cognitive impairment exhibited no significant differences between the two groups in white matter neurodegeneration in motor bundles. However, differences between the two groups were observed in cognitive bundles. Patients with cognitive impairment exhibited a neurodegeneration pattern, while patients with normal cognition showed white matter diffusivity metrics like those of healthy controls [31]. A comparison between Parkinson’s and Alzheimer disease with mild cognitive impairment revealed that Parkinson’s disease patients achieved better results on cognition tests such as immediate and delayed logical memory, the Mini-Mental State Exam, language measures using the Boston Naming Test, a language measure using the vegetable list and Boston Naming Test, an attention measure using digit span forward, and a processing speed measure using the digit symbol test. Significantly more Alzheimer disease patients developed dementia in 1 year in comparison with Parkinson’s disease patients with mild cognitive impairment [32]. A study was performed of 467 patients to test the predictive validity of the comprehensive (level II) version of the International Parkinson and

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Movement Disorder Society criteria for mild cognitive impairment in Parkinson’s disease. This study indicates a clear contribution of level II criteria to the risk of Parkinson’s disease patients to develop dementia [33]. The stability of mild cognitive impairment was studied in 212 Parkinson’s disease patients in a longitudinal study. After 36 months, 8% of Parkinson’s disease patients with mild cognitive impairment developed dementia, and 18% reverted to normal cognition [34]. A cross-sectional sample of patients with Parkinson’s disease with cognitive impairment, mild cognitive impairment, and dementia was analyzed with a neuropsychological test. This test revealed that cognitive impairment was primarily related to Lewy body disease alone (42%), with Alzheimer (31%), with cerebrovascular disease (13%), and with Alzheimer and cerebrovascular disease (2%) [35]. The possible association of the C allele of the rs11136000 genetic variant of the clusterin gene with early cognitive deterioration in Parkinson’s disease has been investigated. Drug-naive de novo Parkinson’s patients recently diagnosed with lower cognitive scores in function and memory tests showed a high-risk clusterin genotype that showed an increase at the 5-year follow-up. However, more research is required to understand the specific neurodegenerative pathways associated with this genetic association [36].

References [1] Aarsland D, Creese B, Politis M, Chaudhuri KR, Ffytche DH, Weintraub D, Ballard C. Cognitive decline in Parkinson disease. Nat Rev Neurol 2017;13:217e31. [2] Pedersen KF, Larsen JP, Tysnes OB, Alves G. Prognosis of mild cognitive impairment in early Parkinson disease: the Norwegian ParkWest study. JAMA Neurol 2013;70:580e6. [3] Giehl K, Ophey A, Reker P, Rehberg S, Hammes J, Barbe MT, Zokaei N, Eggers C, Husain M, Kalbe E, van Eimeren T. Effects of home-based working memory training on visuo-spatial working memory in Parkinson’s disease: a randomized controlled trial. J Cent Nerv Syst Dis 2020;12. 1179573519899469. [4] Nousia A, Martzoukou M, Tsouris Z, et al. The beneficial effects of computer-based cognitive training in Parkinson’s disease: a systematic review. Arch Clin Neuropsychol 2020;35:434e47. [5] Feng YS, Yang SD, Tan ZX, Wang MM, Xing Y, Dong F, Zhang F. The benefits and mechanisms of exercise training for Parkinson’s disease. Life Sci 2020;245:117345. [6] Hsiu-Chen C, Chiung-Chu C, Jiunn-Woei L, Wei-Da C, Yi-Hsin W, Ya-Ju C, ChinSong L. The effects of dual-task in patients with Parkinson’s disease performing cognitive-motor paradigms. J Clin Neurosci 2020;14. pii: S0967-5868. [7] Delgado-Alvarado M, Gago B, Navalpotro-Gomez I, Jime´nez-Urbieta H, RodriguezOroz MC. Biomarkers for dementia and mild cognitive impairment in Parkinson’s disease. Mov Disord 2016;31:861e81. [8] Galtier I, Nieto A, Lorenzo JN, Barroso J. Mild cognitive impairment in Parkinson’s disease: diagnosis and progression to dementia. J Clin Exp Neuropsychol 2016;38:40e50. [9] Yu RL, Lee WJ, Li JY, Chang YY, Chen CC, Lin JJ, Sung YF, Lin TK, Fuh JL. Evaluating mild cognitive dysfunction in patients with Parkinson’s disease in clinical practice in Taiwan. Sci Rep 2020;10:1014.

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[10] Cholerton B, Poston KL, Tian L, Quinn JF, Chung KA, Hiller AL, Hu SC, Specketer K, Montine TJ, Edwards KL, Zabetian CP. Participant and study partner reported impact of cognition on functional activities in Parkinson’s disease. Mov Disord Clin Pract 2019; 7:61e9. [11] Xu R, Hu X, Jiang X, Zhang Y, Wang J, Zeng X. Longitudinal volume changes of hippocampal subfields and cognitive decline in Parkinson’s disease. Quant Imag Med Surg 2020;10:220e32. [12] Lawrence BJ, Gasson N, Loftus AM. Prevalence and subtypes of mild cognitive impairment in Parkinson’s disease. Sci Rep 2016;6:33929. [13] Delgado-Alvarado M, Gago B, Navalpotro-Gomez I, Jime´nez-Urbieta H, RodriguezOroz MC. Biomarkers for dementia and mild cognitive impairment in Parkinson’s disease. Mov Disord. 2016;31:861e81. [14] Svenningsson P, Westman E, Ballard C, Aarsland D. Cognitive impairment in patients with Parkinson’s disease: diagnosis, biomarkers, and treatment. Lancet Neurol 2012;11: 697e707. [15] Williams-Gray CH1, Evans JR, Goris A, Foltynie T, Ban M, Robbins TW, Brayne C, Kolachana BS, Weinberger DR, Sawcer SJ, Barker RA. The distinct cognitive syndromes of Parkinson’s disease: 5 year follow-up of the CamPaIGN cohort. Brain 2009;132: 2958e69. [16] Peng Z, Dong S, Tao Y, Huo Y, Zhou Z, Huang W, Qu H, Liu J, Chen Y, Xu Z, Wang Y, Zhou H. Metabolic syndrome contributes to cognitive impairment in patients with Parkinson’s disease. Park Relat Disord 2018;55:68e74. [17] Wojtala J, Heber IA, Neuser P, Heller J, Kalbe E, Rehberg SP, et al. Cognitive decline in Parkinson’s disease: the impact of the motor phenotype on cognition. J Neurol Neurosurg Psychiatry 2019;90:171e9. [18] Lee JY, Kim JS, Jang W, Park J, Oh E, Youn J, Park S, Cho JW. Association between white matter lesions and non-motor symptoms in Parkinson disease. Neurodegener Dis 2018; 18:127e32. [19] Wang W, Mei M, Gao Y, et al. Changes of brain structural network connection in Parkinson’s disease patients with mild cognitive dysfunction: a study based on diffusion tensor imaging. J Neurol 2020;267:933e43. [20] Bertrand JA1, Bedetti C, Postuma RB, Monchi O, Ge´nier Marchand D, Jubault T, Gagnon JF. Color discrimination deficits in Parkinson’s disease are related to cognitive impairment and white-matter alterations. Mov Disord 2012;27:1781e8. [21] Vesely´ B, Rektor I. The contribution of white matter lesions (WML) to Parkinson’s disease cognitive impairment symptoms: a critical review of the literature. Park Relat Disord 2016;22(Suppl 1):S166e70. [22] Mak E, Dwyer MG1, Ramasamy DP, Au WL, Tan LC, Zivadinov R, Kandiah N. White matter hyperintensities and mild cognitive impairment in Parkinson’s disease. J Neuroimaging 2015;25:754e60. [23] Bledsoe IO, Stebbins GT, Merkitch D, Goldman JG. White matter abnormalities in the corpus callosum with cognitive impairment in Parkinson disease. Neurology 2018;91: e2244e55. [24] Duncan GW, Firbank MJ, Yarnall AJ, Khoo TK, Brooks DJ, Barker RA, Burn DJ, O’Brien JT. Gray and white matter imaging: a biomarker for cognitive impairment in early Parkinson’s disease? Mov Disord 2016;31:103e10. [25] Dadar M, Zeighami Y, Yau Y, Fereshtehnejad SM, Maranzano J, Postuma RB, Dagher A, Collins DL. White matter hyperintensities are linked to future cognitive decline in de novo Parkinson’s disease patients. Neuroimage Clin 2018;20:892e900.  [26] Agosta F, Canu E, Stefanova E, Sarro L, Tomic A, Spica V, Comi G, Kostic VS, Filippi M. Mild cognitive impairment in Parkinson’s disease is associated with a distributed pattern of brain white matter damage. Hum Brain Mapp 2014;35:1921e9.

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[27] Gao Y, Nie K, Huang B, Mei M, Guo M, Xie S, Huang Z, Wang L, Zhao J, Zhang Y, Wang L. Changes of brain structure in Parkinson’s disease patients with mild cognitive impairment analyzed via VBM technology. Neurosci Lett 2017;658:121e32. [28] Chen B, Wang S, Sun W, Shang X, Liu H, Liu G, Gao J, Fan G. Functional and structural changes in gray matter of Parkinson’s disease patients with mild cognitive impairment. Eur J Radiol 2017;93:16e23. [29] Kim I, Shin NY, Yunjin B, Hyu Lee P, Lee SK, Mee Lim S. Early-onset mild cognitive impairment in Parkinson’s disease: altered corticopetal cholinergic network. Sci Rep 2017;7:2381. [30] Galantucci S, Agosta F, Stefanova E, Basaia S, van den Heuvel MP, Stojkovic T, Canu E, Stankovic I, Spica V, Copetti M, Gagliardi D, Kostic VS, Filippi M. Structural brain connectome and cognitive impairment in Parkinson disease. Radiology 2017;283:515e25. [31] Hanganu A, Houde JC, Fonov VS, Degroot C, Mejia-Constain B, Lafontaine AL, Soland V, Chouinard S, Collins LD, Descoteaux M, Monchi O. White matter degeneration profile in the cognitive cortico-subcortical tracts in Parkinson’s disease. Mov Disord 2018;33:1139e50. [32] Besser LM, Litvan I, Monsell SE, Mock C, Weintraub S, Zhou XH, Kukull W. Mild cognitive impairment in Parkinson’s disease versus Alzheimer’s disease. Park Relat Disord 2016;27:54e60. [33] Hoogland J, Boel JA, de Bie RMA, Geskus RB, et al. MDS study group “validation of mild cognitive impairment in Parkinson disease”. Mild cognitive impairment as a risk factor for Parkinson’s disease dementia. Mov Disord 2017;32:1056e65. [34] Lawson RA, Yarnall AJ, Duncan GW, Breen DP, Khoo TK, Williams-Gray CH, Barker RA, Burn DJ, ICICLE-PD study group. Stability of mild cognitive impairment in newly diagnosed Parkinson’s disease. J Neurol Neurosurg Psychiatry 2017;88:648e52. [35] Smith CR, Cullen B, Sheridan MP, Cavanagh J, Grosset KA, Grosset DG. Cognitive impairment in Parkinson’s disease is multifactorial: a neuropsychological study. Acta Neurol Scand 2020;141:500e8. [36] Sampedro F, Marı´n-Lahoz J, Martı´nez-Horta S, Pe´rez-Gonza´lez R, Pagonabarraga J, Kulisevsky J. CLU rs11136000 promotes early cognitive decline in Parkinson’s disease. Mov Disord 2020;35:508e13.

D.1.9. Parkinson’s disease dementia and dementia with Lewy bodies Parkinson’s disease progression in advanced stages of the disease will develop into dementia, although there is different information about the prevalence of dementia in the disease, from to 70% to 90% of patients. However, the prevalence of dementia in late stages of the disease will increase due to increased life expectancy in developed countries [1]. The diagnosis of Parkinson’s disease dementia is based on the following criteria: (1) a Parkinson’s disease diagnosis based on the Queen Square Brain Bank; (2) patient development of Parkinson’s disease prior to dementia; (3) a patient score on the Mini-Mental State Exam below 26; (4) patient presentation of severe cognitive deficits that impact daily living activities that cannot depend on autonomic or motor symptoms; and (5) impairment in at least two of the following tests: lexical fluency or clock drawing, months reversed or seven backward, Mini-Mental State Exam pentagons, and three-word recall. These criteria are considered

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level I testing to determine a diagnosis of Parkinson’s disease dementia. The level II testing provides a deeper characterization of pattern and severity that it is important for pharmacological trials, clinical monitoring, and research studies. The Level II evaluation is based on four domains: (1) assessment of global cognitive efficiency; (2) assessment of subcorticale frontal features; (3) assessment of instrumental functions (language, visuoconstructive, visuospatial, and visuoperceptual); and assessment of neuropsychiatric functions (apathy, depression, visual hallucinations, and psychosis) [2]. A retrospective study of Parkinson’s disease patients with dementia with a disease duration of over 20 years was performed using data from the United Kingdom and Australia (more than 2000 cases). Only 36 patients had a disease duration of at least 20 years, and only seven cases were considered Parkinson’s disease with dementia. These patients, in comparison with Parkinson’s disease without dementia, presented more severe motor symptoms and had lower levels of educational achievement. Of those who had the disease for 20 years or more, 94% did not exhibit significant differences between PD patients without and with dementia in disease duration, age onset, age, sex distribution, and dopaminergic medication [3]. A comparison of olfactory functional connectivity in Parkinson’s and Alzheimer disease revealed that Parkinson’s disease dementia patients have lower functional connectivity in striatal-thalamic-frontal regions from the olfactory bulb and orbitofrontal cortex with striatal-frontal regions and with left fronto-temporal areas than that of Alzheimer patients [4]. Patients with Parkinson’s disease with dementia presented cortical Lewy bodies and Lewy neurite pathology and APOE4 genotype that can independently increase risk for dementia in Parkinson’s disease patients [5]. A study of 155 Parkinson’s disease postmortem brains (109 clinically diagnosed with dementia) revealed the presence of cortical Lewy bodies, b-amyloid plaque deposition, and neuronal TAU-inclusions that were more abundant in the brain of the demented patient group. They found a positive correlation between the severity of neurofibrillary tangles, b-amyloid plaque deposition, and cortical Lewy body scores [6]. It has been proposed that the development of Parkinson’s disease dementia depends on an important dopamine deficit in combination with cognitive decline and synergism between alpha-synuclein and Alzheimer disease pathology. In addition, a marked loss of cortical and limbic projecting dopamine, acetylcholine, serotonin, and noradrenaline neurons has been observed [7]. A slow and progressive cognitive decline and a predominant dysexecutive syndrome in combination with symptoms such as depression, hallucinations, excessive daytime sleepiness, and anxiety are involved in

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the development of dementia in Parkinson’s disease [8]. Male sex was found to be a predictive factor in the evolution from no cognitive impairment to Parkinson’s disease dementia and mild cognitive impairment because males progressed more rapidly than females [9]. As we mentioned before, 70% to 90% of patients in advanced stages of the disease develop dementia. According to the Braak Parkinson’s disease scale, stages 5 and 6 are the most advanced stages of the disease. A study that compared the relationship between alpha-synuclein depositions with clinical symptoms such as dementia and extrapyramidal symptoms used 226 brains that presented alpha-synuclein immunopositive depositions from a total of 2000 brains. Alpha-synuclein immunopositive depositions were detected in the substantia nigra, dorsal motor nucleus of vagus, and basal forebrain. Interestingly, 55% of the alpha-synuclein immunopositive patients did not present clinical signs of dementia or extrapyramidal symptoms [10]. Lewy body-related neurodegenerative disorders include Parkinson’s disease dementia and dementia with Lewy bodies, which present with common neuropathological and clinical findings. It is controversial whether Parkinson’s disease dementia and dementia with Lewy bodies are the same disease. The prevalence of dementia with Lewy bodies is about 1% to 2% in the population over 65 years old, and it is the second disorder inducing dementia after Alzheimer disease, affecting around 5% of dementia patients 75 years of age or older [11]. Meanwhile, in Parkinson’s disease dementia, the prevalence of Parkinson’s disease patients surviving more than 10 and 20 years is 75% and 83%, respectively [12,13]. Similar morphological hallmarks of Parkinson’s disease dementia and dementia with Lewy bodies such as subcortical and cortical alphasynuclein/Lewy body, beta amyloid, and TAU pathologies have been observed. The clinical findings of both disorders include parkinsonism, cognitive impairment, rapid eye movement sleep behavior disorder, frontal executive dysfunction, mild language impairment, visuoconstructive impairment, mood disturbances (depression, anxiety), and neuroleptic sensitivity. No differences in dopaminergic and striatal and cortical cholinergic deficits have been observed with multitracer positronemission tomography. Cholinesterase inhibitors have shown significant benefits in neuropsychiatric symptoms, cognition, and global function in Parkinson’s disease dementia and dementia with Lewy bodies. Some clinical dissimilarities between Parkinson’s disease dementia and dementia with Lewy bodies have been observed. Dementia with Lewy bodies has more frequent spontaneous visual hallucinations, poorer balance and slower walk, less frequent tremor, more frequent orthostatic hypotension, more severe frontal/temporal-associated cognitive subsets, faster cognitive decline, greater deficiencies of attention, lower episodic verbal memory tasks, more frequent attentional fluctuation, and

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delusions. In Parkinson’s disease, dementia visual hallucinations occur after L-dopa therapy, and dementia onset is earlier. Extrapyramidal symptoms precede dementia I Parkinson’s disease dementia. However, it is very difficult to distinguish between Parkinson’s disease dementia and dementia with Lewy bodies when the clinical features are fully developed. Postmortem studies and intravital positron-emission tomography have shown increased cortical and limbic Lewy body pathologies, prominent cortical atrophy, and higher TAU and amyloid beta loads in striatum and cortex in dementia with Lewy bodies compared with Parkinson’s disease dementia. However, cerebrospinal fluid biomarkers and functional and structural neuroimaging can also be used in the diagnosis of these diseases. The cognitive tests that are sensitive in discriminating Parkinson’s disease dementia from dementia with Lewy bodies are semantic fluency and the ReyeOsterrieth complex figure test [14e16]. Some laboratory observations that may be similar or different can be used to distinguish between Parkinson’s disease dementia and dementia with Lewy bodies. The findings present in both disorders include a decrease in dopamine transporter binding in putamen, comparable metabolic decrease in cerebral cortex, decreased cardiac scintigraphy binding using metaiodobenzylguanidine labeled to Iodine-131 or Iodine131, relative preservation of medial temporal lobe, similar EEG abnormalities, occipital hypoperfusion, and beta-glucocerebrosidase mutations. The findings that are different in both disorders including dementia with Lewy bodies: more amyloid binding, more frequent and severe gray matter cortical atrophy, more severe TAU positron-emission tomography imaging, more severe and frequent white matter hyperintensities in temporal lobe, decreased dopamine transporter binding in caudate connected to functional impairment, and a more common cerebrospinal fluid Alzheimer disease profile. Other findings that differ between the two disorders include size and asymmetry substantia nigra sonography, some genetic differences such as APOE and TFAM, alpha-synuclein cerebrospinal fluid oligomers increased in Parkinson’s disease dementia. Different functional connectivity and corticostriatal disruptiondin dementia with Lewy bodies, occipital and parietal disruption, and in Parkinson’ s disease dementia, frontal cortical disruption [16]. Although Parkinson’s disease dementia and dementia with Lewy bodies are sporadic diseases, some genetic factors may contribute to developing these disorders. A genome-wide association study to identify genetic risk factors for dementia with Lewy bodies confirmed a reported association with APOE, alpha-synuclein, and beta-glucocerebrosidase genes. In addition, they reported an association to microtubule-associated protein TAU loci and estimated that the heritable component of this disorder is about 36% alpha-synuclein expression in Parkinson’s disease

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dementia, and dementia with Lewy bodies exhibited an overlap of alphasynuclein biology, suggesting that they have different genetic etiologies. Dementia with Lewy bodies was found to be associated with the APOE gene, whereas Parkinson’s disease dementia is associated with APOE4 [17]. A multicenter study performed in 11 centers with around 721 cases with dementia with Lewy bodies and 151 cases diagnosed with Parkinson’s disease dementia and 1962 controls concluded that glucocerebrosidase-1 mutations are a significant risk to develop dementia with Lewy bodies. These mutations are also present in Parkinson’s disease dementia, but glucocerebrosidase-1 mutations play a more important role in the genetic etiology of dementia Lewy bodies than in Parkinson’s disease dementia [18,19]. It has been suggested that glucocerebrosidase mutations are associated with aggressive cognitive decline in Parkinson’s disease dementia [20]. Another study suggests that glucocerebrosidase mutations predict more rapid progression of motor symptoms and cognitive decline in Parkinson’s disease patients [21]. Glucocerebrosidase and E326K mutations are associated with a greater impact on memory/executive functions and visuospatial skills in Parkinson’s patients, where E326K in combination with glucocerebrosidase mutation induces a more severe cognitive decline [22]. Neurofibrillary tangles express the cytoskeletal protein microtubule-associated protein TAU, and TAU-H1 haplotype has been associated with a risk factor for dementia with Lewy bodies [23]. The caspase-2 cleavage of TAU protein to Dtau314 has been associated with mild cognitive impairment and Alzheimer disease in humans. An association has been suggested between dementia in Lewy body disease and Dtau314 [24]. Tau-H1 haplotypic variant has been associated with increased risk for Parkinson’s disease and accelerated dementia progression [25]. A total of 1713 cases of Parkinson’s disease were analyzed in a genome-wide association study and revealed two genes with a strong association to this disease, alpha-synuclein and microtubule-associated protein TAU [26]. A study performed with many diagnosed dementias with Lewy bodies revealed no evidence for alpha-synuclein gene multiplication [27]. In another study that included 65 patients diagnosed with dementia with Lewy bodies, no alpha-synuclein multiplications were observed [28]. The role of three genetic mutations (APOE ε2/ε3/ε4 alleles, alphasynuclein rs356219, and tau-H1/H2 haplotypes) on cognitive decline in Parkinson’s disease were determined using several psychometric tests such as the letter-number sequencing test and trail making test to assess attention and executive function, the Hopkins Verbal Learning Test-Revised [HVLT-R] to assess memory, semantic and phonemic verbal fluency tests of language processing, the Benton Judgment of Line Orientation test for visuospatial abilities, and the Montreal Cognitive

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Assessment for global cognitive function. No association of alphasynuclein rs356219 and tau-H1/H2 haplotype mutations with cognitive decline was observed in this study. This study suggests that the APOE ε4 allele is an important risk factor of cognitive function in Parkinson’s disease [29]. A study of three Parkinson’s disease patients with alpha-synuclein E46K mutation was performed as a longitudinal neuropsychological study using the following scales: Benton Visual Retention Test, semantic and phonemic verbal fluency tests, clock drawing test, Mattis Dementia Rating Scale, Stroop Test, WAIS III Letter and Number sequencing, Benton Judgment of Line Orientation Test, Rey Auditory Verbal Learning Test, semistructured interviews, and Unified Parkinson’s Disease Rating Scale III. Two patients presented mild to moderate motor symptoms with cognitive decline, while the other did not show cognitive decline. This study concluded that at early stages fluctuating frontal impairment is observed [30]. Over half of alpha-synuclein duplication cases presented early-onset parkinsonism with nonmotor symptoms such as rapid eye movement sleep behavior disorder, dysautonomia hallucinations, and cognitive impairment leading to dementia. Half the autopsied alpha-synuclein duplication cases presented neuronal loss in the hippocampal cornu ammonis 2/3 regions [31]. Alpha-synuclein triplications were observed in a Swedish-American family presented with early-onset Parkinson’s disease dementia [32]. Fifty-nine families with parkinsonism caused by alpha-synuclein multiplications were analyzed and genotyped using an Illumina MEGA high-density genotyping array to detect copy number variants. The copy number variant analysis detected 49 patients with heterozygous alpha-synuclein duplications, 7 with gene triplication, and 2 homozygous duplications. Interestingly, no association with motor symptom onset or dementias could be related to multiplication size or number of genes according to this study [33]. Alpha-synuclein duplications induce variable clinical features as revealed by a study of two families with autosomal-dominant Parkinson’s disease associated with alpha-synuclein duplications. One family exhibited early-onset Parkinson’s disease associated with cognitive deficits, dysautonomia, and psychiatric troubles, while the other family exhibited clinical features from late-onset Parkinson’s disease with mild cognitive impairment to early-onset Parkinson’s disease dementia with psychiatric troubles [34]. Homozygous Lewy body disease patients with alpha-synuclein duplications presented earlier age onset of and earlier death accompanied with extra-severe cognitive deficiency than were observed with heterozygous patients [35]. Fig. 1.8.

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FIGURE 1.8 Clinical features in Parkinson’s disease dementia and dementia with Lewy bodies. Parkinson’s disease dementia and dementia with Lewy bodies have common neuropathological and clinical findings. Similar morphological hallmarks with no differences in dopaminergic and striatal and cortical cholinergic deficits have been observed. However, some clinical dissimilarities between Parkinson’s disease dementia and dementia with Lewy bodies have also been observed. Extrapyramidal symptoms precede dementia I Parkinson’s disease dementia. However, it is very difficult to distinguish between Parkinson’s disease dementia and dementia with Lewy bodies when the clinical features are fully developed.

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References [1] Gratwicke J, Jahanshahi M, Foltynie T. Parkinson’s disease dementia: a neural networks perspective. Brain 2015;138:1454e76. [2] Dubois B, Burn D, Goetz C, et al. Diagnostic procedures for Parkinson’s disease dementia: recommendations from the movement disorder society task force. Mov Disord 2007; 22:2314e24. [3] Szeto JYY, Walton CC, Rizos A, Martinez-Martin P, Halliday GM, Naismith SL, Chaudhuri KR, Lewis SJG. Dementia in long-term Parkinson’s disease patients: a multicenter retrospective study. NPJ Parkinsons Dis 2020;6:2. [4] Lee YH, Bak Y, Park CH, Chung SJ, Yoo HS, Baik K, Jung JH, Sohn YH, Shin NY, Lee PH. Patterns of olfactory functional networks in Parkinson’s disease dementia and Alzheimer’s dementia. Neurobiol Aging 2019;(19):30450e6. pii: S0197-4580. [5] Irwin DJ, White MT, Toledo JB, Xie SX, Robinson JL, Van Deerlin V, et al. Neuropathologic substrates of Parkinson disease dementia. Ann Neurol 2012;72:587e98. [6] Horvath J, Herrmann FR, Burkhard PR, Bouras C, Kovari E. Neuropathology of dementia in a large cohort of patients with Parkinson’s disease. Park Relat Disord 2013;19:864e8. [7] Halliday GM, Leverenz JB, Schneider JS, Adler CH. The neurobiological basis of cognitive impairment in Parkinson’s disease. Mov Disord 2014;29:634e50. [8] Caballol N, Martı´ MJ, Tolosa E. Cognitive dysfunction and dementia in Parkinson disease. Mov Disord 2007;22:S358e66. [9] Cholerton B, Johnson CO, Fish B, et al. Sex differences in progression to mild cognitive impairment and dementia in Parkinson’s disease. Park Relat Disord 2018;50:29e36. [10] Parkkinen L, Pirttila¨ T, Alafuzoff I. Applicability of current staging/categorization of alpha-synuclein pathology and their clinical relevance. Acta Neuropathol 2008;115: 399e407. [11] Hogan DB, Fiest KM, Roberts JI, Maxwell CJ, Dykeman J, Pringsheim T, et al. The prevalence and incidence of dementia with Lewy bodies: a systematic review. Can J Neurol Sci 2016;43:S83e95. [12] Aarsland D, Kurz MW. The epidemiology of dementia associated with Parkinson disease. J Neurol Sci 2010;289:18e22. [13] Hely MA, Reid WG, Adena MA, Halliday GM, Morris JG. The Sydney multicenter study of Parkinson’s disease: the inevitability of dementia at 20 years. Mov Disord 2008;23:837e44. [14] Sezgin M, Bilgic B, Tinaz S, Emre M. Parkinson’s disease dementia and Lewy body disease. Semin Neurol 2019;39:274e82. [15] Martini A, Weis L, Schifano R, et al. Differences in cognitive profiles between Lewy body and Parkinson’s disease dementia. J Neural Transm 2020;127:323e30. [16] Jellinger KA, Korczyn AD. Are dementia with Lewy bodies and Parkinson’s disease dementia the same disease? BMC Med 2018;16:34. [17] Guerreiro R, Ross OA, Kun-Rodrigues C, et al. Investigating the genetic architecture of dementia with Lewy bodies: a two-stage genome-wide association study. Lancet Neurol 2018;17:64e74. [18] Clark LN, Kartsaklis LA, Wolf Gilbert R, Dorado B, Ross BM, Kisselev S, et al. Association of glucocerebrosidase mutations with dementia with Lewy bodies. Arch Neurol 2009;66:578e83. [19] Nalls MA, Duran R, Lopez G, et al. A multicenter study of glucocerebrosidase mutations in dementia with Lewy bodies. JAMA Neurol 2013;70:727e35. [20] Liu G, Boot B, Locascio JJ, et al. Specifically neuropathic Gaucher’s mutations accelerate cognitive decline in Parkinson’s. Ann Neurol 2016;80:674e85. [21] Davis MY, Johnson CO, Leverenz JB, et al. Association of GBA mutations and the E326K polymorphism with motor and cognitive progression in Parkinson disease. JAMA Neurol 2016;73:1217e24.

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[22] Mata IF, Leverenz JB, Weintraub D, Trojanowski JQ, Chen-Plotkin A, Van Deerlin VM, et al. GBA variants are associated with a distinct pattern of cognitive deficits in Parkinson’s disease. Mov Disord 2016;31:95e102. [23] Labbe` C, Heckman MG, Lorenzo-Betancor O, Soto-Ortolaza AI, Walton RL, Murray ME, et al. MAPT haplotype H1G is associated with increased risk of dementia with Lewy bodies. Alzheimers Dement 2016;12:1297e304. [24] Smith BR, Nelson KM, Kemper LJ, et al. A soluble tau fragment generated by caspase-2 is associated with dementia in Lewy body disease. Acta Neuropathol Commun 2019;7:124. [25] Winder-Rhodes SE, Hampshire A, Rowe JB, et al. Association between MAPT haplotype and memory function in patients with Parkinson’s disease and healthy aging individuals. Neurobiol Aging 2015;36:1519e28. [26] Simon-Sanchez J, Schulte C, Bras JM, Sharma M, Gibbs JR, Berg D, et al. Genome-wide association study reveals genetic risk underlying Parkinson’s disease. Nat Genet 2009; 41:1308e12. [27] Lockhart PJ, Kachergus J, Lincoln S, Hulihan M, Bisceglio G, Thomas N, et al. Multiplication of the alpha-synuclein gene is not a common disease mechanism in Lewy body disease. J Mol Neurosci 2004;24:337e42. [28] Johnson J, Hague SM, Hanson M, Gibson A, Wilson KE, Evans EW, et al. SNCA multiplication is not a common cause of Parkinson disease or dementia with Lewy bodies. Neurology 2004;63:554e6. [29] Mata IF, Leverenz JB, Weintraub D, Trojanowski JQ, Hurtig HI, Van Deerlin VM, et al. APOE, MAPT, and SNCA genes and cognitive performance in Parkinson disease. JAMA Neurol 2014;71:1405e12. [30] Somme JH, Gomez-Esteban JC, Molano A, Tijero B, Lezcano E, Zarranz JJ. Initial neuropsychological impairments in patients with the E46K mutation of the alphasynuclein gene (PARK 1). J Neurol Sci 2011;310:86e9. [31] Konno T, Ross OA, Puschmann A, Dickson DW, Wszolek ZK. Autosomal dominant Parkinson’s disease caused by SNCA duplications. Parkinsonism Relat Disord. 2016; 22(Suppl 1):S1e6. [32] Fuchs J, Nilsson C, Kachergus J, et al. Phenotypic variation in a large Swedish pedigree due to SNCA duplication and triplication. Neurology 2007;68:916e22. [33] Book A, Guella I, Candido T, et al. A meta-analysis of a-synuclein multiplication in familial parkinsonism. Front Neurol 2018;9:1021. [34] Elia AE, Petrucci S, Fasano A, et al. Alpha-synuclein gene duplication: marked intrafamilial variability in two novel pedigrees. Mov Disord 2013;28:813e7. [35] Ikeuchi T, Kakita A, Shiga A, et al. Patients homozygous and heterozygous for SNCA duplication in a family with parkinsonism and dementia. Arch Neurol 2008;65:514e9.

D.1.10. Orthostatic hypotension Another nonmotor symptom of Parkinson’s disease is orthostatic hypotension. An association between orthostatic hypotension and Parkinson’s disease has been suggested from epidemiological studies but with some controversy. A meta-analysis based on publications between 2003 and 2017 with 1620 patients showed a prevalence of orthostatic hypotension in Parkinson’s disease of 27% in comparison with 7.9% for healthy controls. This study concluded that there is a significant association between orthostatic hypotension and increased risk of Parkinson’s disease [1]. Orthostatic hypotension has been found to be associated with lower body mass index. A study showed that lower body mass index was decreased in patients with orthostatic hypotension than individuals

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without this symptom and suggests that lower body mass index in elderly patients with Parkinson’s disease is a predisposing factor of orthostatic hypotension [2]. Cognitive impairment and orthostatic hypotension are nonmotor symptoms of Parkinson’s disease, and it has been suggested that they are interrelated. Cerebral hypoperfusion is the result of hypotension that induces anoxic damage to brain regions related to cognitive function. Magnetic resonance imaging studies have revealed an association between a postural drop in blood pressure in Parkinson’s disease patients and white matter hyperintensities. Noradrenergic and cardiac denervation in Parkinson’s disease observed in cardiac imaging studies suggest poorer cognition. It has been proposed that neurogenic orthostatic hypotension depends on deficient norepinephrine release from sympathetic terminals upon standing. Neurogenic orthostatic hypotension has been observed in Parkinson’s disease with synucleinopathies [3]. The principal source of noradrenaline in the brain is the locus coeruleus, and the existence of Lewy bodies has been reported in the locus coeruleus. The level of norepinephrine in the locus coeruleus is decreased in Parkinson’s disease patients with dementia [4]. A study performed with 110 nondemented patients with early de novo Parkinson’s disease explored the possible relationship between high norepinephrinergic orthostatic hypotension and central sympathetic denervation. This study revealed that Parkinson’s disease patients with high norepinephrinergic orthostatic hypotension have impaired vasopressin release and early cognitive decline [5]. The possible relationship between noradrenaline reductions in the locus coeruleus, rapid eye movement sleep behavior disorder, orthostatic hypotension, and cognitive decline was investigated using magnetic resonance imaging to measure noradrenaline transporter availability and neuromelanin in the locus coeruleus in combination with polysomnography to study rapid eye movement sleep behavior disorder and a neuropsychological test battery to determine cognitive function. This study revealed that Parkinson’s disease patients exhibited a decreased locus coeruleus neuromelanin signal on magnetic resonance imaging, reduced norepinephrine transport binding, rapid eye movement sleep behavior disorder without atonia, cognitive decline, and orthostatic hypotension [6]. Noradrenaline decrease in locus coeruleus under orthostatic hypotension motivated the use droxidopa (L-threo-3,4-dihydroxy phenylserine), a norepinephrine precursor to improve neurogenic orthostatic hypotension symptoms. Droxidopa is converted to noradrenaline in cells that have expression of L-aromatic-amino-acid decarboxylase. Droxidopa crosses the bloodebrain barrier and can be converted to noradrenaline in noradrenergic neurons, but renal proximal

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tubular cells also express L-aromatic-amino-acid decarboxylase. Therefore, it seems plausible that noradrenaline produced in the kidneys may contribute to the pressor effect of droxidopa [7]. The treatment of six patients with droxidopa that did not present significant cognitive deficit or symptoms before treatment resulted in cognitive and behavioral symptoms, deficits in memory, mania, confusion, and irritability soon after treatment [8].

References [1] Mu F, Jiao Q, Du X, Jiang H. Association of orthostatic hypotension with Parkinson’s disease: a meta-analysis. Neurol Sci 2020;41:1419e26. [2] Nakamura T, Suzuki M, Ueda M, Hirayama M, Katsuno M. Lower body mass index is associated with orthostatic hypotension in Parkinson’s disease. J Neurol Sci 2017;372: 14e8. [3] Palma JA, Kaufmann H. Orthostatic hypotension in Parkinson disease. Clin Geriatr Med 2020;36:53e67. [4] McDonald C, Newton JL, Burn DJ. Orthostatic hypotension and cognitive impairment in Parkinson’s disease: causation or association? Mov Disord 2016;31:937e46. [5] Umehara T, Oka H, Nakahara A, Matsuno H, Toyoda C. High norepinephrinergic orthostatic hypotension in early Parkinson’s disease. Park Relat Disord 2018;55:97e102. [6] Sommerauer M, Fedorova TD, Hansen AK, Knudsen K, Otto M, Jeppesen J, Frederiksen Y, Blicher JU, Geday J, Nahimi A, Damholdt MF, Brooks DJ, Borghammer P. Evaluation of the noradrenergic system in Parkinson’s disease: an 11C-MeNER PET and neuromelanin MRI study. Brain 2018;141:496e504. [7] Lamotte G, Holmes C, Sullivan P, Goldstein DS. Substantial renal conversion of L-threo3,4-dihydroxyphenylserine (droxidopa) to norepinephrine in patients with neurogenic orthostatic hypotension. Clin Auton Res 2019;29:113e7. [8] McDonell KE, Shibao CA, Biaggioni I, Hartman A, Robertson D, Claassen DO. Cognitive and behavioral changes in patients treated with droxidopa for neurogenic orthostatic hypotension: a retrospective review. Cognit Behav Neurol 2019;32:179e84.

D.1.11. Visual disturbances Visual disturbances are common symptoms in Parkinson’s disease and Parkinson’s disease dementia. These disturbances include hallucinations, diplopia (double vision), difficulty reading, and illusions. A study of 64 Parkinson’s disease and 26 Parkinson’s disease dementia patients showed that 17% of Parkinson’s disease patients, and 89% of patients with Parkinson’s disease dementia have complex visual hallucinations. The authors of this study suggested that diverse hallucinatory experiences in Parkinson’s disease do not need to have similar pathophysiological mechanisms [1]. It has been suggested that pupil reactivity, motion perception, visual acuity, color discrimination, contrast sensitivity, saccadic and pursuit eye movements, visual fields, and visual processing speeds can be involved in Parkinson’s disease visual dysfunction. Rapid eye movement sleep

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behavior disorder in combination with stereopsis, pupil reactivity, visuomotor adaptation, and pursuit eye movement can be considered early features of Parkinson’s disease. Parkinson’s disease dementia is associated with enhanced visual hallucinations, visuospatial deficits, and eye movement problems [2]. Parkinson’s disease patients with psychosis present visual and nonvisual hallucinations and delusions [3]. It has been proposed that the retinal disturbance in Parkinson’s disease is a consequence of the loss of dopaminergic cells in the retina as a consequence of alpha-synuclein aggregation in the intraretinal region [4]. Microglia react in the presence of injury or neurodegeneration by inducing morphological and immunological changes, migration, and proliferation and production of inflammatory cytokines. An excessive inflammatory response can induce neurodegenerative lesions. Neuroinflammation in the brain can induce an inflammatory response in the retina because the retina is an extension of the brain. Parkinson’s disease is a neurodegenerative disease in which both the brain and the retina are affected when activated microglia release inflammatory factors [5]. The correlation between Parkinson’s disease and incidental Lewy body disease with phosphorylated alpha-synuclein deposits in the retina was studied in postmortem brains. All Parkinson’s disease brains and 75% of brains of incidental Lewy body disease have phosphorylated alphasynuclein deposits in ganglion cell perikarya, axons, and dendrites. Some of these alpha-synuclein deposits were like brain Lewy neurites and Lewy bodies. These results contrast with the absence of alpha-synuclein deposits in the retinas of controls [6]. The existence of alpha-synuclein deposits like brain Lewy bodies and Lewy neurites is controversial because a study has reported alphasynuclein expression in the neuroretina with an absence of prominent deposits [7]. Postmortem studies of Parkinson’s disease eyes have shown that deposition of alpha-synuclein inclusions is restricted to the inner retina [8]. It has been proposed that ocular changes observed in Parkinson’s disease such as lens opacity, pupil abnormality, visual dysfunction, and retinal neuronal loss and dysfunction are related to dopamine deficiency and alpha-synuclein depositions in the retina, suggesting that the eye can be used as a noninvasive window to observe what happens in brain neurodegeneration [9]. The idea that alpha-synuclein deposits and dopamine deficits are a consequence of Parkinson’s disease has more support, but other scientists have suggested that retinal alpha-synuclein aggregates can be used a biomarker for Parkinson’s disease but more evidence supporting this idea are required [10]. It has been reported that microglia preferentially

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phagocytosed alpha-synuclein fibrils instead of alpha-synuclein oligomers and monomers. Alpha-synuclein activates microglia, resulting in the production of proinflammatory cytokines, such as interleukin-1 beta and tumor necrosis factor alpha, that stimulate Parkinson’s disease progression [11]. Ferritin is a protein that stores iron in the cells, and alpha-synuclein impairs ferritin autophagy, inducing the accumulation of ferritin in the outer retina. Autophagy dysfunction seems to be dependent on lysosome dysfunction induced by alpha-synuclein because overexpression of Rab1a restores ferritin autophagy [7]. A meta-analysis revealed that Parkinson’s disease patients have an important thinning of the inner retinal layers, suggesting that they can be used as a biomarker in Parkinson’s disease and other neurodegenerative disorders [12]. A study of 118 patient-participants revealed a relationship between cognitive deficit and variations in the thickness of the brain cortex and retina [13].

References [1] Archibald NK, Clarke MP, Mosimann UP, Burn DJ. Visual symptoms in Parkinson’s disease and Parkinson’s disease dementia. Mov Disord 2011;26:2387e95. [2] Armstrong RA. Visual dysfunction in Parkinson’s disease. Int Rev Neurobiol 2017;134: 921e46. [3] Schneider RB, Iourinets J, Richard IH. Parkinson‘s disease psychosis: presentation, diagnosis and management. Neurodegener Dis Manag. 2017;7:365e76. [4] Mohana Devi S, Mahalaxmi I, Aswathy NP, Dhivya V, Balachandar V. Does retina play a role in Parkinson’s disease? Acta Neurol Belg 2020;120:257e65. [5] Ramirez AI, de Hoz R, Salobrar-Garcia E, Salazar JJ, Rojas B, Ajoy D, Lo´pez-Cuenca I, Rojas P, Trivin˜o A, Ramı´rez JM. The role of microglia in retinal neurodegeneration: Alzheimer’s disease, Parkinson, and Glaucoma. Front Aging Neurosci 2017;9:214. [6] Ortun˜o-Lizara´n I, Beach TG, Serrano GE, Walker DG, Adler CH, Cuenca N. Phosphorylated a-synuclein in the retina is a biomarker of Parkinson’s disease pathology severity. Mov Disord 2018;33:1315e24. [7] Baksi S, Singh N. a-Synuclein impairs ferritinophagy in the retinal pigment epithelium: implications for retinal iron dyshomeostasis in Parkinson’s disease. Sci Rep 2017;7:12843. [8] Bodis-Wollner I1, Kozlowski PB, Glazman S, Miri S. a-synuclein in the inner retina in Parkinson disease. Ann Neurol 2014;75:964e6. [9] Guo L, Normando EM, Shah PA, De Groef L, Cordeiro MF. Oculo-visual abnormalities in Parkinson’s disease: possible value as biomarkers. Mov Disord 2018;33:1390e406. [10] Veys L, Vandenabeele M, Ortun˜o-Lizara´n I, Baekelandt V, Cuenca N, Moons L, De Groef L. Retinal a-synuclein deposits in Parkinson’s disease patients and animal models. Acta Neuropathol 2019;137:379e95. [11] Hoffmann A, Ettle B, Bruno A, et al. Alpha-synuclein activates BV2 microglia dependent on its aggregation state. Biochem Biophys Res Commun 2016;479:881e6. [12] Chrysou A, Jansonius NM, van Laar T. Retinal layers in Parkinson’s disease: a metaanalysis of spectral-domain optical coherence tomography studies. Park Relat Disord 2019;64:40e9. [13] Litvinenko IV, Dynin PS, Trufanov AG, Gimadutdinov RF, Maltsev DS. Eye as an object of investigation of cognitive impairment in Parkinson’s disease. Zh Nevrol Psikhiatr Im S S Korsakova 2018;118:105e14.

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D.2. Motor symptoms The loss of dopaminergic neurons of the nigrostriatal system induces motor symptoms when most of these neurons are degenerated. There are several estimations in the literature about the percentage of dopaminergic neurons lost in this neurodegenerative process when motor symptoms are evident, which range between 70% and 80%. The most well-known motor symptom in Parkinson’s disease is resting tremor, but it does not always initiate with resting tremor. I know of two diagnosed Parkinson’s disease patients who had 4 and 10 years of progression before the appearance of resting tremors. Other motor symptoms include bradykinesia, global slowness of voluntary movements, muscular rigidity, and postural instability. These are cardinal motor symptoms in Parkinson’s disease [1] in which the degeneration of nigrostriatal neurons plays an essential role in the development of motor symptoms. The mechanisms involved in the loss of dopaminergic neurons containing neuromelanin involve aggregation of alpha-synuclein to neurotoxic oligomers, protein degradation dysfunction, mitochondrial dysfunction, oxidative stress, neuroinflammation, and endoplasmic reticulum stress.

D.2.1. Alpha-synuclein role in degeneration of nigrostriatal neurons Alpha-synuclein is a monomeric protein of 140 amino acids missing both tryptophan and cysteine residues. Alpha-synuclein is expressed in the nervous system and localized to the nucleus in high concentration at presynaptic terminals [2]. It is a soluble protein but is also found in membrane-associated brain fractions, and it has been estimated to make up 1% of total brain cytosol proteins. It has been proposed that alphasynuclein occurs mainly as a tetramer-rich a-helical structure. However, a study showed that alpha-synuclein in the central nervous system is found mainly as an unfolded monomer [3]. Three regions of alpha-synuclein can be observed: (1) amino acids 1e60 coding for amphipathic a-helices and containing four imperfect repeats of 11-amino acid with KTKEGV motif; (2) amino acids 61e95 coding for the highly amyloidogenic non-amyloid-b component and hydrophobic region and also containing three KTKEGV repeats; and (3) amino acids 96e140 coding for a sequence of the C-terminal region that contain plenty of acidic residues and prolines. Depending on conditions, alpha-synuclein can be converted to diverse conformational states such as fibrils, various oligomeric forms, a partially folded state, a helical membrane-bound form, and amorphous aggregates [4].

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Knockout of alpha, beta, and gamma-synuclein induces decreased survival, age-dependent neuronal dysfunction, and changes in synaptic structure and transmission. Synucleins knockout diminish excitatory synapse size, suggesting that synucleins play an important role in presynaptic terminals. Synuclein knockout induces changes in axonal structure and synaptic protein composition [5]. Isolation of synaptic vesicles using antibodies against synaptobrevin or synaptophysin revealed the presence of alpha-synuclein. Subfractionation of synaptic vesicles using sucrose gradient centrifugation showed that synaptophysin and synaptobrevin were present in all fractions, whereas alphasynuclein is only present in one fraction. Based on these findings, the authors of this study suggested that alpha-synuclein may have a function in synaptic transmission by mediating vesicle cycling [6]. A study performed with immunogold electron microscopy to determine alpha-synuclein subcellular localization in the brain showed alphasynuclein positive staining that was denser in the presynaptic terminals and nucleus than in the axons and cytoplasm. The immunopositive staining was different across brain regions. The cortical neurons exhibited much higher immunopositive staining than hippocampal, striatal, and substantia nigral neurons [7]. A study performed using electron microscopy, optical imaging, and slice electrophysiology revealed that synucleins are necessary for the fast kinetics of synaptic vesicle endocytosis. They suggest that synucleins are necessary at early steps of synaptic vesicle endocytosis [8]. A mouse overexpressing a specific human alpha-synuclein mutation (A53T) in the absence of neurodegeneration was used to study striatal dysfunction. An increased level of dopamine in the striatum of old mice revealed that transmission was dysfunctional and synaptic plasticity was impaired based on the findings that dopamine receptors were upregulated and catechol-o-methyltransferase was transcriptionally downregulated and altered transcript levels of the dopamine inducible genes Cb1, Atf2, Homer1, Freq, and Pde7b [9]. Different forms of alpha-synuclein can enter cells, and inside the cells generates high molecular aggregates stored in intracellular inclusions. Dynamin inhibitors block alpha-synuclein internalization, suggesting a role of the endocytic pathway in alpha-synuclein internalization. Internalized alpha-synuclein was found to be colocalized with Rab GTPase proteins Rab5A and Rab7 [10]. Alpha-synuclein in the presence of lipid membranes can assume an alpha-helical conformation that is important for aggregation and synaptic plasticity. Alpha-synuclein’s ability to aggregate depends on lipid chemical properties and the lipideprotein ratio [11]. A study combining solution and solid-state used NMR spectroscopy was performed to determine conformations of alpha-synuclein bound to lipid membranes imitating the physical properties and

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composition of synaptic vesicles. Three alpha-synuclein regions with different dynamical and structural properties were determined: (1) a central region that determines the affinity to membrane binding; (2) an N-terminal helical sequence that acts as a membrane anchor; and (3) a C-terminal section weakly linked with the membrane [12]. Neurotransmitter releases at presynaptic terminals occur repeatedly and require assembly and disassembly of soluble N-ethylmaleimidesensitive factor attachment protein receptor (SNARE) complex and continuous production of reactive complex-protein intermediates. This study showed that alpha-synuclein promotes the soluble Nethylmaleimide-sensitive factor attachment protein receptor-complex assembly [13]. Because of alpha-synuclein’s interaction with synaptic proteins and presynaptic localization, it has been proposed that alphasynuclein plays a role in dopamine metabolism, neurotransmitter release, vesicle trafficking, synaptic activity and plasticity, learning, and synaptic vesicle pool maintenance [14]. Synapsin III, a neuronal phosphoprotein, is a fundamental regulator of synaptic function in dopaminergic neurons. Changes in the organization of synaptic vesicle pools and selective redistribution and increase in synapsin III were observed in dopaminergic neurons of mice carrying a spontaneous deletion of the alpha-synuclein locus. These changes generate an amplified locomotor response to the stimulation of synapsin-dependent dopamine excess [15]. The expression of a GTPase-deficient Rab3a mutant, Hsp90 inhibitors, geldanamycin and radicicola, and a dominant-negative GDP dissociation inhibitor mutant induced accumulation of membrane-bound alphasynuclein. Alpha-synuclein sequestration on intracellular membranes increased with Rab3a recycling inhibition. Alpha-synuclein membrane dissociation during synaptic activity is regulated by GDP dissociation inhibitor$Hsp90 complex that controls Rab3a membrane dissociation [16]. Alpha-synuclein can interact with different proteins. Wild-type alphasynuclein overexpression induces a decrease in protein and mRNA expression of vesicular monoamine transporter-2 and decreased dopamine uptake [17]. It has been reported that dopamine transporter and alpha-synuclein transporter have a transient interaction at the plasma membrane. Alpha-synuclein membrane localization is increased by dopamine transporter-induced membrane depolarization that increases dopamine transporter localization in cholesterol-rich membrane microdomains and increases dopamine efflux [18]. In dopamine transport expressing cells, alpha-synuclein induces Cl-sensitive and Na þ independent inward current that is inhibited by the selective dopamine reuptake inhibitor GBR12935 [19]. Alpha-synuclein also interacts with serotonin transporter to decrease serotonin uptake and the serotonin level in the plasma [20]. Wild-type human alpha-synuclein expression induces a decrease in the expression

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of tyrosine hydroxylase protein and mRNA expression without affecting the proliferation and growth of transfected cells [21]. Another study reported that overexpression of A53T or wild-type alpha-synuclein did not significantly change tyrosine hydroxylase protein levels, but a significant reduction in tyrosine hydroxylase activity was observed [22]. Microarrays and quantitative RT-PCR were used to study transcriptional changes in neuroblastoma cells transfected with wild-type or mutant alphasynuclein. This study revealed that wild-type alpha-synuclein induces mRNA downregulation of tyrosine hydroxylase, aromatic acid decarboxylase, sepiapterin reductase, and GTP cyclohydrolase [23]. It has been proposed that alpha-synuclein plays a role in fatty acid metabolism. A study to determine the role of alpha-synuclein in fatty acid uptake and metabolism was performed in alpha-synuclein gene-impaired mice treated with palmitic acid by measuring turnover kinetics and uptake in brain phospholipids. A 35% reduction in palmitic acid uptake was observed in mice with alpha-synuclein impairment. The fractional turnover and incorporation rate of palmitic acid in phospholipid classes was decreased in mice with alpha-synuclein impairment, while an increase in the fractional turnover and incorporation rate of palmitic acid in the choline glycerophospholipids was observed. It is important to mention that this effect was brain specific [24]. A more general study on the effect of alpha-synuclein deletion on neutral lipids, phospholipids, and phospholipids acyl chain composition in the brain was performed. Phosphatidylglycerol and cardiolipin were found to be decreased, while brain triacylglycerol, cholesteryl ester, and cholesterol were increased. Interestingly, alpha-synuclein depletion did not affect the expression of cholesterogenic enzymes [25]. Cardiolipin, a mitochondria-specific phospholipid, is decreased in mice with alphasynuclein deletion, while cardiolipin acyl side chains were found to be increased in saturated fatty acids. These findings are correlated with mitochondria activity alterations [26]. The phospholipid cardiolipin in mitochondrial membranes increases the interaction of lipids with alpha-synuclein, promoting the formation of pores in the presence of alpha-synuclein oligomers [27]. Electrophoretic analysis of wild-type human alpha-synuclein and A53T showed an unusual migration above 17 KDa in denaturating gels. In addition, it was demonstrated that alpha-synuclein binds oleic acid, suggesting that alpha-synuclein is a fatty acid-binding protein and probably has a function in fatty acid transport [28]. Apolipoproteins and synuclein lipid interaction are mediated by a sequence repeat composed of 11 amino acids that generate amphipathic helices. Apolipoprotein A-1, alpha-synuclein, and beta-synuclein can develop membrane curvature and transform large vesicles into curved membrane vesicles and tubules. Curvature induction of apolipoproteins

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and synucleins is mediated by wedging of amphipathic helices and membrane insertion alone. Alpha-synuclein’s capacity to generate membrane curvatures to create vesicle budding agrees with a role of alphasynuclein in endocytosis enhancement and vesicle trafficking [29]. A proteomic analysis performed on the brain of a triple knockout of alpha-, beta-, and gamma-synucleins was performed to study their role in membrane curvature. An increase in membrane curvature-related proteins was observed, especially N-BAR protein endophilin A1, suggesting reciprocal regulation of the levels of endophilin A1 and synucleins. Interestingly, tetrameric alpha-synuclein cannot induce membrane curvature; only the monomeric form is able to perform this action. Alphasynuclein with the A30P mutation has poor membrane curvature ability. The authors of this work suggest that synucleins should be considered as proteins that sense and induce membrane curvature [30]. Another study reported that alpha-synuclein-induced membrane remodeling depends on a helical structure and anionic phospholipids. Alpha-synuclein induces vesicle deformation into tubules [31]. Alpha-synuclein’s ability to remodel vesicles was studied using cryoelectron microscopy in combination with electron paramagnetic resonance. Large spherical vesicles were transformed into cylindrical micelles in incubations containing alpha-synuclein/lipid (1: 5e40 ratio). The geometrical relationship between micelle and alpha-synuclein suggests that curvature and tube formation depend on the wedging of its alpha-helix into the micelle outer membrane [32]. The binding of monomeric alpha-synuclein to membranes induces an area expansion correlated with membrane lipid composition [33]. Alphasynuclein N-terminal membrane-binding domain induces tubulation of POPG vesicles. The removal of the hydrophobic nucleus of the nonamyloid component domain of alpha-synuclein resulted in reduction in tubulation and binding affinity to POPG vesicles [34]. Arachidonic acid incorporation into phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine was inhibited by alpha-synuclein [35]. Alpha-synuclein aggregation has been proposed to be involved in Parkinson’s disease. Two different mechanisms have been proposed. (i) Formation of Lewy bodies One of the more remarkable features in Parkinson’s disease is the generation of Lewy bodies and Lewy neurites in dopaminergic neurons that can be observed in postmortem brains [36,37]. Lewy bodies are protein aggregates mainly composed of phosphorylated alpha-synuclein distributed throughout the central and peripheral nervous systems [38e41].

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Lewy bodies are also composed of ubiquitin [42e44], which is a regulatory protein involved in the proteosomal protein degradation system among other cellular processes. Ubiquitination of a protein will determinate the destiny of the protein where ubiquitin has been attached to protein lysine residues or their N-terminal. The ubiquitination process is mediated by proteosomal system enzymes E1, E2, and E3. Ubiquitin is also able to form poly-ubiquitin chains by attaching to itself. Certain ubiquitination will be a signal or protein degradation by 26S proteasome [45]. A mutation of parkin, an E3-enzyme of the proteasomal system, is associated with a juvenile form of genetic Parkinson’s disease [46]. Lewy bodies also comprise neurofilaments found in the cytoplasm of neurons as part of the neuronal cytoskeleton together with microfilaments and microtubules [42,47,48]. Synapsin III, a neuronal phosphoprotein that modulates a-syn aggregation and controls dopamine release, was detected in Lewy bodies [49]. Positive immunostaining of microtubuleassociated protein 5 was observed in Lewy bodies in the forebrain regions and brainstem [50]. Chromogranin and synaptophysin, two marker proteins for neuronal secretory vesicles, were detected in Lewy bodies [51]. Mature Lewy bodies had a concentric structure and rounded shape. Comparison of the localization of neurofilaments and synaptophysin and alpha-synuclein revealed that positive staining of neurofilaments in the peripheral layer of Lewy bodies was closer to the nucleus, and the localization of alphasynuclein and synaptophysin is similar [52]. Lewy bodies are present not only in Parkinson’s disease but also in dementia with Lewy bodies and Parkinson’s disease with dementia [37]. Mutations of homo- and heterozygous glucocerebrosidase-1 mutations have been proposed as an important genetic risk factor for the appearance of synucleinopathies as dementia with Lewy bodies and Parkinson’s disease [53]. Lewy bodies containing alpha-synuclein aggregates as the main component were found to be widespread both in brain regions and in the autonomic nervous system [54,55]. According to the Braak Parkinson’s disease stage hypothesis, the formation of Lewy bodies plays a key role in the development and propagation of the disease from different regions. However, it has been suggested that the aggregation of alpha-synuclein to fibrils and the formation of Lewy bodies are probably a harmless pathway because (1) incidental Lewy body disease can be detected in normal brains more than 60 years of age without neurological pathology. A total of 235 brains were analyzed to detect incidental Lewy bodies using alpha-synuclein immunohistochemistry and following Braak Parkinson’s disease stages. Only 34 brains presented incidental Lewy body disease, and just one brain matched the Braak Parkinson’s disease stage hypothesis [56].

