Frontiers in Clinical Drug Research - Alzheimer Disorders Volume 6 [1 ed.] 9781681083391, 9781681083407

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Frontiers in Clinical Drug Research - Alzheimer Disorders (Volume 6) Edited by Atta-ur-Rahman, FRS

Kings College, University of Cambridge, Cambridge, UK

Frontiers in Clinical Drug Research - Alzheimer Disorders Volume # 6 Editor: Atta-ur-Rahman ISSN (Online): 2214-5168 ISSN (Print): 2451-8743 ISBN (Online): 978-1-68108-339-1 ISBN (Print): 978-1-68108-340-7 ©2017, Bentham eBooks imprint. Published by Bentham Science Publishers – Sharjah, UAE. All Rights Reserved. Reprints and Revisions: First published in 2017.

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CONTENTS PREFACE ................................................................................................................................................ i LIST OF CONTRIBUTORS .................................................................................................................. Li CHAPTER 1 THE TREATMENT OF BRAIN INFLAMMATION IN ALZHEIMER’S DISEASE. CAN TRADITIONAL MEDICINES HELP? .................................................................... James David Adams INTRODUCTION .......................................................................................................................... Anti-inflammatory Agents in AD ........................................................................................... Risk Factors for Developing AD ............................................................................................ Prevention of AD .................................................................................................................... Ceramide and AD ................................................................................................................... The Blood Brain Barrier and AD ............................................................................................ Visfatin and AD ...................................................................................................................... Traditional Plant Medicines for AD ....................................................................................... The Modern Approach to Curing AD ..................................................................................... CONCLUDING REMARKS ......................................................................................................... CONFLICT OF INTEREST ......................................................................................................... ACKNOWLEDGEMENT ............................................................................................................. REFERENCES ............................................................................................................................... CHAPTER 2 STEM CELL STRATEGIES FOR THE MODELING AND THERAPY OF ALZHEIMER’S DISEASE ..................................................................................................................... Haigang Gu 1. INTRODUCTION ...................................................................................................................... 2. NEUROPATHOLOGY OF AD: KEYS TO DRUG DISCOVERY AND ANIMAL MODELS ......................................................................................................................................... The β-amyloid Hypothesis of AD ........................................................................................... The Hyper-phosphorylated Tau Protein Hypothesis of AD ................................................... The cholinergic hypothesis of AD .......................................................................................... 2.1. Drug Discovery of AD ..................................................................................................... 2.1.1. Treatment of Amyloid Pathology ........................................................................ 2.1.2. Treatment of Tau Pathology ................................................................................ 2.1.3. Treatment of Synaptic Dysfunction ..................................................................... 2.1.4. Neurotrophic Factors (NTFs) ............................................................................. 2.1.5. Cell Transplantation ........................................................................................... 2.2. Animal Models of AD ..................................................................................................... 2.2.1. Transgenic Animal Models of AD ....................................................................... 2.2.2. Selective Cholinergic Lesion Animal Models of AD ........................................... 3. STEM CELLS AS USEFUL TOOLS FOR CELL TRANSPLANTATION, DRUG DISCOVERY AND AD MODELING .......................................................................................... 3.1. Neural Stem/Progenitor Cells (NP/SCs) .......................................................................... 3.2. Mesenchymal Stem Cells (MSCs) ................................................................................... 3.3. Embryonic Stem Cells (ESCs) ......................................................................................... 3.4. Induced Pluripotent Stem Cells (IPSCs) .......................................................................... 3.5. In Situ Generation of Neurons in the Brain ..................................................................... 3.6. Modeling and Therapy of AD with Genome Editing ...................................................... 4. PERSPECTIVES ........................................................................................................................ ABBREVIATIONS ......................................................................................................................... CONFLICT OF INTEREST ......................................................................................................... ACKNOWLEDGEMENT .............................................................................................................

1 1 2 3 4 5 6 6 6 11 11 11 12 12 20 21 23 23 23 24 24 24 25 25 26 27 27 28 28 29 31 32 33 35 37 39 41 42 43 44

REFERENCES ............................................................................................................................... 44 CHAPTER 3 RETINAL NEURODEGENERATION IN ALZHEIMER’S DISEASE .................. L. Guo, M. Pahlitzsch, F. Javaid and M.F. Cordeiro INTRODUCTION .......................................................................................................................... THE RETINA – AN INTEGRAL PART OF THE BRAIN ....................................................... VISUAL CHANGES IN AD .......................................................................................................... Visual Abnormalities .............................................................................................................. Pupil Abnormalities ................................................................................................................ RETINAL CHANGES IN AD ....................................................................................................... Retinal Histopathologic Abnormalities ................................................................................... Retinal in vivo Abnormalities ................................................................................................. Retinal Nerve Abnormalities ......................................................................................... Retinal Vasculature Abnormalities ............................................................................... Retinal Cellular Abnormalities – RGC Apoptosis ........................................................ NON-RETINAL OCULAR CHANGES IN AD ........................................................................... AD-RELATED CHANGES IN RETINAL DISEASES .............................................................. AD-related Changes in Glaucoma .......................................................................................... AD-related Changes in AMD ................................................................................................. TARGETING OF AMYLOID-ß IN TREATMENT OF GLAUCOMA AND AMD ............... CONCLUSION ............................................................................................................................... CONFLICT OF INTEREST ......................................................................................................... ACKNOWLEDGEMENTS ........................................................................................................... REFERENCES ............................................................................................................................... CHAPTER 4 PATHOPHYSIOLOGY OF ALZHEIMER DISEASE: CURRENT DRUG THERAPY ................................................................................................................................................ Sumeet Gupta and Vikas Jhawat INTRODUCTION .......................................................................................................................... PATHOPHYSIOLOGY OF ALZHEIMER’S DISEASE ........................................................... HYPERTENSION AND ALZHEIMER’S DISEASE ................................................................. ROLE OF RENIN ANGIOTENSIN SYSTEM (RAS) IN ALZHEIMER’S DISEASE ........... GENETIC POLYMORPHISM AND AD .................................................................................... TREATMENTS FOR ALZHEIMER’S DISEASE ..................................................................... HERBAL DRUGS FOR THE TREATMENT OF AD ............................................................... CONCLUSION ............................................................................................................................... ABBREVIATIONS: ....................................................................................................................... CONFLICT OF INTEREST ......................................................................................................... ACKNOWLEDGEMENTS ........................................................................................................... REFERENCES ............................................................................................................................... CHAPTER 5 BIOLOGICAL MASS SPECTROMETRY FOR DIAGNOSIS OF ALZHEIMER'S DISEASE .................................................................................................................................................. Hani Nasser Abdelhamid and Hui-Fen Wu INTRODUCTION .......................................................................................................................... Requirements of Alzheimer's Disease Diagnosis ................................................................... Application of Mass Spectrometry for Alzheimer's Disease .................................................. Imaging Mass Spectrometry for Alzheimer's Disease ............................................................ Advantages and disadvantages of Mass Spectrometry ........................................................... CONCLUSION ............................................................................................................................... CONFLICT OF INTEREST ......................................................................................................... ACKNOWLEDGEMENTS ...........................................................................................................

56 57 57 60 60 61 62 62 63 63 66 66 68 69 69 71 72 73 74 74 74 87 87 88 92 92 94 95 99 101 102 102 102 103 110 111 112 113 118 118 120 120 120

REFERENCES ............................................................................................................................... 120 CHAPTER 6 THE STRUCTURE-ACTIVITY RELATIONSHIP OF MELANIN AS A SOURCE OF ENERGY DEFINES THE ROLE OF GLUCOSE TO BIOMASS SUPPLY ONLY, IMPLICATIONS IN THE CONTEXT OF THE FAILING BRAIN ................................................. Arturo Solís Herrera INTRODUCTION .......................................................................................................................... Basal Brain Energy Metabolism ............................................................................................. The Role of Pyridine Nucleotides and the Abnormal Expression of Genes .................. REMARKS AND CONCLUSION ................................................................................................ CONFLICT OF INTEREST ......................................................................................................... ACKNOWLEDGEMENTS ........................................................................................................... REFERENCES ............................................................................................................................... CHAPTER 7 NEURO-PROTECTIVE PROPERTIES OF THE FUNGUS ISARIA JAPONICA: EVIDENCE FROM A MOUSE MODEL OF AGED-RELATED DEGENERATION ................... Koichi Suzuki, Masaaki Tsushima, Masanobu Goryo, Tetsuro Shinada, Yoko Yasuno, Eiji Nishimura, Yasuo Terayama, Yuki Mori and Yoshichika Yoshioka INTRODUCTION .......................................................................................................................... IJE Improves Nerve Function in Aged Mouse Brain .............................................................. 1. Neuroprotective Effects of IJE .................................................................................. 2. Histochemical Observation ....................................................................................... 3. Assessments of Acute and Sub-acute Toxicity ........................................................... NMR Analyses in the I. Japonica Extract ............................................................................... 1. Chemical Component of I. Japonica ......................................................................... 2. Biologically Active Substances ................................................................................. 3. NMR and Mass Study of Water Extract of I. Japonica ............................................. Visualization of the Physiological and Pathological Alterations in the Central Nervous System using MRI and MRS .................................................................................................. 1. Fine Imaging Using Ultra-high Field MRI ............................................................... 2. Magnetic Resonance Spectroscopy ........................................................................... 3. Brain Temperature Estimation Using MRS .............................................................. CONCLUDING REMARKS ......................................................................................................... CONFLICT OF INTEREST ......................................................................................................... ACKNOWLEDGEMENTS ........................................................................................................... REFERENCES ...............................................................................................................................

127 127 138 142 149 151 152 152 154 155 156 156 159 160 162 162 165 170 171 172 175 178 180 180 181 181

SUBJECT INDEX .................................................................................................................................. 

i

PREFACE The book series, “Frontiers in Clinical Drug Research – Alzheimer Disorders”, is intended to present the important advancements in the field in the form of cutting edge reviews written by experts. Volume 6 of this eBook series is a compilation of seven well written chapters contributed by prominent researchers in the field. It includes the treatment of brain inflammation, stem cell strategies, retinal neurodegeneration, pathophysiology of Alzheimer disease, and a number of other related areas. Chapter 1 by Adams discusses the use of plant medicines as an alternative treatment to decrease the progression of Alzheimer’s disease (AD). In chapter 2, Haigang Gu describes the recent progress of stem cell strategies for AD modeling and therapy. Cordeiro et al. in chapter 3 focus on the retinal neurodegeneration in AD. The pathological similarities between AD and eye diseases are also discussed. In Chapter 4, Gupta & Jhawat highlight the pathophysiology of Alzheimer disease with respect to the current drug therapy. In chapter 5, Abdelhamid and Wu present the use of biological mass spectrometry for the diagnosis of Alzheimer’s disease. This review also highlights the recent developments in disease diagnosis using mass spectrometry. Chapter 6 by Herrera emphasizes the structureactivity relationship of melanin as a source of energy. The last chapter by Suzuki et al., discusses the neuro-protective properties of the fungus Isaria japonica (IJ). The results showed that products derived from IJ may prevent or decrease the impact of dementia, especially AD. The 6th volume of this book series represents the results of a huge amount of work by many eminent researchers. I am grateful to the authors for their excellent contributions. I would also like to express my gratitude to the editorial staff of Bentham Science Publishers, particularly Mr. Mahmood Alam (Director Publication), Mr. Shehzad Naqvi (Senior Manager Publications) and Ms. Fariya Zulfiqar (Assistant Manager Publications) for their hard work and persistent efforts.

Prof. Atta-ur-Rahman, FRS Kings College University of Cambridge Cambridge UK

ii

List of Contributors Arturo Solís Herrera Eiji Nishimura F. Javaid Haigang Gu Hani Nasser Abdelhamid Hui-Fen Wu

James David Adams Koichi Suzuki L. Guo M. Pahlitzsch M.F. Cordeiro

Masaaki Tsushima Masanobu Goryo Sumeet Gupta Tetsuro Shinada Vikas Jhawat Yasuo Terayama Yoko Yasuno Yoshichika Yoshioka Yuki Mori

Human Photosynthesis® Research Center, Sierra del Laurel, 212, Bosques del Prado Norte, CP 20127, Aguascalientes, México Graduate School of Science, Osaka City University, Osaka, Japan Glaucoma & Retinal Degeneration Research Group, Visual Neurosciences, UCL Institute of Ophthalmology, Bath Street, London, EC1V 9EL, UK Department of Pediatrics, Northwestern University Feinberg School of Medicine, Lurie Children's Hospital Research Center, Chicago, IL 60614, USA Department of Chemistry, Assuit University, Assuit, 71515, Egypt Department of Chemistry and Center for Nanoscience and Nanotechnology, National Sun Yat-Sen University, Kaohsiung, 70, Lien-Hai Road, Kaohsiung, 80424, Taiwan School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, 807, Taiwan Institue of Medical Science and Technology, National Sun Yat-Sen University, Kaohsiung, 70, Lien-Hai Road, Kaohsiung, 80424, Taiwan Doctoral Degree Program in Marine Biotechnology, National Sun Yat-Sen University and Academia Sinica, Kaohsiung, 80424, Taiwan School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90089, USA Organization for Research Promotion, Iwate University, Morioka, Iwate, Japan Glaucoma & Retinal Degeneration Research Group, Visual Neurosciences, UCL Institute of Ophthalmology, Bath Street, London, EC1V 9EL, UK Glaucoma & Retinal Degeneration Research Group, Visual Neurosciences, UCL Institute of Ophthalmology, Bath Street, London, EC1V 9EL, UK Glaucoma & Retinal Degeneration Research Group, Visual Neurosciences, UCL Institute of Ophthalmology, Bath Street, London, EC1V 9EL, UK Western Eye Hospital, Imperial College Healthcare Trust, London, UK Organization for Research Promotion, Iwate University, Morioka, Iwate, Japan Faculty of Agriculture, Iwate University, Morioka, Iwate, Japan Department of Pharmacology, M. M. College of Pharmacy, M. M. University, Mullana, (Ambala), Haryana, India Graduate School of Science, Osaka City University, Osaka, Japan Department of Pharmacology, M. M. College of Pharmacy, M. M. University, Mullana, (Ambala), Haryana, India Division of Neurology and Gerontology, Department of Internal Medicine, Iwate Medical University, Morioka, Iwate, Japan Graduate School of Science, Osaka City University, Osaka, Japan Biofunctional Imaging Laboratory, Immunology Frontier Research Center, Osaka University, Osaka, Japan Biofunctional Imaging Laboratory, Immunology Frontier Research Center, Osaka University, Osaka, Japan

Frontiers in Clinical Drug Research - Alzheimer Disorders, 2017, Vol. 6, 1-19

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CHAPTER 1

The Treatment of Brain Inflammation in Alzheimer’s Disease. Can Traditional Medicines Help? James David Adams* School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90089, USA Abstract: The blood brain barrier degenerates in many people as they age. This degeneration can lead to inflammation, amyloid accumulation, neuron loss, tangle accumulation and dementia. Damage to the blood brain barrier may involve oxygen radical production through a visfatin mediated mechanism. Several plant medicines have been traditionally used to decrease the progression of Alzheimer’s disease. Antioxidant mechanisms of action have been described for these medicines that may protect the blood brain barrier. These plant medicines provide alternative treatments for Alzheimer’s disease.

Keywords: Alzheimer’s disease, Anti-inflammatory prevention, Plant medicines. INTRODUCTION Alzheimer’s disease (AD) involves neurodegeneration induced by amyloidβ. This neurodegeneration results in loss of neurons, plaque and tangle formation and ultimately in dementia. Many AD patients are treated with acetylcholinesterase inhibitors to slow the progression of mild AD. Eventually, most AD patients die from pneumonia and not neurodegeneration. The current consensus is that AD is caused by amyloidβ toxicity in the brain [1]. It is clear that extracellular amyloidβ is toxic to neurons. Amyloidβ aggregates into fibrils, sheets and plaques. Some intermediate amyloid protein aggregates in the plaque formation process are toxic to neurons. The role of inflammation in the pathophysiology of AD is well established [1]. Inflammation in AD can be secondary to amyloidβ accumulation. In other words, amyloidβ causes inflammation in the brain. Inflammation can also occur early in Corresponding author James David Adams: School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90089, USA; Tel: 323-442-1362; Fax: 323-442-1681; E-mail: [email protected] *

Atta-ur-Rahman (Ed.) All rights reserved-© 2017 Bentham Science Publishers

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the disease process and initiate amyloidβ accumulation and AD pathology [2]. This inflammation involves microglial cells, astrocytes, perivascular macrophages and monocytes that infiltrate into the brain [2]. There are a number of different inflammatory molecules that are produced in the brain in this inflammatory process and as a consequence of amyloidβ production including chemokines, complement molecules, cytokines, inflammatory and acute phase proteins, cyclooxygenase-2, and free radicals [2 - 5]. Tau phosphorylation leading to tangle formation may occur as the result of amyloidβ oligomer toxicity [1]. Microglial and astrocytic activation are also involved in alteration of tau phosphorylation [1]. Neurofibrillary tangles are frequently found in AD brains. The question that remains unanswered is why does amyloidβ production increase in the brains of people who will develop AD? This question can be avoided by claiming that 100% of people will develop AD if they live long enough. In other words, amyloidβ accumulation is a natural process in the brain that cannot be avoided. However, many very old people do not develop AD. Anti-inflammatory Agents in AD Several epidemiological studies have examined the use of anti-inflammatory drugs in patients and have found that the use of these drugs may decrease the induction of AD. These studies have been critically reviewed [2, 5, 6]. The use of indomethacin was reported to slow the progression of AD [7]. This finding was later disputed [8]. Patients suffering from arthritis have a decreased risk of developing AD, perhaps because of their use of anti-inflammatory agents [9]. Several other reports have failed to show a protective effect of anti-inflammatory agents in the progression or development of AD. In addition, several attempts to slow the progression of AD with various anti-inflammatory drugs have failed to show an effect. It must be remembered that oral nonsteroidal anti-inflammatory agents (NSAIDs) are very toxic, especially to the elderly. NSAIDs have effects on prostaglandins, lipoxins, resolvins, thromboxanes and other lipid metabolites. NSAIDs cause strokes, heart attacks, kidney damage and ulcers. They cause 42,000 or more deaths in the US every year. NSAIDs should be avoided in trials that hope to delay the progression of AD. Steroids damage the hippocampus and should also be avoided [10]. Perhaps the choice of anti-inflammatory agent has been inappropriate so far. In addition, the doses chosen may have been inappropriate in past studies. The doses chosen were probably too high and induced too much toxicity.

Brain Inflammation

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Risk Factors for Developing AD If all people get AD with age, then the only risk factor for developing AD should be age. However, there are other risk factors that increase the chance of developing AD. The risk factors for developing AD are age, head trauma, high blood pressure, high blood cholesterol, diabetes, cardiovascular disease, atrial fibrillation, apolipoprotein E4, thrombosis, peripheral inflammatory factors, decreased muscle mass and high alcohol consumption [11 - 13]. Women are more likely to develop AD than men [11 - 13]. Brain trauma can cause gliosis, inflammation and deleterious changes to the brain that may be important in AD. Peripheral inflammatory factors cause high blood pressure, high blood cholesterol, type 2 diabetes, cardiovascular disease, atrial fibrillation and thrombosis [14]. These peripheral inflammatory factors include adipokines made in visceral and ectopic fat that are released into the blood. Inflammatory adipokines include visfatin, leptin, resistin, tumor necrosis factor α, IL-6 and others. As people age, visceral and ectopic fat deposits develop. Toxic lifestyles, including lack of exercise and over eating, cause fat accumulation. Ectopic fat is fat that surrounds arteries, infiltrates muscles and other sites. Visceral fat accumulates in the peritoneal cavity. Therefore risk factors for AD are probably high blood levels of inflammatory adipokines released by visceral and ectopic fat. Obesity has increased greatly since the 1980s as reported by the Centers for Disease Control (www.cdc.gov). The incidence of AD has also increased greatly since 1980, in parallel with the increase in visceral obesity [15]. According to the Centers for Disease Control, among the entire US population, 93,500 people died while affected with AD in 2014. The entire US population, age adjusted death rate from AD increased by 39% from 2000 through 2010. Several studies found the incidence of AD decreased over the last 25 years or more by about 25% [16 - 19], in spite of the increases in obesity and type 2 diabetes. These studies were done in selected populations and point to better education and better treatment of heart disease as ways to prevent AD. This indicates that patients who are educated enough about risk factors for AD to seek out better health care and other healthy lifestyle practices have a decreased risk. Weight reduction can be part of a healthy lifestyle. All of these studies advise that patients who practice healthy lifestyles have a decreased risk of developing AD. Is the incidence of AD actually decreasing in the US? The answer is clearly that the incidence of AD is increasing in the total US population. Apolipoprotein E4 transports lipids inside the brain, including cholesterol and triglycerides. When triglycerides accumulate, the alternative fat ceramide is made

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in greater amounts. Apolipoprotein E4 is made in astrocytes and transports lipids to neurons by interacting with receptors in the low density lipoprotein receptor family. Since apolipoprotein E4 is a risk factor for developing AD, lipids are probably important in the mechanism of induction of AD. Muscles produce myokines such as adiponectin, irisin, IL-6, IL-8 and IL-15 [20]. These myokines stimulate lipolysis, decrease atherosclerosis and are antiinflammatory. Muscle is also responsible for clearing some insulin and glucose from the blood. Loss of muscle tissue causes insulin levels to increase leading to insulin resistance, also known as type 2 diabetes. Loss of anti-inflammatory myokines may be important in the induction of AD. Alcohol consumption leads to visceral fat and ectopic fat accumulation since alcohol activates sterol regulatory element binding protein [21]. Alcohol is an obesogen and can cause alcohol induced dementia, which is very similar to AD. In order to live long enough to develop AD, patients must not drink enough to result in death from a heart attack, stroke or cirrhosis. Prevention of AD Factors that decrease the onset and progression of AD include: regular physical activity, coffee consumption, moderate wine consumption, smoking and diets low in fat, high in fruit and vegetables [12, 22 - 24]. Diets high in fruit and vegetables have a major effect on gut bacteria that have a major effect on health. Physical activity decreases visceral fat, ectopic fat in the muscles, increases glucose and insulin clearance from the blood and promotes heart health. Muscle health improves with physical activity. Healthy muscles secrete anti-inflammatory myokines. Exercise also stimulates stem cell growth in every organ including the adult human brain [25]. It is not entirely clear why coffee decreases the likelihood of developing AD. Caffeine has been shown to be neuroprotective in patients older than 65 [26]. Moderate wine consumption can improve heart health that may decrease the chances of developing AD. It is not clear why smoking decreases AD. Nicotine is toxic to the heart and arteries, and stimulates atherosclerosis. A study found that smoking may actually increase the risk of developing AD [27]. However, nicotine is also an appetite suppressant. Perhaps smokers have less visceral and ectopic fat than nonsmokers. It is also possible that smokers die of other things, like cancer and heart disease, before they develop AD. Nicotine has been shown to be neuroprotective in laboratory animal experiments [28].

Brain Inflammation

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Diets high in fruit and vegetables and low in fat can help decrease the likelihood of developing visceral fat [29]. Eating a good diet in combination with regular exercise is the basis of living in balance, a traditional concept. Living in balance allows the body to heal itself [30]. Prevention of AD with education, proper diet, proper weight and regular exercise is the best medicine for AD. As people age, exercise becomes more difficult due to loss of muscle tissue, a normal aspect of aging. However, gentle exercise such as walking can still be done. During aging, the body switches from making subdermal fat to making visceral and ectopic fat. Eating less and eating better food becomes critical at this time. Aging patients, after the age of 60 or so, should weigh less than when they were 20 or so, due to loss of muscle tissue. Aging causes loss of muscle, brain and bone tissue. Prevention of AD should become the normal medicine for everyone. Ceramide and AD Lipids and lipid metabolites can be pharmacologically active. Ceramide is a lipid that is pharmacologically active and becomes much more abundant in the body during visceral obesity [14]. High serum ceramide levels increase the risk of developing AD [31]. Ceramide levels are high in AD brain compared to control brain [32]. Ceramide increases in astrocytes and microglial cells in proximity to capillary amyloid deposits in AD [33]. Amyloidβ activates the production of ceramide in some neurons, which implies that ceramide may be involved in the downstream mechanism of amyloidβ toxicity [32]. Ceramide stabilizes βsecretase, the enzyme that makes amyloidβ [34]. Amyloidβ then activates sphingomyelinases to increase cellular ceramide levels even more [34]. Therefore, ceramide may cause the formation of amyloidβ, or amyloidβ may cause the formation of ceramide. It is most likely that visceral and ectopic fat increase ceramide throughout the body and the brain, leading to increased amyloidβ production. Ceramide induces nitric oxide synthase, both the endothelial (eNOS) and inducible (iNOS) forms [14]. However, the induced iNOS and eNOS also dysfunction in the presence of ceramide leading to oxygen radical and peroxynitrite formation. This oxidative stress damages astrocytes and endothelial cells, leading to a damaged blood brain barrier that allows monocytes and neutrophils to penetrate into the brain. Ceramide increases amyloidβ in the brain that induces NADPH oxidase (NOX) on macrophages, monocytes and neutrophils that penetrate the blood brain barrier. NOX forms extracellular hydrogen peroxide that damages neuronal DNA, causes cell death and activates neutral sphingomyelinase, which makes more ceramide.

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The Blood Brain Barrier and AD The blood brain barrier is formed by endothelial cells that restrict the entry of many molecules into the brain and are joined by tight junctions. Astrocytes wrap their foot processes around the endothelial cells and are involved in maintaining the blood brain barrier. Pericytes are contractile cells involved in sustaining the blood brain barrier as well. It is clear that atherosclerosis of arteries in the blood brain barrier and other sites increases with aging and visceral adiposity, as does the incidence of AD [14]. Pericytes in the blood brain barrier degenerate in AD and a mouse model of AD, resulting in decreased clearance of amyloidβ from the brain [35]. The blood brain barrier becomes leaky and allows serum proteins and inflammatory cells to enter the brain. These inflammatory cells are mostly monocytes [2]. Glucose enters into the brain mostly due to the actions of glucose transporters, such as GLUT1, on endothelial cells. AD patients have decreased GLUT1 activity in their cerebral microvessels [36]. This means there is diminished glucose entry into the brain in AD. Decreased GLUT1 causes the induction of sterol regulatory element binding protein2 in the brain, which decreases amyloidβ clearance from the brain [36]. Visfatin and AD How does the blood brain barrier become damaged in AD? Visfatin is an inflammatory adipokine that increases in AD patients [37]. Blood born visfatin and xanthine dehydrogenase found on endothelial cells of the blood brain barrier catalyze the formation of oxygen radicals and hydrogen peroxide at the blood brain barrier [37]. This and ceramide induced oxygen radical formation damage endothelial cells and pericytes. Some plant derived compounds, such as quercetin and resveratrol, inhibit the release of visfatin from adipocytes [38]. This may decrease blood brain barrier damage. Other adipokines are present in the blood, including monocyte chemoattractant protein-1, which causes monocytes to stick to damaged endothelial cells. Visfatin also induces monocyte chemoattractant protein-1 [39]. Monocytes secrete adhesion proteins that cause more monocytes to adhere to the blood brain barrier. These cells then penetrate into the brain and increase the inflammatory response. Traditional Plant Medicines for AD Before the advent of modern medicine, people suffered from AD [40]. Healers found plant medicines that helped old patients with short-term memory loss

Brain Inflammation

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remain productive awhile longer. Table 1 presents a list of several of these plant medicines [41]. Several anti-inflammatory plant medicines are discussed below. Many people have used these plant medicines and still developed AD. These plant medicines must be used in combination with lifestyle changes to prevent or delay the progression of AD. Angelica sinensis, Angelica pubescens and other Angelica species are used in the treatment of AD. Angelica species contain several coumarins including umbelliferone, umbelliferone 6-carboxylic acid, scopoletin, isoscopoletin, 7-methoxy coumarin, 2’-isopropyl psoralene, scoparone, scopolin and esculetin [42]. Umbelliferone 6-carboxylic acid and esculetin inhibit acetylcholinesterase and β-site amyloid precursor protein cleaving enzyme 1 also called β-secretase 1 [42]. This means that Angelica plant medicines are useful for treating AD since they may enhance brain acetylcholine and decrease brain amyloidβ. Angelica also contains ligustilide that is anti-inflammatory, decreases cortical and hippocampal nerve damage, decreases astrocyte activation and protects the blood brain barrier [43, 44]. Ferulic acid is also found in Angelica plants, inhibits amyloid fibril formation and is an antioxidant, free radical scavenger [45]. Angelica plants contain furanocoumarins that can cause photosensitivity [46]. These plants are used daily by many people in China with no reports of adverse effects. Angelica is also present in Benedictine and other liqueurs that are consumed daily by many people around the world. Table 1. Traditional plant medicines used in Alzheimer’s disease. Plant Name

Active Compound

Mechanism of Action

Preparation

Angelica sinensis danggui

Ligustilide, ferulic acid

Cholinergic, anti-inflammatory

Root

Angelica pubescens duhuo

Coumarins

Anti-inflammatory

Root

Astragalus propinquus huangqi

Cycloastragenol, astragaloside

Telomerase activator, antiinflammatory

Root

Codonopsis pilosula, C. tangshen dangshen

Hesperidin, atractylenolide

Anti-inflammatory, cholinergic

Root

Crocus sativus xihonghua

Crocin

Anti-inflammatory, cholinergic

Pistils

Dipsacus asper xuduan

Saponins

Anti-inflammatory

Root

Glycyrrhiza glabra G. uralensis gancao

Phytosterols, saponins

Anti-inflammatory

Root and rhizome

Heteromeles arbutifolia California holly, toyon

Betulin, icariside E4, farrerol

Anti-inflammatory

Fruit

Indigofera tinctoria true indigo

Indirubins

Inhibit tau phosphorylation

Leaf

Lycium barbarum gouqizi

Polysaccharides

Inhibit tau phosphorylation

Fruit, bark

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(Table 1) contd.....

Plant Name

Active Compound

Mechanism of Action

Preparation

Paeonia alba P officinalis P lactiflora chishao

Phenolics and ursolic acid

Anti-inflammatory

Whole plant

Rhodiola crenulata hongjingtian

Salidroside

Adaptogen, anti-inflammatory

Root and rhizome

Schisandra chinensis wuweizi

Schisandrin

Anti-inflammatory, cholinergic

Fruit

Astragalus propinquus is used in China to treat AD. Astragalus extracts improve learning and memory in a mouse model of AD [47]. Astragaloside IV purified from the plant protects against amyloidβ toxicity by protecting mitochondria and protects the blood brain barrier [48, 49]. Extracts of the plant have been found to enhance telomerase activity in patients [50]. Cycloastragenol found in the plant is a telomerase activator. Telomerase lengthens telomeres. There is some evidence that telomeres may be short in AD patients. However, telomerase knock out mice are protected from amyloidβ pathology [51]. Plant medicines made from Astragalus are used daily in doses up to 40 grams with no reports of adverse effects. Fermented Codonopsis pilosula, dang shen, can enhance learning and memory in rats [52]. Extracts of plants of the Codonopsis genus have been shown to inhibit acetylcholinesterase [53]. Codonopsis contains hesperidin and atractylenolide [54]. Hesperidin is an inhibitor of β-secretase, prevents amyloid fibril formation [55] and protects against aluminum chloride induced cognitive dysfunction [56]. Hesperidin also attenuates learning and memory deficits and suppresses inflammation by activation of Akt/Nrf2 and inhibition of RAGE/NFkB [57]. Plant medicines made from Codonopsis are used daily, safely by many people. However, the medicine can interfere with blood clotting in some patients [58]. Crocus sativus, saffron, is comparable to memantine in the treatment of moderate to severe AD [59]. It is also comparable to donepezil in the treatment of mild to moderate AD [60]. Crocin, an anti-inflammatory ingredient from the plant, increases long-term potentiation in hippocampal neurons and prevents amyloid fibril formation [61]. Crocin is also neuroprotective and anti-inflammatory by inhibition of sphingomyelinase, which decreases ceramide production [62]. This implies that Crocus plant medicines may protect the blood brain barrier by decreasing ceramide. Crocin also decreases microglial cell activation and inflammatory cytokine production in the brain through inhibition of Notch signaling [63]. Crocetin, another active ingredient, may inhibit acetylcholinesterase [61]. Saffron is a spice that is consumed daily by many people around the world with no reports of toxicity. Plant medicines made from saffron are safe.

