Purinergic Signaling in Neurodevelopment, Neuroinflammation and Neurodegeneration 3031269446, 9783031269448

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Henning Ulrich Peter Illes Talita Glaser   Editors

Purinergic Signaling in Neurodevelopment, Neuroinflammation and Neurodegeneration

Purinergic Signaling in Neurodevelopment, Neuroinflammation and Neurodegeneration

Henning Ulrich  •  Peter Illes  •  Talita Glaser Editors

Purinergic Signaling in Neurodevelopment, Neuroinflammation and Neurodegeneration

Editors Henning Ulrich Department of Biochemistry Institute of Chemistry University of São Paulo São Paulo, Brazil

Peter Illes Pharmacology and Toxicology Leipzig University Leipzig, Sachsen, Germany

Talita Glaser Department of Biochemistry Universidade de São Paulo São Paulo, Brazil

ISBN 978-3-031-26944-8    ISBN 978-3-031-26945-5 (eBook) https://doi.org/10.1007/978-3-031-26945-5 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Foreword

Although the major focus of research in the function and dysfunction of the brain is still centered on the role of the main excitatory and inhibitory transmitter systems, there is an increased recognition that an adequate fine-tuning of these excitatory and inhibitory pathways by different neuromodulation systems is critical for a correct information processing by neuronal networks. Without dwarfing the importance of these ON/OFF excitatory/inhibitory systems mainly operated by glutamate and GABA signaling, evidence is accumulating to pinpoint that alterations of neuromodulation systems are paramount for the onset and correction of brain dysfunction in different neuropsychiatric conditions. Notably, these neuromodulation systems can directly fine-tune neuronal activity, but they can also act indirectly by impacting the communication between neurons and other brain cell types, which coordination is essential for optimal information processing in brain circuits. The purinergic system emerges as a particularly relevant neuromodulation system in the control of brain function, in view of its quadruple mode of action – direct control of neuronal excitability and communication, control of neuron-astrocyte communication, control of neuroinflammation and control of neuro-vascular coupling – through the engagement of two parallel and intertwined signals – ATP and adenosine. These two purinergic signals can be released by all cell types in the brain, either locally as a function of the on-going activity or more globally as signals of cellular stress reflecting an imbalance between metabolic support and imposed workload, which can degenerate into cellular damage in different brain diseases. The pleiotropy of these purinergic signals is testified by their numerous receptors, namely at least six ionotropic receptors for ATP, eight metabotropic receptors for ATP/ADP and four metabotropic receptors for adenosine, most of them engaged in the formation of heteromeric complexes that further increase their diversity. Each of these receptors has different defined cellular and subcellular localizations matching the different sources of released purine signals to ensure mediating the adequate direct and indirect fine-tuning of information flow of neuronal networks. The complexity of this system entails that several possible modifications of its various constituents may be at the origin of different dysfunctions of neuronal circuits, typified in the emergence of different neuropsychiatric diseases, either v

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associated with neurodevelopment, aging or due to external factors altering neuronal functioning. However, the complexity of this system also offers the necessary plasticity to allow exploring the manipulation of its different constituents with the aim of therapeutically interfering to correct different brain dysfunctions. This book compiles a series of examples of studies documenting the importance of the purinergic system in the control of various brain processes (Chap. 3), namely neurodevelopment (Chap. 4) and neurogenesis (Chap. 5), that open new perspectives to harness the purinergic system to achieve neuroregeneration. This information is complemented by a careful analysis of the involvement of the purinergic system in neurodegeneration processes (Chap. 2), detailing its importance, both as a cause and as a therapeutic opportunity in various neuropsychiatric diseases such as autism (Chap. 6), depression (Chap. 7), Alzheimer’s disease (Chap. 8), Parkinson’s disease (Chap. 9), amyotrophic lateral sclerosis (Chap. 10), epilepsy (Chap. 11), neuroinflammation (Chap. 12) and brain tumors (Chap. 13). Finally, there is a presentation of new advances in medical chemistry to optimize tools to directly interfere with the various constituents of the purinergic system (Chap. 14), as well as new strategies to indirectly manipulate the purinergic system, namely with the use of acupuncture (Chap. 15). All these subjects are developed by specialists of recognized competence in their area of research, presenting a systematic and updated view of the current state of knowledge. Indeed, reading this book attests to the relevance of the purinergic system to understand the genesis of neuropsychiatric diseases, as well as the interest in manipulating this system to prevent and treat these diseases. It is expected that this compilation effort carried out by Henning Ulrich, Talita Glaser and Peter Illes will ignite the advancement of knowledge in this scientific area, contributing to a better preservation of brain function, as witnessed by the increase in healthspan with aging offered by caffeine (an antagonist of purinergic receptors for adenosine), and bolster the therapeutic options based on the manipulation of the purinergic system, hitherto still limited to the use of istradefylline (an adenosine receptor antagonist) in the management of Parkinson’s disease. University of Coimbra Coimbra, Portugal