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Another study reported that half an analyzed brain followed Braak Parkinson’s disease stages, but an important percentage of brains did not fit the Braak staging system [57]. Stages five and six of the Braak Parkinson’s disease stages are associated with cognitive impairment. The correlation between the clinical severity of the disease and Braak Parkinson’s disease last stages is doubtful because between 30% and 55% of elderly subjects with deposits of alpha-synuclein in Lewy bodies do not present with neuropsychiatric symptoms [58]. Fifty-five percent of cases in another study of extensive alpha-synuclein depositions classified as stage five to six of the Braak Parkinson’s disease stages did not present with dementia [59]. A total of 904 brains with alphasynuclein aggregation in Lewy bodies in the dorsal motor nucleus of substantia nigra, vagus, and/or basal forebrain nuclei were included in a study that revealed that only 30% of those with alpha-synuclein positive pathology exhibited a neurodegenerative disease [60]; and (2) a neuropathological analysis of postmortem brains of a family with Parkinson’s disease in five generations with typical clinical features of idiopathic Parkinson’s disease exhibited degeneration of dopaminergic neurons containing neuromelanin, but no Lewy bodies were observed [61]. A Parkinson’s disease patient carrying G2019S leucine-rich repeat kinase-2 mutation with a 14-year history of the disease presented mild neuronal loss of dopaminergic neurons in the substantia nigra without Lewy body inclusions [62]. Alpha-synucleinopathy with Lewy bodies was not observed in a family with leucine-rich repeat kinase-2 G2019S mutation with slowly progressive parkinsonism [63]. A study of three patients with parkinsonism showed that two patients had Lewy bodies where one of these patients presented pathological features related to Alzheimer disease, whereas the third patient did not present with Lewy bodies [64]. A patient with parkinsonism with leucine-rich repeat kinase-2 G2019S mutation with degeneration of the nigrostriatal neurons and responsive to levodopa treatment presented TAU pathology but in the absence of Lewy bodies [65]. Patients with parkin mutation and juvenile onset of Parkinson’s disease presented neural degeneration and gliosis in substantia nigra with neurofibrillary tangles in the brainstem nuclei and cerebral cortex, but Lewy bodies were not observed [66]. A postmortem study of a patient with juvenile Parkinson’s disease with parkin mutation responsive to levodopa presented degeneration of the nigrostriatal system without the presence of Lewy bodies [67]. A postmortem study of one member of a family with juvenile onset of Parkinson’s disease revealed neuronal loss and gliosis in substantia nigra pars compacta without Lewy bodies [68]. Five patients with heterozygous parkin mutations with parkinsonism were compared with confirmed patients with Parkinson’s disease and controls. These patients presented motor fluctuations, freezing of gait, and postural deformity with a mean age of

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FIGURE 1.9 Alpha-synuclein deposits in Lewy body formation. The Braak Parkinson’s disease stages hypothesis proposes that the formation of Lewy bodies plays a key role in the development and propagation of the disease from different regions.

34 years at onset. Dementia or cognitive impairment was not observed. Patients presented a severe loss of neurons in substantia nigra pars compacta like that seen in Parkinson’s disease patients. No Lewy bodies were

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observed in three patients with parkin mutation, while in two patients, a few Lewy bodies were observed in the cortex and brainstem [69]. Most Parkinson’s patients with parkin mutation have a severe degeneration of nigrostriatal neurons that is responsive to levodopa, but they do not develop Lewy bodies. This brings up questions about the need for Lewy body development in Parkinson’s disease diagnosis and the loss of nigral neurons [70]. A patient with parkin mutation with Parkinson’s disease onset at 32 years old presented bradykinesia, rigidity, impaired postural reflex, a dystonic gait, and good response to levodopa treatment. The patient lacked Lewy bodies measured with alphasynuclein immunohistochemistry and presented degeneration of nigral neurons of substantia nigra pars compacta [71]. Three siblings with juvenile Parkinson’s disease presented rigidity, rest tremor, and bradykinesia where levodopa treatment improved motor symptoms. These patients with parkin mutation exhibited degeneration of dopaminergic neurons containing neuromelanin, but Lewy bodies and neurofibrillary tangles were not observed [72]. The neuropathological studies performed with postmortem brain were the most reliable methodology to diagnose Parkinson’s disease. These studies were based on the detection of alpha-synuclein immune-reactive Lewy bodies. A review of the literature of postmortem studies of genetic parkinsonism concluded that Lewy bodies are not the best marker for Parkinson’s disease because this disease can be diagnosed in the absence of Lewy bodies, as they are not specific for Parkinson’s disease [73] Fig. 1.9.

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[50] Gai WP, Blumbergs PC, Blessing WW. Microtubule-associated protein 5 is a component of Lewy bodies and Lewy neurites in the brainstem and forebrain regions affected in Parkinson’s disease. Acta Neuropathol 1996;91:78e81. [51] Nishimura M, Tomimoto H, Suenaga T, Nakamura S, Namba Y, Ikeda K, Akiguchi I, Kimura J. Synaptophysin and chromogranin A immunoreactivities of Lewy bodies in Parkinson’s disease brains. Brain Res 1994;634:339e44. [52] Voronkov DN, Salkov VN, Anufriev PL, Khudoerkov RM. Lewy bodies in Parkinson’s disease: histological, immunohistochemical, and interferometric examinations. Arkh Patol 2018;80:9e13. [53] Franco R, Sa´nchez-Arias JA, Navarro G, Lanciego JL. Glucocerebrosidase mutations and synucleinopathies. Potential role of sterylglucosides and relevance of studying both GBA1 and GBA2 genes. Front Neuroanat 2018;12:52. [54] Braak H, Del Tredici K, Ru¨b U, de Vos RA, Jansen Steur EN, Braak E. Staging of brain pathology related to sporadic Parkinson’s disease. Neurobiol Aging 2003;24: 197e211. [55] Braak H, Mu¨ller CM, Ru¨b U, Ackermann H, Bratzke H, de Vos RA, Del Tredici K. Pathology associated with sporadic Parkinson’s diseaseewhere does it end? J Neural Transm Suppl 2006;70:89e97. [56] Frigerio R, Fujishiro H, Ahn TB, et al. Incidental Lewy body disease: do some cases represent a preclinical stage of dementia with Lewy bodies? Neurobiol Aging 2011; 32:857e63. [57] Zaccai J, Brayne C, McKeith I, Matthews F, Ince PG. Patterns and stages of alphasynucleinopathy: relevance in a population-based cohort. Neurology 2008;70:1042e8. [58] Jellinger KA. A critical reappraisal of current staging of Lewy-related pathology in human brain. Acta Neuropathol 2008;116:1e16. [59] Parkkinen L, Pirttila T, Alafuzoff I. Applicability of current staging/categorization of alpha-synuclein pathology and their clinical relevance. Acta Neuropathol 2008;115: 399e407. [60] Parkkinen L, Kauppinen T, Pirttila T, Autere JM, Alafuzoff I. Alpha-synuclein pathology does not predict extrapyramidal symptoms or dementia. Ann. Neurol. 2005;57: 82e91. [61] Hasegawa K, Kowa H. Autosomal dominant familial Parkinson disease: older onset of age, and good response to levodopa therapy. Eur Neurol 1997;38(Suppl 1):39e43. [62] Gaig C, Marti MJ, Ezquerra M, Rey MJ, Cardozo A, Tolosa E. G2019S leucine rich repeat kinase-2 mutation causing Parkinson’s disease without Lewy bodies. J Neurol Neurosurg Psychiatry 2007;78:626e8. [63] Rajput A, Dickson DW, Robinson CA, Ross OA, Dachsel JC, Lincoln SJ, Cobb SA, Rajput ML, Farrer MJ. Parkinsonism, leucine rich repeat kinase-2 G2019S, and tau neuropathology. Neurology 2006;67:1506e8. [64] Giasson BI, Covy JP, Bonini NM, et al. Biochemical and pathological characterization of Lrrk2. Ann Neurol 2006;59:315e22. [65] Ling H, Kara E, Bandopadhyay R, et al. TDP-43 pathology in a patient carrying G2019S leucine rich repeat kinase-2 mutation and a novel p.Q124E MAPT. Neurobiol Aging 2013;34. 2889.e5e2889.e2.889E9. [66] Mori H, Kondo T, Yokochi M, Matsumine H, Nakagawa-Hattori Y, Miyake T, Suda K, Mizuno Y. Pathologic and biochemical studies of juvenile parkinsonism linked to chromosome 6q. Neurology 1998;51:890. [67] Yamamura Y, Kuzuhara S, Kondo K, et al. Clinical, pathologic and genetic studies on autosomal recessive early-onset parkinsonism with diurnal fluctuation. Park Relat Disord 1998;4:65e72. [68] Takahashi H, Ohama E, Suzuki S, et al. Familial juvenile parkinsonism: clinical and pathologic study in a family. Neurology 1994;44:437e41.

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[69] Doherty KM, Silveira-Moriyama L, Parkkinen L, et al. Parkin disease: a clinicopathologic entity? JAMA Neurol 2013;70:571e9. [70] (a) Fishman PS, Oyler GA. Significance of the parkin gene and protein in understanding Parkinson’s disease. Curr Neurol Neurosci Rep 2002;2(4):296e302. (b) Giasson BI, Covy JP, Bonini NM, Hurtig HI, Farrer MJ, Trojanowski JQ, Van Deerlin VM. Biochemical and pathological characterization of Leucine rich repeat kinase-2. Ann Neurol 2006; 59:315e22. [71] Hayashi S, Wakabayashi K, Ishikawa A, et al. An autopsy case of autosomal-recessive juvenile parkinsonism with a homozygous exon 4 deletion in the parkin gene. Mov Disord 2000;15:884e8. [72] Gouider-Khouja N, Larnaout A, Amouri R, et al. Autosomal recessive parkinsonism linked to parkin gene in a Tunisian family. Clinical, genetic and pathological study. Park Relat Disord 2003;9:247e51. [73] Schneider SA, Alcalay RN. Neuropathology of genetic synucleinopathies with parkinsonism: review of the literature. Mov Disord 2017;32:1504e23.

(ii) Alpha-synuclein aggregation to neurotoxic oligomers Alpha-synuclein aggregation to oligomers has been related to synaptic dysfunction, endoplasmic reticulum stress, autophagy dysregulation, mitochondrial dysfunction, and oxidative stress [1]. The role of alphasynuclein aggregation in the formation of neurotoxic oligomers was observed when several mutations of alpha-synuclein were associated with the familial form of Parkinson’s disease [2]. Alpha-synuclein fibril structure (1e121 amino acids) determination ˚ revealed polar using cryoelectron microscopy at a resolution of 3.4 A fibrils composed of staggered b-strands. The amino acids 50e70 are associated with familial synucleinopathies containing three mutation sites and contribute to fibril stability by forming the link between the two protofilaments [3]. Posttranslational stabilization of alpha-synuclein protein explains the increase in this protein during aging in mature neurons in the human substantia nigra. Human A53T showed increased stability resulting in greater levels of the mutant protein in transgenic mice [4]. The alpha-synuclein sequence between amino acids 61e95 has been identified to correspond to the non-b-amyloid component of Alzheimer disease senile plaques called the NAC [5]. The NAC is a central hydrophobic region in alpha-synuclein that plays an essential role in alpha-synuclein aggregation to neurotoxic oligomers and is important in membrane vesicle fusion [6]. Low expression of alpha-synuclein induced exocytosis, while its high expression resulted in a slight increase. Alphasynuclein mutants of A30P perturb the helical structure in the helix 1 region of membrane-bound protein, but this is not the case for other mutations associated with the familial form of Parkinson’s disease [7]. The C-terminal of alpha-synuclein (96e140) is highly acidic and disordered, and its acidic property protects the NAC region from

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fibrillation. The N-terminal (1e61) of alpha-synuclein presents a more helical organization, and its interaction with the C-terminal also protects the NAC region from aggregation [8]. Preaggregated NAC regions were found to be neurotoxic in in vitro studies with nerve growth factor differentiated PC12 dopaminergic cell line differentiated with nerve growth factor and in vivo by inducing selective loss of dopaminergic neurons [9]. Aggregation of the NAC region of alpha-synuclein induces apoptotic cell death in human neuroblastoma SH-SY5Y cells [10]. Methyl amidation and acetylation of a peptide corresponding to alpha-synuclein NAC 71e82 amino acids aggregate, presenting a b-sheet secondary structure such as a cross-b structure detected in prion proteins [11]. The beta-sheet secondary structure of NAC (3e18 amino acids) and NAC scrambled sequence (1e18 amino acids) aggregates were found to be toxic in aqueous solution, suggesting that an intermediate of alpha-synuclein in the formation of fibrils is responsible for neurotoxic properties of alphasynuclein the NAC region [12]. A study performed using atomic force microscopy-infrared spectroscopy revealed that the spherical oligomeric species show significantly different forms in their secondary structures. The secondary structure of spherical oligomeric species always show higher antiparallel b-sheet content in comparison with the fibrillar species, suggesting that the antiparallel b-sheet structure is involved in alpha-synuclein oligomer neurotoxicity [13]. Alpha-synuclein aggregation can induce disruption of synaptic functional homeostasis because this protein is abundant in synaptic sites and interacts with cytoskeletal components, lipid membranes, monoamine transporters, chaperones, and synaptic vesicle-associated proteins [14] (Fig. 1.10). It has been proposed that alpha-synuclein aggregation to oligomers is the most toxic event affecting neurotransmitter release because of oligomer impairment of synaptic vesicle trafficking; protein redistribution of the presynaptic SNARE complex that results in deficient docking, tethering, priming, and fusion of synaptic vesicles at the active zone; and a decrease in protein levels in the active zone of synaptic vesicles [15]. Alpha-synuclein aggregation to oligomers has been associated with neurodegeneration by inducing mitochondrial dysfunction, endoplasmic reticulum stress, synaptic dysfunction, autophagy/lysosomal dysregulation, and oxidative stress [1]. A study on the role of charge and length variation on alpha-synuclein aggregation revealed that both the length of the C-terminal and its charge play a significant role at the initial fibril formation stage, while the length of the C-terminal extension plays a role in fibril elongation [16]. The identification of a new alpha-synuclein mutation (H50Q) inducing the familial form of Parkinson’s disease suggests a role of histidine-50 in alpha-synuclein’s normal conformation and function. In vitro studies have demonstrated that substitution of histidine-50 with alanine, aspartic acid,

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FIGURE 1.10

Alpha-synuclein structure and properties. The N-terminal region of alphasynuclein plays a very important role in protein aggregation to neurotoxic oligomers where we can find the mutations associated with the familial form of Parkinson’s disease. The NAC region corresponds to the non-b-amyloid component of Alzheimer’s disease senile plaques. The C-terminal region of alpha-synuclein is highly acidic and protects the NAC region from fibrillation.

or glutamine induces alpha-synuclein aggregation. However, substitution of histidine-50 with the positively charged amino acid arginine prevents alpha-synuclein aggregation. At neutral pH, histidine has a partial positive charge, suggesting that histidine-50 plays a protective role in the alpha-synuclein sequence by preventing its aggregation under normal conditions [17].

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A study performed using bioinformatics, mutation, and NMR techniques identified a sequence in the NAC region of seven amino acids (residues 36e42) that play an essential role in the regulation of alphasynuclein aggregation, because its deletion prevents alpha-synuclein aggregation at pH 7.5. They also reported that pre-NAC region 45e57 amino acids together with NAC region 36e42 amino acids prevented alpha-synuclein aggregation at low pH. Deletion of NAC region 36e42 amino acids or deletion of both regions (NAC 36e42 and pre-NAC 45e57) prevents alpha-synuclein aggregation, toxicity, and vesicle fusion in Caenorhabditis elegans experimental model [18]. One of the major components of the synaptic vesicle membrane is cholesterol, and it reportedly acts as a modulator of alpha-synuclein affinity to synaptic vesicles by decreasing the affinity of the region of residues 65e97 in alpha-synuclein NAC component [19]. A study performed with transgenic mice did not show important changes in the axonal transport of alpha-synuclein in older presymptomatic A53T-transgenic mice, suggesting that the early stages of alpha-synucleinopathy in A53T-transgenic mice are not connected with changes in slow axonal transport, but aging induces a significant slowdown in axonal transport [20]. A study performed in human neurons expressing high levels of alpha-synuclein as a consequence of gene duplication revealed that alpha-synuclein oligomers induced a reduction in axonal mitochondrial transport. Inhibition of alpha-synuclein oligomerization prevents impairment of anterograde axonal transport of mitochondria. In addition, the formation of alpha-synuclein oligomers was linked to reduction of cellular ATP levels and subcellular relocalization of transport-regulating proteins KLC1, Miro1, and TAU [21]. The role of the NAC region in alpha-synuclein transport within axons through association with membranes was studied. The axonal transport of synaptic proteins was disturbed because of alpha-synuclein accumulation as wild-type or with A53T, generating synaptic morphological defects. Alpha-synuclein accumulation and axonal blockage were prevented by deletion of the NAC region (71e82 amino acids). The authors suggested that the NAC region is crucial for alpha-synuclein association with membranes, and its aggregation perturbs axonal transport [22]. Three alpha-synuclein mutations associated with familial forms of Parkinson’s disease have been shown to accelerate alpha-synuclein aggregation. However, the A30P mutation induces the fastest formation of oligomers and the longest delay in the conversion of oligomers to fibril conversion from oligomers to fibrils [23,24]. Alpha-synuclein aggregation has been proposed as a response to (1) apoptosis induced by staurosporine, 6-hydroxy-dopamine, or 1-methyl-4-phenylpyridinium or (2) alpha-synuclein interaction with proaggregant nuclear factor has been linked to disruption of nuclear envelope integrity. Alpha-synuclein

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aggregates increase when lamin B1, a key nuclear envelop constituent protein, is knocked down [24]. A study suggested that the nuclear proaggregant factors related to alpha-synuclein aggregation in brain and apoptotic neurons are histones. Alpha-synuclein aggregates induced by histones included both protofibrils and fibrils [25]. Metal induces alpha-synuclein fibrillation proceeding at the nucleation stage of aggregation, stabilizing higher-order oligomers and disaggregating preformed alpha-synuclein fibrils to comparable types of higher-order oligomers [26]. Alpha-synuclein has specific sequences for metal binding, such as 1-MDVFM-5 that binds copper (II and III), 48VVHGV-52 that binds copper (II and III), zinc(II), and iron (II and III), and 119-DPDNEA-125 that binds Cu(II), zinc(II), iron (II and III), cobalt(II), nickel(II), and manganese(II). The binding of copper(I) to the alpha-synuclein N-terminal region stabilizes the alpha-helical secondary structure, which seems to play a role in its association with membranes and vesicle trafficking. However, calcium (II) binding to the C-terminal region of alpha-synuclein induces a negative modulatory effect on protein membrane-binding properties [27]. Alpha-synuclein aggregation is promoted by copper binding, and there is a search for metal-protein attenuating compound, which are chelators that interrupt interactions between metals and proteins. It has been reported that N-acylhydrazones such as 1-methyl-1H-imidazole-2-carboxaldehyde isonicotinoyl hydrazine have affinity for copper (II) and compete with alpha-synuclein to bind copper (II) [28]. Posttranslational modifications of alpha-synuclein have been proposed as a factor that induces its aggregation to oligomers. Phosphorylation is one of the most studied posttranslational modifications of alphasynuclein, especially phosphorylation of S129. Lewy bodies contain phosphorylated alpha-synuclein S129 deposits, but soluble alphasynuclein is also phosphorylated at S129 [29,30]. The role of alpha-synuclein phosphorylation is controversial because it has been reported that phosphorylation S129 stimulates toxicity in cellular models and formation of alpha-synuclein inclusions [31]. A study with the aim to test the neurotoxic role of alpha-synuclein phosphorylation at serine 129 in rats expressing human wild-type or mutant alphasynuclein with serine 129 revealed no significant loss of dopaminergic neurons or decrease in nigrostriatal terminal density [32]. Less than 4% of total alpha-synuclein in normal brain is found phosphorylated at serine 129. However, around 90% of alpha-synuclein accumulation in Lewy bodies is phosphorylated at serine 129 and suggests that this phosphorylation leads to the development of Lewy bodies [33]. Phosphorylation of alpha-synuclein seems to play a modulating role in its function, structure, toxicity, and degradation. A study on binding affinity of a single-domain antibody fragment to alpha-synuclein that

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binds to the C-terminal region of a-synuclein at nanomolar affinity showed that serine 129 phosphorylation has an insignificant effect on binding affinity, whereas tyrosine 125 phosphorylation diminished binding affinity 400-fold [34]. Overexpression of human mutant alphasynuclein in the olfactory bulb of rats induced a decrease in tyrosine hydroxylase neurons and their fibers with concomitant phosphorylation of alpha-synuclein at serine 129 [35]. It has been reported that phosphorylation at tyrosine 125 of human alpha-synuclein plays a protective role because the level of soluble oligomer diminishes tyrosine 125 phosphorylation but increases serine 129 phosphorylation. This observation was supported by the fact that tyrosine 125 phosphorylation is diminished in Parkinson’s disease patients with synucleinopathy dementia with Lewy bodies [36]. Alpha-synuclein phosphorylation at tyrosine 125 was induced under hyperosmotic stress catalyzed by the protein tyrosine kinase Pyk2/ RAFTK [37]. Alpha-synuclein binding to heat shock protein 70 increased protein phosphorylation at tyrosine 136 phosphorylation. Dopaminergic neuronal cell death and rotarod performance in an animal model for Parkinson’s disease improved when protein tyrosine phosphatase 1B inhibitor was present, suggesting that regulation of tyrosine 136 phosphorylation of alpha-synuclein promotes the turnover of lysosomal degradation of a-synuclein [38]. Alpha-synuclein aggregation, neurobehavioral deficits, and neuropathology were reduced when the gene encoding the nonreceptor tyrosine kinase c-Abl was deleted in a transgenic mouse expressing A53T. On the other hand, overexpression of the nonreceptor tyrosine kinase c-Abl increases alpha-synuclein aggregation, neuropathology, and neurobehavioral insufficiencies, leading to an agedependent increase in phosphorylation of tyrosine-39. An accumulation of tyrosine 39 in alpha-synuclein was observed in human postmortem samples in comparison with control brains [39]. Alpha-synuclein phosphorylated at tyrosine-39 enables interconversion of the protein to the broken-helix state from the vesicle-bound extended-helix state. Phosphorylation of tyrosine-39 leads to diminished binding of a region matching to helix-2 of the broken-helix state in the presence of lipid vesicles. These results indicate that phosphorylation of tyrosine-39 may modulate functional characteristics of alpha-synuclein and its aggregation to oligomers [40]. Alpha-synuclein is localized principally in presynaptic nerve terminals, but a small fraction is present in neuronal nuclei. The increase in alpha-synuclein expression induced DNA strand breaks that increased in the presence of iron when the protein was localized in the nucleus. Alphasynuclein together with iron induced neuronal cell death [41,42]. The effect of alpha-synuclein in the nucleus, and the role of its phosphorylation were studied. Severe transcriptional deregulation that induced

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alpha-synuclein in the nucleus was observed that involves downregulation of genes related to cell cycle. A reduction in alpha-synuclein binding capacity to DNA was observed during transcriptional deregulation. The presence of alpha-synuclein in the nucleus induces the accumulation of high-molecular-weight aggregates that change gene expression and decrease neurotoxicity. Phosphorylation of alphasynuclein at serine-129 modulates its effect on gene expression, nuclear localization, and toxicity [43]. A study on gene expression analysis was performed using RNAsequencing and revealed overexpression of mutant A30P or wild-type alpha-synuclein that induced important changes in gene expression including downregulation of important genes required for DNA repair. Increased alpha-synuclein expression induces a decreased level of acetylated histone 3 [44]. Another study performed with transgenic mice overexpressing mutant A30P and wild-type alpha-synuclein showed that this protein increases DNA binding and induces transcriptional deregulation. Alpha-synuclein-induced upregulation of COL4A2, an important component of basement membranes, in both human mutant A30P and wild type, indicating an important role of collagen-associated genes in alpha-synuclein-dependent neurotoxicity. In dopaminergic cells, mutation A30P in alpha-synuclein increases cell vulnerability to endoplasmic reticulum stress and changes Golgi morphology [45]. Alpha-synuclein aggregation to oligomers induces mitochondrial dysfunction, alteration of endoplasmic reticulum-Golgi transport, formation of pores in membranes, and endoplasmic reticulum stress [46]. A study of human astrocytes demonstrated that when lysosomal degradation of alpha-synuclein oligomers fails, they deposit in the trans-Golgi network, causing swelling of the endoplasmic reticulum and mitochondrial damage. Under these stress conditions, astrocytes transfer alpha-synuclein aggregates to other astrocytes through direct contact and tunneling nanotubes. The transfer of alpha-synuclein oligomers from stressed to healthy astrocytes results in a bidirectional transfer where healthy astrocytes transfer mitochondria as a rescue mechanism for stressed astrocytes [47]. A study showed that alpha-synuclein affects vesicles trafficking from the endoplasmic reticulum to the Golgi. A genome-wide screening revealed that alpha-synuclein affects the expression of Rab guanosine triphosphatase Ypt1p related to cytoplasmic inclusions of alphasynuclein. High expression of Rab guanosine triphosphatase Ypt1p protects against alpha-synuclein-induced loss of dopaminergic neurons in animal models of Parkinson’s disease [48]. In vitro studies and in cell cultures expressing A53E alpha-synuclein mutation show decreased alpha-synuclein aggregation and increased proteasome activity, changing normal proteostasis [49].

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Endoplasmic reticulum plays a very important role in maintaining the regulation of protein homeostasis. Alpha-synuclein oligomers formed in the endoplasmic reticulum of alpha-synuclein-transgenic mice expressing A53T induce neuronal death [50]. Alpha-synuclein oligomers induce endoplasmic reticulum stress by affecting the transport of synaptic vesicles, calcium homeostasis, and intracellular protein trafficking [51]. A study on endoplasmic reticulum transport to Golgi showed that this transport is inhibited when wild-type alpha-synuclein or A53T is overexpressed. This transport was reestablished when endoplasmic reticulum-Golgi arginine soluble N-ethylmaleimide-sensitive factor attachment protein receptors (R-SNAREs) were coexpressed in the same cells, suggesting that alpha-synuclein antagonizes SNARE function to mediate vesicle fusion with membrane [52]. The accumulation of alpha-synuclein in an endoplasmic reticulum generates the formation of oligomers in the lumen of this organelle. Treatment of transgenic mice with expressing A53T with Salubrinal, an inhibitor of endoplasmic reticulum stress, reduces the formation of alphasynuclein oligomers and disease symptoms [53]. Neurons depend on the exchange of metabolites and calcium signals between their organelles. Mitochondria-associated membranes are specialized sites of the endoplasmic reticulum that act as areas of contact between the mitochondria and endoplasmic reticulum. These areas of contact between mitochondria and the endoplasmic reticulum play an important role in calcium signaling, lipid metabolism, autophagy, and mitochondrial dynamics. Mechanisms that affect these areas of contact between mitochondria and endoplasmic reticulum have been proposed as widely involved in neurodegenerative processes [54]. Calcium bidirectional exchange occurs between endoplasmic reticulum vesicles and mitochondria that plays an important function in various neural activities [55]. A study on the effect of alpha-synuclein aggregation on calcium homeostasis showed that this protein aggregation induces a decrease in calcium levels at an early phase followed by an increase under a later phase. In addition, this study found that alpha-synuclein aggregates activate sarco/endoplasmic reticulum Ca2þ-ATPase, and its inhibition with cyclopiazonic acid protects cells against alpha-synuclein toxicity [56]. A study that sought to relate neurite degeneration with functional and morphological remodeling of the endoplasmic reticulum revealed that the endoplasmic reticulum of the neurite neurodegeneration model was more fragmented and complexed with mitochondria. This generated an increase in IP3R-dependent mitochondrial calcium and dysfunction [57]. A study has reported that alpha-synuclein wild type from human brain tissue, cells, or mice is present in mitochondria-associated endoplasmic reticulum membranes and not in mitochondria. In contrast, A30P and A53T presented reduced association with mitochondria-associated

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endoplasmic reticulum membranes and an increase in mitochondrial fragmentation. Interestingly, mitochondrial fragmentation is reversed by overexpression of wild-type alpha-synuclein in cells expressing mutant alpha-synuclein. Overexpression of the mitofusin-2 and dynamin-related protein-1 protein does not reverse mitochondrial fragmentation, suggesting that alpha-synuclein acts downstream of mitochondrial fusion/ fission [58]. Another study on the role of alpha-synuclein A53T and A30P mutations on mitochondriaeendoplasmic reticulum tethering was performed by determining calcium homeostasis and mitochondrial function. This study concluded that these mutations do not change alphasynuclein’s ability to increase mitochondriaeendoplasmic reticulum tethering and mitochondrial calcium transients. However, an increase in alpha-synuclein aggregates induces redistribution of this protein from cytoplasm [59]. A possible role of alpha-synuclein in the control of mitochondrial homeostasis was concluded after studies with alpha-synuclein null mice. The mice lacking alpha-synuclein exhibited diminished mitochondrial respiratory capacity, fragmented mitochondria, protein expression changes related to mitochondrial shape modifications, and altered endoplasmic reticulumemitochondria interactions. These results suggest a physiological role of alpha-synuclein in preserving mitochondrial homeostasis and function [60]. The association between the endoplasmic reticulum and mitochondria is mediated by physical contact between endoplasmic reticulum protein vesicle-associated membrane proteinassociated protein B and outer mitochondrial membrane protein tyrosine phosphatase-interacting protein 5. The association between endoplasmic reticulum and mitochondria mediated by these proteins is lost when wild-type or mutant alpha-synuclein is overexpressed and accompanied by the loss of calcium exchange between these two organelles as well as low production of mitochondrial ATP [61]. The association between endoplasmic reticulum and mitochondria mediated by vesicle-associated membrane protein-associated protein B and tyrosine phosphatase-interacting protein-5 also regulates autophagy. Protein expression decreases endoplasmic reticulum or mitochondrial proteins mediated by siRNA-stimulated autophagy. Overexpression of vesicle-associated membrane protein-associated protein B or tyrosine phosphatase-interacting protein-5 inhibits torin-1 and rapamycininduced autophagy. However, overexpression of these proteins does not have an inhibitory effect on starvation-induced autophagy. The regulatory effect of this association of endoplasmic reticulum and mitochondria on autophagy depends on calcium delivery from the endoplasmic reticulum to mitochondria [62]. The relationship between endoplasmic reticulum stress and autophagy was studied in a manganese exposure model that induces alpha-

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synuclein aggregation to oligomers that are degraded through endoplasmic reticulum stress and autophagy. Inhibition of endoplasmic reticulum stress with 4-phenylbutyric acid resulted in inhibition of autophagy activation. Autophagy activation with rapamycin decreases the formation of alpha-synuclein oligomers, while inhibition of autophagy with 4-phenylbutyric acid increases the formation of alphasynuclein oligomers and activates the PERK signaling pathway that mediates endoplasmic reticulum stress-induced autophagy [63]. Overexpression of mesencephalic astrocyte-derived neurotrophic factor in dopaminergic neurons prevented locomotion defects, decreased progressive neuronal degeneration, and helped with removal of alphasynuclein aggregates. The protective effect of mesencephalic astrocytederived neurotrophic factor is inhibited by silencing endoplasmic reticulum and autophagy linked genes [64]. Alpha-synuclein upregulates PELI1, which induces LAMP2 degradation in activated microglia after treatment with preformed alpha-synuclein fibrils, resulting in autophagy flux impairment [64]. The chaperone-mediated autophagy pathway has been reported to mediate the degradation of wild-type alpha-synuclein. However, mutant alpha-synuclein inhibits other proteins and its own degradation [65]. A study of Parkinson’s disease patients’ neurons revealed that alphasynuclein accumulation induced upregulation of Miro protein levels resulting in delayed mitophagy. Miro protein is removed from impaired mitochondria to stimulate mitochondria degradation via autophagy. Miro protein mediates mitochondrial motility and is located on the outer mitochondrial membrane. The interaction of the N-terminus of alphasynuclein is required for upregulation of Miro protein [66]. It has been reported that microRNA-7 acts on the alpha-synuclein mRNA 30 -untranslated region, inducing alpha-synuclein degradation by promoting autophagy and repressing alpha-synuclein expression [67]. A study that used stimulators/inhibitors of autophagy and proteasome revealed that alpha-synuclein degradation is performed by both lysosomal/autophagy and proteasome systems [68]. Expression of A53T in a cell line induces cell dead because of proteasome and mitochondrial dysfunction, oxidative stress, and increased endoplasmic reticulum stress [69]. Overexpression of retention in endoplasmic reticulum 1 protein in cell culture induces a decrease in the level of wild-type and mutant alpha-synucleins A53T, A30P, and E46K. The use of macroautophagy and proteasomal inhibitors revealed that the degradation of alpha-synuclein induced by the endoplasmic reticulum 1 protein was mainly mediated by the ubiquitin-proteasome system through its interaction with the ubiquitin ligase NEDD4 [70]. A study on the effect of exogenous alpha-synuclein fibrils in microglial cells revealed the involvement of the autophagy proteins optineurin and

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TANK-binding kinase 1 to ubiquitylated lysosomes. Alpha-synuclein fibril-dependent autophagy inhibition induced mitochondrial dysfunction that leads to microglial dell death [71]. Overexpression of the ubiquitin ligase NEDD4 can prevent alpha-synuclein accumulation and the degenerative effects induced by its aggregation to oligomers, while silencing NEDD4 expression using RNAi increases the loss of dopaminergic neurons and locomotor defects [72]. It has been proposed that alpha-synuclein induces neurodegeneration by impairing autophagy regulation and membrane permeability to ions. In addition, alpha-synuclein induces mitochondrial dysfunction by depolarization of the mitochondria membrane, disrupting and releasing cytochrome c [73]. Changes in autophagy/lysosomal markers have been observed when alpha-synuclein inclusion accumulates in the cytosol. Alpha-synuclein inclusion accumulates when autophagy is blocked by chloroquine, while alpha-synuclein clearance increases when autophagy is induced by rapamycin, suggesting an important role of autophagy in the degradation of alpha-synuclein fibrils [74]. A study of alpha-synuclein gene knockout showed that manganese-induced chaperone-mediated autophagy was independent of alpha-synuclein. Chaperone-mediated autophagy is a specific mechanism for degradation of unfolded/misfolded proteins where protein HSC70 detects chaperone-mediated autophagy substrate protein and carries them for Lamp 2A-mediated degradation [75]. 2-Cyano-3-[5-(2,5-dichlorophenyl)-2-furanyl]-N-5quinolinyl-2-propenamide increases degradation of alpha-synuclein aggregates by stimulating autophagy in a mTOR-independent manner [76]. A study revealed that high levels of alpha-synuclein in nigral dopaminergic neurons was related to a decrease in lysosome function because of low levels of transcription factor EB, an important transcriptional regulator of autophagy. Overexpression of transcription factor EB induces alpha-synuclein degradation and neuroprotection, while repression of transcription factor EB expression mediated by microRNA-128 induces alpha-synuclein oligomer neurotoxicity [77]. Inflammasomes are intracellular sensors of both pathogens and aberrant protein aggregates such as alpha-synuclein that activates the innate immune response to release inflammatory mediators [78]. Alphasynuclein induces the formation of nitric oxide and proinflammatory cytokines, and autophagy inducers such as rapamycin and trehalose downregulate the formation of these proinflammatory factors. The inhibition of autophagy at early stages reversed the effects of rapamycin and trehalose. In addition, alpha-synuclein induces the formation of interleukins 10 and 12p70 [79]. The level of interleukin-1b expression, alpha-synuclein aggregates, and their phosphorylated form was found to be increased in Parkinson’s disease patients’ peripheral blood, while transforming growth factor-a and interleukin-6 expression were

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unchanged. Alpha-synuclein oligomers induced the activation of pyrin domain-containing protein-3 inflammasome and increase interleukin-1b, caspase-1, and Atg5 expression in mouse astrocytes. The autophagy inhibitor 3-methyladenine inhibited increases in the expression levels of pyrin domain-containing protein-3, caspase-1, and interleukin-1b [80]. A decrease in the proinflammatory factor glia maturation factor resulted in a decrease in interleukin-1b and improvement of behavioral anomalies in a mouse model of Parkinson’s disease. The downregulation of glia maturation factor induced a decrease in the nod-like receptor pyrin domain-containing protein-3 inflammasome that is a major contributor to neuroinflammation [81]. A study focused on inflammation and alphanuclein expression revealed that a combination of interleukin 1-b and tumor necrosis factor-a or lipopolysaccharide induced a decrease in alpha-synuclein expression in primary culture of the enteric nervous system. Downregulation of alpha-synuclein was prevented in the presence of inhibitors of the p38 signaling pathway that regulate the formation of inflammatory mediators such as including interleukin 1-b, tumor necrosis factor-a TNF-alpha, IL-1-beta, and cyclooxygenase-2. However, interleukin 1-b/tumor necrosis factor-a and lipopolysaccharide had no effect on alpha-synuclein expression in cultures derived from rat central nervous system [82]. It has been proposed that alpha-synuclein proteolysis mediated by low levels of the serine protease plasmin prevents its aggregation to neurotoxic oligomers. Inflammation and extracellular alpha-synuclein aggregates increase the expression of the serine protease inhibitor plasminogen activator inhibitor-1 that decrease the level of plasmin, resulting in diminution of alpha-synuclein proteolysis [83]. Dopaminergic degeneration in the absence or presence of alpha-synuclein aggregates triggers the activation of the microglial NLR family pyrin domain-containing protein-3 inflammasome. In the substantia nigra of Parkinson’s disease patients, the level of the inflammasome adaptor protein apoptosis-linked speck-like protein containing a C-terminal caspase recruitment domain and cleaved caspase-1 is elevated. NLR family pyrin domain-containing protein-3 activation mediated by alpha-synuclein fibrils induces activation of inflammasome, resulting in the release of extracellular interleukin1b and the C-terminal caspase recruitment domain. The inhibition of NLR family pyrin domain-containing protein-3 prevents alpha-synuclein fibril-dependent inflammasome activation and nigrostriatal dopaminergic degeneration and decreases motor deficits and the accumulation of alpha-synuclein aggregates [84]. Alpha-synuclein expression and trafficking has been observed in primary astrocyte cultures. Overexpression of wild-type and mutant A30P and A53T in a cell line inhibited autophagy by decreasing LC3-II and increasing p62 protein levels. A loss of mitochondrial membrane potential

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was observed when cells expressing mutant alpha-synuclein were incubated with rotenone [85]. Induced pluripotent stem cell-derived ventral midbrain dopaminergic neurons and astrocytes from Parkinson’s disease patients carrying leucine-rich repeat kinase-2 G2019S mutation and healthy individuals were developed. The coculture of ventral midbrain dopaminergic neurons derived from control individuals with astrocytes carrying leucine-rich repeat kinase-2 G2019S induced morphological alterations and alpha-synuclein accumulation and significantly diminished cell survival in neurons compared with cocultures with astrocytes derived from healthy individuals. Astrocytes derived from Parkinson’s disease carrying leucine-rich repeat kinase-2 G2019S mutation exhibited progressive a-synuclein accumulation, impaired macroautophagy, and dysfunctional chaperone-mediated autophagy [86]. Formation of astroglial inclusions containing alpha-synuclein aggregation has been observed. Knockdown of the heat shock protein alphaB-crystallin increases autophagy and clearance of preformed alpha-synuclein fibrils in astrocytes. Overexpression of alphaB-crystallin inhibits autophagy, resulting in the accumulation of alpha-synuclein aggregates in the brain of mice expressing mutant A30P alpha-synuclein [87]. AlphaB-crystallin found in Lewy bodies inhibits alpha-synuclein fibrillization in vitro of both wild-type and mutant forms, generating large irregular aggregates of alphaB-crystallin and alpha-synuclein [88]. It has been reported that astrocyte primary cultures surrounded and degraded extracellular human alpha-synuclein aggregates. Alphasynuclein degradation in astrocyte primary cultures was performed by both lysosomal and proteasomal systems because alpha-synuclein clearance was inhibited by autophagy inhibitor 3-methyladenine and proteasome inhibitor MG132 [89]. The role of oxidative stress on alphasynuclein neurotoxicity was studied by overexpressing nuclear factor erythroid 2erelated factor 2, which is a redox-sensitive transcription factor in astrocytes from a mouse expressing A53T. The overexpression of this nuclear factor reduced oxidative stress, gliosis and alpha-synuclein aggregate levels, and phosphorylation, while motor neuron survival increased, and macroautophagy and chaperone-mediated autophagy dysfunction were delayed [90]. The response of astrocytes to dopaminergic degeneration was studied by injecting rats with 6-hydroxydopamine. Dopaminergic denervation induced by this neurotoxin generates free spheroids containing the dopamine transporter protein, tyrosine hydroxylase, and LC3autophagosomes corresponding to the initial stage of autophagy but not to late components of autophagy such as Lamp1/Lamp2 lysosomes. Free spheroid components were observed within astrocytes after 2e5 days of denervation of dopaminergic neurons, suggesting that astrocytes play an important role in removing degenerated dopaminergic neuronal debris

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[91]. Astrocyte exposure to alpha-synuclein oligomers increases glutamate uptake by increasing the level of expression of glutamate transporters GLAST and GLT-1. This effect was reverted when the transforming growth factor beta-1 pathway was inhibited [92]. The existence of alpha-synuclein inclusions in astrocytes, microglia, oligodendrocytes, and pericytes has been observed in the olfactory nucleus of Parkinson’s disease patient olfactory bulbs and the olfactory bulbs of healthy individuals [93]. It has been proposed that activation of T cells in Parkinson’s disease is mediated by astrocytes. Postmortem studies have shown that patient brains expressed high levels of major histocompatibility class II protein correlated with phosphorylated alpha-synuclein and colocalized with astrocyte markers. CD4þ T cells were surrounded by astrocytes expressing major histocompatibility class II. Deposits containing alpha-synuclein/major histocompatibility class II is transferred between astrocytes using tunneling nanotubes to spread inflammation and alphasynuclein aggregates [94]. The innate immune system is activated by alpha-synuclein accumulation resulting in the formation of inflammatory chemokines and cytokines. A study that used heterozygous reporter knockin where the CeC chemokine receptor type-2 was replaced with fluorescent reporters revealed that alpha-synuclein expression stimulates infiltration of the substantia nigra by proinflammatory CeC chemokine receptor type-2 peripheral monocytes. Deletion of CeC chemokine receptor type-2 prevents alpha-synuclein-stimulated monocyte infiltration, decreases major histocompatibility class II protein expression, and inhibits subsequent degeneration of dopamine neurons [95]. It has been reported that human alpha-synuclein fibrils induce the formation and secretion of proinflammatory cytokines in microglia. Microglia phagocytes prefer alpha-synuclein fibrils over the monomeric and oligomeric forms [96]. A study using knockout mice of class II transactivator gene, a transcriptional coactivator required for major histocompatibility complex II, revealed that the class II transactivator reduced alpha-synucleininduced neuroinflammation and neurodegeneration [97]. One of the neurotoxic actions of alpha-synuclein in nigrostriatal neurons is its aggregation to oligomers to induce mitochondrial dysfunction. It has been proposed that mitochondrial dysfunction is induced by mutations in mitochondrial DNA, bioenergetic defects, altered movement of mitochondria, alterations in trafficking or transport, mutation of nuclear DNA gene related to mitochondrial proteins, changes in dynamics of the mitochondria, and changes in morphology and size [98]. Alpha-synuclein aggregation induces mitochondrial dysfunction [99]. The expression of A53T in mice induces nuclear condensation, axonal swellings, somal chromatolytic changes, and mitochondrial DNA damage. Inclusions like Lewy bodies were observed in the cytoplasm of spinal

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motor neurons and cortical neurons that were positive to human alphasynuclein. Electron microscopy revealed the existence of alphasynuclein-positive cytoplasmic inclusions in axons and dendrites with damaged mitochondria [100]. It has been proposed that alpha-synuclein aggregation to oligomers induces the formation of plasma membrane pores that change membrane permeability to ions and induce mitochondrial depolarization, cytochrome c release, and calcium dyshomeostasis, thus resulting in mitochondrial dysfunction and impairment of autophagy regulation [71]. A study showed that the addition of alpha-synuclein fibrils to primary cultures of neurons induces the formation of phosphorylated aggregates other than fibrils that would be the result of their incomplete degradation in autophagy. These SDS-resistant aggregates of 12.5 kDA induce depolarization of mitochondria, release cytochrome C, and cause mitochondrial fragmentation. This study suggests that these alpha-synuclein aggregates induce mitochondrial dysfunction by triggering mitochondrial damage, fission, energetic stress, and mitophagy [101]. The role of alpha-synuclein in mitochondrial dynamics and autophagy was studied by overexpressing wild-type alpha-synuclein in a cell model. This study revealed that wild-type alpha-synuclein induced mitochondrial fragmentation in a dynamin-related protein-1-dependent manner and stimulated mitophagy. However, overexpression of optic atrophy-1 protein inhibited mitochondrial fragmentation and neurotoxicity induced by wild-type alpha-synuclein [102]. Alpha-synuclein induces actin filament network reorganization by interacting with spectrin to alter actin cytoskeleton and mitochondria dysfunction by altering related protein-1 localization [103]. Mitochondria comprises more than 1000 different proteins, of which only 13 are encoded by mitochondrial DNA. Therefore, most mitochondrial proteins are encoded by nuclear DNA and must be imported into mitochondria. The best-known system for importing nuclear proteins into mitochondria is the recognition of mitochondrial targeting signal by the translocase of the outer membrane-20 receptor. This receptor translocates the nuclear protein through the outer membrane into the inner membrane where the translocase of the inner membrane translocates the nuclear protein into the matrix. In the matrix, the mitochondrial targeting signal is cleaved to generate a mature mitochondrial protein. It has been reported that alpha-synuclein aggregates have high affinity to translocase of the outer membrane-20 receptor, preventing its interaction with the translocase of the outer membrane-22 coreceptor and mitochondrial import of nuclear proteins. Alpha-synuclein oligomer-induced impairment of mitochondrial nuclear protein import finally results in mitochondrial dysfunction. Postmortem studies with Parkinson’s disease brains revealed the association of loss of nuclear protein importation into

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mitochondria and interaction between alpha-synuclein aggregates and translocase of the outer membrane-20 [104]. The expression of A53T decreases the level of expression of ATPdependent Clp protease in induced pluripotent stem cell derived from a patient carrying this mutation. ATP-dependent Clp protease is a protease localized in mitochondrial matrix that is important in mitochondrial protein turnover. Deficiency in ATP-dependent Clp protease results in accumulation of mitochondria, increases mitochondrial oxidative stress, affects mitochondrial respiratory activity, and induces cell death. Overexpression of ATP-dependent Clp protease inhibits accumulation of phosphorylated (129) alpha-synuclein and increases expression of superoxide dismutase-2 [105]. The mitochondrial NAD-dependent deacetylase sirtuin-3 is involved in preventing oxidative stress and maintaining mitochondrial function. The association of alpha-synuclein oligomers with mitochondria decreases mitochondrial sirtuin-3 levels and mitochondrial biogenesis. Alpha-synuclein oligomer-dependent sirtuin 3 downregulation is followed by (1) a decrease in phosphorylation of cAMP-response element binding protein and AMPK; (2) an increase in dynamin-related protein-1 phosphorylation; and (3) a significant decrease in mitochondrial respiration. The administration of 5-Aminoimidazole-4-carboxamide-1b-D-ribofuranoside, an AMPK agonist, diminishes alpha-synuclein, reestablishes sirtuin 3 levels, and recovers mitochondrial function [106]. Preformed phosphorylated (serine-129) alpha-synuclein fibrils were found to be associated with mitochondria, and their accumulation was observed with concomitant deficit of mitochondrial respiration leading to mitochondrial dysfunction [107]. A study showed that a high level of alpha-synuclein oligomers impairs axon normal function in human neurons and affects anterograde axonal transport of mitochondria. The inhibition of formation of alpha-synuclein oligomers reestablishes axonal transport. The formation of alpha-synuclein oligomers was linked to changes in subcellular localization of transport-regulating proteins KLC1, Miro1, and TAU and a reduction in ATP levels that leads to axonal transport impairment [21]. It has been proposed that oxidative stress induces the alpha-synuclein aggregation of oligomers that leads to mitochondrial dysfunction and autophagy impairment [108]. The expression of A53T induces oxidative stress and a decrease in mitochondrial complex 1 and NADH cytochrome C reductase activity and the number of mitochondrial DNA copies. These effects are accompanied by an increase at early stage in nuclear factor erythroid-derived-like 2, peroxisome proliferator-activated receptor-

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gamma coactivator 1a, and cytosolic mitochondrial transcription factor A [109]. The levels of total and mitochondrial reactive oxygen species in the cell were higher in cells overexpressing wild-type alpha-synuclein, probably because of the low level of superoxide dismutases 1 and 2. The exposure of these cells to iron induces oxidative effects independent of alpha-synuclein overexpression [110]. Alpha-synuclein interaction with mitochondria induces cytochrome release, increased nitric oxide and mitochondrial calcium, and oxidative stress [111]. It has been proposed that superoxide dismutase-2 is an important regulator of antioxidant defense in mitochondria, and low levels of this enzyme induce oxidative stress. A transgenic mouse expressing mutant A30P alpha-synuclein with partial deficiency of superoxide dismutase-2 expression induces more advanced signs of synucleinopathy [112]. Preformed alpha-synuclein fibrils induce changes in oxidative stress-linked pathways such as induction of mitochondrial superoxide dismutase-2 [113]. Alpha-synuclein serine 129 phosphorylation and its accumulation were reported to be induced by iron with a concomitant increase in the level of expression of casein kinase 2 and polo-like kinase 2. The antioxidant N-acetylcysteine inhibits these iron-dependent effects, suggesting that oxidative stress plays an important role in iron-dependent alpha-synuclein phosphorylation [114]. Under oxidative stress induced by hydrogen peroxide, the levels of expression of both alpha-synuclein and mitochondrial ferritin are increased. Mitochondrial ferritin knockdown increases the protein level of alpha-synuclein, while mitochondrial ferritin has the opposite effect without affecting the mRNA level [115]. Hydrogen peroxide and calcium alone or in combination induce alpha-synuclein aggregation to oligomers in in vitro and in vivo experiments [116]. An increase in intracellular calcium induces alpha-synuclein that can be blocked by calcium channel blockers and BAPTA-AM. An impotent part of cells with alpha-synuclein aggregates has increased oxidative stress and calcium levels [117]. Hydrogen peroxide induces mitochondrial fragmentation to spherically shaped and hyperpolarized mitochondria called mitospheres that depend on fission factor dynamin-related protein-1. It has been reported that alpha-synuclein prevents mitosphere formation and decreases apoptosis under oxidative stress [118]. Alpha-synuclein in fibrillar and oligomeric forms induces oxidative stress, but only oligomeric alpha-synuclein induces endogenous glutathione and neurotoxicity. However, oligomerinduced oxidative stress was completely dependent on the existence of free metal ions. Metal chelator inhibited alpha-synuclein oligomerinduced oxidative stress generation and blocked oligomer-dependent neuronal death [119]. Fig. 1.11.

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FIGURE 1.11 Alpha-synuclein-induced neurodegeneration. Alpha-synuclein aggregation to oligomers induces neurodegeneration by inducing mitochondrial dysfunction, endoplasmic reticulum stress, autophagy dysfunction, formation of vesicle pores, synaptic dysfunction, disruption of endoplasmic reticulumemitochondrion association, oxidative stress, and disruption of calcium homeostasis.

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[78] Voet S, Srinivasan S, Lamkanfi M, van Loo G. Inflammasomes in neuroinflammatory and neurodegenerative diseases. EMBO Mol Med 2019;11:e10248. [79] Bussi C, Peralta Ramos JM, Arroyo DS, et al. Autophagy down regulates proinflammatory mediators in BV2 microglial cells and rescues both LPS and alphasynuclein induced neuronal cell death. Sci Rep 2017;7:43153. [80] Wang X, Chi J, Huang D, et al. a-synuclein promotes progression of Parkinson’s disease by upregulating autophagy signaling pathway to activate NLRP3 inflammasome. Exp Ther Med 2020;19:931e8. [81] Javed H, Thangavel R, Selvakumar GP, et al. NLRP3 inflammasome and glia maturation factor coordinately regulate neuroinflammation and neuronal loss in MPTP mouse model of Parkinson’s disease. Int Immunopharmacol 2020;83:106441. [82] Prigent A, Gonzales J, Durand T, et al. Acute inflammation down-regulates alphasynuclein expression in enteric neurons. J Neurochem 2019;148:746e60. [83] Reuland CJ, Church FC. Synergy between plasminogen activator inhibitor-1, a-synuclein, and neuroinflammation in Parkinson’s disease. Med Hypotheses 2020;138: 109602. [84] Gordon R, Albornoz EA, Christie DC, et al. Inflammasome inhibition prevents a-synuclein pathology and dopaminergic neurodegeneration in mice. Sci Transl Med 2018; 10:eaah4066. [85] Erustes AG, Stefani FY, Terashima JY, et al. Overexpression of a-synuclein in an astrocyte cell line promotes autophagy inhibition and apoptosis. J Neurosci Res 2018;96: 160e71. [86] di Domenico A, Carola G, Calatayud C, et al. Patient-specific iPSC-derived astrocytes contribute to non-cell-autonomous neurodegeneration in Parkinson’s disease. Stem Cell Rep 2019;12:213e29. [87] Lu SZ, Guo YS, Liang PZ, et al. Suppression of astrocytic autophagy by aB-crystallin contributes to a-synuclein inclusion formation. Transl Neurodegener 2019;8:3. [88] Rekas A, Adda CG, Andrew Aquilina J, et al. Interaction of the molecular chaperone alphaB-crystallin with alpha-synuclein: effects on amyloid fibril formation and chaperone activity. J Mol Biol 2004;340:1167e83. [89] Hua J, Yin N, Xu S, et al. Enhancing the astrocytic clearance of extracellular a-synuclein aggregates by Ginkgolides attenuates neural cell injury. Cell Mol Neurobiol 2019;39:1017e28. [90] Gan L, Vargas MR, Johnson DA, Johnson JA. Astrocyte-specific overexpression of Nrf2 delays motor pathology and synuclein aggregation throughout the CNS in the alphasynuclein mutant (A53T) mouse model. J Neurosci 2012;32:17775e87. [91] Morales I, Sanchez A, Rodriguez-Sabate C, Rodriguez M. Striatal astrocytes engulf dopaminergic debris in Parkinson’s disease: a study in an animal model. PLoS One 2017;12:e0185989. [92] Diniz LP, Araujo APB, Matias I, et al. Astrocyte glutamate transporters are increased in an early sporadic model of synucleinopathy. Neurochem Int 2020;138:104758. [93] Stevenson TJ, Murray HC, Turner C, Faull RLM, Dieriks BV, Curtis MA. a-synuclein inclusions are abundant in non-neuronal cells in the anterior olfactory nucleus of the Parkinson’s disease olfactory bulb. Sci Rep 2020;10:6682. [94] Rostami J, Holmqvist S, Lindstro¨m V, et al. Human astrocytes transfer aggregated alpha-synuclein via tunneling nanotubes. J Neurosci 2017;37:11835e53. [95] Harms AS, Thome AD, Yan Z, et al. Peripheral monocyte entry is required for alphaSynuclein induced inflammation and Neurodegeneration in a model of Parkinson disease. Exp Neurol 2018;300:179e87. [96] Hoffmann A, Ettle B, Bruno A, et al. Alpha-synuclein activates BV2 microglia dependent on its aggregation state. Biochem Biophys Res Commun 2016;479:881e6. [97] Williams GP, Schonhoff AM, Jurkuvenaite A, Thome AD, Standaert DG, Harms AS. Targeting of the class II transactivator attenuates inflammation and

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D.2.2. Mitochondrial dysfunction In the ventral midbrain substantia nigra pars compacta, dopamine neurons play a key role in the control of voluntary movements by releasing the neurotransmitter dopamine. The release of dopamine or other neurotransmitters under neurotransmission by dopaminergic or other neurons requires functional mitochondria where the majority of ATP is produced. Therefore, mitochondrial dysfunction plays an important role in the loss of dopaminergic neurons in Parkinson’s disease. Mitochondrial production of ATP is mediated by the coupling between the electron transport chain and mitochondrial oxidative phosphorylation. The protons translocated from the mitochondrial matrix during the transport of electrons from NADH through mitochondrial complexes to finally reduce oxygen to water return to the matrix through mitochondrial ATPase phosphorylating ADP to ATP (Fig. 1.12). The inhibition of mitochondrial complex I by neurotoxins such as 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine induced neurodegeneration of dopaminergic neurons and severe parkinsonism symptoms in humans who used drugs contaminated with this compound, suggesting an important role of mitochondrial dysfunction in Parkinson’s disease [1]. A study that used gene editing to knock out each of 45 subunits that compose complex I revealed that only 25 subunits are necessary for complex I functional activity, and only one is fundamental for cell viability [2]. Chronic exposure to rotenone, a classic inhibitor of mitochondrial complex I of mitochondrial electron transport chain, induces degeneration of dopaminergic neurons and parkinsonism in animals [3]. A study that compared brain mitochondria of 10 patients with Parkinson’s disease with 12 age-matched healthy individuals revealed a reduction in complex I catalytic activity in frontal cortex mitochondria of Parkinson’s disease patients. An increase in protein carbonyls of complex I of Parkinson’s disease brain mitochondria was also observed, suggesting oxidative damage of this mitochondrial complex [4]. Another study of Parkinson’s disease brains observed only a moderate decrease in complex I function. Remarkably, substantia nigra neurons with a deficiency in complex I presented fewer alpha-synuclein

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FIGURE 1.12 Mitochondrial dysfunction induced by complex I inhibition. Inhibition of mitochondrial complex I blocks the flow of electrons from NADH to reduce oxygen. Electron flow through mitochondrial complexes translocates protons from the mitochondrial matrix to the intermembrane space that return to the matrix via ATPase synthase that catalyzes the phosphorylation of ADP to ATP and inorganic phosphate. Therefore, inhibition of the mitochondrial complex I results in low levels of ATP that generate mitochondrial dysfunction. Neuronal neurotransmission is dependent on the existence of ATP, and therefore low levels of ATP prevent neuronal neurotransmission, resulting in neuronal dysfunction.