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However, consumption of 20 g of saffron can be toxic or even lethal to humans [64]. Dipsacus asper, xuduan, is related to teasel and contains saponins such as akebia saponin D. The total saponins and akebia saponin D protect neuronal cells from amyloidβ toxicity [65]. Akebia saponin D also attenuates the loss of memory in rats, injected intracerebroventricularly (ICV) with amyloidβ [67]. The saponin appears to alter Akt and NFkB pathways [66]. A saponin has been found to protect the blood brain barrier [67]. Although an extract of Dipsacus has been shown to have procoagulant effects on isolated platelets, there are no reports of clot problems in humans that use the medicine [68]. Glycyrrhiza glabra, licorice, water extract protects mice from ICV amyloidβ toxicity [69]. 2,2',4'-Trihydroxychalcone, an active ingredient in the plant, is an inhibitor of β-secretase, improves memory and decreases plaque formation in a mouse AD model [70]. Liquiritin, a flavanone glucoside from the plant, is neuroprotective through modulation of ERK and AKT/GSK-3β pathways [71]. Glycyrrhizic acid is neuroprotective by inhibition of oxidative stress and voltage gated sodium channels in the hippocampus and protects the blood brain barrier [72, 73]. Licorice is a food, candy and spice that is consumed daily by many people with no safety problems. Consumption of very large amounts of licorice results in hypertension and hypokalemia [74]. Heteromeles arbutifolia, toyon or California holly, is a traditional medicine used in the treatment of AD [40, 41]. The plant contains betulin, icariside E4, farrerol and other active compounds [40]. Farrerol protects endothelial cells in the blood brain barrier [75]. Other flavonoids in the plant, such as catechin, stimulate the nonamyloidogenic cleavage of amyloid precursor protein [76]. Betulin prevents sterol regulatory element binding protein activation [77], which may help control perivascular fat in the brain. Icariside compounds protect the blood brain barrier, prevent inflammatory cells from entering the brain and prevent neuronal damage [78]. Toyon is a food that can be consumed in large amounts daily with no adverse reactions. Indigofera tinctoria, true indigo, extracts prevent neuronal death in the hippocampus after ICV injection of amyloidβ into mice [79]. A plant extract improves memory in scopolamine treated mice and has antioxidant activity [80]. Gallic acid, quercitrin and myricetin are found in the plant [81]. Gallic acid, catechin and similar compounds decrease amyloid fibril deposition and decrease brain inflammation in a mouse model of AD [82]. Myricetin, a flavonoid, inhibits β-secretase [83] and is neuroprotective. Some flavonoids have been found to protect the blood brain barrier [84]. The plant medicine made from Indigo is

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widely used in Africa where it is considered a safe remedy. However, the plant medicine was reported to cause multiple organ failure and death in a child [85]. Lycium barbarum, goji berry, extracts protect cultured neurons from amyloidβ toxicity [86]. Polysaccharides may be the protective compounds present in the berries [87] and have been shown to decrease tau phosphorylation [88]. Hyperphosphorylation of tau may lead to tangle formation in AD. Goji berries have been used for centuries in China to treat diseases of old age. The plant medicine made from Lycium is safe and is used daily by many people. However, extracts of the plant can cause hypoglycemia in experimental animals [89]. This suggests that diabetics should use the plant medicine with caution if at all. Paeonia alba, peony, contains paeoniflorin that is an anti-inflammatory monoterpene glycoside. Paeoniflorin decreases plaque formation, downregulates tumor necrosis factorα and interleukin-1β in the brain, decreases activation of microglia and astrocytes in a mouse AD model [90]. Paeoniflorin also protects the blood brain barrier [91]. These effects may involve inhibition of suppressor of cytokine signaling 2 [92]. Ursolic acid is present in the plant and decreases memory deficits caused by ICV injection of amyloidβ [93]. Paeonol, a phenolic compound, is anti-inflammatory due to inhibition of toll-like receptor 2 and 4 [94]. Peony also contains several antioxidant flavonoids. Peony is a very popular remedy in China that is used safely, daily by many people. It can cause mild diarrhea in some cases [95]. Rhodiola crenulata extracts are adaptogens that help balance the body. Salidroside, a monoterpene glucoside found in the plant, has antioxidant activity and protects cells from amyloidβ toxicity [96]. The compound decreases reactive oxygen species, inhibits NADPH oxidase, inhibits the expression of iNOS and COX2, and stimulates JNK and p38 MAP kinase pathways [97, 98]. Salidroside also protects the blood brain barrier [99]. Extracts of the plant protect neural stem cells by an antioxidant mechanism and improve hippocampal neurogenesis [100]. Proanthocyanidins found in the plant inhibit amyloid aggregation [101]. The use of Rhodiola plant medicine has been reported to cause occasional mild adverse reactions, such as indigestion. Schisandra chinensis fruit contains several dibenzocyclooctadiene lignans including schisandrin, schisantherin A, schisandrin B and schisandrin C. The lignans, schisandrin B and C protect against amyloidβ toxicity and decrease reactive oxygen species in cultured neurons, and in mice injected ICV with amyloidβ [102 - 104]. An extract of the fruit protects against ICV amyloidβ induced memory loss in mice, inhibits β-secretase and acetylcholinesterase [105]. Schisandrin B inhibits toll like receptor 4 signaling and decreases microglia-

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induced neuroinflammation [106]. Schisantherin A, schisantherin B, schisandrin and schisandrin A (deoxyschizandrin), used individually, improve memory loss induced by ICV amyloidβ injection in mice, perhaps through antioxidant mechanisms [107 - 110]. Isocubebenol, a sesquiterpene isolated from the fruit, inhibits acetylcholinesterase and is neuroprotective [111]. Schisandra plant medicine has not been reported to cause significant adverse reactions. The Modern Approach to Curing AD Several drug candidates have been tested in AD clinical trials. Active vaccines against amyloidβ have failed [1]. Solanezumab, a monoclonal antibody against soluble amyloidβ, failed [112]. Another monoclonal antibody, aducanumab is in trial as of Spring, 2017. It is not clear how antibodies against amyloid could remove deposits of amyloidβ from the brain, since they do not cross the blood brain barrier. Gamma-secretase inhibitor trials have failed [1]. CHF5074, a gamma-secretase inhibitor, did not improve health in 96 patients with mild cognitive impairment [113]. The beta-secretase inhibitor, verbucestat, failed in clinical trial. Pioglitazone, a peroxisome proliferator activated receptor gamma agonist, improved cognition in 21 patients with mild AD, but suffers from hepatoxicity and can induce bladder cancer [114]. Bexarotene, a retinoid X receptor agonist, did not improve amyloid burden in AD patients [115]. CONCLUDING REMARKS Prevention is the best medicine for AD. Many people live long lives without developing AD, perhaps because they live healthy lifestyles that prevent the disease. Plant medicines have been traditionally used for thousands of years to delay the progression of AD. These plant medicines should be used in the traditional way, as crude plant extracts, not as single purified agents. Thousands of years of experience has shown the proper way to use these medicines. Each plant has several active compounds that may work together to delay disease progression. The mechanisms of action of several plant medicines involve decreasing inflammation and protecting the blood brain barrier. However, the use of single purified drugs, derived from plants, has been successful in delaying the progression of AD, such as galantamine, an acetylcholinesterase inhibitor from Galanthus caucasicus. Many of the compounds discussed are anti-inflammatory agents that are not cyclooxygenase inhibitors and offer a better approach to the treatment of AD. CONFLICT OF INTEREST The author declares no conflict of interest, financial or otherwise.

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CHAPTER 2

Stem Cell Strategies for the Modeling and Therapy of Alzheimer’s Disease Haigang Gu* Department of Pediatrics, Northwestern University Feinberg School of Medicine, Lurie Children's Hospital Research Center, Chicago, IL 60614, USA Abstract: Alzheimer's disease (AD) is the most common form of dementia in aged populations.AD is characterized by a progressive decline in memory and cognitive function, accompanied with behavioral changes such as confusion, irritability and aggression, mood swings, language breakdown and eventually long-term memory loss. The most significantly pathological findings in the brains affected by AD are senile plaques (SP), neurofibrillary tangles (NFT) and neuronal loss or degeneration, particularly in the areas connected to the cerebral cortex and hippocampus.The most prominence among these regions is the basal forebrain cholinergic neurons. Many AD studies and clinical trials focus on inhibiting the formation of extracellular senile plaques and intracellular neurofibrillary tangles to prevent or halt disease progression. For example, the Food and Drug Association (FDA) has approved three acetylcholinesterase inhibitors (AChEIs), donepezil, rivastigmine and galantamine as AD therapy. Elevating the neurotransmitter acetylcholine by AChEIs has been shown to benefit cognitive functions in patients. Excitotoxicity caused by glutamatergic synaptic dysfunction contributes to cognitive AD symptoms. Another FDA-approved AD drug, the N-methyl-D-aspartate (NMDA) receptor antagonist memantine, is thought to alleviate the excitotoxicity. To date, however, none of these treatments have been shown to be safe and effective in clinic. Stem cell therapy is a promising therapeutic strategy, which has been shown to replace the neurodegenerative cholinergic neurons and provide exogenous neurotrophic factors in AD brains. Stem cells have been used as therapy of neurodegenerative diseases to deliver RNAi to the brains and regulate the expression of neprilysin, an amyloid-β (Aβ)-degrading enzyme. More recently, stem cells, especially induced pluripotent stem cells (IPSCs), have been used for AD modeling and drug screening. However, effective drugs or other interventions that stop or delay progression of AD remain elusive. Due to the multifaceted features of AD, further investigations of AD therapies are necessary. This review will discuss the recent progress of stem cell strategies for AD modeling and therapy.

Keywords: Alzheimer’s disease, Drug discovery, Small molecules, Stem cells, Therapy. Corresponding author Haigang Gu: Department of Pediatrics, Northwestern University Feinberg School of Medicine, Lurie Children's Hospital Research Center, Chicago, IL 60614, USA; Tel: +1 (773) 755-7312; E-mails: [email protected]; [email protected] *

Atta-ur-Rahman (Ed.) All rights reserved-© 2017 Bentham Science Publishers

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1. INTRODUCTION The most common type of dementia in aged populations is Alzheimer's disease (AD), which is characterized by a progressive decline in memory and cognitive function. Alzheimer's disease is accompanied with behavioral changes such as confusion, irritability and aggression, mood swings and language breakdown. In the late stage of AD, patients lose the functions of movement, learning and memory [1, 2]. The cause of initiation and progression of AD are not well understood. Previous investigations have shown that the incidence of AD is strongly associated with aging. The most significantly pathological findings in brains affected by AD are senile plaques (SP), neurofibrillary tangles (NFT), neuronal loss or degeneration, particularly in the areas connected to the cerebral cortex and hippocampus.The most prominent among the regions is the basal forebrain (BF) cholinergic neurons [3 - 7]. Cholinergic neurons of BF express both the low affinity neurotrophin receptor (P75NTF) and tropomyosin receptor kinase A (TrkA), and respond to neurotrophic factors (NTFs) by increased activity of choline acetyltransferase (ChAT). Neurotrophic factors are also important in the development of neurons and maintaining normal functions of the nervous system, such as outgrowth of axons and neuritis, pathfinding, synaptic genesis and neural circuit formation. Neurotrophic factors have been extensively used for therapeutic studies in the experimental models of AD [8, 9]. Moreover, NTFs have shown beneficial effects in other neurodegenerative diseases, such Parkinson’s disease (PD), Huntington's disease (HD), spinal cord injury (SCI) and stroke. However, NTFs are macromolecular proteins that do not readily cross the blood-brain barrier (BBB). Efficient delivery of NTFs into the central nervous system remains challenging. Strategies to decrease the degradation of acetylcholine in the central nervous system usually involve increasing cholinergic function and improving cognitive functions in AD patients. Some small molecules have been developed to inhibit the cleavage of acetylcholine. To date, cholinesterase inhibitors, such as donepezil, galantamine and rivastigmine, are available for the treatment of AD [10, 11], but their effects must be further investigated. Many patients do not show functional benefit after cholinesterase inhibitor therapy. Furthermore, medication application does not stop the progression of AD. Although grafting embryonic cholinergic neurons has been shown to increase cholinergic function in animal models of AD, this strategy is not clinically feasible due to the limited availability of fetal tissue and ethical concerns. Due to the self-renewal ability of stem cells, sufficient numbers of neurons can be generated for both research and transplantation therapy within a short period of time. Moreover, stem cells have the potential to differentiate into different types of somatic cells. For example, neural stem cells (NSCs) have been successfully cultured, which solves the

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problem of using human fetal donors. Neural stem cells can generate neurons, astrocytes and oligodendroglia in response to environmental signals, including NTFs, retinoic acid (RA) and growth factors. Stem cell-derived neurons can migrate and integrate with host neurons in the brain and spinal cord. Furthermore, stem cell-derived glial cells can secret NTFs to promote the survival of degenerative neurons [12 - 15]. Induced pluripotent stem cells (IPSCs) allow the development of personalized medicine. For example, a specific patient’s IPSCs could be induced to differentiate into cholinergic neurons. And then, the best drug candidates for this patient can be identified using screening a drug library against their IPSC-derived cholinergic neurons [3, 4]. Although many basic scientific and clinical studies have shown that drug treatment could improve cognitive function and memory of AD patients, delaying and/or stopping neuron loss and degeneration is still a considerable challenge [2, 16]. Due to the multifaceted features of AD, more works remain to be done to explore the novel specific therapeutics (Fig. 1). Combining different therapies must be considered in the future. This review discusses the recent progress in the field of AD, focusing on stem cell therapeutic strategies.

Factors Degeneration

Factors Regeneration

Fig. (1). Factors affect Alzheimer’s disease (AD). Loss of the balance between degeneration and regeneration causes AD.

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2. NEUROPATHOLOGY OF AD: KEYS TO DRUG DISCOVERY AND ANIMAL MODELS The main neuropathological changes in AD are senile plaques, neurofibrillary tangles and neuron loss or degeneration, which are associated with memory and cognitive decline. Senile plaques are composed of amyloid β (Aβ), a cleavage product of amyloid precursor protein (APP), which is sequentially cleaved by βand γ-secretase. Neurofibrillary tangles are composed of hyper-phosphorylated tau protein. Extracellular deposition of senile plaques and intracellular neurofibrillary tangles cause chronic inflammatory and oxidative stress to neurons and lead to neuron loss or degeneration. Targeting the formation of Aβ and hyperphosphorylated tau protein has been used to develop the drugs for AD [2, 16 - 18]. Moreover, increasing both the level and duration of cholinergic signals by an acetylcholinesterase inhibitor (AChEIs) could ameliorate symptoms of AD patients [19, 20]. The β-amyloid Hypothesis of AD The amyloid cascade hypothesis has been questioned, improved and expanded over decades, and remains the leading hypothesis in the field. This hypothesis states that the accumulation of Aβ both precedes and causes the other features of AD, including inflammation, deposition of tau, synaptic deficits, neurodegeneration and dementia. The three genes associated with familial Alzheimer’s disease (FAD) are APP itself and the Presenilin 1 and 2 (PS1 and PS2), enzymes that cleave APP to produce Aβ [21]. The earliest versions of this hypothesis stated that formation of the characteristic extracellular amyloid plaques leads to all other AD pathological features [18, 22]. However, it has now been demonstrated that Aβ oligomers have detrimental effects on synaptic function even in the absence of plaque deposition [23]. Although little is known about endogenous functions of APP and Aβ, there is considerable evidence that Aβ damages synapses and disrupts the neural network [17, 24]. The relative contributions of different forms of Aβ to disease progression are still controversial. Some suggest that plaque deposition is protective because it lowers the level of soluble, toxic Aβ oligomers, while others posit that inflammation caused by the plaques is central to the progression of the disease [23]. Although the consensus is that FAD and sporadic AD do share a common, amyloid-dependent pathway, a competing hypothesis states that tau pathology precedes and causes Aβ accumulation in sporadic AD [25]. The Hyper-phosphorylated Tau Protein Hypothesis of AD Neurofibrillary tangles, another characteristic marker of AD, are composed of

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microtubule associated protein tau [26]. Alzheimer’s disease is the most common tauopathy, a family of neurodegenerative diseases characterized by the accumulation of tau. Neurofibrillary tangles are correlated with the severity of cognitive impairment in AD, unlike amyloid plaques [27]. Tau pathology is hypothesized by some to be the primary driver of AD and similar diseases, such as frontotemporal dementia. According to this hypothesis, Aβ accumulation is downstream of tau and is a symptom rather than the cause of AD [21, 28, 29]. The role of tau to AD progression is consistent with the amyloid hypothesis. There is strong evidence that Aβ accumulation occurs first, and then cognitive impairment begins before tau pathology and neuron loss based on post-mortem studies [2]. Aberrant tau deposition occurs later in AD progression, but misfolded, hyper-phosphorylated tau is highly toxic. Tau stabilizes microtubules and is predominantly found in axons. Misfolded, hyper-phosphorylated tau accumulates in cell bodies and neurites in AD. Hyper-phosphorylated tau does not bind and stabilize microtubules. Some evidence suggests that tau is required for the neuronal loss that occurs in late AD. Additionally, neurofibrillary tangles are correlated with the severity of cognitive impairment, unlike amyloid plaques [27]. Tau’s role in the irreversible, progressive neuronal loss of AD makes tau an attractive target for intervention. The cholinergic hypothesis of AD Although other types of neurons are also degenerative in AD, loss of cholinergic neurons in the basal forebrain is the most critical cause of cognitive deficit in AD. Previous studies have shown that lesions of the fimbria-fornix pathway, which is initiated from the basal forebrain and projected to the hippocampus, cause the impairment of learning and memory in animals. Selective cholinergic lesions of basal forebrain cholinergic neurons have been widely used as animal models for the studies of AD. Moreover, increasing both the level and duration of cholinergic signals by intracerebral applications of NTFs and AChEIs could ameliorate symptoms in AD patients [19, 20]. 2.1. Drug Discovery of AD Based on the above hypotheses, current drug discovery targeting AD mainly focuses on the reduction of Aβ and hyper-phosphorylated tau and the increase of acetylcholine levels in the brain. 2.1.1. Treatment of Amyloid Pathology Amyloid β and its associated pathways are perhaps the most attractive targets for treatment of AD, based on the amyloid hypothesis [30 - 32]. Anti-Aβ therapies

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could hypothetically stop disease progression and serve as a preventative medicine. Secretase inhibitors, used to prevent the cleavage of APP and the release of Aβ, have been troublesome. Beta- and γ-secretases cleave important proteins throughout the body and brain, including Notch. Thus, side effects of secretase inhibitors can be severe and substrate-specific secretase inhibitors are now being developed. Furthermore, compounds designed to disrupt Aβ aggregation have been developed. Active and passive Aβ immunizations have been successful in mouse models but unsuccessful in humans. The use of transplanted cells to improve clearance of Aβ is discussed below. 2.1.2. Treatment of Tau Pathology Microtubule-associated protein tau dysfunction leads to buildup of insoluble NFTs that eventually kill neurons. Multiple strategies are used to treat tau dysfunction. Specific kinase inhibitors may restore microtubule function and decrease aggregation by preventing hyper-phosphorylation. As with anti-Aβ strategies, small molecules intended to bind tau and prevent its aggregation could ameliorate AD symptoms. Importantly, such drugs would be designed to block only pathological deposition and would not interfere with normal behavioral function. Agents that promote degradation of tau could prevent buildup of tau and potentially break down the insoluble NFTs. Finally, agents that stabilize tau and restore the functionality of microtubules would help preserve neurons [5, 28, 33]. Anti-tau monoclonal antibodies have been used to treat mouse models of AD expressing human tau protein with the P301S mutation. After 3 months of treatment, hyper-phosphorylated, aggregated, insoluble tau and microglial activation were dramatically decreased. Cognitive impairments of AD mice were markedly improved [34]. 2.1.3. Treatment of Synaptic Dysfunction Early stage AD is characterized by impairment of cognitive function.This type of cognitive impairment is caused by subtle synaptic dysfunction [31]. Previous studies have shown that synaptic dysfunction is highly correlated with memoryrelated synaptic dysfunctions in AD. Mounting evidence suggests that accumulation of Aβ damages the synapses and disrupts the neural network [35 38]. Previous studies with AD animal models suggest that soluble oligomeric Aβ plays a critical role in triggering synaptic dysfunction, neuronal death and the impairment of learning and memory. Strong correlations of Aβ, synaptic dysfunction and cognitive impairment have been investigated in transgenic animal models of AD. Long-term potentiation (LTP) in neurons incubation with Aβ is repressed. On the contrary, long-term depression (LTD) is enhanced in AD [38]. Inhibiting Aβ formation by beta-secretase 1 (BACE1) can reverse synaptic

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dysfunction in AD [10]. Levetiracetam (LEV) is an anti-epileptic drug that binds synaptic vesicle glycoprotein SV2A and regulates synaptic activities through presynaptic calcium channels. In transgenic animal models of AD, LEV treatment could reduce abnormal spike activity, reverse synaptic dysfunction and improve learning and memory [39]. 2.1.4. Neurotrophic Factors (NTFs) Previous studies have shown that imbalance of NTFs in the brain and lack of neurotrophic support causes neuronal atrophy and death. Neurotrophic factors are a family of proteins responsible for the growth and survival of developing neurons and the maintenance of mature neurons. Knockout of nerve growth factor (NGF) in adult transgenic mice leads to severe neuronal death in basal forebrain cholinergic neurons. Intracerebroventricular (ICV) administration of NGF completely prevents retrograde degeneration of cholinergic neurons and increases learning and memory in the animal model of AD [40]. Administration of ciliary neurotrophic factor (CNTF) fully recovers cognitive impairments of AD by reducing Aβ oligomer-induced synaptic damages [41]. There are four families of NTFs: the neurotrophin superfamily, the GDNF superfamily, the Neurokine superfamily and non-neuronal growth factors (Table 1). Table 1. Families of neurotrophic factors (NTFs). Families

Neurotrophic Factor

Receptors

References

Neurotrophin family

Nerve growth factor (NGF), Brain-derived neurotrophic factor (BDNF) Neurotrophin 3 (NT-3) Neurotrophin 4/5 (NT-4/5) Neurotrophin 6 (NT-6)

Trk receptors p75NTR

[8, 42 - 46]

GDNF superfamily

Glial cell line-derived neurotrophic factor GFRα receptors [8, 42, 47] (GDNF) Neurturin (NRTN) Artemin (ARTN) Persephin (PSPN)

Neurokine superfamily

Ciliary neurotrophic factor (CNTF) Leukemia inhibitory factor (LIF) Interleukin-6 (IL-6) Cardiotrophin-1 (CT-1) Oncostatin-M

LIF receptor-β gp130

Non-neuronal growth factors

Acidic fibroblast growth factor (aFGF) Basic fibroblast growth factor (bFGF) Bone morphogenetic protein (BMP) Epidermal growth factor (EGF) Insulin-like growth factor (IGF) Hepatocyte growth factor (HGF)

FGFR [8, 42, 49, 50] EGFR IGF-1R BMP receptors Met receptor

[8, 41, 42, 48]

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Neurotrophin factors are large proteins that do not easily cross the BBB. Delivery of NTFs to the brain poses a major challenge for clinical application [51, 52]. Intracerebral ventricular administration of NGF has been used in clinical trials. Three AD patients were treated with murine NGF injection and showed certain beneficial effects. However, two adverse effects, back pain and weight reduction, occurred after NGF treatment [2]. Development of other NGF delivery options remains challenging. Viral vector methods have been widely used to deliver proteins for in vivo studies. Retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV) and herpes simplex virus (HSV) have been used to deliver NTFs for the treatment of AD [9]. However, major concerns about the safety of viral vectors for clinical applications remain unresolved. Non-viral methods, such as plasmids, micro/nanoparticles, implants and films, have also been used to deliver NTFs to the brain. Although non-viral methods are safer than viral methods, they remain impractical for clinical use. 2.1.5. Cell Transplantation Cell transplantation is a promising AD therapeutic strategy due to recent advances in stem cell technology. Cell transplantation therapy for the central nervous system is also known as cell replacement therapy. Transplanted cells can substitute for dead neurons caused by injury or neurodegenerative disease. Moreover, transplanted cells can induce an intrinsic response in the brain to reestablish neural circuit connections and promote survival of degenerative neurons. Furthermore, transplanted cells could supply extra glial cells as neurotoxin cleaners and minipumps to deliver NTFs and neurotransmitters. As such, cell transplantation is a promising novel therapy for AD [53 - 56]. Transplantation of cholinergic neurons has been used to replace dead neurons and has shown functional recovery in AD model. However, the availability of embryonic tissue for transplantation is limited. Furthermore, transplantation of embryonic tissue causes ethical concerns. Rapid advances in stem cell biology provide an alternative and prospective treatment for AD. Stem cells are highly expandable and multipotent. Tissue specific neural progenitor cells (NPCs) and neurons have been successfully generated from stem cells including neural stem cells (NSCs), mesenchymal stem cells (MSCs), embryonic stem cells (ESCs) and induced pluripotent stem cells(IPSCs). Such stem cell-derived NPCs and neurons have been used for the modeling and treatment of neurodegenerative diseases, including AD, PD, HD and amyotrophic lateral sclerosis (ALS) [57 - 59]. 2.2. Animal Models of AD Animal models have been widely used in preclinical studies for AD drug discovery. The most commonly used animal models of AD are transgenic and

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selective cholinergic lesion models. The latter includes mechanical lesioning of the fimbria-fornix pathway to mimic the cholinergic deficit of septohippocampal pathway loss in AD and chemical lesioning of cholinergic neurons in extensive brain regions by ICV infusion of 192 IgG-saporin. Both of these models are widely used for testing potential AD therapies. 2.2.1. Transgenic Animal Models of AD Mutations in three genes lead to FAD: APP, PS1 and PS2. The most common AD mouse model overexpresses human APP with one or more mutations that drastically increase Aβ production. For example, the popular strain Tg2576 overexpresses human APP with the KM670/671NL (Swedish) mutation and the J20 strain overexpresses human APP with both the Swedish and V717F (Indiana) mutations [60, 61]. Other models express human mutant Presenilins (γ-secretase catalytic subunit), such as an exon 9 deletion (PS1-ΔE9) in PS1 [62]. This mutation increases γ-secretase cleavage of APP and therefore also increases Aβ production. These transgenic mice typically have an AD-like phenotype that increases with age and may include increased soluble Aβ levels, plaque deposition, neuronal morphology changes, synaptic dysfunction and cognitive deficits. However, the tau in AD patients is absent in these FAD mouse model. Most APP overexpression models lack the severe neuronal loss that AD patients suffer. In order to recapitulate both Aβ and tau pathology in vivo, Frank LaFerla’s lab created 3xTg, the most widely used of these strains [63]. In addition to the FAD transgenes APP and PS1, the 3xTg model has MAPT P301L, a mutation associated with another tauopathy known as frontotemporal dementia. One concern with using transgenic APP-overexpressing mice for drug discovery is that FAD is rare; the vast majority of AD patients have late-onset AD. Apolipoprotein E4 (ApoE4) is by far the strongest genetic risk factor for lateonset AD. Most people are homozygous for ApoE3, the most common allele. One copy of the ApoE4 allele triples the risk of developing AD and two ApoE4 alleles increase the risk 12-fold [64]. Research and drug discovery efforts using transgenic ApoE4 mice may lead to treatments that can prevent or delay the development of late-onset AD. FAD models harboring ApoE4 develop AD earlier than those homozygous for ApoE3 [65]. 2.2.2. Selective Cholinergic Lesion Animal Models of AD Cholinergic neurons in the basal forebrain include medial septum nucleus (MS), vertical diagonal band nucleus (VDB), horizontal diagonal band nucleus (HDB) and nucleus basalis of Meynert (NBM), which project to the hippocampus,

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amygdale and entire cerebral cortex. Among these cholinergic neurons, the septohippocampal pathway connecting basal forebrain and hippocampus is related to the cognitive deficit in AD. Selective cholinergic lesion animal models mimic the loss of cholinergic neurons in AD patients. There are two types of animal models with selective cholinergic lesion; one is the mechanical lesioning animal model of AD and the other is the chemical lesioning animal model of AD. Mechanical lesioning with fimbria-fornix transaction selectively damaged. The septohippocampal pathway, which causes cholinergic neuronal degeneration in both the basal forebrain and hippocampus. Cognition, learning and memory are impaired in animal models of AD with fimbria-fornix transaction [40, 52, 55, 66, 67]. Other types of neurons projecting to the hippocampus, such as gammaaminobutyric acid (GABA) neurons in the basal forebrain, are also injured with fimbria-fornix transaction. Chemical lesioning animal models of AD use 192 IgG-saporin, which targets neurons expressing p75NTR. Saporin, also called ribosome-inactivating protein (RIP),is a protein studied in behavioral research. Saporin is one of the most toxic molecules. After conjugated to a monoclonal antibody against NGF receptors, it can be used to kill p75NTR-expressing cells. In the 1990s, Dr. Wiley’s lab at Vanderbilt University developed 192 IgG-saporin to target and eliminate cholinergic neurons [68, 69]. After injection of 192 IgG-saporin in the brain, cholinergic neurons around the injected regions selectively die. If 192 IgG-saporin is chronically injected into cerebral ventricle, cholinergic neurons in basal forebrain and hippocampus are selectively killed. The 192 IgG-saporin must be injected repeatedly into the brain because it cannot cross the BBB. While chemical lesioning models are more expensive than mechanical lesioning models, 192 IgG-saporin is more specific for damaging cholinergic neurons. 3. STEM CELLS AS USEFUL TOOLS FOR CELL TRANSPLANTATION, DRUG DISCOVERY AND AD MODELING Embryonic basal forebrain cholinergic neurons have been transplanted into the hippocampus of animal models of AD. After transplantation, abilities of learning and memory in AD models have significantly improved [70, 71]. This strongly suggests that cell replacement therapy is a promising method for the therapy of neurodegenerative diseases. For clinical applications, transplantation of embryonic mean basal forebrain cholinergic neurons (BFCNs) is infeasible due to the limitation of available embryonic tissue and ethical concerns. Recent progress of stem cell technology makes it possible to overcome the limitations of using embryonic tissue.

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Stem cells have self-renewal and multipotent properties (Table 2). Stem cells are divided into pluripotent stem cells (PSCs) and multipotent stem cells. Pluripotent stem cells include ESCs and IPSCs. Multipotent stem cells, also called adult stem cells, such as NSCs, mesenchymal stem cells (MSCs), liver stem cells (LSCs), cardiac stem cells (CSCs) and hematopoietic stem cells (HSCs) [72 - 79]. Mesenchymal stem cells include adipose-derived MSCs, bone marrow-derived MSCs and umbilical cord blood-derived MSCs based on the tissues used for isolating MSCs. Among adult stem cells, NSCs and MSCs have been widely used for the therapy of neurodegenerative diseases. Stem cells can be expanded to a considerable number of cells and induced to differentiate into specific neurons, such as cholinergic neurons, dopaminergic neurons and GABA neurons. Moreover, IPSCs derived from AD patients may offer a promising tool to develop novel personalized therapy or serve as preventative medicine (Fig. 2). SVZ and SGZ

Inner cell mass

Fibroblasts

Bone marrow Adipose tissue Umbilical cord blood

NSCs

ESCs

IPSCs

MSCs

Neurons

Drug screening

NPCs

Modeling of diseases

Cell transplantation

Fig. (2). The strategies of stem cells for drug discovery, disease modeling and cell transplantation. SVZ, subventricular zone; SGZ, subgranular zone; NSCs, neural stem cells; NPCs, neural progenitor cells; ESCs, embryonic stem cells; IPSCs, induced pluripotent stem cells; MSCs, mesenchymal stem cells.