Rodrigo Cunha

Preface

This book is dedicated to the recently deceased Prof. Geoffrey Burnstock, whose crucial contributions along many decades has turned purinergic signaling into a flourishing field of science. The hypothesis of purinergic signaling proposing ATP as a transmitter in non-­ adrenergic and non-cholinergic nerves, formulated by Prof. Geoffrey Burnstock, was initially highly contested, as many researchers found it difficult to accept that ATP, as an intracellular energy storing molecule, could also have signaling functions, when released by cells into the extracellular space. Since the cloning and structural/pharmacological characterization of purinergic receptors made major advances, the field of research on the receptors activated by nucleotides or nucleosides has greatly expanded. In addition, a whole plethora of ectoenzymes degrading ATP into adenosine has also been recognized. Intensive efforts have been undertaken to study the mechanisms of purinergic signaling in basic science, and it has become evident that dysfunctions of the receptors involved and related signaling mechanisms may underlie many diseases, including brain disorders, which are the subject of this book. Neurodegeneration occurs in the context of neuroinflammation, which contributes to neuronal tissue loss and impaired neuronal repair, caused by a decrease in neurogenesis. The reader becomes introduced to the topic of purinergic signaling and its importance for brain physiology by the three initial chapters; this is followed by topics on nucleoside and nucleotide signaling, being important for neurotrophic actions and neurogenesis, neuroinflammation, and brain repair. Besides dealing with the role of purinergic signaling in normal brain physiology and neurotransmission, this book discusses the functions of the purinergic system in diverse neurologic and psychiatric disorders, including autism, major depression, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, epilepsy, and brain tumors. Autism as a psychiatric disorder with neurodevelopmental traits revealed dysfunctions of purinergic signaling and disturbance of purine metabolism as well as proinflammatory conditions. Pharmacological inhibition of purinergic receptor activity, such as by suramin for the treatment of Asperger Syndrome, confirms the role of defects in the purinergic system as an etiological factor. On the other hand, neuroprotective effects were seen upon enhancement of vii

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P1 receptor activity. Purinergic receptors are also causally involved in mood disorders, like depression. Purinergic signaling regulates neuroplasticity, whose dysfunction together with impaired neurogenesis and increased neuroinflammation contributes to the progress of Alzheimer’s disease and age-related dementia. Similar features were observed in basal ganglia diseases, such as Parkinson’s disease, and amyotrophic lateral sclerosis, where the P2X7 and further purinergic P2 and P1 receptors are involved in the progression of neuronal loss. Another important point for the understanding of the mechanisms of purinergic receptors comes from the study of epilepsy, in which the amount of ATP release was associated with a diminished seizure threshold. In addition, the overexpression of P2X receptors in the epileptic brain indicates that dysfunctional purinergic neurotransmission directly contributes to seizures and neuronal excitation/inhibition imbalance. Various P1 and P2 receptors may regulate microglial activity to establish a pro- or anti-brain tumor microenvironment and metabolism. Purinergic receptors have also been suggested to contribute to brain tumor-associated memory impairment. In summary, this book covers the recent advancements of the exciting field of purinergic signaling in brain dysfunction and neurodegenerative diseases. It provides flourishing ideas for new research projects in the field of purinergic signaling, and the discovery of novel targets and therapeutic interventions into purinergic signaling pathways. More recently, selective P1 and P2 receptor agonists and antagonists have been developed by rational design, which are currently tested in clinics. Acupuncture procedures have been established for treatment of neuroinflammation caused by P2X4 and P2X7 receptor activity. It is expected that soon drugs targeting purinergic signaling will be available for treating inflammation-related brain disorders. São Paulo, Brazil Leipzig, Sachsen, Germany São Paulo, Brazil

Henning Ulrich Peter Illes Talita Glaser

Contents

1

Purinergic Signaling: An Overview��������������������������������������������������������    1 Talita Glaser and Henning Ulrich

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Adenosine A2A Receptor-Containing Heteromers and Neuroprotection��������������������������������������������������������������������������������   11 Rafael Franco, Rafael Rivas-Santisteban, Alejandro Lillo, Jaume Lillo, Iu Raïch, Catalina Pérez-Olives, Claudia Llinas del Torrent, Gemma Navarro, and Irene Reyes-Resina

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 Purinergic Signaling in Brain Physiology����������������������������������������������   23 Talita Glaser and Henning Ulrich

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Neurotrophic Actions of Adenosine and Guanosine: Implications for Neural Development and Regeneration? ����������������������������������������   41 Filipa F. Ribeiro, Joaquim A. Ribeiro, and Ana M. Sebastião

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 Purinergic Signaling in Neurogenesis and Neural Fate Determination: Current Knowledge and Future Challenges������������������������������������������   69 Roberta Andrejew, Natalia Turrini, Qing Ye, Yong Tang, Peter Illes, and Henning Ulrich

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 Purinergic Signaling in Autism Spectrum Disorder ����������������������������   97 Iohanna Deckmann, Júlio Santos-Terra, and Carmem Gottfried