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aggregations. The authors concluded that complex I deficiency in Parkinson’s disease probably does not have a role in the neurodegenerative process and happens independently of mitochondrial DNA damage [5]. A study performed with postmortem Parkinson’s disease brain frontal cortex, putamen, and substantia nigra using the Southern blot technique revealed that the seven subunits of complex I coded by mitochondrial DNA did not exhibit any deletion [6]. Therefore, dependence on functional mitochondrial protein import machinery to import nuclearencoded proteins for a functional complex I is fundamental. A study of postmortem Parkinson’s disease brains suggest impairment of the mitochondrial protein import system because of downregulation of translocase of the inner membrane 23 and translocase of the outer membrane 20. In addition, the level of expression NADHeubiquinone oxidoreductase enzyme, a key component of complex I, was decreased [7]. Alpha-synuclein impairs mitochondrial protein nuclear-encoded by binding to translocase of the outer membrane 20 [8]. A study of postmortem brains with Parkinson’s disease and Parkinson’s disease dementia revealed that mitochondrial complex I and mitochondrial DNA levels in the prefrontal cortex were significantly decreased in Parkinson’s disease dementia, but no significant changes were observed in the Parkinson’s disease patient prefrontal cortex [9]. A study on mitochondrial complex I deficiency performed in different brain regions, blood platelets, and skeletal muscle from Parkinson’s disease patients showed that this is limited to the brain and substantia nigra [10]. Mitochondrial complex I deficiency was found to be specific for Parkinson’s disease substantia nigra, and there is no correlation between complex I deficiency and L-3,4-dihydroxyphenylalanine therapy [11]. A meta-analysis on mitochondrial complex I downregulation performed in the substantia nigra brain of Parkinson’s disease patients confirmed this observation, but the studies on impairment assembly of complex I in frontal cortex were not confirmed. Meta-analysis on studies performed in blood and skeletal muscle were confirmed with some exceptions [12]. The effect on complex I and regulation of mitochondrial biogenesis in Parkinson’s disease frontal cortex were studied by quantifying the expression of seven mitochondrial DNA-encoded, 33 nuclear DNAencodes complex I genes as well as several mitobiogenesis genes and six assembly factors for complex I. They found mitobiogenesis signals that were maintained but downregulated and were correlated with reduced mitochondrial NADH-driven electron transfer [13]. A study that used a mice Ndufs4GT/GT encoding a structural complex I protein exhibited a reduction in mitochondrial complex I in the hippocampus and alterations in tricarboxylic acid cycle metabolism, such as lower concentrations of isocitrate, citrate, and cis-aconitate. Ndufs4GT/ GT mice also showed increased alanine concentrations in the brain that

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have been suggested as a marker of mitochondrial dysfunction [14]. The subunit of complex I Ndufs4 mutation induces deficits in complex I activity [15]. A study performed with Ndufs4 knockout mouse model observed an important ubiquitin protein increase in substantia nigra. In addition, neurofilaments in general 20S proteasome activities were significantly diminished, while ubiquitinated neurofilaments increased in substantia nigra of knockout animals. The authors suggest that mitochondrial complex I dysfunction induces proteasome inhibition that increases ubiquitinated neurofilaments and decreases the expression of neurofilaments [16]. Tumor necrosis factor alpha, a proinflammatory cytokine, changes mitochondrial complex I activity, decreases ATP levels, and increases autophagy and reactive oxygen species levels. In addition, tumor necrosis factor alpha decreases the level of nuclear-encoded transcript of mitochondrial complexes [17]. The tricarboxylic acid cycle plays an important role in the formation of NADH and FADH2, substrates of the mitochondrial electron transport chain, to generate ATP. One of the components of tricarboxylic acid cycle is part of the mitochondrial electron transport chain. Succinate dehydrogenase, also known as succinate-coenzyme Q reductase or mitochondrial complex II, is a flavoenzyme containing FAD that is reduced to FADH2, which transfers electrons to ubiquinone in the respiratory electron transport chain. In the cerebellum of Parkinson’s disease patients, the level of expression of one of the enzymes of this cycle, alphaketoglutarate dehydrogenase complex, was at normal level, but its enzymatic activity was significantly reduced [18]. It was reported that many deletions in pigmented neurons located in the substantia nigra were observed in a study of human postmortem brains of the elderly. The number of mitochondrial deletions was higher in neurons that were cytochrome C oxidaseedeficient compared with cytochrome C oxidaseepositive neurons, suggesting a role of cytochrome C oxidase deletions that could be directly related to dysfunction of cellular respiration [19]. A study that compared Parkinson’s disease cerebrospinal fluid with healthy controls revealed that the metabolic profile of patients showed an association between mitochondrial dysfunction and the tricarboxylic acid cycle, fatty acid/lipid metabolism, and glutathione metabolism [20]. A study related to mutations in mitochondrial DNA revealed that both older controls and Parkinson’s disease patients had a high level of deletions associated with dysfunction of mitochondrial respiration. These authors propose that this high level of mitochondrial DNA mutations is important both in neuronal loss in Parkinson’s disease and in aging of the brain [21]. A meta-analysis on mitochondrial complex IV, also known as cytochrome C oxidase, deficiency in Parkinson’s disease confirmed a decrease in complex IV in substantia nigra, frontal cortex, cerebellum, and

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peripheral blood [12]. A study performed to identify genes associated with Parkinson’s disease of a young Caucasian women using functional genomics and whole-exome sequencing showed that the homozygous variant in CHCHD2, a mitochondrial protein of unknown function, leads to deficiencies in complex I and IV [22]. The analysis of the mitochondrial electron transport chain organization in Parkinson’s disease patients carrying a Pink-1 mutation showed that complex IV was significantly lost. This observation was confirmed in studies with cultured neurons from Pink1/ mice [23]. A reduction in mitochondrial electron transport chain complex I, II, III, and IV activity in angular gyrus and frontal cortex area 8 was observed in Parkinson’s disease dementia [24]. Mitochondrial complex I and IV activity were found to be significantly decreased in idiopathic Parkinson’s disease patient leukocytes [25]. A link between extracellular vesicle trafficking neuroinflammation and mitochondrial dysfunction has been proposed. Parkinson’s disease patients have more circulation extracellular vesicles, and analysis of circulating extracellular vesicles of Parkinson’s disease patients showed that they have lower mitochondrial markers ATP5A, NDUFS3, and SDHB and tetraspanin protein markers CD9 and CD63 in comparison with controls. Seven molecules were identified as relevant in circulating extracellular vesicles in 94% of Parkinson’s disease and 67% of control samples including C-reactive protein, CD9, fibroblast growth factor 21, NDUFS3, tumor necrosis factor alpha, interleukin 9, and macrophage inflammatory protein 1b [26]. Mitochondrial dynamics dysfunction involves p38 MAPK activation induced phosphorylation of dynamin-related protein-1 at serine 616, activating dynamin-related protein-1 that increases mitochondria fission and results in mitochondrial dysfunction [27]. The brain is completely dependent on energy (ATP) supply for normal functions such as neurotransmission. The major source of energy is the mitochondria, and the maintenance of healthy and functional mitochondria is essential for the brain. Mitochondrial damage occurs under different stress situations, but there is a mechanism for the maintenance of healthy and functional mitochondria by undergoing mitochondrial fission and fusion [28]. Under mitochondrial fission/fusion, the tubular architecture is preserved, but cellular stress induces an excessive mitochondrial fission that results in mitochondria division into daughter mitochondria [29]. The impaired daughter mitochondria induce mitochondrial autophagy to remove damaged mitochondria that lose their normal function [30]. The protein guanosine triphosphatase family, composed of dynamin-related proteins and proteins involved in constriction and scission of the double membranes, regulates mitochondrial fission [31]. Dynamin-related protein-1 plays a crucial role in mitochondrial fission. Under mitochondrial damage, dynamin-related protein-1 is moved from

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cytosol to the outer mitochondrial membrane to induce mitochondrial constriction and scission. Dynamin-related protein-1 binds to its receptors such as mitochondrial dynamic 51 kDa protein and 49 kDa protein or mitochondrial fission factor and fission-1. Other important components of mitochondrial division are dynamin 1 and dynamin 2 [32]. Dynaminrelated protein-1 is involved in several steps of mitochondrial fission such as its movement from cytosol to the outer mitochondrial membrane, GTP hydrolysis, high self-assembly, and eventual disassembly [33]. Different dynamin-related proteinsdfor example, dynamin-related protein-1dplay an important role in maintaining mitochondrial homeostasis by mediating mitochondrial fission, fusion, and mitophagy to prevent neurodegeneration occurring in Parkinson’s disease. Interestingly, dynamin-related protein-1 can be involved in abnormal mitochondrial fission and mitochondrial autophagy leading to neuronal death [34]. Dynamin-related protein-1-dependent mitochondrial fragmentation was induced by alpha-synuclein, but overexpression of optic atrophy-1 protein prevents alpha-synuclein-induced cytotoxicity and mitochondrial fragmentation. In addition, alpha-synuclein expression activates mitochondrial autophagy [35]. Alpha-synuclein overexpression affects mitochondrial morphology and increases dynamin-related protein-1 translocation to mitochondria [36]. However, another report shows that dynamin-related protein-1 is not required for mitochondrial fragmentation when alpha-synuclein interacts directly with mitochondrial membranes [37]. Mitochondrial fusion is controlled by the dynaminrelated GTPases mitofusin-1, optic atrophy-1, and mitofusin 2, and their activation is performed by the binding of two mitochondria to form a structure in the contact point between the outer membranes; GTP hydrolysis induces the fusion of the outer membranes and finally the fusion of the inner membranes [38]. Optic atrophy-1 is a mitochondrial dynamin-like GTPase involved in fusion of inner membrane mitochondria controlled by proteolytic cleavage mediated by metalloendopeptidase-1. Genetic deletion of p32/ C1QBP activates metalloendopeptidase-1, and cleaving of optic atrophy-1 results in mitochondrial fragmentation, swelling, respiration, and decreased lipid utilization [39]. The mitochondrial ATP-sensitive potassium channel was found to be involved in the regulation of mitochondrial fission/fusion and biogenesis. The pore subunits of Kir6.1, an important component of the mitochondrial ATP-sensitive potassium channel, was an important contributor to mitochondrial dynamics. The authors suggest that the mitochondrial ATP-sensitive potassium channel regulates mitochondrial dynamics [40]. Overexpression of the wild-type DJ-1 gene induced elongated mitochondria, while overexpression of the mutant DJ-1 gene induced significant DNA fragmentation and mitochondrial dysfunction and increased

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susceptibility to oxidative stress. A significant decrease in dynaminrelated protein-1 was observed in wild type DJ-1, while it was increased in mutant DJ-1. Interestingly, knockdown of dynamin-related protein-1 in mutant DJ-1 cells prevents the impaired mitochondria morphology and all related mitochondria/neuronal dysfunctions [41]. The 13-kDa protein overexpression induces apoptosis and mitochondrial dysfunction, while the knockdown of 13-kDa protein decreases neurotoxin-dependent apoptosis and mitochondrial dysfunction in a cell line. The knockout of 13-kDa protein in mice prevents neurotoxin-induced loss of dopaminergic neurons and motor deficiencies [42]. Mitochondria play an important role in cellular calcium homeostasis by taking up calcium to balance rapid influx of extracellular calcium or cellular calcium increase. Mitochondrial uptake of calcium is performed by mitochondrial calcium uniporter where calcium release from mitochondria is performed by sodium/calcium antiporter. A study revealed that the expression of mutant leucine-rich repeat kinase-2 gene induced an increase in the mitochondrial calcium uptake 1 protein and mitochondrial calcium uniporter levels, while the mitochondrial calcium uptake 1 protein level was unchanged. This increase in the upregulation of mitochondrial calcium uptake 1 protein and mitochondrial calcium uniporter levels was also observed in patient fibroblasts with this mutation and in sporadic Parkinson’s disease postmortem brain accompanied with increased mitochondrial calcium levels. The upregulation of mitochondrial calcium uptake 1 protein and mitochondrial calcium uniporter involved activation of the ERK1/2 pathway where the inhibition of this pathway prevented leucine-rich repeat kinase-2-induced neurite shortening. The inhibition or knockdown of mitochondrial calcium uniporter diminished leucine-rich repeat kinase-2-induced mitochondrial calcium uptake and neuritic/dendritic shortening [43]. PTEN-induced kinase 1 mutation induces mitochondrial calcium dysregulation by phosphorylating the leucine zipper-EF-hand containing transmembrane protein-1 that is a Ca2þ/Hþ antiporter. Expression of the phosphorylated leucine zipper-EF-hand containing transmembrane protein-1 prevents mitochondrial calcium dysregulation induced by PTEN-induced kinase-1 mutation [44]. Calcium overload seems to play an important role in Parkinson’s disease. It has been proposed that neuronal calcium sensor-1, involved in the regulation of dopamine dependent receptor desensitization in normal brain, may play a role in neurodegeneration in Parkinson’s disease, but data supporting this idea are missing [45]. It has been proposed that the mechanisms involved in calcium homeostasis of nigrostriatal neurons play an important role in dopamine release and modulation of lysosomal and mitochondrial function and their susceptibility to metabolic stress and neuronal degeneration [46].

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D.2.3. Protein degradation dysfunction role in degeneration of nigrostriatal neurons One of the hallmarks of Parkinson’s disease is the formation of proteinaceous inclusions such as Lewy bodies and neurites. These protein inclusions are mainly composed of cytosolic alpha-synuclein aggregates, cytoskeleton neurofilaments, synaptophysin, and other ubiquitinated proteins [1]. Ubiquitination is not a condition for the generation of these aggregates, while C-terminal truncation probably prevents alphasynuclein degradation by lysosomal or proteasomal degradation [2]. (i) Proteasomal system The ubiquitineproteasome system is composed of two steps: (1) ubiquitination of a protein to be degraded mediated by three enzymes, ubiquitin activating enzyme, ubiquitin conjugating enzyme, and ubiquitin ligase such as parkin, where a mutation of this enzyme is associated with a familial form of Parkinson’s disease; and (2) protein degradation performed by the 26S proteasome complex. The proteasome complex is composed of a 19S subunit where the polyubiquitin chain is removed before protein digestion into peptide fragments performed by a 20S subunit that is the final step of ubiquitinated proteins [3]. Ubiquitin is a protein composed of 76 amino acids that acts as signal for protein degradation in the proteasomal system. Interestingly, ubiquitin can also be ubiquitinated on N-terminal or seven lysine residues, changing the signal and its fate. Other ubiquitin posttranslational modifications include lysin phosphorylation, acetylation, or sumoylation, which involves addition of small

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ubiquitin-like modifiers [4]. The addition of ubiquitin to the protein is performed by the formation of a covalent binding between side-chain lysine of the protein to degrade and the C-terminal glycine of ubiquitin [5]. Proteasomal protein degradation is performed in three different ways, but only one pathway is ubiquitin dependent (26S ubiquitin-dependent proteasome). There are two ubiquitin-independent proteasomal pathways, 26S ubiquitin-independent proteasome and 20S ubiquitinindependent proteasome [6,7]. Parkin and ubiquitin C-terminal hydrolase L1, two enzymes of the ubiquitin-proteasomal system, have been found to be associated with familial form of Parkinson’s disease [8,9]. In sporadic Parkinson’s disease, the alpha-subunit of 26/20S proteasome was found to be lost in dopaminergic neurons, and the enzymatic activity of 20S proteasome and PA700 proteasome activator were decreased in the substantia nigra pars compacta [10]. The parkin unfolded receptor induces endoplasmic reticulum stress when it accumulates in dopaminergic neurons with a concomitant increase in carboxyl terminus of the Hsc70-interacting protein. The carboxyl terminus of the Hsc70-interacting protein is a cochaperone molecule with E3 ubiquitin ligase activity. Interestingly, the carboxyl terminus of the Hsc70-interacting protein increases parkin’s ability to inhibit unfolded Pael receptor-induced cell death [11]. Carboxyl terminus of the Hsc70-interacting protein promotes leucinerich repeat kinase-2 degradation mediated by ubiquitin-proteasomal system. Mutant LARRK2-induced toxicity is prevented by carboxyl terminus of HSP70-interacting protein overexpression, while its knockdown increases these toxic effects. Cell viability is increased when heat shock protein-90 chaperone activity is inhibited, resulting in an increase in leucine-rich repeat kinase-2 degradation [12]. The carboxyl terminus of Hsp70-interacting protein is a component of Lewy bodies colocalizing with Hsp70 and alpha-synuclein. Alpha-synuclein inclusion generation and a decrease in alpha-synuclein levels were observed when carboxyl terminus of Hsp70-interacting protein was overexpressed. Alpha-synuclein degradation was mediated by carboxyl terminus of Hsp70-interacting protein where the tetratricopeptide repeat domain is necessary for alpha-synuclein proteasomal degradation [13]. Monoubiquitination and polyubiquitination of alpha-synuclein was performed by carboxyl terminus of Hsp70-interacting protein. Alphasynuclein, carboxyl terminus of Hsp70-interacting protein, and cochaperone bcl-2-associated athanogene-5 were found to form a kind complex in the brain [14]. Tumor necrosis factor receptoreassociated factor-6 is an E3 ubiquitin ligase that associates with alpha-synuclein and PARK7/DJ-1. Tumor necrosis factor receptoreassociated factor-6 induces

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accumulation of cytoplasmic aggregates composed of insoluble and polyubiquitinated mutant DJ-1. In postmortem brain of Parkinson’s disease patients, tumor necrosis factor receptoreassociated factor-6 is found in Lewy bodies colocalized with alpha-synuclein [15]. Impairment of mitochondrial membrane potential stabilizes PTENinduced putative kinase-1 that recruits parkin from cytosol to mitochondria to activate autophagy to degrade damaged mitochondria. The formation of complex between PTEN-induced putative kinase-1, tumor necrosis factor receptoreassociated factor-6, and sterile a and TIR motif containing-1 is important for PTEN-induced putative kinase-1 import in the outer membrane and its stabilization on impaired mitochondria [16]. The gene coding the enzyme ubiquitin C-terminal hydrolase-L-1 expresses an enzyme with ubiquitin ligase activity that is different from hydrolase activity. Alpha-synuclein degradation is affected by these two opposing enzymatic activities. Ubiquitin C-terminal hydrolase-L-1, ligase, and hydrolase activity may be involved in proteosomal protein degradation [17]. The inhibition of 26/20S proteasomal function by lactacystin induces degeneration of dopaminergic neuron mesencephalic cultures with concomitant accumulation of ubiquitin and alpha-synuclein and formation of immunopositive inclusions in the cytoplasm. An increase in alphasynuclein accumulation and loss of ubiquitin immunoreactivity was observed when ubiquitin C-terminal hydrolase was inhibited [18]. A study on proteasome structural integrity where alpha- and beta-subunits are the catalytic core of 26/20S proteasomes. A selective and important loss of alpha subunits in dopaminergic neurons in the substantia nigra pars compacta of sporadic Parkinson’s disease patients that affects proteasome stability, assembly and enzymatic activity [19]. The exposure of adult rats to proteasome inhibitors induces rigidity, tremor, progressive parkinsonism with bradykinesia, and abnormal posture. Positronemission tomography revealed degeneration of the nigrostriatal neurons. This treatment also induced degeneration of dopaminergic neurons, neuroinflammation, apoptosis in the substantia nigra, and dopamine depletion in the striatum. Inclusion containing ubiquitin and alpha-synuclein was observed [20]. Ubiquitin-proteasomal system dysfunction was induced by expression of A53T accompanied with accumulation of autophagy vesicles and lysosomal protein degradation dysfunction [21]. Overexpression of ubiquitin carboxy-terminal hydrolase-L-1, a deubiquitylating enzyme, induces ribbonlike aggresomes when proteasome is inhibited. These aggresomes colocalize with gamma-tubulin, ubiquitylated proteins, and heat shock protein-70 [22].

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(ii) Lysosomal system Functional mitochondria are essential for dopaminergic neuron survival in Parkinson’s disease, and damaged mitochondria are removed in the lysosomal protein degradation system mitophagy. Mitophagy is initiated by the activation of ubiquitin kinase PTEN-induced kinase-1 that activates ubiquitin ligase parkin by phosphorylating ubiquitin. Parkin adds a ubiquitin chain to the mitochondrial outer membrane, recruiting autophagy receptors. A study of five knockout autophagy receptors revealed that optineurin and NDP52 are receptors for parkin- and PTENinduced kinase-1-mediated mitophagy. Later, optineurin and NDP52 recruit autophagy factors WIPI1, DFCP1, and ULK1 to mitochondria [23]. A study performed with skin fibroblasts from a young Parkinson’s disease patient with parkin mutation showed abnormal morphology and acidification of the late endocytic compartments, lysosomal dysfunction, autophagy flux impairment, and mitochondrial biogenesis dysfunction [24]. Autophagy-related 7 gene is essential for autophagy because a mouse lacking autophagy-related 7 gene in brain induced a decrease in coordinated movement, had abnormal limb-clasping reflexes, and did not survive more than 28 weeks. These animals did not present alterations in the proteasomal system, suggesting that autophagy has a very important role in neuron survival [25]. The lack of autophagy-related 5 gene in a mouse induced progressive impairment of in motor functions and accumulation of inclusions bodies, suggesting that autophagy plays a very important role in the prevention of accumulation of protein aggregates [26]. A study performed by expressing mutant and wild-type alpha-synuclein in the presence of inhibitors and stimulator of autophagy and proteasome concluded that alpha-synuclein is degraded both with proteasomal and lysosomal/ autophagy systems. Inhibition of autophagy results in the accumulation of autophagy vesicles containing alpha-synuclein aggregates, while stimulation of autophagy by rapamycin induces clearance of this protein [27]. Chaperone-mediated autophagy is a lysosomal protein degradation mode of specific substrates, and the knockdown of lysosomal-associated membrane protein 2A impairs chaperone-mediated autophagy by changing the expression of dihydropyrimidinase-related protein, a microtubule-binding phosphoprotein, and protein deglycase DJ-1 associated with a familial form of Parkinson’s disease [28]. Chaperone-mediated autophagy is involved in wild-type alphasynuclein degradation in lysosomes, while mutant alpha-synuclein inhibits autophagy [29]. Ubiquitination of the carboxyl terminus of alpha-synuclein catalyzed by the ubiquitin ligase NEDD4 promotes its degradation in the endosomal-lysosomal pathway. Overexpression of ubiquitin ligase NEDD4 increases alpha-synuclein ubiquitination and

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clearance by the lysosomal system [30]. Leucine-rich repeat kinase 2 has been reported to be involved in the regulation of autophagy, intracellular vesicle traffic, and lysosomal function [31]. A study performed in astrocyte cell model with the aim to study the possible role of leucine-rich repeat kinase-2 in macroautophagy regulation revealed that leucine-rich repeat kinase-2 inhibition stimulates macroautophagy in a noncanonical way independent of mammalian target of rapamycin and unc-51-like kinase 1. However, this stimulation was dependent on the activation of Beclin 1-containing class III PI3-kinase [32]. A study performed with surviving dopaminergic neurons of Parkinson’s disease patient brain tissue revealed that inhibition of leucine-rich repeat kinase-2 improves lysosomal function and endosomal maturation [33]. The expression of leucine-rich repeat kinase-2 G2019S mutation induces lysosome enlargement and lysosomal pH reduction and increases the expression of lysosomal ATPase ATP13A2. The inhibition of leucinerich repeat kinase-2 prevents lysosomal morphology defects and dysfunctions [34]. The association of form of autosomal-recessive juvenile-onset Parkinson’s disease with the mutation of F-box protein-7 has been reported. F-box protein-7 acts as stress response protein that can play both neurotoxic and cytoprotective roles. Wild-type F-box protein-7 protects cells by facilitating mitophagy when stress conditions lead to F-box protein-7 upregulates and concentrate into mitochondria [35]. Changes in mitochondrial membrane potential activate phosphorylation of mitochondrial polyubiquitin catalyzed by PTEN-induced kinase1 that facilitates parkin activation leading to mitophagy activation to remove phospho-polyubiquitin-tagged mitochondria. This phosphopolyubiquitin signal is more important in tyrosine hydroxylase immunopositive neurons [36]. Membrane-linked leucine-rich repeat kinase-2 colocalized to autophagosome membranes, and lack of endogenous leucine-rich repeat kinase-2 expression leads to autophagy impairment and the accumulation of macroautophagy substrates [37]. The accumulation of glucosylceramide as a consequence of deficient activity of acid b-glucosidase is observed in Gaucher disease, a lysosomal storage disease. In Parkinson’s disease, heterozygous mutation of acid b-glucosidase has been linked to more progressive and earlier onset Parkinson’s disease. Accumulation of ubiquitin and alpha-synuclein accompanied with brain glucosylceramide accumulation and progressive neurological manifestations has been observed in homozygous mouse for acid beta-glucosidase [38]. Cells expressing mutated acid beta-glucocerebrosidase gene showed decreased glucocerebrosidase protein expression and activity and lysosomal defects. Alpha-synuclein and neuronal calcium-binding protein-2 levels were increased, and calcium homeostasis was impaired [39]. The beta-glucocerebrosidase L444P heterozygous mutation induces mitochondrial dysfunction by

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mitochondrial priming and inhibition of autophagy in cell cultures. Postmortem studies in Parkinson’s disease brain having this mutation showed impaired autophagy [40]. Deficiency of the lysosomal enzyme cathepsin D induces accumulation of alpha-synuclein, macroautophagy impairment, and reduction in proteasomal activity [41]. Parkin regulates endosome structure and function because loss of Parkin function results in a decrease in endosomal tubulation, mannose 6 phosphate receptor, membrane association of vacuolar protein sorting nexin 1 and sorting 35. Parkin mutant increases the release of exosomes by increasing the formation of intraluminal vesicles. Parkin ubiquitination on lysine 38 residue regulates the levels and activity of late-endosomal GTPase Rab7 [42]. The expression of a mutation of vacuolar protein sorting-35, a crucial protein for endosome-to-Golgi recovery of membrane proteins, induces a reduction in alpha-synuclein degradation done by chaperone-mediated autophagy by impair endosome-to-Golgi recovery of Lamp2a that results in alpha-synuclein accumulation with concomitant degeneration of dopamine neurons [43]. The vacuolar protein sorting-35 D620N mutation impairs autophagy in part by inducing autophagy protein ATG9A abnormal trafficking [44]. The mutation of the ATP13A2 gene encoding a lysosomal type 5 P-type ATPase has been associated with an autosomal-recessive familial form of Parkinson’s disease. The mutation ATP13A2 gene induces reduction in the degradation of lysosomal substrates, impairment of lysosomal acidification, reduction in lysosomal-dependent clearance of autophagosomes, and reduction in proteolytic processing of lysosomal enzymes [45]. The knockdown of ATP13A2 increases alpha-synuclein misfolding [46]. Mutant ATP13A2 induces lysosomal dysfunction resulting in alphasynuclein accumulation and neurotoxicity, and knockdown of the alphasynuclein gene reduces neurotoxicity induced by ATP13A2 mutation [47]. The possible role of ATP13A as a regulator of the autophagylysosome system was studied by measuring the level of expression of synaptotagmin-11 gene, a Parkinson’s diseaseeassociated gene. ATP13A depletion downregulates synaptotagmin-11 at transcriptional and posttranslational levels, resulting in the impairment of autophagosome degradation and lysosomal dysfunction [48]. ATP13A2, a lysosomal polyamine exporter, promotes polyamines endocytosis and transport into the cytosol, remaking the role of endolysosomes in polyamine uptake into cells. Mutant ATP13A2 are unable to perform this function [49]. Overexpression of functional ATP13A2 induced lipid homeostasis disruption and decreased the total content of cholesterol, triglycerides, and lipids droplets. The authors of this study suggest that the increase in normal ATP13A2 changes the endolysosomal system to vesicle secretion [50]. RABGEF1 factor is carried to damaged mitochondria via ubiquitin binding of parkin and promotes downstream to damaged mitochondria

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of Rab proteins, RAB5, and RAB7A. Depletion of the endosomal Rab GTPase RAB7A prevented ATG9A vesicle assembly and mitochondria encapsulation by autophagic membranes, suggesting an important role of endosomal Rab cycles in mitophagy [51]. The impairment of mitochondrial respiratory chain disables AMP-activated protein kinase and inhibits lysosomal normal function. The disabling of AMP-activated protein kinase during respiratory chain impairment depends on the increased level of AMP-activated protein kinase -inhibiting protein. AMP-activated protein kinase disabling during mitochondrial respiratory chain prevents lysosomal normal function by diminishing lysosomal Ca2þ channel activity [52]. Parkin knockout neurons exhibited changes in lysosomal morphology such as enlarged lysosomes, an increase in the number of lysosomes and autophagy-lysosomal pathway impairment [53]. The loss of ATP13A2 function in dopaminergic neurons derived from a patient exhibited impaired lysosomal exocytosis, increased cytosolic calcium, reduced lysosomal calcium storage, and impaired lysosomal calcium homeostasis. Alpha-synuclein accumulates because of lysosomal exocytosis impairment but the induction of the lysosomal calcium channel transient receptor potential mucolipin-1 upregulated lysosomal exocytosis and prevented the accumulation of alpha-synuclein. The authors suggest that lysosomal exocytosis in human dopaminergic neurons regulates intracellular alpha-synuclein levels [54]. The activation of the lysosomal b-glucocerebrosidase gene in induced human midbrain dopaminergic neurons derived from Parkinson’s disease patients with alpha-synuclein mutation resulted in a reduction in alpha-synuclein accumulation in the cell body and synaptic localization of this protein [55]. The knockin of leucine-rich repeat kinase-2 mutation LRRK2R1441G in mice showed that the accumulation of alpha-synuclein oligomers in the striatum and cortex, accumulation of proteins heat shock protein 8 and chaperon mediated autophagy-specific lysosomalassociated membrane protein 2A, and lysosomal dysfunction [56]. A study performed with peripheral blood mononuclear cells from Parkinson’s disease patients showed a reduction in autophagy degradation pathway and lysosomal dysfunction [57]. Mitochondrial Rho GTPase-1 has been linked to mitochondrial-endoplasmic reticulum contact sites that play a crucial role in autophagy initiation and cellular calcium homeostasis. Mitochondrial Rho GTPase-1 mutation induces changes in mitochondrial dynamics, increased reactivity to calcium stress, and impaired autophagy flux [58].

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D.2.4. Role of oxidative stress in degeneration of nigrostriatal neurons For a long time, oxidative stress has been proposed to be involved in the degeneration of the nigrostriatal dopaminergic system [1]. The brain is

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completely dependent on oxygen and glucose for normal functioning because dopamine release under neurotransmission requires ATP. The main source of ATP is the mitochondria, where glucose oxidation to NADH and FADH2 generates ATP by coupling the mitochondrial electron transport chain and oxidative phosphorylation. Under prolonged starvation longer than 1 week, general metabolism changes by replacing an important part of glucose-dependent ATP production for ketonic body-dependent ATP formation. In the brain, glucose- or ketonic bodydependent ATP production requires oxygen. The main ATP production is done in the mitochondria by coupling the mitochondrial electron chain to oxidative phosphorylation. The electron transport chain transfers electrons from NADH to oxygen, reducing one molecule of oxygen to water in the presence of two molecules of hydrogens. In this electron transfer between NADH to oxygen the electron passes through mitochondrial complex I, III, and IV translocating protons from the mitochondrial matrix to intermembrane space. The protons accumulated in the mitochondrial intermembrane space will return to the mitochondrial matrix through mitochondrial ATPase phosphorylating ADP to ATP in the presence of inorganic phosphate. The mitochondrial electron transport chain is an important and permanent source of oxidative stress by generating superoxide that can dismutase to hydrogen peroxide, which can generate more reactive oxygen radicals such as hydroxyl radicals [2]. One possible way to generate superoxide is under the reduction of ubiquinone (coenzyme Q or also known as Q10) to ubiquinone semiquinone radical catalyzed by mitochondrial complexes I and II. Normally, the electrons of ubiquinone semiquinone radical electrons are transferred to the mitochondrial complex III. However, it is also possible that small amounts of ubiquinone semiradical transfer electrons to oxygen, generating superoxide. The formation and accumulation of superoxide was blocked when complex I was inhibited with rotenone, a classical inhibitor of this complex [3]. The formation of superoxide in isolated mitochondria was dependent on high proton-motive force and mitochondria is not generating ATP and coenzyme Q (ubiquinone) is reduced, and the ratio NADH/NADþ in the mitochondrial matrix is high [4]. Fig. 1.13. It has been reported that mitochondrial complex I generates reactive oxygen species through the flavin mononucleotide group, a component of complex I (NADH: ubiquinone oxidoreductase). The formation of reactive oxygen species was dependent on succinate that transfers electrons to mitochondrial complex II (succinate dehydrogenase, succinate: ubiquinone oxidoreductase). Normally, succinate transfers electrons to complex II reducing its flavin adenine dinucleotide that it is oxidized by transferring electrons to finally reducing ubiquinone. In general, flavin dinucleotide and flavin mononucleotide can donate or accept two protons and electrons. The single electron and proton transfer generate the

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FIGURE 1.13 Oxidative stress induced in the mitochondrial electron transport chain. Ubiquinones play an important role in the mitochondrial electron transport chain by accepting electrons from mitochondrial complexes I and II and donating electrons to mitochondrial complex III. During this electron transfer, it is possible that ubiquinone semiquinone radical or ubiquinol reduces oxygen to generate superoxide radicals. Two superoxide radicals dismutate to hydrogen peroxide in the presence of two protons. Hydrogen peroxidase is the precursor of hydroxyl radicals that induce oxidative stress.

semiquinone intermediate both when the oxidized flavin mononucleotide is one-electron reduced or the fully reduced flavin mononucleotide is one-electron oxidized. Flavin mononucleotide semiquinone or flavin dinucleotide semiquinone can reduce dioxygen to superoxide. Therefore, the formation of reactive oxygen species supported by succinate can be

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explained by the formation of succinate:ubiquinone oxidoreductase flavin dinucleotide semiquinone of during one-electron and one-proton transfer from succinate that partially reduce dioxygen to superoxide or the leakage of electrons, which were intended to reduce ubiquinone, in a reverse transfer reduce the flavin mononucleotide from complex I to semiquinone that also ends up reducing dioxygen to superoxide [5]. The inhibition of ubiquinone reduction site of complex II induces the formation of reactive oxygen species under subsaturating succinate concentrations. This inhibition generates mainly hydrogen peroxide and less superoxide, but we must remember that two molecules of superoxide dismutase to hydrogen peroxide spontaneously or enzymatically catalyzed by superoxide dismutase. The formation of reactive oxygen species also was observed when the flux of electrons in the respiratory chain was inhibited in other complexes [6]. A study of submitochondrial particles revealed that mitochondrial complex I generates superoxide in an independent manner of the protonmotive force that it is blocked by flavin-site inhibitors of complex I, while inhibitors of ubiquinone reduction have no effect on superoxide production. The reverse electron transfer from complex II to complex I is driven by succinate oxidation and the proton-motive force generated during ATP hydrolysis, resulting in flavin reduction, dioxygen reduction and formation of NADþ. Superoxide generation induced by reverse electron transfer is blocked by ubiquinone reduction and flavin-site inhibitors. Superoxide formation during NADH-dependent electron transfer in respiratory electron chain to ubiquinone and the reverse electron transfer from complex II to complex I are dependent on complex I flavin [7]. The antioxidant resveratrol decreases the formation of malondialdehyde and intracellular reactive oxygen species and increases the reduced glutathione level. Resveratrol enhanced mitochondrial complex I activity and inhibit the formation of reactive oxygen species generated in the mitochondria by enhancing DJ-1 protein expression and its mitochondrial translocation. Resveratrol induces the direct binding of complex I subunits NDUFS4 and ND1 [8]. Studies with genetically modified mitochondria have shown that the free mobility of Rieske iron-sulfur protein head domain is required for electron transfer from ubiquinol to complex III (cytochrome bc1 complex). The inhibition of mitochondrial electron chain with antimycin A result in leakage of single electrons to dioxygen, generating superoxide [9]. The role hydrogen gas (H2) on mitochondrial dependent formation of superoxide, electron transfer direction, and mitochondrial membrane potential was studied. Hydrogen gas changed the direction of electron transfer depending on the level of NADþ, and inhibit complex I-dependent superoxide generation and reduce mitochondrial membrane potential [10].

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Another permanent source of reactive oxygen species is the formation of hydrogen peroxide during dopamine degradation in the cytosol. Monoamine oxidase catalyzes dopamine oxidative deamination to 3,4-dihydroxyphenylacetaldehyde, hydrogen peroxide, and ammonia. Hydrogen peroxide is a potent biological oxidizing agent, and it is the precursor of Fenton reaction that generates hydroxyl radicals that are one of the most harmful reactive specie, suggesting a possible role of oxidative stress in the loss dopaminergic neurons of the nigrostriatal system observed in Parkinson’s disease. This idea was supported by postmortem studies performed in Parkinson’s disease brain that showed evidence of oxidative stress such as protein oxidation, DNA oxidation and glutathione depletion, an important endogenous antioxidant [11]. A postmortem study showed that reduced glutathione decreased in Parkinson’s disease brain, however, no differences in oxidized glutathione level between control brains and patient brains were observed [12] Fig. 1.14. The presence of reactive oxygen species induces lipid peroxidation. The level of malondialdehyde, a metabolite generated during lipid peroxidation, increased, while polyunsaturated fatty acid levels decrease in brain regions of Parkinson’s disease patients [13]. The 8hydroxyguanosine and 8-hydroxy-2 deoxyguanosine have been used as markers of DNA oxidation induced by oxidative stress. A study performed with 44 Parkinson’s disease patients showed a significative increase in 8-hydroxyguanosine and 8-hydroxy-2 deoxyguanosine in cerebrospinal fluid [14]. Fenton reaction is catalyzed by reduced iron (II), and it has been reported that the iron level is increased in the substantia nigra of Parkinson’s disease patients [15]. Significant increase in iron (III) was observed in a study of Parkinson’s disease postmortem substantia nigra in comparison with age-matched controls [16]. A reduction in reduced glutathione in substantia nigra was observed in multiple-system atrophy, Parkinson’s disease, and progressive supranuclear palsy, but only in Parkinson’s disease substantia nigra was it significantly decreased [17]. Although the majority found was as iron (III) as ferritin, transferrin and lactoferrin, it is completely feasible that biological reducing agents reduce iron (III) to iron (II), which catalyzes the Fenton reaction. Ascorbic acid release was observed after the acute administration of ethanol in rat striatum determined by microdialysis. This study revealed that the increase in ascorbic acid level in the striatum was accompanied with a reduction in the hydroxyl radical level [18]. One-electron reduction of iron (III) to iron (II) was catalyzed by the flavoenzyme NAD(P)H-dependent flavoenzyme lipoyl dehydrogenase determined using electron paramagnetic resonance spectrometry using NAD(P)H as electron donator that reduces the enzyme flavine. The oxidation state of iron (III) was unchanged when the flavoenzyme was inhibited, inactivated, or in the absence of NAD(P)H in the reduced

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FIGURE 1.14

Oxidative stress induced during dopamine degradation. Monoamine oxidase catalyzes the oxidative deamination of dopamine, generating ammonia, 3,4dihydroxyphenylacetaldehyde, and hydrogen peroxide. Hydrogen peroxide is a powerful oxidative agent and hydroxyl radical precursor that induces oxidative stress in the presence of iron (II).

state [19]. One of the most important endogenous antioxidants is glutathione in the reduced state that it is found in the cell at millimolar level in tissues and micromolar in fluids. Glutathione plays an important role in brain preventing lipid peroxidation, oxidative stress, and scavenging free radicals. Glutathione is found in the reduced state in healthy individuals, but the level of reduced glutathione is significantly decreased in Parkinson’s disease brain [20]. Based on the evidence of oxidative stress observed in Parkinson’s disease such as lipid peroxidation, low level of reduced glutathione,

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increase in iron levels, DNA, and protein oxidation and the fact monoamine oxidase-dependent dopamine degradation produce permanently hydrogen peroxide, the precursor of hydroxyl radicals, the use of monoamine oxidase inhibitors in Parkinson’s disease therapy was proposed. The monoamino oxidase B inhibitor L-deprenyl was proposed as neuroprotective therapy in Parkinson’s disease [21]. The use of antioxidants such as vitamin E, iron chelators, lipid peroxidation inhibitors were also proposed as consequence of the evidence of oxidative stress in Parkinson’s disease [22]. Expression of parkin gene in cells induced an increase in proteasomal activity, a level reduction in ubiquitinated protein, 3-nitrotyrosine-containing proteins, and protein carbonyl levels. While expression of mutant parkin gene induced an increase in lipid peroxidation and protein carbonyls [23]. The inhibition of proteasomal system induced a significant increase in the level of lipid peroxidation, protein carbonyls, and 3-nitrotyrosine, a marker for reactive nitrogen species-induced damage. Higher concentration of proteasome inhibitor induced a decrease in reduced glutathione and an increase in nitric oxide and in the level of 8-hydroxyguanine, a marker for oxidative DNA damage [24]. 26S Proteasome activity is lost when oxidative stress induces the dissociation of the 19S regulatory particle from the 20S core particle and ubiquitinated proteins accumulate. Hydrogen peroxide increases the association of 19D particle with the proteasome-interacting protein Ecm29. Deletion of Ecm29 blocked 26S proteasome disassembly. Oxidative stress-induced 26S proteasome disassembly was prevented by Ecm29 [25]. 26S proteasome dysfunction impairs the nuclear factor erythroid 2e related factor 2 that mediates the antioxidant response by inducing the transcription of antioxidant enzymes and Kelch-like ECH-associated protein 1 pathway. Autophagy was also impaired by diminishing the level of crucial autophagy proteins LC3B and ATG9 [26]. 26S Proteasomal depletion increased the expression of peroxiredoxin 6, an enzyme that has phospholipase A2 and antioxidant peroxidase activities, suggesting that proteasomal dysfunction can induce oxidative stress in astrocytes [27]. Hydrogen peroxide-induced oxidative stress induces aggregation of parkin, the ligase 3 enzyme of the proteasome, to high molecular insoluble aggregates. Hydrogen peroxide also induces aggregation of others ubiquitin E3 ligases, suggesting that oxidative stress impairs the proteasomal system [28]. Oxidative stress potentiates alpha-synuclein aggregation to oligomers induced by proteasomal inhibition [29].

References [1] Monzani E, Nicolis S, Dell’Acqua S, et al. Dopamine, oxidative stress and proteinquinone modifications in Parkinson’s and other neurodegenerative diseases. Angew Chem Int Ed Engl 2019;58:6512e27.

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[2] Treberg JR, Braun K, Zacharias P, Kroeker K. Multidimensional mitochondrial energetics: application to the study of electron leak and hydrogen peroxide metabolism. Comp Biochem Physiol B Biochem Mol Biol 2018;224:121e8. [3] Sun L, Liao K, Wang D. Honokiol induces superoxide production by targeting mitochondrial respiratory chain complex I in Candida albicans. PLoS One 2017;12:e0184003. [4] Murphy MP. How mitochondria produce reactive oxygen species. Biochem J 2009;417: 1e13. [5] Liu Y, Fiskum G, Schubert D. Generation of reactive oxygen species by the mitochondrial electron transport chain. J Neurochem 2002;80:780e7. [6] Siebels I, Dro¨se S. Q-site inhibitor induced ROS production of mitochondrial complex II is attenuated by TCA cycle dicarboxylates. Biochim Biophys Acta 2013;1827:1156e64. [7] Pryde KR, Hirst J. Superoxide is produced by the reduced flavin in mitochondrial complex I: a single, unified mechanism that applies during both forward and reverse electron transfer. J Biol Chem 2011;286:18056e65. [8] Zhang Y, Li XR, Zhao L, Duan GL, Xiao L, Chen HP. DJ-1 preserving mitochondrial complex I activity plays a critical role in resveratrol-mediated cardioprotection against hypoxia/reoxygenation-induced oxidative stress. Biomed Pharmacother 2018;98: 545e52. [9] Staniek K, Gille L, Kozlov AV, Nohl H. Mitochondrial superoxide radical formation is controlled by electron bifurcation to the high and low potential pathways. Free Radic Res 2002;36:381e7. [10] Ishihara G, Kawamoto K, Komori N, Ishibashi T. Molecular hydrogen suppresses superoxide generation in the mitochondrial complex I and reduced mitochondrial membrane potential. Biochem Biophys Res Commun 2020;522:965e70. [11] Reale M, Pesce M, Priyadarshini M, Kamal MA, Patruno A. Mitochondria as an easy target to oxidative stress events in Parkinson’s disease. CNS Neurol Disord Drug Targets 2012;11:430e8. [12] Sofic E, Lange KW, Jellinger K, Riederer P. Reduced and oxidized glutathione in the substantia nigra of patients with Parkinson’s disease. Neurosci Lett 1992;142:128e30. [13] Dexter DT, Carter CJ, Wells FR, et al. Basal lipid peroxidation in substantia nigra is increased in Parkinson’s disease. J Neurochem 1989;52:381e9. [14] Gmitterova´ K, Gawinecka J, Heinemann U, Valkovic P, Zerr I. DNA versus RNA oxidation in Parkinson’s disease: which is more important? Neurosci Lett 2018;662:22e8. [15] Sofic E, Paulus W, Jellinger K, Riederer P, Youdim MB. Selective increase of iron in substantia nigra zona compacta of parkinsonian brains. J Neurochem 1991;56:978e82. [16] Sofic E, Riederer P, Heinsen H, et al. Increased iron (III) and total iron content in post mortem substantia nigra of parkinsonian brain. J Neural Transm 1988;74:199e205. [17] Sian J, Dexter DT, Lees AJ, et al. Alterations in glutathione levels in Parkinson’s disease and other neurodegenerative disorders affecting basal ganglia. Ann Neurol 1994;36:348e55. [18] Huang M, Liu W, Li Q, Wu CF. Endogenous released ascorbic acid suppresses ethanolinduced hydroxyl radical production in rat striatum. Brain Res 2002;944:90e6. [19] Petrat F, Paluch S, Dogruo¨z E, et al. Reduction of Fe(III) ions complexed to physiological ligands by lipoyl dehydrogenase and other flavoenzymes in vitro: implications for an enzymatic reduction of Fe(III) ions of the labile iron pool. J Biol Chem 2003;278:46403e13. [20] Owen JB, Butterfield DA. Measurement of oxidized/reduced glutathione ratio. Methods Mol Biol 2010;648:269e77. [21] Youdim MB, Ben-Shachar D, Riederer P. The role of monoamine oxidase, iron-melanin interaction, and intracellular calcium in Parkinson’s disease. J Neural Transm Suppl 1990;32:239e48. [22] Youdim MB, Lavie L. Selective MAO-A and B inhibitors, radical scavengers and nitric oxide synthase inhibitors in Parkinson’s disease. Life Sci 1994;55:2077e82. [23] Hyun DH, Lee M, Hattori N, et al. Effect of wild-type or mutant Parkin on oxidative damage, nitric oxide, antioxidant defenses, and the proteasome. J Biol Chem 2002; 277:28572e7.

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[24] Lee MH, Hyun DH, Jenner P, Halliwell B. Effect of proteasome inhibition on cellular oxidative damage, antioxidant defences and nitric oxide production. J Neurochem 2001;78:32e41. [25] Wang X, Yen J, Kaiser P, Huang L. Regulation of the 26S proteasome complex during oxidative stress. Sci Signal 2010;3:ra88. [26] Ugun-Klusek A, Tatham MH, Elkharaz J, et al. Continued 26S proteasome dysfunction in mouse brain cortical neurons impairs autophagy and the Keap1-Nrf2 oxidative defence pathway. Cell Death Dis 2017;8:e2531. [27] Elkharaz J, Ugun-Klusek A, Constantin-Teodosiu D, et al. Implications for oxidative stress and astrocytes following 26S proteasomal depletion in mouse forebrain neurones. Biochim Biophys Acta 2013;1832:1930e8. [28] LaVoie MJ, Cortese GP, Ostaszewski BL, Schlossmacher MG. The effects of oxidative stress on parkin and other E3 ligases. J Neurochem 2007;103:2354e68. [29] Lev N, Melamed E, Offen D. Proteasomal inhibition hypersensitizes differentiated neuroblastoma cells to oxidative damage. Neurosci Lett 2006;399:27e32.

D.2.5. Neuroinflammation’s role in degeneration of nigrostriatal neurons It has been proposed that the loss of dopaminergic neurons is dependent on the increase in the level of cytokines and/or decrease in the levels of neurotrophins that induce programmed cell death. In the cerebrospinal fluid and in the nigrostriatal dopamine system of Parkinson’s disease patients has been observed an increase in the levels of cytokines, such as interleukins 1-beta, 2, 4, and 6, tumor necrosis factor alpha, transforming growth factors alpha, 1 beta, and beta2, accompanied with a decrease in neurotrophins, such as nerve growth factor and brain-derived neurotrophic factor [1,2]. In the brain, microglia cells (brain macrophages) are the first line of defense against pathological threats through the induction of inflammatory processes. Postmortem studies have revealed signs of neuroinflammation such as HLA-DR-positive reactive microglia in the substantia nigra of Parkinson’s disease patients [3]. The level of interleukins 2, 4, 6, and 10 and interferon gamma and tumor necrosis factor alpha in the serum levels of patients with idiopathic Parkinson’s disease with cardiovascular risk factor and atypical parkinsonism were increased in comparison with age-matched controls [4]. The level of interleukins 2, 1 beta, and 6 were significantly increased in de novo Parkinson’s disease patient cerebrospinal fluid in comparison with control group [5]. Extreme microglial activation can increase the production of interleukin-1-b, tumor necrosis factor alpha, interferon gamma, interleukin, reactive oxygen species, inducible nitric oxide synthase, and nitric oxide. In addition, excessive microglial activation also increases microglial phagocytosis, migration and lymphocyte infiltration [6]. Microglia activation is mediated by chemokines, cytokines, and other inflammatory

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inducers. The expression of the chemokine C-X-C motif chemokine 12 and its receptor CXCR4 was increased in Parkinson’s disease brain in comparison with control brains. In animal studies the upregulation of receptor CXCR4 was related to the loss of dopaminergic neurons [7]. The extracellular factor S100B involves receptor for advanced glycation end products in the upregulation of expression of the proinflammatory cyclooxygenase 2 and interleukin 1 beta [8]. The nuclear factor-kB transcription factor is an important regulator of apoptosis and inflammation that it is composed by several subunits including RelA, which it has been found to be increased in dopaminergic neurons and glia cells of Parkinson’s disease. RelA inhibition results in neuroprotection against 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine and its metabolite 1-methyl-4phenylpyridinium [9]. It has been proposed that microglia necroinflammatory responses accelerates neurodegeneration. It has been shown that the transfer of T cells from Copolymer-1-immunized mouse provide protection against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurodegeneration. The transfer of CD3 activated T cells to 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine-treated mice protect against this neurotoxin-induced neurodegeneration with concomitant upregulation of transforming growth factor beta and glial cell-derived neurotrophic factor [10]. A study to investigate the role of the immune system in Parkinson’s disease showed that circulating lymphocytes in patients. It was observed a decrease in helper T cells and B cells, and increase in activated, CD4(þ) CD25(þ) lymphocytes [11]. A study of peripheral blood lymphocytes showed that an increase in effector/memory T cells was associated with progressive Unified Parkinson’s Disease Rating Scale III scores [12]. Protein kinase C delta activation play an important role in dopaminergic neuronal loss. Proinflammatory inducers, such as tumor necrosis factor alpha, alpha-synuclein, and lipopolysaccharide, upregulates protein kinase C delta. The upregulation of protein kinase C delta was also observed in Parkinson’s patients’ microglia of the ventral midbrain region in comparison with control brains [13]. Alpha-synuclein induces microglia activation and the formation of multi-epitope-specific autoantibodies in Parkinson’s disease patients. However, these patients did not develop specific autoantibodies against gamma-synuclein or beta-synuclein [14]. Alpha-synuclein autoantibodies against monomeric alpha-synuclein detected using Biacore surface plasmon resonance, Western blot, and ELISA were significantly higher in Parkinson’s disease patients blood sera in comparison with control group. Autoantibodies against alpha-synuclein oligomers were no detected [15]. 6-Hydroxydopamine-dependent lesions in the nigrostriatal neurons induced major histocompatibility complex class I and II antigen expression in monocytes/macrophages was observed at the injection site [16].

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6-Hydroxydopamine-induced loss of dopaminergic neurons and motor deficits decreased when a lentiviral vector encoding dominant-negative tumor necrosis factor was injected into rat substantia nigra [17]. 6Hydroxydopamine lesion induces rotational behavior and microglial and astroglial activation in Wistar rats [18]. The administration of exogenous neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine induces a strong gliosis in substantia nigra pars compacta accompanied with inducible nitric oxide synthase upregulation. Interestingly, the mice lacking expression of inducible nitric oxide synthase were more resistant to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxic effects [19]. The antiinflammatory and immunosuppressive effects of glucocorticoids are mediated by glucocorticoid receptors. A transgenic mouse expressing glucocorticoid receptors antisense increases 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine-induced neurotoxic effects on dopaminergic neurons. In addition, an increase in inducible nitric oxide synthase was observed in activated microglia and astrocytes in comparison with wildtype mouse, and the inhibition of inducible nitric oxide synthase decrease 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced effect on transgenic mouse [20]. Mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine generate antibodies to nitrated and native alpha-synuclein. Mice immunization with nitrotyrosine-modified alpha-synuclein induces a strong T cell proinflammatory secretory and proliferative responses, which it was specific for the modified alpha-synuclein. T cells from nitrotyrosinemodified alpha-synuclein immunized mice induced a strong neuroinflammatory response accompanied with an accelerated dopaminergic neuron loss [21]. A study of 67 patients showed that alphasynuclein aggregates induces cytotoxic T and helper cell response in Parkinson’s disease [22]. The accumulation of phosphorylated a-syn inclusions, loss of substantia nigra pars compacta dopaminergic neurons and major histocompatibility complex II expression and reactive microglia was observed after intrastriatal administration of preformed alpha-synuclein fibrils [23]. An acute neuroinflammation was observed in wild-type and transgenic mice overexpression A53T was observed after injection of lipopolysaccharide. However, chronic degeneration of the nigrostriatal dopaminergic neurons, persistent neuroinflammation, accumulation of alpha-synuclein aggregates, nitrated alpha-synuclein, and formation of protein inclusions like Lewy bodies was only observed in the transgenic mice overexpressing mutant alpha-synuclein [24]. Intracerebral injection of human alpha-synuclein oligomers induce a neuronal cell loss accompanied with microglia activation that attenuated by inhibiting MAP kinase [25]. Alpha-synuclein intracerebral injection to striatum induces microglia activation and an increase in the expression of

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interferon gamma, interleukin 1 alpha, and tumor necrosis factor alpha. Intracerebral injection of alpha-synuclein into substantia nigra induces an increase in mRNA level of striatal transforming growth factor beta, but no effect was observed on the mRNA level of striatal glial fibrillary acidic protein [26]. Lipopolysaccharide intranigral injection induced an inflammatory response by increasing the gene expression of inducible nitric oxide synthase, nuclear factor-kB, and gp91phox protein as well as glial activation. This inflammatory response was accompanied by an increase in NADPH oxidase activity and reactive oxygen species. The inhibition of NADPH oxidase reduces glial activation and oxidative stress [27]. Lipopolysaccharide induces an increase in glucose-6-phosphate dehydrogenase expression, the rate-limiting enzyme of the pentose phosphate pathway, accompanied with microglial activation. The knockdown or inhibition of microglial glucose-6-phosphate dehydrogenase decrease lipopolysaccharide-induced degeneration of dopaminergic neurons. A study of postmortem brain of Parkinson’s disease patients showed dysregulation of glucose-6-phosphate dehydrogenase. The authors of this study suggest that an increase in glucose-6-phosphate dehydrogenase expression will increase NADPH production stimulating the formation of reactive oxygen species mediated nu NADPH oxidase [28]. The knockdown of leucine-rich repeat kinase 2 gene in microglia cells inhibited resulted in the inhibition of proinflammatory response. The knockout of leucine-rich repeat kinase 2 gene in rats prevented lipopolysaccharide-induced degeneration of dopaminergic neurons and neuroinflammation [29]. Leucine-rich repeat kinase-2 genetic deletion or inhibition induces a reduction in cyclooxygenase-2 and interleukin-1b expression in lipopolysaccharide-dependent inflammation. The authors suggest that leucine-rich repeat kinase-2 play a role in neuroinflammation, microglia activation and controlling of nuclear factor kappa-B p50 inhibitory signaling [30]. Microglia primary cell cultures showed toll-like receptor 4 stimulation in microglia primary microglia cultures and an increase in leucine-rich repeat kinase-2 expression and activity during inflammation. Leucine-rich repeat kinase-2 activity inhibition or knockdown of protein decrease nitric oxide synthase induction and tumor necrosis factor alpha secretion [31]. The toll-like receptorinduced immune response is regulated by pleckstrin homology-like domain family A member-1. The knockdown of pleckstrin homologylike domain family A member-1 diminish 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine-induced motor impairment and neuroinflammation. In lipopolysaccharide-induced pleckstrin homology-like domain family A member-1 deficiency the expression of interleukin-1beta, tumor necrosis-alpha, cyclooxygenase-2, and inducible nitric oxide synthase were reduced [32].

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Analysis of inflammatory indexes in Parkinson’s disease compared with healthy controls showed that the patients presented significantly higher levels of neutrophils, monocytes, and high-sensitivity C-reactive protein. The authors suggest that C-reactive protein can be a possible biomarker for disease progression [33]. The transcription factor Nurr1 has crucial role in the maintenance and generation of dopaminergic neurons but Nurr1 has also an antiinflammatory function. In lipopolysaccharideinduced inflammation Nurr1 regulates the expression of RasGRP1 gene that control the Ras-Raf-MEK-ERK signaling cascade in lipopolysaccharide-induced neuroinflammation [34]. Alpha-synuclein aggregation induces the transcriptional upregulation of the microglial voltage-gated potassium channel Kv1.3 in microglial cultures, animals and in Parkinson’s disease’s postmortem brain. The microglial Kv1.3 posttranslational modification and transcriptional upregulation was also mediated by the kinase Fyn. The knockout or inhibition of Kv1.3 in primary microglia cultures inhibited neuroinflammation and neurodegeneration [35]. The deregulation of microRNAs has been associated with neurodegeneration in Parkinson’s disease. The expression of microRNAs related to inflammation and neurodegeneration were analyzed in serum of idiopathic Parkinson’s disease patients carrying leucine-rich repeat kinase-2 compared with controls. The microRNAs-146a, -335-3p, and -335-5p were found to be downregulated in idiopathic and leucine-rich repeat kinase-2 patients, whereas the microRNA-155 was found to be upregulated in leucine-rich repeat kinase-2 mutation patients in comparison with idiopathic Parkinson’s disease [36]. The FBXO7, a component of ubiquitin E3 ligase receptor, induces inflammation and mitochondrial damage by promoting PTEN-induced kinase-1 cellular degradation. The molecule BC1464 prevents the FBXO7 association to PTEN-induced kinase-1 resulting in the increase in PTEN-induced kinase-1 activity and concentration, inflammation decrease and reduction mitochondrial damage [37]. The inflammasome nucleotide-binding oligomerization domainleucine-rich repeat and pyrin domain-containing protein-3 has been reported to be an inflammatory complex present in microglia. The activation of this inflammasome induces the secretion of interleukin-1b/18 and promotes pyroptosis, a kind of cell death that break microglia to release interleukin-1b [38]. The nucleotide-binding oligomerization domain and leucine-rich repeat pyrin-3 domain inflammasome is constituted by a sensor, the caspase-1 protease, and a caspase recruitment domain. The activation of nucleotide-binding oligomerization domain and leucine-rich repeat pyrin-3 domain inflammasome in mouse microglia by alpha-synuclein fibrils induced secretion of interleukin-1b and caspase recruitment domain release in the absence of pyroptosis [39].

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The noncoding RNA GAS5 promotes the inflammatory response, and it was found to be upregulated in lipopolysaccharide-activated microglia and tissue of animal model for Parkinson’s disease. The knockdown of GAS5 inhibited Parkinson’s disease progression in vivo by upregulating the expression of nucleotide-binding oligomerization domain and leucine-rich repeat pyrin 3 domain [40]. The inhibition of histone deacetylase 6 by tubastatin A decreased nucleotide-binding oligomerization domain and leucine-rich repeat pyrin-3 domain-induced inflammatory response, decrease oxidative stress and improved acetylation levels of peroxiredoxin 2 [41]. An important activation and assembly of the nucleotide-binding oligomerization domain and leucine-rich repeat pyrin-3 domain inflammasome was induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and ATP, resulting in neuron loss in a neuron-microglia coculture [42]. The deficiency of glia maturation factor, a proinflammatory mediator, decreased interleukin-1-beta in mouse model of Parkinson’s disease. Caspase-1 and the nucleotide-binding oligomerization domain and leucine-rich repeat pyrin-3 domain inflammasome were significantly increased in Parkinson’s disease brain in comparison with control brain. Alpha-synuclein and glia maturation factor colocalized in reactive astrocytes of Parkinson’s disease midbrain. The translocation of glia maturation factor and nucleotide-binding oligomerization domain and leucine-rich repeat pyrin-3 domain to mitochondria was induced by the activation of the nucleotide-binding oligomerization domain and leucinerich repeat pyrin-3 domain in microglia [43].