Stem cell transplantations are a recent interesting target for AD. Stem cells can be modified with genes for gene therapy and serve as a model for studying neural development. Increasing numbers of investigators have used stem cells as potential treatment for neurological disorders. Stem cell treatments have been successfully used in bone marrow transplants for years to treat aplastic anemia and blood cancers. Multiple studies have provided strong evidence that stem cells are a good cell source for cell replacement therapy in human disorders. Most

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advances in stem cell research have been focused in treating neurodegenerative diseases, such as AD, PD, SCI and stroke [6, 54, 80 - 82]. Stem cell research for the treatment of nervous system disorders is promising yet challenging to demonstrate this therapy can restore lost function. Stem cells play an important role in the development of new medical techniques, drug screening and cell replacement therapy of neurological disorders. Furthermore, recent progress of reprogramming and genome editing allow the generation of functional neurons and genetic correction in situ. 3.1. Neural Stem/Progenitor Cells (NP/SCs) In the brain, neural stem/progenitor cells (NS/PCs) are located in the neurogenic regions, including the subventricular zone (SVZ) and the subgranular zone (SGZ) of the hippocampal dentate gyrus. Neural stem cells can be easily harvested from these two brain areas. Neural stem/progenitor cells are self-renewing, multipotent progenitors in the nervous system that can be induced to differentiate into the three phenotypes in the nervous system (neurons, astrocytes and oligodendrocytes) under appropriate conditions. Neural stem cells represent an ideal tool for studying neurogenesis and cellular treatment of nervous system disorders. Behavior of NSCs is controlled by intrinsic and extrinsic factors. Many transcriptional factors have been shown to control the proliferation and differentiation of NSCs in vitro and in vivo. Neural stem cells can be isolated from embryonic and adult mammalian brains in the neurosphere assay [83 - 85]. Proliferation and differentiation of NS/PCs are associated with the induction of the NTFs in vitro and in vivo. LIM homeobox 8/L3 (Lhx8/L3) has been used to enhance cholinergic neuronal differentiation from NS/PCs. Overexpression of Lhx8/L3 in NS/PCs significantly increase the percentage of ChAT-positive cells, but not that of microtubule-associated protein 2 (MAP2)-positive cells [86]. This provides evidence that Lhx8/L3 is a marker for cholinergic neurons. Neurotrophic tyrosine kinase type 1 (NTRK1) also promotes cholinergic neuronal differentiation in NS/PCs [87]. Neural stem/progenitor cells have been widely used to study neural development, regeneration and cell replacement therapy of neurodegenerative diseases [7, 15]. Our previous studies showed that NS/PCs can migrate into adjacent brain tissue and locally differentiate into neurons and astrocytes after transplantation. Transplanted NS/PCs did improve learning and memory in the rat model of AD [55]. Dr. Wu’s group has developed a novel in vitro priming protocol to generate highly pure neurons from NS/PCs, called primed NS/PCs. After transplantation, primed NS/PCs (but not unprimed NS/PCs) could differentiate into cholinergic neurons in the brain [88]. A recent study showed that transplantation of human

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NSCs into 3xTG-AD mice enhanced endogenous synaptogenesis and improved spatial memory. However, the pathology of Aβ and hyper-phosphorylated tau is not changed in the brains of these transgenic mice [89]. Neural stem/progenitor cells have been widely used to deliver NTFs for the therapy of neurological diseases and drug screening [90]. Transfection of neurotrophin-3 (NT-3) dramatically increases cholinergic neuronal differentiation of NS/PCs. After transplantation, survival of NT-3-transfected NS/PCs is significantly increased compared to controls [58]. Neural stem cells expressing insulin-like growth factor-I (IGF-I) enhance the neuroprotection in animal models of AD after transplantation. Although most of the transplanted NSCs expressing IGF-I differentiate into GABA neurons, they produce vascular endothelial growth factor (VEGF), which plays a role in neuroprotection in vitro and in vivo [91]. Garavaglia et al. established NSCs as a reproducible high-throughput screening tool to evaluate drug efficacy [92]. A library of pharmacologically active compounds (LOPAC) from Sigma and GlaxoSmithKline’s Cell & Pathway (C&P) containing 8438 compounds has been used to screen potent small molecules with NPCs to promote NSC differentiation into neurons by using a quantitative whole-well immunofluorescence assay. Garavaglia et al. identified a group of Glycogen synthase kinase 3 beta (GSK3β) inhibitors were potent inducers of neuronal differentiation. The function of these GSK3β inhibitors was validated in both mouse and human NSCs [93]. To study the safety of common FDA-approved drugs, 2,000 compounds have been tested with NSCs-derived IPSCs. Around 100 compounds, including some cardiac glycosides, have shown toxicity to human NSCs [94]. 3.2. Mesenchymal Stem Cells (MSCs) Mesenchymal stem cells have been found in almost all adult organs, such as bone marrow, adipose tissue, umbilical cord blood, periosteum and skeletal tissue. Mesenchymal stem cells are multipotent cells that can self-renew, proliferate and differentiate into multiple cell lineages including osteoblasts, chondrocytes, myoblasts, adipocytes, fibroblasts and neuron-like cells. Transplants of MSCs have been shown to have considerable promise for the therapy of AD. Mesenchymal stem cells have unique properties that distinguish them from other types of stem cells [95]. 1. Mesenchymal stem cells can be easily harvested from patients. 2. Mesenchymal stem cells possess high migratory capacity. After transplantation, MSCs are able to migrate to injured sites, such as injured brain or myocardial infarction. 3. Mesenchymal stem cells are able to secrete growth factors and cytokines.

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4. Mesenchymal stem cells have been shown to modulate immunological functions. 5. Mesenchymal stem cells are easily infected and transfected. MSCs are the ideal tool for delivery of growth factors. Mesenchymal stem cells have been used for the therapy of PD, SCI and ischemia stroke and have shown beneficial functional recovery in animal models. Kim and colleagues showed that transplantation of human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) could reduce Aβ plaques in vivo through up-regulation of neprilysin, an Aβ-degrading enzyme [96]. The MSC therapy of AD has been tested in the 5xFAD mouse model. Their results showed that transplanted MSCs effectively reduce learning deficits in an AD model and reduce the level of amyloid-β42 (Aβ42) in the mouse brain [97]. Mesenchymal stem cells improve the working memory in the 3xTg mouse model of AD [98]. Bone marrow mesenchymal stem cells (BMSCs) from rhesus monkeys have been successfully differentiated into cholinergic neurons in the presence of RA and Sonic Hedgehog (SHH). Moreover, these cholinergic neurons express synapsin and acetylcholine [99]. We have differentiated MSCs to neurons in threedimensional (3D) biodegradable scaffolds and the nanotopological surface of nanotubes, indicating that MSCs are reliable cell source for neural tissue engineering [100, 101]. Mesenchymal stem cells is an ideal platform for drug screening. A siRNA library targeting 5,000 human genes has been tested with human MSCs to identify repressors of osteogenic differentiation. Twelve suppressors have been confirmed to suppress osteogenic differentiation in MSCs [102]. A library of compounds targeting epigenetic factors has been screened with MSCs. Inhibitors of focal adhesion kinase (PF-573228) and insulin-like growth factor-1R/insulin receptor signaling (NVP-AEW51) have been shown to significantly decrease abexinostatmediated adipocytic differentiation. Moreover, an inhibitor of Wnt (XAV939) and transforming growth factor-beta (TGF-β) (SB505124) reduced abexinostatmediated osteogenic differentiation [103]. Improving the survival of transplanted MSCs (and NSCs) remains challenging. Moreover, the properties of MSCs-derived neurons,such as action potential, miniature excitatory postsynaptic currents (mEPSCs), miniature inhibitory postsynaptic currents (mIPSCs) and synaptic maturation, have not yet been extensively studied. 3.3. Embryonic Stem Cells (ESCs) Embryonic stem cells are derived from the inner cell mass of blastocysts. Human embryos reach the blastocyst stage 4–5 days post fertilization and consist of

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50–150 cells. Embryonic stem cells are pluripotent stem cells and can differentiate into all derivatives of the three primary germ layers: ectoderm, mesoderm and endoderm. They can develop into more than 200 cell types of the adult body. Previous studies have demonstrated that ESCs are suitable candidates too for cell replacement therapy in human diseases, such as neurological disorders, heart failure, lung injury and bone repair. In 2009, FDA-approved oligodendrocyte progenitor cells derived from ESCs were used for clinical trials in patients with spinal cord injuries. Previous reports have described that ESCs are suitable candidates for neuronal differentiation and cell replacement therapy [6, 15, 54, 73, 74, 80, 82, 104 - 106]. By using genetic modification, Manabe and colleagues reported that overexpression of L3/Lhx8 promoted cholinergic neuronal differentiation from ESCs; suppression of L3/Lhx8 in ESCs by siRNA dramatically decreased ChAT-positive neuronal differentiation. This study showed that L3/Lhx8 is an important factor for cholinergic neuronal differentiation from ESCs [107]. In 2011, Dr. Kessler’s group generated cholinergic neurons from human ESCs by overexpression of Lhx8 and Gbx1. Cholinergic neurons derived from human ESCs showed functional electrophysiological properties and formed synaptic connection with host neurons in ex vivo brain slice cultures [108]. Embryonic stem cells have been used for cell replacement therapy of AD and drug screening. Neuronal precursor cells are derived from mouse ESCs with NGF, sonic hedgehog (SHH), RA and interleukin-6 (IL-6). Embryonic stem cells derived NPCs have a significantly increased functional recovery in memory deficits of the ibotenic acid-lesioned rat model of AD, according to Morris watermaze and spatial-probe testing after transplantation [109]. Dr. Zhang and his colleagues generated medial ganglionic eminence (MGE)-like cells from human ESCs. In the presence of NGF, BDNF, BMP9 and SHH, the MGE cells further differentiated into basal forebrain cholinergic neurons and GABA interneurons in vitro. After transplantation, MGE cells differentiated into cholinergic neurons and GABA neurons in the mouse brain. Furthermore, transplanted MGE cells improved learning and memory deficits in amouse model of AD [53]. A library of 2880 compounds was screened by Dr. Studer’s group to identify potent small molecules to maintain the pluripotency and early lineage differentiation of human ESCs. Although failure rate with this screen was relatively high, human ESCs are still a reliable platform for drug screening [110]. Human ESC-derived neural cells have been used to screen 6,984 compounds with luciferase assay. Based on screening results, X5050 is the most potent compound. After intraventricular infusion of X5050, the expression of BDNF- and REST-regulated genes were increased in the prefrontal cortex of mice with quinolinate-induced striatal lesions [111].

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Clinical applications of ESCs are still challenge. First, isolating ESCs from fertilized human embryos has major ethical concerns. Second, the potential adverse effects of ESCs applications in clinic include formation of teratomas and other cancers. Third, donor-host rejection is an additional problem. Fourth, synaptic functions of human stem cell-derived neurons have not yet been studied extensively. We are able to compare the synaptic functions of stem cells-derived neurons with that of the isolated neurons from transgenic animals [112]. It is impractical to perform similar studies in human stem cell-derived neurons due to the ethical concerns of using human embryos. Furthermore, it is still challenging to genetically modify human neurons generated from ESCs. 3.4. Induced Pluripotent Stem Cells (IPSCs) In 2006, a research group leads by Dr. Yamanaka at Kyoto University in Japan used a reprogramming technique to generate IPSCs from mouse fibroblasts using four transcriptional factors: Octamer-binding transcription factor 4 (Oct4), sex determining region Y-box 2 (Sox2), Kruppel-like factor 4 (Klf4) and c-Myc [113]. Induced pluripotent stem cells have shown identical properties to ESCs. One year later, Dr. Yamanaka’s group used the same combinations of transcriptional factors to generate IPSCs from human fibroblasts [114]. At the same time, another group led by Dr. Thomson at University of WisconsinMadison in the USA used another cocktail of transcriptional factors (Oct4, Sox2, Lin28 and Nanog) to generate IPSCs from human fibroblasts [115]. Induced pluripotent stem cells promise to be an ideal candidate for regenerative medicine and personalized therapy. Since this initial discovery, considerable efforts have been done to develop, improve and optimize this technique by using different combinations of transcriptional factors and small molecules. For example, Dr. Deng’s group at Peking University in China found that lineage specifiers, which suppress ESC identity, can be used to generate IPSCs from mouse fibroblasts [116, 117]. Moreover, Dr. Deng’s group found that IPSCs can be generated with a cocktail of small molecules [118]. To date, IPSCs have been generated from many kinds of somatic cells, such as fibroblasts, liver and neural stem cells. Furthermore, many laboratories around the globe have used similar techniques to reprogram cells from patients with various diseases, including neurodegenerative disorders such as AD, PD, and HD. Today, an increased number of investigators use IPSCs for cell replacement therapy. Many reports have shown that IPSCs can be induced to differentiate into functional neurons and astrocytes. Induced pluripotent stem cells transplantation with fibrin glue has shown a significant decrease in the size of infarct and improved the motor function in rats with middle cerebral artery occlusion four weeks after transplantation. Moreover, IPSCs were used for modeling neurological disorders and screening the drugs [73, 81].

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Recently, IPSCs have been derived from somatic cells of patients. Patient-derived IPSCs allow the study of genetic changes and the development of diseases. Moreover, specific types of cells differentiated from patient-derived IPSCs have been widely used to screen drugs. In 2011, IPSCs have been generated from FAD patients carrying PS1 (A246E) and PS2 (N141I) mutations by using the five factors, Oct4, Sox2, Klf4, Lin28 and Nanog. Familial Alzheimer's disease-IPSCs have been induced to differentiate into neurons. In this study, the authors found that Aβ42 secretion from neurons derived from FAD-IPSCs carrying PS1 and PS2 mutations were much higher comparing to the control. This phenomenon is consistent with Aβ42 secretion in AD patients’ brains carrying PS mutations. Amyloid β42 secretion dramatically decreased after the culture was treated with a γ-secretase inhibitor, Compound E [119]. In 2012, Israel and colleagues in USA generated IPSCs from two AD patients carrying the duplication of the Aβ precursor protein gene (APP(Dp)) by using retroviruses encoding Oct4, Sox2, Klf4 and c-Myc. Levels of Aβ (1-40), hyper-phosphorylated tau (Thr 231) and active GSK-3β were higher in IPSC-derived neurons from patients carrying APP(Dp) mutation than that of controls. Interestingly, similar phenomena were observed in IPSC-derived neurons from sporadic AD patients. The properties of IPSC-derived neurons from one of the sporadic AD patients had similar phenotypes with FAD samples [120]. A year later, Kondo et al. generated IPSCs from FAD patients carrying E693Δ mutation and sporadic AD using episomal vectors (Sox2, Klf4, Oct4, c-Myc, Lin28 and small hairpin RNA for p53), and then induced IPSCs to differentiate into cortical neurons. They found increased levels of Aβ oligomers in IPSC-derived neurons and astrocytes carrying the E693Δ mutation. Accumulated Aβ oligomers caused endoplasmic reticulum and oxidative stress, which was reversed after applied docosahexaenoic acid (DHA) [121]. Cortical neurons were also generated from Down syndrome-IPSCs (DSIPSCs) and Down syndrome-ESCs (DS-ESCs). Cortical neurons derived from DS-IPSCs had increased extracellular accumulation of pathogenic Aβ42 in cortical neurons derived from DS-IPSCs compared to the control in late stage of cell culture (after day 70). Thioflavin T analog 1 (BTA1)-labeled amyloid showed intracellular and extracellular aggregates of amyloid in DS-IPSC-derived cortical neurons. To verify this observation, they generated cortical neurons from DSESCs. Extracellular and intracellular Aβ42 aggregation was observed in cortical neurons derived from DS-ESCs. Furthermore, the distribution of Aβ42 aggregation in cultured cortical neurons derived from DS-ESCs was similar with that in cortical neurons derived from DS-IPSCs. These studies provided evidence that IPSC-derived neurons from AD patients can be used to analyze pathological changes and screen drugs for clinical applications [122]. Dr. Koliatsos’s group generated IPSCs from patients carry the FAD PS1 mutation A246E with episomal vectors to express the OCT4, SOX2, NANOG, KLF4, MYC, LIN28 and SV40LT

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reprogramming factors and differentiated functional neurons. The Aβ42/Aβ40 ratio was increased in neurons derived from FAD-IPSCs [123]. The contribution of the Sortilin Related Receptor 1 (SORL1) gene for sporadic AD (SAD) has been investigated by using IPSCs. Human neurons carrying SORL1 variants showed a reduced SORL1 expression and APP processing after adding BDNF in culture. This provides evidence that SORL1 is associated with an increased SAD risk [124]. Although patient specific neurons have certain neuropathological properties, mature phenotypic and physiological properties are usually normal in most of studies. IPSCs have been used as a platform to explore the possibility for personalized cell therapy and drug testing. Dr. Zhang’s group generated NS/PCs from rhesus monkey IPSCs. These rhesus monkey IPSC-derived NS/PCs survived around six months and differentiated into neurons, astrocytes and oligodendrocytes in the brains of the rhesus monkey model of PD [125]. A3D human culture of NS/PCs derived from FAD-IPSCs has been used for modeling AD and drug testing by Dr. Kim’s group. Extracellular deposition of Aβ peptide and Aβ plaques was increased in neurons with FAD mutations in β-APP and PS 1compared to controls in this 3D culture system. Aggregated phosphorylated tau (hyper-phosphorylated tau) was also increased in the soma and neurites of 3D differentiated neurons. After treatment with β- or γ-secretase inhibitors, levels of both Aβ and hyperphosphorylated tau were decreased [126]. A library containing 6,912 smallmolecule compounds was tested with IPSCs-derived neural crest precursors from patients with familial dysautonomia. In these patients, expression of I-κ-B kinase complex-associated protein (IKBKAP) is remarkably reduced due to a single point mutation in the gene. Eight molecules were identified to rescue the expression of IKBKAP [127]. Clinical applications of IPSCs still have safety concerns. Cell therapy of IPSCs has the potential to form teratomas and other cancers. Induced pluripotent stem cells retain an epigenetic memory. Whether such changes have adverse effects or not is still unknown. The first clinical trial of IPSC cell replacement therapy was performed in a patient with age-related macular degeneration by Dr. Masayo Takahashi and colleagues at RIKEN Center for Developmental Biology in Japan. This clinical trial was halted due to safety concerns. Furthermore, Yamanaka’s group in Japan found two genetic changes in patient-derived IPSCs [128]. 3.5. In Situ Generation of Neurons in the Brain In 2010, Dr. Wernig and colleagues used a cocktail of transcriptional factors, achaete-scute complex homolog-like 1 (Ascl1), POU domain, class3, transcription factor 2 (Brn2) and myelin transcription factor 1 like (Myt1l), to successfully

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generate functional neurons from mouse fibroblasts [129]. One year later, this group generated functional neurons from human fibroblasts [130]. Since then, several groups reported that functional neurons can be generated from human somatic cells. Moreover, fibroblasts and astrocytes can be reprogrammed into proliferative neural progenitors. Reprogramming technology has been used to directly generate functional neurons in the brain. Dr. Chen’s group demonstrated that NeuroD1 transduction can reprogram reactive astroglial cells into functional neurons in a transgenic AD mouse model [131]. In situ-generated neurons will offer a novel avenue for therapy of neurodegenerative diseases, such as AD. Dr. Cheng’s group showed that Ascl1 could convert astrocytes into functional neurons in the dorsal midbrain [132]. Astrocytes were converted into neurons in injured spinal cords by a single transcription factor, SOX2 [133]. SOX2 converted astrocytes into proliferative neural progenitors in the animal brain [134]. Furthermore, IPSCs expressing Oct4, Sox2, Klf4 and c-Myc can generate teratomas in mice [135]. All of these promising studies provide a novel method for in situ replacement of dead neurons for the therapy of neurological disorders. Table 2. Glossary of stem cell term. Term

Definition

References

Stem cells

Stem cells are undifferentiated cells that have the capability [73, 77 - 79, 81, of self-renewal and differentiation. Stem cells produce 113, 114, 135, 151 other stem cells through systematic cell division or stem 156] cells and precursors through asystematic cell division. Stem cells can become specific types of cells through differentiation. Stem cells can be generated or reprogrammed in vitro and in vivo.

Pluripotent stem cells Pluripotent stem cells can give rise to all types of tissues, [54, 77 - 79, 81, (PSCs) but not an entire organism. 106, 113, 114, 151 156] Embryonic stem cells Embryonic stem cells are pluripotent stem cells isolated [54, 81, 106, 113, (ESCs) from the inner cell mass of early embryos or fetal tissue. 114, 151, 152] Induced pluripotent stem Induced pluripotent stem cells are pluripotent stem cells [54, 77 - 79, 81, cells (IPSCs) generated from somatic cells or adult stem cells through 106, 113, 114, 151 reprogramming. The properties of induced pluripotent stem 156] cells are similar with those of embryonic stem cells. Neural stem/progenitor Neural stem cells are stem cells generated from embryonic [56, 83 - 85, 88, cells (NS/PCs) or adult nervous system tissues. They can differentiate into 157 - 160] all three types of cells found in the nervous system: neurons, astrocytes and oligodendrocytes.

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(Table ) contd.....

Term

Definition

References

Mesenchymal stem cells Mesenchymal stem cells are stem cells that have been found [75, 76, 100, 101, (MSCs) in most adult organs, such as bone marrow, adipose tissue, 161 - 166] blood, periosteum and skeletal tissue and are capable of differentiation into multiple cell lineages including osteoblasts, chondrocytes, myoblasts, adipocytes, fibroblasts and neurons. Differentiation

Differentiation is the process by which stem cells lose [73, 77 - 79, 81, pluripotency or multipotency and become a more 113, 114, 135, 151 specialized cell type. 156]

Dedifferentiation

Dedifferentiation is a reverse process of differentiation. A [73, 77 - 79, 81, mature cell becoming immature, or a somatic cell becoming 113, 114, 135, 151 a stem cell. 156]

Transdifferentiation

Transdifferentiation is a process by which one type of [73, 135, 151] [77 somatic cell becomes another type of somatic cell. For 79, 81, 113, 114, example, blood cells becoming neurons. 152 - 156] Transdifferentiation is also called lineage reprogramming.

Reprogramming

Reprogramming is a process by which epigenetic marks of a [73, 77 - 79, 81, cell are erased and the cell becomes another type of cell. 113, 114, 135, 151 Reprogramming can happen naturally or artificially. 156]

3.6. Modeling and Therapy of AD with Genome Editing Recent advances in genome editing make it possible to specifically generate genetic mutations, deletions, insertion and corrections in stem cells and somatic cells from patients [136]. Site-specific DNA nucleases include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins, such as CRISPR associated protein 9 (Cas9) and Centromere and Promoter Factor 1 (Cpf1). Among them, the CRISPR-Cas9 system is easily designed and highly efficient. An increased number of investigators are using this technology for genome editing [137 - 140]. Dr. Zhang’s and Dr. Church’s groups simultaneously reported that the CRISPR-Cas9 system is an ideal genetic targeting tool in human cells [141, 142]. Furthermore, the CRISPR-Cas9 system has shortened the procedure of creating transgenic animals from months or years to several weeks [143, 144]. Such techniques are reproducible tools for drug discovery, and for modeling and gene therapy of neurodegenerative diseases (Fig. 3) [136, 145, 146]. The CRISPR-Cas9 system has been successfully used for gene correction. The CRISPR-Cas9 system has been used to correct the function of dystrophin protein in Duchenne muscular dystrophy (DMD) patient-derived IPSCs (DMD-IPSCs). After differentiation of the DMD-IPSCs to skeletal muscle cells, dystrophin

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protein was successfully detected [147]. The CRISPR methodology has also been tested in the mdx mouse model of DMD. A gene-editing approach with CRISPRCas9 corrected the frame-disrupting DMD mutation and recovered dystrophin expression. These results demonstrate that CRISPR-Cas9-modified cells could potentially recover muscle function in the mdx mouse muscle [148]. The hemoglobin subunit beta (HBB) gene mutation in thalassemias has been corrected by using the CRISPR-Cas9 system in IPSCs derived from patients carrying HBB mutations. Comparing with non-corrected clones, the ratio of embryoid bodies (EBs) formation and the percentage of hematopoietic progenitor positive cells were significantly increased [149]. Furthermore, Cas9 has a high efficiency for genome editing in post-mitotic neurons in the mouse brain [150].

A

Donor plasmid

APP locus

5’ arm

Promotor

Targeted locus

sgRNA1

Promotor

Donor plasmid

APP locus

sgRNA2

Mutated APP

5’ arm

Endogenous APP

Promotor

3’ arm

Mutated APP

(WT Cas9)

Targeted locus

3’ arm

Endogenous APP

(WT Cas9)

B

Mutated APP

Promotor

sgRNA1

sgRNA2

Correctd APP

Fig. (3). Strategies of modeling and therapy of AD by using CRISPR-Cas9 technology. A, Generating cell lines containing mutated APP from normal cells. Normal stem cells or somatic cells are targeted by the CRISPR-Cas9 system and endogenous APP is replaced with mutated APP carrying a point mutation, deletion or frame shift. Stem cells differentiate into neurons for modeling AD or drug testing. Somatic cells can be induced to form IPSCs and then differentiate into neurons. Somatic cells can be directly converted to neurons. B, Genetic correction of cell lines containing mutated APP. Mutated stem cells or somatic cells are targeted by the CRISPR-Cas9 system and mutated APP is replaced with endogenous APP. Stem cells differentiate into neurons for testing genetic correction and function recovery. Somatic cells can be induced to form IPSCs and then differentiate into neurons. Alternatively, somatic cells can be directly converted to neurons.

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4. PERSPECTIVES Recent progress in basic science and molecular diagnostics provides possibilities of personalized therapy. For example, high-through sequencing techniques, such as RNA-sequencing and ChIP-sequencing, and genetic approaches can identify whether a patient has specific genetic changes and how effective those genetic changes are for therapy. Patient-derived stem cells can be generated. Patientderived stem cells and neurons are used to screen patient-specific effective drugs. It is debatable whether personalized therapy is practical or not. First, current techniques to develop personalized therapy are still challenging. Second, the cost of developing personalized therapy will be much higher than that of conventional therapy and most patients will not be able to afford it. Third, the procedure may take a long time. By the time a personalized therapy is developed for a particular AD patient, the best therapeutic window may have already passed. With recent advances such as C-11 Pittsburgh compound B, a radioactive analog of thioflavin T, which can be used to image amyloid plaque burden positron emission tomography (PET) molecular imaging, the potential for early diagnosis of late-onset AD has increased. Genetic testing for ApoE4 and familial AD mutations are available but rarely performed outside of study recruitments. There are currently no disease-modifying treatments for AD, and the diagnosis can lead to depression, particularly in FAD patients. Furthermore, ApoE4 carriers may experience unnecessary emotional fear although many will never develop the disease. However, the potential benefits of diagnosis should not be ignored. Earlier treatment may increase quality of life and delay the need for full-time caregivers. If AD patients can remain independent for a longer period of time, the caregiver burden and considerable expenses can be reduced. Patients would be given the opportunity earlier in their disease to make legal arrangements and informed decisions on whether or not to have children. Due to the multifaceted features of AD, specific therapeutic agents must be further investigated. Multitype drug treatment may be more beneficial for AD in the clinic. Although the mechanisms of AD neuropathy are far from understood and no curative treatments are available, increasing efforts are ongoing to elucidate the secret of AD and develop more specific drugs. Alzheimer’s disease was first described more than a hundred years ago. Recent discovery of the protein structure of γ-secretase opens a new vista on drug development for the therapy of AD [167, 168]. Stem cell-derived whole cerebral organoids have 3D spatial architecture of the brain in culture, which provide a better understanding of human brain development and how neural toxins affect the functions of human neurons [169, 170]. The hope is that we could cure or at least slow down AD by using personalized therapy with novel therapeutic drugs.

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ABBREVIATIONS AAV

= Adeno-associated virus

AChEI

= Acetylcholinesterase inhibitor

AD

= Alzheimer's disease

aFGF

= Acidic fibroblast growth factor

APP

= Amyloid precursor protein

ARTN

= Artemin

Aβ

= Amylold β

BBB

= Blood-brain barrier

BDNF

= Brain-derived neurotrophic factor

BF

= Basal forebrain

bFGF

= Basic fibroblast growth factor

BMP

= Bone morphogenetic protein

ChAT

= Choline acetyltransferase

CNTF

= Ciliary neurotrophic factor

CRISPR = Clustered regularly interspaced short palindromic repeats CT-1

= Cardiotrophin-1

DHA

= Docosahexaenoic acid

DMD

= Duchenne muscular dystrophy

EB

= Embryoid body

EGF

= Epidermal growth factor

ESCs

= Embryonic stem cells

FAD

= Familial Alzheimer’s disease

GABA

= γ-aminobutyric acid

GDNF

= Glial cell line-derived neurotrophic factor

HD

= Huntington's disease

HDB

= Horizontal diagonal band

HGF

= Hepatocyte growth factor

HSV

= Herpes simplex virus

ICV

= Intracerebroventricular

IGF

= Insulin-like growth factor

IGF-I

= Insulin-like growth factor-I

IL-6

= Interleukin-6

IPSCs

= Induced pluripotent stem cells

Klf4

= Kruppel-like factor 4

Haigang Gu

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Frontiers in Clinical Drug Research - Alzheimer Disorders, Vol. 6 43

LEV

= Levetiracetam

LIF

= Leukemia inhibitory factor

LTD

= Long-term depression

LTP

= Long-term potentiation

mEPSCs = Miniature excitatory postsynaptic currents MGE

= Medial ganglionic eminence

mIPSCs = Miniature inhibitory postsynaptic currents MS

= Medial septum

MSCs

= Mesenchymal stem cells

NBM

= Nucleus basalis of Meynert

NFT

= Neurofibrillary tangles

NGF

= Nerve growth factor

NPCs

= Neural progenitors

NRTN

= Neurturin

NSCs

= Neural stem cells

NT-3

= Neurotrophin 3

NT-4/5

= Neurotrophin 4/5

NT-6

= Neurotrophin 6

NTFs

= Neurotrophic factors

Oct4

= Octamer-binding transcription factor 4

PD

= Parkinson's disease

PSPN

= Persephin

RA

= Retinoic acid

SCI

= Spinal cord injury

SHH

= Sonic Hedgehog

Sox2

= Sex determining region Y-box 2

SP

= Senile plaques

TALENs = Transcription activator-like effector nucleases VDB

= Vertical diagonal band

VEGF

= Vascular endothelial growth factor

ZFNs

= Zinc finger nucleases

CONFLICT OF INTEREST The author declares no conflict of interest, financial or otherwise.

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CHAPTER 3

Retinal Neurodegeneration in Alzheimer’s Disease L. Guo1, M. Pahlitzsch#, 1, F. Javaid#, 1 and M.F. Cordeiro*, 1, 2 Glaucoma & Retinal Degeneration Research Group, Visual Neurosciences, UCL Institute of Ophthalmology, Bath Street, London, EC1V 9EL, UK 2 Western Eye Hospital, Imperial College Healthcare Trust, London, UK 1

Abstract: Alzheimer’s disease (AD) is the most common cause of dementia globally. The prevalence has increased dramatically with an aging population. Although considerable progress has been made over the last few decades in understanding the pathophysiology of AD, early and accurate diagnosis of the disorder is still a formidable challenge, and there is currently no effective treatments available to slow down disease progression. The fundamental issue on this disadvantage is largely due to a lack of reliable biomarkers for neurodegeneration in the brain. However, mounting evidence has shown that except the brain, the eye, particularly the retina, is also affected in AD. Because of its transparent nature and ease of accessibility, the eye can serve as a ‘window’ into the brain. Advanced imaging technologies enable observation of changes in the retina in real time, e.g. measurement of thickness of the retinal nerve fibre layer (RNFL) by coherence tomography (OCT), detection of changes in the optic nerve head (ONH) by confocal scanning laser ophthalmoscopy (cSLO), and monitoring of retinal neuronal apoptosis by DARC (Detection of Apoptosing Retinal Cells). In addition to the ocular structural changes in AD patients, similar pathological mechanisms identified in the brain have also been established in the retina, including increased amyloid-ß (Aß) deposition and tau pathology. Furthermore, AD-related changes in the retina have also been observed in eye diseases, including glaucoma and age-related macular degeneration (AMD), and targeting of Aß has been demonstrated to be neuroprotective for those eye diseases. This review focuses on the recent advances in ocular changes, particularly retinal neurodegeneration in AD, discusses pathological similarities between AD and eye diseases, and highlights the potential of retinal imaging in identification of promising biomarkers for early AD.