References [1] Nagatsu T, Mogi M, Ichinose H, Togari A. Changes in cytokines and neurotrophins in Parkinson’s disease. J Neural Transm Suppl 2000;60:277e90. [2] Mogi M, Harada M, Riederer P, Narabayashi H, Fujita K, Nagatsu T. Tumor necrosis factor-alpha (TNF-alpha) increases both in the brain and in the cerebrospinal fluid from parkinsonian patients. Neurosci Lett 1994;165:208e10. [3] McGeer PL, Itagaki S, Boyes BE, McGeer EG. Reactive microglia are positive for HLADR in the substantia nigra of Parkinson’s and Alzheimer’s disease brains. Neurology 1988;38:1285e91. [4] Brodacki B, Staszewski J, Toczyłowska B, et al. Serum interleukin (IL-2, IL-10, IL-6, IL4), TNFalpha, and INFgamma concentrations are elevated in patients with atypical and idiopathic parkinsonism. Neurosci Lett 2008;441:158e62. [5] Blum-Degen D, Mu¨ller T, Kuhn W, Gerlach M, Przuntek H, Riederer P. Interleukin-1 beta and interleukin-6 are elevated in the cerebrospinal fluid of Alzheimer’s and de novo Parkinson’s disease patients. Neurosci Lett 1995;202:17e20. [6] Zhang QS, Heng Y, Yuan YH, Chen NH. Pathological a-synuclein exacerbates the progression of Parkinson’s disease through microglial activation. Toxicol Lett 2017;265: 30e7. [7] Shimoji M, Pagan F, Healton EB, Mocchetti I. CXCR4 and CXCL12 expression is increased in the nigro-striatal system of Parkinson’s disease. Neurotox Res 2009;16: 318e28.

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[8] Bianchi R, Giambanco I, Donato R. S100B/RAGE-dependent activation of microglia via NF-kappaB and AP-1 Co-regulation of COX-2 expression by S100B, IL-1beta and TNFalpha. Neurobiol Aging 2010;31:665e77. [9] Bellucci A, Bubacco L, Longhena F, et al. Nuclear factor-kB dysregulation and a-synuclein pathology: critical interplay in the pathogenesis of Parkinson’s disease. Front Aging Neurosci 2020;12:68. [10] Reynolds AD, Banerjee R, Liu J, Gendelman HE, Mosley RL. Neuroprotective activities of CD4þCD25þ regulatory T cells in an animal model of Parkinson’s disease. J Leukoc Biol 2007;82:1083e94. [11] Bas J, Calopa M, Mestre M, et al. Lymphocyte populations in Parkinson’s disease and in rat models of parkinsonism. J Neuroimmunol 2001;113:146e52. [12] Saunders JA, Estes KA, Kosloski LM, et al. CD4þ regulatory and effector/memory T cell subsets profile motor dysfunction in Parkinson’s disease. J Neuroimmune Pharmacol 2012;7(4):927e38. [13] Gordon R, Singh N, Lawana V, et al. Protein kinase Cd upregulation in microglia drives neuroinflammatory responses and dopaminergic neurodegeneration in experimental models of Parkinson’s disease. Neurobiol Dis 2016;93:96e114. [14] Papachroni KK, Ninkina N, Papapanagiotou A, et al. Autoantibodies to alphasynuclein in inherited Parkinson’s disease. J Neurochem 2007;101:749e56. [15] Yanamandra K, Gruden MA, Casaite V, Meskys R, Forsgren L, Morozova-Roche LA. a-synuclein reactive antibodies as diagnostic biomarkers in blood sera of Parkinson’s disease patients. PLoS One 2011;6:e18513. [16] Akiyama H, McGeer PL. Microglial response to 6-hydroxydopamine-induced substantia nigra lesions. Brain Res 1989;489:247e53. [17] McCoy MK, Ruhn KA, Martinez TN, McAlpine FE, Blesch A, Tansey MG. Intranigral lentiviral delivery of dominant-negative TNF attenuates neurodegeneration and behavioral deficits in hemiparkinsonian rats. Mol Ther 2008;16:1572e9. [18] Rodrigues RW, Gomide VC, Chadi G. Astroglial and microglial reaction after a partial nigrostriatal degeneration induced by the striatal injection of different doses of 6hydroxydopamine. Int J Neurosci 2001;109:91e126. [19] Liberatore GT, Jackson-Lewis V, Vukosavic S, et al. Inducible nitric oxide synthase stimulates dopaminergic neurodegeneration in the MPTP model of Parkinson disease. Nat Med 1999;5:1403e9. [20] Morale MC, Serra PA, Delogu MR, et al. Glucocorticoid receptor deficiency increases vulnerability of the nigrostriatal dopaminergic system: critical role of glial nitric oxide. FASEB J 2004;18:164e6. [21] Benner EJ, Banerjee R, Reynolds AD, et al. Nitrated alpha-synuclein immunity accelerates degeneration of nigral dopaminergic neurons. PLoS One 2008;3:e1376. [22] Sulzer D, Alcalay RN, Garretti F, et al. T cells from patients with Parkinson’s disease recognize a-synuclein peptides [published correction appears in Nature. 2017 Sep 13; 549(7671):292]. Nature 2017;546:656e61. [23] Duffy MF, Collier TJ, Patterson JR, et al. Lewy body-like alpha-synuclein inclusions trigger reactive microgliosis prior to nigral degeneration [published correction appears in J Neuroinflammation. 2018 May 29;15(1):169]. J Neuroinflammation 2018; 15:129. [24] Gao HM, Zhang F, Zhou H, Kam W, Wilson B, Hong JS. Neuroinflammation and a-synuclein dysfunction potentiate each other, driving chronic progression of neurodegeneration in a mouse model of Parkinson’s disease. Environ Health Perspect 2011;119: 807e14. [25] Wilms H, Rosenstiel P, Romero-Ramos M, et al. Suppression of MAP kinases inhibits microglial activation and attenuates neuronal cell death induced by alpha-synuclein protofibrils. Int J Immunopathol Pharmacol 2009;22:897e909.

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[26] Sznejder-Pachołek A, Joniec-Maciejak I, Wawer A, Ciesielska A, Mirowska-Guzel D. The effect of a-synuclein on gliosis and IL-1a, TNFa, IFNg, TGFb expression in murine brain. Pharmacol Rep 2017;69:242e51. [27] Sharma N, Nehru B. Apocyanin, a microglial NADPH oxidase inhibitor prevents dopaminergic neuronal degeneration in lipopolysaccharide-induced Parkinson’s disease model. Mol Neurobiol 2016;53:3326e37. [28] Tu D, Gao Y, Yang R, Guan T, Hong JS, Gao HM. The pentose phosphate pathway regulates chronic neuroinflammation and dopaminergic neurodegeneration. J Neuro inflammation 2019;16:255. [29] Daher JP, Volpicelli-Daley LA, Blackburn JP, Moehle MS, West AB. Abrogation of a-synuclein-mediated dopaminergic neurodegeneration in LRRK2-deficient rats. Proc Natl Acad Sci U S A 2014;111:9289e94. [30] Russo I, Berti G, Plotegher N, et al. Leucine-rich repeat kinase 2 positively regulates inflammation and down-regulates NF-kB p50 signaling in cultured microglia cells [published correction appears in J Neuroinflammation. 2016;13(1):70]. J Neuro inflammation 2015;12:230. [31] Moehle MS, Webber PJ, Tse T, et al. LRRK2 inhibition attenuates microglial inflammatory responses. J Neurosci 2012;32:1602e11. [32] Han C, Yan P, He T, et al. PHLDA1 promotes microglia-mediated neuroinflammation via regulating K63-linked ubiquitination of TRAF6. Brain Behav Immun 2020;S08891591(19). 31575-2. [33] Jin H, Gu HY, Mao CJ, Chen J, Liu CF. Association of inflammatory factors and aging in Parkinson’s disease. Neurosci Lett 2020:135259. [34] Oh M, Kim SY, Gil JE, et al. Nurr1 performs its anti-inflammatory function by regulating RasGRP1 expression in neuro-inflammation. Sci Rep 2020;10(1):10755. [35] Sarkar S, Nguyen HM, Malovic E, et al. Kv1.3 modulates neuroinflammation and neurodegeneration in Parkinson’s disease. J Clin Invest 2020;130:4195e212. [36] Oliveira SR, Dionı´sio PA, Correia Guedes L, et al. Circulating inflammatory miRNAs associated with Parkinson’s disease pathophysiology. Biomolecules 2020;10:E945. [37] Liu Y, Lear TB, Verma M, et al. Chemical inhibition of FBXO7 reduces inflammation and confers neuroprotection by stabilizing the mitochondrial kinase PINK1. JCI Insight 2020;5(11):e131834. [38] Wang S, Yuan YH, Chen NH, Wang HB. The mechanisms of NLRP3 inflammasome/ pyroptosis activation and their role in Parkinson’s disease. Int Immunopharmacol 2019;67:458e64. [39] Gordon R, Albornoz EA, Christie DC, et al. Inflammasome inhibition prevents a-synuclein pathology and dopaminergic neurodegeneration in mice. Sci Transl Med 2018; 10:eaah4066. [40] Xu W, Zhang L, Geng Y, Liu Y, Zhang N. Long noncoding RNA GAS5 promotes microglial inflammatory response in Parkinson’s disease by regulating NLRP3 pathway through sponging miR-223-3p. Int Immunopharmacol 2020;85:106614. [41] Yan S, Wei X, Jian W, et al. Pharmacological inhibition of HDAC6 attenuates NLRP3 inflammatory response and protects dopaminergic neurons in experimental models of Parkinson’s disease. Front Aging Neurosci 2020;12:78. [42] Lee E, Hwang I, Park S, et al. MPTP-driven NLRP3 inflammasome activation in microglia plays a central role in dopaminergic neurodegeneration. Cell Death Differ 2019; 26(2):213e28. [43] Javed H, Thangavel R, Selvakumar GP, et al. NLRP3 inflammasome and glia maturation factor coordinately regulate neuroinflammation and neuronal loss in MPTP mouse model of Parkinson’s disease. Int Immunopharmacol 2020;83:106441.

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D.2.6. Role of endoplasmic reticulum stress in degeneration of nigrostriatal neurons The endoplasmic reticulum is an organelle where the maintenance of calcium homeostasis, synthesis and export of proteins occurs. In this organelle occurs translation, folding and transport of newly synthetized proteins where correct folded proteins are transported to the Golgi apparatus from the rough endoplasmic reticulum. The unfolded proteins in the endoplasmic reticulum are eliminated by inducing expression of endoplasmic reticulum chaperones. The impairment of endoplasmic function result in the accumulation of unfolded proteins that induces the unfolded protein response. Under endoplasmic reticulum stress the cell triggers a response to promotes cell survival called unfolded protein response. The unfolded protein response is induced by the activation of three sensors such as activation of transcription factor-6, double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase; and (3) inositol-requiring transmembrane kinase-1. The unfolded protein response dysfunction plays a relevant role in the degeneration of the nigrostriatal system in Parkinson’s disease [1]. The failure the unfolded protein response to stop endoplasmic reticulum stress leads to apoptosis and neurodegeneration of the nigrostriatal neurons in Parkinson’s disease [2]. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyran-dependent endoplasmic reticulum stress induces apoptosis by activation of caspase-12 and inositol-requiring transmembrane kinase-1 [3]. Endoplasmic reticulum stress induces the unfolded protein response to promote the survival of the cell but under certain conditions endoplasmic reticulum stress finally induces apoptosis and cell death. A study to understand what trigger the signaling switch from cell protection to apoptosis was performed using tetrachlorobenzoquinone-induced endoplasmic reticulum stress. Tetrachlorobenzoquinone induced X-boxbinding protein-1 splicing, increased inositol-requiring kinase/endonuclease 1-alpha phosphorylation, increased C/EBP homologous protein expression, and increased caspase 12 activation. This study concluded that protein disulfide isomerase-A1/A3 is a signal that switch endoplasmic reticulum stress from cellular protection to apoptosis and cell death when excessive or constant endoplasmic reticulum stress trigger protein disulfide isomerase-A1/A3 release from endoplasmic reticulum lumen to induce mitochondrial outer membrane permeabilization [4]. Expression of A53T induced mitochondrial dysfunction, a decrease in proteasome activity, an increase oxidative stress, cell death, an increase in endoplasmic reticulum stress and caspase-12 activity. An inhibitor of

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endoplasmic reticulum provided a protective effect on A53T induced cell death [5]. The expression of A53T in a transgenic mouse induces endoplasmic reticulum chaperones without activation of phospho-eIF2alpha, suggesting that alpha-synuclein can induce cell death by generating an abnormal unfolded protein response. Alpha-synuclein aggregation was associated with endoplasmic reticulum stress by interacting with endoplasmic reticulum chaperones, suggesting that alpha-synuclein has a direct effect on endoplasmic reticulum function. The inhibition of endoplasmic reticulum stress decrease A53T-dependent neurodegeneration [6]. In astrocytes the A30P and A53T induce endoplasmic reticulum stress activating in astrocytes triggered ER stress via the protein kinase RNAlike endoplasmic reticulum stress kinase/eukaryotic translation initiation factor 2-alpha signaling pathway, leading to astrocyte apoptosis [7]. The expression of the unfolded protein response-linked activating transcription factor 4/cAMP-responsive element-2 was increased when alpha-synuclein aggregates accumulate. Alpha-synuclein was found to be bound to the glucose-regulated protein 78/immunoglobulin heavy chain-binding protein, a misfolded protein-sensor/unfolded protein response activator [8]. Endoplasmic reticulum-Golgi transit of COPII vesicles is inhibited by the accumulation of alpha-synuclein aggregates. ATF6, the protective part of unfolded protein response, is handled via COPII mediated endoplasmic reticulum-Golgi transit. Alpha-synuclein decreased endoplasmic reticulum-associated degradation because of alpha-synuclein inhibition of ATF6 handling [9]. The activation of unfolded protein response in the substantia nigra of Parkinson’s Disease patient was detected in dopaminergic neurons containing neuromelanin using immunoreactivity against two unfolded protein response markers such as phosphorylated eukaryotic initiation factor 2alpha and phosphorylated pancreatic endoplasmic reticulum kinase. Interestingly, phosphorylated pancreatic endoplasmic reticulum kinase immunoreactivity colocalized with alpha-synuclein aggregates immunoreactivity in dopamine neurons [10]. The model neurotoxin used for experimental Parkinson’s disease, 6-hydroxydopamine, induces genes involved in endoplasmic reticulum stress such as c-Jun, BiP, elements of the ubiquitin-proteasome system, X-Box binding protein1 and endoplasmic reticulum chaperones [11]. Oxidative and endoplasmic reticulum stress induces the activating transcription factor 4 that it is increased in dopaminergic neurons containing neuromelanin in Parkinson’s disease substantia nigra. The knockdown of activating transcription factor 4 increased the cell dead in cell cultures exposed to 6-hydroxidopamine or 1-methyl-4phenylpyridinium neurotoxins used as cell model for Parkinson’s disease, while the over expression of activating transcription factor 4

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decreased the cell death. The effect of activating transcription factor-4 was associated with parkin because 6-hydroxidopamine or 1-methyl-4phenylpyridinium neurotoxins decrease the level of parkin protein and the knockdown of activating transcription factor-4 increase the reduction of parkin protein level while the overexpression of activating transcription factor-4 induced the opposite effect [12]. An increase in parkin-specific protein and mRNA levels was observed during endoplasmic reticulum stress that it is mediated by a transcription factor of the unfolded protein response (activating transcription factor-4). The activating transcription factor-4 acts on a specific CREB/ATF site inside of the parkin promoter while c-Jun acts on the same site, but it works as transcriptional repressor of parkin expression. The same study observed that endoplasmic reticulum stress leading to unfolded protein response and upregulation of parkin was induced by mitochondrial damage. Parkin prevents mitochondrial damage that was independent on the proteasome [13]. The neurotoxin 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine induces ATF6alpha activation, oxidative stress, increase in endoplasmic reticulum chaperones and endoplasmic reticulum-associated degradation. ATF6 deficient mice exhibited proteins inclusions positive against ubiquitin and loss of dopaminergic neurons after 1-Methyl-4-phenyl-1,2,3,6tetrahydropyridine treatment. In cell experiments 1-methyl-4phenylpyridinium induces oxidative stress, phosphorylation of p38 mitogen-activated protein kinase, and increase the activity of transcriptional activator transcriptional activator ATF6alpha because of ATF6alpha interaction with interaction phosphorylated p38 mitogen-activated protein kinase. These results suggest a relationship between endoplasmic reticulum stress and oxidative stress [14]. ATF6alpha knockout mice exhibited a strong suppression of astrocyte activation, reduction of brain-derived neurotrophic factor, heme oxygenase-1 after 1-Methyl-4phenyl-1,2,3,6-tetrahydropyridine/probenecid. The decrease in BDNF was linked to a decrease in the expression of ATF6alpha-dependent molecular chaperone GRP78 in the endoplasmic reticulum and the decrease in heme oxygenase-1 was related to a decrease in the expression of the pro-apoptotic gene CHOP, which is ATF4-dependent. The activation of unfolded protein response reversed the observed effects in 1Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid treated AFT6alpha knockout mice [15]. The knockdown of X-Box binding protein 1, a regulator of the unfolded protein response, induces chronic endoplasmic reticulum stress and degeneration of dopaminergic neurons. The delivery of an active form of X-Box binding protein 1 using gene therapy reduces striatal denervation in 6-hydroxydopamine-treated animals, supporting unfolded protein response role in the protein homeostasis in dopaminergic neurons [16].

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A study about the role of inositol-requiring protein-1 alpha- X-Box binding protein-1 pathway under the effect of proteasome inhibitors and the neurotoxin 1-methyl-4-phenylpyridinium (MPPþ) showed that the expression of the active form of X-Box binding protein-1 exhibited protective effects. The neurodegeneration in a mouse experimental model induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine was suppressed by X-Box binding protein-1 overexpression [17]. The transplantation of neural stem cells transfected with X-box-binding protein-1 into the right lateral ventricles of rats treated with rotenone induced an increase in tyrosine hydroxylase, 3,4-dihydroxyphenylacetic acid and dopamine levels in the substantia nigra, a decrease in alphasynuclein expression and neurological behaviors induced by the neurotoxin [18].The homologous of human HSPC117 protein in C. elegans is RTCB-1 that protects neurons from human alpha-synuclein-dependent degeneration because the knockdown of RTCB-1increased alphasynuclein-dependent degeneration. HSPC117 is a crucial subunit of the human tRNA splicing ligase complex and splicing of HAC1, a transcription factor involved in the unfolded protein response, is intermediated by a tRNA ligase. RTCB-1 is required for X-box binding protein1, the C. elegans homologous of HAC1, mRNA splicing and RTCB-1 ligase activity is necessary for its neuroprotective role that it is intermediated through X-box binding protein-1 in the unfolded protein response pathway [19]. Mild endoplasmic reticulum stress (“preconditioning”) has a neuroprotective effect in experimental models of Parkinson’s disease. The presence of apoptotic signals together with mild endoplasmic reticulum stress induces autophagy. Mild endoplasmic reticulum stress-induced protection is lost when autophagy is impaired [20]. Mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine induced upregulation of RNF13, p-c-jun N-terminal kinase/c-jun N-terminal kinase, p-inositol-requiring enzyme-1/inositol-requiring enzyme-1, apoptosis signal-regulating kinase-1, TNF receptor-associated factor-2. The silencing of RNF13 gene decrease motor dysfunctions induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and dopaminergic neurons degeneration by inhibiting endoplasmic reticulum stress-induced inositol-requiring enzyme-1-TNF receptor-associated factor-2-apoptosis signal-regulating kinase-1-c-jun N-terminal kinase pathway [21]. It has been reported that microRNA-34a-5p target the unfolded protein response sensor inositol-requiring enzyme-1alpha, X-box binding protein-1 and binding immunoglobulin protein. MicroRNA-34a-5p overexpression induces an important reduction in X-box binding protein-1 and inositol-requiring enzyme-1alpha increasing caspase activity and cytotoxicity [22]. A study to determine the effect of

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astragaloside-IV on endoplasmic reticulum stress-induced apoptosis in an in vitro experimental model of Parkinson’s disease where the cells were treated with 1-methyl-4-pehnyl-pyridine. Astragaloside-IV, an extract from astragalus membranous, inhibit the long noncoding RNA lincRNAp21 expression that bind CHOP protein, an apoptotic marker of endoplasmic reticulum stress. A decrease in lincRNA-p21 expression increase CHOP protein ubiquitination accelerating its degradation, protecting endoplasmic reticulum stress-induced apoptosis [23]. Manganese induces unfolded protein response sensor inositolrequiring enzyme-1 and autophagy. Autophagy was downregulated when Jun N-terminal kinase was inhibited. The knockdown of inositolrequiring enzyme-1 inhibited the expression of p-Jun N-terminal kinase, inositol-requiring enzyme-1, apoptosis signal-regulating kinase-1, and TNF receptor-associated factor 2 [24]. Leucine-rich repeat kinase 2-G2019S mutation in astrocytes induces sarco-/endoplasmic reticulum Ca2þ-ATPase dysfunction leads to calcium depletion in the endoplasmic reticulum, which induces mitochondrial dysfunction because of the formation of endoplasmic reticulum-mitochondria contacts and calcium overload in mitochondria [25]. A study shows that parkin knockdown induced a strong endoplasmic reticulum stress in astrocytes, cytokine release, c-Jun N-terminal kinases activation and an increase in nucleotideoligomerization domain receptor 2 [26]. Glia maturation factor, an inflammatory protein, induces degeneration of dopaminergic neurons. 1Methyl-4-phenylpyridinium and glia maturation factor increased mitogen-activated protein kinase, inositol-requiring enzyme 1alpha, endoplasmic reticulum stress, phospho-eukaryotic translation initiation factor 2 alpha kinase-3 and the mammalian target of rapamycin [27]. Four endoplasmic reticulum stressors that activates unfolded protein response, such as endoplasmic reticulum-Golgi protein transport inhibitor brefeldin A, N-glycosylation inhibitor tunicamycin, calcium pump inhibitor pump inhibitor thapsigargin and the reducing agent 2-mercaptoethanol, activate chaperone-mediated autophagy [28].

References [1] Xiang C, Wang Y, Zhang H, Han F. The role of endoplasmic reticulum stress in neurodegenerative disease. Apoptosis 2017;22:1e26. [2] Hetz C. The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat Rev Mol Cell Biol 2012;13:89e102. [3] Wang H, Dou S, Zhu J, Shao Z, Wang C, Cheng B. Ghrelin protects dopaminergic neurons against MPTP neurotoxicity through promoting autophagy and inhibiting endoplasmic reticulum mediated apoptosis. Brain Res 2020:147023. [4] Liu Z, Wang Y, Wang Y, et al. Effect of subcellular translocation of protein disulfide isomerase on tetrachlorobenzoquinone-induced signaling shift from endoplasmic reticulum stress to apoptosis. Chem Res Toxicol 2017;30:1804e14.

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[5] Smith WW, Jiang H, Pei Z, et al. Endoplasmic reticulum stress and mitochondrial cell death pathways mediate A53T mutant alpha-synuclein-induced toxicity. Hum Mol Genet 2005;14:3801e11. [6] Colla E, Coune P, Liu Y, et al. Endoplasmic reticulum stress is important for the manifestations of a-synucleinopathy in vivo. J Neurosci 2012;32:3306e20. [7] Liu M, Qin L, Wang L, et al. a synuclein induces apoptosis of astrocytes by causing dysfunction of the endoplasmic reticulum Golgi compartment. Mol Med Rep 2018; 18:322e32. [8] Bellucci A, Navarria L, Zaltieri M, et al. Induction of the unfolded protein response by a-synuclein in experimental models of Parkinson’s disease. J Neurochem 2011;116: 588e605. [9] Credle JJ, Forcelli PA, Delannoy M, et al. a-Synuclein-mediated inhibition of ATF6 processing into COPII vesicles disrupts UPR signaling in Parkinson’s disease. Neurobiol Dis 2015;76:112e25. [10] Hoozemans JJ, van Haastert ES, Eikelenboom P, de Vos RA, Rozemuller JM, Scheper W. Activation of the unfolded protein response in Parkinson’s disease. Biochem Biophys Res Commun 2007;354:707e11. [11] Holtz WA, O’Malley KL. Parkinsonian mimetics induce aspects of unfolded protein response in death of dopaminergic neurons. J Biol Chem 2003;278:19367e77. [12] Sun X, Liu J, Crary JF, et al. ATF4 protects against neuronal death in cellular Parkinson’s disease models by maintaining levels of parkin. J Neurosci 2013;33:2398e407. [13] Bouman L, Schlierf A, Lutz AK, et al. Parkin is transcriptionally regulated by ATF4: evidence for an interconnection between mitochondrial stress and ER stress. Cell Death Differ 2011;18:769e82. [14] Egawa N, Yamamoto K, Inoue H, et al. The endoplasmic reticulum stress sensor, ATF6a, protects against neurotoxin-induced dopaminergic neuronal death. J Biol Chem 2011;286:7947e57. [15] Hashida K, Kitao Y, Sudo H, et al. ATF6alpha promotes astroglial activation and neuronal survival in a chronic mouse model of Parkinson’s disease. PLoS One 2012; 7:e47950. [16] Valde´s P, Mercado G, Vidal RL, et al. Control of dopaminergic neuron survival by the unfolded protein response transcription factor XBP1. Proc Natl Acad Sci U S A 2014; 111:6804e9. [17] Sado M, Yamasaki Y, Iwanaga T, et al. Protective effect against Parkinson’s diseaserelated insults through the activation of XBP1. Brain Res 2009;1257:16e24. [18] Si L, Xu T, Wang F, Liu Q, Cui M. X-box-binding protein 1-modified neural stem cells for treatment of Parkinson’s disease. Neural Regen Res 2012;7:736e40. [19] Ray A, Zhang S, Rentas C, Caldwell KA, Caldwell GA. RTCB-1 mediates neuroprotection via XBP-1 mRNA splicing in the unfolded protein response pathway. J Neurosci 2014;34:16076e85. [20] Fouillet A, Levet C, Virgone A, et al. ER stress inhibits neuronal death by promoting autophagy. Autophagy 2012;8:915e26. [21] Ji M, Niu S, Guo J, Mi H, Jiang P. Silencing RNF13 alleviates mice models with Parkinson’s disease via regulating endoplasmic reticulum stress-mediated IRE1a-TRAF2ASK1-JNK pathway. J Mol Neurosci 2020;70:1977e86. [22] Krammes L, Hart M, Rheinheimer S, et al. Induction of the endoplasmic-reticulumstress response: MicroRNA-34a targeting of the IRE1a-branch. Cells 2020;9:1442. [23] Ge B, Li SL, Li FR. Astragaloside-IV regulates endoplasmic reticulum stress-mediated neuronal apoptosis in a murine model of Parkinson’s disease via the lincRNA-p21/ CHOP pathway. Exp Mol Pathol 2020;115:104478. [24] Liu C, Yan DY, Wang C, et al. IRE1 signaling pathway mediates protective autophagic response against manganese-induced neuronal apoptosis in vivo and in vitro. Sci Total Environ 2020;712:136480.

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[25] Lee JH, Han JH, Kim H, Park SM, Joe EH, Jou I. Parkinson’s disease-associated LRRK2G2019S mutant acts through regulation of SERCA activity to control ER stress in astrocytes. Acta Neuropathol Commun 2019;7:68. [26] Singh K, Han K, Tilve S, Wu K, Geller HM, Sack MN. Parkin targets NOD2 to regulate astrocyte endoplasmic reticulum stress and inflammation. Glia 2018;66:2427e37. [27] Selvakumar GP, Iyer SS, Kempuraj D, et al. Molecular association of glia maturation factor with the autophagic machinery in rat dopaminergic neurons: a role for endoplasmic reticulum stress and MAPK activation. Mol Neurobiol 2019;56:3865e81. [28] Li W, Yang Q, Mao Z. Signaling and induction of chaperone-mediated autophagy by the endoplasmic reticulum under stress conditions. Autophagy 2018;14:1094e6.

D.3. Diagnosis of idiopathic Parkinson’s disease For a long time, clinical diagnosis of idiopathic Parkinson’s disease was focused on motor symptoms. However, the Movement Disorder Society proposed new criteria for clinical diagnosis of idiopathic Parkinson’s disease that include nonmotor symptoms. The diagnosis has been suggested to be done in two steps. First, determine whether the patient has parkinsonism, defined as bradykinesia, rigidity, and/or rest tremor, and second, determine whether this parkinsonism is a consequence of idiopathic Parkinson’s disease [1]. According to Movement Disorder Society diagnosis criteria, once the patient has the diagnosis of parkinsonism, it should then be determined whether this parkinsonism is due to idiopathic Parkinson’s disease. To be diagnosed with idiopathic Parkinson’s disease requires the following: (1) Lack of absolute exclusion criteria. The exclusion criteria according to the Movement Disorder Society include (1) lack of response to levodopa treatment; (2) cerebellar abnormalities, such as limb ataxia, cerebellar gait, or cerebellar oculomotor abnormalities; (3) treatment with a dopamine-depleting drug or a dopamine receptor blocker; (4) parkinsonian features restricted to the lower limbs for more than tree; (5) diagnosis of probable primary progressive aphasia or behavioral variant frontotemporal dementia; (6) normal functional neuroimaging of the presynaptic dopaminergic system; (7) unequivocal progressive aphasia or cortical sensory loss; (8) selective slowing of downward vertical saccades or downward vertical supranuclear gaze palsy; and (9) existence of an alternative condition known to produce parkinsonism. (2) At least two supportive criteria such as (1) excellent response to dopaminergic therapy resulting in near normal function; (2) occurrence of levodopa-induced dyskinesia; (3) rest tremor of a limb; (4) positive results of olfactory loss test or cardiac sympathetic denervation test [1]. (3) No red flags such as (1) rapid evolution of gait damage needing regular use of wheelchair; (2) complete lack of advance of motor

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symptoms; (3) early bulbar dysfunction, such as severe dysarthria, dysphonia or severe dysphagia; (4) dysfunction of inspiratory respiratory; (5) severe autonomic failure (6) recurrent falls as a consequence of impaired balance with; (7) contractures of hand or feet or anterocollis; (8) absence common nonmotor symptoms (9) mysterious pyramidal tract signs; and (10) bilateral symmetric parkinsonism [1].

Reference [1] Postuma RB, Berg D, Stern M, Poewe W, Olanow CW, Oertel W6, Obeso J, Marek K8, Litvan I, Lang AE1, Halliday G, Goetz CG, Gasser T, Dubois B, Chan P, Bloem BR, Adler CH, Deuschl G. MDS clinical diagnostic criteria for Parkinson’s disease. Mov Disord 2015;30:1591e601.

E. Genetic Parkinson’s disease The discovery of gene mutations associated with a familial form of Parkinson’s disease has been a very important input in Parkinson’s disease research because it opens up several lines of basic research for understanding the role of these proteins in the sporadic form of Parkinson’s disease. The first gene associated with the familial form of the disease was the discovery of autosomal-dominant mutation in alphasynuclein in Italy [1]. This discovery generated an intensive research about alpha-synuclein role in sporadic form of the disease, resulting in the identification of alpha-synuclein as one of the most important components of Lewy bodies and Lewy neurites and has been a very important hallmark in Parkinson’s disease [2].

E.1. Autosomal-dominant mutations Alpha-synuclein. Alpha-synuclein mutations are one of the autosomal-dominant genes that can cause a familial form of Parkinson’s disease. The A53T point mutation was reported in Italian and Greek families [1,3]. This a very rare mutation because a study of 100 Parkinson’s disease patients with or without family history with disease onset before 51 years of age did not find this alpha-synuclein gene mutation [4]. Patients carrying this mutation presented typical clinical signs observed in idiopathic Parkinson’s disease such as rest tremor, hypokinesia, rigidity and postural instability but the age of onset was early, and the disease progression was more rapid [5]. The mutation A30P in alpha-synuclein gene was found in a German family with Parkinson’s disease [6]. Patients with the A30P mutation

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present similar phenotype to that of sporadic idiopathic Parkinson’s disease [7]. A postmortem study of a Spanish family with autosomaldominant parkinsonism and dementia revealed the heterozygous nonconservative E46K mutation in alpha-synuclein gene [8]. The mutation H50Q was detected in a patient with Parkinson’s disease and dementia responsive to L-dopa [9]. The missense mutations in alpha-synuclein gene that have been associated with a familial form of Parkinson’s disease includes A53T, A53E, A30P, E46K, G51D, and H50Q point mutations. Interestingly, these point mutations are localized in the N-terminal region of alpha-synuclein gene. Two hypotheses about how these point mutations lead to degeneration have been proposed: (1) the mutation in this gene region impairs alphasynuclein ability to bind membranes lipids; and (2) the mutations increase alpha-synuclein ability to aggregate to oligomers [10]. Alpha-synuclein gene multiplications are more common than point mutations and have been reported in families worldwide [11]. The first reported alpha-synuclein gene multiplication was a triplication in a Parkinson’s disease with dementia with an average age of onset at 34 years old. This alpha-synuclein triplication expressed functional DNA copies and the over dosage of this gene probably explain the early onset of the disease [12]. The genetic triplication of alpha-synuclein results in early onset of Parkinson’s disease and a study showed that alpha-synuclein triplication generates the double amount of alpha-synuclein protein in blood, resulting in an increase of depositions of aggregated alphasynuclein [13]. Alpha-synuclein gene duplication was also reported in familial Parkinson’s disease but the phenotype was different in comparison with gene triplication since the clinical phenotype was like idiopathic patients with late age onset, slow progress and note presented dementia or cognitive decline. These results suggest that the disease progression and alphasynuclein dosage are in direct relationship [14]. A study reported patients carrying alpha-synuclein duplication was reported. Interestingly, two patients presented sporadic Parkinson’s disease, and one patient presented a familial form of the disease. The patient with familial form of the disease developed an early onset with rapid progression, dysautonomia and cognitive impairment [15]. A study of 113 patients with autosomal-dominant hereditary Parkinson’s disease and 200 patients with sporadic Parkinson’s disease patients detected two families with alpha-synuclein gene duplication in the group of patients with autosomal-dominant hereditary form of the disease. One of these patients presented dementia [16]. A study to screen alpha-synuclein genomic multiplications performed with more than 200 families with autosomal-dominant Parkinson’s disease showed that 1.5% presented gene duplications [17].

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A study performed with five families with Parkinson’s disease carrying multiplication of alpha-synuclein gene showed that the clinical severity of symptoms of the disease are correlated with alpha-synuclein dosage without influence of other genes [18]. A meta-analysis that included 59 families with Parkinson’s disease carrying alpha-synuclein multiplications. A copy number variants analysis showed 49 patients with heterozygous alpha-synuclein duplication, seven with triplication mutation, two with homozygous duplication. The disease onset is correlated with with alpha-synuclein dosage and dementia was observed in 50.9% of analyzed cases. This study concluded that alpha-synuclein multiplications are globally distributed and rare [19]. Leucine-rich repeat kinase-2. Leucine-rich repeat kinase-2 gene mutations are also autosomal dominant that are the most frequent mutation in the familial form of Parkinson’s disease. The most common mutation in leucine-rich repeat kinase-2 gene is the heterozygous point mutation G2019S in the 6.6% of autosomal-dominant Parkinson’s disease patients from Portugal, Italy, and Brazil [20]. More than 100 leucine-rich repeat kinase-2 mutations have been reported but only G2019S, R1441 C/G/H, I2020T and Y1699C mutations have been associated with familial form of Parkinson’s disease [21]. A meta-analysis that included more than 30000 Parkinson’s disease patients carrying leucine-rich repeat kinase-2 mutation showed that women have higher prevalence of G2019S mutation [22]. The highest frequency of leucine-rich repeat kinase-2 2019S mutation has been observed in North-African Arab populations where the 42% of familial form of Parkinson’s disease and 35.7% of sporadic Parkinson’s disease carry this mutation [23]. A study about the penetrance of leucine-rich repeat kinase-2 G2019S mutation in Ashkenazi Jewish Parkinson’s disease patients from New York, USA, and Tel Aviv, Israel was 26% [24]. A meta-analysis that includes 66 studies that reported clinical features of Parkinson’s disease patients carrying G2019S, R1441G, R1628P and G2385R leucine-rich repeat kinase-2 mutations. The most relevant clinical features of leucine-rich repeat kinase-2 G2019S mutation were early onset, family history, good response to L-dopa, developed dyskinesia and motor fluctuations and female sex [25]. Leucine-rich repeat kinase-2 (LRRK2) G2385R variant has been associated with Asian population. A study of around 1000 Chinese Parkinson’s disease patients showed that the clinical features of this mutation were postural instability and gait difficulty dominant, higher rapid eye movements sleep behavior disorder and motor fluctuations [26]. A comparison between two leucine-rich repeat kinase-2 mutations was performed using 199 Parkinson’s disease patients with G2385R mutation, 516 PD patients with the G2019S mutation and 790 patients with idiopathic Parkinson’s disease. The motor fluctuations frequency and UPDRS

160

1. Parkinson’s disease

part II or III scores were higher in the G2385R mutation patients in comparison to patients with G2019S mutation. Idiopathic Parkinson’s disease patients showed significant higher UPDRS part III scores than patients with G2019S mutation. Idiopathic Parkinson’s patients had lower problem with postural instability gait disorder [27]. Parkinson’s disease patients in North Africa carrying leucine-rich repeat kinase-2 G2019S mutation is the most common cause of the disease. These patients have similar clinical features in comparison with idiopathic patients but leucine-rich repeat kinase-2 G2019S mutation carriers have significant higher mini-mental state examination scores, UPDRS-III scores were lower, no sex difference but men has better cognition, less tendency to depression, and higher education level [28]. Vacuolar protein sorting-35. Exome sequencing of an Austrian family revealed a missense mutation, p.Asp620Asn, in vacuolar protein sorting35 in all family members with Parkinson’s disease that participate in this study. This is an autosomal-dominant mutation with an age onset of 53 years. vacuolar protein sorting-35 is involved in retrograde transport between the trans-Golgi network and endosomes [29]. A multicenter worldwide study of more than 15,000 individuals showed that the mutation p.Asp620Asn in vacuolar protein sorting-35 gene was present in five familial and two sporadic Parkinson’s disease patients but this mutation was not found in healthy controls [30]. The next-generation sequencing technologies was used to study a Swiss family with autosomal-dominant late-onset Parkinson’s disease that was L-dopa responsive and tremor as the predominant symptom. This study suggested that the p.Asp620Asn missense mutation in vacuolar protein sorting-35 gene is associated with Parkinson’s disease. Vacuolar protein sorting-35 gene play an essential role in membrane protein recycling and endosome-trans-Golgi trafficking and it is an important part of the retromer cargo-recognition complex. The p.Asp620Asn missense mutation in vacuolar protein sorting-35 gene was present in all family members [31]. A study on vacuolar protein sorting-35 mutation in Japanese population with autosomal-dominant and sporadic Parkinson’s disease reported that p.D620N mutation was detected in three patients with autosomal Parkinson’s disease and in on sporadic patient [32]. However, a study performed with 1011 sporadic Parkinson’s disease patients did not observe significant difference in vacuolar protein sorting35 gene mutations with healthy controls in the Chinese population [33].

E.2. Autosomal-recessive mutations Parkin. The first autosomal-recessive gene discovered was parkin, an E3 ligase enzyme of the proteasomal system. A deletion in parkin gene

1. Parkinson’s disease

161

between D6S1937 and AFMa155td9 was observed in this gene for autosomal-recessive juvenile familial form of Parkinson’s disease [34]. The mean age at onset of the familial juvenile form of Parkinson’s disease was 23 years and these patients were responsive to L-dopa treatment [35]. A study of 26 Japanese Parkinson’s disease patients carrying parkin mutation showed that these patients were predominantly female, and the features of the disease were slow progression, L-dopa responsive, wearing-off phenomenon and dyskinesia induced by L-dopa. The typical symptoms of Parkinson’s disease bradykinesia, tremor, rigidity were mild but postural instability, hyperreflexia and dystonic posture were more prominent than in the idiopathic form of the disease [36]. A study of 73 patients with early onset revealed that 49% carried a parkin mutation. The age onset of the disease in 77% of the patients were less than 20 years and only 3% of the patients have an age onset after 30 years [37]. A study of 37 Spanish patients with autosomal-recessive disease or either early-onset or autosomal-recessive pattern of inheritance detected parkin homozygous mutation in six patients [38]. A study of 35 European families with the familial form of early-onset autosomal-recessive Parkinson’s disease showed a deletion of exon 4 in one family with homozygous parkin mutation. The study of the remaining 34 families revealed eight not-described point heterozygous or homozygous mutations in 20 patients [39]. A study performed with 34 patients from 18 families with parkin mutation showed four homozygous deletions in exon 4, exon 3, exons 3 to 4, and exon 5 in 10 patients. Two point mutations in exon 5 were also detected in two patients belonging a two families [40]. The possible existence of genetic mutation in with 23 sporadic Chinese Parkinson’s disease patients with early onset before 45 year was performed using next-generation sequencing and multiplex ligationdependent amplification. This study revealed that 10 patients carried 12 parkin mutations, one in vacuolar protein sorting-13 homolog C and one in leucine-rich repeat kinase-2. Parkin was the most frequent mutation (34%) [41]. The rearrangement of parkin exon in near 7% of patients in a study performed with 150 Parkinson’s disease patients with early onset was observed. A parkin gene heterozygous duplication was also observed [42]. PTEN-induced kinase-1. The second identified autosomal-recessive gene associated with the familial form of Parkinson’s disease was PTEN-induced kinase-1 (PARK6) in a Sicilian family in Italy. Four family members were affected with a parkinsonism of slow progression and responsive to L-dopa that were not related to a parkin mutation. The family was analyzed with a genome-wide homozygosity screen that revealed a mutation in PTEN-induced kinase-1 gene [43]. The locus of PTEN-induced kinase-1 mutation was in chromosome 1p36. Two missense mutation were detected in three consanguineous PTEN-induced

162

1. Parkinson’s disease

kinase-1 families, a truncation nonsense mutation and a missense mutation at a conserved amino acid region. Studies performed with cell cultures revealed that PTEN-induced kinase-1 was located at mitochondria and played a protective role in mitochondrial because PTEN-induced kinase-1 mutation induced cellular stress [44]. PTEN-induced kinase-1 normal function is to induce selective autophagy of impaired mitochondria, by activating the E3 ubiquitin ligase parkin. It has been proposed that parkin translocation to impaired mitochondria is mediated by PTEN-induced kinase-1-dependent phosphorylation of both parkin and ubiquitin at serine 65. The expression of ubiquitin S65A mutant prevents both PTEN-induced kinase-1-dependent ubiquitin phosphorylation and parkin translocation to impaired mitochondria, inhibiting autophagy-dependent of impaired mitochondria degradation [45]. A study performed in Italy with sporadic and familial Parkinson’s disease patients to detect PTEN-induced kinase-1 mutation identified a homozygous mutation in four sporadic patients, and a heterozygous truncating mutation in four sporadic patients. The patients with homozygous mutation presented an early-onset, good response to L-dopa, slow progression, dystonia at onset, symmetric onset, and sleep benefit [46]. Another study performed in 51 families with autosomal-recessive Parkinson’s disease to detect PTEN-induced kinase-1 mutations reported one homozygous missense C388R mutation and one homozygous deletion 13516-18118del. The patients with 13516-18118del mutation presented dementia symptoms suggesting that early-onset Parkinson’s disease with dementia may be associated with PTEN-induced kinase-1 mutation [47]. A study of 77 Parkinson’s disease families participates in a study in Turkey that analyzed 86 patients reported to new PTENinduced kinase-1 point mutations P416L and L31X [48]. The prevalence of gene mutations in early-onset Parkinson’s disease in United kingdom showed that the most prevalent mutations were parkin gene with 8.6% in parkin gene, PTEN-induced kinase-1 gene, with 3.7%, and the protein deglycase-1 gene, with 0.4%. PTEN-induced kinase-1 mutation is more common in patients of Asian origin [49]. A study of 187 early-onset Dutch Parkinson’s disease patients showed that parkin was the most prevalent mutation > Protein deglycase-1 > PTEN-induced kinase-1 > Leucine-rich repeat kinase-2 [50]. PTEN-induced kinase-1 mutations are most single nucleotide change, but it has been reported that a patient with sporadic Parkinson’s disease carry a deletion of the complete gene. This patient generates various aberrant mRNA as consequence of a splice site mutation (g.15445_15467del23) [51]. A study on PTEN-induced kinase-1 mutations with 177 families with autosomal-recessive Parkinson’s disease identified 10 pathogenic mutations that included three frameshift deletion

1. Parkinson’s disease

163

mutations, five missense and two nonsense. These mutations were homozygous or heterozygous. The clinical features of 12 patients were indistinguishable from Parkinson’s disease patients carrying a parkin mutation, including increased reflexes and dystonia at onset [52]. Protein deglycase-1. Another autosomal-recessive mutation in early onset of the familial form of Parkinson’s disease was reported in the Netherlands, and Italy. The protein glycase-1 (PARK7) locus was mapped in chromosome 1p36 [53]. A study of 185 Parkinson’s disease with early onset was performed to detect protein deglycase-1 mutation. Only one homozygous and one heterozygous mutation was detected and the reported 14Kbp deletion was not observed in patients or controls [54]. Two brothers with a homozygous frameshift mutation in protein deglycase-1 presented a familial form of Parkinson’s disease associated with hypomimia and high-pitched voice at 29 years old were L-dopa responsive [55]. It has been suggested that protein deglycase-1 function is related to a protective role against oxidative stress where a cysteine at C106 play a role [56]. It has been reported that protein deglycase-1 is a dimeric protein with protease activity that it is blocked with the C106A mutation. The mutation L166P impair protein deglycase-1 folding generating a spontaneous unfolded protein that it is not able to form homodimer with itself or heterodimer with wild-type protein deglycase-1 [57]. Protein deglycase-1 dimerization seems to be an important feature in this protein function where its dimers are stabilized under oxidative stress. However, protein deglycase-1 mutations such as L10P, M26I, L166P, and P158D prevent the formation of homodimers. However, the protein deglycase-1 E64D mutation can form homodimers but E64D mutant inhibit the formation of homodimers of wild-type protein deglycase-1. In addition, E64D mutation stimulate the formation of aggresome containing protein deglycase-1 [58]. Protein deglycase-1 mutation P158DEL, L166P, and L10P presented a significant reduced stability, which were degraded by proteasomal system. The proteins of P158DEL and L10P mutation was able to dimerize with protein deglycase wild type, but these mutant proteins were no able to form homodimers [59]. Protein deglycase-1 D149A, L166P and R98Q mutations induce a significant increase in fragmented mitochondria while the wild-type protein induced elongated mitochondria. The overexpression of protein deglycas1 mutation induces mitochondrial dysfunction and increased neurons susceptibility to oxidative stress. The regulator of mitochondrial fission dynamin-like protein-1 was increased in cell expressing protein deglycase-1 mutation while decreased in wild-type cells. The knockdown of protein deglycase-1 prevented mitochondria dysfunction and the formation of abnormal mitochondria [60]. ATPase cation transporter 13A2. Reported as a mutation in P-type ATPase gene, ATP13A2, in a Chilean family with early-onset

164

1. Parkinson’s disease

Parkinson’s disease that has similar symptoms of Kufor Rakeb syndrome. This mutation induced a loss of function, mainly neuronal P-type ATPaseP13A2 (PARK9). This mutation was autosomal-recessive in early onset of familial form of Parkinson’s disease with dementia and pyramidal degeneration [61]. The protein P-type ATPaseP13A2 is a lysosomal type 5 P-type ATPase that play an important role in the maintenance of lysosomal pH. The degradation of proteins and damaged organelles in the lysosomes is dependent on the activation of hydrolytic enzymes that require an acidic luminal pH. Lysosomes acidic luminal pH is depending on vacuolar-type H þ -ATPase, such as P-type ATPaseP13A2, that pumps protons to the lumen with concomitant a counterion flux. Fibroblast derived from Parkinson’s disease patients carrying P-type ATPaseP13A2 mutation exhibited decreased proteolytic activity of lysosomal enzymes, impaired lysosomal acidification, decreased degradation of lysosomal substrates, and reduced lysosomal-dependent clearance of autophagosomes. The knockdown of P-type ATPaseP13A2 induces lysosomal impairment and cell death while normal P-type ATPaseP13A2 levels restores lysosomal function [62]. P-type ATPaseP13A2 mutations, including homozygous and heterozygous, have been identified in independent families. The heterozygous mutation has been detected in sporadic Parkinson’s disease. P-type ATPaseP13A2 seems to play a role in zinc (II) homeostasis where disruption of zinc (II) homeostasis result in mitochondrial dysfunction, lysosomal impairment, and alpha-synuclein accumulation [63]. Kufor Rakeb syndrome is an autosomal-recessive Parkinson’s disease form where patients carrying P-type ATPaseP13A2 mutation have a juvenile onset of the disease accompanied other clinical features, including dementia, supranuclear gaze palsy, and generalized brain atrophy [64]. Phospholipase A2 group VI- Phospholipase A2 group VI (PARK12) gene is coding a calcium-independent group VI phospholipase A2 involved in brain iron accumulation, the related Karak syndrome and the infantile neuroaxonal dystrophy [65]. A mutation in Phospholipase A2 group VI gene was identified in three patients with adult-onset dystoniaparkinsonism that were L-dopa responsive. The clinical symptoms included pyramidal and cognitive/psychiatric features, and cerebellar and cerebral atrophy detected with magnetic resonance imaging but without iron in the basal ganglia [66]. Sequencing analysis of phospholipase A2 group VI gene in an Iranian patient with consanguineous dystonia-parkinsonism find a R632W mutation [67]. A study of 29 patients with early-onset Parkinson’s disease that presented other clinical features such as dystonia, psychosis, mental retardation/dementia, and hyperreflexia showed two new heterozygous phospholipase A2 group VI gene mutations, p.F72L/p.R635Q and

1. Parkinson’s disease

165

p.Q452X/p.R635Q, in three patients. The clinical features of these patients with phospholipase A2 group VI mutation were early-onset Parkinson’s disease with relatively rapid progression, L-dopa-responsive, dementia and frontotemporal lobar atrophy. Iron accumulation in the striatum and substantia nigra was observed in one patient using magnetic resonance image technique [68]. A study on phospholipase A2 group VI mutations on three early-onset Parkinson’s disease showed that one patient with dystonia-parkinsonism with major cognitive decline and depression presented a heterozygous mutation c.991G > T (p.D331Y)/c.1077G > A (M358IfsX). The other patients with early-onset parkinsonism that were responsive to L-dopa presented a homozygous c.991G > T (p.D331Y) mutations in phospholipase A2 group VI gene. The analysis of these three patients with other 32 cases reported before showed that 17 cases had early-onset Parkinson’s disease, 14 cases had dystonia-parkinsonism, three cases had hereditary spastic paraparesis, and one case had hereditary spastic paraparesis. Patients presenting homozygous p.D331Y mutation did not have brain iron accumulation [69]. F-box protein-7- F-box protein-7 (PARK15) mutation was detected in two families with early onset, pyramidal tract signs, and progressive parkinsonism. F-box protein 7 homozygous truncating mutation (Arg498Stop) was detected in an Italian family while a heterozygous missense Thr22Met mutation and a splice site IVS7 þ 1G/T mutation were detected in a Dutch family [70]. A study on F-box protein-7 mutations in Chinese early-onset parkinsonism patients performed with 135 patients showed no pathogenetic mutations in this population [71]. A new homozygous F-box protein-7 c.1492C > T, p.Arg498* mutation was reported in a Turkish family. The clinical features observed in this family included mental retardation, L-dopa responsive, but without pyramidal signs. L-dopa treatment induced dyskinesias and psychiatric side effects [72]. F-box only protein 7 gene express two isoforms overexpressed mutant F-box only protein 7 proteins R498X, T22M, and R378G have reduced stability in comparison with wild-type protein [73]. It has been reported that F-box protein-7 is involved in mitochondrial maintenance by interacting with PTEN-induced kinase-1 and parkin, participating in parkin-dependent mitophagy. Decreased level of expression of F- box protein-7 resulted in insufficient parkin translocation to damaged mitochondria, mitophagy, and ubiquitination of mitofusin-1 [74]. The wild-type F-box protein-7 plays an important role in in cellular mitophagy while mutant F-box protein-7 inhibits mitophagy. Under stress conditions wild-type F-box protein-7 upregulates, accumulates into mitochondria, and F-box protein-7 aggregates are formed. F-box protein-7 mutations become worse with its aggregation effects in mitochondria. F-box protein-7 aggregation and toxicity can be decreased in the presence

OMIM

PARK gene

Protein

Gene/locus

Autosomal

Onset

Cytogenetic location

168601

PARK1

Alpha-synuclein

SNCA

Dominant

Late

4q22.1

600116

PARK2

Parkin

PRKN

Recessive

Juvenile

6q26

602404

PARK3

Parkinson disease 3

e

Dominant

605543

PARK4

Alpha-synuclein triplication

SNCA

Dominant

Early

4q22.1

613643

PARK5

Ubiquitin carboxylterminal esterase L1

UCHL1

Dominant

Late

4p13

605909

PARK6

PTEN-induced kinase 1

PINK1

Recessive

Early

1p36.12

606324

PARK7

Protein deglycase

DJ1

Recessive

Early

1p36.23

607060

PARK8

Leucine-rich repeat kinase 2

LRRK2

Dominant

Late

12q12

610513

PARK9

ATPase cation transporting 13A2

ATP13A2

Recessive

Juvenile

1p36.13

606852

PARK10

Parkinson disease 10/ AAOPD

LOC200008

Late

1p32

607688

PARK11

GRB10-interacting GYF protein 2

GIGYF2

Dominant

Late

2q37.1

300557

PARK12

Parkinson disease 12

e

e

Late

Xq21-q25

606441

PARK13

HtrA serine peptidase 2

HTRA2

e

e

2p13.1,

612953

PARK14

phospholipase A2 group VI

PLA2G6

Recessive

Early

22q13.1

166

TABLE 1.1 Genes associated with familial form of Parkinson’s disease.

2p13

1. Parkinson’s disease

PARK15

F-box protein 7

FBXO7

Recessive

Early

22q12.3

613164

PARK16

Parkinson disease 16

e

e

e

1q32

614203

PARK17

Vacuolar protein sorting 35

VPS35

Dominant

Late

16q11.2

614251

PARK18

Eukaryotic translation initiation factor 4 gamma 1

EIF4G1

Dominant

Late

3q27.1

615528

PARK19A

Putative tyrosineprotein phosphatase auxilin

DNAJC6

Recessive

Juvenile

1p31.3

615528

PARK19B

Putative tyrosineprotein phosphatase auxilin

DNAJC6

Recesive

Early

1p31.3

615530

PARK20

Synaptojanin-1

SYNJ1

Recessive

Early

21q22.11

616361

PARK21

DnaJ homolog subfamily C member 13

DNAJC13

Dominant

Late

3q22 þ G25

616710

PARK22

Coiled-coil-helixcoiled-coil-helix domain-containing protein 2

CHCHD2

Dominant

Late

7p11.2

616840

PARK23

Vacuolar protein sorting-associated protein 13C

VPS13C

Recessive

Early

15q22.2

1. Parkinson’s disease

260300

167

168

1. Parkinson’s disease

of coenzyme Q10, glutathione, and proline, while an inhibitor of mitochondrial protease increases F-box protein-7 deleterious aggregation in mitochondria [75]. F-box protein-7 is an adaptor protein in the ubiquitin E3 ligase complex SCFFBXO7 with the function to recognize and mediate non-degradative or degradative ubiquitination of substrates. F-box protein-7 can regulate proliferation, cell cycle, proteasomal and mitochondrial functions by interacting with multiple target proteins. Wild-type F-box protein-7 has both deleterious and protective functions while mutant F-box protein-7 is toxic. Cellular stress promotes F-box protein-7 translocation into mitochondria from the nucleus and induces F-box protein-7 deleterious aggregation in mitochondria [76] Table 1.1.

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[40]

[41]

[42]

[43]

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[50] [51] [52] [53] [54] [55]

[56]

[57]

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Disease Genetics Study Group and the European Consortium on Genetic Susceptibility in Parkinson’s Disease. Hum Mol Genet 1999;8:567e74. Hattori N, Kitada T, Matsumine H, et al. Molecular genetic analysis of a novel Parkin gene in Japanese families with autosomal recessive juvenile parkinsonism: evidence for variable homozygous deletions in the Parkin gene in affected individuals. Ann Neurol 1998;44:935e41. Jiang Y, Yu M, Chen J, et al. Parkin is the most common causative gene in a cohort of mainland Chinese patients with sporadic early-onset Parkinson’s disease. Brain Behav 2020:e01765. Lin Y, Zeng YF, Cai NQ, Lin XZ, Wang N, He J. Analysis of exon dosage using multiplex ligation-dependent probe amplification in Chinese patients with early-onset Parkinson’s disease. Eur Neurol 2019;81:246e53. Valente EM, Bentivoglio AR, Dixon PH, et al. Localization of a novel locus for autosomal recessive early-onset parkinsonism, PARK6, on human chromosome 1p35-p36. Am J Hum Genet 2001;68:895e900. Valente EM, Abou-Sleiman PM, Caputo V, et al. Hereditary early-onset Parkinson’s disease caused by mutations in PINK1. Science 2004;304:1158e60. Kane LA, Lazarou M, Fogel AI, et al. PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity. J Cell Biol 2014;205:143e53. Bonifati V, Rohe´ CF, Breedveld GJ, et al. Early-onset parkinsonism associated with PINK1 mutations: frequency, genotypes, and phenotypes. Neurology 2005;65:87e95. Li Y, Tomiyama H, Sato K, et al. Clinicogenetic study of PINK1 mutations in autosomal recessive early-onset parkinsonism. Neurology 2005;64:1955e7. Lohmann E, Dursun B, Lesage S, et al. Genetic bases and phenotypes of autosomal recessive Parkinson disease in a Turkish population. Eur J Neurol 2012;19:769e75. Kilarski LL, Pearson JP, Newsway V, et al. Systematic review and UK-based study of PARK2 (parkin), PINK1, PARK7 (DJ-1) and LRRK2 in early-onset Parkinson’s disease. Mov Disord 2012;27:1522e9. Macedo MG, Verbaan D, Fang Y, et al. Genotypic and phenotypic characteristics of Dutch patients with early onset Parkinson’s disease. Mov Disord 2009;24:196e203. Marongiu R, Brancati F, Antonini A, et al. Whole gene deletion and splicing mutations expand the PINK1 genotypic spectrum. Hum Mutat 2007;28:98. Iba´n˜ez P, Lesage S, Lohmann E, et al. Mutational analysis of the PINK1 gene in earlyonset parkinsonism in Europe and North Africa. Brain 2006;129:686e94. Bonifati V, Rizzu P, Squitieri F, et al. DJ-1(PARK7), a novel gene for autosomal recessive, early onset parkinsonism. Neurol Sci 2003;24:159e60. Abou-Sleiman PM, Healy DG, Quinn N, Lees AJ, Wood NW. The role of pathogenic DJ1 mutations in Parkinson’s disease. Ann Neurol 2003;54:283e6. Stephenson SE, Djaldetti R, Rafehi H, et al. Familial early onset Parkinson’s disease caused by a homozygous frameshift variant in PARK7: clinical features and literature update. Park Relat Disord 2019;64:308e11. Canet-Avile´s RM, Wilson MA, Miller DW, et al. The Parkinson’s disease protein DJ-1 is neuroprotective due to cysteine-sulfinic acid-driven mitochondrial localization. Proc Natl Acad Sci U S A 2004;101:9103e8. Olzmann JA, Brown K, Wilkinson KD, et al. Familial Parkinson’s disease-associated L166P mutation disrupts DJ-1 protein folding and function. J Biol Chem 2004;279: 8506e15. Repici M, Straatman KR, Balduccio N, Enguita FJ, Outeiro TF, Giorgini F. Parkinson’s disease-associated mutations in DJ-1 modulate its dimerization in living cells. J Mol Med (Berl) 2013;91:599e611. Ramsey CP, Giasson BI. L10p and P158DEL DJ-1 mutations cause protein instability, aggregation, and dimerization impairments. J Neurosci Res 2010;88:3111e24.