Keywords: Aß, Alzheimer’s disease, AMD, DARC, Glaucoma, Retinal imaging, Retinal neurodegeneration, Tau. Corresponding author M. Francesca Cordeiro: Glaucoma & Retinal Degeneration Research Group, Visual Neurosciences, UCL Institute of Ophthalmology, Bath Street, London, EC1V 9EL, UK; Tel/Fax: (+44) 0207 608 6938; E-mail: [email protected] # These authors contributed equally to the work. *

Atta-ur-Rahman (Ed.) All rights reserved-© 2017 Bentham Science Publishers

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INTRODUCTION Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive cognitive decline and memory impairment [1]. AD is the most common cause of dementia, and there is currently no known treatment to delay its progression. It has been estimated there is 44 million people affected by dementia globally in 2010 with the cost of over US$600 billion, and the prevalence of AD worldwide is anticipated to triple by 2050 [2]. The hallmark lesions in AD are amyloid-ß (Aß) plaques and neurofibrillary tangles (NFTs), composed of tau protein, both causing neuronal degeneration and synaptic failure in the brain [3, 4]. Although the first case of AD was reported a century ago [5], early and accurate diagnosis of the disease still remains a formidable challenge. Currently, the diagnosis of AD is based on clinical neurological and psychiatric examinations in addition to distinguishing pathological features from the medical and family history [6]. Over the last decade however, neuroimaging of biomarkers has been investigated in multicentre clinical trials worldwide [7, 8], aimed to find the validated tools for the early diagnosis of AD, the tracking of disease progression, and the evaluation of novel therapeutic strategies. The outcomes have been encouraging, and a recent comprehensive review from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) [8] has reported that cerebrospinal fluid (CSF) biomarkers, ß-amyloid 42 and tau, as well as amyloid positron emission tomography (PET) may reflect the earliest signs in AD and that longitudinal magnetic resonance imaging (MRI) is proved most highly predictive of disease progression and has great potential for improving novel drug development, but none of them is a mature biomarker yet [9 - 11]. The main difficulty in the early detection of AD is possibly the incapacity of direct observation of microscopic and cellular changes in life time in the brain [12]. This however, is easily performed non-invasively through the medium of the eye [13 - 16]. Evolving imaging techniques now enable direct detection of changes in the retina and the optic nerve disc, as well as changes in single retinal neurons and their axons. Mounting evidence suggests that there are visual and ocular manifestations of AD, thus supporting the concept that the eye is indeed a window to the brain [17 - 21]. Tracking of retinal changes in real time may further facilitate improved understanding of the neuropathological mechanisms in AD, which implicates development of diagnostic methodologies in addition to providing parameters in assessment of novel therapeutic strategies. THE RETINA – AN INTEGRAL PART OF THE BRAIN The retina is part of the brain in the central nervous system (CNS) Embryologically, both the retina and the brain are derived from the neural tube, a

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precursor of the CNS during development. Anatomically, the retina connects to the brain through a collection of fibres – the optic nerve. The retina converts light into nerve signals to allow us to see the world. The neural retina consists of three layers of nerve-cell bodies which are connected by two layers of plexiform (Fig. 1). The nerve-cells in the most outer layer are the light receptors called photoreceptors (the rods and cones) and that in the most inner layer are the retinal ganglion cells (RGCs). The middle layer of the retina contains three types of nerve cells which are bipolar cells, horizontal cells, and amacrine cells. On the layer of RGCs, their axons run across the surface of the retina, collect in a bundle at the optic disc, and leave the eye to form the optic nerve. RPE

ONL

OPL BC

HC

INL AC

Light entrance

PR Signal transmission

PR

IPL GCL

GC

Fig. (1). Retinal structure and light transmission. The retina consists of three layers of nerve-cell bodies and two layers of plexiform, which are responsible for the transmission of light signals from the retina to the brain. The nerve-cells in the most outer layer (outer nuclear layer, ONL) are the light receptors called photoreceptors (PR) and that in the most inner layer (GCL) are the retinal ganglion cells (GC). The middle layer of the retina (inner nuclear layer, INL) contains three types of nerve cells - bipolar cells (BC), horizontal cells (HC), and amacrine cells (AC). On the layer of RGCs, their axons pass across the surface of the retina, collect in a bundle at the optic disc, and leave the eye to form the optic nerve. The light enters the eye from the inner surface of the retina via GCL, and passes through all the layers before being detected by PR (light entrance arrow). PR transduces the visual signals to GC via the three intermediate neurons and their synapses in the two platforms (IPL and OPL) (signal transmission arrow). RPE: retinal pigmental epithelium.

Light enters the eye and gets onto the inner surface of the retina after passing through the transparent media, i.e. the corner, lens and vitreous. Light then further

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travels to the photoreceptors through the various cellular and non-cellular layers (Fig. (1), Light entrance arrow). Photoreceptors detect the light and transduce the visual signals to RGCs via the intermediate nerve cells (bipolar cells, horizontal cells, amacrine cells) (Fig. (1), Signal transmission arrow). The RGCs then transmit the visual information to the lateral geniculate nucleus (LGN) in the brain by the optic nerve, so called the visual pathway (Fig. 2).

Optic Radiations

Optic Nerve Optic Chiasm

6

Optic Tract

5 4

Parvocellular

3

LGN Superior Colliculus

2 1

Magnocellular

Optic Radiations Optic Tract

Primary Visual Cortex

Fig. (2). Lateral geniculate nucleus (LGN) in the visual pathway. The LGN in the brain receives visual information from the retina via the optic nerve fibres. After leaving the retina, the optic nerve fibres partially cross at the optic chiasm and further travel along the optic tract before reaching to the two LGNs. From the LGN, the visual information is relayed to the primary visual cortex via the optic radiations. In mammalian, the LGN is structured into six primary layers, in which magnocellular neurons located in ventral layer 1-2, parvocellular neurons in dorsal layers 3-6, and koniocellular neurons intercalated between the primary LGN layers.

In the visual pathway, the LGN of the thalamus provides the main input to the primary visual cortex in both the primate and human brain (Fig. 2). The LGN is therefore recognised as the primary processor of retinal-derived visual signals, transmitting colour and object information from the retina to the visual cortex. There are three distinct visual pathways in the LGN, which are the magno-, parvo-, and konio-cellular pathways. In the parvocellular pathway, colour and object information is transmitted whereas in the magnocelllular pathway, signals involved in spatial recognition and motion are passed through [22]. In the koniocellular pathway, blue/yellow chromatic information is processed before relaying to the visual cortex [23]. The human LGN is structured into six primary layers, in which, magnocellular neurons are located in the ventral layers 1-2 and

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parvocellular neurons in the dorsal layers 3-6, while koniocellular neurons are distributed in an intralaminar fashion between the primary LGN layers [24] (Fig. 2). The neurons in the LGN layers share the same name with corresponding types of RGCs in the retina, i.e. magno-, parvo-, and konio-RGCs. In addition, the LGN layers are side-specific, with layers 2, 3, and 5 connected to the ipsilateral eye, and layers 1, 4, and 6 to the contralateral eye [25, 26]. From the LGN, visual information is further carried to the primary visual cortex via the optic radiation, for higher level processing (Fig. 2). VISUAL CHANGES IN AD Visual Abnormalities Visual complaints are common in early AD, well in advance of an established diagnosis [19, 27]. Visual disturbances include difficulties in reading and finding objects [28, 29], depth perception [28, 30 - 32], perceiving structure from motion [28, 29, 32], colour recognition [28, 33], and impairment in spatial contrast sensitivity [30, 34, 35]. Previously, the visual abnormality in AD was believed to be only associated to impairment in the higher visual pathway, i.e. the primary visual cortex [36, 37]. However, mounting evidence suggests that pre-cortical degeneration is also involved in the disease process [38, 39]. Indeed, the visual defects in AD can be attributed to alterations which could take place at any level of the visual pathway (Table 1). Histological findings in AD patients show that large diameter RGCs and axons are more vulnerable [40]. Due to their important role in contrast sensitivity, especially at low spatial and high temporal frequencies [41], large RGC injury could damage contrast sensitivity at an early stage of AD [28, 40]. In addition, Selective loss of large RGCs is also related to motion damage in AD [28, 32]. Table 1. Association of visual abnormality and visual pathway in AD. Visual Pathway Primary/higher Visual cortex

Specific Area in the Brain

Difficulties in

References

Lateral occipital complex

Reading and finding objects [28, 29, 32]

V4

Colour recognition

[28, 33]

Fronto-parietal area

Visual attention and memory

[29, 30]

V1, MT or V5*

Depth and motion perception

[29 - 32]

Retinal ganglion cells and axons * MT or V5: medial temporal visual area

Spatial contrast sensitivity

[30, 34, 35]

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Pupil Abnormalities Studies have indicated that both light and mydriatic eye drops result in specific changes within the pupils of AD patients. In 1994, it was first noted by Scinto et al. that there was a marked hypersensitivity when patients with probable AD were administered with the cholinergic (ACh) antagonist, tropicamide (0.01%) [42]. Pharmacologically, Tropicamide is classified as a muscarinic acetylcholine receptor antagonist that binds to muscarinic receptors. In AD, it is established that there is an abnormality in ACh pathways, and AD patients possess ACh receptors that may be more sensitive to very dilute tropicamide than normal subjects. Following this preliminary report, numerous studies have confirmed statistical significance between the antagonistic pupil response in AD patients as compared to normal controls, indicating that it may be used as a potential diagnostic marker for AD [43, 44]. Additionally, Scinto has further suggested that Apolipoprotein E (ApoE) allelic variability, a factor linked to late-onset, sporadic AD, is the cause of this difference in pupil response, and suggests that it is influenced by tau hyperphosphorylation [45]. Furthermore, Scinto published a prospective longitudinal study that indicated there was higher risk in the development of significant cognitive impairment with a hypersensitive pupil response memory, and attention and language areas are known to be targeted in particular, consistent with the established pattern displayed in pre-clinical AD. The risk is further increased with ApoE allelic variability [46]. Abnormal pupillary responses in AD have further been associated with poor problem solving abilities, as a result of cognitive impairment. Pupil size correlates with mental activity during problem solving, and has therefore been suggested as a direct measure of cognitive capacity [47]. The pupillary response to light has also been suggested to be abnormal in AD. Following assessment of the pupil reaction to light using a single flash, the pupil reaction to light in AD patients was found to significantly differ from that of control subjects with a shorter latency and lower amplitude of maximum reaction. These findings suggest that dynamic pupillometry could prove as a useful tool to assist clinicians in the early diagnosis of AD [48, 49]. Given these findings, the development of a pupillometer system was introduced with an infrared charge coupled device camera and a personal computer for the analysis of pupil area based on video images, in addition to the programme to calculate the pupil dilation rate [44]. Most recently, using a Compact Integrated Pupillograph, a study further demonstrated that patients with AD and MCI displayed an amplitude increase and less pronounced pupil size decrease over time than controls. Pupil size increase was also shown to correlate with cerebrospinal fluid markers in AD [50].

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RETINAL CHANGES IN AD A list of retinal abnormalities in AD is shown in Table 2, by reviewing literatures. Table 2. Retinal abnormalities in AD. Retina

Described Abnormalities

References

RGC soma

Aß deposition, correlated with RGC apoptosis & loss

RGC axons

Marked reduction in RNFL thickness

[14, 40, 51 - 53] [54 - 58]

Optic disc

Increased CDR, reduced neuroretinal rim volume and area

[59 - 61]

Vasculature

Narrowed blood vessels, reduced blood flow

RPE

Increased APP immunoreactivity

[56, 62, 63] [53]

Retinal Histopathologic Abnormalities Histopathological evidence of retinal degeneration was first reported by Hinton et al., thirty years ago [40]. Analysis of post-mortem AD retinae displayed widespread degeneration of the axons within the optic nerves, in addition to reduced thickness of the retinal nerve fibre layer (RNFL), and fewer number of RGCs [40]. It was noted that cells particularly affected were the large diameter RGCs and axons [40, 64]. Blanks et al. went on to provide further extent evidence of RGC degeneration in the patients with AD using ultrastructural analysis, displaying a range of intracellular injuries. The cell cytoplasm was noted to be pale with mitochondria and endoplasmic reticulum swollen. Additionally, the nuclei were also noted to be pale with dispersed chromatin in early stages, and vacuolated cytoplasm and clumped chromatin in late stages [51]. Additional studies demonstrated that although extensive neuronal loss was detected in the entire AD retina, the superior and inferior quadrants displayed the largest neuronal reduction [52]. Moreover, the number of astrocytes was increased with the ratio of astrocytes to neurons significantly higher in AD subjects as compared to controls [52]. Central retinal RGC loss was also noted to be extensive in AD, and the most significant reduction occurred in the temporal foveal region [65]. There are however different findings from other studies conflicting with the results described, which may be attributed to differences in methodology [36, 37]. The characteristic neuropathological marker in AD is the deposition of Aß, resulted by abnormal processing of amyloid precursor protein (APP) in the brain. APP and Aß immunoreactivity are prominent in RGCs and the RNFL in the human retina and are indicated to increase with age [19, 66, 67]. Recent data from transgenic AD models (APP transgenic mouse (Tg 2576), APP/PS-1 double transgenic mouse, and APPswe/PS1ΔE9 transgenic mouse), have shown increased

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immunoreactivity of Aß and APP in the retina, including RGCs, the RNFL, and the inner plexiform when compared to wild-types [18, 20, 21, 53, 67 - 69]. Furthermore, these studies also established that these developments were agedependent, and correlated with Aß plaque load in the brain. More importantly, Aß deposition in the Tg-AD retina is found to precede Aß plaques in the brain [66, 70]. Moreover, tau protein abnormality identified in the brain is also seen in the retina and optic nerve in transgenic AD mice [71 - 74]. The AD-related Aß deposition and tauopathy in the retina showed to be correlated with RGC apoptosis and retinal structural and functional impairment [18, 20, 53, 68]. AD causes complex morphological and functional transformation within astroglial cells in the brain, which contributes to the development of amyloid plaques and outcome of neuropathological process [75, 76]. Evidence shows that astroglial atrophy associates with early stages of neurodegenerative processes, causing disruptions in synaptic connectivity and neuronal death in AD; and increased astroglial activation in the later stages contribute to the neuroinflammatory component of neurodegeneration [75 - 77]. Recent evidence suggests that Muller cells and astrocytes in the AD retina are remodelled in a similar way to astrocyte cells in the AD brain [78, 79]. In a triple transgenic AD mouse model (3xTg-AD), overexpressing APPSwe and tauP301L, and carrying a PS1M146V knock-in mutation, abnormal glial morphology and activation is observed as early as 9 months old in the retina and increased with age [78]. Like Aß deposition in the retina, the glial activation in the 3xTg-AD mice appears earlier in the retina than in the brain, where astroglial atrophy does not become significant until 12-18 months in hippocampus [80, 81]. Astrocytes have been recognised as central players in the cellular phase of AD, attributable to their properties of neuronal plasticity, clearance function, lipid metabolism and immune responses; the astroglia population however, has been extremely under-investigated in AD research [82]. Investigation of the role of astrocytes in AD retina is also essential as not only could this enhance our understanding of disease mechanisms, but could also facilitate the development of novel diagnostic tests as well as drug interventions. Retinal in vivo Abnormalities The visualisation of retinal changes has greatly advanced with the development of modern imaging techniques. This has provided researchers with a valuable tool and allowed for advancement in early diagnosis and monitoring of disease progression and drug efficacy in eye diseases, and also in AD. Retinal Nerve Abnormalities Retinal nerve changes can be detected by advanced imaging technologies that use

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a non-invasive, easily assessed approach in routine ophthalmological exploration. In particular, the optical coherence tomography (OCT) plays a fundamental role in the diagnosis of retinal nerve fibre abnormalities. The OCT is an optical imaging system that utilises light waves to capture cross-sectional images of different ocular structures. In 1991, it was first used by ophthalmologists [83], and over the last two decades has become increasingly applied in clinical practice and research in order to assess the RNFL thickness [84] and other parameters of the eye [85]. The technology is rapidly advancing due to its reproducibility, and Fourier Domain OCT (FD-OCT) has been replaced by Spectral Domain OCT (SD-OCT, the third generation technique). The two key elements of the SD-OCT is a broadband light source and a high speed spectrometer that generate a mean imaging range of ~3.0 μm, a fast sensitivity fall-off (~6 dB/1.5 mm) and a 120 kHz scan rate [86]. Another type approached the market is called swept source OCT (SS-OCT), using a laser system equipped with a high speed generator and a photodetector for spectral interferogram detection. SS-OCT offers a longer imaging distance (>5 mm), a decreased system sensitivity fall-off (~6dB/3mm), a higher penetrating depth (using 1 μm wavelength), faster speed (>100kHz A-line rate) and an increase in axial resolution in air (~10 μm) [86]. In addition, a recently developed functional OCT modality allows imaging of changes in cell functional behaviour with a stimulating input through the entire retinal layers in a time-dependant manner [87, 88], including quantification of the blood flow in the retina implementing by colour Duplex OCT [89] and oxygen saturation of the retina by employing both, FD-OCT and SD-OCT [90]. One of the recent outstanding improvements is the ability of the SD-OCT to analyse the RGC sublayers, including ganglion cell-inner plexiform layer (GC-IPL) for imaging of the RGC dendrites [91, 92]. The key factor of this ability is in the high spatial resolution of the SD-OCT [91, 92]. Additionally, software of the SD-OCT, such as enhanced depth imaging (EDI) technique is used to assess choroidal thickness by using the 1.05µm wavelength [86, 93, 94]. Although controversy exists in evaluating the retinal nerve fiber changes in AD, the attention of neuroimaging in AD is recently turned towards the exclusion of any other pathology caused by a neurodegenerative process [95, 96]. Using SDOCT, the parapapillary retinal nerve fiber layer thickness showed a strong decrease in Alzheimer’s patients in comparison to healthy subjects [54, 55], and this reduction appears to be already included in patients with mild cognitive impairment (MCI), thus early stages of the disease. However, thinning of the peripapillary RNFL has also been detected in other diseases, including glaucoma, multiple sclerosis, neuromyelitis optica, and Parkinson’s disorder [97, 98]. Evidence has also suggested that the reduction of the retinal nerve fiber layer thickness was considerably prominent in the superior part in early stages of the Alzheimer disease [56, 57], consistent with symptoms of inferior visual field loss.

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Pattern electroretinogram (PERG) results displayed abnormal readings as a result of the retinal dysfunction related to the demonstrated structural changes in the retinal nerve fiber layer [99, 100]. This PERG anomaly observed in AD may reflect degeneration and dysfunction in RGCs and their axons [101, 102], as ERG signals are reported to present electrophysiological activity of RGCs [103]. Additionally, neural conduction can be recorded by the retinocortical time (RCT), defined as the difference between P100-wave latency in pattern VEP and a specific parameter of the pattern ERG (P50-wave implicit time) [104]. Electrophysiological examinations can be useful in early phases of the disease along with normal tests in standard ophthalmological diagnostics, when RGC dysfunction and optic nerve alterations are already present [104]. Additionally to the defects in the peripapillary area, OCT imaging in AD patients has detected a substantial decrease in thickness and volume of the macula, exhibiting significant correlation with severity of disease [105]. The macular thickness is anatomically composed of approximately 35% of retinal ganglion cells and their fibers. The significant thinning of the macula and reduction in volume potentially identifies considerable reduction of RGCs in the fovea. This is consistent with histopathological findings by Blanks et al., where 25% reduction of the neuronal network in the retinal ganglion cell layer was found in the macular retina in human AD [106]. In support, one recent study shows a reduced thickness of the macular GC-IPL in line with the RNFL thinning in AD patients [57, 58]. Thus, the macular GC-IPL thinning is suggested as a new marker for detection of neurodegenerative damage in early AD and MCI [57]. Controversially however, a recent study failed to show any difference in the central subfield retinal thickness and peripapillary area between AD and healthy controls [107]. Moreover, to date there is no correlation found between the thinning of the RNFL and the severity of dementia, and also no correlation of other OCT parameters, including optic disc excavation and macular thickness, with either AD-specific CSF data or MiniMental Status Examination (MMSE score [108, 109]. The morphology of the optic nerve head (ONH) can be described as a circular, small yellowish-orange region located in the back pole of the retina, which concentrates all RGC axons for their descent through the lamina cribrosa – a perforated osseous structure. The optic nerve is defined as the conglomerate of all these single nerve fibres transporting the information from the eye towards the cerebral structures. Confocal scanning laser ophthalmoscopy (cSLO) has been used to identify changes in the ONH in AD patients in comparison to healthy subjects. The changes are similar to that found in glaucoma, including a strong reduction in the retinal nerve fibre layer, neuroretinal rim, and increased vertical cup-to-disc ratio, supporting the assumption of a decreased number of nerve fibres merged in the ONH [59]. Although a recent study failed to demonstrate

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significant difference of the ONH between AD and controls, it successfully differentiated AD patients from glaucoma using the cSLO, suggesting the ability to recognize specific neurodegenerative conditions [60]. Accordingly, a significant correlation of the optic neuropathy with the onset, severity and duration of AD has been previously reported [110]. Similar findings in human AD have also been observed using fundus photography [110, 111]. Retinal Vasculature Abnormalities The development of cognitive failure is notably the early sign of AD and is attributed to abnormalities in cerebral blood flow [112, 113]. Because of the fact that the retinal and cerebral microvascular system develops from a similar embryological origin, sharing anatomical and physiological features (e.g., non anastomotic end arteries, blood-brain and blood-retina barriers), researchers have looked into the retinal vascular changes in AD [62, 63]. Using digital fundus photography with a semi-automated software, an altered retinal microvascular system, including narrower retinal venules and sparser and more tortuous retinal vessels, is identified in AD patients, compared with age-matched controls, suggesting that these retinal alterations may reflect similar pathophysiological processes in the cerebral microvasculature in AD [63]. In support, a recent study has not only demonstrated abnormal retinal parameters on vascular structures, but also associated the retinal vascular changes to neocortical amyloid plaque burden [114]. The findings indicate that the widely used retinal photography offers a sensitive, noninvasive method for detecting preclinical AD, which might be useful for population screening [114]. Previous research has identified retinal blood column narrowing, by Doppler retinal blood flow measurements, in addition to a considerable decrease in the blood flow rate of the retina in the patients with AD, compared to control subjects [56]. This again suggests that changes in the retinal blood flow may be associated with that in the brain, where reduced perfusion is believed to affect ATP synthesis in addition to causing oxidative stress and neuronal death – the mechanisms preceding clinical dementia [113]. Retinal Cellular Abnormalities – RGC Apoptosis Although individual RGCs cannot be real-time visualised without a marker, RGC apoptosis can be seen by novel imaging technology of DARC (Detection of Apoptotic Retinal Cells). Over the last decade, our group has established the DARC and demonstrated it is a valuable tool to monitor RGC apoptosis in vivo in experimental animal models [13 - 15, 115, 116]. DARC employs fluorescein labelled annexin V and a confocal scanning laser ophthalmoscopy. Annexin V, administered either locally or systemically, binds to phosphatidylserine (PS) which translocate from intracellular to extracellular membrane upon initiation of

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apoptosis. DARC enables us to visualise single RGCs undergoing apoptosis, which is recognised as the earliest sign of glaucoma [13]. Because RGC apoptosis can be monitored in the same living eye over time, DARC imaging is also a useful device to assess treatment effectiveness and to screen new drugs [14, 16, 115, 117] (Fig. 3).

A

Control

B

Abab

C

Triple Abab

% Reduction of RGC Apoptosis Compared to Control

100.00

90.00

80.00

70.00

60.00

50.00 Abab

Triple Abab

At 3 weeks after IOP Elevation

Fig. (3). Targeting of amyloid-ß is neuroprotective in experimental glaucoma. In vivo DARC images (A-C) show that glaucoma-induced RGC apoptosis (A, control, white spots) was considerably inhibited by either single treatment (B, Aßab: anti-Aβ antibody only) or combined treatment (C, Triple Aßab: Aβab, Congo red and β-secretase inhibitor) at 3 weeks. Statistical analysis demonstrated that both the single and combination therapies significantly reduced RGC apoptosis compared to control (p=0.007 and 0.002 respectively), but Triple Aβab was more effective than Aβab alone (pT polymorphism may be associated with higher risk of AD. Homozygous alleles of IL-1A and IL-1B have been identified as a risk factor of AD [63, 64]. Another protein Cystatin C is a co localizing amyloidogenic protein with beta-amyloid in arteriolar walls in the brains of AD patients. A synergistic association was seen between CST3-A allele, APO E4 and AD in patients of age 60 -74 years. However some studies found no correlation between Cystatin C gene (CST 3) polymorphism and risk of AD [65 - 67]. Study revealed that the rs266729 GG and rs1501299 TT genotypes and GT and CG haplotypes of adiponectin genes are at a greater risk of LOAD [68]. Many other protein have been found involved in AD inflammation including clusterin (CLU), complement receptor 1 (CR1), C reactive protein (CRP), tumor necrosis factor a (TNF-a), the interleukins 1a, Interleukin-6, Interleukin-10 and cyclo-oxygenase 2 (COX-2) [69]. TREATMENTS FOR ALZHEIMER’S DISEASE AD (dementia) is an age related progressive and irreversible neurodegenerative disorder which occurs at older ages mostly after 65 years. Currently there is no proper cure or treatment is available for this disease and these available treatments

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only delay the progression of the disease with temporary symptomatic relief. Current treatment strategies mainly target on the antihypertensive therapies with special focus on ACE inhibitors and ARBs. Other drugs therapies are also available for AD which are briefly described below (Table 1): Table 1. Various therapies available for AD. Sr. No.

Drug Class

Examples

1.

Antihypertensive drugs

ACE inhibitors- Perindopril, Captopril ARBs- Telmisartan, Valsartan, Losartan, olmesartan and Candesartan CCBs- Nimodipine, nilvadipine, and Nitrendipine

2.

Anti amyloid therapy

Memoquin

3.

AchE inhibitors

Donepezil, Galantamine, Rivastigmine, Tacrine

4.

NMDA antagonist

Memantine

5.

Antioxidants

Vitamin E, Vitamin C, MAO (selegiline), phenolic and polyphenolic compounds, tannins

6.

Anti inflammatory agents NSAIDs like Celecoxib, rofecoxib, flurbiprofen and ibuprofen

7.

Vaccines

AN-1792

8.

Statins

Atorvastatin, Fluvastatin, Lovastatin, Pitavastatin, Pravastatin, Rosuvastatin and Simvastatin

9.

Antipsychotics

Olanzapine, Risperidone

10.

Other therapies

Estrogen therapy- Raloxifene TNF-α blocker- Etanercept

1. Antihypertensive Drugs: Antihypertensive drugs especially ACE inhibitors are proven to be very effective in the management of AD and improve cognitive stability for longer duration. ACE enzyme produces Ang II by enzymatic conversion from Ang I. Ang II block the release of neurotransmitter Ach which is necessary for the nerve conduction and proper functioning of the brain. Therefore, ACE inhibitors along with ARB are very useful drugs candidate for the treatment of AD. ACE inhibitors reduce the inflammation associated with vascular system and in the brain local ACE reduce the neuronal inflammation and benefit in 50% of Alzheimer’s patients and slow the progression of AD. ACE inhibitors works by reducing the level of Ang II, increasing Ach and breaking down β-amyloid. Ang II found to reduce Ach level which interfere memory function [70 - 72]. Studies also have found reduced risk of occurrence of AD in patients taking ACE inhibitors than those not taking ACE inhibitor therapies. ACE inhibitors also delay the progression of cognitive decline and AD. The brain penetrating ACE inhibitors which easily cross the blood brain barrier have more pronounced beneficial effects in cognitive decline as compared to other non brain penetrating antihypertensive

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drugs. This beneficial effect of brain penetrating drugs might be due to direct action of drug on the brain’s local RAS system due to more bioavailability in brain. However, no significant difference was observed on the blood pressure lowering effect of brain penetrating and non penetrating drugs. The centrally acting ACE inhibitor, especially perindopril, is found associated with slowing in the decline rate of brain functions but the effect on mood and behavior were not significant [73, 74]. Calcium channel blockers (CCBs) are also found beneficial in AD and people taking CCBs are less prone to develop AD. But all studies of CCBs were on hypertensive patients only, so effect of CCBs on non hypertensive is unknown. ARBs block the action of Ang II via binding to AT1 receptor and reduce the risk and progression of AD [75]. Pre-clinical study of Ang II receptor blocker on mice showed significant improvement in cognitive and memory functions after administration of low oral dose of olmesartan. Olmesartan pretreatment also prevented vascular dysregulation induced by β amyloid formation. Another pre-clinical study suggests that candesartan and perindopril prevents β amyloid deposition and memory related impairments caused by free radical damage. Even one study found that ARB prevent progression of dementia and AD more significantly as compared to ACE inhibitors and other antihypertensive agents but primarily in male population. The AD associated pathology was also found less in ARB treated patient’s autopsy evaluations [76 - 79]. 2. Anti-Amyloid Therapy: Amyloid and plaques are the major factors behind the pathophysiology of AD. Beta amyloid produced by γ-secretage enzymes from the degradation of amyloid precursor protein (APP) forms the plaques and insoluble amyloids which starts degradation of brain neurons leading to AD. The anti-amyloid therapy mainly focuses on (a) clearance of Aβ (b) Inhibit production and aggregation of Aβ [80]. 3. Acetylcholinestrase (AchE) Inhibitors: AchE inhibitors act by inhibiting the enzymes AchEs which degrade the neurotransmitter Ach so that the effect of Ach prolongs. Therefore, AchEs are the first line treatment for AD [81 - 83]. But AchE use is limited because of their shorter half life and systemic cholinergic actions. Along with cholinesterases anticholinergic drugs are also prescribed for the management of AD but these produce undesirable cognitive side effects. 4. N-methyl-D-aspartate Antagonist (NMDA Antagonist): NMDA antagonist prevents the glutamate associated excitotoxicity during signal transduction. Memantine is the drug which prevent the over activation of NMDA glutamate receptors by binding NMDA receptor in preference to glutamate resulting in cognition improvement [84]. Memantine is a suitable candidate drug of this class as it only prevents excess stimulation of NMDA receptors via glutamate and maintains normal function of the neurotransmitter which is necessary for

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5.

6.

7.

8.

9.

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maintaining the elasticity of nerves [85]. The reported adverse events in case of memantine include dizziness, headache and confusion. A few patients developed agitation [86]. Antioxidants: Antioxidants prevents damage from free radicals and oxidative stress to neurons by scavenging the oxidants and free radicals. Vitamin E and C and monoamine oxidase inhibitors (MAO) are the agents which help to scavenge the oxidants. Vitamin E donates the hydrogen atom and scavenges or reduces the reactive species and stabilizes them with unpaired electrons. Vitamin C helps in recycling or regeneration of vitamin E from its oxidized state to further make it active. It is very difficult to identify the extent of benefit of vitamin E therapy for AD but it may be used as prophylactic therapy [87, 88]. Anti-Inflammatory Agents: Beta amyloid and tangles deposited in the brain and dead neuronal cells may induce inflammation as a natural cell defense mechanism via microglial cells by releasing cyto-toxic pro-inflammatory molecules. Long term studies proved the effectiveness of NSAIDs in prevention of AD. Consumption of NSAIDs for more than 5 years reduces the risk of developing AD. But longer duration consumption of NSAIDs produces gastrointestinal symptoms and kidney and liver toxicity [89]. However some studies showed that treatment of AD with NSAIDs is disappointing and these drugs are only effective for prophylaxis only have doesn’t have any cognitive beneficial effects [90, 91]. Vaccination: Vaccines boost up the immune system to fight against the pathogenic microorganism which may cause damage to the brain neurons. Vaccine is administered in beta amyloid form that clears the plaques and improves the AD. Immunotherapy works in several ways in the treatment of AD such as direct dissolution of plaques by antibodies induced conformational changes, activation of microglial cells induced by antibodies, phagocytosis of protein deposits, neutralization of toxic soluble oligomers, clearance of circulating Aβ cell-mediated immune responses, immunoglobulin M (IgM) mediated hydrolysis of beta amyloid and plaques [92]. Statins: Cholesterol is considered as the risk for AD but exact relationship is not fully clear between serum cholesterol level and risk and progression of AD. Statins do not affect the production of β-amyloid in humans but in vitro study in mice has shown the effect. Therefore statins may show neuroprotective and anti-inflammatory effects instead of prevention of βamyloid formation [93, 94]. Antipsychotics: These are mainly used for treatment of behavioral symptoms. Olanzapine and risperidone are suggested to reduce aggression and psychosis in Alzheimer’s patients. Use of atypical antipsychotics in patients with Alzheimer disease to treat behavioral symptoms generally should be avoided

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because of possibility of adverse effects [95, 96]. 10. Other Therapies: Other than the above therapies there are number of therapies which may be beneficial in the treatment of AD. Estrogen therapy is suggested to improve cognition after menopause in women. Raloxifene is a potent estrogen receptor modulator which may reduce the risk of AD. Implantation of healthy neuron can also be beneficial in AD. Insulin resistance can make brain deficient in energy for cell maintenance and nerve synaptic connections leading to cell death. Also hyperinsulinemia is reported to induce inflammation. Nasal insulin is found to reach brain cells very quickly and improve verbal memory [8]. In AD patients the level of Tumor Necrosis Factor-alpha (TNF-α) has been found increased which is an indication of inflammation so etanercept (TNF-α blockers) found to show cognitive improvement [97]. HERBAL DRUGS FOR THE TREATMENT OF AD No treatment with allopathic medicines available till date to treat AD. All the available therapies provide symptomatic relief only for shorter duration. The condition of the AD patients worsens with age and intensity of disease, ultimately leading to death. Therefore a lot of research is been going on throughout the world on herbal medicines to find out the cure for the disease. Many studies have shown promising result of herbs in AD treatment because of their cognitive benefits and their mechanisms of action with respect to disease pathophysiology. The potential mechanism of action of these herbs not confined to the inhibition of AChE but also include the modification of Aβ processing, protection against apoptosis and oxidative stress and anti-inflammatory effects with minimum or negligible adverse effects. Herbal plats have been used in traditional Chinese and Indian medicinal system to treat memory loss, cognition impairments, enhancement of brain activity and slowing down the degenerative processes in AD patients. Various herbal plants used for the treatment of AD are as follows: ●

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Ginkgo biloba: The clinical studies have shown that Ginkgo biloba extract have the activity for the treatment of AD equivalent to the drug therapies such as donepezil and tacrin. The Ginko extract have shown activities such as antioxidant, neuroprotective and cholinergic. Galanthus caucasicus: The alkaloid galantamine have shown its benefit in the treatment of mild to moderate AD and memory impairments. The activity of galantamine is found comparable to AchE inhibitors. Clinical studies have proven the beneficial effect of this plant over placebo in AD patient when taken for 6 months. Huperzia serrata: It is a natural alkaloidal cholinesterase inhibitor derived from the Chinese herb Huperzia serrata. Because of its antioxidant and neuro-

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protective action it is sold as dietary supplements for memory loss and mental impairment. Therefore it is a component of herbal remedies for treatment of AD [98 - 101]. Catharanthus roseus: Vinpocetin is an alkaloid found in Catharanthus roseus. It is neuro-protective and enhances the cerebral blood flow. Therefore it is used in memory loss and age related memory impairment. Melissa officinalis: It reduces agitation and enhances cognitive function in mild to moderate AD patients. Euphorbia royleana boiss (Source of Shilajit): Shilajit is a dark, viscous and sticky substance which is obtained from rocks. Latex of Euphorbia royleana Boiss is the source of Shilajit. It is supposed to affect some parts in cortical and basal forebrain cholinergic signal transduction cascade in brain. Withania somnifera (Ashwagandha): It is regarded as nervine tonic and used in AD and other type of dementias as it slows stops, reverses and removes neuritic atrophy and synaptic loss. Bacopa monniera (Neer Bramhi) and Centella asiatica (Mandookparni/ Bramhi): Both these are not the same plant but still known as brahmi due to overlapping of their properties. These are neuro-protective and are beneficial in age related cognitive decline. Therefore useful in memory improvement and intelligence [98 - 101]. Curcuma longa (Haldi): Curcuma longa has a very good anti inflammatory activity and nerve inflammation is one of the major pathological factors for AD. Therefore Curcuma longa may have role in the treatment of AD. It has been found to prevent oxidative and inflammation induced formation of Aβ and plaques and improve in neurological deficit. Panax ginseng: It has been shown to improve cognitive performance and protective functions in Alzheimer patients. Celastrus paniculatus (Malkangni): It has been used in indigenous medicinal system for memory and braid related disease. It is administered as nervine stimulant, sedative, rejuvenant, tranquilizer and diuretic. It helps in memory enhancement and stimulates intellect [98 - 101]. Glycyrrhiza glabra (The Licorice Root): A dose of 150 mg/kg of aqueous extract of Glycyrrhiza globra has been found to improve learning and memory by decreasing production of β amyloid and formation of plaques in animal study. Hypericum perforatum: It helps in improving learning and memory function by reducing oxidative stress and also acts as antidepressant. The antioxidant activity is due to presence of quercetin and quercitrin which scavenge free radicals present in brain. Lepidium meyenii (Maca): Maca improves memory and learning in animal studies. It reduce AchE activity, therefore helps in maintaining level of

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neurotransmitter Ach. Prunella vulgaris: It increase memory and learning via restoring the action of cholinergic system neurotransmitter and methyl D aspartate signaling. Its antioxidant property is additional to above one and also acts as antidepressant [98 - 101]. Cyperus rotundus: This plant also improves cholinergic system because of its anti AchE activity and helps in restoring learning and memory activities. Zizyphus jujube: The flavonoid content of this herb has histamine release, activity of AChE and cyclooxygenase I and II inhibitory activity. Improve in ADsymptom is due to increase in the cholinergic activity. Morinda citrifolia: It has analgesic, anti-inflammatory and antioxidant activity and ethyl acetate extract of this plant reduce oxidative stress and enhance memory. Polygala tenuifolia: In Chinese medicine system it is prescribed for mind soothing effect and indicated for insomnia, mental confusion and disorientation. Preclinical studies suggest that it prevents reduction in cholinergic activity by inhibiting the secretion of Aβ from cultured cells [98 - 101].