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[60] Wang X, Petrie TG, Liu Y, Liu J, Fujioka H, Zhu X. Parkinson’s disease-associated DJ-1 mutations impair mitochondrial dynamics and cause mitochondrial dysfunction. J Neurochem 2012;121:830e9. [61] Ramirez A, Heimbach A, Gru¨ndemann J, et al. Hereditary parkinsonism with dementia is caused by mutations in ATP13A2, encoding a lysosomal type 5 P-type ATPase. Nat Genet 2006;38:1184e91. [62] Dehay B, Ramirez A, Martinez-Vicente M, et al. Loss of P-type ATPase ATP13A2/ PARK9 function induces general lysosomal deficiency and leads to Parkinson disease neurodegeneration. Proc Natl Acad Sci U S A 2012;109:9611e6. [63] Park JS, Blair NF, Sue CM. The role of ATP13A2 in Parkinson’s disease: clinical phenotypes and molecular mechanisms. Mov Disord 2015;30:770e9. [64] Park JS, Sue CM. Hereditary parkinsonism-associated genetic variations in PARK9 locus lead to functional impairment of ATPase type 13A2. Curr Protein Pept Sci 2017;18: 725e32. [65] Morgan NV, Westaway SK, Morton JE, et al. PLA2G6, encoding a phospholipase A2, is mutated in neurodegenerative disorders with high brain iron. Nat Genet 2006;38: 752e4. [66] Paisan-Ruiz C, Bhatia KP, Li A, et al. Characterization of PLA2G6 as a locus for dystonia-parkinsonism. Ann Neurol 2009;65:19e23. [67] Sina F, Shojaee S, Elahi E, Paisa´n-Ruiz C. R632W mutation in PLA2G6 segregates with dystonia-parkinsonism in a consanguineous Iranian family. Eur J Neurol 2009;16: 101e4. [68] Yoshino H, Tomiyama H, Tachibana N, et al. Phenotypic spectrum of patients with PLA2G6 mutation and PARK14-linked parkinsonism. Neurology 2010;75:1356e61. [69] Chu YT, Lin HY, Chen PL, Lin CH. Genotype-phenotype correlations of adult-onset PLA2G6-associated Neurodegeneration: case series and literature review. BMC Neurol 2020;20:101. [70] Di Fonzo A, Dekker MC, Montagna P, et al. FBXO7 mutations cause autosomal recessive, early-onset parkinsonian-pyramidal syndrome. Neurology 2009;72:240e5. [71] Luo LZ, Xu Q, Guo JF, et al. FBXO7 gene mutations may be rare in Chinese early-onset Parkinsonism patients. Neurosci Lett 2010;482:86e9. [72] Yalcin-Cakmakli G, Olgiati S, Quadri M, et al. A new Turkish family with homozygous FBXO7 truncating mutation and juvenile atypical parkinsonism. Park Relat Disord 2014;20:1248e52. [73] Zhao T, De Graaff E, Breedveld GJ, et al. Loss of nuclear activity of the FBXO7 protein in patients with parkinsonian-pyramidal syndrome (PARK15). PLoS One 2011;6:e16983. [74] Burchell VS, Nelson DE, Sanchez-Martinez A, et al. The Parkinson’s disease-linked proteins Fbxo7 and Parkin interact to mediate mitophagy. Nat Neurosci 2013;16:1257e65. [75] Zhou ZD, Xie SP, Sathiyamoorthy S, et al. F-box protein 7 mutations promote protein aggregation in mitochondria and inhibit mitophagy. Hum Mol Genet 2015;24:6314e30. [76] Zhou ZD, Lee JCT, Tan EK. Pathophysiological mechanisms linking F-box only protein 7 (FBXO7) and Parkinson’s disease (PD). Mutat Res 2018;778:72e8.

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C H A P T E R

2 Parkinson’s pharmacological therapy Pharmacological treatment of Parkinson’s disease is palliative because it does not cure the underlying causes that induce symptoms. The 1957 discovery that motor symptoms are related to the loss of dopaminergic neurons containing neuromelanin has been very important in pharmacological treatment of the disease. The drop in dopamine levels caused by degeneration of the nigrostriatal system generates an imbalance between dopamine levels with respect to other neurotransmitters such as glutamate and GABA. This concept led to the development of a therapy to restore dopamine levels using its precursor L-dihydroxyphenylalanine (Ldopa). In 1967 L-dopa was introduced in the treatment of Parkinson’s disease, and to this day it remains a star among drug treatments even though its chronic use induces dyskinesia. The pharmacological treatments of Parkinson’s disease are classified in the rest of this chapter.

Dopaminergic drugs L-dopa

Since 1967, L-dopa has been the gold-standard drug for treating Parkinson’s disease because it produces shocking positive effects over a period of 4 to 6 years of chronic treatment during which patients recover many of their motor deficits. The synthesis of dopamine in dopaminergic neurons is catalyzed by aromatic amino acid decarboxylase that catalyzes the decarboxylation of L-dopa to dopamine and CO2. The problem is that L-dopa decarboxylation to dopamine is not a reaction exclusive to the braindthe peripheral nervous system also catalyzes L-dopa conversion to

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dopamine. L-dopa metabolism outside the central nervous system generates a marked difference in dose-to-dose plasma concentration, which induces the motor fluctuations that are a complication of treatment with Ldopa. Therefore, L-dopa is administered together with inhibitors of catecholo-methyltransferase such as entacapone or an aromatic amino acid decarboxylase such as carbidopa and benserazide to achieve the therapeutic concentration in the brain. It has been reported that the catechol-omethyl-transferase inhibitor opicapone in combination with L-dopa is a good alternative to control motor fluctuations in Parkinson’s disease [1]. However, chronic treatment with L-Dopa generates severe side effects such as dyskinesia. L-DOPA-induced dyskinesia is brought about by a loss of synaptic depotentiation, which is an incapacity to converse previously induced long-term depression [2]. Other side effects of L-dopa include “wearing off” or “oneoff” fluctuations as well as disabilities in posture, gait, speech, and balance. Cognitive impairment, hallucination, and orthostatic hypotension have also been reported [3e6]. Dopamine replacement therapy in Parkinson’s disease induces impulse control disorders in which some patients present with pathological behaviors such as compulsive eating, gambling, shopping, and disinhibited sexual behaviors [7].

Dopamine agonists Parkinson’s disease treatment with dopaminergic drugs includes dopamine agonists such as pergolide, bromocriptine, pramipexole, cabergoline, ropinirole, rotigotine, dihydroergocryptine mesylate, piribedil, and apomorphine. Treating Parkinson’s disease motor symptoms with dopamine agonists is less effective than L-dopa treatment. Postsynaptic dopamine receptors are stimulated by dopamine agonists [8,9]. Chronic treatment of Parkinson’s disease with dopamine induces a decrease in striatal dopamine receptor type-2 in both the caudate and the putamen. L-dopa treatment does not induce a decrease in striatal dopamine receptor type-2 [10]. A study on working memory function was performed with the D1þD2 receptor agonist pergolide and the D2þD3 receptor agonist pramipexole. Visual-object, visual-spatial, and verbal working memory was studied in 19 “de novo” Parkinson’s disease patients. In patients with low performance, all work-memory tasks improved when pergolide and pramipexole were administered, but no effect was observed in patients with high performance [11]. A study with the aim to determine whether pramipexole, with more affinity for the dopamine D3 receptor, was more effective than pergolide in treating tremor in Parkinson’s disease showed no significative difference between the two treatments [12].

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Monoamine oxidase inhibitors The aim of replacement therapy with L-dopa is to reach a therapeutic concentration in the brain, and therefore, L-dopa is administered with catechol-o-methyltransferase or aromatic amino acid decarboxylase inhibitors. Another way to increase the level of dopamine is to inhibit dopamine degradation catalyzed by monoamine oxidase-B, which converts dopamine to 3,4-dihydroxyphenylacetaldehyde, ammonia, and hydrogen peroxideda potent oxidizing agent and the precursor of the harmful hydroxyl radical [13]. Based on this idea, monoamine oxidase B inhibitors were developed as a new therapeutic alternative. Twenty-seven publications were used to perform a meta-analysis on monoamine oxidase B inhibitors safinamide, selegiline, and rasagiline. Their effects as monotherapy or together with dopamine agonist or Ldopa were compared. Evaluated on the Unified Parkinson’s Disease Rating Scale, these monoamine oxidase inhibitors obtained an improvement in treatment when used as monotherapy and in combination with Ldopa [14]. The first selective irreversible monoamine oxidase B inhibitor used in Parkinson’s disease therapy was L-deprenyl (selegiline) [13]. Selegiline is used in early Parkinson’s disease as monotherapy or together with L-dopa in more advanced stages of the disease. Selegiline treatment exhibited symptomatic benefits such as a delay in L-dopa therapy initiation and a decrease in disability [15]. The monoamine oxidase B inhibitor safinamide is a reversible and selective inhibitor with glutamatergic and dopaminergic properties. In Europe, it is approved for use in Parkinson’s disease with mid- to latestage fluctuations and in combination therapy with stable L-dopa doses. In placebo-controlled clinical trials with Parkinson’s disease patients with motor fluctuations, safinamide increased the daily “on” time without dyskinesia [16]. It has been reported that safinamide tolerability, safety, and clinical handling are better than those of L-dopa or dopamine agonists. Safinamide reduces L-dopa doses and dopamine agonists during extensive treatment of Parkinson’s disease [17]. Rasagiline is an irreversible inhibitor of monoamine oxidase B that is a bifunctional compound because it exerts a neuroprotective-antiapoptotic effect in Parkinson’s disease as a monoamine oxidase inhibitor, but rasagiline also has a functional group that acts as a cholinesterase inhibitor [18]. A clinical trial focusing on early treatment of Parkinson’s disease with 1 mg rasagiline induced improvements, but a dose of 2 mg did not provide benefits [19]. Therapy with 1 mg rasagiline provides an improvement as determined by the Unified Parkinson’s Disease Rating Scale score but is associated with a low incidence of adverse behavioral and cognitive events. A 2 mg rasagiline dose does not have beneficial effects [20].

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Anticholinergic drugs Cholinergic degeneration is involved in cognitive and gait impairments, rapid eye movement sleep disturbances, and psychosis, among other symptoms [21]. Anticholinergic drugs reestablish the balance between the dopamine and acetylcholine systems in the striatum by blocking the postsynaptic muscarinic receptors to antagonize hyperactive cholinergic transmission. Anticholinergic compounds were the first pharmacological treatment in Parkinson’s disease and include biperiden, benztropine, orphenadrine, diphenhydramine, procyclidine, ethopropazine, and trihexyphenidyl [22]. Anticholinergic drugs are used for the treatment of cognitive decline and gait dysfunction caused by deficits in cholinergic transmission. A meta-analysis that included 941 Parkinson’s disease patients showed that anticholinergic drugs were effective in cognitive impairment treatment in patients with Parkinson’s disease [23]. A study on cholinesterase inhibitors that included 10 trials showed that cholinesterase inhibitors increase cognitive function in Parkinson’s disease patients suffering from cognitive impairment and dementia [24]. A meta-analysis of Parkinson’s disease patients with cognitive impairment, dementia, and dementia with Lewy bodies showed that cholinesterase inhibitors improved cognitive function [25]. A study performed with 6-hydroxydopamine hemi-lesioned rats to investigate the effect of acetylcholine muscarinic receptor antagonists (tropicamide, telenzepine, and PD-102807) on L-dopa-induced dyskinesia showed that their intrastriatal administration alleviated dyskinesia and inhibited striatal glutamate and nigral GABA release. The authors suggest that M1 acetylcholine muscarinic receptors antagonists facilitate dyskinesia, while the potentiation of striatal M4 acetylcholine muscarinic receptor transmission attenuates dyskinesia [26]. The anticholinergic drug trihexyphenidyl has been used in Parkinson’s disease for spasms, tremors, weak muscle control, and stiffness. Trihexyphenidyl is also used to treat drug-induced parkinsonism resulting from use of the central nervous system drugs chlorpromazine, haloperidol, and fluphenazine [27]. A study on the effect of antiparkinsonian anticholinergic drugs on dementia risk was performed using the Taiwan National Health Insurance Research Database, which included around 30,000 patients newly diagnosed with Parkinson’s disease. A significant increase in dementia risk was observed in patients treated with anticholinergic drugs for six months or more [28]. Rivastigmine inhibits cholinesterase, increases cholinergic tone, and is an effective drug against dementia [16]. Parkinson’s disease patient falls have been associated with loss of projections in the cholinergic system as well as cognitive impairment. Donepezil, an acetylcholinesterase inhibitor, together with idalopirdine, a

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5HT6 receptor antagonist, decrease fall frequency in dual-lesioned rats. The administration of acetylcholine esterase, butyrylcholinesterase inhibitor rivastigmine, and idalopirdine significantly improved the performance of dual-lesioned rats [29]. Experimental studies performed in rats to study the role of the cholinergic system of the retina in corporal movement showed that anticholinergic systems in the retina can control movement [30].

Other pharmacological treatments Melatonin secretion gradually diminishes with advanced age, and in Parkinson’s disease this decrease is related to an increase in oxidative stress and degeneration of dopaminergic neurons. Pinealectomized and dopamine-deficient pinealectomized rats were treated with three antiparkinsonian drugs in a study on psychiatric and motor functiondthe results showed that altered pineal function plays an important role in Parkinson’s disease symptoms [31]. The pineal hormone melatonin’s physiological functions in the brain include suppressing neuroinflammation, regulating circadian rhythms, and inhibiting biomolecular oxidation and oxidative stress [32]. The circadian role in nigrostriatal dopamine function in Parkinson’s disease was studied by administering antiparkinsonian drugs into the vitreus humor, which is adjacent to the retina. L-dopa administration improved motor symptoms in late degeneration stages, while administration of ML-23, a melatonin receptor antagonist, improved motor symptoms in the early stages of Parkinson’s disease and in the dark phase of the cycle [33]. The treatment of autonomic dysfunction in Parkinson’s disease includes drugs for constipation such as laxatives, prokinetic agents, and prucalopride in addition to a high-fiber diet. Midodrine and droxidopa are promising new drugs in the treatment of orthostatic hypotension. The treatment of bladder dysfunction includes b-agonists and the selective b-3 agonist mirabegron. The treatment of sialorrhea is with the botulinum toxin [34].

New targets and disease-modifying drugs for Parkinson’s disease treatment in phase 3 Adenosine antagonists A new target for Parkinson’s disease treatment is adenosine receptors. Adenosine induces a multitude of physiopathological effects and regulates cardiovascular, central nervous, peripheral, and immune systems by

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targeting four G-protein-coupled receptors: A1, A2A, A2B, and A3. The source of intracellular adenosine is dephosphorylation ATP, ADP, and AMP generated during cellular metabolism [35]. The adenosine A2A receptor subtype is expressed solely in the striatum. Adenosine A2A receptor is coexpressed with dopamine D2 receptor and enkephalin and is expressed principally by neurons projecting to the globus pallidus. However, adenosine A2A receptors are sporadically expressed by neurons projecting to the substantia nigra with dopamine D1 receptor and substance P [36]. Adenosine A2A receptors are localized to the basal ganglia in the indirect output pathway, providing the opportunity to modulate output from the striatum. Adenosine A2A antagonists have the capacity to modulate basal ganglia neurotransmission, improving motor function in preclinical models of Parkinson’s disease based on exogenous neurotoxins. These positive results in preclinical models suggested adenosine A2A antagonists as a potential drug for Parkinson’s disease treatment without inducing dyskinesia [37]. Caffeine chronic treatment prevents nigrostriatal dopaminergic neuron methamphetamine-induced neurotoxicity. Caffeine is a nonselective adenosine antagonist that inhibits both A1 and A2 adenosine receptors. However, caffeine did not prevent neurotoxic effects of 1-methyl-4phenyl-1,2,3,6- tetrahydropyridine [38]. An increase in locomotor activity was observed in 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine-treated animals exposed to KW-6002 ((E)-1,3diethyl-8-(3,4-dimethoxystyryl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione), a selective adenosine A2A antagonist, suggesting that adenosine A2A receptor may represent an opportunity for the treatment of Parkinson’s disease [39]. A study on the effect of the selective adenosine A2A receptor antagonist KW-6002 in combination with L-dopa and selective D1 or D2 dopamine receptor agonists showed that administration of KW-6002 in combination with a low dose of L-dopa induced an additive effect that improved motor impairment in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated animals. A similar effect was observed when 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine-treated animals were exposed to the combination of KW-6002 and the D1 receptor agonist SKF80723 or the selective dopamine D2 receptor agonist quinpirole [40]. Adenosine A2A receptor knockout mice treated with 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine exhibited less degeneration and improved motor activity. Adenosine A2A receptors are specifically located with any dopamine D2 receptors [41]. Dopamine receptor D2 knockout induces impairment of locomotion and coordinated movements, but adenosine receptor A2A reverses these effects, suggesting that dopamine receptor D2 and adenosine receptor A2A have independent and antagonistic activities in controlling motor and neuronal functions in the basal ganglia [42].

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Rotenone-treated mice increased adenosine A2A receptor gene expression. Administration of adenosine A2A antagonist ZM241385 in combination with curcumin and niacin restores the level of adenosine A2A receptor gene expression [43]. Istradefylline, an adenosine A2A antagonist, decreases “off” time in Parkinson’s disease patients [37]. The score on the Unified Parkinson’s Disease Rating Scale Part III suggested an improvement in the motor function of Parkinson’s disease patients and increased time without dyskinesia. It was approved for Parkinson’s disease treatment in Japan in 2013 and in the United States in 2019 [44]. The possible benefit of using istradefylline to treat gait difficulties in Parkinson’s disease has been evaluated in 31 Parkinson’s disease patients in a multicenter study. Patients treated with istradefylline showed a significant improvement in postural stability, gait, and freezing of gait [45].

Glucagon-like peptide-1 receptor agonists Glucagon-like peptide-1 receptor agonists stimulate receptors in the pancreas to release insulin to maintain the glucose level in the blood. Glucagon-like peptide-1 receptors are also expressed in the brain, and it has been observed that insulin signaling in the brain plays a role in neuronal repair, metabolism, and synaptic efficiency. However, in Parkinson’s disease, brain insulin signaling is desensitized. Clinical studies on the neuroprotective role of glucagon-like peptide-1 receptors in Parkinson’s disease have been performed. A double-blind study was performed with exenatide, a glucagon-like peptide-1 receptor agonist versus placebo, with 62 participants who received exenatide or placebo for 48 weeks. A single-blind trial was performed with 45 participants who received exenatide versus no additional treatment randomized for 12 months. The authors concluded that exenatide may improve motor impairment in Parkinson’s disease [46]. The possible increase in Akt signaling and in braineinsulin pathways was studied in participants in the Exenatide-Parkinson’s disease trial. Increased tyrosine phosphorylation of insulin receptor substrate-1 and expression of downstream substrates such as total Akt and phosphorylated mechanistic target of rapamycin (mTOR) were observed at 48 weeks in patients treated with exenatide [47]. Neuroprotective effects in preclinical models of Parkinson’s have been observed with the incretin glucose-dependent insulinotropic polypeptide and glucagon-like peptide. The dual agonist DA-JC4, an agonist of both glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1 and receptor, improved motor dysfunction, reduced mitochondrial stress, and protected dopaminergic neurons induced by rotenone in rats. The protective effect of DA-JC4 was dependent on the

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AKT/JNK signal pathway because the AKT inhibitor or JNK activator abolished the DA-JC4-induced protective effect [48]. The dual agonist DA-JC1 was also neuroprotective in a preclinical model of Parkinson’s disease, such as the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model and the 6-hydroxydopamine rat model. DA-JC1 increased the levels of pAkt/CREB cell signaling and the growth factor GDNF. DA-JC1 also activated the autophagy marker Beclin-1 in a 6-hydroxydopamine preclinical model of Parkinson’s disease [49].

Memantine The ability of memantine, an N-methyl-D-aspartate receptor antagonist, to inhibit alpha-synuclein cellecell transmission is investigated in a phase 3 study using magnetic resonance imaging (ClinicalTrials.gov Identifier: NCT03858270). Lewy bodies are protein aggregates mainly composed by phosphorylated alpha-synuclein. It has been proposed that the formation of Lewy bodies plays a key role in the development and propagation of the disease from different regions [50]. A randomized controlled study of memantine versus placebo in the United Kingdom, Norway, and Sweden was performed with 72 patients with Parkinson’s disease dementia and dementia Lewy bodies and showed improved speed by the memantine group on attentional tasks [51]. A clinical study with 199 patients with dementia Lewy bodies and mild to moderate Parkinson’s disease dementia from Germany, the United Kingdom, Austria, Spain, France, Greece, Italy, and Turkey showed an improvement in neuropsychiatric-inventory scores in patients with dementia Lewy bodies but not in Parkinson’s disease dementia [52]. A study with 30 Parkinson’s disease patients with gait disturbances was performed to evaluate the efficacy of memantine. Those patients who received memantine in combination with antiparkinsonian drugs presented an improvement in their gait cycle phase ratios in comparison with control group [53]. It has been shown that the noncompetitive N-methyl-D-aspartate receptor antagonist decreases levodopa-induced dyskinesia and parkinsonian symptoms in 6-hydroxydopamine-lesioned mice treated with L-dopa [54]. A study of the role of glutamate in dyskinesia in Parkinson’s disease patients used N-methyl-3(thiomethylphenyl)cyanamide positronemission tomography as a marker of activated N-methyl-D-aspartate receptor ion channels. Dyskinetic patients in the “on” condition presented higher activation of N-methyl-D-aspartate receptor ion channels in the putamen, caudate, and precentral gyrus in comparison with patients without dyskinesia. The authors suggest that Parkinson’s disease patients with dyskinesia may have abnormal glutamatergic transmission in motor areas following levodopa administration [55].

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Memantine induces a significant reduction of L-dopa-induced dyskinesia in 6-hydroxydopamine-lesioned rats during the first few days of administration, but memantine’s antidyskinesia effect disappeared after the fifth administration. The authors suggest that an increase in the synaptic GluN2A/GluN2B ratio at striatal synaptic membranes may be associated with loss of the anti-dyskinetic effect of memantine, suggesting that Nmethyl-D-aspartate receptor inhibition may be only transiently effective against L-dopa-induced dyskinesia in Parkinson’s disease patients [56].

Ganoderma lucidum Ganoderma lucidum is a herb with nutritional and medicinal properties. Ganoderma has been used for thousands of years in traditional Chinese medicine, and preclinical phase 2 studies have demonstrated that Ganoderma is well tolerated and safe, and it improved symptoms when used with L-dopa. A multicenter, double-blind, randomized, delayed-start, placebo-controlled phase 3 clinical study with 288 untreated Parkinson’s disease patients is in progress to evaluate Ganoderma effects on modifying disease (ClinicalTrials.gov Identifier: NCT03594656). Ganoderma is a neuroprotective agent from oxidative stress. In studies with mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine as a preclinical model for Parkinson’s disease, Ganoderma increased tyrosine hydroxylase expression and improved locomotor performance. In studies with a cell line treated with the 1-methyl-4-phenylpyridinium ion, Ganoderma protected against mitochondrial dysfunction, PINK1/Parkin and AMPK/mTOR signaling pathways, cytochrome C release, and activation of caspase-9 and caspase-3 [57]. In a study with microglia activated with the 1-methyl-4-phenylpyridinium ion, Ganoderma prevented the formation of cytotoxic and proinflammatory factors such as interleukin 1b, nitric oxide, and tumor necrosis factor-alpha [58].

Drugs in clinical trials A long list of clinical trials for Parkinson’s disease are in progress in different stages (phases 1, 2, and 3), and we can classify the drugs as either disease-modifying drugs that can change the course of the disease by inhibiting or slowing its development or drugs that treat symptoms of the disease but do not change the course of the disease. A study of clinical trials for Parkinson’s disease showed that 145 clinical trials are in progress, but only 39% are clinical studies with disease-modifying drugs. Only 3 clinical trials are in phase 3 for disease-modifying drugs (exenatide, memantine, and Ganoderma), while 36 drugs are in phase 2 and 18 drugs are in phase 2. The majority of clinical studies for Parkinson’s disease are performed with drugs for disease symptoms, with the majority focused

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on motor symptoms and the rest on nonmotor symptoms such as Parkinson’s disease dementia, neurogenic orthostatic hypertension, general nonmotor symptoms, pain, anxiety, constipation, impulse control disorders, Parkinson’s disease psychosis, hallucinations, depression, fatigue, and neuropsychiatric symptoms [59]. Several clinical trials in phase 2 are evaluating disease-modifying drugs related to alpha-synuclein. In general, it is accepted that alpha-synuclein plays a role in the degeneration of nigrostriatal neurons. Alpha-synuclein aggregation to neurotoxic oligomers or fibrils that generates alphasynuclein deposits in Lewy bodies seems to play a central role in the disease. A clinical study using monoclonal antibodies against alpha-synuclein aggregates is in progress (ClinicalTrials.gov Identifier: NCT03318523). Cinpanemab (BIIB054) antibodies were successfully tested in a phase 1 trial that demonstrated it to be safe and well tolerated [60]. However, in the last 2020 investors report, the Biogen company has reported that the development of Cinpanemab has been discontinued due to the negative results of this clinical phase (https://investors.biogen.com/static-files/e8fb597e5179-4279-8ba1-48df48152d86). Mannitol induces alpha-synuclein clearance in an mThy1-human alphasynuclein transgenic animal model, inhibiting alpha-synuclein accumulation. Mannitol at lower concentrations prevents alpha-synuclein fibril formation, while at higher concentration it reduces tetramers and highmolecular-weight oligomer formation [61]. A phase 2 clinical study on mannitol is in progress to test tolerability, safety, and efficacy. Mannitol or placebo (dextrose) in escalating doses will be tested in Parkinson’s disease patients for 36 weeks (ClinicalTrials.gov Identifier: NCT03823638). Ambroxol is in a phase 2 clinical study for Parkinson’s disease dementia to improve both motor and cognitive symptoms. Ambroxol increases the level of beta-glucocerebrosidase, which decreases alpha-synuclein levels, improving cognition in animal models (ClinicalTrials.gov Identifier: NCT02914366). The level of dopamine transporter and tyrosine hydroxylase, mitochondrial complex I activity, and glucocerebrosidase activity were restored in rats treated with 6-hydroxydopamine, suggesting that ambroxol induces the regeneration of dopaminergic neurons [62].

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dementia, and dementia with Lewy bodies: systematic review with meta-analysis and trial sequential analysis. J Neurol Neurosurg Psychiatry 2015;86:135e43. Meng YH, Wang PP, Song YX, Wang JH. Cholinesterase inhibitors and memantine for Parkinson’s disease dementia and Lewy body dementia: a meta-analysis. Exp Ther Med 2019;17:1611e24. Brugnoli A, Pisano` CA, Morari M. Striatal and nigral muscarinic type 1 and type 4 receptors modulate levodopa-induced dyskinesia and striato-nigral pathway activation in 6-hydroxydopamine hemilesioned rats. Neurobiol Dis 2020;144:105044. Jilani TN, Sabir S, Sharma S. Trihexyphenidyl. In: StatPearls. Treasure Island (FL): StatPearls Publishing; July 10, 2020. Hong CT, Chan L, Wu D, Chen WT, Chien LN. Antiparkinsonism anticholinergics increase dementia risk in patients with Parkinson’s disease. Park Relat Disord 2019;65: 224e9. Koshy Cherian A, Kucinski A, Wu R, de Jong IEM, Sarter M. Co-treatment with rivastigmine and idalopirdine reduces the propensity for falls in a rat model of falls in Parkinson’s disease. Psychopharmacology 2019;236:1701e15. Willis GL, Freelance CB. The effect of intravitreal cholinergic drugs on motor control. Behav Brain Res 2018;339:232e8. Willis GL. The therapeutic effects of dopamine replacement therapy and its psychiatric side effects are mediated by pineal function. Behav Brain Res 2005;160:148e60. Chen D, Zhang T, Lee TH. Cellular mechanisms of melatonin: insight from neurodegenerative diseases. Biomolecules 2020;10:E1158. Willis GL. Intraocular microinjections repair experimental Parkinson’s disease. Brain Res 2008;1217:119e31. Kulshreshtha D, Ganguly J, Jog M. Managing autonomic dysfunction in Parkinson’s disease: a review of emerging drugs. Expet Opin Emerg Drugs 2020;25:37e47. Borea PA, Gessi S, Merighi S, Vincenzi F, Varani K. Pharmacology of adenosine receptors: the state of the art. Physiol Rev 2018;98:1591e625. Schiffmann SN, Vanderhaeghen JJ. Adenosine A2 receptors regulate the gene expression of striatopallidal and striatonigral neurons. J Neurosci 1993;13:1080e7. Jenner P, Mori A, Hauser R, Morelli M, Fredholm BB, Chen JF. Adenosine, adenosine A 2A antagonists, and Parkinson’s disease. Park Relat Disord 2009;15:406e13. Delle Donne KT, Sonsalla PK. Protection against methamphetamine-induced neurotoxicity to neostriatal dopaminergic neurons by adenosine receptor activation. J Pharmacol Exp Therapeut 1994;271(3):1320e6. Kanda T, Tashiro T, Kuwana Y, Jenner P. Adenosine A2A receptors modify motor function in MPTP-treated common marmosets. Neuroreport 1998;9:2857e60. Kanda T, Jackson MJ, Smith LA, et al. Combined use of the adenosine A(2A) antagonist KW-6002 with L-dopa or with selective D1 or D2 dopamine agonists increases antiparkinsonian activity but not dyskinesia in MPTP-treated monkeys. Exp Neurol 2000;162: 321e7. Salmi P, Chergui K, Fredholm BB. Adenosine-dopamine interactions revealed in knockout mice. J Mol Neurosci 2005;26:239e44. Aoyama S, Kase H, Borrelli E. Rescue of locomotor impairment in dopamine D2 receptor-deficient mice by an adenosine A2A receptor antagonist. J Neurosci 2000;20: 5848e52. Motawi TK, Sadik NAH, Hamed MA, Ali SA, Khalil WKB, Ahmed YR. Potential therapeutic effects of antagonizing adenosine A2A receptor, curcumin and niacin in rotenone-induced Parkinson’s disease mice model. Mol Cell Biochem 2020;465:89e102. Paton DM. Istradefylline: adenosine A2A receptor antagonist to reduce “OFF” time in Parkinson’s disease. Drugs Today 2020;56:125e34.

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[45] Iijima M, Orimo S, Terashi H, et al. Efficacy of istradefylline for gait disorders with freezing of gait in Parkinson’s disease: a single-arm, open-label, prospective, multicenter study. Expet Opin Pharmacother 2019;20:1405e11. [46] Mulvaney CA, Duarte GS, Handley J, et al. GLP-1 receptor agonists for Parkinson’s disease. Cochrane Database Syst Rev 2020;7(7):CD012990. [47] Athauda D, Gulyani S, Karnati HK, et al. Utility of neuronal-derived exosomes to examine molecular mechanisms that affect motor function in patients with Parkinson disease: a secondary analysis of the exenatide-PD trial. JAMA Neurol 2019;76:420e9. [48] Li T, Tu L, Gu R, et al. Neuroprotection of GLP-1/GIP receptor agonist via inhibition of mitochondrial stress by AKT/JNK pathway in a Parkinson’s disease model. Life Sci 2020;256:117824. [49] Jalewa J, Sharma MK, Gengler S, Ho¨lscher C. A novel GLP-1/GIP dual receptor agonist protects from 6-OHDA lesion in a rat model of Parkinson’s disease. Neuropharmacology 2017;117:238e48. [50] Braak H, Del Tredici K, Ru¨b U, de Vos RA, Jansen Steur EN, Braak E. Staging of brain pathology related to sporadic Parkinson’s disease. Neurobiol Aging 2003;24:197e211. [51] Aarsland D, Ballard C, Walker Z, et al. Memantine in patients with Parkinson’s disease dementia or dementia with Lewy bodies: a double-blind, placebo-controlled, multicentre trial. Lancet Neurol 2009;8:613e8. [52] Emre M, Tsolaki M, Bonuccelli U, et al. Memantine for patients with Parkinson’s disease dementia or dementia with Lewy bodies: a randomised, double-blind, placebocontrolled trial. Lancet Neurol 2010;9:969e77. [53] Miliukhina IV, Gracheva EV. Vliyanie Akatinola Memantina na narusheniya khod’by na razvernutykh stadiyakh bolezni Parkinsona [The use of Akatinol Memantine in the treatment of gait disturbances in Parkinson’s disease]. Zh Nevrol Psikhiatr Im S S Korsakova 2020;120:27e33. [54] Ogawa M, Zhou Y, Tsuji R, Kasahara J, Goto S. Intrastriatal memantine infusion dampens levodopa-induced dyskinesia and motor deficits in a mouse model of hemiparkinsonism. Front Neurol 2019;10:1258. [55] Ahmed I, Bose SK, Pavese N, et al. Glutamate NMDA receptor dysregulation in Parkinson’s disease with dyskinesias. Brain 2011;134:979e86. [56] Tronci E, Fidalgo C, Zianni E, et al. Effect of memantine on L-dopa-induced dyskinesia in the 6-OHDA-lesioned rat model of Parkinson’s disease. Neuroscience 2014;265: 245e52. [57] Ren ZL, Wang CD, Wang T, et al. Ganoderma lucidum extract ameliorates MPTPinduced parkinsonism and protects dopaminergic neurons from oxidative stress via regulating mitochondrial function, autophagy, and apoptosis. Acta Pharmacol Sin 2019;40:441e50. [58] Ding H, Zhou M, Zhang RP, Xu SL. Sheng Li Xue Bao 2010;62:547e54. [59] McFarthing K, Buff S, Rafaloff G, Dominey T, Wyse RK, Stott SRW. Parkinson’s disease drug therapies in the clinical trial pipeline: 2020. J Parkinsons Dis 2020;10:757e74. [60] Kuchimanchi M, Monine M, Kandadi Muralidharan K, Woodward C, Penner N. Phase II dose selection for alpha synuclein-targeting antibody cinpanemab (BIIB054) based on target protein binding levels in the brain. CPT Pharmacometrics Syst Pharmacol 2020;9: 515e22. [61] Shaltiel-Karyo R, Frenkel-Pinter M, Rockenstein E, et al. A blood-brain barrier (BBB) disrupter is also a potent a-synuclein (a-syn) aggregation inhibitor: a novel dual mechanism of mannitol for the treatment of Parkinson disease (PD). J Biol Chem 2013;288: 17579e88. [62] Mishra A, Krishnamurthy S. Neurorestorative effects of sub-chronic administration of ambroxol in rodent model of Parkinson’s disease. Naunyn-Schmiedeberg’s Arch Pharmacol 2020;393:429e44.

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3 Dopamine synthesis 3-Hydroxytyramine (dopamine) is a neurotransmitter of dopaminergic neurons; in the central nervous system, it plays an essential role in body motor functions. Dopamine formation is part of catecholamine synthesis in which the amino acid tyrosine is finally converted to epinephrine (adrenaline). Dopamine synthesis occurs mainly in dopaminergic systems of the central nervous system, but dopamine is synthetized in other tissues as well, such as the enteric nervous system where tyrosine hydroxylase-immunoreactivity has been observed [1]. Dopamine synthesis includes two reactions, the hydroxylation of the amino acid tyrosine to L-3,4-dihydroxyphenylalanine (L-dopa) catalyzed by tyrosine hydroxylase and the decarboxylation of L-dopa to dopamine catalyzed by aromatic amino acid decarboxylase.

Tyrosine hydroxylase The conversion of tyrosine to L-3,4-dihydroxyphenylalanine (L-dopa) is the first step and the rate-limiting reaction in catecholamine synthesis that is catalyzed by the enzyme tyrosine hydroxylase. Tyrosine hydroxylase, tryptophan hydroxylase, and phenylalanine hydroxylase constitute a family of monooxygenases that use tetrahydropterin as substrate. Tyrosine hydroxylase is a homotetramer composed of four identical subunits having three domains. The C-terminal contains a short alpha helix domain that plays a role in its tetramerization. The N-terminal comprises 150 amino acids and is a regulatory domain, while the central domain is the catalytic domain [2]. The conversion of tyrosine to L-dopa catalyzed by tyrosine hydroxylase requires 5,6,7,8-tetrahydrobiopterin and oxygen. Purification of rat tyrosine hydroxylase showed that this is a monooxygenase that contains iron without the heme group [3]. Fig. 3.1. The tyrosine hydroxylase gene is composed of 14 exons in humans that express four isoforms using the same gene but with alternative mRNA splicing. Tyrosine hydroxylase isoform 1 enzymatic activity is regulated

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FIGURE 3.1 L-dopa synthesis. Synthesis of L-dopa. The amino acid tyrosine is hydroxylated to L-dopa in a reaction catalyzed by tyrosine hydroxylase. This reaction requires tyrosine, dioxygen, and 5,6,7,8-tetrahydrobiopterin, and the products are L-dopa, water, and tetrahydrobiopterin-4a-carbinolamine. Tyrosine hydroxylase is a monooxygenase that contains iron without the heme group, although the role of iron in this reaction is not completely clear.

by phosphorylation or dephosphorylation of serines 19 and 40. Phosphorylation of other isoforms also plays a role in tyrosine hydroxylase regulation [4]. Tyrosine hydroxylase is phosphorylated by cAMPdependent protein kinase A, which influences the inhibitory effect of L-dopa and dopamine. Phosphorylation of tyrosine hydroxylase isoform 1 increases twofold the dissociation constant of L-dopa to the enzyme. Phosphorylation decreases enzyme affinity to dopamine [5]. Tyrosine hydroxylase phosphorylation of serine-40 is catalyzed by cyclic AMP-dependent protein kinase and the calmodulin-dependent multiprotein kinase and protein kinase C, while serine-19 is also phosphorylated by the calmodulin-dependent multiprotein kinase. Other phosphorylation of tyrosine hydroxylase occurs at serines 8 and 31. Interestingly, all phosphorylation occurs in the amino terminal region [6]. An in vitro study with tyrosine hydroxylase showed that the oxidation state of iron in the active site plays a central role in regulation of the enzyme. Anaerobic rapid freeze-quench EPR and anaerobic stopped-flow spectroscopy showed that tyrosine hydroxylase UV-absorbance changed after the addition of a reducing agent such as tetrahydrobiopterin, where Fe3þ is reduced to Fe2þ. No radical formation was detected, and reducing agents such as glutathione and ascorbate reduced iron but at a rate that was much slower [7]. Tyrosine hydroxylase exhibits high enzymatic activity in the pH range of 5.8e7.4, where epinephrine and norepinephrine decrease this activity. Tyrosine hydroxylase is inhibited by dopamine and activated in the presence of an ATP-generating system by the catalytic subunit of cyclic AMP-dependent protein kinase [8]. Inhibition of the ubiquitin-proteasomal protein degradation system (20S or 26S proteasome) results in accumulation of phosphorylated tyrosine hydroxylase [9]. A study on the possible role of ubiquitin-proteasome and lysosomal systems in tyrosine hydroxylase turnover was performed using proteasome and lysosome inhibitors. Inhibition of the proteasome system

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by lactacystin induces a significant increase in the tyrosine hydroxylase level, while inhibition of the lysosomal degradation system with bafilomycin did not change the tyrosine hydroxylase level [10]. Alpha-synuclein regulates dopamine synthesis by binding to and inhibiting tyrosine hydroxylase phosphorylation. An increase in the alphasynuclein level reduces the serine-40 phosphorylation that plays an important role in tyrosine hydroxylase activation and dopamine synthesis. Tyrosine hydroxylase phosphorylation and dephosphorylation are catalyzed by cyclic AMP-dependent protein kinase PKA and protein phosphatase. Alpha-synuclein overexpression did not affect the level of these enzymes, but the increase in alpha-synuclein expression was accompanied by an increase in protein phosphatase enzymatic activity. Protein phosphatase activity inhibition increased tyrosine hydroxylase serine-40 phosphorylation, but this occurred only when alpha-synuclein was overexpressed [11].

Aromatic amino acid decarboxylase Aromatic amino acid decarboxylase (EC 4.1.1.28) catalyzes the conversion of L-Dopa to dopamine and L-5-hydroxytryptophan to serotonin. This enzyme has 480 amino acids and is dimeric. Pyridoxal 50 -phosphate is the cofactor of aromatic amino acid decarboxylase involved in the decarboxylation of L-dopa. However, pyridoxal 50 -phosphate is highly reactive and can perform other reactions such as oxidative deamination, transamination, racemization, and elimination. It has further been reported that some pyridoxal 50 -phosphate-dependent enzyme decarboxylases can consume oxygen to convert an amino acid to a carbonyl compound [12]. Fig. 3.2. This enzyme is expressed in the brain and other tissues. The expression of aromatic amino acid decarboxylase in peripheral tissues constitutes a problem for L-dopa therapy in Parkinson’s disease because of the peripheral conversion of L-dopa to dopamine before it crosses the bloode brain barrier [13]. Two promoters and 16 exons have been reported in the rat aromatic Lamino acid decarboxylase gene. The cDNA sequence of pheochromocytoma

FIGURE 3.2 Dopamine synthesis. L-dopa is converted to dopamine in a reaction cata-

lyzed by aromatic amino acid decarboxylase where pyridoxal 50 -phosphate is the cofactor required for dopamine formation.

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and liver are different at the 50 -untranslated region. The third exon has two splicing acceptors specific to the first and second exons, which code the 50 untranslated sequence of the liver cDNA and pheochromocytoma [14]. Aromatic amino acid decarboxylase is inhibited by carbidopa and benserazide, which are administered together with L-dopa to Parkinson’s disease patients to prevent L-dopa conversion to dopamine before it crosses the bloodebrain barrier. The active metabolite of benserazide, trihydroxybenzylhydrazine, irreversibly binds both aromatic amino acid decarboxylase and pyridoxal 50 -phosphate-dependent enzymes, and free pyridoxal 50 -phosphate and pyridoxal 50 -phosphate-dependent enzymes [15]. Deficiency of aromatic amino acid decarboxylase is an extremely rare autosomal-recessive disorder that results in decreased synthesis of dopamine and other neurotransmitters. The symptoms of aromatic amino acid decarboxylase deficiency are hypotonia, movement disorders, autonomic dysfunction, and behavioral disorders [16]. It was reported that mRNA isoforms spliced with a new exon called exon 6a in normal and c.714þ4A > T lymphoblastoid cells. The proteins SRSF9 and SRSF6 and ciselements involved in the splicing of this mutated transcript were identified. Interestingly, antisense oligonucleotides restore the normal mRNA splicing and increase the level of aromatic amino acid decarboxylase protein [17]. Aromatic amino acid decarboxylase is one of the enzymes involved in norepinephrine synthesis, and lack of epinephrine release results in hypotension. Droxidopa, L-dihydroxyphenylserine, is decarboxylated by aromatic amino acid decarboxylase to finally generate norepinephrine. The administration of droxidopa to patients with hypotension in a clinical study with double-blind placebo-controlled trials and crossover showed an improvement in standing ability and amelioration of symptoms of orthostatic hypotension [18]. Dopamine synthesis occurs in the cytosol, where the enzymes tyrosine hydroxylase and aromatic amino acid decarboxylase are localized. It has been reported that dopamine in the cytosol in the presence of oxygen can be oxidized to neuromelanin, generating various neurotoxic ortho-quinones. It has been proposed that one of these ortho-quinones, aminochrome, is responsible for triggering the degenerative process in dopaminergic neurons containing neuromelanin that results in mitochondrial dysfunction, addition of alpha-synuclein to neurotoxic oligomers, protein degradation dysfunction of both the lysosomal and proteasomal systems, endoplasmic reticulum stress, neuroinflammation, and oxidative stress [19e21]. Interestingly, it has been reported that tyrosine hydroxylase and aromatic amino acid decarboxylase do not act alone in the cytosol, but rather, these enzymes are associated with vesicular monoamine transporter-2 localized in the membrane of monoaminergic vesicles [22]. Vesicular monoamine transporter-2 transports dopamine from the cytosol to monoaminergic vesicles, where dopamine accumulates for use in neurotransmission. This is very important because dopamine is completely stable inside monoaminergic vesicles, which have an acidic pH and prevent dopamine oxidation to neuromelanin (Fig. 3.3).

Aromatic amino acid decarboxylase

FIGURE 3.3

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Association of enzymes to a kind of complex to prevent free cytosolic dopamine. A kind of complex between tyrosine hydroxylase, aromatic amino acid decarboxylase, and vesicular monoamine transporter-2 is formed to prevent free dopamine in the cytosol. Tyrosine is converted to L-dopa in this complex and immediately converted to dopamine that is transported into the monoaminergic vesicle for neurotransmission by vesicular monoamine transporter-2, which is located in the membrane of these vesicles. This complex prevents the synthesis of free cytosolic dopamine that can oxidize to neuromelanin to generate neurotoxic ortho-quinones, of which aminochrome is the most neurotoxic.

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References [1] Wakabayashi K, Takahashi H, Takeda S, Ohama E, Ikuta F. Lewy bodies in the enteric nervous system in Parkinson’s disease. Arch Histol Cytol 1989;52(Suppl. l):191e4. [2] Fitzpatrick PF. Tetrahydropterin-dependent amino acid hydroxylases. Annu Rev Biochem 1999;68:355e81. [3] Ramsey AJ, Hillas PJ, Fitzpatrick PF. Characterization of the active site iron in tyrosine hydroxylase. Redox states of the iron. J Biol Chem 1996;271:24395e400. [4] Nagatsu T. Tyrosine hydroxylase: human isoforms, structure and regulation in physiology and pathology. Essays Biochem 1995;30:15e35. [5] Sura GR, Daubner SC, Fitzpatrick PF. Effects of phosphorylation by protein kinase A on binding of catecholamines to the human tyrosine hydroxylase isoforms [published correction appears in J Neurochem. 2004;90:1280] J Neurochem 2004;90:970e8. [6] Campbell DG, Hardie DG, Vulliet PR. Identification of four phosphorylation sites in the N-terminal region of tyrosine hydroxylase. J Biol Chem 1986;261:10489e92. [7] Frantom PA, Seravalli J, Ragsdale SW, Fitzpatrick PF. Reduction and oxidation of the active site iron in tyrosine hydroxylase: kinetics and specificity [published correction appears in Biochemistry. 2006;45(13):4338] Biochemistry 2006;45:2372e9. [8] Okuno S, Fujisawa H. A new mechanism for regulation of tyrosine 3-monooxygenase by end product and cyclic AMP-dependent protein kinase. J Biol Chem 1985;260: 2633e5. [9] Kawahata I, Tokuoka H, Parvez H, Ichinose H. Accumulation of phosphorylated tyrosine hydroxylase into insoluble protein aggregates by inhibition of an ubiquitinproteasome system in PC12D cells. J Neural Transm 2009;116:1571e8. [10] Congo Carbajosa NA, Corradi G, Verrilli MA, Guil MJ, Vatta MS, Gironacci MM. Tyrosine hydroxylase is short-term regulated by the ubiquitin-proteasome system in PC12 cells and hypothalamic and brainstem neurons from spontaneously hypertensive rats: possible implications in hypertension. PloS One 2015;10:e0116597. [11] Peng X, Tehranian R, Dietrich P, Stefanis L, Perez RG. Alpha-synuclein activation of protein phosphatase 2A reduces tyrosine hydroxylase phosphorylation in dopaminergic cells. J Cell Sci 2005;118:3523e30. [12] Bisello G, Longo C, Rossignoli G, Phillips RS, Bertoldi M. Oxygen reactivity with pyridoxal 5’-phosphate enzymes: biochemical implications and functional relevance. Amino Acids 2020;52:1089e105. [13] Bertoldi M. Mammalian dopa decarboxylase: structure, catalytic activity and inhibition. Arch Biochem Biophys 2014;546:1e7. [14] Hahn SL, Hahn M, Kang UJ, Joh TH. Structure of the rat aromatic L-amino acid decarboxylase gene: evidence for an alternative promoter usage. J Neurochem 1993;60: 1058e64. [15] Montioli R, Voltattorni CB, Bertoldi M, Parkinson’s Disease. Recent updates in the identification of human dopa decarboxylase inhibitors. Curr Drug Metabol 2016;17:513e8. [16] Himmelreich N, Montioli R, Bertoldi M, Carducci C, Leuzzi V, Gemperle C, Berner T, Hyland K, Tho¨ny B, Hoffmann GF, Voltattorni CB, Blau N. Aromatic amino acid decarboxylase deficiency: molecular and metabolic basis and therapeutic outlook. Mol Genet Metabol 2019;127:12e22. [17] Tsai CR, Lee HF, Chi CS, Yang MT, Hsu CC. Antisense oligonucleotides modulate dopa decarboxylase function in aromatic l-amino acid decarboxylase deficiency. Hum Mutat 2018;39:2072e82. [18] Kaufmann H. L-dihydroxyphenylserine (Droxidopa): a new therapy for neurogenic orthostatic hypotension: the US experience. Clin Auton Res 2008;18(Suppl. 1):19e24. [19] Segura-Aguilar J. On the role of endogenous neurotoxins and neuroprotection in Parkinson’s disease. Neural Regen Res 2017;12:897e901.

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[20] Herrera A, Mun˜oz P, Steinbusch HWM, Segura-Aguilar J. Are dopamine oxidation metabolites involved in the loss of dopaminergic neurons in the nigrostriatal system in Parkinson’s disease? ACS Chem Neurosci 2017;8:702e11. [21] Segura-Aguilar J, Paris I, Mun˜oz P, Ferrari E, Zecca L, Zucca FA. Protective and toxic roles of dopamine in Parkinson’s disease. J Neurochem 2014;129:898e915. [22] Cartier EA, Parra LA, Baust TB, Quiroz M, Salazar G, Faundez V, Egan˜a L, Torres GE. A biochemical and functional protein complex involving dopamine synthesis and transport into synaptic vesicles. J Biol Chem 2010:1957e66.

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4 Dopamine storage and release The neurotransmission of dopamine requires monoaminergic vesicles containing dopamine. In Dopaminergic neurons, dopamine comes from two sources: (1) de novo synthesis of dopamine that converts the amino acid tyrosine to dopamine in a sequential reaction (tyrosine to L-dopa and Ldopa to dopamine) catalyzed by tyrosine hydroxylase and aromatic amino acid decarboxylase, respectively; and (2) reuptake of dopamine released during neurotransmission mediated by dopamine transporter. Uptake of dopamine into monoaminergic vesicles is mediated by vesicular monoamine transporter-2. Vesicular monoamine transporter-2 mediates transport monoamines such as dopamine, serotonin, norepinephrine, and histamine from neuronal cytoplasm into monoaminergic vesicles. Vesicular monoamine transporter2 has 517 amino acids. The change of cysteine 126 or 333 reduces serotonin transport, suggesting that disulfide bonds play an important role in monoamine transport. Vesicular monoamine transporter-2 has two posttranslational modifications, glycosylation at asparagine residues 84 and 91 and phosphorylation at serines 511 and 513 (1,2, UniProtKB-Q05940). Vesicular monoamine transporter-2 regulation is mediated by its posttranslational modifications, phosphorylation and glycosylation. Phosphorylation of two serines plays an important role in the regulation of vesicular monoamine transporter-2 because this phosphorylation is required for its localization to large monoaminergic vesicles. Serine dephosphorylation induces the removal of vesicular monoamine transporter-2 from large monoaminergic vesicles [1]. Glycosylation of vesicular monoamine transporter-2 plays an important role in its trafficking to large monoaminergic vesicles for neurotransmission. Inhibition of vesicular monoamine transporter-2 glycosylation by 1-deoxymannojirimycin, a specific alpha-mannosidase I inhibitor, induces vesicular monoamine transporter-2 traffic to synaptic-like microvesicles [2]. It has been reported that methamphetamine affects vesicular monoamine transporter-2 function by decreasing dopamine uptake into monoaminergic vesicles and dihydrotetrabenazine binding, suggesting

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that methamphetamine will increase free cytosolic dopamine that can oxidize to neuromelanin [3]. In the nerve terminals, methamphetamine decreases dopamine uptake mediated by vesicular monoamine transporter-2. Methamphetamine also decreases vesicular dopamine uptake in the hippocampus [4]. It has been proposed that amphetamine and substituted amphetamines act by redistributing catecholamines such as dopamine from synaptic vesicles to the cytosol and inducing reverse transport of transmitters through plasma membrane uptake transporters [5]. An increase in free cytosolic dopamine will result in its oxidation to neuromelanin through the formation of ortho-quinones that can be neurotoxic under certain circumstances. Vesicular monoamine transporter-2 is expressed in all monoaminergic neurons, and immunohistochemical studies have revealed that this transporter is localized along soma, axons, and dendrites, suggesting that dopamine and other monoamines can be released at these cellular domains [6]. The heterozygote knockout of vesicular monoaminergic transporter-2 induced a significant increase in mice treated with 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine in comparison with control mice, suggesting that the significant decrease in this neurotoxin neurotoxicity depends on vesicular monoamine transporter-2-dependent uptake of the neurotoxin into monoaminergic vesicles [7]. The effect of 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine on dopamine transporter and vesicular monoamine transporter-2 loss was compared at different times of neurotoxin administration. These studies showed that losses of dopamine transporter localized in the neuronal membrane and vesicular transporters did not occur during the same time frame [8]. Inhibition of vesicular monoamine transporter-2 induces depletion of dopamine in the nerve terminals, and it has been used to treat Huntington chorea and tardive dyskinesia. Tetrabenazine, deutetrabenazine, and valbenazine are three inhibitors of vesicular monoamine transporter-2 that especially induce depletion of dopamine but also serotonin and norepinephrine in the nerve terminals, resulting in a reduction of spontaneous jerklike movements of the extremities, face, trunk, and neck that are typical symptoms in Huntington disease [9]. Tetrabenazine, a reversible inhibitor of vesicular monoamine transporter-2, reduces morphine-induced hyperlocomotion in mice without stereotypic behavior, suggesting a possible antagonistic action of tetrabenazine on some opiate activity [10]. Vesicular monoamine transporter-2 has been proposed to prevent the occurrence of free cytoplasmatic dopamine by transporting dopamine into monoaminergic vesicles. Free cytoplasmatic dopamine oxidizes to neuromelanin by generating three potential neurotoxic ortho-quinones, of which aminochrome is the most neurotoxic. Dopamine in the

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monoaminergic vesicles is completely stable and accumulates because the pH inside the vesicles is slightly low [11e13]. Adenoviral-dependent overexpression of synaptic vesicular monoamine transporter-2 inhibited neuromelanin formation by decreasing the free cytosolic dopamine level. The iron chelator desferrioxamine inhibited neuromelanin synthesis, suggesting oxidation of cytosolic dopamine to ortho-quinones involved in neuromelanin synthesis [14]. Uptake of dopamine and other monoamines into monoaminergic vesicles mediated by vesicular monoamine transporter-2 is dependent on generation of an electrochemical proton gradient that consumes ATP, suggesting coupling between monoamine uptake and proton efflux. Monoamine influx and proton efflux are inhibited by reserpine, an inhibitor of vesicular monoamine transporter-2 in isolated chromaffin ghosts. The generation of ATP-dependent proton gradient resulted in an acidic inside of isolated chromaffin ghosts [15]. Synaptogyrin-3, a synaptic vesicle protein, was found to colocalize with dopamine transporter at synaptic terminals in immunoprecipitation experiments. This interaction between dopamine transporter and synaptogyrin-3 was also observed in live neurons by using fluorescence resonance energy transfer microscopy. Interestingly, dopamine transporter N-terminal was found to bind isolated brain synaptic vesicles. Reserpine, an inhibitor of vesicular monoamine transporter-2, inhibited the synaptogyrin-3 effect on dopamine transporter activity. The formation of a kind of complex between dopamine transporter, synaptogyrin-3, and vesicular monoamine transporter-2 was observed [16]. A study performed to determine cytosolic dopamine level using intracellular patch electrochemistry showed that L-dopa induced a cytosolic dopamine increase in substantia nigra that is higher than in the ventral tegmental area [17]. In the cytosol at physiological pH, dopamine oxidizes to ortho-quinones in a sequential manner that finally polymerize to form neuromelanin. However, under certain conditions these orthoquinones, especially aminochrome, can be neurotoxic. Aminochrome induces mitochondrial dysfunction, alpha-synuclein aggregation to neurotoxic oligomers, protein degradation dysfunction of both lysosomal and proteasomal systems, endoplasmic reticulum stress, neuroinflammation, and oxidative stress. There are two sources of dopamine in dopaminergic neurons, de novo dopamine synthesis and dopamine reuptake after neurotransmission mediated by dopamine transporter. The slightly acidic pH inside monoaminergic vesicles plays a very important role in preventing dopamine oxidation to neuromelanin, because dopamine at this pH is stable and accumulates for neurotransmission. Vesicular monoamine transporter-2 is associated with tyrosine hydroxylase and aromatic amino acid decarboxylase to form a kind of complex that prevents the formation of free dopamine. Interestingly, the vesicular

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monoamine transporter-2 is also associated with dopamine transporter and the synaptic protein synaptogyrin-3 to form a kind of complex to prevent the existence of free cytosolic dopamine. A postmortem study in human brain to determine the relationship between vesicular monoamine transporter-2 and neuromelanin level in the ventral tegmental area and in the ventral substantia nigra was performed. This study showed an inverse relationship between vesicular monoamine transporter-2 expression and neuromelanin levels in dopaminergic neurons. Ventral tegmental area dopaminergic neurons have more expression of vesicular monoaminergic transporter-2 than substantia nigra dopaminergic neurons do. Dopaminergic neurons that are more vulnerable to neurodegeneration in the substantia nigra than ventral tegmental area have more neuromelanin pigment and lower vesicular monoamine transporter-2 expression [18]. These results support the important role of vesicular monoamine transporter-2 in preventing dopamine oxidation to neuromelanin, which under certain circumstances generates neurotoxic ortho-quinones and neurodegeneration. A study with postmortem brain of Parkinson’s disease patients supporting the protective role of vesicular monoamine transporter-2 was performed by isolating monoaminergic vesicles from the striatum. This study revealed that dopamine uptake into monoamine vesicles was significantly reduced, and the binding of dihydrotetrabenazine to vesicular monoamine transporter-2 was also decreased in isolated monoamine vesicles from Parkinson’s disease patients [19]. Lower expression of vesicular monoamine transporter-2 implies that the cytosolic free dopamine level will increase, resulting in more dopamine oxidation to neuromelanin with formation of potentially neurotoxic ortho-quinones, especially aminochrome. Normal function of dopamine neurons requires normal dopamine release that it is dependent on a functional vesicular monoamine transporter-2 in two ways. First, a functional vesicular monoamine transporter-2 will accumulate dopamine in monoamine vesicles for successful neurotransmission to control body motor activity; and second, a functional vesicular monoamine transporter-2 will prevent the loss dopaminergic neurons that will affect normal body motor function. Low vesicular monoamine transporter-2 expression or dysfunction will increase the free cytosolic dopamine that can be oxidized to neuromelanin with the formation of neurotoxic ortho-quinones such as aminochrome. The deficiency or dysfunction of the enzymes DT-diaphorase and glutathione transferase M2-2, which prevent aminochromic neurotoxicity, will result in mitochondrial dysfunction, alpha-synuclein aggregation to neurotoxic oligomers, protein degradation dysfunction of both lysosomal and proteasomal systems, endoplasmic reticulum stress, neuroinflammation, and oxidative stress (Fig. 4.1).

FIGURE 4.1 Vesicular monoamine transporter-2-mediated mechanism of dopamine uptake into monoamine vesicles. Vesicular monoamine transporter-2 is localized on the membrane of the synaptic monoamine vesicles used for neurotransmission. Vesicular monoamine transporter-2 (VMAT-2) is associated with tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase (AADC) in a kind of complex that prevents the formation of free cytosolic dopamine during de novo dopamine synthesis from tyrosine. Another source of dopamine in dopaminergic neurons is dopamine reuptake mediated by dopamine transporter (DAT) after neurotransmission. Interestingly, dopamine transporter, the synaptic protein synaptogyrin-3 (Snp3), and vesicular monoamine transporter-2 are also associated in a kind of complex to prevent the occurrence of free cytosolic dopamine. Dopamine inside monoamine vesicle dopamine is completely stable and accumulates for neurotransmission due to dopamine uptake mediated by vesicular monoamine transporter-2 coupled with a proton pump that decreases the pH inside synaptic monoaminergic vesicles. The pumping of protons into synaptic monoaminergic vesicles as well as dopamine release requires ATP. The figure has been modified from a paper (PMID: 32864581). From Segura-Aguilar, Sulzer, Zucca, F., Zecca, L. Overexpression of vesicular monoamine transporter-2 may block neurotoxic metabolites from cytosolic dopamine: a potential neuroprotective therapy for Parkinson’s disease. Clin Pharmacol Transl Med 2019;3:10.

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References [1] Waites CL, Mehta A, Tan PK, Thomas G, Edwards RH, Krantz DE. An acidic motif retains vesicular monoamine transporter 2 on large dense core vesicles. J Cell Biol 2001; 152:1159e68. [2] Yao J, Hersh LB. The vesicular monoamine transporter 2 contains trafficking signals in both its N-glycosylation and C-terminal domains. J Neurochem 2007;100:1387e96. [3] Brown JM, Hanson GR, Fleckenstein AE. Methamphetamine rapidly decreases vesicular dopamine uptake. J Neurochem 2000;74:2221e3. [4] Rau KS, Birdsall E, Volz TJ, Riordan JA, Baucum 2nd AJ, Adair BP, Bitter R, Gibb JW, Hanson GR, Fleckenstein AE. Methamphetamine administration reduces hippocampal vesicular monoamine transporter-2 uptake. J Pharmacol Exp Ther 2006;318:676e82. [5] Sulzer D, Sonders MS, Poulsen NW, Galli A. Mechanisms of neurotransmitter release by amphetamines: a review. Prog Neurobiol 2005;75:406e33. [6] Hoffman BJ, Hansson SR, Mezey E, Palkovits M. Localization and dynamic regulation of biogenic amine transporters in the mammalian central nervous system. Front Neuroendocrinol 1998;19:187e231. [7] Gainetdinov RR, Fumagalli F, Wang YM, Jones SR, Levey AI, Miller GW, Caron MG. Increased MPTP neurotoxicity in vesicular monoamine transporter 2 heterozygote knockout mice. J Neurochem 1998;70:1973e8. [8] Kilbourn MR, Kuszpit K, Sherman P. Rapid and differential losses of in vivo dopamine transporter (DAT) and vesicular monoamine transporter (VMAT2) radioligand binding in MPTP-treated mice. Synapse 2000;35:250e5. [9] LiverTox: Clinical and research information on drug-induced liver injury [internet]. Bethesda (MD): National Institute of Diabetes and digestive and kidney diseases; 2012e. Vesicular monoamine transporter 2 (VMAT2) inhibitors. April 2, 2019. PMID: 31643515. [10] Kitanaka N, Kitanaka J, Hall FS, Kandori T, Murakami A, Muratani K, Nakano T, Uhl GR, Takemura M. Tetrabenazine, a vesicular monoamine transporter-2 inhibitor, attenuates morphine-induced hyperlocomotion in mice through alteration of dopamine and 5-hydroxytryptamine turnover in the cerebral cortex. Pharmacol Biochem Behav 2018;172:9e16. [11] Herrera A, Mun˜oz P, Steinbusch HWM, Segura-Aguilar J. Are dopamine oxidation metabolites involved in the loss of dopaminergic neurons in the nigrostriatal system in Parkinson’s disease? ACS Chem Neurosci 2017;8:702e11. [12] Segura-Aguilar J, Paris I, Mun˜oz P, Ferrari E, Zecca L, Zucca FA. Protective and toxic roles of dopamine in Parkinson’s disease. J Neurochem 2014;129:898e915. [13] Segura-Aguilar, Sulzer D, Zucca FA, Zecca L. Overexpression of vesicular monoamine transporter-2 may Block neurotoxic Metabolites from cytosolic dopamine: a potential neuroprotective Therapy for Parkinson’s disease. Clin Pharmacol Transl Med 2019;3: 143e8. [14] Sulzer D, Bogulavsky J, Larsen KE, Behr G, Karatekin E, Kleinman MH, Turro N, Krantz D, Edwards RH, Greene LA, Zecca L. Neuromelanin biosynthesis is driven by excess cytosolic catecholamines not accumulated by synaptic vesicles. Proc Natl Acad Sci U S A 2000;97:11869e74. [15] Johnson RG, Carty SE, Scarpa A. Proton: substrate stoichiometries during active transport of biogenic amines in chromaffin ghosts. J Biol Chem 1981;256:5773e80. [16] Egan˜a LA, Cuevas RA, Baust TB, Parra LA, Leak RK, Hochendoner S, Pen˜a K, Quiroz M, Hong WC, Dorostkar MM, Janz R, Sitte HH, Torres GE. Physical and functional interaction between the dopamine transporter and the synaptic vesicle protein synaptogyrin-3. J Neurosci 2009;29:4592e604.

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[17] Mosharov EV, Larsen KE, Kanter E, Phillips KA, Wilson K, Schmitz Y, Krantz DE, Kobayashi K, Edwards RH, Sulzer D. Interplay between cytosolic dopamine, calcium, and alpha-synuclein causes selective death of substantia nigra neurons. Neuron 2009; 62:218e29. [18] Liang CL, Nelson O, Yazdani U, Pasbakhsh P, German DC. Inverse relationship between the contents of neuromelanin pigment and the vesicular monoamine transporter-2: human midbrain dopamine neurons. J Comp Neurol 2004;473:97e106. [19] Pifl C, Rajput A, Reither H, Blesa J, Cavada C, Obeso JA, Rajput AH, Hornykiewicz O. Is Parkinson’s disease a vesicular dopamine storage disorder? Evidence from a study in isolated synaptic vesicles of human and nonhuman primate striatum. J Neurosci 2014; 34:8210e8.

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5 Dopamine oxidative deamination Dopamine is an essential neurotransmitter that plays an essential role in the regulation and control of motor activity in the body, but the dopamine catechol structure converts dopamine into a potential source of neurotoxic ortho-quinones during its oxidation to neuromelanin. Therefore, it is very important to prevent the occurrence of free cytosolic dopamine, and the enzyme monoamine oxidase catalyzes dopamine oxidative deamination in order to degrade excess dopamine in the cytosol.

Monoamine oxidases Monoamine oxidase has two isoforms (monoamine oxidases A and B) that possess 73% amino acid sequence similarity, but the two isoforms differ in their substrate specificity, tissue distribution, and inhibitor selectivity. Monoamine oxidase-A endogenous substrates includes serotonin, norepinephrine, tyramine, and dopamine, while monoamine oxidase-B uses only the substrates dopamine and tyramine. Monoamine oxidase-A is expressed in heart, placenta, and intestines, while monoamine oxidase-B is expressed in brain, liver cells, and platelets. The structure of human monoamine oxidase-A is a monomer with 527 amino acids, while monoamine oxidase-B is a dimer with a primary structure comprising 520 amino acids. Monoamine oxidases A and B are both flavoenzymes and contain a molecule of FAD [1]. The nucleotide sequences of monoamine oxidases A and B reveal that both isoforms contain five amino acids (Ser-Gly-Gly-Cys-Tyr) where the cofactor FAD is covalently bound to cysteine and play a key role in their catalytic activity [2]. The active site of monoamine oxidase-B has a very hydrophobic site for amine binding near the flavin bound to cysteine 397 [3]. Monoamine oxidases are localized in the outer mitochondrial membrane, which allows the enzyme to degrade cytosolic monoamines [4].

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Monoamine oxidase-B The oxidative deamination of dopamine in dopaminergic neurons is catalyzed by monoamine oxidase-B generating 3,4-dihydroxyphenylacetaldehyde, ammonia, and hydrogen peroxide. This reaction generates two molecules that potentially can be involved in neurotoxic reactions: ammonia and hydrogen peroxide. Liver or urea cycle dysfunction increases ammonia levels and can be neurotoxic in the brain. Hydrogen peroxide is the precursor of hydroxyl radicals, and in the presence of iron(II), it can be a potential source of oxidative stress. 3,4-Dihydroxyphenylacetaldehyde is oxidized to 3,4-dihydroxyphenylacetic acid by aldehyde dehydrogenase-1. However, the intracerebral injection of 3,4-dihydroxyphenylacetaldehyde into substantia nigra and the ventral tegmental area induces neurodegeneration in substantia nigra dopaminergic neurons [5]. Intracerebral injection of 3,4dihydroxyphenylacetaldehyde induced loss of striatal terminals and contralateral behavior like that observed with 6-hydroxydopamine, while non-dopaminergic neurons of the pars reticulata were not affected [6]. Alpha-synuclein interacts with 3,4-dihydroxyphenylacetaldehyde, resulting in the formation of small oligomers that inhibit the formation of alpha-synuclein fibrils [7]. The role of 3,4-dihydroxyphenylacetaldehyde on N-terminally acetylated alpha-synuclein aggregation to oligomers was studied in alpha-synuclein mutants A53T, A30P, G51D, E46K, and H50Q. The formation of alpha-synuclein large oligomers was increased in N-terminally acetylated alpha-synuclein mutants E46K, A53T, and H50Q. Alpha-synuclein binding to synaptic-like small unilamellar vesicles prevents 3,4-dihydroxyphenylacetaldehyde effects, and N-terminal acetylation of alpha-synuclein increased protection against 3,4dihydroxyphenylacetaldehyde [8]. The major product of alpha-synuclein reaction with 3,4dihydroxyphenylacetaldehyde is a dicatechol pyrrole lysine adduct [9]. The autoxidation of dicatechol pyrrole lysine rings stimulates its decomposition, generating an intermediate product (dicatechol isoindole lysine). 3,4-Dihydroxyphenylacetaldehyde-induced alpha-synuclein oligomers occur in two steps: formation of the dicatechol pyrrole lysine adduct with alpha-synuclein and cross-linking of alpha-synuclein molecules [10]. Reactive oxygen species such as superoxide promote 3,4-dihydroxyphenylacetaldehyde oxidation that it is inhibited by superoxide dismutase. Superoxide dismutase prevents the formation of dicatechol pyrrole adducts with lysine and the formation of cross-linking alpha-synuclein [11]. 3,4-Dihydroxyphenylacetaldehyde induces alpha-synuclein methionine oxidation and the formation of two different types of alpha-

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synuclein oligomers (dimers and trimers). The mutation of methionines 5, 116, and 127 to valine increases the formation of large alpha-synuclein oligomers, and stabilization of large alpha-synuclein oligomers increases 3,4-dihydroxyphenylacetaldehyde neurotoxicity [12]. It has been proposed that 3,4-dihydroxyphenylacetaldehyde-induced alphasynuclein oligomers provide the link between alpha-synucleinopathy and dopamine neuron loss in Lewy body diseases [13]. Divalent metal ions such as copper (II), iron (II), and Mn (II) increase 3,4dihydroxyphenylacetaldehyde-induced alpha-synuclein oligomerization in vitro and in a cell system [14]. It has been observed that cytosolic aldehyde dehydrogenase-1 activity and gene and protein expression decrease in the substantia nigra of Parkinson’s disease patients, while the mitochondrial aldehyde dehydrogenase-2 increases in the putamen of Parkinson’s disease patients [15]. Aldehyde dehydrogenases 1 and 2 null mice showed age-dependent deficits in motor activity determined by gait analysis and rotarod. Administration of L-DOPA plus benserazide reduces deficits in motor activity. An increase in 3,4-dihydroxyphenylacetaldehyde and 4hydroxynonenal, a marker for oxidative stress, was also observed [16]. Gene expression profiling of sporadic Parkinson’s disease substantia nigra pars compacta reveals impairment of aldehyde dehydrogenase, ubiquitin-proteasome subunits, and chaperone HSC-70 [17]. Monoamine oxidase-B inhibitors have been used in Parkinson’s disease therapy to inhibit dopamine degradation and hydrogen peroxide formation. L-deprenyl (selegiline) is an irreversible monoamine oxidase-B inhibitor that has been used as monotherapy and in combination with L-dopa [18]. A bifunctional irreversible inhibitor of monoamine oxidase-B is rasagiline, which is an inhibitor of this enzyme and has a neuroprotective-antiapoptotic effect [19]. Safinamide is a reversible and selective monoamine oxidase inhibitor with glutamatergic and dopaminergic properties [20]. A study done with neonatal and adult cardiomyocytes showed that monoamine oxidase activation induces a decrease in mitochondrial membrane potential in the presence of the ATP synthase inhibitor oligomycin. The addition of monoamine oxidase-B inhibitor pargyline inhibits mitochondrial dysfunction [21]. A study on the increase in monoamine oxidase-dependent hydrogen peroxide production showed that N1methylhistamine, the major catabolite of histamine, is a substrate of this enzyme. The addition of the monoamine oxidase inhibitor pargyline resulted in N1-methylhistamine accumulation and reduction of oxidative stress [22]. Fig. 5.1.

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FIGURE 5.1 Dopamine oxidative deamination. Free cytosolic dopamine is degraded by removing the dopamine amino group by monoamine oxidase- B enzyme, a dimeric flavoenzyme containing flavin adenine dinucleotide (FAD) that converts dopamine to 3,4dihydroxyphenylacetaldehyde. This reaction requires dioxygen and a molecule of water. The other products generated during this reaction are ammonia and hydrogen peroxide, which can potentially be neurotoxic. 3,4-Dihydroxyphenylacetaldehyde is oxidized to 3,4dihydroxyphenylacetic acid Aldehyde dehydrogenase (ADH) with concomitant formation of NADH.

References [1] Manzoor S, Hoda N. A comprehensive review of monoamine oxidase inhibitors as Anti-Alzheimer’s disease agents: a review. Eur J Med Chem 2020;206:112787. [2] Bach AW, Lan NC, Johnson DL, Abell CW, Bembenek ME, Kwan SW, Seeburg PH, Shih JC. cDNA cloning of human liver monoamine oxidase A and B: molecular basis of differences in enzymatic properties. Proc Natl Acad Sci U S A 1988;85:4934e8. [3] Edmondson DE, Binda C, Mattevi A. Structural insights into the mechanism of amine oxidation by monoamine oxidases A and B. Arch Biochem Biophys 2007;464:269e76. [4] Segura-Aguilar J, Paris I. Mechanisms of dopamine oxidation and Parkinson’s disease. In: Kostrzewa R, editor. Handbook of neurotoxicity. New York, NY: Springer; 2014. https://doi.org/10.1007/978-1-4614-5836-4_16. [5] Burke WJ, Li SW, Williams EA, Nonneman R, Zahm DS. 3,4Dihydroxyphenylacetaldehyde is the toxic dopamine metabolite in vivo: implications for Parkinson’s disease pathogenesis. Brain Res 2003;989:205e13. [6] Panneton WM, Kumar VB, Gan Q, Burke WJ, Galvin JE. The neurotoxicity of DOPAL: behavioral and stereological evidence for its role in Parkinson disease pathogenesis.

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PLoS One 2010;5:e15251. https://doi.org/10.1371/journal.pone.0015251. Erratum in: PLoS One. 2011;6(2). Follmer C, Coelho-Cerqueira E, Yatabe-Franco DY, Araujo GD, Pinheiro AS, Domont GB, Eliezer D. Oligomerization and membrane-binding properties of covalent adducts formed by the interaction of a-synuclein with the toxic dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL). J Biol Chem 2015;290:27660e79. Lima VA, do Nascimento LA, Eliezer D, Follmer C. Role of Parkinson’s disease-linked mutations and N-terminal acetylation on the oligomerization of a-synuclein induced by 3,4-dihydroxyphenylacetaldehyde. ACS Chem Neurosci 2019;10:690e703. Werner-Allen JW, DuMond JF, Levine RL, Bax A. Toxic dopamine metabolite DOPAL forms an unexpected dicatechol pyrrole adduct with lysines of a-synuclein. Angew Chem Int Ed Engl 2016;55:7374e8. Werner-Allen JW, Monti S, DuMond JF, Levine RL, Bax A. Isoindole llinkages provide a pathway for DOPAL-mediated cross-llinking of a-synuclein. Biochemistry 2018;57: 1462e74. Werner-Allen JW, Levine RL, Bax A. Superoxide is the critical driver of DOPAL autoxidation, lysyl adduct formation, and crosslinking of a-synuclein. Biochem Biophys Res Commun 2017;487:281e6. Carmo-Gonc¸alves P, do Nascimento LA, Cortines JR, Eliezer D, Roma˜o L, Follmer C. Exploring the role of methionine residues on the oligomerization and neurotoxic properties of DOPAL-modified a-synuclein. Biochem Biophys Res Commun 2018;505: 295e301. Goldstein DS, Kopin IJ, Sharabi Y. Catecholamine autotoxicity. Implications for pharmacology and therapeutics of Parkinson disease and related disorders. Pharmacol Ther 2014;144:268e82. Jinsmaa Y, Sullivan P, Gross D, Cooney A, Sharabi Y, Goldstein DS. Divalent metal ions enhance DOPAL-induced oligomerization of alpha-synuclein. Neurosci Lett 2014;569: 27e32. Gru¨nblatt E, Riederer P. Aldehyde dehydrogenase (ALDH) in Alzheimer’s and Parkinson’s disease. J Neural Transm (Vienna) February 2016;123(2):83e90. Wey MC, Fernandez E, Martinez PA, Sullivan P, Goldstein DS, Strong R. Neurodegeneration and motor dysfunction in mice lacking cytosolic and mitochondrial aldehyde dehydrogenases: implications for Parkinson’s disease. PLoS One 2012;7:e31522. Mandel S, Grunblatt E, Riederer P, Amariglio N, Jacob-Hirsch J, Rechavi G, Youdim MB. Gene expression profiling of sporadic Parkinson’s disease substantia nigra pars compacta reveals impairment of ubiquitin-proteasome subunits, SKP1A, aldehyde dehydrogenase, and chaperone HSC-70. Ann N Y Acad Sci 2005;1053:356e75. Youdim MBH. Monoamine oxidase inhibitors, and iron chelators in depressive illness and neurodegenerative diseases. J Neural Transm (Vienna) 2018;125:1719e33. Youdim MB, Fridkin M, Zheng H. Bifunctional drug derivatives of MAO-B inhibitor rasagiline and iron chelator VK-28 as a more effective approach to treatment of brain ageing and ageing neurodegenerative diseases. Mech Ageing Dev 2005;126:317e26. Blair HA, Dhillon S. Safinamide: a review in Parkinson’s disease. CNS Drugs 2017;31: 169e76. Kaludercic N, Carpi A, Nagayama T, Sivakumaran V, Zhu G, Lai EW, Bedja D. Monoamine oxidase B prompts mitochondrial and cardiac dysfunction in pressure overloaded hearts. Antioxid Redox Signal 2014;20:267e80. Costiniti V, Spera I, Menabo` R, Palmieri EM, Menga A, Scarcia P, Porcelli V, Gissi R, Castegna A, Canton M. Monoamine oxidase-dependent histamine catabolism accounts for post-ischemic cardiac redox imbalance and injury. Biochim Biophys Acta Mol Basis Dis 2018;1864:3050e9.

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6 Dopamine methylation Dopamine is stable in monoaminergic vesicles because the pH inside vesicles is slightly acidic where the dopamine catechol structure is strongly protonated, preventing dopamine oxidation to ortho-quinones that are potentially neurotoxic. Dopamine derived ortho-quinones can induce mitochondrial dysfunction, alpha-synuclein aggregation to neurotoxic oligomers, protein degradation dysfunction of both lysosomal and proteasomal, endoplasmic reticulum stress, neuroinflammation and oxidative stress. Oxidative deamination of dopamine and dopamine methylation are two mechanism to prevent dopamine oxidation to ortho quinones and their potential neurotoxic effects.

Catechol ortho-methyltransferase Catechol ortho-methyltransferase catalyzes the degradation catechol structure of catecholamines such as dopamine, norepinephrine, and catechol estrogen. Catechol ortho-methyltransferase catalyze the methylation of dopamine by introducing a methyl group in one of the hydroxyl groups of the dopamine catechol structure. This reaction requires the methyl donor S-adenosyl-L-methionine and generates 3methoxytyramine and S-adenosyl-L-homocysteine. Catechol orthomethyltransferase also catalyzes methylation of dopamine metabolite 3,4-dihydroxyphenylacetic acid to homovanillic acid, a final product of dopamine metabolism [1]. Catechol ortho-methyltransferase, a magnesium-dependent enzyme, is coded by one gene that codes for two isoforms, a cytosolic form and a membrane-bound form attached to the inner side of the cell plasma membrane in brain and other tissues [2]. A study in rats showed catechol ortho-methyltransferase activity in liver outer mitochondrial membranes from rat liver. It also found that the soluble form of catechol orthomethyltransferase is the major form in all analyzed tissues [3].

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Dopamine released from dopaminergic neurons under neurotransmission binds dopamine receptors located in postsynaptic neurons, which are then released to the intersynaptic space, where the dopamine transporter of dopaminergic neurons mediates dopamine reuptake. However, in brain areas with a low density of dopaminergic neuron terminals, such as the frontal cortex, dopamine is removed by low-affinity transporters such as the plasma membrane amine transporter or organic cation transporters 1, 2, and 3 localized in glia cells and neurons. Dopamine degradation in nondopaminergic postsynaptic neurons or glia cells is performed by catechol ortho-methyltransferase and monoamine oxidase [2]. For more than 50 years, L-dopa has been the most effective therapy in Parkinson’s disease despite the severe effects induced by this drug. Oral administration of L-dopa requires that this drug crosses the bloodebrain barrier, and in the brain it is converted to dopamine by aromatic amino acid decarboxylase. Only 1% of L-dopa administered to patients reaches the brain due to extensive drug metabolism before L-dopa cross the bloode brain barrier. L-dopa undergoes decarboxylation, ortho-methylation, oxidation, and transamination. Catechol ortho-methyltransferase catalyzes the conversion of L-dopa to 3-methyldopa [1]. L-dopa peripheral decarboxylation is inhibited by benserazide or carbidopa, and its ortho-methylation is inhibited by tolcapone and entacapone, two selective and reversible nitrocatechol-type catechol ortho-methyltransferase inhibitors. A study with human volunteers showed that both tolcapone and entacapone inhibit catechol orthomethyltransferase activity in erythrocytes, decreasing L-dopa elimination of levodopa, increasing L-dopa bioavailability, and inhibiting the formation of 3-ortho-methyldopa [4]. Entacapone is a catechol ortho-methyltransferase inhibitor that improves L-dopa availability and efficacy. Entacapone is administered in commination with carbidopa, an inhibitor of aromatic amino acid decarboxylase, and L-dopa to increase L-dopa availability and inhibit Ldopa degradation [5]. L-dopa administration is combined with aromatic amino acid decarboxylase and catechol ortho-methyltransferase inhibitor to prevent peripheral L-dopa degradation. The half-life and bioavailability of L-dopa is increased by catechol-O-methyltransferase inhibitors [6]. Fluctuation in motor performance is one symptom observed in Parkinson’s disease treatment after 2 years, and catechol orthomethyltransferase inhibitors play an important role in treating these fluctuations. Opicapone is a third-generation catechol orthomethyltransferase inhibitor [7] (Fig. 6.1).

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FIGURE 6.1 Dopamine degradation by catechol ortho-methyltransferase. Dopamine degradation is catalyzed by catechol ortho-methyltransferase, which transfers one methyl group into one hydroxyl of the catechol structure of dopamine in a reaction that requires Sadenosyl-L-methionine, generating 3-methoxytyramine and S-adenosyl-L-homocysteine. 3-Methoxytyramine is converted to 3-methoxy-4-hydroxyphenylacetaldehyde by monoamine oxidase-B, and 3-methoxy-4-hydroxyphenylacetaldehyde is finally converted to homovanillic acid, a final product of dopamine metabolism. 3,4-Dihydroxyphenylacetic acid, a metabolite generated by monoamine oxidase-B during dopamine degradation, is also methylated by catechol ortho-methyltransferase to homovanillic acid.

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References [1] Bonifa´cio MJ, Palma PN, Almeida L, Soares-da-Silva P. Catechol-O-methyltransferase and its inhibitors in Parkinson’s disease. CNS Drug Rev 2007;13:352e79. [2] Finberg JPM. Inhibitors of MAO-B and COMT: their effects on brain dopamine levels and uses in Parkinson’s disease. J Neural Transm (Vienna) 2019;126:433e48. [3] Grossman MH, Creveling CR, Rybczynski R, Braverman M, Isersky C, Breakefield XO. Soluble and particulate forms of rat catechol-O-methyltransferase distinguished by gel electrophoresis and immune fixation. J Neurochem 1985;44:421e32. [4] Kaakkola S. Clinical pharmacology, therapeutic use and potential of COMT inhibitors in Parkinson’s disease. Drugs 2000;59:1233e50. [5] Entacapone/levodopa/carbidopa combination tablet: Stalevo. Drugs R D 2003;4:310e1. [6] Tambasco N, Romoli M, Calabresi P. Levodopa in Parkinson’s disease: current status and future developments. Curr Neuropharmacol 2018;16:1239e52. [7] Salamon A, Za´dori D, Szpisjak L, Klive´nyi P, Ve´csei L. Opicapone for the treatment of Parkinson’s disease: an update. Expert Opin Pharmacother 2019;20:2201e7.

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7 Dopamine oxidation to neuromelanin and neurotoxic metabolites Dopamine oxidation to neuromelanin depends on the stability of the dopamine catechol group. Under acidic conditions, such as inside the monoamine vesicles, the protons of hydroxyl groups are strongly bound to oxygen, while at the physiological pH of the cytoplasm, the protons are weakly bound to oxygen. Therefore, dopamine accumulates in monoamine vesicles and is resistant to catechol oxidation. Dopamine oxidation to neuromelanin proceeds through the formation of three ortho-quinoneedopamine ortho-quinones, aminochrome, and 5,6indolequinoneethat can be neurotoxic [1e3] Fig. 7.1.

Dopamine ortho-quinone The oxidizing agents that catalyze dopamine oxidation to dopamine ortho-quinone include dioxygen at physiological pH in the dopaminergic neuronal cytosol, metals such as manganese (III) under both aerobic and

FIGURE 7.1 Dopamine oxidation to neuromelanin. The hydroxyl groups of the dopamine catechol structure oxidize to an ortho-quinone structure, generating three orthoquinones (dopamine ortho-quinone, aminochrome, and 5,6-indolequinone) that are potentially neurotoxic. This oxidation is catalyzed by metals such as iron, copper, and manganese as well as enzymes with peroxidase activity and oxidizing agents such as oxygen. The most stable and most studied ortho-quinone is aminochrome.

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anerobic conditions, copper (II), iron (III), and enzymes that act as a peroxidase, such as prostaglandin H synthase, cytochrome P450, lactoperoxidase, xanthine oxidase, tyrosinase, and dopamine betamonooxygenase, using peroxidase activity [4e11]. Hypochlorous acid is generated during inflammation by the innate immune system, and it has been reported that hypochlorous acid oxidizes dopamine to dopamine ortho-quinone with a typical yellow color. Calcium cation affects dopamine oxidation, which is probably due to the chelation of the semiquinone formed during dopamine oxidation [12]. Dopamine ortho-quinone is very unstable at physiological pH in cytosol because the stable pH level for this quinone is below 2.0 [10]. Dopamine ortho-quinone at physiological pH cyclizes instantly in two steps to aminochrome at a rate of 0.15 s
1 [13]. Dopamine ortho-quinone formed protein adducts in experiments done with isolated mitochondria and cell lines. The oxidation of dopamine to dopamine ortho-quinone with tyrosinase in the presence of mitochondria induced the formation of dopamine ortho-quinone adducts with proteins such as mitochondrial complexes I, III, and IV, isocitrate dehydrogenase, ubiquitin C-terminal hydrolase-L1, protein deglycase, mitochondrial voltage-dependent anion channel 1, actin gamma, superoxide dismutase 2, heat shock protein 60 and mortalin/GRP75/mtHSP70, creatine kinase, and other mitochondrial proteins [14]. In the same study but performed with SH-SY5Y incubated with dopamine alone, only some proteins that formed adducts with dopamine in isolated mitochondria formed adducts with dopamine in SH-SY5Y cells. Dopamine inside SH-SY5Y cells oxidize to dopamine ortho-quinone that forms adducts with protein deglycase, ubiquitin C-terminal hydrolase-L1, actin, and mortalin/GRP75/mtHSP70 [14]. The difference observed in isolated mitochondria and SH-SY5Y cells can be explained by noting that the formation of dopamine ortho-quinone in SH-SY5Y cell cytosol has two possible paths to follow: one, to react with the closest protein, and two, to cyclize to form aminochrome. Therefore, it is possible to think that the adducts formed by dopamine oxidation in SH-SY5Y cells can be generated by dopamine ortho-quinone or aminochrome. Dopamine ortho-quinone has been reported for adducts in a dopaminergic cell line expressing parkin when these cells were incubated with dopamine. The formation of adducts was increased by the addition of hydrogen peroxide. Covalent addition of dopamine ortho-quinone to parkin inactivates parkin E3 ligase activity [15]. Dopamine ortho-quinone has been reported to form adducts with human dopamine transporter by reacting with cysteine residues [16]. Tyrosine hydroxylase is inactivated by the covalent addition of dopamine ortho-quinone inactivated in the presence of reduced glutathione, dithiothreitol, or NADH [17] (Fig. 7.2).

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FIGURE 7.2 Dopamine ortho-quinone (dopamine o-quinone) neurotoxicity. Dopamine o-quinone is a transient metabolite that undergoes cyclization immediately at the physiological pH of dopaminergic neurons because dopamine o-quinone is stable at a much lower pH of 2.0. Dopamine o-quinone is neurotoxic by forming adducts with parkin, dopamine transporter, tyrosine hydroxylase, and mitochondrial proteins such as complexes I, III, and IV, protein deglycase-1, ubiquitin carboxy-terminal hydrolase-L1, isocitrate dehydrogenase, voltage-dependent anion-selective channel-1, actin gamma, superoxide dismutase 2, heat shock protein 60, mortalin/GRP75/mtHSP70, and other mitochondrial proteins that induce mitochondrial dysfunction.

Aminochrome In contrast to dopamine ortho-quinone, aminochrome is the most stable ortho-quinone generated during dopamine oxidation to neuromelanin. It was reported that aminochrome was stable around 40 min in an nuclear magnetic resonance (NMR) in vitro experiment that detected aminochrome structure changes [18]. The rate of aminochrome rearrangement to 5,6indolequinone is 0.06 m1- [19].

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Aminochrome can be one-electron reduced by one-electron transfer flavoenzymes to the leukoaminochrome o-semiquinone radical, which is very unstable in the presence of oxygen [20]. The leukoaminochrome o-semiquinone radical reduces dioxygen to superoxide radicals with concomitant autoxidation to aminochrome, generating a redox cycling between aminochrome and leukoaminochrome o-semiquinone radical. This redox cycling runs until NADH or dioxygen is depleted in the cytosol to generate both NADþ and superoxide radicals as products. Superoxide radicals are enzymatically catalyzed by superoxide dismutase or spontaneously converted to hydrogen peroxide as a precursor for the formation of harmful hydroxyl radicals and oxidative stress. Another consequence of redox cycling between aminochrome and the leukoaminochrome o-semiquinone radical is cytosolic NADH depletion. The glycolysis generates NADH for ATP formation in the mitochondria during the coupled electron transport chain and oxidative phosphorylation. Therefore, this redox cycling decreases mitochondrial ATP formation in dopaminergic neurons [10,21]. Alternatively, aminochrome is able to form adducts with proteins such as mitochondrial complex I and inactivating an important part of the electron transport chain, because the majority of mitochondrial ATP are dependent on NADH, which transfers its electron through complex I. Mitochondrial complex II is the other site of entry for electrons in the mitochondrial electron transport chain, but the majority of electrons introduced into to this electron transport chain are from NADH. Therefore, mitochondrial complex I inactivation by aminochrome results in mitochondrial dysfunction [21e24]. Aminochrome induces the formation of alpha-synuclein aggregation to neurotoxic oligomers when DT-diaphorase is silenced in a cell line expressing a stable si-RNA against this enzyme. Aminochrome-alphasynuclein oligomers induce a significant increase in cell death, apoptosis, and DNA fragmentation; scyllo-inositol, an antioligomer compound, significantly decreases these neurotoxic effects [25]. Aminochrome induces lysosomal dysfunction by inhibiting the vacuolar H-type ATPase localized in lysosome membrane, resulting in a significant decrease in lysosome pH determined by using acridine orange [26]. Another study showed that aminochrome induces autophagy dysfunction, resulting in the accumulation of autophagic vacuoles containing undigested proteins and cellular components, a decrease in lysosome pH, and an increase in cell death [27,28]. It has been reported that aminochrome inhibits the proteasomal protein degradation system, where neither superoxide dismutase nor catalase prevented aminochrome-dependent proteasome inhibition [29]. A study showed that aminochrome inhibits 20/26S proteasome and activates endoplasmic reticulum stress [30].

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Microtubules play an important role in intracellular trafficking and cell division and are the major components of the cytoskeleton. Aminochrome interferes with microtubule assembly and stability and induces microtubule network disruption leading to changes in cell morphology and cell death in a time- and dose-dependent manner [31]. Aminochrome induces dramatic changes in cell morphology such as a reduction in cell size, and the elongated cell shape becomes spherical. Aminochrome induces the aggregation of alpha- and beta-tubulin and actin around the cell membrane. Actin aggregations in the connections between cells in the cell culture were also observed, suggesting that aminochrome can disrupt axonal transport [32]. Aminochrome induced cell death in microglial primary culture and primary mesencephalic neuron-glia cultures accompanied by reactive gliosis, an increase in the number of NF-kB p50 immunoreactive cells, morphological changes in GFAPþ and Iba1þ cells, and an increase in the number of OX-42þ cells, suggesting that aminochrome induces microglia and astrocyte activation [33]. A study with organotypic cultures showed that aminochrome induced reduction of GDNF and NGF mRNA levels and a decrease in GFAP expression as well as an increase in both IL-1b and TNF-a mRNA and protein levels and morphological changes in Iba1þ cells [34]. These results suggest that aminochrome induces neuroinflammation. Aminochrome induces nuclear DNA damage demonstrated by aminochrome-induced nuclear DNA lesions and fragmentation and [35]. Another study showed that aminochrome also induces mitochondrial DNA damage [24]. Aminochrome is neurotoxic in cell cultures by inducing mitochondrial dysfunction, alpha-synuclein aggregation to neurotoxic oligomers, protein degradation dysfunction of both lysosomal and proteasomal systems, endoplasmic reticulum stress, neuroinflammation, oxidative stress, DNA oxidative damage, and disruption of cytoskeleton architecture by inducing actin and alpha- and beta-tubulin aggregation and interfering in microtubule stability and assembly. However, the unilateral injection of aminochrome into striatum induces the typical contralateral behavior observed when in 6-hydroxydopamine lesions but without significant loss of dopaminergic neurons in striatum and substantia nigra. Aminochrome induces a neuronal dysfunction by significantly reduction of dopamine release and increase in GABA release. Aminochrome generates an imbalance in the levels of the neurotransmitters dopamine and GABA. The number of presynaptic vesicles decreases dramatically, explaining the decrease observed in dopamine release, while isolated monoaminergic vesicles exhibit a significant increase in dopamine concentration as a compensatory mechanism. The number of damaged mitochondria increases significantly, and the striatum ATP level decreases significantly, suggesting that aminochrome in vivo induces mitochondrial dysfunction. ATP is essential both for dopamine release from synaptic monoaminergic vesicles and for vesicle anterograde axonal transport.

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Aminochrome induced dramatic morphological changes called cell shrinkage. These results suggest that aminochrome induces dysfunction of dopaminergic neurons where the contralateral behavior can be explained by the aminochrome-induced ATP decrease required for both anterograde transport of synaptic vesicles and dopamine release. Aminochrome could be implemented as a new model neurotoxin to study Parkinson’s disease. The contralateral behavior increased over time, suggesting that aminochrome induces progressive neuronal dysfunction [36] (Fig. 7.3).

5,6-Indolequinone Aminochrome is stable for around 40 min before starting its rearrangement to 5,6-indolequinone at a rate of 0.06 mine1. 5,6-Indolequinone is also unstable and polymerize to pigment called neuromelanin. The polymerization of 5,6-indolequinone increases when the pH and its concentration increase. 5,6-Indolequinone can react with both glutathione and NADH, and it is high unstable, explaining why it does not accumulate and cannot be isolated, because 5,6-indolequinone is polymerized immediately by auto-condensation [2,19,37,38]. A study performed using scanning tunneling microscopy with melanin synthetized from tyrosine revealed that the melanin structure is composed of 5,6-indolequinone units assembled by van der Waals interactions [39]. Alpha-synuclein forms adducts in vitro with 5,6indolequinone, preventing the formation of neuromelanin, but the properties of these alpha-synuclein-5,6-indolequinone oligomers are unknown [19]. 5,6-Indolequinone forms adducts with Nurr1, a nuclear receptor essential for dopaminergic neuron development, maintenance, and survival by forming adducts with Nurr1 cysteine 566, which was detected using X-ray crystallography and biophysical assays. Experiments with 5,6-indolequinone and cell cultures showed that this ortho-quinone stimulates Nurr1 activity such as the transcription of genes required for dopamine homeostasis [40] (Fig. 7.4).

Dopaminochrome It has been reported that dopamine oxidation generates the formation of dopaminochrome that inhibits the formation of alpha-synuclein fibrils by forming alpha-synuclein spherical oligomers. The mutation of alphasynuclein amino acids 125e129 prevents dopaminochrome-dependent spherical oligomers, suggesting that this region is essential for dopaminochrome adduct formation with alpha-synuclein [41].

Dopaminochrome

219

FIGURE 7.3 Aminochrome neurotoxicity. In the cytosol of dopaminergic neurons, aminochrome can be one-electron reduced by flavoenzymes that use NADH as an electron donor to transfer one-electron generating leukoaminochrome ortho (o)-semiquinone radicals. The leukoaminochrome ortho-semiquinone radical is very reactive with oxygen and autoxidizes to aminochrome with concomitant reduction of oxygen to superoxide. Two molecules of superoxide react spontaneously or enzymatically in the presence of two protons generating hydrogen peroxide, a precursor of hydroxyl radicals in the presence of reduced iron. Hydroxyl radicals are very harmful oxygen radicals that induce oxidative stress. Aminochrome can form adducts with mitochondrial complex I, generating mitochondrial dysfunction. Aminochrome induces the formation of neurotoxic alpha-synuclein oligomers, proteasome and autophagy dysfunction, neuroinflammation, cytoskeleton architecture disruption, and endoplasmic reticulum stress. Finally, aminochrome induces neurodegeneration of dopaminergic neurons containing neuromelanin by generating progressive neuronal dysfunction.

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FIGURE 7.4 5,6-Indolequinone neurotoxicity. 5,6-Indolequinone is a very unstable ortho-quinone that is difficult to analyze for its neurotoxic effects. In vitro studies showed that 5,6-indolequinone forms adducts with alpha-synuclein and nuclear receptor related-1 protein (Nurr1), a nuclear receptor of intracellular transcription factor that plays a crucial role in the maintenance of dopaminergic neurons.

Dopaminochrome forms adducts with beta-synuclein, inhibiting the formation of fibrils but generating neurotoxic oligomers and thus potentiating beta-synuclein neurotoxicity in rodent and human cell cultures. Mitochondrial outer membrane integrity prevents beta-synuclein oligomer neurotoxicity [42]. Dopaminochrome induces apoptotic cell death characterized without caspase-3 activation and with a lack of effect of a pan-caspase inhibitor, Z-VAD-fmk, suggesting caspase-independent apoptosis. Dopaminochrome

Neuromelanin

221

also induces oxidative DNA damage [43]. Dopaminochrome induces oxidative stress in cell cultures by increasing superoxide formation, and administration of N-acetylcysteine prevents dopaminochrome neurotoxicity. Depletion of reduced glutathione by addition of cells by buthionine sulfoximine increased the neurotoxic effects of dopaminochrome [44]. Dopaminochrome induces the formation of hydrogen peroxide at mitochondrial complex I in the presence of calcium [45]. Intranigral injection of dopaminochrome into rats induced slow and progressive degeneration of dopaminergic neurons, contrasting with the very rapid progression in animals lesioned with the 1-methyl-4phenylpyridinium ion [46]. The structure of dopaminochrome has not yet been determined. Dopaminochrome cannot be dopamine orthoquinone or 5,6-indole quinone due to their high instability. Neither can it be aminochrome, since it has a different absorption spectrum from that of dopaminochrome. The structure of aminochrome has been determined with NMR and has an absorption maximum of 280 and 475 nm [32], while dopaminochrome has an absorption maximum of 303 and 479 nm [47] (Fig. 7.5).

Neuromelanin Neuromelanin is a dark pigment that it is formed when free dopamine in the cytosol oxidizes to ortho-quinones (dopamine o-quinone, aminochrome, and 5,6-indolequinone) that are not stable and finally polymerizes to a structure composed of 5,6-indolequinone units [39]. It is important to note that neuromelanin formation is driven by the existence of free cytosolic dopamine because dopamine inside monoaminergic vesicles is stable, preventing neuromelanin formation [48]. Human substantia nigra neuromelanin is composed of two different forms of neuromelanin, pheomelanin and eumelanin [2]. Eumelanin is formed during dopamine oxidation to 5,6-indolequinone, while pheomelanin is formed from 5-S-cysteinyl-dopamine and 2-S-cysteinyl-dopamine [49]. Melanin formation in the skin is driven by the enzyme tyrosinase, but in substantia nigra, this enzyme is not responsible for neuromelanin formation despite the existence of some publications that have reported the expression of tyrosinase in the brain [2]. The possible role of tyrosinase in substantia nigra neuromelanin formation is not feasible because neuromelanin is a very slow process that takes years to accumulate neuromelanin pigment. In the human, neuromelanin at 1 year old is undetectable, and this pigment slowly increases with age, and between 50 and 90 years old, the substantia nigra pars compacta contains 2.3e3.7 mg/mg of tissue [50].

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FIGURE 7.5 Dopaminochrome neurotoxicity. The dopaminochrome structure is still unknown, and it has been reported that it induces the formation of alpha- and beta-synuclein oligomers, oxidative stress, and oxidative DNA damage.

Neuromelanin accumulates in a specific double-membrane organelle that can be observed with transmission electron microscopy [22,51], where pheomelanin is in the core of these organelles surrounded by eumelanin [52,53]. Interestingly, neuromelanin can bind peptides because the catechol structure of 5,6-indolequinone is able to form cysteine peptide amino acid adducts. The total content of peptides in neuromelanin corresponds to 12%e15% of total neuromelanin weight [54]. Neuromelanin also binds to lipids through 14e22 isoprenic units of lipids, where the most common lipid observed in neuromelanin is dolichol [55e58]. Analysis of the total content of lipids in neuromelanin showed that 18% of the total weight of neuromelanin corresponds to lipids [59].

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Neuromelanin is also considered a scavenger of metals that catalyze the formation of reactive oxygen species. A study performed in isolated neuromelanin, substantia nigra, and putamen revealed that iron was the metal with the highest concentration both in this pigment and in these brain regions but iron concentration in neuromelanin was tenfold higher than in the substantia nigra and putamen. Other metals observed in neuromelanin include Zn, Se, Cr, Sr, Sb, Co, Ni, Ce, Hg, Au, Ag, Ta, and Sc, which are in much higher concentrations than in the substantia nigra and putamen. Reduced iron can catalyze a Fenton reaction to generate hydroxyl radicals, which are a potent inducer of oxidative stress [60e64]. A study performed with primary mesencephalic cultures showed that neuromelanin induces dopaminergic neurodegeneration by reducing the number of, and shortening, dendrites and decreasing dopamine uptake [65]. Neuromelanin induces mitochondria-mediated apoptosis by releasing cytochrome c, collapsing mitochondrial membrane potential, and activating caspase 3. However, overexpression of Bcl-2 did not suppress apoptosis [66]. Extracellular neuromelanin induces microglial activation with concomitant production of proinflammatory factors, nitric oxide, superoxide, and hydrogen peroxide. Neuromelanin induces neurodegeneration in primary ventral midbrain cultures in a microgliadependent manner [67]. These neurotoxic effects of neuromelanin are induced by the addition of free pigment to cell cultures. Neuromelanin contains bound metals and a free catechol structure of 5,6-indolequinone polymerized units that can induce oxidative stress or form adducts with proteins, respectively. However, neuromelanin is accumulated inside a double-membrane organelle that prevents the neurotoxic effect of neuromelanin. The clearest evidence that neuromelanin plays a protective role in dopaminergic neurons containing neuromelanin is the fact that the substantia nigra of healthy seniors contains intact dark dopaminergic neurons with this pigment [50].

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[56] Zecca L, Bellei C, Costi P, Albertini A, Monzani E, Casella L. New melanic pigments in the human brain that accumulate in aging and block environmental toxic metals. Proc Natl Acad Sci U S A 2008;105:17567e72. [57] Fedorow H, Pickford R, Hook JM, Double KL, Halliday GM, Gerlach M, Riederer P, Garner B. Dolichol is the major lipid component of human substantia nigra neuromelanin. J Neurochem 2005;92:990e5. [58] Zucca FA, Basso E, Cupaioli FA, Ferrari E, Sulzer D, Casella L, Zecca L. Neuromelanin of the human substantia nigra: an update. Neurotox Res 2014;25:13e23. [59] Engelen M, Vanna R, Bellei C, Zucca FA, Wakamatsu K, Monzani E, Ito S, Casella L, Zecca L. Neuromelanins of human brain have soluble and insoluble components with dolichols attached to the melanic structure. PloS One 2012;7:e4849063. Zecca L, Pietra R, Goj C, Mecacci C, Radice D, Sabbioni E. Iron and other metals in neuromelanin, substantia nigra, and putamen of human brain. J Neurochem. 1994;62:1097e1101. [60] Zucca FA, Segura-Aguilar J, Ferrari E, Mun˜oz P, Paris I, Sulzer D, Sarna T, Casella L, Zecca L. Interactions of iron, dopamine and neuromelanin pathways in brain aging and Parkinson’s disease. Prog Neurobiol 2017;155:96e119. [61] Zecca L, Tampellini D, Costi P, Rizzio E, Giaveri G, Gallorini M. Combined biochemical separation and INAA for the determination of iron and other metals in Neuromelanin of human brain Substantia Nigra. J Radioanal Nucl Chem 2001;249:449e54. [62] Zecca L, Gallorini M, SchVunemann V, Trautwein AX, Gerlach M, Riederer P, Vezzoni P, Tampellini D. Iron, neuromelanin and ferritin content in the substantia nigra of normal subjects at different ages: consequences for iron storage and neurodegenerative processes. J Neurochem 2001;76:1766e73. [64] Brooks J, Everett J, Lermyte F, Tjendana Tjhin V, Sadler PJ, Telling N, Collingwood JF. Analysis of neuronal iron deposits in Parkinson’s disease brain tissue by synchrotron xray spectromicroscopy. J Trace Elem Med Biol 2020;62:126555. [65] Zhang W, Zecca L, Wilson B, Ren HW, Wang YJ, Wang XM, Hong JS. Human neuromelanin: an endogenous microglial activator for dopaminergic neuron death. Front Biosci 2013;5:1e11. [66] Naoi M, Maruyama W, Yi H, Yamaoka Y, Shamoto-Nagai M, Akao Y, Gerlach M, Tanaka M, Riederer P. Neuromelanin selectively induces apoptosis in dopaminergic SH-SY5Y cells by deglutathionylation in mitochondria: involvement of the protein and melanin component. J Neurochem 2008;105:2489e500. [67] Zhang W, Phillips K, Wielgus AR, Liu J, Albertini A, Zucca FA, Faust R, Qian SY, Miller DS, Chignell CF, Wilson B, Jackson-Lewis V, Przedborski S, Joset D, Loike J, Hong JS, Sulzer D, Zecca L. Neuromelanin activates microglia and induces degeneration of dopaminergic neurons: implications for progression of Parkinson’s disease. Neurotox Res 2011;19:63e72.

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8 Neuroprotective mechanisms against dopamine oxidationdependent neurotoxicity Vesicular monoamine transporter-2 Dopamine oxidation to neuromelanin proceeds through the formation of three orthoquinones that induce neurotoxicity in dopaminergic neurons. Therefore, the first protective mechanism against dopamine oxidation-dependent neurotoxicity is to prevent dopamine oxidation in the cytosol due to dopamine inside monoaminergic vesicles having its protons strongly attached to hydroxyl groups. The transport of dopamine into monoaminergic vesicles is mediated by vesicular monoamine transporter-2 localized in the monoaminergic vesicle membrane and coupled to an Hþ-ATPase that pumps electrons into the vesicles. Therefore, the pH inside monoaminergic vesicles is slightly acidic, thus preventing dopamine hydroxyl group oxidation to ortho-quinones [1e3]. There are two sources of dopamine in dopaminergic neurons, dopamine synthesized from the amino acid tyrosine and dopamine released during neurotransmission that is transported from intersynaptic space back to dopaminergic neurons through the action of the dopamine transporter. Interestingly, vesicular monoamine transporter-2 is associated with both tyrosine hydroxylase and aromatic amino acid decarboxylase in a kind of complex to prevent free dopamine in the cytosol during dopamine synthesis from tyrosine as well as dopamine transporter and synaptogyrin-3 during dopamine reuptake [3]. It has been demonstrated that a high level of expression of vesicular monoaminergic transporter-2 results in a low observed level of neuromelanin in dopaminergic neurons [4e6], which supports the

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neuroprotective role of vesicular monoamine transporter-2 against dopamine oxidation neurotoxicity. Therefore, high expression of vesicular monoamine transporter-2 prevents dopamine oxidation to neuromelanin, which requires the formation of potentially neurotoxic ortho-quinones. Although dopamine oxidation normally a harmless process because neuromelanin is present in intact dopaminergic neurons in healthy senior dopaminergic neurons in the substantia nigra [7], there is a potential risk of ortho-quinones neurotoxicity. The enzymes DT-diaphorase and glutathione transferase M2-2, however, prevent ortho-quinone neurotoxicity during dopamine oxidation to aminochrome.

DT-diaphorase The most studied ortho-quinone formed during dopamine oxidation to neuromelanin is aminochrome because it is more stable than dopamine ortho-quinone and 5,6-indolequinone. DT-diaphorase (NAD(P)H: quinone oxidoreductase, NQO1; EC 1.6.99.2) catalyzes the two-electron reduction of aminochrome to the hydroquinone leukoaminochrome [8]. DT-diaphorase is expressed in various tissues such as brain, liver, kidney, and intestine. In the brain, DT-diaphorase is expressed in the substantia nigra, striatum, hypothalamus, hippocampus, frontal cortex, and cerebellum. Flavoenzymes reduce quinones by transferring one or two electrons to semiquinone or hydroquinone, respectively. The electron donators for quinone reduction by flavoenzymes are NADH or NADPH, but only DT-diaphorase transfers two electrons from both NADH and NADPH. The regional distribution of quinone reductase activity in the rat brain showed that DT-diaphorase catalyzes 80%e85% of total quinone reductase activity in the striatum, hippocampus, and frontal cortex in cytosol, while in substantia nigra DT-diaphorase catalyzes 95% of total quinone reductase activity in the substantia nigra [9]. DT-diaphorase is a flavoenzyme containing the cofactor flavin adenine dinucleotide flavoenzyme, and seven isoforms have been separated using FPLC-chromatofocusing. The isoforms have different isoelectric points and posttranslational glycosylation. DT-diaphorase is a dimer and contains one flavin adenine dinucleotide molecule in each subunit. DTdiaphorase catalyzes various quinones such as vitamin K1, menadione, benzoquinones, and naphthoquinones [10]. DT-diaphorase catalyzes the two-electron reduction of aminochrome to leukoaminochrome that is slowly autoxidized in the presence of oxygen to generate superoxide. However, leukoaminochrome autoxidation in the presence of manganese is inhibited by superoxide dismutase and the chelator DETAPAC [8]. Leukoaminochrome formation can be followed

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spectrophotometrically, whereas leukoaminochrome ortho-semiquinone cannot. The detection of leukoaminochrome ortho-semiquinone requires electron spin resonance stabilization, while dopamine orthosemiquinone is nearly undetectable because of its high instability and low yield. One-electron reduction of aminochrome catalyzed by NADPH cytochrome P450 reductase catalyzes the formation of leukoaminochrome ortho-semiquinone, which is detected with electron spin resonance [11]. In the cytosol, the presence of superoxide dismutase and glutathione peroxidase neutralizes the formation of superoxide and hydrogen peroxide during leukoaminochrome slow autoxidation. DT-diaphorase inhibition by stable expression of an siRNA induces cell death [12]. Several lines of evidence demonstrate that DT diaphorase inhibition induces mitochondrial dysfunction. The inhibition of DTdiaphorase induces a significant decrease in mitochondrial membrane potential, swelling, and disruption of outer and inner mitochondrial membranes, which can be observed using transmission electron microscopy [13]. Another study with transmission electron microscopy showed that inhibition of DT-diaphorase induces mitochondrial damage by disrupting the external mitochondrial membrane and cristae architecture as well as disorganization of the mitochondrial matrix [14]. DT-diaphorase inhibition induces damage of mitochondrial DNA, disruption of mitochondrial membrane potential, release of cytochrome C, and mitochondria damage as determined by electron transmission microscopy [15,16]. Ninefold and sevenfold overexpression of DT-diaphorase and vesicular monoamine transporter-2 decreases cell death and mitochondrial innermembrane remodeling and reestablishes ATP levels. An in vitro study with purified human alpha-synuclein showed that DT-diaphorase prevents aminochrome-induced formation of alphasynuclein oligomers as determined by thioflavin T fluorescence, circular dichroism, western blot, and transmission electron microscopy. The silencing of expression of DT-diaphorase by an siRNA induces alphasynuclein aggregation to neurotoxic oligomers. Alpha-synuclein oligomer neurotoxicity decreases significantly in the presence of the antioligomer compound scyllo-inositol [17]. DT-diaphorase inhibition induces autophagy dysfunction by autophagosomal fusion ability and motility, leading to accumulation of autophagosomes and consequently aminochrome-induced beta-tubulin aggregation [18]. An intact microtubule network is required for the formation of autophagosome and fusion between endosomes and autophagosomes. Autophagosome motility also depends on microtubules [19]. DT-diaphorase induces beta-tubulin aggregation that disrupts the cytoskeleton architecture [20], and aminochrome induces the formation of tubulin-aminochrome adducts that inhibit tubulin polymerization and microtubule disassembly [21]. The silencing of DT-diaphorase expression

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through siRNA stable expression induces lysosomal dysfunction [22]. Aminochrome induces proteasomal dysfunction Inhibition of DTdiaphorase induces proteasome inhibition that neither catalase nor superoxide dismutase can restore. The addition of DT-diaphorase prevents aminochrome-induced proteasomal dysfunction [23]. Aminochrome one-electron reduction catalyzed by flavoenzymes using NADH or NADPH generates the formation of the leukoaminochrome ortho-semiquinone radical, which is extremely reactive with oxygen. In the presence of oxygen, leukoaminochrome ortho-semiquinone autoxidizes to aminochrome with concomitant formation of superoxide that is spontaneously or enzymatically catalyzed by superoxide dismutase to form hydrogen peroxide. In the presence of iron(II), hydrogen peroxide is converted to hydroxyl radicals that are very reactive, thus generating oxidative stress. Inhibition of DT-diaphorase induces oxidative stress when a cell line is incubated with copperedopamine complex. An electron spin resonance spectrum has revealed the presence of a C-centered radical when DT-diaphorase is inhibited [24]. The incubation of cells with aminochrome and inhibition of DTdiaphorase induce the formation of hydroperoxides and hydroxyl radicals; electron spin resonance determined that hydroxyl radicals are stabilized using spin trapping agents [13]. One-electron reduction of a mixture of aminochrome and dopamine ortho-quinone generated by dopamine oxidation in the presence of tyrosine in an electron spin resonance experiment using spin trapping agents revealed that NADPHcytochrome P450 reductase catalyzes the formation of both leukoaminochrome ortho-semiquinone and dopamine ortho-semiquinone [11]. Fig. 8.1.

Glutathione transferase-M2-2 Human glutathione transferase M2-2 catalyzes glutathione conjugation of aminochrome to 4-S-glutathionyl-5,6-dihydroxyindoline, which is resistant to biological oxidizing agents such as oxygen, hydrogen peroxide, and superoxide. This conjugate appears to be a final product, because glutathione transferase M2-2 prevents the formation of reactive oxygen species during one-electron reduction of aminochrome to leukoaminochrome orthosemiquinone catalyzed by NADPH cytochrome P450 reductase [25]. Glutathione transferase is expressed in astrocytes of human substantia nigra and is the most active isoform of glutathione transferase in aminochrome conjugation, conjugating dopachrome, noradrenochrome, and adrenochrome [25,26].

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FIGURE 8.1 DT-diaphorase protects dopaminergic neurons against aminochrome neurotoxicity. DT-diaphorase catalyzes the two-electron aminochrome reduction to hydroquinone leukoaminochrome that slowly autoxidizes, reducing oxygen to superoxide, but superoxide dismutase and glutathione peroxidase prevent the generation of oxidative stress. DT-diaphorase prevents aminochrome one-electron reduction to leukoaminochrome orthosemiquinone, a radical that it is highly reactive with oxygen, to generate a redox cycling that depletes the oxygen and NADH required for ATP production and induces oxidative stress. DT-diaphorase also prevents aminochrome-induced mitochondrial dysfunction, endoplasmic reticulum stress, alpha-synuclein aggregation to neurotoxic oligomers, autophagy dysfunction, proteasomal dysfunction, cytoskeleton disruption, and neuroinflammation.