CONCLUSION AD is a neurodegenerative disease in which patient’s memory and learning processes are impaired. It mainly happens after the age of 65 years. Various pathophysiological conditions are found associated with the etiology of AD. These may include the one which is directly associated with brain and nervous system like brain physiology, death of neurons, degradation of neurotransmitters, ionic disturbance which impair nerve conduction, excitotoxic effect of some chemicals and formation of β-amyloid and plaques. While other physiological factors include the conditions which indirectly affect the brain and neurons such as autoimmunity, viral and microbial invasion, toxin accumulation, inflammation, oxidative stress, genetics and problems related to vascular system. Brain has its own RAS system which is other than systemic RAS. The components of local RAS in brain includes ACE, Angiotensin peptides I-IV, and AGT receptors which act as neuromodulators in the brain and control hormone secretion and neurotransmitter release. RAS components also induce inflammation and oxidative stress in nerve cells via AT1R receptors which create problems in nerve impulse transmission and degeneration of neurons. Genetic polymorphism may affect the pathophysiological processes of AD which may induce or worsen the conditions of this disease. Various genes have been identified which may have an impact on AD intensity or prevalence. Different treatment strategies have been practiced to treat or improve this disease based on the pathophysiological condition causing the disease. Restoration of neuron function, augmentation of neurotransmitters for better nerve conduction, prevention of oxidative stress and

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inflammation are the goals of treatment therapy. Various herbal medicines are also available which are used for the treatment of nervous system related ailments since ages in different indigenous system of medicines of India and China like homeopathic system, herbal system and ayurvedic system. Although we have different therapies and medicines available for the treatment of AD but all are not so effective and provide only symptomatic relief, therefore, there a need of more research in this area to find out the better cure of AD. ABBREVIATIONS: ACE

Angiotensin Converting Enzymes

Ach

Acetylcholine

AchE

Acetylcholinestrase

AD

Alzheimer’s disease

AGT

Angiotensinogen

Ang I-IV Angiotensin I-IV APOE

Apolipoprotein E

ARB

Angiotensin Receptor Blockers

AT1R

Angiotensin 1 receptor

Aβ

Beta Amyloid

CCB

Calcium Channel Blockers

COX-1

Cycloxygenase-1

COX-2

Cycloxygenase-2

CRP

C Reactive Protein

LOAD

Late Onset Alzheimer’s disease

MAO

Mono Amine Oxidase

NMDA

N-methyl-D-aspartate

NSAIDs Non Steroidal Anti Inflammatory Drugs RAS

Renin Angiotensin System

ROS

Reactive Oxygen Species

TNF-α

Tumor Necrosis Factor alpha

CONFLICT OF INTEREST The authors declare no conflict of interest, financial or otherwise. ACKNOWLEDGEMENTS Declared none.

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CHAPTER 5

Biological Mass Spectrometry for Diagnosis of Alzheimer's Disease Hani Nasser Abdelhamid2,* and Hui-Fen Wu1,3,4,5,* Department of Chemistry and Center for Nanoscience and Nanotechnology, National Sun YatSen University, Kaohsiung, 70, Lien-Hai Road, Kaohsiung, 80424, Taiwan 2 Department of Chemistry, Assuit University, Assuit, 71515, Egypt 3 School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, 807, Taiwan 4 Institue of Medical Science and Technology, National Sun Yat-Sen University, 80424, Taiwan 5 Doctoral Degree Program in Marine Biotechnology, National Sun Yat-Sen University and Academia Sinica, Kaohsiung, 80424, Taiwan 1

Abstract: Mass spectrometry (MS) has advanced the diagnosis of Alzheimer's disease. In the present chapter, applications of mass spectrometry for the diagnosis of Alzheimer's disease were summarized. Mass spectrometry showed new exciting results, offered high sensitivity (in the femtomolar range), showed high selectivity, has better accuracy, offered high throughput, were extremely rapid (the entire process required few minutes) and can be used for quantitative, qualitative and imaging. Recent mass spectrometry techniques based on nanotechnologies replaced some of the classical MS techniques. These new technologies improved the diagnosis of Alzheimer's disease. Mass spectrometry covered wide range of Alzheimer's disease biomarkers such as amyloid β, total tau protein (t-tau), α-synuclein, posttranslational modification (phosphorylated tau protein, protein S-nitrosation (SNO), racemization, methylation, chlorination and others) and metals ions. From the analytical point of view, mass spectrometry offered detection of large number of biomarkers in a single test. Mass spectrometry has significantly advanced Alzheimer's diagnosis of living patient and postmortal. Monitoring Alzheimer's biomarkers using MS is very promising for the diagnosis in early stages of the disease. However, the proper interpretation of MS profiling is critical and requires careful investigations. Furthermore, the identification of the biomarkers using MS profile is affected by many key variables that have to be considered during the analysis. Corresponding author Hani Nasser Abdelhamid: Department of Chemistry, Assuit University, Assuit, 71515, Egypt; Tel: 00201279744643; Fax: 0022342708; E-mail: [email protected], [email protected]; Hui-Fen Wu: Department of Chemistry and Center for Nanoscience and Nanotechnology, National Sun Yat-Sen University, Kaohsiung, 70, Lien-Hai Road, Kaohsiung, 80424, Taiwan; Tel: 886752520003955; Fax: 88675253908; E-mail: [email protected] *

Atta-ur-Rahman (Ed.) All rights reserved-© 2017 Bentham Science Publishers

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Keywords: Alzheimer's disease, Amyloid β, Biomarkers mass spectrometry imaging, Mass spectrometry, Tau protein. INTRODUCTION Mass spectrometry (MS) is an attractive and invaluable analytical technique that can be applied for wide analytes [1, 2]. Mass spectrometry (MS) measured the mass to charge ratio (m/z) of ions to identify and quantify the target molecules. It has been applied for many fields such as proteomics [3, 4], metabolomics [5], biology [6], nanotoxicology [7 - 10], and others [11 - 14]. It has advanced the field of molecular medicine and provided revolution in the diseases diagnosis. It has many subclass based on ionization methods. Thus, these techniques provided a practical analyzer for biomarkers, diagnosis and screening of many diseases. Mass spectrometry potentially outperformed the other traditional methods [15 - 17]. Mass spectrometry (MS) was used in diagnosis and screening for Alzheimer's disease [17 - 20], heart disease [21], inherited metabolic diseases [22], newbornscreening programs [23], inborn errors of metabolism [24], heart diseases and clinical proteomics [25], diabetes mellitus [26], and others [27 - 29]. Mass spectrometry is potentially promising in clinical chemistry for identification of disease's biomarkers [30]. Disease biomarkers can be identified by mass spectrometry analysis. The analysis can be in combination with separations techniques and identification is simple by using fingerprinting (Peptide Mass Fingerprinting, PMF) or peptide sequence tag (PST). Database of protein, peptide and other biomolecules biomarker can be used for further identification and confirmation. Alzheimer’s disease is dementia type disease that belongs to neuropathological and neurodegenerative disorder affecting >5% of the population over the age of 65. Alzheimer’s disease affects the patient's memory, language, thinking, mood, and behavior (difficulty speaking, confusing about events, and walking). Alzheimer's disease is mainly pathological alterations in the brain of patients due to unknown reasons. It may be due to β-amyloid deposition and hyperphosphorylation of τ protein [31], oxidative stress [32], mitochondrial dysfunction [33], metal dyshomeostasis [34], and lipid dysregulation [35]. The main challenge of this disease is that their symptoms usually develop slowly. The symptoms become worse over the time and are enough to affect the daily tasks. Thus, early diagnosis of the disease is highly demanded. Among the different analytical techniques, mass spectrometry is promising for Alzheimer's disease diagnosis.

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This chapter discussed the applications of mass spectrometry for the diagnosis of Alzheimer's disease. The requirements of the diagnosis in the early stages were discussed. The recent achievement of the disease diagnosis using mass spectrometry was reviewed. The examples cited here highlighted the contribution of mass spectrometry for Alzheimer's disease. Mass spectrometry offered several advantages such as fast diagnosis, high sensitivity, high selectivity, accurate and are easy to combine with other separation techniques. Requirements of Alzheimer's Disease Diagnosis There are several requirements for diagnosis and screening of Alzheimer's disease. The analysis should be (i) fast to analysis many organs, tissues and body samples in a short time; (ii) offer high accuracy to avoid errors and misconclusion; (iii) have high sensitivity to detect the disease in the early stages; (iv) offer high selectivity toward the target biomarker to give clear indication without any confusion; (v) sample preparation should show minimum loss of the biomarker or cause no artefacts; (vi) provide high resolution in order to analysis complex and real sample such as body fluids, organs or tissues; (vii) sample pretreatment such as preconcentration or separation method should be simple; (viii) the device should be simple to handle, easy to clean and can be recondition fast for next measurement and (ix) interfering species cause no effect on the separation procedure. Among different analytical techniques, mass spectrometry fulfilled almost all the previous criteria as discussing in this chapter. Thus, it has been applied for many diseases such as Alzheimer's disease. Mass spectrometry consists of five parts as shown in Fig. (1); sample inlet, sample analyzer, mass analyzer that separate ions based on m/z, detector and vacuum system [36 - 41]. The investigated species are ionized in the mass analyzer before the separation based on mass to charge in the analyzer. The ionized species are detected in the detector and a plot of the intensity versus the mass to charge ration is obtained. To avoid the lost of the ions charge, vacuum is used. Analyte Insertion

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Fig. (1). Mass spectrometry consists of five parts; sample inlet, ion source, mass analyzer, detector and high vacuum.

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Application of Mass Spectrometry for Alzheimer's Disease Numerous hypotheses were proposed to explain the neurodegenerative mechanisms that occur in AD. Alzheimer's disease could be due to overexpression of the mutant proteins, Aβ, tau, and α-synuclein [42]. The disease disorder of the protein begins in specific regions of the brain. Then, it spreads to other areas in the patient brain. The appearance of amyloid (neuritic) plaques and neurofibrillary tangles in the brain is a characteristic feature of Alzheimer's disease. These plaques cause of progressive intellectual failure in aged human [43]. The filamentous lesions that define AD occur within neurons (neurofibrillary tangles), in extracellular cerebral deposits (amyloid plaques), and in meningeal and cerebral blood vessels (amyloid angiopathy) [44]. The amyloid β (Aβ) protein with a molecular weight of c.a. 4,000 Da is the subunit of vascular and plaque amyloid filaments for AD [45, 46]. A simple approach for shotgun mass spectrometry was reported based on the labeling using isotope dimethyl labeling for the brain proteome in the temporal neocortex (Fig. 2) [19]. Authors identified and quantified 827 unique proteins between Alzheimer’s disease (AD) patients and non-AD individual. Extraction

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Lewczuk et al. investigated cerebrospinal fluid (CSF) from patients with AD and nondemented control subjects using surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) [47]. They observed three amyloid beta (Aβ) peptides with molecular masses of 4525.1 Da, 4846.8 Da, and 7755.8 Da besides the well-known Aβ. Authors noticed that the signal to noise ratio of two known AD biomarkers, Aβ1-40 and β1-42, were significantly decreased and unaltered in AD, respectively. These specific changes in the expression of peptides in the cerebrospinal fluid (CSF) were mainly used as indications in Alzheimer’s disease [48]. Carrette et al. [49] used surface-enhanced laser desorption/ionization (SELDI-MS) to describe the evaluation of biomarkers for Alzheimer’s disease as shown in Fig. (3). They reported five polypeptides (13.4, 11.78, 11.98, 4.82 kDa) as biomarkers for the disease. They also observed differential expression of these biomarkers. Mori et al. reported that Aβ1-40 is the major species of Aβ protein in AD cerebral cortex [50]. Alzheimer's disease may be due to the oxidative stress of proteins, lipids or DNA. Lyras et al. investigated the oxidative analysis of proteins, lipid or DNA from seven different brain areas [51]. They observed no difference in lipid oxidation, but they noticed an increase of the protein carbonyls and DNA oxidation in frontal, occipital, parietal, temporal lobe, middle temporal gyrus and hippocampus. They found a significant difference in the parietal lobe [51]. Smith et al. reported the protein oxidation products (carbonyl) and the activities of two enzymes; glutamine synthetase and creatine kinase for postmortem frontal and occipital-pole brain samples [52]. They observed that the content of carbonyl increase exponentially with age. They noticed that the rate is duplicated in the frontal pole compared to the occipital pole. In other side, they found that in young patients both aged groups (AD and age-matched controls) have higher carbonyl content and lower activities for glutamine synthetase and creatine kinase activities. These results indicate that protein oxidation products were accumulated in the brain and thus decrease of the enzyme activities (glutamine synthetase and creatine kinase) [52]. In the same context, the analysis of biomacromolecules bound carbonyls at hippocampal tissues of Alzheimer's disease patients and controls using immunocytochemical technique showed that carbonyls were increases in neuronal cytoplasm and nuclei of neurons and glia [53]. Analysis of Alzheimer's disease (AD) biomarker using electrospray ionization mass spectrometry (ESI-MS) was reported. Analysis of the alterations in ethanolamine plasmalogen in cellular membranes from different regions of human body showed a dramatic decrease in plasmalogen content (up to 40% (mol) of total plasmalogen) [54]. Data showed a correlation of the deficiency in gray matter plasmalogen content with the AD clinical dementia ratings (CDR) CDR 10% (mol) of deficiency at CDR 0.5 (very mild dementia) to 30% (mol) of

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deficiency at CDR 3 (severe dementia). There is an absence of alterations of plasmalogen content and molecular species in cerebellar gray matter at any CDR despite dramatic alterations of plasmalogen content in cerebellar white matter [54].

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Fig. (3). (A) SELDI-TOF-MS protein profiles of CSF samples from two different AD patients (AD1-AD2) and two controls (C1-C2), and (B) Average intensities of the five significant (p < 0.05) clusters differentially expressed between the two groups. Figure reprinted with permission from [49]. Copyright permission belongs to WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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The alteration of tau protein is another hypothesis that may explain causes of Alzheimer's disease [55, 56]. Tau protein is abundant and soluble microtubule associated protein (MAP) in neurons of the central nervous system. Tau protein stabilizes microtubules by the interaction between its isoforms and phosphorylation tau protein. The defective and no longer stabilize tau microtubules properly cause pathologies and dementias of the nervous system i.e. Alzheimer's disease and Parkinson's disease. Furthermore, excessive or abnormal phosphorylation of tau protein caused to PHF-tau (paired helical filament) and NFTs (neurofibrillary tangles) can be pathology of Alzheimer's disease. Hanger et al. solubilized and purified PHF-tau using Mono Q chromatography (anion exchanger packed with MonoBeads in a Tricorn column) and reversed-phase high performance liquid chromatography (HPLC) [57]. The purified protein was subjected to proteolysis digestion and phosphorylation sites were analyzed using nanoelectrospray mass spectrometry. Authors recorded and identified 22 phosphorylation sites in PHF-tau [57]. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSA-PAGE) was used to purify tau proteins from Sarkosyl insoluble pellet of brain homogenates [58]. Data revealed that the peptide maps of PHF-tau and normal tau before and after dephosphorylation showed three anomalously eluted peaks which contained abnormally phosphorylated peptides, residues 191-225, 226-240, 260-267, and 386-438 (numbering is sorted according to the longest tau isoform) [59]. Mass spectrometry was used for the analysis of the protein sequence localized Thr-231 and ser-235 as the abnormal phosphorylation sites of tau protein. Data revealed that each tau 1 site (residues 191-225) and the most carboxyl-terminal portion of the protein (residues 386-438) carries more than two abnormal phosphates [60]. Authors observed that Ser-262 was the phosphorylated fraction of PHF-tau. They noticed that not only phosphorylation, but also modifications were occurred. The protein undergoes removal of the initiator methionine, and Nu-acetylation at the amino terminus and deamidation at 2 asparaginyl residues were found in PHF-tau in normal tau [60]. Abnormal posttranslational modification of protein is another pathway for neurodegeneration. A chemical proteomic strategy for the identification of protein S nitrosation (SNO protein) based on SNO trapping by triaryl phosphine (SNOTRAP) followed by mass spectrometry was reported [61]. Nitrosylation of cysteine residue using SNOTRAP specified 313 endogenous SNO sites in 251 proteins in the mouse brain. Author observed 135 SNO proteins only during neurodegeneration. Mass spectrometry was also used to cover other posttranslational modification such as chlorination [62], lysine methylation [63], racemization and isomerization of N-terminal amyloid β [64] and others [65]. Mass spectrometry provided fast, simple and simultaneous detection of several modification in single test.

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Fig. (4). Schematic representation of Laser ablation electrospray ionization (LAESI-MS) (top) Isotope peak distributions (down) of (a) [hIAPP+4H]4+, (b) [hIAPP + Cu(II) + 2H]4+, (c) [hIAPP + 3H]3+, and (d) [hIAPP + Cu(II) + H]3+. Images were reprinted with permission from [68]. Copyright permission belongs to American Chemical Society (ACS).

Brain homeostasis of transition metals such as zinc (Zn (II)), copper (Cu (II)), aluminum (Al (III) and iron (Fe (III), Fe (II)) has an effect in Alzheimer's disease

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(AD) [66]. It was reported that zinc and copper extracellular pooling in amyloid and intraneuronal accumulation of iron. The role of metals in AD and metalloproteomics using analytical techniques such as mass spectrometry were recently reviewed [67]. Laser ablation electrospray ionization mass spectrometry (LAESI-MS) combined with ion mobility separation (IMS) (LAESI-IMS-MS) was used to probe amylin–copper (II) interactions as shown in Fig. (4) [68]. Data revealed that copper (II) ions disrupt the association pathway to the formation of β-sheet rich amylin fibrils. Electrospray ionization mass spectrometry (ESI-MS) was used for metal-peptide complexes [69]. Aluminum ions were also reported particularly for its possible role in the etiology of Alzheimer’s disease [16, 60, 70]. Aluminum ions cause accumulation relatively with high quantities in the brains of Alzheimer's disease patients [70, 71]. Other technique such as flame atomic absorption spectrometry (FAAS) was also used for the detection of trace levels of aluminum (Al (III)) in scalp hair samples of Alzheimer’s (AD) patients [72]. Data support the proposal effect of Al (III) on Alzheimer's disease. Authors observed a significant higher level in scalp hair samples of AD male patients [72]. Recently (2016), the screen of mouse brain lysates showed that synaptotagmin 1 (Syt1) is Ca2+-sensitive Alzheimer's disease-associated presenilin 1 (PS1) [73]. The results indicated the importance of Sty1 as Ca2+-sensitive PS1 modulator that may regulate synaptic Aβ. Application of direct infusion electrospray mass spectrometry as global vision for Alzheimer's disease was reported [17]. Direct infusion electrospray mass spectrometry provided information about membrane destabilization processes, oxidative stress, hypometabolism, or neurotransmission alterations [17]. Imaging Mass Spectrometry for Alzheimer's Disease Mass spectrometry imaging (IMS) visualize the spatial distribution of chemical compositions e.g. compounds, biomarkers, metabolites, peptides or proteins in organs or tissues according to the molecular masses (mass to charge, m/z). IMS offers clear distribution without ambiguity, is fast and is easy approach for the analysis of metabolite and biomolecules distributions within whole-body tissue simultaneously [74]. IMS techniques were used to study the role of Cu [75] (Fig. 5), Al, Be, Cd, Co, Cr, Hg, Mn, Ni, Pb and V [76] for Alzheimer's disease. Advantages and disadvantages of Mass Spectrometry Mass spectrometry offered many advantages for the diagnosis of Alzheimer's disease. It provided fast analysis (< 5 min), offered high throughput [77], are easily combined with other techniques to reduce the system complexity [78], are cost effective, can be automatized [79], offered high sensitivity compared to other techniques, and showed high selectivity and better accurate. Mass spectrometry is

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easy to combine with other separation techniques such as high-performance liquid chromatography (HPLC), gas chromatography (GC) and capillary electrophoresis (CE) [80]. These combination were coined as hyphenated techniques. These combination has the ability to resolve isobaric compounds and offered reliable quantification, as well as improved identification of metabolites through MS/MS experiments and prediction of retention/migration times [81]. They also offered multi-analysis for several species simultaneously. For instance, CE-MS can be used for the analysis of choline, creatinine, asymmetric dimethyl-arginine, homocysteine-cysteine disulfide, phenylalanyl-phenylalanine, acylcarnitines, asparagine, methionine, histidine, carnitine, acetyl-spermidine, and C5-carnitine. The technique can also differentiate between the species that increase (choline, creatinine, asymmetric dimethyl-arginine, homocysteine-cysteine disulfide, phenylalanyl-phenylalanine, and different medium chain acylcarnitines) and species that decreased (asparagine, methionine, histidine, carnitine, acetylspermidine, and C5-carnitine) [80]. Mass spectrometry showed also high selectivity using immunoproteomic assay that employs monoclonal antibodies (mAbs) on Preactivated Surface (PS20) chip array [82]. Fe

Zn

Mn

Subchronic MPTP 7d 2h 28 d

Control

Cu

0

mg g-1

10

0

mg g-1

10

0

mg g-1

30

0

mg g-1

0.4

Fig. (5). Quantitative metal images of Cu, Zn, Fe, Mn representative of each group (control, 2 h, 7 d, and 28 d after the last of five daily 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin (MPTP) injections). Figure reprinted with permission from Reference [75]. Copyright permission belongs to American Chemical Society (ACS).

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However, mass spectrometry suffers from disadvantages such as; 1) protein analysis requires often other technique such as separation or preconcentration to reduce the sample complexity or to increase the analyte concentration; 2) sometime, expensive technique such as stable isotope dilution [83], or antibodies are required; 3) the large dynamic range of the protein analyte requires high sensitivity and certain precaution; 4) interference due to the combination with other technique such as gas chromatography mass spectrometry that cause extra oxygen oxidation of DNA oxidation leads to misconclusion [84 - 85]; 5) protein proteolysis may cause artifactual effect in the data analysis [50]; and 6) low throughput of the separation technique decrease the productivity of MS. Thus, new separation techniques are highly required to avoid the major drawback such as the low sample throughput for the conventional separation methods. It is important to stress that there are many parameters such as biomarker types and concentration, type of matrix (CSF and plasma), analytical technique and data interpretation should be considered for better analysis of Alzheimer's disease [86]. CONCLUSION Mass spectrometry (MS) showed significant effect in the diagnosis of Alzheimer's disease. Mass spectrometry offered better analysis for amyloid, tau protein, metals, metalloprotein and other biomarkers. The technique was easy for sample preparation, provided high throughput analysis, showed high sensitivity, provided high accuracy and was able to combine with other techniques. Techniques based on mass spectrometry are promising for the discovery of early diagnostic biomarkers of Alzheimer's disease. Mass spectrometry is easy and are able to extend to real investigation in hospitals and clinical chemistry of Alzheimer's disease. CONFLICT OF INTEREST The authors declare no conflict of interest, financial or otherwise. ACKNOWLEDGEMENTS H.F. Wu is grateful to the Ministry of Science and Technology of Taiwan for financial support. H.N. Abdelhamid thanks Assuit University and Ministry of Higher Education, Egypt for the support. REFERENCES [1]

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CHAPTER 6

The Structure-Activity Relationship of Melanin as a Source of Energy Defines the Role of Glucose to Biomass Supply Only, Implications in the Context of the Failing Brain Arturo Solís Herrera* Human Photosynthesis® Research Center, Sierra del Laurel, 212, Bosques del Prado Norte, CP 20127, Aguascalientes, México Abstract: Decreasing brain metabolism is a substantive cause of cognitive abnormalities in Alzheimer´s Disease (AD), although this hypo-metabolism is poorly understood, i.e. is not known if it is primary or secondary. Neuron ion homeostasis and thereby synapsis are a crucial and highly energy demanding processes, and one of the hallmarks of AD is the loss of synapsis in defined regions of the brain. Until today, alterations in mitochondrial energy supply have been considered the main concern due to in aging rat neuron model, mitochondria are both chronically depolarized and produce more reactive oxygen species with age. Thereby, impoverished mitochondrial function has been actively studied trying to reverse and recover ATP generation. Today, after more than 100 years that Alois Alzheimer described Augusta D., patients still die in the same way, in spite multiple treatments, multiple theories, multiple studies and unfruitful clinical trials. We believe that the unraveling of the unsuspected intrinsic property of melanin to transform visible and invisible light into chemical energy through the dissociation of the water molecule, as chlorophyll in plants, will mark a before and after, this is: a new frontier, in the understanding and treatment of the nightmare of the XXI century: Alzheimer´s Disease.

Keywords: Alzheimer, Energy, Hydrogen, Light, Melanin, Neurodegeneration, Synapsis. INTRODUCTION Alzheimer´s Disease is characterized by a progressive deterioration of cognitive function with memory loss. The most affected regions of brain in AD include the Corresponding author Arturo Solís Herrera: Human Photosynthesis® Research Center Sierra del Laurel, 212, Bosques del Prado Norte, CP 20127, Aguascalientes, México; Tel/Fax: +524492517232; E-mail: [email protected].

*

Atta-ur-Rahman (Ed.) All rights reserved-© 2017 Bentham Science Publishers

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basal forebrain, amygdaloidal body, hippocampus, entorhinal cortex neocortex, and brain stem nuclei [1] (Fig. 1). Most cases are sporadic with no known genetic linkage. Basal Forebrain

Amygdala

Hippocampus

Entorhinal Cortex

Neocortex

Brainstem nucleus

Fig. (1). Arrows show approximate location of the brain tissue that seems especially affected in AD, functional and anatomically.

Despite the many existing histopathological descriptions to date, the cause of Alzheimer's Disease remains in the incognito [2]. The presence of extracellular βamyloid peptide-containing neuritic plaques, intracellular neurofibrillary tangles (NFT) and the loss of synapses in more or less defined regions of the brain are the hallmarks associated with AD in post-mortem pathology. Amyloid (starch-like) deposits contain extremely insoluble protein fibrils with similar morphologic features with many, if not all, neurodegenerative disorders [3]. These 80-150 Å length fibrils comprise many different proteins with no obvious sequence similarity. Abnormal protein aggregation characterize

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Alzheimer´s Disease (AD), Parkinson´s Disease (PD), Creutzfeld-Jakob Disease (SP, Spongiform Encephalopathy, prion protein deposits), Motor Neuron Diseases, the large group of polyglutamine disorders (tri-nucleotide repeat diseases), including Huntington´s Disease, as well as diseases of peripheral tissue like Familial Amyloid Polyneuropathy (FAB) [4], Amyotrophic Lateral Sclerosis, and Tautopathies (Progressive Supranuclear Palsy, Pick´s disease, corticobasal degeneration, Familial Frontotemporal Dementia, Parkinson-linked to chromosome 17). Abnormal protein-protein interactions that result in the formation of intracellular and extracellular aggregates of proteinaceous fibrils are a common neuropathological feature of several neurodegenerative diseases. It has been suggested that abnormal protein-protein interactions and/or the lesions that result from the aggregation of these proteins could play a mechanistic role in the dysfunction and death of neurons in several common (and rare) neurodegenerative diseases. Lewy bodies (LB) are intracytoplasmic neuronal inclusions observed very frequently in PD, however, they also occur commonly in the brains of patient with clinical and pathological features of AD. Numerous cortical LBs are found in Dementia with Lewis bodies (DLB), which is similar to AD clinically, but pathologically distinct NFTs and senile plaques (SPs) are rare or completely absent in DLB brains. The precise molecular composition of LBs is unclear, also their role in the degeneration of neurons in PD, and DLB. Synuclein was identified in rat brain in 1991, subsequently, a fragment of the 140 amino acid long human α-synuclein protein was reported to be present in some amyloid plaques of AD brains. The normal functions of α-synuclein in neurons are poorly understood. The biochemical changes that predispose this normally soluble and randomly structured α-synuclein protein to aggregate or interact aberrantly with itself or other proteins, are unknown. The widespread presence of α-synuclein in perikaryal LBs, and in dystrophic neuronal processes of brains of patients with PD and DLB, and immunohistochemical studies with antibodies to α-synuclein reveal a much more extensive network of dystrophic processes, suggesting a generalized failure more than a punctual alteration. The state of the art in relation to pathological findings and the clinical picture in AD, PD and other neuro-degenerations are has become so intricate, that even is has failed to discern if the correlation and co-location of fibrillar proteins and the affected tissue suggests that fibrillization contributes to cell death or if it is an

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inseparable epiphenomenon. But before proceeding with more analysis of the numerous and complex biochemical changes described in the literature about neuronal degeneration, I think it would be more productive to focus on what can be considered a generalized failure of cell biochemistry. And it is that widespread failure is characteristic of energy, and this in any system, not just eukaryotic cell. In the light of the discovery of the unsuspected inherent capacity of melanin transform the visible and invisible light to chemical energy through the dissociation of the molecule of water, such as chlorophyll in plants; glucose as energy source hitherto dogmatic role calls into question. In short, the chemical reaction that happens inside melanin [5] is this one: 2H2O(liquid) → 2H2(gas) + O2(gas) → 2H2O(liquid) + 4e-

This is: melanin is able to dissociate the water molecule, and astonishing also is able to support the opposite reaction: the back-bonding of the molecule, and for each 2 molecules of re-formed water, then 4 high energy electrons are generated. In chlorophyll the reaction is irreversible, due to oxygen is expelled to atmosphere. 2H2O(liquid) → 2H2(gas) + O2(gas)

Thereby, chlorophyll cannot re-form the water molecule, so water must be applied frequently to plant´s leaves to replenishment of the substrate. Melanin, to dissociate and re-shape the water molecule, makes our body highly efficient in the management and use of water, because otherwise, our daily water needs would be enormous. Therefore, it is convenient to redefine the functions of the two main components of the CNS: cerebrospinal fluid (Fig. 2) water as a substrate in cellular bio-energy processes, and the seemingly passive role of the neuro-melanin (Fig. 4). An old concept that also must be modified is that eukaryote cells are energy independent (Fig. 3), because so far they considered as glucose-dependent. So what Physiology seemed to revolve around blood vessels, but the generation and distribution of energy from the melanin, it is conceptualized differently by the way in which melanin releases chemical energy (Fig. 5), this is: as growing spheres.

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Fig. (2). Magnetic resonance images allowed us to understand some things, and raise doubts in others. According to our finding that eukaryotic cell is able to take energy from the visible and invisible light, through the water molecule dissociation; thus, the purpose of brain grooves would be to allow that the neuron is in direct contact with its source of energy: water of CSF. On the other hand, the neuro-melanin substantia nigra, is possible to appreciate it in MRI, located immediately above pons.

Fig. (3). The drawing comes to illustrate the concept that we are formed by cells energetically independent, as each one, without exception; it has the amount of melanin adequate to supply the chemical energy (H2) required so that each cell can carry out their functions, according to the shape and location. In the case of the Central Nervous System (CNS), the anatomy is adapted so that any neuron can be in constant and direct contact with their main source of energy: water of the cerebrospinal fluid.

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Substantia Nigra

Fig. (4). The location of the substantia nigra is strategic, given that melanin releases energy in symmetric form, in all directions.

Fig. (5). The yellow sphere represents the approximate area where the tissue damage (both functional and anatomical) is observed in AD. Neuro-melanin is especially abundant in substantia nigra (pars compacta), and locus ceruleus.

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The role of hydrogen as an energy carrier, according to as it does in the universe, changed radically the current concepts about the bio-energetic processes of eukaryotic cells [5]. Currently, explanations that tries to explain the metabolism of the glucose, have numerous inconsistencies (Fig. 9), because any molecular change requires energy, and they are not usually considered all the changes that happens only in glucose molecule itself. Aggregation of proteins is easily visible in other parts of body when metabolism is altered. A good example is the lens of the eye. It has no blood capillaries, so the aqueous humor is responsible of the nutrition of the lens and the disposal of metabolic products. So far, is believed that the energy necessary for the lens is provided mainly through anaerobic glycolysis, the Krebs cycle, located in peripheral cells, only provide about 5% of the necessary energy. Pentose phosphate cycle is also another important metabolic pathway in lens since it provides NADPH necessary for the maintenance of the redox status of the lens proteins. The majority of the proteins in the lens are alpha, beta and gamma crystallines. They should maintain transparent environment, so they should be in a native, nonaggregate state. Some disturbances, as changes in the redox states of these proteins or changes in osmolarity in the lens can produce loss of the native state and aggregation of these proteins. Thereby, cataracts result from changes in solubility and aggregation of the crystalline proteins. However, the lens is immersed in water and surrounded by densely pigmented tissues, the iris and choroid (Fig. 6). The proteins that normally must not be aggregated, are in the cornea, the lens and the vitreous body. The chemical energy required to keep the transparency comes from melanin which is located in the iris, ciliary body and choroid layer, and its concentration is usually 40% more than the skin. The vicinity of densely pigmented tissues to the lens, is not taken into account, currently from the metabolic point of view, but with the discovery of unsuspected intrinsic property of melanin transform light energy into chemical energy by means of the dissociation of the molecule of water, such as chlorophyll in plants; then we can infer that the transparency of the surrounding tissues, these are: cornea [6], lens and vitreous humor, it reflects a stages of high energy, both in humans and mammals and other species that have the equivalent to the human eye.

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Sclerae

Cornea

Vitreous Body Pupillary Space

Iris Choroidal Layer Ciliary Body

Fig. (6). The yellow sphere represents the approximate area where the tissue damage (both functional and anatomical) is observed in AD. Neuro-melanin is especially abundant in substantia nigra (pars compacta), and locus ceruleus.

We may think that power requiring transparent tissues to maintain transparency, is supplied by the tissues so pigmented that surround them (Fig. 7), and the glucose that comes from blood vessels in the choroid, is used by tissues only as building blocks. In other words: transparency is a high energy state. Growing spheres of Energy that comes from melanin.

Fig. (7). The melanin in the eye, had assigned the role to absorb excess light that penetrates the eye, so that the images were of better quality, avoiding internal reflections, but a so passive role is far from their true function, which is to transform the visible and invisible light into chemical energy, by means of the water molecule dissociation; as the chlorophyll in plants.

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The choroid layer of the eye, as part of uveal tract, from the Greek -grape color -, has a provision found in other parts of the body and the CNS (choroid plexuses). The choroid typically presents an enormous amount of tiny blood vessels, surrounded by densely pigmented cells; and its function is similar in wherever you are, i.e.: in the eye, ciliary body is the production of aqueous humor, and in the CNS, in the choroidal plexuses, the production of cerebrospinal fluid. But the capacity of melanin to dissociate and reform the water molecule, allows us to assign a new function to the ciliary body and choroid plexus: the production and resorption of water, then to dissociate it produces chemical energy that is transported by molecular hydrogen (H2), but the re-form it produces liquid water and high energy electrons (e-). The presence of numerous vessels in the choroid and ciliary body can be explained by the need of tissue of molecules that can be used as building blocks of the biomass, being the main carbohydrates; as well as the handling of CO2 mostly. One subtle proof that both the production and resorption of cerebrospinal fluid and aqueous humor are processes that rely on light energy is located in the observed fact of the variations that occur throughout the day both in the aqueous humor and cerebrospinal fluid. In the case of the aqueous humor, the maximum production of aqueous humor is around six in the afternoon, the minimum is about 5 in the morning; and in relation to the mechanisms of absorption of aqueous humor, which also depends on the light, they follow a similar variation, is the minimal reabsorption in the morning 5 am-, and the maximum reabsorption is around six in the afternoon. The cell uses energy in many ways, and the organs and the body also. In the specific case of the eye, which contains three important elements whose function depends on the transparency thereof, namely: cornea, lens, and vitreous body. The energy that emanates from the melanin in the form of molecular hydrogen and high energy electrons, tissues and cells, use they for many things, but one of them is keep the form, and in the case of the eye, to maintain transparency [7]. I.e., maintain the structural elements in its optimum state for its function, because when proteins tend to aggregate themselves, the function is impoverished. It is conceivable that similar things happen in other organs, such as CNS (Fig. 8). In the case of the structures of the CNS, is would not speak of transparency, but instead of a precise, exact, anatomical provision both macro how microscopic.

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Fig. (8). Blue sphere represents the approximate area that molecular hydrogen coming from neuro-melanin, would cover in a consistent fashion. H2 emanates mainly from pigmented structures as substantia nigra, and locus ceruleus, however melanin is also placed in choroidal plexuses, meninges, etc.

This is: both the organellos and the molecules inside the cell, are placed in an accurate, exact, which by no means is random; and it is determined from the beginning, since the beginning of time for the generation and distribution of energy that comes from the melanin (Fig. 9). The yellow and blue spheres can be overlapped, as an example of what happens in the normal patient, i.e.: the usual generation of molecular hydrogen by melanin properly covers the energy needs of the structures of the CNS, this is: both spheres have approximately the same size, the same volume, thereby the CNS structures will not show damage of some kind, neither functional nor anatomic. But if melanin is damaged, for example, metals, pesticides, herbicides, anesthetic agents, drugs, ageing, cold, plastics, repetitive head blunt trauma -boxers, football players- etc., then the transformation of visible and invisible light into chemical energy through the dissociation of the molecule of water, such as chlorophyll in plants, will be impoverished, and the blue sphere will be smaller than the yellow sphere, which will result in structures within the yellow area displayed morphological changes consistent with low chemical energy levels. When the levels of chemical energy from melanin turn down suddenly, i.e. head trauma, intoxication by drugs, and toxic fumes, tissues will show edema and hemorrhage, however, when the chemical energy levels are diminished in a chronic form,

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tissues will show protein aggregation that in liver is called cirrhosis, and in brain dementia, and some cases will show unorderly mitosis.

Fig. (9). The blue field represents molecular hydrogen that is released by the melanin under pathological circumstances, so cannot cover adequately needs of tissues usually affected in AD (yellow sphere). When the energy that carries the hydrogen is not sufficient to meet the energy needs of the surrounding tissues, then cells fails in a generalized way and is manifested by malfunctions of various types, as well as by alterations in the morphology of the cells, for example, the accumulation of substances that normally cell handles well, i.e.: beta amyloid.

It is conceivable that the decrease in the generation and distribution of energy that comes from the melanin (Fig. 10) causes a fault in the operation of the neuron, which manifests itself in various ways both clinical and histologic. And our limited knowledge and exploration tools limit further delve into them. Is it unavailable methods histological that they will allow us to demonstrate each and every one of the biochemical alterations that occur in the neuron, we can even measure the molecular hydrogen. That is why that has not had detected energy requiring eukaryotic cell is transported by molecular hydrogen, because currently there is no practical or easy way to measure or at least detect it. The limited descriptions of neuronal biochemical alterations in AD patients, reflect a widespread failure; this has not been possible to concatenate them and form an understandable layout. Thereby, it is not rare, that the underlying cause for selective neuronal loss in AD remains unclear, being a significant obstacle in the understanding and managing

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of disease. Several hypotheses, mainly around β-amyloid (starch-like) accumulation, intracellular neurofibrillary tangles, and the loss of synapses; comprising misfolded proteins, Ubiquitin-proteasome system dysfunction; excitotoxic reactions, oxidative and Nitrosative stress, mitochondrial failure, synaptic failure, altered metal homeostasis; tau phosphorylation, and misoperation of chaperones [8], hypotheses all, not have been able to develop a therapeutic strategy successful, or at least slowing, so far, the inexorable progression of the disease.

Fig. (10). Exact differences from the biochemical point of view between the melanin in the iris and the neuro-melanin in the CNS does not have been firmly established, because melanin is a highly complex molecule, which is almost impossible to study in the laboratory. But expected their function is the same where want to that is find: generation of chemical energy through the dissociate of the water molecule.

There is enough evidence that these aspects all, are associated with AD, and probably play a role in the disease process as whole; however, no single one of these theories is sufficient to explain the spectrum of abnormalities found in the disease. Basal Brain Energy Metabolism In normal brain, the energy metabolism homeostasis requires (theoretically), production and delivery of energy-rich phosphoryl and NAD+ oxidizing power [9]. Until now, it is considered that mitochondria contribute ≈ 90% of the required energy for cellular functions [10]. Apparently, the brain has low levels of stored glycogen, and thereby is highly dependent on oxidative metabolism. The basal rate of glucose utilization in astrocytes is higher than in neurons [11], but in light of finding of melanin as source of energy, then astrocytes glucose utilization is for biomass synthesis mainly. Concept as glucose is the obligatory energy substrate for the brain [12] seems as out of discussion, however, glucose, in spite to be an

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extraordinary molecule, it’s not able to provide the energy required for its own metabolism (Fig. 11). O

Cytosol

Pyruvic acid from glycolysis OH

O

Mitochondrial outer and inner membranes O

Acetic acid

OH

Matrix

NH2 O

Acetyl CoA

S

N H

O

O N H

O

P

HO O P O O

N O

O OH

OH

HO HO

P

N

N N

O OH O

O

O O

H 2O

OH

HO O

Oxaloacetic acid

OH OH

HO

O HO

Malic acid

O

OH O

Citric acid OH

OH

O

HO O HO

Fumaric acid

OH

Isocitric acid

O HO

OH

OH O

O

Alpha-ketoglutaric O acid

O

O

HO

OH

HO

Succinic acid

O

OH O

O -ooc

S O

Co A

Succinyl CoA

Fig. (11). The diagram shows the already described biochemical steps of the Krebs cycle, which was first published in 1950. Yellow stars we add to highlight changes that suffers molecules and by natural reason are energy-required processes, since this is defined as that which produces a change. The stars are for the attention that in the bioenergetics of the cell, currently are not taken into account numerous stages that indeed require energy, i.e. in the case of cell membrane, to keep the shape. This is: chemical energy is required to maintain the form and function.

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Fatty acids, ketone bodies, acetoacetate, β-hydroxybutyrate, pyruvate, and lactate, are molecules supposedly involved in energy production; but they are, in reality just useful metabolic substrates for biomass replenishment aims, and only if formed inside brain parenchyma [13]. Besides that, their formation, maintenance and metabolism depend completely of the generation and distribution of energy that comes from melanin (Fig. 12).

Fig. (12). Outline represents the cell membrane in orange, the cell nucleus and the nucleolus in blue, the black points that surround the nucleus are granules of melanin (melanosomes) in its usual location, the perinuclear space; from which emanates the chemical energy in the form of growing areas that are spreading throughout the cell cytoplasm. In single figure represent growing areas of energy of a single granule of melanin (melanosomes), but the case for each of the granules.

Mitochondrial electron transport is far to be perfect. Even under ideal conditions, there is superoxide anion production and other reactive oxygen nitrogen species; further, these deleterious processes can increase significantly with aging. Mitochondria are positioned within axons, dendrites, and synaptic terminals; supposedly to provide ATP, oxidizing power by means of NAD+, and calcium buffering for these compartments. Synaptic areas, especially dendritic spines, are the sites where the neurodegenerative process occurs early in AD [14]. However, based in these deep rooted concepts, it is difficult to explain why synapses are the sites where the neuro-degenerative process occurs early in AD [15]. The dentate gyrus, in its outer part, has a reduced synaptic density in AD, and synapses are completely lost within the dense amyloid core of a classic senile plaque. It is very clear that synapses degeneration correlates strongly with cognitive decline, being the current explanations mainly theoretical, thereby, basic question remains: where does synaptic pathology start?

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Despite great number of publications, studies, and experiments, the puzzle cannot be solved. However, a slight change in our concepts before analyze data (melanin as source of energy) can modify substantially the interpretation of results. Let´s see some examples: There is a considerable evidence suggesting that synapses are primary sites of calcium deregulation in AD, this is: chemical energy available is not sufficient to regulate calcium flow as has been done million times, million years by the synapsis. Does synaptic loss occur because of excess release of glutamate or increased susceptibility to glutamate levels released by young neurons? The level of chemical energy (from melanin) must be within the range that has been during the evolution. If this level is not adequate, then, the synapsis will show a generalized failure, typical of energy failure. Glutamate levels are determined by the levels of chemical energy from melanin. Synaptic plasticity is dependent of neuron ionic homeostasis, but in turn, this homeostasis is totally dependent of generation and distribution of energy (by melanin). The events associated with the balance between mass (from glucose) and energy (from melanin), indeed would impact synaptic and cognitive function. If glucose is the energy source of the neuron, the abnormally high levels of blood glucose, would run as a factor of protection against neuro-degeneration, as if the energy levels are right, the neuron operates as it has done for millions of years, millions of times. Brain derived neurotrophic factor has been found to facilitate synaptic plasticity through a variety of mechanisms, however, every single one requires energy. Further: energy is defined as everything that produces a change. This concept fits very well with our finding that cell uses energy in many ways. And so it should be, since not only cell requires energy to carry out each and every one of the functions that are required to form what we call life, but also requires energy to retain the shape. There is (theoretically) reactive oxygen species (ROS) production during increased energy consumption. Perhaps a better explanation is: when chemical energy (from melanin) is impoverished, by cold, metals, plastics, pesticides, herbicides, fertilizers, aging, etc., then, the levels of diatomic hydrogen (H2) are impaired, thereby the problem is, at least; double. By one side, molecular hydrogen is the energy carrier per excellence, and secondly: H2 is the best antioxidant, man ever known.

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When available in the form of molecular hydrogen chemical energy levels decrease significantly, altering the astonishing accuracy with which happens the myriad of chemical reactions inside the cell, whether in temporality, space and location; and on the other hand the important antioxidant effect of hydrogen decreases, with which corrosion is present, or at least is accelerated. Neuronal- astrocytic interactions are highly complex processes finely tuned along 4 billion years of evolution, and work very well when levels of chemical energy (hydrogen) are within the range that it has been during all evolution, for all of creation. Impairment of brain energy metabolism can lead to diffuse neuronal damage, due to chemical energy is needed and used in many ways by cells and tissues. Thereby, reduced brain glucose metabolism can be explained because the chemical energy levels (from melanin) are not enough to support normal brain carbohydrates metabolism. Increased oxygen consumption also observed in AD patients, is explained by the low level of hydrogen and its antioxidant effect, therefore, the oxygen begins to combine as usually, randomly, which produces significant deleterious effect in neuron form and function. The Role of Pyridine Nucleotides and the Abnormal Expression of Genes The generation and distribution of energy (from melanin, and not from glucose) in a consistent, continues, unceasing way, is vital for cellular survival. Energy cannot be stored, so the maintenance of cellular energy reserves is wrongful, oppositely, carbon chains or biomass can be truly stored in several ways. Virtually any molecule present in the interior of the cell depends on full of the energy that emanates from the melanin, called nucleotides, enzymes, organelles, etc. And dependence could be called absolute, because they depend on it since its origin, formation, and then also for its conservation and right functions. In the case of genes, it is not exception, because they depend on the continued presence of energy that carries the molecular hydrogen from melanin. When chemical energy levels are not adequate, the genes cannot express themselves properly, but it may also be that since the genes are formed in the individual when H2 levels were insufficient, which would indicate that the nucleotides that comprise them could be altered. The genetic code also has millions of years of evolution, and has the same amazing perfection than the rest of the systems that make up our body. It is,

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therefore, that no has been demonstrated, so far; a correlation between genetic alterations and Alzheimer's Disease. It simply does not exist, because gene expression also depends on the normal levels of chemical energy, from melanin, not glucose. If fundamental, basic chemical energy for the cell or neuron in this case, is adequate, then the system works well, as it has millions of years. When there is an imbalance between biomass from glucose and energy of melanin, then begin to occur diffuse and unpredictable alterations, and among them the incorrect expression or even altered sequence of one or more genes. This is consistent with findings that only takes 10% of the DNA mitochondrial to maintain normal respiration, and whereas about 90% of the DNA is affected so that the activity of the complex IV is compromised [16]. Any body and cellular function depends on chemical energy in the form of molecular hydrogen, mainly, that melanin release to dissociate the water molecule. High energy electrons generated at the re - shape it, also have an important role, although most located because the electrons usually have low penetration. If growing energy spheres of each melanosome represent in the drawing, the drawing would be unintelligible, but if we do mentally, we can see how the energy that emanates from the melanin has spread across entire cell, following the laws of simple diffusion. Even the fact that the melanosomes cover completely the cell nucleus, explains the source of energy of the largest cellular organelle, as does not contain mitochondria or not ATP. Nor do we represent the cellular organelles, but also depend on the chemical energy that comes from the melanin. Only glucose is source of carbon chains that the cell uses to build and replenish the biomass. In summary: foods are source of biomass, but our body takes the energy of the light through the dissociation of the molecule of water, being our chlorophyll melanin. The structures and molecules involved in the formation of ATP, also depend entirely on energy from melanin. The coenzyme NAD+, is involved in many metabolic processes as an essential cofactor for enzyme-catalyzed oxidation; as major electrons donor for mitochondrial electron transport to power (¿?) oxidative phosphorylation, and as important contributor to ATP production. NAD+ is located within the mitochondrial matrix, NAD+ and NADP are key molecules involved in signal transduction, transcription, DNA repair, glutathione metabolism, and the NADPH-dependent thioredoxin system, which are important for the maintenance of the cellular anti-oxidant system and detoxification reactions [17].

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No doubt, that the functions of the NAD+ are transcendent, and highly relevant, but just as substantive metabolic intermediary and not as a form of storing energy, because energy cannot be stored. It is said that a reduction in the levels of NAD+ may stimulate the neurodegeneration. But when the chemical energy levels of melanin are within proper range, which have had throughout the evolution of creation, then the levels of NAD+ are also suitable. In our opinion, the depletion of NAD+, are only one example of the several detectable biochemical imbalances that can be observed when the equilibrium between the mass (from glucose) and energy (of melanin) are not within the relatively narrow ranges that it has had throughout the evolution of creation. Isn’t that when NAD+ is low, thereafter energy is bad, instead, when the energy is not adequate, thereafter NAD+ will decline, because the synthesis and maintenance of levels and function of NAD+, require, in turn, of the chemical energy (of melanin). Again, the cell uses energy in many ways. It is congruous that both acute and chronic neurodegenerative disease have been linked to the loss of NAD+ (biomass) stores, because the biomass (NAD+ and any biomolecule) depends of adequate chemical energy levels, thus allowing their consistent and adequate replenishment. When chemical energy (H2) levels are impoverished, for example: pain, aging, cold, toxics in water, in the air, shock, sadness, anesthetic agents; etc., then the surprisingly accurate biochemical cellular system, begins to collapse; and this can be detected through the elevation of ROS and Ca2+ levels, as well as the levels of mitochondrial NAD+; even it seems that they are inter-dependent, but the primary event are the low levels of chemical energy coming from melanin; the events that can be subsequently detected only are a reflection, a consequence of the main event. In any system, when the fault is widespread; We must first think about energy. And this no doubt, as with a generation and power distribution right (coming from the melanin); the cell works well, as has done it millions of years, millions of times (Fig. 13). An univocal sign that the fault in the system is widespread when the generation and distribution of energy is impaired, is the observed fact that wherever we explore will find significant imbalances, i.e.: decline in mitochondrial enzymes, reduced activity of hexokinase, an enzyme that catalyzes the phosphorylation of glucose; the regulation of Glut3 (neuronal glucose transporter) deficit; also in the complex (PDHC) pyruvate dehydrogenase; the complex dehydrogenase, αketoglutarate (KGDHC) and cycle-oxygenase (COX).

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In the light of the knowledge that the main source of energy of the CNS is by melanin, and not by glucose, as up to now it was firmly believed; the interpretation of some of the phenomena observed in the patients with neuronal degeneration, can be interpreted in a more useful way (Fig. 13).

Fig. (13). Melanin releases chemical energy in a very orderly and consistent way both day and night, and so that it does not require living within an entity alive, because it performs the same function both inside and outside the cell, which explains the origin of life.

Knowing that the energy chemical that it is generated by the dissociation and reform of the water molecule is mainly carried by molecular hydrogen (H2) and electrons of high energy (e-); Mitochondrial alterations (depolarization with age, leakage of the protons) can be understood as one of many possible manifestations, due to the decrease in the ability of the body to transform the light, visible and invisible; into chemical energy; which can be seen since the 26 years, approximately 10% each decade, and after fifties goes into free fall. Other example is the dysregulation of calcium dynamics; this is: activation of mitochondrial matrix dehydrogenases [18], and production of NADH are alterations that can be interpretated as low levels of chemical energy; thereby, calcium regulation is impaired in spite the cell has done it million times, million years. The altered homeostasis of calcium in AD and other neuro-degenerations, leads to other major or at least more widened metabolic disorders, in part derived from the dysregulation of calcium dynamics and in main part because the levels of chemical energy (H2) produced, of itself; a fault in which all intracellular processes shall have substantive artifacts. The abnormal influx of calcium and much-damaging overload to the inside of the cell, are not isolated or exquisite phenomena, they are actually alterations relatively easy to detect, but in fact they are one of many manifestations of the imbalance derived from an depauperated level of chemistry energy, from melanin. It is not so much alterations which we

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could call pinpoint to have an effect on cascade, and they have it, undoubtedly, but not so crucial, important or as marked as those that result of low molecular hydrogen levels in the cell cytoplasm. A good sample is the primary ROS generated mainly in mitochondria that is O2-., which is rapidly converted to H2O2 in AD patients [19], by mitochondrial manganese superoxide dismutase or cytosolic copper/zinc superoxide dismutase enzymes. The resulting H2O2 is oxidized to water by glutathione peroxidase or catalase. But this processes have questions that are answered easily arising chemical energy from the water molecule dissociation: where do the O2-. takes molecular hydrogen (H2) that is required to form hydrogen peroxide (H2O2)? Any molecule yields one or several of its components in exchange for nothing, the interchange requires energy invariably. For example, water from the cytoplasm does not yield molecular hydrogen disassembling the water molecule, yields it unless water has been dissociated in advance (by the melanin), because in that case, the H2, which does not combines with water, then it is taken up by the O2-., and the chemical energy required so that O2 be reduced to hydrogen peroxide (rapidly) It is very likely that it is provided by the molecular hydrogen itself. On the other hand, the enzymes mentioned above, only accelerate the process, but do not change the need for available chemical energy to make the reaction happen. Thus the steps of O2-. to H2O2 and later to H2O, they require imperatively; available chemical energy; and the H2 is the perfect actor. Oxidative Stress H2O2, in the presence of reduced transition metals, is converted to the highly reactive hydroxyl radical (.OH), some metals, especially Iron, Cadmium, Mercury; etc., they all are able to poison the melanin itself, so the unexpected ability to dissociate the water molecule, is impoverished; which means that the formation of hydroxyl radicals (¯OH) comes since the very generation of energy. Oxidative stress and free radical damage have been consistently associated with AD pathogenesis [20], but the intracellular processes are so amazingly accurate, that any change in the levels of chemical energy (from melanin), will produce a unpredictable, widespread damage, for example: the formation of beta-amyloid, and other abnormal protein aggregations (tangles); It is therefore that to date not is has been able to define if either oxidative stress and/or protein aggregation is Neurodegeneration initial event; and now we can say that neither of them, as both are initiated by a decline in levels of chemical energy coming of melanin (Fig 14).

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Fig. (14). Schematic representation of the way that melanin releases energy in form of a growing spheres. In this example, the melanin of the choroid plexuses of one side.

The extention of damaged neuron tissue described in AD, were expected, as in any system, due to power failure; so It produces a widespread and at all levels dysfunction. The fact that seem to interplay each other increasing the damage to the system, is very logical; but the initial problem is in the generation and distribution of energy, from melanin (Fig. 15), of course. Analyses of tissue homogenates from postmortem brain tissue never display the very orderly and consistent levels of hydrogen diatomic and high-energy electrons (from melanin), which are the basis of cellular functioning. To be able to detect them, it is necessary to study them in the patient alive, in this moment, a formidable challenge. The so limited memantine´s clinical effects can be explained by the wrong concept of overactivation of NMDA receptors, in our opinion must be considered as dysregulation of NMDA receptors, that seems as overactivation, but the problem it is more complex, with multiple factors involved; and finally depends (them all) of the levels of chemical energy that comes from melanin (molecular Hydrogen and high energy electrons); this is: the factors that regulate the overactivation of NMDA receptors are dysfunctioning, also by a low level of chemical energy from melanin. In short, when the levels of chemical energy within the cell, are not adequate, the whole system is distorted, and the multiple and complex interactions between components (or not known) that comprise it; also modify negatively, that is, at the

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end; a perverse circle (Fig. 16).

Fig. (15). Diagrammatic representation of the overlapping of growing spheres of energy, that comes from melanin in choroid plexuses, of both sides.

Fig. (16). Schematic representation of overlapping of the spheres of energy (Hydrogen from melanin), in this case; from the substantia nigra -both sides-, in mesencephalon. Is conceivable where converge the growing fields, generated an area of high energy, which is congruent with areas such as the midbrain, which predictably requires a considerable amount of chemical energy available to carry out its function.

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REMARKS AND CONCLUSION Alzheimer's disease seems to widely exceed our capacity for abstraction, as does not have significant progress to date; despite the strenuous efforts of researchers, doctors and patients who continue to die in the same way that Auguste D. Currently it is thought that the dementia of Alzheimer's disease is due to deposits or aggregates of amyloid-β in the extracellular space of the brain cortex. This model has been criticized because there is little or no correlation between deposits and dementia. Others have questioned whether the amyloid-β is actually neurotoxic [21] Acetyl cholinesterase inhibitors and memantine are the only drugs approved for the treatment of Alzheimer's disease and have been on the market for many years. Its efficiency is statistically significant, but is of little or no clinical relevance [22]. The majority of present and past clinical trials do not include patients in advanced stages of Alzheimer's Disease, since the only approved treatments are indicated for mild and moderate forms of AD [23]. Individuals with high levels of education are simply paths other than cognitive impairment before the diagnosis of AD, with a sharp fall of cognitive function in the years immediately following the diagnosis of Alzheimer's [24]. The regulation of each and every one of the neural functions, starts from the energy that drives them, including from the how chemical energy is generated. The way in which melanin transduce the visible and invisible, light into chemical energy through the dissociation of the molecule of water, as the plants [25] it is surprisingly accurate, as it is very consistent, continuous, incessant, as it happens both day and night. It is what chemistry is called directed synthesis (Fig. 17). Melanin absorbs light, and the absorbed energy it is dissipated separating the water molecule. It’s the small great detail that gives rise to life, as it takes place both inside and outside of a living body [26]. It is amazing that despite the amount of light, whether much or little, the energy that emanates from the melanin is preserved within relatively narrow ranges, as if the light energy is too strong, decreases it melanin, and if it is low, the melanin tends to increases it. The physical characteristics of light are remarkably stable, but when they are transduced by the melanin into chemical energy through dissociation and re-form of water molecule, they seem to refine an extraordinary manner and the chemical energy that melanin produced, then becomes the ideal energy both to generate

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life, as well as to hold it, supporting CNS functions (Fig. 18).

Fig. (17). The energy that emanates from the melanin, mainly hydrogen diatomic form; since electrons of high energy that are generated in the re-forming of the water molecule, is considered to have low penetration; It is used in many ways by the surrounding tissues. For example: to preserve the shape, because even to retain shape, power is required.

Given that is the energy that explains the origin of life; It is understandable that it has been the same throughout all creation, of all evolution. And the ranks that is generated and distributed to the inside of the cell have not changed since the beginning of time. Is for this reason that the variations of the chemical energy that emanate from the melanin in the form of molecular hydrogen and high energy electrons, when they come out of the range; they produce some imbalance, and the clinical manifestation of the same call it disease. The processes of our body are surprisingly accurate, very precise; reflecting at all times, the energy that gave him birth. Alterations in CNS pigmented system (Figs. 15-18) tend to cause alterations, as the chemical energy levels not have the same levels that have been since the beginning of time.

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We believe that the discovery of the intrinsic property of the melanin of transforming light into chemical energy, begins to bear fruit, as it allows us to structure a logic proposal about the pathogenesis of anatomical lesions observed in Alzheimer's disease, by correlating its location with the neuromelanin and the way in which the chemical energy diffuses through tissues following the laws of simple diffusion. Even it is proposed that the intensification of the dissociation of the water molecule, is a possible therapy for Parkinson's patients [27]. “I don´t believe God put the melanin granule in the central nervous system for nothing. It must be doing something. Something big” G.C. Cotzias Brilliance 6 Ex: 15925 Se: 4 Im: 36 DFOV 200.0 mm

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Fig. (18). With drawing, try to give idea about how increased melanin energy spheres would be, and which come from the different structures of the CNS, in which there is accumulation of the molecule of melanin, which previously was considered something like a trash can where CNS placed waste; but we now know that its function is completely different and fundamental, because it is nothing less than transform the visible and invisible light into chemical energy through the dissociation of the molecule of water, such as chlorophyll in plants. The areas where growing spheres overlapped, the levels of chemical energy are higher.

CONFLICT OF INTEREST The author declares no conflict of interest, financial or otherwise.