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Glutathione transferase M2-2 can also catalyze glutathione conjugation of aminochrome precursor dopamine orthoquinone to 5-S-glutathionyldopamine [27]. Interestingly, 5-glutathionyl-dopamine is not the final product because the glutathione, tripeptide g-L-Glu-L-Cys-Gly, is degraded to cysteine conjugate. Therefore, 5-glutathionyl-dopamine is finally degraded to 5-cysteinyl-dopamine, which has been detected in human cerebrospinal fluid, brain, and neuromelanin, suggesting that glutathione transferase M2-2 plays an important role in detoxification of aminochrome neurotoxic metabolism [28e30]. The silencing of glutathione transferase M2-2 by stable expression of siRNA induces cell death and autophagy dysfunction as revealed by the accumulation of autophagic vacuoles containing undigested cellular components as determined using transmission electron microscopy. A significant decrease in lysosome pH and an increase in LAMP2 immunostaining has been observed, suggesting lysosomal dysfunction [31]. Glutathione transferase M2-2 silencing also induces cell death and mitochondrial dysfunction by decreasing mitochondrial membrane potential and ATP levels. Interestingly, the inhibition of lysosomal vacuolartype Hþ-ATPase decreases cell death and restores mitophagy, ATP levels, and mitochondrial membrane potential, suggesting that aminochromeinduced mitochondrial dysfunction is dependent on autophagy dysfunction. Glutathione transferase M2-2 prevents both mitochondrial and autophagy dysfunction, therefore suggesting a very important role in the prevention of aminochrome neurotoxicity [32,33]. Fig. 8.2.

Astrocytes neuroprotection against aminochrome neurotoxicity Several lines of evidence support the idea that astrocytes protect neurons. It has been proposed that astrocytes play an important role in regulating brain metabolism mediated by the astrocyteeneuron lactate shuttle based on lactate transfer between astrocytes and neurons [34]. This idea is controversial, and it has been proposed that astrocyte release of glutamate is the source of lactate released after neuronal activation, but increased lactate does not provide energy for neuronal activity [35]. It has been proposed that the astrocyte-neuron lactate shuttle, where astrocytes take up the glutamate released by neurons coupled with sodium that activates Na(þ)-K(þ) ATPase, further activates the transport of glucose to astrocytes. This glucose is metabolized through the glycolytic process of astrocytes, generating lactate that is released by astrocytes. This lactate is used to generate the energy in neurons that is required for synaptic activity [36].

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FIGURE 8.2 Glutathione transferase M2-2 prevents aminochrome neurotoxicity. Glutathione transferase M2-2 prevents aminochrome neurotoxicity by conjugating aminochrome to 4-S-glutathionyl-5,6-dihydroxyindoline, which is resistant to biological oxidants such as oxygen, hydrogen peroxide, and superoxide. Glutathione transferase M2-2 also conjugates the precursor of aminochrome dopamine ortho-quinone to 5-S-glutathionyl dopamine, which is degraded to 5-cysteinyl dopamine and has been detected in human neuromelanin and cerebrospinal fluid.

Astrocyteeneuron interaction plays an important role in neuron activity by trafficking metabolites from astrocytes to neurons, such as energy substrates, cholesterol, neurotransmitters such as glutamate, and the amino acids L-glutamic acid, L-cysteine, and glycine required for glutathione synthesis [37]. It has been reported that noradrenaline increases the intracellular glutathione concentration in astrocytes by stimulating b3 -adrenoceptor, and the addition of noradrenaline to mixed cultures containing astrocytes and neurons attenuates hydrogen peroxide neurotoxicity. The inhibition of b3 -adrenoceptor and glutathione synthesis abolishes the observed protective effects of noradrenaline. The inhibition of glutathione secretion from astrocytes mediated by multidrugresistance-associated protein 1 also prevents noradrenaline neuroprotection [38]. A coculture of astrocytes and neurons was shown to be more resistant to 6-hydroxydopamine neurotoxicity, suggesting a protective role of astrocytes [39].

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Astrocytes surrounding dopaminergic neurons can take up dopamine released during neurotransmission. Astrocytes express dopamine transporter and nonspecific dopamine transporters such as norepinephrine transporter, organic cation transporter 3, and plasma membrane monoamine transporter [31,40,41]. Glutathione transferase M2-2 is expressed in human astrocytes, but aminochrome is formed in dopaminergic neurons containing neuromelanin during dopamine oxidation to neuromelanin. However, astrocytes can take up dopamine by dopamine transporter or other nonspecific transporters such as norepinephrine transporter, organic cation transporter 3, and plasma membrane monoamine transporter [31,40,41]. A new astrocyte neuroprotective mechanism has been proposed for dopaminergic neurons. Dopamine uptake into astrocytes can result in dopamine oxidation to aminochrome inside astrocytes. Aminochrome increases the expression of glutathione transferase M2-2 in astrocytes that secrete this enzyme into conditioned media, and dopaminergic neurons take up this enzyme into the cytosol, protecting these neurons against aminochrome neurotoxicity [42,43]. Glutathione transferase M2-2 is secreted from astrocytes by forming exosomes that cross over the cell membrane into intersynaptic space, and later, exosomes cross over dopaminergic neuron cell membranes to deliver the enzyme into neuronal cytosol [44]. This astrocyte-mediated neuroprotective mechanism against aminochrome appears to be a very important mechanism for generating neuromelanin without inducing aminochrome neurotoxicity. Aminochrome induces mitochondrial dysfunction, protein degradation dysfunction of both the lysosomal and the proteasomal system, alphaesynuclein aggregation to neurotoxic oligomers, endoplasmic reticulum stress, neuroinflammation, disruption of cytoskeleton architecture, oxidative stress, and progressive neuronal dysfunction. We must remember that dopamine oxidation to neuromelanin is a harmless pathway in healthy seniors who have intact dopaminergic neurons containing neuromelanin in the substantia nigra. Neuromelanin is formed and accumulates in the substantia nigra throughout one’s lifetime [7] without destroying the dopaminergic neurons that contain neuromelanin because of this neuroprotective mechanism against aminochrome neurotoxicity. This neuroprotective mechanism is mediated by the enzymes of DTdiaphorase expressed in dopaminergic neurons and glutathione transferase M2-2 expressed in astrocytes and secreted through exosomes into dopaminergic neurons where aminochrome is formed during neuromelanin synthesis (Fig. 8.3).

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FIGURE 8.3 Astrocyte neuroprotective mechanism against aminochrome neurotoxicity. Astrocytes express dopamine transporter (DAT) and nonspecific dopamine transporter, such as organic cation transporter 3 (OCT3) and plasma membrane monoamine transporter (PMAT) that take up dopamine released under neurotransmission into astrocytic cytosol. Dopamine at physiological pH autoxidizes to aminochrome, but astrocytes express glutathione transferase M2-2 (GSTM2). Glutathione transferase catalyzes glutathione conjugation of both dopamine ortho-quinone and aminochrome, thus preventing aminochrome neurotoxicity. Aminochrome increases expression of the glutathione transferase M2-2 gene, resulting in an increase in the protein level encapsulated in exosomes. Astrocytes secrete glutathione transferase M2-2econtaining exosomes into synaptic interspace where exosomes cross over the dopaminergic neuron membrane to deliver this enzyme into the cytosol. Glutathione transferase M2-2 in dopaminergic neuronal cytosol conjugates dopamine orthoquinone and aminochrome to 5-glutathionyl dopamine and 4-S-glutathionyl-5,6dihydroxyindoline, respectively. 5-Glutathionyl dopamine is degraded to 5-cysteinyl-dopamine, which has been detected in the human brain, neuromelanin, and cerebrospinal liquid. 4-S-glutathionyl-5,6-dihydroxyindoline is resistant to biological oxidizing agents such as oxygen, hydrogen peroxide, and superoxide. Dopaminergic neurons express DT-diaphorase that prevent aminochrome neurotoxicity by preventing that together with glutathione transferase M2-2, which prevents the neurotoxic event that occurs with the oxidation of dopamine to neuromelanin.

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9 Exogenous neurotoxins as a preclinical model for Parkinson’s disease The exogenous neurotoxins 6-hydroxydopamine, 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP), and rotenone have been the most used preclinical models for studying the mechanisms of Parkinson’s disease and testing potential drugs for use in disease therapy.

6-Hydroxydopamine The discovery that the loss of dopaminergic neurons and reduction in dopamine levels observed in Parkinson’s disease were associated with motoric symptoms of the disease led to investigation of the degenerative process of the nigrostriatal system. It was later reported that intracerebral injection of 6-hydroxydopamine induced degeneration of the noradrenergic and dopaminergic systems. Injection of 6-hydroxydopamine into the substantia nigra induced anterograde degeneration of the nigrostriatal system. This discovery had an enormous impact on the investigation of the mechanisms of the disease and on the testing of possible new drugs for treating Parkinson’s disease [1]. One important feature of 6-hydroxydopamine is that it induces oxidative stress because of its high instability in the presence of oxygen. 6Hydroxydopamine autoxidizes to 6-hydroxidopamine quinone by reducing oxygen to superoxide, which enzymatically or nonenzymatically generates hydrogen peroxide. In the presence of iron(II), hydrogen peroxide is converted to the hydroxyl radical, which is a potent free radical that induces oxidative stress [2,3]. Reactive oxygen species are formed in the presence of oxygen both during 6-hydroxydopamine autoxidation to 6-hydroxydopamine quinone

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and during one-electron reduction of 6-hydroxydopamine quinone to 6hydroxydopamine semiquinone catalyzed by flavoenzymes that catalyze one-electron transfer from NADH in the cytosol. One-electron reduction of 6-hydroxydopamine quinone appears to be an important part of the neurotoxicity and oxidative stress induced by 6-hydroxydopamine and caused by silencing of the expression of DT-diaphorase, a unique flavoenzyme that catalyzes two-electron reduction of quinones, which significantly increases 6-hydroxydopamine neurotoxicity and oxidative stress [4,5]. Antioxidants, such as glutathione, ascorbic acid, N-acetylcysteine, and cysteine, prevent 6-hydroxydopamine-induced oxidative stress during its autoxidation in the presence of oxygen [6]. The endogenous carnosine dipeptide, which has metal ion chelation and antioxidant action, prevented 6-hydroxydopamine-induced oxidative stress, cell death, and cJun amino-terminal kinase activation [7]. The antioxidant sulfuretin significantly inhibited 6-hydroxydopamine-dependent neuronal cell death, apoptosis, neurotoxicity, and the formation of reactive oxygen species. Sulfuretin also decreased 6-hydroxydopamine-dependent mitochondrial dysfunction and phosphorylation of c-Jun N-terminal kinase [8]. 6-Hydroxydopamine autoxidation induces the formation of reactive oxygen species and the release of iron(II) from proteins that potentiate the generation of harmful hydroxyl radicals [9]. The flavone naringenin, which has antioxidant properties, decreased 6-hydroxydopaminedependent oxidative stress and 6-hydroxydopamine-induced reduction of mitochondrial membrane potential [10]. 6-Hydroxydopamine induces neuroinflammation because the nonselective cyclooxygenase inhibitor ibuprofen protects dopaminergic neurons. The use of selective inhibitors of cyclooxygenases 1 and 2 revealed that only cyclooxygenase-2 was involved in 6-hydroxydopamine neurotoxicity [11]. The cyclooxygenase-2 product prostaglandin E2 is increased in Parkinson’s disease, and it has been reported that prostaglandin E2 receptor-1 is involved in 6-hydroxydopamine-induced neurotoxicity [12]. 6-Hydroxydopamine induces an mRNA level of proinflammatory mediators such as interleukin-1b, tumor necrosis factor alpha, and interleukin6 [13]. Another study reported that 6-hydroxydopamine increased the expression of proinflammatory mediators such as inducible nitric oxide synthase, tumor necrosis factor alpha, and cyclooxygenase-2, which was inhibited by suppressing the p38 MAPK pathway [14]. 6-Hydroxydopamine induces mitochondrial dysfunction by inhibiting mitochondrial complex I activity, but this inhibitory effect is not mediated by 6-hydroxydopamine-induced oxidative stress [15,16]. 6Hydroxydopamine impairs the mitochondrial membrane [17]. 6Hydroxydopamine induces the release of TNF receptor-associated protein 1, a mitochondrial molecular chaperone, from mitochondria

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to cytosol in a study that used MN9D dopaminergic neuronal cells [18]. 6-Hydroxydopamine injection induces mitochondrial dysfunction by inhibiting complexes I, IV, and V, resulting in decreased Na þ -K þ ATPase activity, increased caspase-3 and caspase-9 activity, and increased motor, spatial memory, and olfactory impairment [19]. 6-Hydroxydopamine induces endoplasmic reticulum stress by increasing the expression of BiP and the C/EBP-homologous proteins [20]. Another study showed that 6-hydroxydopamine upregulated the expression of alpha-synuclein, high-temperature requirement protein A2, glucoseregulated protein 78,/EBP-homologous binding protein, and active caspase-3, and it reduced the levels of XIAP [21]. 6-Hydroxydopamine induced apoptosis and autophagy-lysosomal pathway dysfunction [22]. The relationship between apoptosis and endoplasmic reticulum stress was studied by determining the autophagy regulator beclin-1 and specific marker for autophagy P62, the regulator for endoplasmic reticulum stress glucose-regulated protein 78, and the marker of unfolded protein-response glucose-regulated protein 78. 6-Hydroxydopamine induced increased expression of glucose-regulated protein 78, C/EBP homologous-binding protein, and beclin-1 and decreased the expression of autophagy receptor P62, suggesting a possible link between endoplasmic reticulum stress and autophagy [23]. 6-Hydroxydopamine increases alpha-synuclein 129phosphorylation at sub-lethal doses in a nonapoptotic subpopulation of cells suggesting that alpha-synuclein phosphorylation precedes 6hydroxydopamine-induced apoptosis [24].

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine MPTP is an exogenous neurotoxin that has been used extensively to study Parkinson’s disease mechanisms and test potential drugs in Parkinson’s disease treatment. MPTP has been reported to induce severe parkinsonism in just 3 days in drug addicts who have used a synthetic drug known as China white that contained MPTP as a contaminant. MPTP’s extremely rapid inducement of parkinsonism in just 3 days contrasts with the extremely slow development of Parkinson’s disease both before and after motor symptoms. MPTP crosses the bloodebrain barrier, and monoamine oxidase-B catalyzes its conversion to the MPTP ion, which has a high affinity to dopamine transporter. This high affinity to dopamine transporter explains its neurotoxic action on dopaminergic neurons that have high expression of this transporter [25e28]. The 1-methyl-4-phenylpyridinium ions inside dopaminergic neurons accumulate in mitochondria and induce mitochondrial dysfunction by inhibiting complex I, which impairs mitochondrial ATP synthesis [29,30].

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Low concentrations of the 1-methyl-4-phenylpyridinium ion induced an increase in lactate caused by mitochondrial respiratory electron chain inhibition induced by this neurotoxin. Glucose oxidation to pyruvate cannot continue in the mitochondria due to inhibition of the respiratory electron chain, and pyruvate is converted to lactate in the cytosol, increasing glycolytic-dependent ATP formation. The inhibition of glycolysis increases 1-methyl-4-phenylpyridinium ion-induced cell death [31]. The 1-methyl-4-phenylpyridinium ion induces mitochondrial dysfunction by activating mitochondrial bioenergetics and biogenesis via activation of SIRT1, an enzyme that contributes to cellular regulation [32]. MPTP induces oxidative stress, dopamine depletion in the striatum, iron deposition, and apoptosis in the substantia nigra that are antagonized by lactoferrin. Lactoferrin decreased MPTP-dependent nigral iron accumulation by upregulating ferroportin-1, an iron export protein, and downregulating divalent metal transporter-1, an iron import protein [33]. The hippocampus is directly innervated with dopaminergic neurons that release dopamine in the hippocampus. A study on the role of MPTP on adult hippocampal neurogenesis showed that this neurotoxin induces oxidative stress and neuroinflammation [34]. MPTP induces oxidative stress accompanied with a decrease in the antioxidant enzyme superoxide dismutase and glutathione, PI3K/Akt activation, alpha-synuclein overexpression, and gliosis [35]. Another study showed that MPTP induces accumulation of hydrogen peroxide and nitric oxide as well as the inhibition of catalase, acetylcholinesterase, and glutathione transferase activity [36]. MPTP induces upregulation of NADPH oxidase, formation of reactive oxygen species, and activation of microglia in the substantia nigra pars compacta. A decrease in protein oxidation and loss of dopaminergic neurons was observed when mutant mice expressing a defective NADPH oxidase were treated with MPTP [37]. MPTP increases the level of sequestosome (also known as ubiquitinbinding protein p62), beclin-1, p-AMPK, and p-Ulk1 and decreases the level of p-mTOR, LAMP-2, and LAMP-2A, suggesting that this neurotoxin impairs autophagy [38]. MPTP induces downregulation of brainspecific microRNA-124, a noncoding RNA, accompanied with an increase in the expression of sequestosome 1 and phospho-p38 mitogenactivated protein kinases. The study concluded that microRNA-124 could inhibit neuroinflammation by targeting sequestosome 1, phospho-p38 mitogen-activated protein kinases, and autophagy [39]. MPTP decreases the expression of brain-specific microRNA-124 with a concomitant increase in calpain 1 and 2 expression that is modulated by microRNA-124 in vivo. In a cell line, the 1-methyl-4-phenylpyridinium ion increased expression of calpain p25, cyclin-dependent kinase 5, and p35 cleavage products. Knockdown of microRNA-124 resulted in increased hydrogen peroxide and reactive oxygen species [40].

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The 1-methyl-4-phenylpyridinium ion induces endoplasmic reticulum stress, activation of caspase-3, and cell death. The endoplasmic reticulum stress-related factor C/EBO homologous protein was expressed in a cell model [41]. The effect of the 1-methyl-4-phenylpyridinium ion on endoplasmic reticulum stress was studied in an in vivo model by intracerebral injection of this neurotoxin. Loss of dopaminergic neurons as well as oxidative DNA damage were observed and accompanied with endoplasmic reticulum stress response intermediated by NF-kappa B and ATF6 activation that leads to activation of GADD 153. Endoplasmic reticulum stress was accompanied with c-Jun N-terminal kinase and caspase-3 activation [42]. The 1-methyl-4-phenylpyridinium ion induces an increase in alphasynuclein expression and aggregation that depends on natural resistance-associated macrophage protein-1 downregulation [43]. Another study suggests that 1-methyl-4-phenylpyridinium ion-induced alpha-synuclein accumulation depends on nuclear factor erythroid 2related factor 2/heme oxygenase-1 pathway dysfunction [44]. MPTP induces alpha-synuclein aggregation mediated by cyclin-dependent kinase 5 activation in monkey brain because the knockdown of cyclin-dependent kinase 5 reduces alpha-synuclein aggregation [45].

Rotenone Rotenone is the classical inhibitor of mitochondrial complex I of electron transport chain that has been used to induce mitochondrial dysfunction and has been used as a preclinical model for Parkinson’s disease. Rotenone induces inhibition of mitochondrial complex I, loss of nigrostriatal neurons, rigidity, hypokinesia, and accumulation of fibrillar inclusions that contain alpha-synuclein and ubiquitin [46]. Rotenone induces neuronal loss associated with oxidative stress, ATP depletion, and endoplasmic reticulum stress and the expression of activating transcription factor 4, phospho-PERK (pancreatic endoplasmic reticulum kinase/ PKR-like endoplasmic reticulum kinase), immunoglobulin heavy-chain binding protein, and DNA damage-inducible protein/C/EBP homologous protein [47]. It has been reported that rotenone induces oxidative stress, alphasynuclein aggregate accumulation, autophagy dysfunction, and endoplasmic reticulum stress. Pretreatment of cells with geraniol before the addition of rotenone improved cell viability accompanied with a decrease in intracellular redox, autophagy flux, endoplasmic reticulum stress, and alpha-synuclein expression as well as improvement of mitochondrial membrane potential [48]. A protective effect against rotenone-induced endoplasmic reticulum stress was exerted by candesartan cilexetil, a

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high-affinity and selective Ang II receptor antagonist, by downregulating DNA damage-inducible protein/C/EBP homologous protein and activating transcription factor 4 [49]. Rotenone induces cell death by increasing the intracellular free calcium ion level and activating CaMKII, resulting in mTOR signaling inhibition and neuronal apoptosis. Rotenone-dependent mTOR inhibition and cell death were attenuated by BAPTA/AM, a chelator of calcium, inhibiting CaMKII and preventing extracellular Ca2þ influx with EGTA [50]. A microarray study performed in rotenone-treated cultured neocortical neurons showed that this neurotoxin induces major DNA changes in biological processes involved in increased expression of apoptotic genes, mitochondrial dysfunction, activation of calcium signaling, the ubiquitinproteasome system, and autophagy [51]. Rotenone induces an increase in oxidized proteins, oxidative stress, and proteasome system dysfunction by modifying the 20S beta subunit [52]. Rotenone inhibited tyrosine hydroxylase and parkin expression while it increased alpha-synuclein and PINK1 expression involved in mitochondrial dysfunction. Rotenone also impaired the expression level of AMP-activated protein kinase, mTORC, ULK1, and ATG13 involved in autophagy dysfunction. Changes in the expression of tethering proteins involved in mitochondria-associated endoplasmic reticulum contacts and signals of endoplasmic reticulum stress have been observed [53].

Exogenous neurotoxin preclinical models for Parkinson’s disease The development of new pharmacological treatments to halt the progress of Parkinson’s disease requires the use of preclinical models that replicate disease processes. Most clinical studies are based on a successful preclinical model that uses exogenous neurotoxins. The problem is that 50 years after the introduction of L-dopa in Parkinson’s disease therapy, a disease-modifying drug is still missing. All attempts to transfer successful preclinical studies based on exogenous neurotoxins to clinical studies and new drugs that can modify or halt the progress of Parkinson’s disease have failed. L-dopa continues to be the most effective drug despite severe side effects that occur following 4e6 years of chronic use [54,55]. Studies performed with 6-hydroxydopamine and MPTP with glial cell line-derived neurotrophic factor showed very promising therapeutic effects for Parkinson’s disease because this neurotropic factor regenerated damaged tissue, increased dopamine levels in the striatum and globus pallidus, and improved L-dopa-induced dyskinesia, rigidity, and bradykinesia [54,55]. However, a clinical study with a glial cell line-derived neurotrophic factor analog, neurturin, failed to demonstrate improvement when compared with control subjects [56].

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The role of oxidative stress in Parkinson’s disease has been antagonized by antioxidants such as coenzyme Q10 in the MPTP preclinical model because coenzyme Q10 prevents dopamine depletion, loss of tyrosine hydroxylase neurons, and induction of alpha-synuclein aggregation and inclusion formation in the substantia nigra pars compacta [57,58]. However, these promising studies have not translated into successful clinical trials [59]. The antagonist of adenosine 2A receptor preladenant showed very promising results against dyskinesia in preclinical models based on 6hydroxydopamine and MPTP. However, no significant differences from placebo treatment were observed in a clinical study [60e63]. Another study focused on the treatment of L-dopa-induced dyskinesia was performed using creatine. Creatine decreased L-DOPA-induced dyskinesia in preclinical models based on MPTP and 6-hydroxydopamine [64e66]. However, clinical studies did not show beneficial effects of creatine in Parkinson’s disease [67]. Neuroinflammation is considered a mechanism involved in the neurodegeneration of nigrostriatal dopaminergic neurons and peroxisome proliferator-activated receptor ligands. Pioglitazone, a peroxisome proliferator-activated receptor gamma agonist, demonstrated a successful antiinflammatory effect in an MPTP preclinical model by decreasing the activation of microglia, induction of iNOS-positive cells, and number of glial fibrillary acidic protein positive cells in both the substantia nigra pars compacta and the striatum. A multicenter, randomized trial and doubleblind clinical study with pioglitazone did not observe modified progression in early Parkinson’s disease and did not recommend a larger trial in patients [68e70]. The question is why we cannot translate successful preclinical studies based on exogenous neurotoxins to new disease-modifying therapy in Parkinson’s disease. The failure of clinical studies attempting to find new therapies [71e73] to modify the progression of Parkinson’s disease has opened a discussion in the scientific community about finding and understanding the reasons for these failures. Many questions have been raised, such as whether Parkinson’s disease is one or more disorders or whether alpha-synuclein aggregated in Lewy bodies by neurites are essential in the etiology of all synucleinopathies. Parkinson’s disease is considered a synucleinopathy whereby the formation of Lewy bodies spreads from one region of the brain to another according to the Braak scale. However, there are studies that do not show a correlation between the Braak scale and dopaminergic neuron density in the substantia nigra. There are patients with familial Parkinson’s with parkin or leucine-rich repeat kinase 2 mutations who do not have Lewy bodies [74]. Regardless of whether Parkinson’s disease is several disorders under the same name, all those affected develop motor symptoms caused by the loss of dopaminergic neurons in the nigrostriatal system. All preclinical

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models with exogenous neurotoxins are based on the loss of dopaminergic neurons from the nigrostriatal system. The question is what triggers the loss of dopaminergic neurons in the nigrostriatal system that contain neuromelanin. Exogenous neurotoxins induce mitochondrial dysfunction, the addition of alpha-synuclein to neurotoxic oligomers, the dysfunction of both lysosomal and proteasomal protein degradation systems, endoplasmic reticulum stress, neuroinflammation, and oxidative stress that finally kill dopaminergic neurons that contain neuromelanin. The problem lies in determining what triggers these neurotoxic mechanisms in the dopaminergic neurons of the nigrostriatal system that end in the loss of dopaminergic neurons that contain neuromelanin. Are they aggregations of alpha-synuclein that spread from other regions to the nigrostriatal system, or are they metals or environmental pollutants that trigger these neurotoxic mechanisms? The fulminant effect of MPTP in humans who have ingested illicit drugs contaminated with this compound, and who have developed parkinsonism in just 3 days [26], suggests that the neurotoxin that triggers the loss of dopaminergic neurons in the nigrostriatal system cannot be exogenous because the progress of the disease takes years. This suggests that the loss of dopaminergic neurons in the nigrostriatal system must be induced by an endogenous neurotoxin that triggers neurotoxic mechanisms that end in the death of a single neuron. Being an endogenous neurotoxin, its neurotoxic effects are not expansive to affect other neurons as exogenous neurotoxins do. This implies that the selective loss of a single neuron would require years to accumulate enough neuronal loss to generate motor symptoms that occur as they do in Parkinson’s disease. To have a preclinical model that represents the process of disease progression in Parkinson’s disease that we can use to develop new drugs to halt the development of the disease, it is not enough to have a neurotoxin that induces the loss of the nigrostriatal system and neurotoxic mechanisms. It is critical to determine what triggers these neurotoxic mechanisms. The death of dopaminergic neurons is a chain of events wherein mitochondrial dysfunction, the formation of neurotoxic alphasynuclein oligomers, dysfunction of protein degradation, endoplasmic reticulum stress, neuroinflammation, and oxidative stress ultimately kill these neurons. The problem is that if the drugs do not aim to inhibit or prevent the first event that triggers the neurotoxic mechanisms, therapy ultimately does not have its expected effect on patients. It is useless to have a preclinical model that induces neuronal loss and other neurotoxic mechanisms if the exogenous neurotoxin does not exist in the patient because the preclinical study assumed that a drug applied to dopaminergic neurons in the nigrostriatal system would prevent neuronal death caused by a neurotoxin that will not be present in the clinical study.

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[51] Yap YW, Chen MJ, Peng ZF, Manikandan J, Ng JM, Llanos RM, La Fontaine S, Beart PM, Cheung NS. Gene expression profiling of rotenone-mediated cortical neuronal death: evidence for inhibition of ubiquitin-proteasome system and autophagy-lysosomal pathway, and dysfunction of mitochondrial and calcium signaling. Neurochem Int 2013;62:653e63. [52] Shamoto-Nagai M, Maruyama W, Kato Y, Isobe K, Tanaka M, Naoi M, Osawa T. An inhibitor of mitochondrial complex I, rotenone, inactivates proteasome by oxidative modification and induces aggregation of oxidized proteins in SH-SY5Y cells. J Neurosci Res 2003;74:589e97. [53] Ramalingam M, Huh YJ, Lee YI. The impairments of a-synuclein and mechanistic target of rapamycin in rotenone-induced SH-SY5Y cells and mice model of Parkinson’s disease. Front Neurosci 2019;13:1028. [54] Segura-Aguilar J. The importance of choosing a preclinical model that reflects what happens in Parkinson’s disease. Neurochem Int 2019;126:203e9. [55] Segura-Aguilar J. Neurotoxins as preclinical models for Parkinson’s disease. Neurotox Res 2018;34:870e7. [56] Olanow CW, Bartus RT, Volpicelli-Daley LA, Kordower JH. Trophic factors for Parkinson’s disease: to live or let die. Mov Disord 2015;30:1715e24. [57] Cleren C, Yang L, Lorenzo B, Calingasan NY, Schomer A, Sireci A, Wille EJ, Beal MF. Therapeutic effects of coenzyme Q10 (CoQ10) and reduced CoQ10 in the MPTP model of Parkinsonism. J Neurochem 2008;104:1613e21. [58] Beal MF, Matthews RT, Tieleman A, Shults CW. Coenzyme Q10 attenuates the 1methyl-4-phenyl-1,2,3,tetrahydropyridine (MPTP) induced loss of striatal dopamine and dopaminergic axons in aged mice. Brain Res 1998;783:109e14. [59] Parkinson Study Group QE3 Investigators, Beal MF, Oakes D, Shoulson I, et al. A randomized clinical trial of high-dosage coenzyme Q10 in early Parkinson disease: no evidence of benefit. JAMA Neurol 2014;71:543e52. [60] Hodgson RA, Bedard PJ, Varty GB, Kazdoba TM, Di Paolo T, Grzelak ME, Pond AJ, Hadjtahar A, Belanger N, Gregoire L, Dare A, Neustadt BR, Stamford AW, Hunter JC. Preladenant, a selective A(2A) receptor antagonist, is active in primate models of movement disorders. Exp Neurol 2010;225. 384-3. [61] Hauser RA, Stocchi F, Rascol O, Huyck SB, Capece R, Ho TW, Sklar P, Lines C, Michelson D, Hewitt D. Preladenant as an adjunctive therapy with levodopa in Parkinson disease: two randomized clinical trials and lessons learned. JAMA Neurol 2015; 72(12):1491e500. [62] Pinna A, Ko WK, Costa G, Tronci E, Fidalgo C, Simola N, Li Q, Tabrizi MA, Bezard E, Carta M, Morelli M. Antidyskinetic effect of A2A and 5HT1A/1B receptor ligands in two animal models of Parkinson’s disease. Mov Disord 2016;31:501e11. [63] Stocchi F, Rascol O, Hauser RA, Huyck S, Tzontcheva A, Capece R, Ho TW, Sklar P, Lines C, Michelson D, Hewitt DJ, Preladenant Early Parkinson Disease Study Group. Randomized trial of preladenant, given as monotherapy, in patients with early Parkinson disease. Neurology 2017;88:2198e206. [64] Valastro B, Dekundy A, Danysz W, Quack G. Oral creatine supplementation attenuates L-dopa-induced dyskinesia in 6-hydroxydopamine-lesioned rats. Behav Brain Res 2009;197:90e6. [65] Matthews RT, Ferrante RJ, Klivenyi P, Yang L, Klein AM, Mueller G, KaddurahDaouk R, Beal MF. Creatine and cyclocreatine attenuate MPTP neurotoxicity. Exp Neurol 1999;157:142e9. [66] Beal MF. Neuroprotective effects of creatine. Amino Acids 2011;40:1305e13. [67] Writing Group for the NINDS Exploratory Trials in Parkinson Disease (NET-PD) Investigators, Kieburtz K, Tilley BC, Elm JJ, Babcock D, Hauser, et al. Effect of creatine monohydrate on clinical progression in patients with Parkinson disease: a randomized clinical trial. J Am Med Assoc 2015;313:584e93.

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[68] Breidert T, Callebert J, Heneka MT, Landreth G, Launay JM, Hirsch EC. Protective action of the peroxisome proliferator-activated receptor-gamma agonist pioglitazone in a mouse model of Parkinson’s disease. J Neurochem 2002;82:615e24. [69] Dehmer T, Heneka MT, Sastre M, Dichgans J, Schulz JB. Protection by pioglitazone in the MPTP model of Parkinson’s disease correlates with I kappa B alpha induction and block of NF kappa B and iNOS activation. J Neurochem 2004;88:494e501. [70] NINDS Exploratory Trials in Parkinson Disease (NET-PD) FS-ZONE Investigators. Pioglitazone in early Parkinson’s disease: a phase 2, multicentre, double-blind, randomised trial. Lancet Neurol 2015;14:795e803. [71] Athauda D, Foltynie T. The ongoing pursuit of neuroprotective therapies in Parkinson disease. Nat Rev Neurol 2015;11:25e40. [72] Lindholm D, Ma¨kela¨ J, Di Liberto V, Mudo` G, Belluardo N, Eriksson O, Saarma M. Current disease modifying approaches to treat Parkinson’s disease. Cell Mol Life Sci 2015; 73:1365e79. [73] Park A, Stacy M. Disease-modifying drugs in Parkinson’s disease. Drugs 2015;75: 2065e71. [74] Espay AJ, Kalia LV, Gan-Or Z, Williams-Gray CH, Bedard PL, Rowe SM, Morgante F, et al. Disease modification and biomarker development in Parkinson disease: revision or reconstruction? Neurology 2020;94:481e94.

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10 Preclinical models based on genetic mutations associated with the familial form of Parkinson’s disease The discovery of gene mutations associated with familial forms of Parkinson’s disease has been an enormous new input into this field of research because it has revealed several proteins associated with the disease. The research community has begun to create preclinical models based on mutations associated with the familial form of the disease. Several transgenic animals have been developed from gene mutations associated with the familial form of Parkinson’s disease [1]. A transgenic mouse expressing wild-type human alpha-synuclein developed cytoplasmatic inclusions containing alpha-synuclein and ubiquitin in neurons in the substantia nigra, neocortex, and hippocampus [2]. A mouse with alpha-synuclein deletion resulted in a viable and fertile animal with normal brain architecture, dopaminergic cell bodies, fibers, and synapses. Dopaminergic neurons presented a reduction in striatal dopamine and a decrease in dopamine-dependent locomotor activity, suggesting that alpha-synuclein plays a role in presynaptic activity [3]. The expression of the human alpha-synuclein gene coding for A53T mutation in mice showed neuronal alpha-synucleinopathy, neuronal degeneration, motor impairment with denervation of neuromuscular junctions, and axonal damage [4]. A transgenic mouse expressing alphasynuclein mutation A30P showed its accumulation in neuronal cell bodies and neurites in the brain and its synaptic colocalization with betasynuclein and synaptophysin [5]. A comparison between overexpressing alpha-synuclein human A53T and A30P mutations and wild-type mice showed that human A53T mutant alpha-syn neurons presented Lewy body-like inclusions, nuclear condensation, large axonal swellings, and

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somal chromatolytic changes in brain stem and spinal cord. An extensive depletion of motor neurons and axonal degeneration were observed accompanied with mitochondrial DNA damage and decreased activity of mitochondrial complex IV. Mice expressing human alpha-synuclein A30P mutant exhibited less effects than mice expressing A53T mutation [6]. Another study comparing the effect of alpha-synuclein human A53T and A30P mutations and wild-type revealed that only mice expressing A53T mutation presented postsynaptic dysfunction, long-term potentiation and spatial learning impairment, and tau phosphorylationdependent postsynaptic dysfunction [7]. A proteomic analysis of a mouse overexpressing human A30P*A53T alpha-synuclein mutations showed alteration (upregulation or downregulation) in proteins related to endoplasmic reticulum stress, ubiquitin-proteasome system impairment, mitochondrial dysfunction, and oxidative stress [8]. A transgenic mouse with knockout of parkin and parkin-associated endothelin receptor-like receptor presented progressive and early loss of dopaminergic as well as noradrenergic neurons in the absence of inclusion-body formation. Upregulation of dopamine metabolism, mitochondrial dysfunction, oxidative stress, and progressive activation of unfolded protein response suggesting endoplasmic reticulum stress was also observed [9]. Parkin knockout mice showed an accelerated microtubule acetylation in dopaminergic neurons in the substantia nigra and striatum, changing microtubule stability and inducing alterations in mitochondria transport and mobility [10]. Lack of parkin in Parkinson’s disease-mito-PstI mouse induces early neurodegeneration of dopaminergic neurons, changes in mitochondrial morphology, and double-strand breaks only in the mitochondrial DNA of dopaminergic neurons [11]. Knockout of parkin gene in mice induced lower expression of glial fibrillary acidic protein, class III beta tubulin, and neurofilament, while p21 protein was accumulated in the neural stem cells of parkin knockout mice. c-Jun N-terminal kinase inhibitors reversed decreased p21 ubiquitination and differentiation ability [12]. The expression of C-terminaltruncated human mutant parkin (parkin-Q311X) in a transgenic mouse model induces progressive late-onset hypokinetic motor deficits, loss of dopaminergic neurons in substantia nigra and dopamine terminals in striatum, and reduction of dopamine levels in the striatum [13]. Expression of parkin-Q311X mutation in mice induces a reduction in the expression of the signaling pathway related to the lysosomal regulator transcription factor EB and the master mitochondrial regulator peroxisome proliferator-activated receptor-gamma coactivator and increases PARIS protein expression [14].

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Mice expressing parkin or PTEN-induced kinase 1 gene deletion induce neuroinflammation and accumulation of mitochondrial DNA mutations. Parkin deletion-dependent loss of dopaminergic neurons in substantia nigra is prevented by the loss of the central regulator of the type I interferon (STING) response to cytosolic DNA [15]. Deletion of the PTEN-induced kinase 1 gene in mice showed no differences in tyrosine hydroxylase mRNA expression or catecholamine protein concentrations in the ventral tegmental area, but GABA-related genes were upregulated [16]. This knockout also induces early and progressive motor, alphassynuclein aggregation, and vocal deficits [17]. A proteomic analysis performed in PTEN-induced kinase 1 deficient mice showed a decrease in synaptic plasticity, aberrant cellular signaling, increased oxidative stress, diminished proteostasis networks, altered neuronal structure, reduced neurotransmission, and altered metabolism [18]. The expression of leucine-rich repeat kinase 2 G2019S mutations in mice induces degeneration of dopaminergic and noradrenergic neurons, alpha-synuclein aggregation, reduction in synaptic vesicle number, and an increase of clathrin-coated vesicles in dopaminergic neurons [19]. Another study with mice expressing the same leucine-rich repeat kinase 2 mutation demonstrated a strong degeneration of substantia nigra dopaminergic neuron degeneration; the addition of a selective kinase inhibitor decreases the effect of this mutation. The authors suggest that the animal model is suitable for preclinical studies for disease-modifying therapy [20]. Leucine-rich repeat kinase-2 and kinase-1 deficient mice presented selective neurodegeneration in dopaminergic neurons in the substantia nigra pars compacta and of noradrenergic neurons in the locus coeruleus, an increase in apoptosis and alpha-synuclein levels, and autophagy impairment [21]. Another study with leucine-rich repeat kinase 2 knockout mice exhibited autophagy-lysosomal pathway dysfunction as indicated by lipofuscin granule accumulation and altered levels of LC3-II and p62 [22]. Neuronal ubiquitin-positive inclusions, neurite degeneration, and altered distribution of axonal phosphorylated neurofilaments was observed when leucine-rich repeat kinase-2 G2019S mutation was expressed in rodents [23]. Protein deglycase-1 knockout rats developed a progressive degeneration of dopaminergic neurons in substantia nigra pars compacta with significant loss of dopamine neurons, motor impairment, decreased glutamate release, and increased acetylcholine [24,25]. A study with protein deglycase-1 knockout suggested that its protective response to oxidative stress regulates the expression of uncoupling protein 4 by

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oxidation of protein deglycase and partially via the NF-kB pathway [26]. Knockout of protein deglycase-1 induces accumulation of dysfunctional mitochondria and autophagy impairment [27]. Transgenic animals have also been very useful for understanding the mechanisms involved with some mutations associated with a familial form of Parkinson’s disease such as alpha-synuclein, parkin, leucine-rich repeat kinase 2, protein deglycase-1, and PTEN-induced kinase 1, but they fail to replicate the true pathophysiology occurring in idiopathic Parkinson’s disease, explaining why successful results obtained with these animal models cannot be translated to clinical studies [28]. In idiopathic Parkinson’s disease, we know that the degenerative process of the disease involves mitochondrial dysfunction, oxidative stress, neuroinflammation, endoplasmic reticulum stress, diffusion of both lysosomal and proteasomal protein degradation systems, and aggregation of alpha-synuclein to neurotoxic oligomers, but what triggers these mechanisms is still unknown. The slow progression of the neurodegenerative process and of the disease takes years, suggesting that the neurotoxin that triggers the different mechanisms associated with the neurodegenerative process must be of endogenous origin, because exogenous neurotoxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine induce severe parkinsonism in just 3 days [29]. It seems possible that an endogenous molecule of the same dopaminergic neurons under certain conditions can become an endogenous neurotoxin that causes the death of a single affected neuron without producing an expansive process that kills many neurons around it. Over the years, the loss of dopaminergic neurons containing neuromelanin could accumulate until the motor symptoms appear. Preclinical models based on genetic mutations cannot be useful for testing a new drug that can halt the progression of idiopathic Parkinson’s disease. In the familial form of Parkinson’s disease associated with gene mutations, the cause of this pathology is a mutation that itself finally develops disease symptoms. In the case of familial Parkinson’s associated with mutations in the gene for alpha synuclein, we know that the cause of the disease is a mutation of this gene that induces alpha-synuclein aggregation to oligomers resulting in synaptic dysfunction, endoplasmic reticulum stress, autophagy dysregulation, mitochondrial dysfunction, neuroinflammation, and oxidative stress [30,31]. Therefore, successful preclinical studies based on alpha-synuclein mutations or other genes can never result in successful clinical studies for idiopathic Parkinson’s disease because they do not include what triggers the neurotoxic mechanisms involved in the neurodegeneration Fig. 10.1.

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FIGURE 10.1

Can we use preclinical models based on mutations associated with a familial form of the disease to test new drugs for idiopathic Parkinson’s disease? The preclinical model based on a mutation of alpha-synuclein gene induces the formation of neurotoxic oligomers that develop synaptic dysfunction, endoplasmic reticulum stress, autophagy dysregulation, mitochondrial dysfunction, neuroinflammation, and oxidative stress. These neurotoxic mechanisms degenerate dopaminergic neurons containing neuromelanin that finally develop a familial form of Parkinson’s disease (A). In idiopathic Parkinson’s disease, neurodegeneration and disease progression are very slow, suggesting that an endogenous molecule of dopaminergic neurons containing neuromelanin is converted to an endogenous neurotoxin under certain exceptional conditions. A preclinical model that can produce successful results that can be translated to clinical studies and finally new drugs to halt Parkinson’s disease progression requires that this model includes the neurotoxin that triggers the degeneration of nigrostriatal neurons. A sequence of events occurs before Parkinson’s symptoms develop that includes conversion of an endogenous molecule to an endogenous neurotoxin that triggers alpha-synuclein aggregation to a neurotoxic oligomer, mitochondria dysfunction, endoplasmic reticulum stress, protein degradation dysfunction of both lysosomal and proteasomal systems, neuroinflammation, and oxidative stress. These neurotoxic mechanisms induce loss of a single dopaminergic neuron containing neuromelanin that accumulates over years until motor symptoms appear (B). Therefore, a preclinical model based on mutations associated with the familial form of the disease or exogenous neurotoxins cannot translate successful results to clinical studies and new therapies because these preclinical models have not included the trigger for the mechanisms that induce degeneration of dopaminergic neurons in patients with idiopathic Parkinson’s disease. A preclinical model that can translate successful results to clinical studies and new therapies to halt the disease progression will require the inclusion of the neurotoxin that triggers those neurotoxic mechanisms that end with the loss of dopaminergic neurons containing neuromelanin.

References [1] Koprich JB, Kalia LV, Brotchie JM. Animal models of a-synucleinopathy for Parkinson disease drug development. Nat Rev Neurosci 2017;18:515e29. [2] Masliah E, Rockenstein E, Veinbergs I, Mallory M, Hashimoto M, Takeda A, Sagara Y, Sisk A, Mucke L. Dopaminergic loss and inclusion body formation in alpha-synuclein mice: implications for neurodegenerative disorders. Science 2000;287:1265e9.

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[3] Abeliovich A, Schmitz Y, Farin˜as I, Choi-Lundberg D, Ho WH, Castillo PE, Shinsky N, Verdugo JM, Armanini M, Ryan A, Hynes M, Phillips H, Sulzer D, Rosenthal A. Mice lacking alpha-synuclein display functional deficits in the nigrostriatal dopamine system. Neuron 2000;25:239e52. [4] van der Putten H, Wiederhold KH, Probst A, Barbieri S, Mistl C, Danner S, Kauffmann S, Hofele K, Spooren WP, Ruegg MA, Lin S, Caroni P, Sommer B, Tolnay M, Bilbe G. Neuropathology in mice expressing human alpha-synuclein. J Neurosci 2000;20:6021e9. [5] Kahle PJ, Neumann M, Ozmen L, Muller V, Jacobsen H, Schindzielorz A, Okochi M, Leimer U, van Der Putten H, Probst A, Kremmer E, Kretzschmar HA, Haass C. Subcellular localization of wild-type and Parkinson’s disease-associated mutant alpha -synuclein in human and transgenic mouse brain. J Neurosci 2000;20:6365e73. [6] Martin LJ, Pan Y, Price AC, Sterling W, Copeland NG, Jenkins NA, Price DL, Lee MK. Parkinson’s disease alpha-synuclein transgenic mice develop neuronal mitochondrial degeneration and cell death. J Neurosci 2006;26:41e50. [7] Teravskis PJ, Covelo A, Miller EC, Singh B, Martell-Martı´nez HA, Benneyworth MA, Gallardo C, Oxnard BR, Araque A, Lee MK, Liao D. A53T mutant alpha-synuclein induces tau-dependent postsynaptic impairment independently of neurodegenerative changes. J Neurosci 2018;38:9754e67. [8] Yan J, Zhang P, Jiao F, Wang Q, He F, Zhang Q, Zhang Z, Lv Z, Peng X, Cai H, Tian B. Quantitative proteomics in A30P*A53T a-synuclein transgenic mice reveals upregulation of Sel1l. PLoS One 2017;12:e0182092. [9] Wang HQ, Imai Y, Inoue H, Kataoka A, Iita S, Nukina N, Takahashi R. Pael-R transgenic mice crossed with parkin deficient mice displayed progressive and selective catecholaminergic neuronal loss. J Neurochem 2008;107:171e85. [10] Cartelli D, Amadeo A, Calogero AM, Casagrande FVM, De Gregorio C, Gioria M, Kuzumaki N, Costa I, Sassone J, Ciammola A, Hattori N, Okano H, Goldwurm S, Roybon L, Pezzoli G, Cappelletti G. Parkin absence accelerates microtubule aging in dopaminergic neurons. Neurobiol Aging 2018;61:66e74. [11] Pinto M, Nissanka N, Moraes CT. Lack of parkin anticipates the phenotype and affects mitochondrial morphology and mtDNA levels in a mouse model of Parkinson’s disease. J Neurosci 2018;38:1042e53. [12] Park MH, Lee HJ, Lee HL, Son DJ, Ju JH, Hyun BK, Jung SH, Song JK, Lee DH, Hwang CJ, Han SB, Kim S, Hong JT. Parkin knockout inhibits neuronal development via regulation of proteasomal degradation of p21. Theranostics 2017;7:2033e45. [13] Lu XH, Fleming SM, Meurers B, Ackerson LC, Mortazavi F, Lo V, Hernandez D, Sulzer D, et al. J Neurosci 2009;29:1962e76. [14] Siddiqui A, Bhaumik D, Chinta SJ, Rane A, Rajagopalan S, Lieu CA, Lithgow GJ, Andersen JK. Mitochondrial quality control via the PGC1a-TFEB signaling pathway is compromised by parkin Q311X mutation but independently restored by rapamycin. J Neurosci 2015;35:12833e44. [15] Sliter DA, Martinez J, Hao L, Chen X, Sun N, Fischer TD, Burman JL, Li Y, Zhang Z, Narendra DP, Cai H, Borsche M, Klein C, Youle RJ. Parkin and PINK1 mitigate STING-induced inflammation. Nature 2018;561:258e62. [16] Stevenson SA, Ciucci MR, Kelm-Nelson CA. Intervention changes acoustic peak frequency and mesolimbic neurochemistry in the Pink1-/- rat model of Parkinson disease. PLoS One 2019;14:e0220734. [17] Kelm-Nelson CA, Stevenson SA, Ciucci MR. Atp13a2 expression in the periaqueductal gray is decreased in the Pink1 -/- rat model of Parkinson disease. Neurosci Lett 2016; 621:75e82. [18] Triplett JC, Zhang Z, Sultana R, Cai J, Klein JB, Bu¨eler H, Butterfield DA. Quantitative expression proteomics and phosphoproteomics profile of brain from PINK1 knockout

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mice: insights into mechanisms of familial Parkinson’s disease. J Neurochem 2015;133: 750e65. Xiong Y, Neifert S, Karuppagounder SS, Liu Q, Stankowski JN, Lee BD, Ko HS, Lee Y, Grima JC, et al. Robust kinase- and age-dependent dopaminergic and norepinephrine neurodegeneration in LRRK2 G2019S transgenic mice. Proc Natl Acad Sci U S A 2018; 115:1635e40. Nguyen APT, Tsika E, Kelly K, Levine N, Chen X, West AB, Boularand S, Barneoud P, Moore DJ. Dopaminergic neurodegeneration induced by Parkinson’s disease-linked G2019S LRRK2 is dependent on kinase and GTPase activity. Proc Natl Acad Sci U S A 2020;117:17296e307. Giaime E, Tong Y, Wagner LK, Yuan Y, Huang G, Shen J. Age-dependent dopaminergic neurodegeneration and impairment of the autophagy-lysosomal pathway in LRRKdeficient mice. Neuron 2017;96:796e807.e6. Tong Y, Yamaguchi H, Giaime E, Boyle S, Kopan R, Kelleher 3rd RJ, Shen J. Loss of leucine-rich repeat kinase 2 causes impairment of protein degradation pathways, accumulation of alpha-synuclein, and apoptotic cell death in aged mice. Proc Natl Acad Sci U S A 2010;107:9879e84. Tsika E, Nguyen AP, Dusonchet J, Colin P, Schneider BL, Moore DJ. Adenoviralmediated expression of G2019S LRRK2 induces striatal pathology in a kinasedependent manner in a rat model of Parkinson’s disease. Neurobiol Dis May 2015; 77:49e61. Giangrasso DM, Furlong TM, Keefe KA. Characterization of striatum-mediated behavior and neurochemistry in the DJ-1 knock-out rat model of Parkinson’s disease. Neurobiol Dis 2020;134:104673. Creed RB, Menalled L, Casey B, Dave KD, Janssens HB, Veinbergs I, van der Hart M, Rassoulpour A, Goldberg MS. Basal and evoked neurotransmitter levels in parkin, DJ-1, PINK1 and LRRK2 knockout rat striatum. Neuroscience 2019;409:169e79. Xu S, Yang X, Qian Y, Xiao Q. Parkinson’s disease-related DJ-1 modulates the expression of uncoupling protein 4 against oxidative stress. J Neurochem 2018;145:312e22. Krebiehl G, Ruckerbauer S, Burbulla LF, Kieper N, Maurer B, Waak J, Wolburg H, Gizatullina Z, Gellerich FN, Woitalla D, Riess O, Kahle PJ, Proikas-Cezanne T, Kru¨ger R. Reduced basal autophagy and impaired mitochondrial dynamics due to loss of Parkinson’s disease-associated protein DJ-1. PLoS One 2010;5:e9367. Potashkin JA, Blume SR, Runkle NK. Limitations of animal models of Parkinson’s disease. Parkinsons Dis 2010;2011:658083. Williams A. MPTP toxicity: clinical features. J Neural Transm Suppl 1986;20:5e9. Fields CR, Bengoa-Vergniory N, Wade-Martins R. Targeting alpha-synuclein as a therapy for Parkinson’s disease. Front Mol Neurosci 2019;12:299. Williams GP, Schonhoff AM, Jurkuvenaite A, Thome AD, Standaert DG, Harms AS. Targeting of the class II transactivator attenuates inflammation and neurodegeneration in an alpha-synuclein model of Parkinson’s disease. J Neuroinflammation 2018;15:244.

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11 Preclinical models based on endogenous neurotoxins Clinical studies based on successful preclinical studies with exogenous neurotoxins have failed because the preclinical model does not include the triggers of the degenerative process of neuromelanin-containing dopaminergic neurons of the nigrostriatal system. Exogenous neurotoxins do not exist in the nigrostriatal system, and therefore when a new drug is tested in a preclinical model with exogenous neurotoxins, it is not possible to determine whether the drug can prevent the neurotoxin that triggers the neurodegenerative process. Therefore, preclinical modeling of the degenerative process in the nigrostriatal system must include the neurotoxin that triggers it. To choose a preclinical system that reflects the processes of the disease, the following should be considered: • (1) The exogenous neurotoxin 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine induced severe parkinsonism in a human exposed to this synthetic drug contaminant in only 3 days [1], in contrast to the extremely slow progression of neurodegeneration both before and after motor symptoms, suggesting that the neurotoxin that triggers the neurotoxic mechanisms involved in degeneration of the nigrostriatal system cannot be of exogenous origin. • (2) The slow accumulated loss of neuromelanin-containing dopaminergic neurons, which takes years before reaching the 60%e70% threshold required for motor symptoms to appear, suggests that the endogenous neurotoxin must be generated within the lost dopaminergic neurons. The fact that a molecule of a dopaminergic neuron becomes an endogenous neurotoxin and induces the death of the dopaminergic neuron is an exclusive and nonexpansive event that do not affects neighboring neurons. After the death of this single dopaminergic neuron, the microglia remove this dead neuron. Because these neurotoxic events are not expansive, the accumulation of the loss of dopaminergic neurons

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containing neuromelanin is an extremely slow process that occurs for many years before motor symptoms appear. Possible neurotoxins generated within dopaminergic neurons containing neuromelanin include alpha-synuclein, 3,4-dihydroxyphenylacetaldehyde, and aminochrome [2e5].

Alpha-synuclein Alpha-synuclein is an endogenous neurotoxin when it aggregates, and two models of neurotoxicity have been proposed: alpha-synuclein aggregation to fibrils that accumulate in Lewy bodies that propagate from other brain regions, such as the olfactory bulb [6], to nigrostriatal neurons as well as alpha-synuclein aggregation to neurotoxic oligomers inside dopaminergic neurons that induce synaptic dysfunction, endoplasmic reticulum stress, autophagy dysregulation, mitochondrial dysfunction, and oxidative stress [7].

Alpha-synuclein aggregation and accumulation in Lewy bodies In the Parkinson’s disease-stages hypothesis, the formation of Lewy bodies plays a key role in the development and propagation of the disease from different regions [8,9]. The formation of Lewy bodies begins in the olfactory bulb and is related to premotor symptoms. The formation of these Lewy bodies extends to other regions until they reach the nigrostriatal system to generate premotor symptoms. This has been called the prion-like hypothesis. The authors of the prion-like hypothesis propose that alpha-synuclein assemblies can act as seeds that promote aggregation. In the neuronal assembly, alpha-synuclein is released into extracellular space where other neurons take up the assembled alpha-synuclein and continue its propagation [10,11]. Alpha-synuclein is a monomeric protein localized in neuronal cytosol, but much evidence shows that alpha-synuclein can be found in human cerebrospinal fluids and blood and in both monomeric and oligomeric forms [12e14]. It has been reported that fibrillar alpha-synuclein is internalized by primary neurons and transported in axons using anterograde and retrograde transport [15]. Extracellular alpha-synuclein is internalized into cells through endocytosis, and inhibitors of intracellular endocytosis decrease extracellular uptake into the cells [16,17]. Whether the propagation of alpha-synuclein fibrils from one to another neuron requires neuronal degeneration is controversial because alpha-synuclein fibrils can be transported in both retrograde and anterograde directions and secreted by axons after anterograde transport without degeneration, suggesting that transneuronal propagation occurs in intact healthy neurons [18].

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Neuron-derived exosomes containing alpha-synuclein seem to mediate the propagation of alpha-synuclein from neurons to other neurons, microglia, and astrocytes. Erythrocytes contain around a 1000-fold higher alpha-synuclein concentration than in the cerebrospinal fluid in generating extracellular vesicles containing alpha-synuclein. Extracellular vesicles, probably exosomes, containing alpha-synuclein from erythrocytes can cross the bloodebrain barrier and deliver alphasynuclein to the central nervous system. An increase in microglial inflammatory response was observed when erythrocyte extracellular vesicles crossed over the bloodebrain barrier [19]. 4-Hydroxynonenal, a lipid peroxidation product, induces aggregation of alpha-synuclein and activates the secretion of extracellular vesicles containing neurotoxic alpha-synuclein oligomers [20]. Alpha-synuclein oligomers have been detected in saliva samples from Parkinson’s disease patients [21]. Alphasynuclein aggregates, when their function is lost, are secreted to extracellular space using extracellular vesicles [22]. However, reports suggest that alpha-synuclein propagation is mediated by neuronal degeneration. Intraperitoneal injection of monoclonal antibodies against misfolded alpha-synuclein decreases Lewy bodies and Lewy neurite-like formation and degeneration of dopaminergic neurons in substantia nigra and improves motor deficiency in mice with intrastriatal injection of alphasynuclein synthetic preformed fibrils [23]. Lymphocyte-activation gene-3 promotes alpha-synuclein misfolded preformed fibril endocytosis, transmission, and toxicity. The absence of lymphocyte-activation gene-3 decreases alpha-synuclein misfolded preformed fibril-induced degeneration of dopamine neurons [24]. The transfer of alpha-synuclein to glia cells induces inflammation that may be propagated to neurons and glia cells [25]. The addition of extracellular alpha-synuclein to the culture medium induces cytotoxicity [26,27]. Alphasynuclein aggregates attached to heparan sulfate proteoglycans promote the uptake of these aggregates via endocytosis to new neurons [28]. Extracellular alpha-synuclein induces increased calcium influx, plasma membrane receptor activation, synthesis of nitric oxide, and proinflammatory cytokines triggering apoptosis [29]. There is increasing evidence that the formation of fibrils or its deposits is not involved in alpha-synucleindependent neurotoxicity, while prefibrillar species, oligomers, have been reportedly involved in the neurotoxic effects of alpha-synuclein [30]. The problems with the prion-like hypothesis can be summarized as follows. (1) Fibrils formed by this process eventually constitute the largest component of Lewy bodies. According to the hypothesis, prion-like fibrils are secreted from one neuron and transferred to another neuron and then from the olfactory bulb to other neurons and regions of the brain. This hypothesis is based on studies by Braak carried out on postmortem brains in different stages of progression of Parkinson’s disease where the bodies of

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Lewy and Lewy neurites were detected [9]. However, we must remember that when neurons degenerate, the microglia phagocytize these dead cells and eliminate them. So the question becomes whether the presence of Lewy bodies observed in postmortem studies corresponds to the neurons lost during the neurodegenerative process or to the neurons that survived the degenerative process. Are Lewy bodies a part of the pathology in Parkinson’s disease or a form of neuroprotection in which the aggregates of fibrils are deposited? (2) We must also remember that in the postmortem brains of patients with genetic Parkinson’s disease associated with parkin and LRRK2, gene mutations do not present Lewy bodies [31e33], suggesting that Lewy bodies are not part of the pathology but rather a side effect in which monomeric alpha-synuclein is aggregated to fibrils that polymerize and are deposited in Lewy bodies. (3) The rate of progression of the disease does not match the process of propagation of alpha-synuclein aggregates as prefibrils as the authors of the prion-like hypothesis suggest. The degenerative process of Parkinson’s disease prior to motor symptoms takes years, and after the onset of motor symptoms, progressive degeneration takes many more years. However, the propagation of alphasynuclein fibrils through exosomes [34] or lysis of the degenerated neurons produces a relatively rapid propagative effect that does not match the extremely slow process of Parkinson’s disease because it induces massive and rapid degeneration of dopaminergic neurons.