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ACKNOWLEDGEMENTS This work was supported by Human Photosynthesis® Research Center. REFERENCES [1]

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CHAPTER 7

Neuro-protective Properties of the Fungus Isaria japonica: Evidence from a Mouse Model of Agedrelated Degeneration Koichi Suzuki1,*, Masaaki Tsushima1, Masanobu Goryo2, Tetsuro Shinada3, Yoko Yasuno3, Eiji Nishimura3, Yasuo Terayama4, Yuki Mori5 and Yoshichika Yoshioka5 Organization for Research Promotion, Iwate University, Morioka, Iwate, Japan Faculty of Agriculture, Iwate University, Morioka, Iwate, Japan 3 Graduate School of Science, Osaka City University, Osaka, Japan 4 Division of Neurology and Gerontology, Department of Internal Medicine, Iwate Medical University, Morioka, Japan 5 Biofunctional Imaging Laboratory, Immunology Frontier Research Center, Osaka University, Osaka, Japan 1 2

Abstract: Isaria japonica (IJ), is an entomopathogenic fungus that is grown on pupae of the silkworm Bombyx mori for its medicinal properties. Its extracts have potential neuro-protective effects. An extract reversed astrogliosis in the CA3 area of the hippocampus of aged mice. The CA3 area is responsible for spatial pattern association and completion, detection of novel situations, and short-term memory. This finding led us to the development of treatments to improve age-related impairment of patients with Alzheimer’s disease (AD). Acute and subchronic toxicity and chemical profiling of the extract were conducted for the assessments of medical use. We are now evaluating preclinical trials with AD patients. For the diagnosis of AD, magnetic resonance imaging (MRI) enabled the detection of the previously invisible pathological alterations in a mouse sclerosis model with autoimmune encephalomyelitis. Magnetic resonance spectroscopy (MRS) showed that demyelination regions in some multiple screlosis (MS) patients had increased lactic acid content, suggesting the presence of ischemic events. These results show that products derived from IJ may prevent or reduce the impact of dementia, especially AD, and MRI and MRS could lead widely to the diagnosis of neurological diseases. Corresponding author Koichi Suzuki: Biococoon Institute, Inc., Research and Development Center by Collaboration of Morioka City and Iwate University, Morioka 020-8551, Japan; Tel: +81 19 613 5564; Fax: +81 19 613 5570; E-mail: [email protected]

*

Atta-ur-Rahman (Ed.) All rights reserved-© 2017 Bentham Science Publishers

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Keywords: Aged brain, Alzheimer’s disease, Astrogliosis, Dementia, Entomopathogenic fungus, Isaria japonica, Magnetic resonance imaging and magnetic resonance spectroscopy analyses, Multiple sclerosis, Nuclear magnetic resonance spectroscopy analysis. INTRODUCTION Under natural conditions, the entomopathogenic fungus Isaria sinclairii (= I. cicadae) grows on larvae of the cicada, Meimura opalifera Walker (Hemiptera: Cicadidae). Following the discovery that the culture broth of this fungus had potent immunosuppressive activity [1], a novel synthetic compound (FTY720), with lower toxicity and in vitro and in vivo immunosuppressive activity, was developed from a fungal metabolite as a lead compound, myriocin (= ISP-1) [2]. This compound, named fingolimod, has opened up a new approach to the treatment of MS [3]. Keeping in mind since the brain disease-treated agents are originated from entomopathogenic fungi, we have learnt that Ophiocordyceps, Cordyceps and Isaria spp. are traditionally used as to treat cancer, diabetes, cardiovascular diseases, and neural disorders, albeit without good scientific evidence [4 - 8]. The price of natural products and large-scale harvesting of wild fungi pose problems [9, 10]. Biopharmaceuticals derived from those fungi are anticipated, but the effect of 3’-deoxyadenosine, a cordycepin with potential anti-cancer first described in 1950, has not been tested in clinical trials [11]. Many studies have only shown about pharmaceutical effects of medicinal mushroom and fungi on the experimental animals, but medicinal uses for human have made very little progress so far [12]. There were anti-fatigue ability and higher endurance with the supplement of Ophiocordyceps (= Cordyceps) sinensis [13] and for patients with advanced liver disease and inoperable tumors and treated with 4 natural agents that included O. sinensis, the tumor was found to decrease in size, the tumor marker levels decreased substantially, and the patients survived comfortably [14]. Yet, more experiments are needed to demonstrate sufficient data on the efficacy and safety of entomopathogenic fungi to find new sources for drug discovery [12]. Thus, other sources that do not contribute to the loss of natural entomopathogenic fungi or depend on market forces are being investigated. We have grown I. japonica (IJ = Paecilomyces tenuipes) sourced from a mountain field in Fukusima Prefecture, Japan, on dried silkworm (Bombyx mori) pupae left over from silk extraction (Fig. 1) [15], obviating the need for wild harvesting.

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Fig. (1). Synnemata and conidia of IJ cultured on dried pupae of Bombx mori.

To evaluate the effects of entomopathogenic fungi, mice aged by treatment with ᴅ-Galactose in an aging model for the brain and dosed orally with a hot-water extract of O. sinensis showed a significantly reduced decline of spatial learning and memory ability [16]. The hot-water extract of O. sinensis also prevented structural changes in the hippocampus of aged mice and shortened the mount latency of castrated rats. These findings indicate that the hot-water of O. sinensis has an anti-aging function. Therefore, we tested IJ extract (IJE) for similar effects. We found that IJE improves nerve function in aging mice and may lead to the development of treatments for Alzheimer’s disease (AD) [15]. This comprehensive review discussed neural improvement in the aged brain; nuclear magnetic resonance (NMR) analyses of IJE; towards a goal of complementary and alternative medicines /or medicines originated from the entomopathogenic fungus, and the potential use of MRI and MRS for the diagnosis of neurological diseases. IJE Improves Nerve Function in Aged Mouse Brain IJE reduced astrogliosis and improved memory deficits are the characteristics of serious disorders of the central nervous system such as AD and MS [17, 18]. 1. Neuroprotective Effects of IJE In many studies, ᴅ-Galactose induced [19 - 21] or SAMP8 [22] mice have drawn attention in research on dementia owing to their characteristic learning and memory deficits in old age. ᴅ-Galactose treatment induces learning and memory impairment but causes no neuromuscular dysfunction, and it is effective for testing the neuroprotective effects of chemicals. Thus, chronic systemic exposure of mice to ᴅ-Galactose is a useful model for analyzing the mechanisms of neurodegeneration and neuroprotective drugs and agents [21]. In accordance with

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models for the induction of senescence by ᴅ-Galactose [19 - 21], four groups of mice with 100 mg ᴅ-Galactose /kg-day s. c. were injected for 8 weeks: normal control, aging control and IJE × 5 or 25 mg/kg-day (administrated orally to aging control) [15]. According to an experimental schedule (Fig. 2), the pre-acquisition trial (no foot shock) for habituation to test the equipment was also performed one week before the acquisition trial (with foot shock) to exclude differences in the learning and memory ability of the mice. A Morris water maze test was performed over a 9-day period from 2 days after the end of the step-through passive avoidance test. In the step-through passive avoidance testing, normal control mice showed a normal delayed response; aging control mice showed a significantly reduced response; but, aging control mice dosed with IJE at 25 mg/kg-day showed the same response as normal control mice (Fig. 3).

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The mice were also evaluated in the Morris maze to examine the effects of IJE on the time they took to reach the submerged platform and on the number of crossing they made. The mean swing speed of four groups was similar and did not cause neuromuscular dysfunction (Fig. 4). More remarkably, the mice in the IJE 5 and 25 mg /kg-day groups spent significantly more time in the target quadrant (1) than in quadrants 2 and 4 (Fig. 5). In contrast, mice in the normal control and aging control groups did not spend significantly more time in quadrant 1. These results indicate that IJE improved spatial learning and memory capacity in mice aged by ᴅ-Galactose. Thus, hot-water extracts not only of O. sinensis [16] but also of I. japonica improve spatial learning and memory in aged mice. However, there is a marked difference of both extract concentrations in between the dosed with O. sinensis

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extract at 1 to 4 g/kg-day administrated orally for 6 weeks and those dosed with IJE at 5 or 25 mg/kg-day for 5 weeks. Whether the difference is related to differences in the bioactive compounds between the fungi is not known [15, 16]. Step-through latency (s)

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Fig. (3). Effects of IJE in step-through passive avoidance testing of mice with ᴅ-Galactose-induced aging. NC = normal control (n = 8): injected with saline (0.9%) and dosed with plain water. AC = aging control (n = 8): injected with ᴅ-Galactose at 100 mg/kg-day s.c. for 8 weeks and dosed with plain water for 5 weeks. IJE was given to AC mice at 5 or 25 mg /kg-day (n = 8) for 5 weeks. All values are means ± SEM. *P < 0.05 vs. AC. Adapted from Ref [15].

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Fig. (4). Effects of IJE on swim speeds in the Morris water maze test (day 8) of mice aged by ᴅ-Galactose. All values are means ± SEM. Adapted from Ref [15].

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Fig. (5). Effects of IJE on time spent in target and other quadrants in the Morris water maze test (day 8) by ᴅGalactose. All values are means ± SEM. *P < 0.05, ** P < 0.01, ***P < 0.0501 vs. target quadrant 1 in both groups. N.S. did not spend significantly in target quadrant 1 of two controls. Adapted from Ref [15].

2. Histochemical Observation Morphological observation reveals another outstanding difference between O. sinensis extract and IJE. In the experiment of O. sinensis extract [16], neurons in the hippocampus of aged mice had a slight pyknosis and morphological degeneration, but it prevented these structural changes. By contrast, we observed apparent astrogliosis in the CA3 area of the hippocampus of aged mice, and in mice treated with IJE × 5 mg/kg-day slight astrogliosis was found, but there was no astrogliosis in IJE × 25 mg/kg-day mice (Fig. 6) [15]. In our histochemical observation, hematoxylin-eosin staining revealed that the hippocampal neurons were well conserved in each mouse group (A, C, E, and G in Fig. (6)). When the hippocampal neurons in aged mice were stained with Holzer stain for fibrous components of astrocytes, marked astrogliosis was observed in the CA3 area and not in those of aged mice administrated with a high dose of IJE (D and H in Fig. (6)). Thus, our histochemical finding has been facilitating immunohistochemical analyses labelled with specific markers such as glial fibrillary acidic protein and anti-glutamate transporter [23, 24]. The CA3 area is responsible for spatial pattern association and completion, detection of novel situations, and short-term memory [25]. Reactive gliosis is a universal reaction to brain injury and is instrumental in sealing off the injured tissue, promoting tissue integrity, and restricting inflammation and neural death

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[23, 26]. Hence, our results may aid in neural repair in brain dysfunctions such as AD. A

B

CAl CA3

NC DG

C

D

E

F

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H

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IJE 5

IJE 25

Fig. (6). Effects of IJE on hippocampal structures of mice aged by ᴅ-Galactose. Left column (A, C, E, G): hippocampal structures stained with hematoxylin-eosin stain for neurons (Bars = 40 μm). Right column (B, D, F, H): CA3 areas of the hippocampus stained by Holzer stain for fibrous components of astrocytes (Bars = 40 μm). White arrowheads show gliosis. Adapted from Ref [15].

3. Assessments of Acute and Sub-acute Toxicity We analyzed the acute and subchronic toxicity of IJE. Ophiocordyces and Cordyces (Hypocreales), entomopathogenic fungi that grow parasitically on lepidopteran larvae and pupae, are two genera that are among the most popular fungal nutritional supplements in Asia. The safety of naturally grown O. sinensis has been confirmed, and both cultivated mycelia of O. sinensis and the cultivated

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fruiting body of C. guangdongesis are considered safe for long-term human consumption as traditional Chinese herb [27, 28]. The use of C. militaris has also been shown to be safe [29]. The acute and subchronic toxicities of IJE have been evaluated comprehensively, but host species and chemical profiling have not been reported [30]. Recently, we tested acute toxicity on adult female ICR (Crl: CD1, evaluated from Charles Riber Laboratories) mice and subchronic toxicity with adult female Wistar rats. IJE did not cause either significant visible signs of toxicity or mortality in the mice, and IJE at 25 mg and 500 mg/kg for 28 consecutive days did not cause mortality. Consistent with histopathological observation shown in Fig. (7), we found no significant differences in food or water consumption, hematological parameters, or relative organ weights between the treated and control groups [31]. Thus, IJE appears to be safe when administered orally. Control

25 mg/kg

500 mg/kg

A1

B1

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C7

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Cerebrum

Cerebellum

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Lung

Heart

Liver

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Fig. (7). Histology of the organs of female rats administered 0 (control), 25 or 500 mg/kg of IJE. After the organ samples were fixed in 10% formalin, they were embedded in paraffin. Sections of 4 μm in width were conventionally stained with hematoxylin-eosin stain and examined under a light microscope. Adapted from Ref [31].

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NMR Analyses in the I. Japonica Extract Old remedies so-called traditional medicines are commonly employed worldwide. Traditional medicines have received significant interests as complementary medicines from the view point of promoting the quality of life and maintenance of our health. The role and consumption would be more growing in the era of aging society and the increasing of population. In consideration of the indispensable role of the traditional and complementary medicines, World Health Organization reported the WHO Traditional medicine Strategy 2014-2023 (http://www.who.int/medicines/publications/traditional/trm_strategy14_23/en/) in 2013, aiming to strengthen the role in keeping populations healthy. One of the crucial aspects in this proposal is the establishment of the safety and quality assessments of traditional and complementary medicines to employ them more safely and reliably. Isolation and structure determination of the biologically active compounds in traditional and complementary medicines have been recognized as important research subjects to provide the evidence-based proof of quality, toxicity, and safety of these medicines. In addition, the structurally novel natural products provide opportunity to the drug discovery. Products of entomopathogenic hypocrealean fungi represented by “Winter-Wor-Summer Grass”, “Dong-ChungHaCao” in Korea, “DongChaongXiaCao” in Chinese, and “To-Chu-Kaso” in Japanese have gained much attention due to its promising utility as traditional and complementary medicines in the South East Asia. Many biologically active substances have been isolated from the products of Cordyceps sp. and Isaria sp. The recent progress in the research area has been reported in excellent reviews [5, 7, 32 - 36]. In this section, previous studies focusing on the isolation and structure determination of chemical components of I. japonica (= P. tenuipes, I. tenuipes) are described. NMR and MS analyses of IJE are discussed. These chemical aspects would aid to consider the safety, toxicity, and quality of the traditional medicines originated from the products of entomopathogenic Hypocrealean fungi such as I. japonica and I. cicadae. 1. Chemical Component of I. Japonica 1.1. Living Substances The mass balance of living substances in cultivated fruiting-bodies of P. tenuipes (host: B. mori) was analyzed (Table 1) [37]. This analysis indicates that the major organic compounds in the living substances are fatty acids.

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Table 1. Approximate contents of living substances in the fruiting body of P. tenuipes [37]. Living Substances

%

H2O

57.56

Crude fat

21.76

Crude protein

6.83

Crude fiber

6.20

Crude carbohydrate

3.49

Fig. (8). Fatty acid contents in P. tenuipes and the silkworm products [37].

The contents of fatty acids were analyzed by gas chromatography. This analysis suggested that the total ratio of unsaturated fatty acids [79%] was much higher than that of saturated fatty acids. Linoleic acid, linolenic acid and oleic acid were detected as the unsaturated fatty acid components (Fig. 8). Total amounts of sugar contents were estimated to be 24.0 mg/g (dry weight) (Fig. 9). It is interesting that the amount of the P. tenuipes product was higher than that of a silkworm powder (14.0 mg/g). Glucose and mannitol were the major sugar components in the fungi

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product. Total amounts of amino acids in the fungi product and the silkworm powder were estimated to be 17.1 mg/g and 17.4 mg/g, respectively. Arg, Gly, Pro, Tyr, and Ser were the major amino acid contents in the fungi product (Fig. (10), upper). The amino acid profiling of the silkworm powder was different from those of the fungi product (Fig. (10), lower).

Fig. (9). Structures of sugars in the fungi product. Numbers indicate the dry weight of sugars in the fungi and silkworm products (mg). Square parenthesis for the silkworm powder [37].

Fig. (10). Major amino acids in P. tenuipes (upper) and the silkworm powder (lower). Numbers indicate the dry weight of each amino acid (mg/g) [37].

Cao et al. [38] analyzed the nucleoside contents in the fermentation broth of P. tenuipes (Fig. 11). The amount of each nucleoside was estimated to be as follows: uridine (3.63 mg/g), guanosine (5.55 mg/g), adenosine (0.86 mg/g), inosine (0.46 mg/g), and thymidine (0.23 mg/g). Chen et al. reported that the contents of cytidine, inosine and guanosine are about 3, 7, and 9 times of those in C. sinensis, respectively [39]. Adenosine and uridine are rich in the fermentation product. The amounts of these nucleosides were found to be higher than those of C. sinensis.

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Comparative analysis of adenosine in the fruiting bodies and the corpus of P. tenuipes suggested that adenosine was more abundant in the fruiting bodies [37].

Fig. (11). Structures of nucleosides in P. tenuipes [38].

2. Biologically Active Substances Various biologically active compounds were isolated from I. japonica. The yields were dependent on the cultivation and fermentation conditions, and extraction protocols. The amounts of these compounds in the fungi products were basically much lower than those of the living substances. 2.1. Cyclic Peptides Beauvericin and beauvericin A were isolated by the bioassay-guided fractionation of a mycelia extract of P. tenuipes BCC1614 (Fig. 12) [40]. These natural products displayed moderate antitubercular activity against Micobacterium tuberculosis (H37Ra strain) and antimalarial activity against Plasmodium falciparum (K1 strain). Supothina et al. compared the productivity of beauvericin from three forms ((i) natural specimen, (ii) cultivated synnemata on rice media, and (iii) mycelia from fermentation in liquid media) of the four I. tenuipes strains (BCC 31640, 33299, 35849, and 35850) [41]. A small amount of beauvericin (0.0017~0.036 mg/g) was detected from (i). The degrees of productivity from (ii) and (iii) were much greater than that of (i). For instance, in the fermentation media of the mycelia, the amount of beauvericin was estimated to be 0.49~30.3 mg/g (4.0~100.2 mg/L).

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Fig. (12). Structures and biological activities of beauvericin and beauvericin A [40].

Precursor-induced biosynthesis by feeding of l-isoleucine, d-isoleucine, lalloisoleucine, and d-alloisoleucine to the liquid fermentation media of P. tenuipes BCC1614 was attempted to produce beauvericin analogs (Fig. 13). Under the precursor induced conditions, beauvericin A, beauvericin B, and beauvericin C, and their diastereoisomers named allobeauvericins A~C were obtained from the fermentation broth [42].

Fig. (13). Structures of beauvericins isolated by the precursor induce biosynthesis [42].

Recently, antioomycete activity of beauvericin against the phytopathogens Phytophthora sojae and Aphanomyces ochlioides was reported by Putri et al. [43]. Moreover, many beauvericins and their related natural products were isolated from this class of fungus. The chemical and biological activities of beauvericins are reviewed in the literature [44].

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2.2. Isariotins and Its Related Natural Products Isariotins A~J and TK-57-164A were isolated from the fermentation product of I. tenuipes strain (BCC 7831,12625, 15621, 21283, or 23112 (Fig. 14) [45 - 47]. Isariotin G, F, I, and J displayed moderate antimalarial activities against P. falciparum K1 in a range of IC50 value from 2.10 to 5.51 μM and cytotoxic activities against KB, BC, and NCI-H187 cancer cell lines in a range of IC50 value from 0.64 to 44.86 μM. Isariotin F showed antitubercular activity against M. tuberculosis H37Ra at MIC value of 60.4 μM and antifungal activity against Candida albicans at IC50 value of 13.9 μM. The other isariotins showed much less potent biological activities.

Fig. (14). Structures of isariotins A~J and TK-57-164A [45 - 47].

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2.3. Cyclic Terpenoids Two cytotoxic terpenoids, ergosterol peroxide and 4β-acetoxyscirpenediol were isolated from the methanol extract of the dried carpophores of P. tenuipes (Fig. 15) [48]. The IC50 values of ergosterol peroxide against human gastric tumor, human hepatoma, human colorectal tumor, and murine sarcoma-180 cell lines were 18.7, 158.2, 84.6 and 74.1 μM, respectively. 4β-Acetoxyscirpenediol showed antiproliferation activities against the same cancer cell lines. These IC50 values were estimated to be 1.2, 4.0, 2.2 and 1.9 μM, respectively [48]. Further pharmacological investigations of 4β-acetoxyscirpendiol indicated that it induced apoptosis in human MOLT-4, THP-1 and Jurkut T cell leukaemia in vitro [49, 50] and inhibited 2-deoxy-d-glucose uptake by the human Na+/glucose transporter-1 expressed in Xenopus laveis oocyto and HEK-293 cell [51, 52].

Fig. (15). Structures of cyclic terpenoids isolated from I. japonica [53 - 55].

Kikuchi and Oshima et al. isolated various trichothecanes and their analogs from the cultivated fruiting bodies of P. tenuipes (Fig. 15) [53 - 55]. From this extract, tenuipesin A, paecilomycine A, paecilomycine B, paecilomycine C, spirotenuipesine A, and spirotenuipesine B were isolated as novel trichothecane analogs. Incubation of 1321N1 human astrocytoma cells (glial cell line) with paecilomycine A, paecilomycine B, paecilomycine C, spirotenuipesine A or

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spirotenuipesine B followed by the addition of the mixture to PC-12 cells allowed for the potent neurite outgrowth of PC-12 cell line. These results indicated that these natural products would be a promoter of the neurotrophic factor biosynthesis in glial. 2.4. Others Chen et al. isolated penostatines by the bioassay guided fractionation of the fermentation medium of P. tenuipes, RCF 37776 (Fig. 16) [56]. These natural products revealed inhibitory effects against protein phosphatase 1B in a range of IC50 values of 0.37 ~ 33.65 μM. Among the penostatins, penostatin J was the most potent. It displayed the inhibitory effect at the IC50 value of 0.37 μM.

Fig. (16). Structures of penostatin A, B, C, and J [56].

Hanasanagin was isolated from I. japonica cultivated on silkworm pupae (Fig. 17) [57]. Hanasanagin is a dipeptide like molecule in which l-DOPA and a 3,4diguanidinobutanoyl moiety are linked by an amide bond. This natural product displayed potent free radical scavenging activities.

Fig. (17). Structure of hanasanagin [57].

A chemical epigenetic method using a histone deacetylase inhibitor and a DNA methyltransferase inhibitor was successfully applied to produce three polyketides, tenuipyrone, and cephalosporolide B from the fermentation of I. tenuipes (Fig. 18) [58].

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Polysaccharides of mushrooms have received significant interests because of their potential biological activities such as antioxidant, free radical scavenging, antiviral, hepatoprotective, antifibrotic, antiinflammatory, antidiabetic and hypocholesterolemic activities [59]. Lu et al. analyzed polysaccharide components from P. tenuipes Samson by gel permeation chromatography (Fig. 19) [59]. The average of molecular weight of the polysaccharides was estimated to be 1.02 × 104. Further analyses indicated that the polysaccharide is composed of ᴅ-glucose, ᴅ-Galactose, and ᴅ-mannose (ca. 2:1:1) and these are linked by β-(1 → 6)-ᴅ-glucose for the main chain, and β-(1 → 6)-ᴅ-mannose and β-(2 → 6)-ᴅgalactose for the side chains.

Fig. (18). Structures of cyclic terpenoids isolated from I. tenuipes [58].

Fig. (19). Structures of sugar components in the polysaccharide of P. tenuipes [59].

3. NMR and Mass Study of Water Extract of I. Japonica H-NMR chart of the hot water extract of I. japonica is shown in Fig. (20) [31]. The signal complexity would ascribe to the mixture of several components in the extract. The broad signals from 0 to 5 ppm would be assigned to be the protons of peptides and lipids. Signals between 6.8 to 7.5 ppm would be attributable to the aromatic protons of amino acids, peptides, nucleosides, etc. in the extract. The biologically active molecules isolated from I. japonica such as trichothecenes, spirotenuipesine A and B, cordycepin, hanasanagin, etc. could not be detected due to the following reasons; (i) the lower solubility of these lipophilic compounds under the extraction conditions using hot water, (ii) the lower contents of these compounds than those of the living substances, and/or (iii) the resulting spectral complexity of the crude mixture. It is interesting to note that MALDI-TOF Mass analysis of the extract indicated the presence of a sharp signal at [M+H]+ = 5735.7 1

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which is putatively assigned as a signal of peptides, though the possibility of oligosaccharides could not be ruled out. Further isolation and analysis of the major components in the extract are ongoing. (a) 600 MHz 1H-NMR spectrum tochu-H24-July_1H-presaturation_D2O_temp25 O1P=4.6938 ppm

BRUKER

Current Data Parameters NAME EXPNO PROCNO

nd3 363 1

P2 - Acquisition Parameters 20121002 Date_ Time 8.56 spect INSTRUN 5 mm BBI 1H - BB PROBHD zgpr PULPROG 65536 TD SOLVENT D20 NS 179 DS 4 12376.237 SWH 0.188846 FIDRES AQ 2.6477449 RG 322.5 40.400 DW DE 6.00 298.0 TE D1 4.00000000 0.00002000 d12 1 TD0 -------NUC1 P1 PL1 PL9 SFO1

Hz Hz sec usec usec K sec sec

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F2 - Processing parameters SI 32768 SF 600.1300010 MHz WDW EM SSB 0 LB 0.30 Hz GB 0 PC 0.50

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(b) MALDI TOF MS spectrum Kratos PC Axima CFRplus V2.4.0: Mode linear, Power. 42, P.Ext. @ 5000 (bin 99) %Int

13 mV[sum= 1256 mv] Profiles 1=100 Smooth Av 50 -Baseline 100 2813.8

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90 80 70 2862.6

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2797.4 1155.3 1041.1 991.5 1077.5

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2846.2 4811.6 3724.5 4043.8

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5767.5 5027.3

5755.7

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Fig. (20). Analysis of the hot water extract of I. japonica by NMR and MALDI-TOF Mass: (a) NMR chart, (b) Mass spectrum.

Visualization of the Physiological and Pathological Alterations in the Central Nervous System using MRI and MRS MRI and MRS have been widely used in medical diagnosis and also in pre-

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clinical researches. A Functional MRI (fMRI) is an important tool which is used for the research of the neurosciences. We have applied these techniques in order to assess clearly neurological diseases [60 - 63]. 1. Fine Imaging Using Ultra-high Field MRI MS is an autoimmune disease of the central nervous system, where Th cells (CD4+ T cells) play an important role in the development of inflammation [64, 65]. The MRI in Fig. (21) was obtained from a patient with MS. There are disseminated high intensity regions in the white matter. Those are the plaques of MS. MRI is a popular clinical tool used in diagnosing MS and it is routinely used for the in vivo detection of the corresponding plaque regions. Although the sclerosis lesions are found by MRI as shown in Fig. (21), the lesions sometimes do not correlate with the neurological impairments [66, 67]. This is likely due to the fact that a conventional clinical 3 tesla (T) MRI shows only the smaller restricted regions and damages incurred than those detected by histopathological studies. Furthermore, these high intensity plaques reflect relatively advanced pathogenesis and not earlier symptoms. Recently Nielsen et al. [68] showed the contribution of cortical legion subtypes visualized by 7 T MRI to the physical and cognitive performance in MS. MS is considered to be associated with progressive oligodendrocyte loss, neuronal loss and demyelination. However, its pathogenesis is not so clear, and MS is an intractable disease even now.

Fig. (21). MRI of a patient’s brain with MS at 3 tesla (T). Disseminated high intensity regions in white matter are the plaques of MS.

Arima et al. [69] showed that autoreactive CD4+ T cells accumulate on the dorsal side of the lower lumber spinal cord to pass through the blood-brain barrier and to cause inflammation in the central nervous system of experimental autoimmune

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encephalomyelitis (EAE) mice. EAE mice have been widely used in preclinical researches as human MS models. We tried to visualize the changes that occurred during the progression of the disease using this mouse model. EAE induction was performed as follows [70]. A myelin oligodendrocyte glycoprotein peptide in complete Freund’s adjuvant and pertussis toxin was injected intravenously into C57BL/6 mice. Pathogenic CD4+ T cells were collected from the mice treated above and transferred into other wild type of C57BL/6 mice. EAE was induced about 5 days after the T cell transfer. Fig. (22) shows the axial T2 weighted MRI of the spinal cord from L3 to L6 at 11.7 T. These images were obtained during the progression of the disease from the same mouse. Although we used an 11.7 T MRI scanner, it was not easy to visualize the changes in the spinal cord because the size of the spinal cord at the lower lumber region is very small. The scale in the figure is only 500 μm. New highly sensitive radio frequency (RF) coils for mice were made and used. Significant changes in the spinal cord were found during the development of the disease. The first was the signal intensity of the spinal cord, the second was the size of the spinal cord, and the third was the spinal arteries.

day5

day7

day9

day12

day14

pre

onset

peak

partial remission

complete remission

L3

L4 L5

L6

Fig. (22). Axial T2 weighted MRI of the spinal cord of the same EAE mouse. These images were obtained 5 to 14 days after pathogenic T cell transfer. Clinical stages are also indicated in the figure. The scale bar is 500 μm. The arrows indicate spinal arteries. This figure was made by the modification of Figure 2 of Ref [63].

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The signal intensity of the spinal cord increased from pre-onset to peak, and from peak it returned to remission. The size of the spinal cord increased from pre-onset to peak, and from peak it returned to remission. These phenomena indicate the edematous change of the spinal cord at the lumber region by EAE induction. Our images coincide with the results of Arima et al. [69]. We noticed the change in the spinal arteries. The arteries are the dark spots in Fig. (22) (arrows). The sizes of the spinal arteries were about 100 micrometer. The right and dorsal side arteries got thick at onset (for example, L5 and L6). The dorsal artery of L4 became smaller at the peak. These changes meant an abnormal blood flow in the spinal arteries during EAE induction. Since the changes in the spinal cords and blood vessels were not much compared with their individual differences, it is not easy to find these changes with different mice. mouse A

1mm pre

onset

peak

partial remission

complete remission

onset

peak

partial remission

complete remission

mouse B L3 L

R

L4

L5 pre

1mm

Fig. (23). MRA of mouse spinal arteries. These MRA were obtained from the same A and B mice during EAE induction. This figure was made by the modification of Figure 6 of Ref [63].

The fine magnetic resonance angiography (MRA) of the spinal arteries was obtained using our new highly sensitive coil as shown in Fig. (23). The enlargement and meandering of the dorsal spinal arteries were found at the peak as shown in mouse A (yellow arrows). The occluded vessels were found in the other mice. The occlusion indicates the ischemia of the spinal cord. The lower

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images in Fig. (23) show the branched spinal artery of the same mouse B. The dotted line is the position of the vertebral disk between L3 and L4. The displacement of the branch could be seen. The displacement was large at the peak. The mean value of the displacement was about 2 mm. The displacement of the blood vessels would not be found, if the same mice were not used repeatedly during EAE induction. These changes in the spinal arteries indicate the changes in the blood stream and tissue oxygenation during EAE induction. Therefore, we observed the magnetic resonance spectra of the patient’s brain with MS in order to confirm whether the ischemic event occurred in the human brain or not. Lactate signal could be detected using MRS if the ischemic events occurred. 2. Magnetic Resonance Spectroscopy Fig. (24) shows the MRS of a healthy human brain. The spectrum was obtained from the 8 cm3 gray matter region, which is indicated by a white line. Information about the contents of metabolites and neurotransmitters could be obtained by the spectrum. N-acetylaspartate (NAA), glutamate (Glu), glutamine (Gln), creatine (Crn), choline (Cho), and myo-inositol signals are detectable in a normal healthy brain. The other brain metabolites such as GABA, Glutathione (GSH), and ascorbic acid (vitamin C) can be observed using the special MRS measurement technique [61, 71].

posterior

5

4

N-acetylaspartate - CH3

creatine - CH 2 Glu & Gln (C2) myo-inositol choline - CH 3 creatine - CH3

left

glutamine (C4) glutamate (C4)

right

H 2O

anterior

3 2 d / ppm

1

0

Fig (24). 1H MRS of normal human brain at 3 T. The spectrum was obtained from the gray matter indicated on the left image. The volume of interest is 8 cm3.

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MR spectrum of the plaque region in the patient brain with MS showed a marked increase of lactate and marked decrease of NAA, Crn, and myo-inositol (Fig. 25). The accumulation of lactate suggested the presence of ischemia. This ischemic event coincides with the results of the EAE mice.

creatine

posterior

4

3 2 d / ppm

lactate

choline

left

N-acetylaspartate

right

glutamate & glutamine

anterior

1

0

Fig. (25). 1H MRS of patient brain with MS at 3 T. The spectrum was obtained from the plaque region indicated on the left image. The volume of interest is 8 cm3. This figure was made by the modification of Figure 9 of Ref [64].

Glu levels has been shown to increase in patients with MS [67]. Gln was reported to decrease in Alzheimer’s disease (AD) and to increase in hepatic encephalopathy. However, it is not easy to assess Glu and Gln separately using the human MRS at 3 T. We tried to measure Glu and Gln separately using the echo time (TE) modulated spectra. Glu and Gln contents in the human brain were successfully determined separately at TE = 60 ms. Fig. (26) shows the procedure of the separate quantification of Glu and Gln. Gln increased in the brain of the patient with liver cirrhosis before the pathogenesis of hepatic encephalopathy [60]. In vivo MRS could also be used to assess other human brain metabolites, such as GABA, GSH, and vitamin C. GABA is the major inhibitory neurotransmitter in the brain, and GSH plays an important role in the detoxification of reactive oxygen species [72]. GSH content in the central nervous system should be high in order to maintain the brain function and its neuronal survival [73]. Detection of these metabolites using the conventional point resolved spectroscopy (PRESS) or stimulated echo acquisition mode (STEAM) measurement sequence is not easy at 3 T. We used the MEGA-PRESS sequence (improved PRESS sequence designed

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NAA -CH 3

NAA -CH 2Glu, Gln (C4) Glu (C4)

Cr -CH 3

Cr -CH 2Glu, Gln (C2)

(a) [ human brain ]

Cho- N+ -(CH3)3

by Mescher and Garwood) to detect GABA and GSH [71]. Fig. (27) shows the GSH signal from the human brain, where NMR editing technique is necessary [61, 74]. GSH content of the normal human brain was estimated as 1.9 mM [61]. The content may be useful in assessing neuronal diseases.

(b) [ NAA + Cr ]

(d) [ Glu + Cr ]

Glu (C4)

(c) [ (a) - (b) ]

(f) [ Gln + Cr ]

Gln (C4)

(e) [ (c) - (d) ]

(g) [ (e) - (f) ]

4.0

2.0 3.0 chemical shift / ppm

Fig. (26). Separate determination of Glu and Gln in human brain by using 1H MRS at 3 T. The separate determination could be done using the spectrum obtained at 60 ms of echo time (TE).

Metabolites detected by MRS are important in maintaining human brain homeostasis and function, and therefore we assessed the neuronal degeneration using the information of the brain spectrum. Saito et al. [62] showed that post-

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operative changes in the cerebral metabolites detected by MRS were associated with changes in the cognitive function after carotid endarterectomy.

54F

NAA

GSH

The information about these metabolites detected by MRS may be useful in assessing the effect of IJ powder on the brain.

MEGA-PRESS TE=80ms

4

3

d / ppm

2

1

Fig. (27). GSH signal from human brain. The right figure shows the brain spectrum obtained by the improved MRS sequence designed by Mescher and Garwood (MEGA-PRESS) [71]. GSH signal appears at 2.9 ppm. This spectrum was obtained from the white matter of a normal volunteer indicated on the left image.

3. Brain Temperature Estimation Using MRS MRS also provides some physiological information, such as pH, and has the possibilities of estimating temperatures non-invasively at deep regions. Although the temperatures at deep regions such as the brain are important for metabolism and also for its function, the information about brain temperatures is limited even now. We estimated the brain temperatures of normal volunteers and patients with neurological diseases using MRS [75 - 77]. Fig. (28) shows the short term repeated measurements of brain temperatures in the same normal volunteer. The standard deviation (SD) of our brain temperature measurements was less than 0.1 oC. The human brain temperature distribution could be estimated by this method. Ishigaki et al. [75] applied the technique to patients with unilateral chronic major cerebral artery steno-occlusive disease. A significant correlation was observed between the brain temperature difference (disease side – normal side) and both cerebral blood volume and oxygen extraction fraction ratios. Brain temperature measured by MRS can detect cerebral hemodynamic impairment in patients with unilateral chronic internal carotid or middle cerebral artery occlusive disease.

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NS = 128 (6 min), 5 times (1) 36.86 oC (2) 36.96 oC (3) 36.88 oC (4) 36.85 oC (5) 36.98 oC

right

36.91 +_ 0.06 oC (mean +_ SD)

anterior left

posterior

Fig. (28). Repeated human brain temperature measurements within a short period of time. The subject was a normal healthy volunteer. The temperatures were measured repeatedly 5 times at rest using MRS. SD is the standard deviation.

Fig. (29) shows the dynamical brain temperature change of the healthy volunteer during knee flexion (~ 1 Hz). The brain temperature started to rise just after the start of the task. The brain temperature gradually fell after the end of the task. The physiological dynamic brain temperature change could be detected using MRS [76, 77]. 40 39

T / oC

knee flexion 38

brain

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esophagus 20

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80 60 t / min

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Fig. (29). Dynamical human brain temperature change during knee flexion. Brain and esophagus temperatures were measured by MRS and thermocouple, respectively.

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MRI and MRS provide anatomical, physiological and biochemical information noninvasively such as structure, function, temperature, pH, and metabolism as mentioned above. This diverse information could be used for the assessment of neurological diseases such as MS and dementia (AD). The information could also be useful to assess the therapeutic responses of neurological diseases by IJE. CONCLUDING REMARKS For the discovery of new drugs generated from natural products, we believe that an entomopathogenic fungus, IJ, extract has potential in the neuroprotection and the improvement of cognitive functioning. We have shown that a hot-water extract of IJ reduces astrogliosis in the hippocampal CA3 area of aged mice and is safe to use. We are performing chemical profiling to isolate individual components, and now evaluating IJ powder in a preclinical trial with AD patients. Chemical profiling data will be valuable in establishing the safety of this class of traditional medicines. Yet, we have continued immunohistochemical and molecular analyses, and astrogliosis-improving agents originated from I. japonica extract may be expected for innovating medical drug development of neurological diseases such as AD. Thus, it is possible that I. japonica extract offers attractive potential from complementary and alternative medicine/or medicine to diagnosis for neurological disorders. Meanwhile, to provide a series of the flow of a key I. japonica extract, new MRI techniques enabled that the detection of previously invisible pathological alternations in mice with autoimmune encephalomyelitis, which are used as a model of human MS. We have used MRS to obtain information on neurotransmitters, GSH, and brain temperatures. It showed that demyelinated regions in some MS patients had increased lactic acid content, suggesting the presence of ischemic events or the impairment of blood flow in plaque regions. MRI and MRS provide diverse information about the brain such as its structure, function, temperature, pH, and metabolism. This information could be used for the assessment of MS and dementia. The combination might provide a more sensitive method to assess and understand diseases. Our MRI and MRS techniques could also be used to assess the pharmacological effects of products derived from IJ on patients with neurological disorders. To this end, we will use them to analyze the central nervous system of IJ extract in a clinical trial. CONFLICT OF INTEREST The authors declare no conflict of interest, financial or otherwise.

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ACKNOWLEDGEMENTS This work was supported by JSPS KAKENHI Grant Number 23228001. We thank Chairman Teruo Kagaya (KAGAYA Co. Ltd.) for his encouragement and funds. REFERENCES [1]

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SUBJECT INDEX A Abnormal protein-protein interactions 129 ACE 93, 94, 95, 96 activity 93, 94 enzyme 93, 94, 96 gene 94, 95 gene polymorphism 94, 95 ACE inhibitors 93, 96, 97 penetrating 93, 96 Acetylcholine 21, 33 Acetylcholinesterase 7, 8, 10, 11 inhibitors 1, 11, 20, 23 AchE activity 100, 101 Action 91, 93 neurodegenerative 91 neuro-protective 93 Active compound mechanism 7, 8 Adeno-associated virus (AAV) 27 Adenosine 164, 165 Administration of ciliary neurotrophic factor 26 Allelic variability 61 Alzheimer disease 57, 64, 87, 98, 110, 111, 112 biomarkers 110 diagnosis 111, 112 neuroimaging initiative (ADNI) 57 Amacrine cells (AC) 58, 59, 158 American chemical society (ACS) 113, 117, 119 Amino peptidase A (APA) 94 Amyloid 7, 9, 23, 24, 25, 28, 36, 62, 63, 67, 68, 70, 73, 88, 89, 90, 97, 110, 111, 113, 116, 129 β 23, 24, 36, 110, 111, 113, 116 plaques 24, 63, 68, 89, 113, 129 precursor protein (APP) 7, 9, 23, 25, 28, 36, 62, 63, 67, 70, 73, 88, 90, 97 Analysis, brain tissue 113 Analytical techniques 111, 112, 118, 120 Angelica 7 plants 7 species 7 Angiotensin 92, 93, 94, 95, 96, 97, 101

converting enzyme (ACE) 92, 93, 94, 95, 101 receptor blockers (ARB) 93, 96, 97 Anti-amyloid therapy 97 Anti-Aß antibodies 72 Antihypertensive therapy 93 Anti-inflammatory 1, 98, 99 effects 98, 99 prevention 1 Antioxidant 1, 9, 10, 11, 100, 101, 142 activity 9, 10, 100, 101 effect 142 mechanisms 1, 10, 11 Apolipoprotein E4 3, 4, 28 Apoptotic retinal cells 66 APP locus 40 Aqueous humor 133, 135 Asparagine 119 Astrocytes 2, 4, 5, 6, 10, 22, 31, 35, 36, 37, 38, 62, 63, 159, 160 Astrogliosis 155, 159, 180 Asymmetric dimethyl-arginine 119 AT1R receptors 93, 101 Atherosclerosis 4, 6 Atractylenolide 7, 8 Atrial fibrillation 3

B Basal brain energy metabolism 138 Basal forebrain (BF) 20, 21, 24, 26, 28, 29, 34, 100, 128 cholinergic neurons (BFCNs) 20, 24, 26, 29, 34 Beauvericin 165, 166 amount of 165 Biomarkers mass spectrometry imaging 111 Bipolar cells (BC) 58, 59, 167 Block Aβ aggregation 73 Blood brain barrier 1, 5, 6, 7, 8, 9, 10, 11, 21, 27, 29, 93, 96, 172 damaged 5 catalyze 6 degenerates 1 Blood pressure 3, 92, 97

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high 3, 92 Blood vessels 130, 134, 135, 174, 175 Bone morphogenetic protein (BMP) 26 Brain 3, 7, 26, 34, 37, 89, 90, 91, 93, 97, 98, 101, 113, 116, 117, 131, 137, 140, 142, 149, 159, 160, 177, 178 acetylcholine 7, 90 atrophies 89 cortex 89, 149 dementia 137 -derived neurotrophic factor (BDNF) 26, 34, 37 dysfunctions 160 energy metabolism 142 grooves 131 homeostasis of transition metals 117 homogenates 116 injury 159 neuron cells 91 neurons 91, 97, 98 parenchyma 140 physiology 101 proteome 113 RAS system 93 spectrum 177, 178 trauma 3 Brain metabolites 175, 176 human 176 Brain temperature 178 difference 178 estimation using MRS 178 measurements 178 Brain tissues 31, 90, 91, 92, 128, 147 adjacent 31 invade 90 postmortem 147

C Calcium 97, 145 channel blockers (CCBs) 97 dynamics 145 Carbon chains 142, 143 Cardiac stem cells (CSCs) 30 Catharanthus roseus 100

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Cell(s) 22, 27, 29, 30, 31, 34, 35, 38, 39, 58, 59, 70, 91, 130, 131, 133, 135, 136, 137, 139, 140, 141, 142, 143, 145, 172, 173 amacrine 58, 59 bipolar 58, 59 -derived neurons 22, 35 membrane 139, 140 replacement therapy 27, 29, 30, 31, 34, 35 eukaryotic 58, 59, 70, 91, 130, 131, 133, 137 horizontal 58, 59, 70 neuron 91 transfer 173 transplantation 27, 29, 30 Central nervous system (CNS) 21, 57, 58, 130, 131, 135, 136, 138, 145, 150, 151, 171, 172, 176, 180 Cerebral 20, 21, 29, 68, 114, 178 artery 178 cortex 20, 21, 29, 68, 114 Cerebrospinal fluid 57, 68, 69, 89, 114, 130, 131, 135 Chemical component 162 Chemical energy 130, 131, 133, 134, 135, 136, 138, 139, 141, 142, 143, 144, 145, 146, 147, 149, 150, 151 basic 143 diffuses 151 levels of 136, 141, 142, 144, 145, 146, 147, 150, 151 low levels of 144, 145, 147 Cholesterol 3, 91, 92, 98 high blood 3 Cholinergic activity 101 Cholinergic neurons 20, 21, 22, 24, 26, 27, 28, 29, 30, 31, 33, 34 embryonic 21 generated 34 loss of 24, 29 neurodegenerative 20 Choroidal plexuses 135, 136 Choroid 133, 135, 147, 148 layer 133, 135 plexuses 135, 147, 148 Ciliary 26 neurotrophic factor 26 neurotrophicfactor 26

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Clustered regularly interspaced short palindromic repeats (CRISPR) 39 Codonopsis 8 Cognitive 20, 21, 22, 24, 28, 29, 91, 100, 127, 141, 149, 178 deficits 24, 28, 29 function 20, 21, 22, 91, 100, 127, 141, 149, 178 Cognitive impairment 24, 25, 26, 61, 90, 149 severity of 24 Colocalization 71, 72 Components, neuroinflammatory 63 Confocal scanning laser ophthalmoscopy 56, 65, 66 Contrast sensitivity, spatial 60 Cordyceps 155 Cortical neurons 36 C reactive protein (CRP) 95 Creatinine 119 Cultured cortical neurons 36 Cultured neurons 10 Curcuma longa 100 Cyclic terpenoids 168, 170 Cycloastragenol 7, 8

D Damages 65, 132, 134 neurodegenerative 65 tissue 132, 134 DARC imaging 67 Decreasing brain metabolism 127 Deficit, neurological 100 Degenerative neurons 22, 27 Delay disease progression 11 Detection of Apoptotic Retinal Cells 66 Diagnosis of neurological diseases 154, 156 Disease 3, 4, 41, 56, 63, 64, 65, 74, 87, 88, 91, 92, 95, 99, 101, 111, 112, 127, 129, 138, 155, 173, 177 cardiovascular 3, 92, 155 dementia type 111 eye 56, 63, 74 heart 3, 4, 111 neuronal 177 progressive 88 Disorders 30, 31, 34, 35, 38, 72, 180

neurodenerative 72 neurological 30, 31, 34, 35, 38, 180 Distribution, human brain temperature 178 DLB brains 129 DNA, neuronal 5 Donor plasmid 40 Down syndrome-IPSCs (DSIPSCs) 36 Drug discovery 20, 23, 24, 27, 28, 29, 30, 39, 155, 162 Drug(s) 2, 20, 30, 31, 32, 33, 34, 36, 92, 93, 96, 97, 156 antihypertensive 92, 96 anti-inflammatory 2 neuroprotective 156 penetrating 93, 97 screen 36 screening 20, 30, 31, 32, 33, 34 DS-IPSC-derived cortical neurons 36 Duchenne muscular dystrophy (DMD) 39, 40 Dynamical brain temperature change 179 Dysfunction, neuromuscular 156, 157 Dystrophic neuronal processes 129

E EAE induction 173, 174, 175 Ectopic fat 3, 4, 5 Electrons, molecular hydrogen and high energy 135, 147, 150 Embryoid bodies (EBs) 40 Embryonic 27, 29, 30, 33, 34, 35, 38 basal forebrain 29 stem cells (ESCs) 27, 30, 33, 34, 35, 38 tissue 27, 29 Endogenous APP 40 Endothelial cells 5, 6, 9 Energy, main source of 131, 145 Enhanced depth imaging (EDI) 64 Entomopathogenic fungi 154, 155, 156, 160, 180 Epidermal growth factor (EGF) 26 Episomal vectors 36 Ergosterol peroxide 168 Euphorbia royleana Boiss 100 Evolution of creation 144 Excitotoxicity 20

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F Factors 3, 4, 22, 26, 34, 35, 61, 68, 87, 88, 90, 92, 93, 97, 141, 147 derived neurotrophic 141 discussed 90 line-derived neurotrophic 26 peripheral inflammatory 3 Familial 23, 28, 129 Alzheimer’s disease (FAD) 23, 28 Amyloid polyneuropathy 129 Families of neurotrophic factors 26 Fatty acids 140, 162, 163 Fermentation broth 164, 166 Ferulic acid 7 Fibroblasts 30, 32, 35, 38, 39 human 35, 38 Fibrous components of astrocytes 159, 160 Fimbria-fornix transaction 29 Flame atomic absorption spectrometry (FAAS) 118 Flavonoids 9 Food and drug association (FDA) 20 Formation 5, 6, 7, 8, 9, 10, 20, 23, 35, 40, 87, 91, 92, 97, 100, 101, 118, 140, 142, 143, 146 amyloid 91, 92, 97 amyloid fibril 7, 8 plaque 9, 10, 87, 91 Functional neurons 31, 35, 37, 38 differentiated 37 generated 38 Functions 71, 72, 88, 156 nerve 156 neuronal 88 visual 71, 72 Fungi, entomopathogenic hypocrealean 162

G GABA 30, 32, 34 interneurons 34 neurons 30, 32, 34 Galantamine 11, 20, 21, 96, 99 Gallic acid 9 Ganglion cells (GC) 58, 60, 65, 119 GDNF superfamily 26

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Generated cortical neurons 36 Generation and distribution of energy 130, 136, 137, 140, 141, 142, 144, 147 Genetic changes 36, 37, 41 Genome editing 31, 39, 40 Glaucoma 56, 64, 65, 66, 67, 68, 69, 70, 71, 72, 74 -associated neurodegeneration 70 patients 68, 70 treatment 70, 72 Glaucomatous damage 69 Glutamate 91, 97, 114, 141, 175 levels 141 synthetase 114 Glycyrrhiza glabra 7, 9, 100 Growth factors 22, 26, 32, 33 nerve 26 non-neuronal 26 GSH 176, 177, 178 content 176, 177 signal 177, 178 Guanosine 164

H Hanasanagin 169, 170 Hematopoietic stem cells (HSCs) 30 Hematoxylin-eosin stain 160, 161 Hepatic encephalopathy 176 Hepatocyte growth factor (HGF) 26 Herbal treatment 87 Herpes simplex virus (HSV) 27 Hesperidin 7, 8 High 110, 112, 116, 118, 119, 120, 130, 135, 143, 147, 150 energy electrons 130, 135, 143, 147, 150 performance liquid chromatography (HPLC) 116, 119 selectivity 112, 119 sensitivity 110, 112, 118, 120 Hippocampa 8, 69, 159, 160l neurons 8, 69, 159 structures 160 Histidine 119 Histochemical observation 159 Homeostasis 127, 141, 177 Homocysteine-cysteine disulfide 119

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Homozygous 28, 94, 95 Host neurons 22, 34 Human brain 41, 179 development 41 temperature change 179 Human NSCs 32 Hyper-phosphorylated tau protein 23 Hypertension 9, 92, 93, 94

I ICV injection of amyloidβ 9, 10 IgG-saporin 28, 29 Immunoreactivity 62, 63 Impairment of brain energy metabolism 142 Implantation of healthy neuron 99 Induced pluripotent stem cells (IPSCs) 20, 22, 27, 30, 35, 36, 37, 38, 40 transplantation 35 Inflammation 1, 2, 3, 23, 87, 91, 92, 95, 96, 98, 99, 100, 101, 102, 172 neuronal 96 Inflammatory 3, 6, 9 adipokines 3, 6 cells 6, 9 Information 59, 60 object 59 visual 59, 60 Ingredient, active 8, 9 Inhibitors 25, 32, 33, 97 secretase 25 Inhibitory effects 169 In situ-generated neurons 38 Intracellular processes 145, 146 Intraneuronal accumulation 118 Ion mobility separation (IMS) 118 IPSC-derived 22, 36 cholinergic neurons 22 neurons 36 neurons and astrocytes 36 IPSCs, generated 36 Isariotins 167

K Kinase, creatine 114 Knee flexion 179

L Lactate 140, 176 Late onset Alzheimer’s disease (LOAD) 94, 95 Lateral geniculate nucleus (LGN) 59, 60, 70 Layers 58, 59, 60 dorsal 59, 60 inner 58 of nerve-cell bodies 58 of plexiform 58 primary 59 ventral 59 Leukemia inhibitory factor (LIF) 26 Light energy 133, 135, 149 melanin transform 133 Light 58, 59 entrance arrow 58, 59 receptors 58 Liver stem cells (LSCs) 30 Living substances 162, 163, 165, 170 Local RAS system 87, 92, 93, 97 Locus ceruleus 132, 134, 136 Long-term potentiation (LTP) 8, 25 Loss, neuronal cell 87, 93

M Magnetic resonance angiography (MRA) 174 Magnetic resonance 57, 131, 154, 155, 156, 171, 172, 175, 177, 178, 179, 180 imaging (MRI) 57, 131, 154, 155, 156, 171, 172, 180 resonance spectroscopy (MRS) 154, 155, 156, 171, 175, 177, 178, 179, 180 Markers, characteristic neuropathological 62 Mass analyzer 112 Mass spectrometry 110, 111, 112, 113, 114, 116, 118, 119, 120 applications of 110, 112, 113 direct infusion electrospray 118 electrospray ionization 114, 118 Medial ganglionic eminence (MGE) 34 Medicines 9, 162, 180 complementary 162 traditional 9, 162, 180

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Melanin 127, 130, 131, 136, 138, 140, 141, 142, 143, 144, 145, 146, 147, 149, 151 Melanosomes 140, 143 Memantine 8, 97, 98, 149 Memory 8, 9, 10, 11, 34, 57, 89, 90, 91, 93, 96, 97, 99, 100, 127, 156, 157 ability 156, 157 deficits 8, 10, 34, 156 functions 90, 91, 96, 97, 100 impairment 57, 93, 99, 100, 156 loss 9, 11, 89, 99, 100, 127 Mesenchymal stem cells (MSCs) 27, 30, 32, 33, 39 Metabolism 70, 111, 133, 139, 140, 142, 178, 180 neuronal 70 normal brain carbohydrates 142 reduced brain glucose 142 MGE cells 34 Microglial cells 2, 5, 91, 98 Microtubule associated protein (MAP) 24, 116 Midbrain 38, 148 dorsal 38 Mild cognitive impairment (MCI) 11, 61, 64, 65 Modeling and therapy 20, 39, 40 Models 9, 10, 27, 28, 29, 30, 33, 62, 67, 68, 127, 149, 156, 157, 180 aging rat neuron 127 Moderate wine consumption 4 Molecular hydrogen 135, 136, 137, 141, 142, 143, 145, 146, 147, 150 Monoclonal antibodies 11, 29, 119 Monocyte chemoattractant protein-1 6 Morris water maze test 157, 158, 159 Motor Neuron Diseases 129 Multiple sclerosis 64, 155 Mutations 25, 28, 36, 37, 39, 40, 41, 63, 88, 90 genetic 39, 88, 90 Mycelia 165 Myokines 4 anti-inflammatory 4 Myricetin 9

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N Natural products 155, 162, 165, 166, 167, 169, 180 Neprilysin 20, 33, 68 Nerve 26, 27, 34, 59, 90,91 conduction 90, 96, 101 fibres, optic 59 growth factor (NGF) 26, 27, 34 synapse 90, 91 Nervous 21, 31, 38, 71, 101, 102, 116, 156, 172, 180 system 21, 31, 38, 71, 101, 102, 116, 156, 172, 180 system disorders 31 Network, neuronal 65 Neural 10, 21, 22, 27, 30, 31, 32, 33, 35, 38 development 30, 31 progenitor cells (NPCs) 27, 30, 32 stem cells (NSCs) 10, 21, 22, 27, 30, 31, 32, 33, 35, 38 stem/progenitor cells 31, 32, 38 Neurodegeneration 1, 23, 56, 63, 69, 116, 127, 144, 146, 156 Neurodegenerative 63, 64, 66, 72, 87, 113, 140 age 87 conditions 66, 72 mechanisms 113 process 140 processes 63, 64, 140 Neurodegenerative diseases 20, 21, 24, 27, 29, 30, 31, 38, 39, 94, 101, 129, 144 chronic 144 therapy of 20, 29, 30, 38 Neurodegenerative disorders 35, 57, 95, 111, 128 irreversible 95 Neurofibrillary tangles 2, 20, 21, 23, 24, 57, 71, 113, 116, 128, 138 intracellular 20, 128, 138 Neurogenesis 10, 31 hippocampal 10

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Neuroimaging 57, 64 Neuroinflammation 11, 93 induced 11 Neurokine superfamily 26 Neurological 32, 154, 156, 172, 178, 180 diseases 32, 154, 156, 172, 178, 180 impairment 172 Neuromelanin 151 Neuro-melanin substantia nigra 131 Neuromodulators 93, 101 Neuromyelitis optica 64 Neuronal apoptosis 56 Neuronal- astrocytic interactions 142 Neuronal 9, 25, 26, 28, 34, 63, 66, 73, 89, 90, 114, 129, 144, 176 atrophy 26 cells death 89 cytoplasm 114 death 9, 25, 26, 63, 66, 73 glucose transporter 144 inclusions 129 membrane 90 morphology changes 28 precursor cells 34 survival 176 Neuronal cells 9, 91, 93, 98 dead 91, 98 Neuronal damage 9, 142 diffuse 142 Neuronal degeneration 29, 57, 70, 130, 145, 177 cholinergic 29 Neuronal differentiation 31, 32, 34 cholinergic 31, 32, 34 decreased ChAT-positive 34 promoted cholinergic 34 Neuronal loss 20, 21, 24, 28, 62, 70, 137, 172 progressive 24 selective 137 Neurons 1, 21, 25, 26, 27, 29, 30, 31, 35, 36, 37, 38, 40, 41, 58, 59, 60, 88, 89, 90, 93, 101, 129, 141 cells-derived 35 cortex 89 dead 27, 38 developing 26 differentiated 37 dopaminergic 30

human 35, 37, 41 incubation 25 intermediate 58 isolated 35 koniocellular 59, 60 magnocellular 59 mature 26 parvocellular 59, 60 post-mitotic 40 young 141 Neuropathological 57, 63, 73, 111, 129 changes 73 feature, common 129 mechanisms 57 process 63 Neuropathology 23, 70 Neuropathy 41, 66 optic 66 Neuroprotection 32, 72, 154, 156, 180 effects 156 effects, potential 154 effects of IJE 156 therapies 72 Neurosciences 172 Neurosphere assay 31 Neurotoxic 70, 91, 149 effect 91 fragments 70 insults 70 Neurotoxicity 68, 71, 72, 73 chronic optineurin 71 mediated 72 Neurotoxin cleaners 27 Neurotransmitter(s) 20, 27, 87, 88, 90, 91, 93, 94, 96, 97, 101, 175, 176, 180 acetylcholine 20 Ach 96, 97, 101 activity 87 blockage 94 depletion 87 release 90, 101 inhibitory 176 mediated inhibition Ach 93 release 93 systems 87, 88 Neurotrophic factor(s) (NTFs) 20, 21, 22, 24, 26, 27, 31, 32, 169 biosynthesis 169

194 Frontiers in Clinical Drug Research - Alzheimer Disorders, Vol. 6

exogenous 20 Neurotrophic 26, 31 support 26 tyrosine kinase type 31 Neurotrophin 26, 27 factors 27 family 26 superfamily 26 Neutrophils 5 NFTs, insoluble 25 Nicotine 4 NMDA receptors 91, 97, 147 overactivation of 147 Nuclear magnetic resonance (NMR) 155, 156, 170, 171 spectroscopy analysis 155 Nuclei 59, 128 brain stem 128 lateral geniculate 59

O Occipital-pole brain samples 114 Oligodendrocytes 31, 37, 38 Olmesartan 96, 97 Ophiocordyceps 155 Optic 56, 58, 59, 60, 62, 63, 65, 66, 69 disc 58 nerve 58, 59, 62, 63, 65, 69 nerve head (ONH) 56, 65, 66 radiations 59, 60 Osteogenic differentiation 33 Oxidative stress 9, 23, 66, 67, 68, 87, 91, 92, 93, 99, 101, 111, 114, 118, 146

P Paecilomycine 168 Paeoniflorin 10 Parkinson’s disease (PD) 21, 27, 31, 33, 35, 37, 116, 129 Penostatins 169 Peptide sequence tag (PST) 111 Pericytes 6 Perindopril 97 Peripapillary areas 65 Personalized therapy 30, 35, 41

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Phenylalanyl-phenylalanine 119 Phosphorylation sites 116 Photoreceptors 58, 59, 71 Pigmented epithelium 71 Plant medicines 1, 6, 7, 8, 9, 10, 11 Plaque 23, 28, 172, 176, 180 deposition 23, 28 regions 172, 176, 180 Plasma ACE level 94 Plasmalogen content 114, 115 Plasticity, neuronal 63 Plexiform 58 Pluripotent stem cells (PSCs) 20, 22, 27, 30, 34, 35, 37, 38 Polymorphism 94, 95 Polysaccharides 10, 170 Positron emission tomography (PET) 41, 57 Primary open-angle glaucoma (POAG) 69 Products 114, 154, 162, 163, 164, 167, 180 fermentation 164, 167 protein oxidation 114 silkworm 163, 164 Proliferative neural progenitors 38 Proteins 4, 6, 9, 23, 25, 26, 27, 39, 62, 73, 88, 90, 91, 95, 97, 111, 113, 114, 116, 118, 128, 129, 133, 135 amyloid precursor 9, 23, 62, 73, 88, 97 associated 39 regulatory element binding 4, 6

R Radicals, free 2, 91, 98, 100 Radio frequency (RF) 173 RAS 87, 92, 93, 94, 101 components 93, 94, 101 system 87, 92, 93, 94, 101 Reactive oxygen species (ROS) 10, 91, 93, 127, 141, 144, 176 Receptors 4, 10, 21, 26, 37, 61, 90, 91 muscarinic 61, 90 neurotrophin 21 nicotinic 90 Reduced neuroretinal rim volume 62 Reduction, largest neuronal 62 Regions 20, 21, 31, 93, 113, 114, 127, 128, 172, 178

Subject Index

Frontiers in Clinical Drug Research - Alzheimer Disorders, Vol. 6 195

defined 127, 128 disseminated high intensity 172 neurogenic 31 Renin 92, 93, 94 angiotensin system (RAS) 87, 92, 93, 94 Reprogramming 31, 38, 39 Restoration of neuron function 101 Retinal 56, 57, 58, 59, 60, 62, 63, 65, 66, 67, 69, 70, 71, 72, 73 abnormalities 62 changes 57, 62, 63 diseases 69 ganglion cells (RGCs) 58, 59, 60, 62, 63, 65, 66, 67, 70 imaging 56, 73 nerve fibre layer (RNFL) 56, 62, 63, 65 neuro-degeneration 71 neurons, single 57 Retinoic acid (RA) 22, 33, 34 RGC apoptosis 62, 66, 67, 69, 70, 72 RGC layer 58, 67, 69 Ribosome-inactivating protein (RIP) 29 Rivastigmine 20, 21, 96 RNFL thickness 62, 64, 68, 73

S Salidroside 8, 10 Saponins 7, 8, 9, 10, 11 akebia 9 Schisandrin 8, 10, 11 Schisantherin 10, 11 Senile plaques (SP) 20, 21, 23, 70, 129 Septohippocampal pathway 29 Signal transmission arrow 58, 59 Silkworm powder 163, 164 Sinensis extract 159 SNO proteins 116 Somatic cells 21, 35, 36, 38, 39, 40 Species, reactive oxygen 10, 91, 93, 127, 141, 176 Spheres 130, 147, 148, 151 growing 130, 147, 148, 151 increased melanin energy 151 Spirotenuipesine 168, 169, 170 Stages brain atrophies 89

Stem cell(s) 20, 21, 22, 27, 29, 30, 31, 32, 34, 38, 39, 40 multipotent 30 pluripotent 30, 34, 38 transplantations 30 research 31 Strategies, therapeutic 20, 22, 27, 57, 138 Structures of cyclic terpenoids 168, 170 Subgranular zone 30, 31 substantia nigra 90, 132, 134, 136, 148 Subventricular zone 30, 31 Synapses 25, 58, 127, 140, 141 Synaptic 23, 25, 28, 35, 141 dysfunction 25, 28 functions 23, 35 plasticity 141

T Tangles, neurobrillary 113 Target 157, 159 quadrant 157, 159 Tau 2, 7, 10, 23, 24, 25, 28, 56, 57, 69, 70, 73, 111, 116, 120, 138 neuropathy 70 pathology 23, 24, 25, 28, 56, 73 phosphorylation 2, 7, 10, 138 protein 57, 69, 70, 111, 116, 120 Telomerase activator 7, 8 Tenuipes 162, 163, 164, 165, 167, 168, 169, 170 strains 165, 167 Thickness, macular 65 Thrombosis 3 Tissues 4, 5, 27, 30, 38, 112, 118, 129, 133, 134, 135, 136, 137, 142, 147, 151 damaged neuron 147 muscle 4, 5 pigmented 133 Toxicity 2, 8, 32, 154, 160, 161, 162 subchronic 154, 160, 161 Traditional Plant Medicines 6, 7 Transcription activator-like effector nucleases (TALENs) 39 Transcriptional factors 31, 35, 37 combinations of 35 Transfection of neurotrophin-3 32

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Transplantation 27, 29, 31, 32, 33, 34, 35 of cholinergic neurons 27 Transplanted cells 25, 27 Triglycerides 3 Tropicamide 61

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abnormality 60 cortex 59, 60, 70 cortex primary 59, 60, 70 pathway 59, 60 signals 58, 59 Vitamin 96, 98, 175, 176

U W Umbelliferone 6-carboxylic acid 7

V Vascular 32, 66 changes 66 endothelial growth factor (VEGF) 32 Visceral fat 3, 4 Visfatin 1, 3, 6 Visual 58, 59, 60, 70

Water molecule 127, 130, 131, 133, 134, 135, 136, 138, 143, 145, 146, 149, 150, 151 dissociation 131, 134, 146

Z Zinc finger nucleases (ZFNs) 39