Alpha-synuclein aggregation to neurotoxic oligomers inside dopaminergic neurons of nigrostriatal system Alpha-synuclein aggregation to neurotoxic oligomers induced by gene mutations induces mitochondrial dysfunction, oxidative stress, synaptic dysfunction, endoplasmic reticulum stress, and autophagy dysregulation [7]. The formation of neurotoxic alpha-synuclein oligomers occurs within the dopaminergic neurons of the nigrostriatal system when the gene for these proteins expresses a mutated protein such as A53T that induces familial Parkinson’s disease. However, the use of these mutations of the alpha-synuclein gene as a preclinical model to test new drugs for idiopathic Parkinson’s has the problem that the model does not include the agent or molecule that triggers the aggregation of alpha-synuclein to neurotoxic oligomers in this form of the disease. A drug that slows or halts the development or progress of the disease must prevent formation of the neurotoxic oligomers of alpha-synuclein. In a preclinical model that uses an alpha-synuclein mutation, the drug is unable to demonstrate therapeutic effect because this preclinical model does not include the agent that induces alpha-synuclein aggregation to neurotoxic oligomers in idiopathic Parkinson’s disease (Fig. 11.1).

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FIGURE 11.1 Alpha-synuclein as a preclinical model for sporadic Parkinson’s disease. Under certain conditions, alpha-synuclein aggregates to neurotoxic oligomers that induce protein degradation dysfunction, mitochondrial dysfunction, neuroinflammation, oxidative stress, endoplasmic reticulum stress, and synaptic impairment. These neurotoxic effects induce neurodegeneration that accumulates over many years and finally develops into motor symptoms in Parkinson’s disease patients. To develop a drug that stops the progression of the disease requires that the preclinical model includes the cause of the induced aggregation of alpha-synuclein to neurotoxic oligomers. In familial Parkinson, what induces the formation of neurotoxic oligomers is a mutation. Therefore, testing a drug in a preclinical model that uses mutated alpha-synuclein, or expressing duplications of the gene to produce excess alpha-synuclein, has no effect for a patient with sporadic Parkinson’s, as this model does not include what induces alpha-synuclein aggregation to neurotoxic oligomers.

3,4-Dihydroxyphenylacetaldehyde Excess dopamine is degraded through oxidative deamination of dopamine to 3,4-dihydroxyphenylacetaldehyde, a reaction catalyzed by the enzyme monoamine oxidase. During this reaction, ammonia and hydrogen peroxide are also formed. However, 3,4dihydroxyphenylacetaldehyde is not a final product, and this metabolite is converted to 3,4-dihydroxyphenylacetic acid by the enzyme aldehyde dehydrogenase-1 in the cytosol. Accumulation of 3,4dihydroxyphenylacetaldehyde has been reported to induce neurotoxicity by stimulating oxidative stress and alpha-synuclein aggregation to neurotoxic oligomers [35e37]. It has been reported that 3,4-dihydroxyphenylacetaldehyde is neurotoxic via covalent binding with biomolecules and functional proteins [38]. 3,4-Dihydroxyphenylacetaldehyde forms adduct with tyrosine hydroxylase and L-aromatic-amino-acid decarboxylase [39]. It has been reported that 3,4-dihydroxyphenylacetaldehyde reduces astrocytes viability, decreases mitochondrial function, induces apoptosis, and increases nitrative and oxidative stress in a concentration-dependent manner [40]. It has

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been reported that alpha-synuclein incubation in vitro with 3,4dihydroxyphenylacetaldehyde or dopamine in the presence of tyrosinase detected that the formation of alpha-synuclein adduct was more prominent [41]. Methionine amino acids of alpha-synuclein play an important role in scavenging reactive oxygen species generated by 3,4dihydroxyphenylacetaldehyde. The substitution of alpha-synuclein C-terminal methionine residues (especially methionine 127) by valine increased the formation of 3,4-dihydroxyphenylacetaldehyde-induced large oligomers in comparison with wild-type protein [42]. Aldehydes form adducts with primary and secondary amine groups contained in aminoindan and rasagiline. The formation of Schiff base adducts between 3,4dihydroxyphenylacetaldehyde and rasagiline or aminoindan was observed using mass spectrometry. Aminoindan and rasagiline reduced aggregation of alpha-synuclein of all sizes in both in vitro experiments and cell experiments. However, rasagiline is an inhibitor of monoamine oxidase that catalyzes the formation of 3,4-dihydroxyphenylacetaldehyde [43]. Alpha-synuclein N-terminal acetylation is less prone to form oligomers upon incubation with 3,4-dihydroxyphenylacetaldehyde than is the nonN-terminally acetylated protein [44]. 3,4-Dihydroxyphenylacetaldehyde induces glyceraldehyde-3-phosphate dehydrogenase aggregation [45]. A study performed with aldehyde dehydrogenase 1A1 null mouse did not show negatively affected growth and development of substantia nigra dopaminergic neurons nor changed protein expression levels of tyrosine hydroxylase, dopamine transporter, or vesicular monoamine transporter2. Dopamine levels significantly increased [46]. Another study performed with mice null for aldehyde dehydrogenase 1A1 and aldehyde dehydrogenase-2 showed deficits in motor performance determined by gait analysis and performance on an accelerating rotarod, significant loss of tyrosine hydroxylase neurons in the substantia nigra, and a reduction of dopamine and metabolites in the striatum. An increase in 3,4dihydroxyphenylacetaldehyde and 4-hydroxynonenal was also observed [47]. A study to determine the RNA expression of 137 gene expression was performed with postmortem human substantia nigra pars compacta from Parkinson’s disease patients and revealed that aldehyde dehydrogenase-1 expression was decreased, suggesting that 3,4-dihydroxyphenylacetaldehyde accumulated in these patients. The average age of the patients included in this study was 77 years [48]. Another postmortem study of Parkinson’s disease brain also reported a decrease in aldehyde dehydrogenase-1 expression and neurodegeneration in the ventral aldehyde dehydrogenase-1-positive dopaminergic subpopulations [49].

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Low expression of aldehyde dehydrogenase-1 in postmortem samples of Parkinson’s disease patients results in 3,4-dihydroxyphenylacetaldehyde accumulation that should induce neurodegeneration of nigrostriatal neurons. However, the question is whether the low expression of aldehyde dehydrogenase-1 in neurons of postmortem tissue that survive the degenerative process is an indicator that 3,4-dihydroxyphenylacetaldehyde plays a role in the degenerative process of Parkinson’s disease. Another point to consider is that the low expression of aldehyde dehydrogenase-1 in postmortem tissue of Parkinson’s patients is based on measurement of the total RNA levels of all neurons that survive. This implies that the low expression of this enzyme induces the accumulation of 3,4dihydroxyphenylacetaldehyde and finally neurotoxic effects that should affect most dopaminergic neurons of the nigrostriatal system. This implies that 3,4-dihydroxyphenylacetaldehyde should induce massive degeneration of dopaminergic neurons, which goes against the temporality of the degenerative process that occurs over many years both before and after motor symptoms appear. Therefore, 3,4-dihydroxyphenylacetaldehyde is not a suitable preclinical model for Parkinson’s disease (Fig. 11.2).

Aminochrome Dopamine oxidation to neuromelanin requires the formation of orthoquinones such as dopamine ortho-quinone, aminochrome, and 5,6indolequinone that can be neurotoxic under certain circumstances [2e5]. Dopamine ortho-quinone is unstable at the physiological pH of the cytoplasm of dopaminergic neurons where the amino group spontaneously cyclizes to form aminochrome. The formation of dopamine orthoquinone in the presence of tyrosinase and isolated mitochondria in an in vitro experiment results in the formation of an extensive list of proteins that have adducts with this ortho-quinone, such as mitochondrial complexes I, III, and IV. However, when dopamine ortho-quinone is formed intracellularly in a neuron incubated in the presence of dopamine, the formation of adducts with these mitochondrial complexes of the electron transport chain is not detected [50]. This is explained by the formation of dopamine ortho-quinone in the presence of isolated mitochondria for which there is nothing preventing the formation of dopamine orthoquinone adducts with mitochondrial proteins, while within a cell in the cytoplasm, dopamine ortho-quinone has two alternatives, to form adducts with some protein or to cyclize to aminochrome. 5,6-Indolequinone is also unstable as it polymerizes to form neuromelanin. The polymerization of 5,6-indolequinone to neuromelanin increases as the pH and concentration of 5,6-indolequinone increase.

FIGURE 11.2 3,4-Dihydroxyphenylacetaldehyde as a preclinical model for sporadic Parkinson’s disease. 3,4-Dihydroxyphenylacetaldehyde (DOPAL) is an intermediate formed during dopamine degradation catalyzed by monoamine oxidase. DOPAL is converted to 3,4dihydroxyphenylacetic acid that catechol ortho-methyltransferase later converts to homovanillic acid. This a harmless pathway where excess dopamine is degraded, but low expression of aldehyde dehydrogenase-1 results in DOPAL accumulation. DOPAL in the presence of oxygen oxidizes to DOPAL ortho-semiquinone by reducing dioxygen to superoxide, which spontaneously or enzymatically is converted to hydrogen peroxide. Hydrogen peroxide is the precursor of hydroxyl radicals that induce oxidative stress. DOPAL ortho-semiquinone also reduces oxygen to superoxide. A reduction in aldehyde dehydrogenase-1 activity has been reported in Parkinson’s disease patients, but it is unclear whether this depends on a mutation of the aldehyde dehydrogenase-1 activity gene. To evaluate a drug in a preclinical model for Parkinson’s disease that uses 3,4dihydroxyphenylacetaldehyde requires inclusion of the cause for the induced low activity of aldehyde dehydrogenase-1; otherwise, the results will have no impact for patients with sporadic Parkinson’s disease.

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5,6-Indolequinone forms adducts with alpha-synuclein and the nuclear receptor Nurr1 [51,52]. However, its instability prevents more information about its neurotoxic effects. Aminochrome is the most stable ortho-quinone and therefore the most studied. Aminochrome can be neurotoxic through two mechanisms: (1) its one-electron reduction by flavoenzymes that one-electron transfer to generate leukoaminochrome ortho-semiquinone, which is highly unstable in the presence of oxygen [53]. Leukoaminochrome ortho-semiquinone reduces oxygen to superoxide to generate a redox cycling between aminochrome and leukoaminochrome orthosemiquinone that depletes NADH and accumulates superoxide, which finally induces oxidative stress; and (2) the formation of adducts with proteins such as alpha-synuclein, mitochondrial complex I, cytoskeleton network proteins actin and alpha- and beta-tubulin, and other proteins [54,55]. Alpha-synuclein has been proposed as the endogenous neurotoxin that triggers neurodegeneration of the nigrostriatal system. In familial Parkinson’s, it is a mutation that induces the formation of neurotoxic oligomers, but in idiopathic Parkinson’s, it is not clear what triggers the formation of alpha-synuclein neurotoxic oligomers within dopaminergic neurons. Interestingly, aminochrome is generated within dopaminergic neurons that contain neuromelanin and induces the aggregation of alphasynuclein to neurotoxic oligomers, suggesting that aminochrome is what triggers the formation of alpha-synuclein neurotoxic oligomers in idiopathic Parkinson’s disease [55]. Aminochrome induces mitochondrial dysfunction through inhibition of complex I caused by the formation of adducts with aminochrome and the depletion of NADH when aminochrome is reduced with an electron, generating a redox cyclization between aminochrome and leukoaminochrome ortho-semiquinone that uses NADH to reduce aminochrome [56e59]. Aminochrome induces protein degradation dysfunction by impairing both lysosomal and proteasomal systems. Aminochrome induces lysosomal dysfunction by inhibiting lysosomal vacuolar H-type ATPase, which plays an essential role in maintaining a relatively low pH inside lysosomes [60]. Aminochrome induces autophagy dysfunction by preventing the fusion of autophagosomes and lysosomes that require microtubules. Aminochrome prevents the formation of microtubule assembly by forming adducts with alpha- and beta-tubulin [61e64]. Aminochrome induces dysfunction of proteasome and endoplasmic reticulum stress [65e67]. Aminochrome induces oxidative stress when one-electron transfer flavoenzymes reduce aminochrome to a leukoaminochrome ortho-semiquinone radical that is very unstable in the presence of oxygen [53]. The leukoaminochrome ortho-semiquinone radical reduces oxygen to

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superoxide that is converted to hydrogen peroxide. Finally, hydrogen peroxide is converted to hydroxyl radicals in the presence of reduced iron, generating oxidative stress [56]. It has been reported that aminochrome induces glial activation by increasing the number of NF-kB p50 immunoreactive cells and OX-42þ cells, suggesting that aminochrome induces neuroinflammation [68]. A study that used organotypic midbrain slice cultures demonstrated that aminochrome induces morphological changes in Iba1þ cells, an increase of both IL-1b and TNF-a mRNA and protein levels, reduction of GFAP expression, and decreased NGF and GDNF mRNA levels. These results suggest that aminochrome induces neuroinflammation and downregulation of neurotrophic factors [69]. Interestingly, intracerebral injection of aminochrome into striatum induces contralateral behavior in the absence of significant loss of tyrosine hydroxylase immunopositive neurons in the substantia nigra pars compacta. Aminochrome induces a significant reduction of dopamine synaptic release accompanied with a significant increase in GABA levels, suggesting that aminochrome induces an imbalance between neurotransmitters in the basal ganglia. The significant decrease in dopamine release can be explained by the significant decrease in the number of synaptic vesicles at the synaptic cleft of animals treated with aminochrome [70]. The prominent decrease in synaptic vesicles induced by aminochrome can be explained by aminochrome’s reported disruption of the cytoskeleton architecture because aminochrome induces alpha- and beta-tubulin aggregation and prevents microtubule polymerization [54,61]. Active axonal transport requires an intact cytoskeleton with functional microtubules [71,72]. Intracerebral injection of aminochrome into striatum induces mitochondrial damage resulting in mitochondrial dysfunction with a significant reduction in ATP levels. Aminochrome induced a dramatic morphological change characterized as cell shrinkage, suggesting that aminochrome induced a progressive neuronal dysfunction [70]. Aminochrome is an endogenous neurotoxin formed inside dopaminergic neurons that are lost in Parkinson’s disease during dopamine oxidation to neuromelanin. Aminochrome induces mitochondrial dysfunction, aggregation of alpha-synuclein to neurotoxic oligomers, protein degradation dysfunction of both lysosomal and proteasomal systems, endoplasmic reticulum stress, neuroinflammation, and oxidative stress, but the formation of neuromelanin seems to be a normal and harmless pathway because the majority of healthy seniors do not develop Parkinson’s disease and have intact dopaminergic neurons containing neuromelanin [73]. This apparent paradox, that neuromelanin synthesis requiring dopamine oxidation to aminochrome does not induce neurodegeneration, can be explained by the existence of two enzymes, DT-diaphorase and glutathione transferase M2-2, that prevent aminochrome neurotoxicity in dopaminergic neurons and astrocytes.

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Flavoenzymes reduce quinones by one- or two-electron transfer to semiquinones or hydroquinones, but DT-diaphorase is a unique flavoenzyme that reduces quinones to hydroquinones by two-electron transfer and is expressed in most tissues. In the brain, DT-diaphorase is expressed in different regions such as the substantia nigra, striatum, hypothalamus, hippocampus, frontal cortex, and cerebellum. In the nigrostriatal system, DT-diaphorase is constitutively expressed in dopaminergic neurons and astrocytes. In substantia nigra, DT-diaphorase is responsible for 97% of total quinone reductase activity [74]. DTdiaphorase reduces aminochrome to leukoaminochrome using both NADH and NADPH as electron donators [75]. DT-diaphorase prevents (1) aminochrome-induced cell death [76]; (2) formation of alpha-synuclein oligomers and their neurotoxicity [55]; (3) mitochondrial dysfunction [56,57,77,78]; (4) oxidative stress [62]; (5) lysosomal dysfunction [60]; (6) autophagy dysfunction [62]; (7) proteasomal dysfunction [65,66]; and (8) aggregation of actin and alpha- and beta-tubulin and disruption of the cytoskeleton architecture [54]. DT-diaphorase is also expressed in astrocytes and protects astrocytes against aminochrome neurotoxicity [79]. Glutathione transferases catalyze the detoxication of many xenobiotics including environmental contaminants, carcinogens, anticancer agents, antibiotics, and products of oxidative reactions. Human glutathione transferases M1-1, M2-2, and M3-3 catalyze aminochrome conjugation with glutathione to 4-S-glutathionyl-5,6-dihydroxyindoline, but the isoenzyme M2-2 is 1035-fold and 194-fold more active than M3-3 and M11 isoenzymes. The conjugation of aminochrome to 4-S-glutathionyl-5,6dihydroxyindoline results in the reduction of its quinone structure to a hydroquinone structure that is resistant to autoxidation in the presence of biological oxidizing agents such as oxygen, superoxide, and hydrogen peroxide. Glutathione transferase M2-2 is constitutively expressed in astrocytes and human substantia nigra and catalyzes glutathione conjugation of aminochrome precursor dopamine ortho-quinone to 5-glutathionyl-dopamine [80e82]. Glutathione is a tripeptide (L-gamma-glutamyl-L-cysteinyl-glycine), and glutathione conjugates are normally degraded by removing glutamate and glycine amino acids. 5Glutathionyl-dopamine is degraded to 5-cysteinyl-dopamine, which is a final product because it has been observed to be present in human neuromelanin and cerebrospinal fluid [83e85]. It has been proposed that glutathione transferase M2-2 is a protective enzyme in dopaminergic neurons because this enzyme prevents (1) aminochrome-dependent cell death in astrocytes [69]; (2) aminochromeinduced mitochondrial dysfunction [64,65,70]; (3) aminochromeinduced autophagy dysfunction [62]; (4) aminochrome-dependent formation of alpha-synuclein neurotoxic oligomers [85]; and (5) aminochrome-induced lysosomal dysfunction [62].

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Astrocytes play a protective role for surrounding neurons in modulating neuronal function and survival. Astrocytes release precursors of the antioxidant glutathione [86,87], providing energy by the astrocytee neuron lactate shuttle [88], and release the proteins metalloproteinase inhibitor-1, superoxide dismutase, and thioredoxin peroxidase-2, among others [89]. Astrocytes also release glutathione transferase M2-2 to protect neurons against aminochrome neurotoxicity. Astrocytes constitutively express glutathione transferase, but the presence of aminochrome increases the expression of this enzyme. Interestingly, dopaminergic neurons can internalize glutathione transferase M2-2 secreted by astrocytes and prevent aminochrome neurotoxicity in dopaminergic neurons [90e92]. It has been reported that astrocytes release glutathione transferase M2-2 through the secretion of exosomes containing this enzyme, which explains how dopaminergic neurons are able to internalize the glutathione transferase M2-2 released by astrocytes [93]. This cooperation between astrocytes and dopaminergic neurons plays an important role in preventing the neurotoxic effects of aminochrome on dopaminergic neurons by increasing the neuroprotection provided by DT-diaphorase. This cooperative neuroprotection against aminochrome explains why oxidation of dopamine to neuromelanin is a harmless process for healthy seniors who have intact dopaminergic neurons containing neuromelanin (Fig. 11.3). Aminochrome is the endogenous neurotoxin that seems to be most appropriate for a preclinical model for the following reasons. (1) Aminochrome is an endogenous neurotoxin formed within dopaminergic neurons during dopamine oxidation to neuromelanin that under certain conditions becomes a neurotoxin. (2) Normally, the oxidation of dopamine to neuromelanin proceeds without neurotoxic effects due to the neuroprotective action of the enzymes DT-diaphorase and glutathione transferase M2-2 that therefore prevents the neurotoxic effects of aminochrome. (3) The neurotoxic effects of aminochrome induce the death of a single neuron when the neuroprotection of DT-diaphorase and glutathione transferase is exceeded. The death of this single dopaminergic neuron does not have a propagative effect. The microglial cells degrade the dead dopaminergic neuron by engulfing the cellular remains. This implies that advancement of the aminochrome-induced neurodegenerative process is extremely slow, as in Parkinson’s disease, for which it takes years to degenerate enough dopaminergic neurons containing neuromelanin for motor symptoms to appear. (4) aminochrome triggers mitochondrial dysfunction, aggregation of alpha-synuclein to neurotoxic oligomers, dysfunction of protein degradation of the lysosomal and proteasomal systems, endoplasmic reticulum stress, neuroinflammation, and oxidative stress, all of which are mechanisms reportedly involved in the degenerative process of Parkinson’s disease [94e107] (Fig. 11.4).

FIGURE 11.3 Aminochrome metabolism: neurotoxicity and neuroprotection. Aminochrome is formed inside the dopaminergic neurons that are lost in Parkinson’s disease during neuromelanin synthesis. Dopamine oxidizes to neuromelanin by forming three transients ortho-quinones: dopamine ortho-quinone, aminochrome, and 5,6-indolequinone. However, aminochrome is the most stable of these. Aminochrome is neurotoxic when it is oneelectron reduced by flavoenzymes that one-electron transfer or form adducts with proteins such alpha-synuclein, actin, and alpha- and beta-tubulin. It has been reported that aminochrome induces mitochondrial dysfunction, alpha-synuclein aggregation to neurotoxic oligomers, protein degradation dysfunction of both lysosomal and proteasomal systems, endoplasmic reticulum stress, neuroinflammation, and oxidative stress. Unilateral intracerebral injection of aminochrome into striatum induces (1) progressive neuronal dysfunction by a significative decrease in dopamine release that results in imbalanced neurotransmitter levels; (2) a dramatic morphological change known as cell shrinkage; (3) a significative decrease in the number of synaptic vesicles; and (4) mitochondrial damage resulting in a significative decrease in ATP level and mitochondrial dysfunction. Aminochrome is formed within the dopaminergic neuron under certain circumstances, and its neurotoxic effects affect only one neuron because it has no propagative effects. The dead dopaminergic neuron is removed by the action of microglia that dispose of cellular or aggregated debris. The neurotoxic effects of aminochrome within a dopaminergic neuron affect a single neuron, and the accumulation of enough loss of dopaminergic neurons to develop motor symptoms, as seen in Parkinson’s disease, occurs over an extremely long period of many years. Neuromelanin synthesis is a harmless process, as healthy seniors have intact neuromelanin-containing dopaminergic neurons. This happens because there is a neuroprotective mechanism against the neurotoxic effects of aminochrome composed of two enzymes that prevent the neurotoxic effects of aminochrome and an astrocyte neuroprotective mechanism that protects dopaminergic neurons from the neurotoxic effects of aminochrome. The flavoenzyme DT-diaphorase prevents the neurotoxic effects of aminochrome by reducing it with two electrons to leukoaminochrome. On the other hand, astrocytes secrete glutathione transferase M2-2 through exosomes that cross through the membranes of dopaminergic neurons, releasing this enzyme in the cytosol of these neurons, thus protecting them from the neurotoxic effects of aminochrome. Glutathione transferase M2-2 catalyzes the conjugation of aminochrome and its precursor dopamine ortho-quinone with glutathione. Aminochrome is neurotoxic when the protective capacity of the enzymes DT-diaphorase and glutathione transferase M2-2 is exceeded.

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FIGURE 11.4 Aminochrome as a preclinical model for Parkinson’s disease. The advantages of using aminochrome as a preclinical model for Parkinson’s disease are related to (1) the formation of this endogenous neurotoxic within neurons that are lost in the disease; (2) aminochrome’s inducement of all the mechanisms reported to be involved in the degenerative process of Parkinson’s disease such as mitochondrial dysfunction, alpha-synuclein aggregation to neurotoxic oligomers, protein degradation dysfunction of both lysosomal and proteasomal systems, endoplasmic reticulum stress, neuroinflammation, and oxidative stress; (3) the induction of a slow degeneration mediated by progressive neuronal dysfunction that is similar to the slow advance of the degenerative process observed in the disease; and (4) aminochrome neurotoxicity that is not expansive and only affects the neuron where aminochrome is neurotoxic because the protective capacity of the enzymes DT-diaphorase and glutathione transferase M2-2 has been exceeded. The remains of the dead dopaminergic neuron are removed by action of the microglia.

References

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C H A P T E R

12 Conclusions More than 50 years have passed since the introduction of L-dopa in the treatment of Parkinson’s disease, and it continues to be the gold-standard drug despite the severe side effects that appear from chronic use at between 4 and 6 years. On the other hand, we have seen that successful results obtained by preclinical models using exogenous neurotoxins that have promised great advances in the treatment of the disease have failed. However, the use of preclinical models based on exogenous neurotoxins to study the mechanism of the disease and test new potential treatments continues. I think it is time to reflect on the preclinical models of Parkinson’s disease. There is a minority group of scientists who think that preclinical models based on exogenous neurotoxins should not be used, but a great majority continue to use these models without reflecting on whether they have any utility for Parkinson’s disease. The use of these exogenous toxin-based preclinical models may be useful to study mechanisms of neurodegeneration in general but not those related to Parkinson’s disease. It is difficult to think that we will be successful in clinical studies with Parkinson’s patients when a new drug is discovered that manages to halt development of the disease but has been tested in a preclinical model that does not replicate what happens in the disease. It is useless to use a preclinical model that does not include the triggers to neurotoxic events. To change the course of the disease and halt the degenerative process, a new drug must prevent mitochondrial dysfunction, the addition of alphasynuclein to neurotoxic oligomers, dysfunction of protein degradation, neuroinflammation, endoplasmic reticulum stress, and oxidative stress observed in the disease. Inhibition of these neurotoxic mechanisms by a new drug in a preclinical model based on exogenous neurotoxins does not imply that the drug will achieve the same effect in clinical studies, because the drug’s effectiveness is judged based on preventing the exogenous neurotoxin from triggering these mechanisms. The failure of the drug in clinical studies can be explained by the realization that this exogenous neurotoxin is not responsible for the degenerative process of a

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patient with idiopathic Parkinson’s. It is not enough to use a preclinical model that generates these neurotoxic mechanisms if what triggers these mechanisms does not exist in the dopaminergic neurons that are lost during the degenerative process before and after motor symptoms. A preclinical model for idiopathic Parkinson’s requires that the general features of the disease, such as the extremely slow speed of the degenerative process, are considered both before and after motor symptoms. It takes years to achieve a loss of more than 60%e70% of the dopaminergic neurons that contain neuromelanin, which implies that what triggers the degenerative process of the disease cannot induce a rapid or propagative degeneration. The fact that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine induces severe parkinsonism in as little as 3 days in humans who have abused illicit drugs contaminated with this neurotoxin clearly suggests that exogenous neurotoxins cannot be involved in the slow degenerative process of idiopathic Parkinson’s disease. Therefore, a degenerative process of idiopathic Parkinson’s that takes years to accumulate the 60%e70% loss of dopaminergic neurons containing neuromelanin to develop motor symptoms suggests that the neurotoxin that triggers neurodegeneration of this type of neuron must be endogenous in nature. In addition, the endogenous neurotoxin must be produced within these dopaminergic neurons that contain neuromelanin so that each neurotoxic event affects only a single neuron, because the accumulated loss of these neurons occurs extremely slowly over many years. Three endogenous neurotoxins form within dopaminergic neurons and can degenerate neuromelanin-containing dopaminergic neurons: oligomers of alpha-synuclein, 3,4-dihydroxyphenylacetaldehyde, and aminochrome. A mutation in the alpha-synuclein gene induces the formation of neurotoxic oligomers associated with familial Parkinson’s disease. The question is what induces the formation of neurotoxic oligomers of alpha-synuclein in idiopathic Parkinson’s. Using a preclinical model that overexpresses a mutated alpha-synuclein will not demonstrate the success of a new drug in a preclinical model of idiopathic Parkinson’s if this drug cannot prevent the formation of neurotoxic oligomers. 3,4-Dihydroxyphenylacetaldehyde is another endogenous neurotoxin that is formed within dopaminergic neurons during monoamine oxidase catalyzed degradation of dopamine. 3,4-Dihydroxyphenylacetaldehyde is converted to 4-dihydroxyphenylacetic acid by the enzyme aldehyde dehydrogenase-1. The accumulation of 3,4-dihydroxyphenylacetaldehyde has been suggested as a neurotoxic that induces oxidative stress and alphasynuclein aggregation. A study carried out with postmortem samples of substantia nigra from patients with Parkinson’s disease has demonstrated low expression of the aldehyde dehydrogenase-1 gene, suggesting that 3,4dihydroxyphenylacetaldehyde accumulates because of the low expression

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of this protein. The question is what generates low expression of this enzyme that affects all dopaminergic neurons of the substantia nigra. The fact that there is generalized low expression in dopaminergic neurons of the substantia nigra suggests that the neurotoxic effects of 3,4dihydroxyphenylacetaldehyde are not focused on a single neuron, implying that the degenerative effects should be rapid, which differs from what happens in the disease. Aminochrome is generated during the oxidation of dopamine to neuromelanin within dopaminergic neurons and is transformed into an endogenous neurotoxin when the neuroprotective system composed of the enzymes DT-diaphorase and glutathione transferase M2-2 is surpassed. Aminochrome induces all the mechanisms involved in the neurodegeneration of dopaminergic neurons such as mitochondrial dysfunction, the addition of alpha-synuclein to neurotoxic oligomers, dysfunction of protein degradation, neuroinflammation, endoplasmic reticulum stress, and oxidative stress. Aminochromic neurotoxicity does not induce a propagative effect, and its neurotoxic effects will only affect the dopaminergic neuron where aminochrome was converted into a neurotoxin. The remains of this degenerated dopaminergic neuron are removed by microglia. Therefore, aminochrome-induced neuronal death is consistent with the extremely slow degeneration observed in the disease, where the loss of dopaminergic neurons containing neuromelanin accumulates for years, having reached 60%e70% by the time that motor symptoms appear. It is time to reflect on the preclinical models we use to study Parkinson’s disease mechanisms and test new drugs. These models give the impression that the failures observed in translating successful results from preclinical studies based on exogenous neurotoxins to clinical studies and new drugs to slow the development of the disease are indifferent to most scientists who continue to use these preclinical models to study the mechanisms of this disease and test potential new drugs.

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Index ‘Note: Page numbers followed by “f” indicate figures and “t” indicate tables’

A Adenosine A2A receptors, 178 Adenosine antagonists, 177e179 Akinetic-rigid Parkinson’s disease, 62e63 Alcohol withdrawal, 47 Aldehyde dehydrogenase-1 activity, 205 Alpha-synuclein, 23, 182 aggregations, 32 amino acid sequence, 92 apolipoproteins and synuclein lipid interaction, 83e84 autosomal-dominant mutations A30P mutation, 157e158 A53T point mutation, 157 copy number variants analysis, 159 gene duplication, 158 gene multiplications, 158 missense mutations, 158 coding, 80 C-terminal, 92e93 dopamine synthesis, 189 dynamical and structural properties, 81e82 expression, 80 fatty acid metabolism, 83 fibril structure, 92 GTPase-deficient Rab3a mutant, 82 human alpha-synuclein mutation, 81 immunogold electron microscopy, 81 knockout, 81 Lewy bodies formation alpha-synuclein deposits, 87f alpha-synucleinopathy, 86 Braak Parkinson’s disease stage hypothesis, 85e86 chromogranin and synaptophysin, 85 glucocerebrosidase-1 mutations, 85 neurofilaments, 85 parkin mutation, 86e88 ubiquitin, 85 membrane localization, 82 membrane remodeling, 84 monoubiquitination and polyubiquitination, 126e127 neurodegeneration, 109f

287

neurotoxic oligomers actin filament network reorganization, 106 alphaB-crystallin, 104 alpha-synuclein null mice, 100 A30P mutation, 95e96 astrocyte exposure, 104e105 astrocytes, 98 A53T expression, 101, 107 A53T-transgenic mice, 95 autophagy, 100e101 calcium homeostasis, 99 chaperone-mediated autophagy pathway, 101 DNA strand breaks, 97e98 dopaminergic degeneration, 104e105 endoplasmic reticulum, 99 endoplasmic reticulum stress, 100e101 ferritin knockdown, 108 gene expression analysis, 98 glia maturation factor downregulation, 103 histidine-50 substitution, 93e94 inflammasomes, 102e103 leucine-rich repeat kinase-2 G2019S mutation, 103e104 metal binding, 96 mitochondrial fragmentation, 99e100 mitochondrial NAD-dependent deacetylase sirtuin-3, 107 mitochondrial nuclear protein import, 106e107 mitofusin-2 and dynamin-related protein-1 protein overexpression, 99e100 neurodegeneration, 93 nigral dopaminergic neurons, 102 nigrostriatal neurons, 105e106 NLR family pyrin domain-containing protein-3 activation, 103 oxidative stress, 104 plasma membrane pores, 106 posttranslational modifications, 96 protein homeostasis, 99 proteolysis, 103

288

Index

Alpha-synuclein (Continued) serine 129 phosphorylation, 96e97 superoxide dismutase-2 expression, 108 T cells activation, 105 tyrosine 125 phosphorylation, 97 ubiquitin ligase NEDD4 overexpression, 101e102 vesicle-associated membrane proteinassociated protein B overexpression, 100 vesicles trafficking, 98 neurotransmitter release, 82 non-b-amyloid component, 92e93, 95 N-terminal membrane-binding domain, 84 phosphatidylglycerol and cardiolipin, 83 posttranslational stabilization, 92 preclinical models extracellular alpha-synuclein, 264 Lewy bodies formation, 264 lymphocyte-activation gene-3, 265 neuromelanin-containing dopaminergic neuron loss, 263e264 neuron-derived exosomes, 265 neurotoxic oligomers, 266 prion-like hypothesis, 264e266 protein interaction, 82 proteomic analysis, 84 structure and properties, 94f synaptic transmission, 81 wild-type, 82e83 Alpha-synucleinopathy, 86 Ambroxol, 182 Aminochrome, 215e218 dopamine oxidation, 285 neurodegeneration, 285 neurotoxicity, 12 astrocytes neuroprotection, 234e236 mechanisms, 271 preclinical model, 276f astrocytes, 274 dopamine ortho-quinone, 269 DT-diaphorase, 273 glutathione transferases, 273 5,6-indolequinone, 269e271 mitochondrial dysfunction, 271 neuromelanin synthesis, 272 neurotoxicity and neuroprotection, 275f neurotoxic mechanisms, 271 oxidative stress, 271e272 progressive neuronal dysfunction, 272 protein degradation dysfunction, 271

Aminoindan, 268 Amiodarone, 5f Amisulpride, 5f Anhedonia, 38, 43 Anticholinergic drugs, 176e177 Anxiety balance and gait disturbance, 59 Beck Anxiety Inventory motor aspects, 57e58 cognitive dysfunction, 58 depression, 57 diagnotic test, 56 dopaminergic neurons, 57e58 dorsal anterior cingulate cortex impairment, 58 functional imaging studies, 58 glucose metabolism, 58 insomnia, 55 motor symptoms, 56 nonmotor symptoms, 56e58 prevalence, 57 restless legs syndrome, 59 stress, 57 treatment-dependent side effects, 56 Aromatic amino acid decarboxylase, 189e190 Astrocytes, 274 astrocyteeneuron interaction, 235 neuroprotection against aminochrome neurotoxicity, 236, 237f astrocyteeneuron interaction, 235 brain metabolism, 234 glutathione transferase M2-2 expression, 236 ATPase cation transporter 13A2, 163e164 Attention-deficit/hyperactivity disorder (ADHD), 40 Autophagosome motility, 231e232 Autophagy, 8, 154 Autophagy-related 7 gene, 128

B Beck Anxiety Inventory, 57e58 Benton Judgment of Line Orientation test, 71e72

C Calcium channel antagonists, 5f Calcium homeostasis, 122 Carnosine dipeptide, 242 Catechol ortho-methyltransferase

Index

catechol structure degradation, 209 dopamine degradation, 211f inhibitor, 210 isoforms, 209 Cerebral hypoperfusion, 76 Chaperone-mediated autophagy, 101, 128e129 Cholinergic degeneration, 176 Cholinesterase inhibitors, 69e70 Chromogranin and synaptophysin, 85 Cognitive decline akinetic-rigid Parkinson’s disease, 62e63 anxiety, 58 biomarkers, 61 Chinese Parkinson’s disease patients, 61e62 cholinergic deficiency, 64 computer-based cognitive training, 60e61 dementia, 60, 62 exercise training, 61 metabolic syndrome, 62 mild cognitive impairment, 60e61 orthostatic hypotension, 76 predictive validity, 64e65 prevalence, 62 self-rating, 61e62 white matter lesions, 63 Constipation alcohol withdrawal, 47 alpha-synuclein aggregations, 46 alpha-synuclein expression, 49 anxiety, 47 colon biopsies, 47e48 enteric nervous system, 46 enteric neuronal system, 48 glia cells, 49 gut microbiota, 47 microbiota composition, 48 MitoPark mouse model, 47 neuronal cell bodies, 46 probiotics, 48e49 sympathetic neurons, 47 vagus nerve stimulation, 46e47 Copper-induced parkinsonism aminochrome neurotoxicity, 12 ATP7B gene mutation, 10e12 dopamine-copper complex, 12 dopamine reuptake, 10e12 mechanism, 11f mutations, 10 Wilson’s disease, 10 Corticotropin-releasing hormone, 39 C-reactive protein, 146

289

D DA-JC4 agonist, 179e180 Dementia and dementia with Lewy bodies alpha-synuclein duplication, 72 immunopositive depositions, 69 Alzheimer disease, 68 Braak Parkinson’s disease scale, 69 clinical features, 73f cognitive tests, 69e70 cortical Lewy bodies, 68 diagnosis, 67e68 E326K mutations, 71 extrapyramidal symptoms, 69e70 genetic factors, 70e71 genetic mutations, 71e72 genome-wide association study, 71 glucocerebrosidase-1 mutations, 71 laboratory observations, 70 longitudinal neuropsychological study, 72 morphological hallmarks, 69e70 olfactory functional connectivity, 68 postmortem studies and intravital positron-emission tomography, 69e70 predominant dysexecutive syndrome, 68e69 prevalence, 67, 69 retrospective study, 68 Depression anhedonia, 38 anxiety, 57 attention-deficit/hyperactivity disorder, 40 depressive disorders, 38 dopamine hypothesis anhedonia, 43 animal experiment, 42e43 ketamine, 43 postlesion stress, 43 stress, 42e43 yoga and resistance training, 43 glucocorticoid receptors, 39e40 insomnia, 55 molecular mechanisms, 39e40 serotonin hypothesis abnormal serotonin levels, 40 anxious behavior, 42 selective serotonin reuptake inhibitor, 40e42 serotonin transporter promotor polymorphism, 39

290 Depression (Continued) stress, 39, 41f 3,4-Dihydroxyphenylacetaldehyde, 284e285 aldehyde dehydrogenase-1 expression, 268e269 glyceraldehyde-3-phosphate dehydrogenase aggregation, 268 oxidative deamination, 267 preclinical model, 270f RNA expression, 268 tyrosine hydroxylase, 267e268 Donepezil, 176e177 Dopamine cytosolic dopamine level, 197e198 methylation, 209e212 neurotransmission, 195e201 oxidative deamination, 203e207, 206f storage and release, 195e201 vesicular monoamine transporter-2. See Vesicular monoamine transporter-2 Dopamine agonists, 174 Dopamine oxidation, 213e227 aminochrome actin aggregations, 217 adducts, 216 alpha-synuclein aggregation, 216 autophagy dysfunction, 216 cell death, 217 lysosomal dysfunction, 216 microtubules, 217 neuronal dysfunction, 217e218 neurotoxicity, 219f nuclear DNA damage, 217 proteasome inhibition, 216 redox cycling, 216 dopaminochrome alpha-synuclein mutation, 218 apoptotic cell death, 220e221 beta-synuclein neurotoxicity, 220 neurotoxicity, 222f 5,6-indolequinone, 218 neuromelanin, 221e223 ortho-quinone hypochlorous acid, 214 neurotoxicity, 215f oxidizing agents, 213e214 physiological pH, 214 SH-SY5Y cells, 214 tyrosine hydroxylase inactivation, 214 Dopamine replacement therapy, 174 Dopaminergic drugs

Index

dopamine agonists, 174 173e174 Dopaminergic neurons, 229 Dopamine synthesis aromatic amino acid decarboxylase, 189f deficiency, 190 norepinephrine synthesis, 190 promoters and exons, 189e190 pyridoxal 5’-phosphate, 189 cytosol, 190, 191f enzymes, 191f tyrosine hydroxylase alpha-synuclein, 189 enzymatic activity, 188 isoforms, 187e188 L-3,4-dihydroxyphenylalanine conversion, 187, 188f oxidation, 188 phosphorylation, 187e188 proteasome system inhibition, 188e189 Droxidopa, 76e77, 190 Drug-induced parkinsonism age at onset, 4e6 DAT-SPECT imaging, 2e3 drugs, 4, 5f erroneous diagnoses, 2e3 neuroleptic exposure, 4 vs. Parkinson’s disease, 4 prevalence, 4e6 symptoms, 4 transcranial sonography, 3e4 trimetazidine, 4 DT-diaphorase dopaminergic neurons protection, 233f human alpha-synuclein, 231 inhibition, 231e232 isoforms, 230 leukoaminochrome autoxidation, 230e231 ortho-quinone, 230 oxidative stress, 232 proteasomal dysfunction inhibition, 231e232 silencing, 231e232 Dynamin-related protein-1-dependent mitochondrial fragmentation, 120e121 Dyskinesia glutamate, 180 L-dopa, 174 L-dopa,

Index

E Endoplasmic reticulum stress. See also Nigrostriatal neuron degeneration 6-hydroxydopamine, 243 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP), 245 rotenone, 245e246 Entacapone, 174, 210 Enteric nervous system, 46 Epworth Sleepiness Scale score, 52e53 Eszopiclone, 55 Eumelanin, 221 Excessive daytime somnolence, 35 brain white matter microstructure changes, 53 dopaminergic impairment, 52 light therapy, 52e53 neurological disorders, 51e52 nocturnal sleep, 51e52 pharmacological drugs, 52 prevalence, 52

F F-box protein-7, 129, 146 Ferritin autophagy, 79 Ferroptosis, 8 Flavoenzymes, 273 Fluoxetine, 5f, 40e42

G Ganoderma lucidum, 181 Gaucher disease, 129e130 Genetic Parkinson’s disease autosomal-dominant mutations alpha-synuclein, 157 leucine-rich repeat kinase-2, 159 vacuolar protein sorting-35, 160 autosomal-recessive mutations ATPase cation transporter 13A2, 163e164 parkin, 160e161 phospholipase A2 group VIPhospholipase A2 group VI (PARK12), 164 protein deglycase-1, 163 PTEN-induced kinase-1, 161e162 P-type ATPaseP13A2 mutations, 164 gene mutations, 157 genes associated, 166te167t Glia cells, 49

291

Glia maturation factor, 103, 154 Glucagon-like peptide-1 receptor agonists, 179e180 Glucocerebrosidase-1 mutations, 71, 85 Glucocorticoid receptors, 39e40 Glutathione transferase-M2-2 glutathione conjugation, 232, 234 silencing, 234 Glutathione transferases, 273 5-Glutathionyl-dopamine, 234

H Haloperidol, 5f Hopkins Verbal Learning Test-Revised [HVLT-R], 71e72 6-Hydroxydopamine, 176 alpha-synuclein 129-phosphorylation, 243 antioxidants, 242 autoxidation, 241e242 discovery, 241 endoplasmic reticulum stress, 243 mitochondrial dysfunction, 242e243 neuroinflammation, 242 oxidative stress, 241 proinflammatory mediators, 242 6-Hydroxydopamine-dependent lesions, 143e144 4-Hydroxynonenal, 265 Hypochlorous acid, 214 Hyposmia, 26e28 Hypothalamus-pituitary-adrenal axis activity, 39

I Idalopirdine, 176e177 Idiopathic Parkinson’s disease alpha-synuclein, 23 development, 23, 24f diagnosis, 156e157 inclusion-body pathology, 23 motor symptoms, 23 nonmotor symptoms, 23 stages, 23, 24f 5,6-Indolequinone, 218, 269e271 Inflammasomes, 102e103 Insomnia chronic, 54e55 mood disorder symptoms, 55 nutritional supplementation and dietary modification, 55

292 Insomnia (Continued) polychromatic light exposure, 55 symptoms, 54 Intestine inflammation, 48 Istradefylline, 179 Itopride, 5f

K Kayser-Fleischer rings, 10

L L-dopa,

173e174, 283 half-life and bioavailability, 210 peripheral decarboxylation inhibition, 210 synthesis, 188f Lenalidomide, 4 Leucine-rich repeat kinase-2, 159 G2019S mutation, 86, 257 induced mitochondrial calcium uptake, 122 Leukoaminochrome autoxidation, 230e231 ortho-semiquinone, 271 o-semiquinone radical, 216 Levomepromazine, 5f Lipid peroxidation, 138 Lipopolysaccharide-induced pleckstrin homology-like domain family, 145 Lysosomal system, protein degradation dysfunction. See Protein degradation dysfunction

M Maneb, 8 Manganese-induced parkinsonism aminochrome, 15 chronic manganese exposure, 13e15 glutamatergic neurotransmission, 14 magnetic resonance imaging brain scanning, 13 manganese depositions, 14e15 mechanism, 16f motor and sensory disorders, 14 neuronal degeneration, 13 occupational activities, 13 positron-emission tomography study, 14 redox ability, 15 welding fumes exposure, 14 Mannitol, 182 Memantine, 180e181 Memory, 58

Index

Mesencephalic astrocyte-derived neurotrophic factor overexpression, 100e101 Methylation, dopamine, 209e212 1-Methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) endoplasmic reticulum stress, 245 fulminant effect, 248 induced parkinsonism biochemical and histological studies, 19 dopamine nigrostriatal system loss, 20 L-dopa-induced dyskinesias, 18e19 marmosets treatment, 19 neurodegeneration, 20 neurotoxic action, 19e20 neurotoxicity, 21f nonmotor symptoms, 18e19 mitochondrial dysfunction, 243e244 oxidative stress, 244 parkinsonism, 243 preclinical model, 246e248 sequestosome level, 244 Microglia necroinflammatory responses, 142e143 Mild cognitive impairment, 60e61 Alzheimer disease, 64 biomarkers, 61 diffusion tensor magnetic resonance imaging, 63e64 predictive model, 61e62 prevalence, 62 structural brain connectome, 64 voxel-based morphometry techniques, 64 white matter hyperintensities, 63 Mini-Mental State Exam, 67e68 Miro protein, 101 Mitochondrial dysfunction calcium homeostasis, 122 complex I deficiency, 118 complex I inhibition, 116, 117f complex IV deficiency, 119e120 dopaminergic neuron loss, 116 extracellular vesicle trafficking neuroinflammation, 120 gene editing, 116 mitochondrial DNA mutations, 119 mitochondrial fission/fusion, 120e121 Ndufs4GT/GT mice, 118e119 optic atrophy-1, 121 postmortem Parkinson’s disease, 116e118

Index

PTEN-induced kinase 1 mutation, 122 tricarboxylic acid cycle, 119 wild-type DJ-1 gene overexpression, 121e122 MitoPark mouse model, 47 Mitophagy, 128 Monoamine oxidase inhibitors, 175 isoforms, 203 monoamine oxidase-A, 203 monoamine oxidase-B, 203 aldehyde dehydrogenase-1 activity, 205 alpha-synuclein reaction, 204 3,4-dihydroxyphenylacetaldehyde oxidation, 204 inhibitors, 205 nucleotide sequences, 203 oxidative deamination, 204 Montreal Cognitive Assessment score, 61e62 Multiple-system atrophy, 138

N Neuroinflammation alpha-synuclein autoantibodies, 143 and glia maturation factor, 147 oligomers injection, 144e145 6-hydroxydopamine, 242 6-hydroxydopamine-dependent lesions, 143e144 immune system, 143 inflammatory indexes, 146 interleukins, 142 leucine-rich repeat kinase 2 gene knockdown, 145 lipopolysaccharide intranigral injection, 145 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine neurotoxic effects, 143e144 microglial activation, 142e143 microglia primary cell cultures, 145 microRNAs deregulation, 146 noncoding RNA GAS5, 147 nucleotide-binding oligomerization domain-leucine-rich repeat, 146 proinflammatory inducers, 143 pyrin domain-containing protein-3, 146 Neuromelanin, 236 dopaminergic neurodegeneration, 223 lipids content, 222

293

melanin formation, 221 neurotoxic effects, 223 reactive oxygen species, 223 substantia nigra, 221 Neuron-derived exosomes, 265 Neuroprotective mechanisms DT-diaphorase, 230e232 glutathione transferase-M2-2, 232e234 vesicular monoamine transporter-2, 229e230 Neurotoxicity aminochrome, 219f dopaminochrome, 221, 222f 5,6-indolequinone, 220f neuromelanin, 223 ortho-quinone, 215f Neurturin, 246 Nigrostriatal neuron degeneration endoplasmic reticulum stress astragaloside-IV, 153e154 A53T induced mitochondrial dysfunction, 150e151 COPII mediated endoplasmic reticulum-Golgi transit, 151 manganese, 154 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine, 152 microRNA-34a-5p overexpression, 153e154 neural stem cells transplantation, 153 oxidative and endoplasmic reticulum stress, 151e152 parkin protein level, 151e152 phosphorylated pancreatic endoplasmic reticulum kinase immunoreactivity, 151 stress-induced protection, 153 tetrachlorobenzoquinone, 150 unfolded protein response, 150 X-Box binding protein 1 knockdown, 152e153 neuroinflammation. See Neuroinflammation oxidative stress, 136f alpha-synuclein aggregation, 140 ATP production, 134e135 dopamine degradation, 139f flavin mononucleotide, 135e137 hydrogen peroxide, 138 lipid peroxidation, 138 multiple-system atrophy, 138 one-electron reduction, 138e139

294 Nigrostriatal neuron degeneration (Continued) parkin gene expression, 140 reactive oxygen species, 135e137 resveratrol, 137 26S proteasome dysfunction, 140 submitochondrial particles, 137 superoxide generation, 135, 137 protein degradation dysfunction. See protein degradation dysfunction Noradrenaline, 76 Norepinephrine hypothesis of depression, 40 synthesis, 190 Nurr1, 218

O Obstructive sleep apnea, 33e34 Olfactory dysfunction cholinergic dysfunction, 27 cognitive impairment, 27e28 comorbid depression, 29 delirium, 27 gray matter volume, 28 hyposmia, 26e28 lateralized microstructural changes, 29 nigrostriatal dopaminergic function, 26e27 odor-identification capacity, 26 progression, 29 quantitative analysis, 28 raphe nuclei, 27 smell identification test, 26 ventral glomerular insufficiency, 28 white matter microstructure, 28 Opicapone, 210 Optic atrophy-1 protein, 106, 121 Ortho-quinone, 213e214 Orthostatic hypotension cerebral hypoperfusion, 76 cognitive impairment, 76 lower body mass index, 75e76 neurogenic, 76 noradrenaline, 76e77 prevalence, 75e76 Oxidative stress, 104 6-hydroxydopamine, 241 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP), 244 nigrostriatal neuron degeneration. See Nigrostriatal neuron degeneration rotenone, 245e246

Index

P Paraquat-induced parkinsonism agriculture, 7 cardiac contractile dysfunction, 8 endoplasmic reticulum stress-dependent cell death, 8 glutamate efflux-starting excitotoxicity, 7 increased glucose uptake, 8 lung damage, 8 maneb exposure, 8 mitochondrial dysfunction, 7 oxidative stress, 7 protein dysfunction, 8 symptoms, 7 transport, 7 Pargyline, 205 Parkin-Q311X mutation, 256 Parkinsonism copper-induced, 10, 11f, 12 drug-induced, 2e6 manganese-induced. See Manganeseinduced parkinsonism 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine-induced, 18e20 paraquat-induced, 7e8 Parkinson’s disease age risk, 1e2 classification, 2, 3f epidemiology, 1 familial form, 2 Health Improvement NetworkÆs primary care database, 25 idiopathic, 2, 23 incidence, 1 motor symptoms, 25 mitochondrial dysfunction. See Mitochondrial dysfunction nigrostriatal neurons degeneration. See Alpha-synuclein protein degradation dysfunction. See Protein degradation dysfunction neuropsychiatric disorders, 25 nonmotor symptoms anxiety. See Anxiety cognitive decline. See Cognitive decline constipation. See Constipation dementia and dementia with Lewy bodies. See Dementia and dementia with Lewy bodies depression. See Depression excessive daytime somnolence. See Excessive daytime somnolence

Index

insomnia, 54e55 olfactory dysfunction, 26e29 orthostatic hypotension, 75e76 rapid eye movement sleep behavior disorder. See Rapid eye movement sleep behavior disorder visual disturbances, 77e78 parkinsonism, 2e6 pharmacological treatment, 173e185 preclinical models. See Preclinical models prevalence, 1e2 sex differences, 1 Pharmacological treatment adenosine antagonists, 177e179 anticholinergic drugs, 176e177 clinical trials, 181e182 dopaminergic drugs dopamine agonists, 174 L-dopa, 173e174 monoamine oxidase inhibitors, 175 Ganoderma lucidum, 181 glucagon-like peptide-1 receptor agonists, 179e180 melatonin receptor antagonist, 177 memantine, 180e181 midodrine and droxidopa, 177 Pheomelanin, 222 Pioglitazone, 247 Pittsburg Sleep Quality Index, 52e55 Preclinical models, 283e284 endogenous neurotoxins, 284 alpha-synuclein, 264e266 aminochrome, 269e274 3,4-dihydroxyphenylacetaldehyde, 267e269 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine, 263 exogenous neurotoxins, 283e284 adenosine 2A receptor, 247 disease progression, 248 glial cell line-derived neurotrophic factor, 246 6-hydroxydopamine, 241e243 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine, 243e245 motor symptoms, 247e248 neuroinflammation, 247 neurotoxic mechanisms, 248 oxidative stress, 247 rotenone, 245e246 synucleinopathy, 247 gene mutations, 259f

295

alpha-synuclein human A53T and A30P mutations, 255e256 discovery, 255 idiopathic Parkinson’s disease, 258 leucine-rich repeat kinase 2 G2019S mutations, 257 neurodegenerative process, 258 parkin knockout mice, 256 parkin-Q311X mutation, 256 protein deglycase-1 knockout rats, 257e258 PTEN-induced kinase 1 gene deletion, 257 transgenic animals, 258 idiopathic Parkinson’s, 284 Prion-like hypothesis, 264e266 Promazine, 5f Proteasomal system, protein degradation dysfunction. See Protein degradation dysfunction Protein deglycase-1, 163 Protein degradation dysfunction lysosomal system ATP13A2 gene mutation, 130e131 chaperone-mediated autophagy, 128e129 glucosylceramide accumulation, 129e130 knockout autophagy receptors, 128 leucine-rich repeat kinase-2 G2019S mutation, 129 lysosomal b-glucocerebrosidase gene activation, 131 mitophagy, 128 Parkin mutation, 130 RABGEF1 factor, 130e131 Rho GTPase-1 mutation, 131 proteasomal system alpha-synuclein, 126e127 Hsc70-interacting protein, 126 leucine-rich repeat kinase-2 degradation, 126 PTEN-induced putative kinase-1, 127 26/20S proteasomal function inhibition, 127 tumor necrosis factor receptorassociated factor-6, 126e127 ubiquitineproteasome system, 125e126 Protein phosphatase activity inhibition, 189

296 PTEN-induced kinase-1 (PARK6) F-box protein-7, 165e168 homozygous mutation, 162 mutation, 122 parkin gene mutation, 162 ubiquitin S65A mutant, 162

R Raphe nuclei, 27 Rapid eye movement sleep behavior disorder adult population, 31 alpha-synuclein aggregations, 32 atonia, 31 balance and gait impairments, 34 dopamine transporter positron-emission tomography, 32e33 excessive daytime sleepiness, 35 gender, 32 meta- and meta-regression analysis, 31 neurobiological pathways, 33 neurodegeneration, 32 neuroinflammation, 34e35 neuromelanin imaging, 33 obstructive sleep apnea, 33e34 prevalence, 31 resting-state electroencephalography, 34e35 screening questionnaire, 34e35 sleep and circadian phenotypes, 35e36 sleep-disordered breathing, 34 striatal dopamine transporter dysfunction, 33 substantia nigra damage, 33 synucleinopathies, 32, 35 Rasagiline, 175, 268 RelA inhibition, 142e143 Reserpine, 197 Restless legs syndrome, 59 Resveratrol, 137 Rieske iron-sulfur protein, 137 Rotenone cell death, 246 endoplasmic reticulum stress, 245e246 neuronal loss, 245 oxidative stress, 245e246 tyrosine hydroxylase inhibition, 246

S Safinamide, 175, 205 Selective serotonin reuptake inhibitor, 40e42

Index

Selegiline, 175, 205 Sequestosome level, 244 Serotonin hypothesis, depression, 40 Sertraline, 5f Smell identification test, 26 Sodium oxybate, 52 Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, 82 Striatal dopamine transporter dysfunction, 33 Succinate dehydrogenase, 119 Sulfuretin, 242 Sulpiride, 5f Sympathetic neurons, 47 Synapsin III, 85 Synaptogyrin-3, 197 Synucleinopathies, 32, 35

T Tacrolimus, 4 Tetrabenazine, 196 Tetrachlorobenzoquinone-induced endoplasmic reticulum stress, 150 Tolcapone, 210 Transcranial sonographic substantia nigra, 3e4 Transcription factor Nurr1, 146 Tricarboxylic acid cycle, 119 Trihexyphenidyl, 176 Trimetazidine, 4 Tumor necrosis factor receptor-associated factor-6, 126e127 Tunicamycin, 154 Tyrosine hydroxylase, 187e189

U Ubiquitination, 85 Ubiquitineproteasome system, 125e126 Unified Parkinson’s Disease Rating Scale, 27, 175

V Vacuolar protein sorting-35, 160 Vagus nerve stimulation, 46e47 Vesicular monoamine transporter-2, 195 adenoviral-dependent overexpression, 196e197 amino acids, 195 amphetamine, 196 dopamine uptake, 198, 199f glycosylation, 195

297

Index

heterozygote knockout, 196 inhibition, 196 methamphetamine, 195e196 monoaminergic vesicle isolation, 198 neuromelanin, 229e230 and neuromelanin level, 198 neuroprotective mechanisms, 229e230 posttranslational modifications, 195 synaptogyrin-3 effect, 197 vesicular monoamine transporter-2, 195 Visual disturbances alpha-synuclein aggregation, 78 alpha-synuclein deposits, 78e79 ferritin autophagy, 79 incidental Lewy body disease, 78 microglia, 78

neuroinflammation, 78 psychosis, 78 rapid eye movement sleep behavior disorder, 77e78 symptoms, 77

W Wilson’s disease, 10

X X-Box binding protein-1 knockdown, 152e153 pathway, 152e153

Y Yoga program, 43

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