7

 Purinergic Signaling in Depression��������������������������������������������������������  129 Deidiane Elisa Ribeiro, Manuella P. Kaster, Henning Ulrich, Sabrina F. Lisboa, and Sâmia Joca

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 Roles of Purinergic Receptors in Alzheimer’s Disease ������������������������  191 Cécile Delarasse and David Blum

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 Purinergic Signaling in Parkinson’s Disease ����������������������������������������  203 Jean Bezerra Silva, Ana Flávia Fernandes Ferreira, Talita Glaser, Henning Ulrich, and Luiz Roberto G. Britto

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10 A  Step in the ALS Direction: Lessons from the Purinome������������������  223 Cinzia Volonté, Justin J. Yerbury, and Ronald Sluyter 11 Purinergic  P2 Receptors in Epilepsy������������������������������������������������������  259 Jonathon Smith and Tobias Engel 12 Purinergic  Signaling in Neuroinflammation ����������������������������������������  289 Beatriz de Andrade de Faria, Ágatha Oliveira-Giacomelli, Mariusz Z. Ratajczak, and Henning Ulrich 13 Purinergic  Signaling in Brain Tumors ��������������������������������������������������  309 Carolina A. Bento, Lara M. F. Guimarães, Vanessa F. Arnaud-­Sampaio, Izadora L. A. Rabelo, Guilherme A. Juvenal, Henning Ulrich, and Claudiana Lameu 14 Development  of Purinergic Receptor Agonists and Antagonists ��������  339 Kenneth A. Jacobson 15 Acupuncture  for Counteracting P2X4 and P2X7 Receptor Involvement in Neuroinflammation ������������������������������������������������������  359 Yong Tang, Patrizia Rubini, Hai-Yan Yin, and Peter Illes Index������������������������������������������������������������������������������������������������������������������  375

Chapter 1

Purinergic Signaling: An Overview Talita Glaser and Henning Ulrich

Abstract  This chapter summarizes some of the evidence that originated the purinergic signaling discovery from the first identification of purines and pyrimidines to their roles as extracellular signaling molecules in physiology and pathophysiology. Briefly, we describe the history of the purinergic signaling establishment and its composition followed by the modern main findings and challenges in this field of research, serving as the base reference for the following chapters. Keywords  Adenosine · ATP · History · Non-adrenergic neurotransmission · Non-cholinergic neurotransmission

Discovery of the Purinergic System The detection of purines and pyrimidines dates back to 1776, when the pharmacist Carl Wilhelm isolated uric acid from bladder stones (Scheele, 1776 in Burnstock, 2012). In the 1850s, the discovery of most of the purines (adenine, xanthine, and hypoxanthine) and pyrimidines (thymine, cytosine, and uracil) took place by Ludwig Karl Martin Leonhard Albrecht Kossel (1853–1927 (in Burnstock, 2012)). The original Kossel report appeared in Chem. Ber., 1885, 18, 79 (Burnstock, 2012), followed by the clarification of the cellular nucleus composition by nucleic acids (Miescher 1874; Hoppe-Seyler 1871 in Burnstock, 2012). The name purine, from the Latin purum uricum (pure uricum), was given by Emil Fischer after his studies of the structure and synthesis of caffeine and similar compounds, for which he was T. Glaser Department of Biochemistry, Universidade de São Paulo, São Paulo, Brazil H. Ulrich (*) Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, Brazil e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 H. Ulrich et al. (eds.), Purinergic Signaling in Neurodevelopment, Neuroinflammation and Neurodegeneration, https://doi.org/10.1007/978-3-031-26945-5_1

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awarded with the Nobel Prize in 1902 (Franzen & Tois, 2012). Pyrimidines were named by Pinner in 1885 (Burnstock, 2012), after his studies on the determination of the sugar part of nucleosides and nucleotides, followed by the nucleotide isolation by Phoebus Aaron Levene (Levene & Jacobs, 1908; Levene & Tipson, 1931). Later, the discoveries in the field focused on the detection of the different purines and pyrimidines in the tissues. The first evidence of purine’s presence and action was done in the cardiovascular field, when Bass (Bass 1914 in Burnstock, 2012) detected adenine, probably as 5′-monophosphate (AMP), in the cardiac tissue. Meanwhile, (Thannhauser & Bommes, 1914  in Abbracchio & Williams, 2001) showed that adenine was not toxic when injected subcutaneously in men. In 1927, Gustav Embden and Margarete Zimmermann described the detection of adenosine monophosphate in skeletal muscle (Embden and Zimmermann, 1927, in Burnstock, 2012). Even though the field was enlightened by many findings, the discovery of adenosine 5′-triphosphate (ATP), which is the main player of the modern purinergic system, was simultaneously reported in 1929 by two independent groups (Fiske & Subbarow, 1929; Lohmann, 1929). Twelve years later, Fritz Lipman established the concept of the high-energy phosphate bond, describing ATP as the main cell energetic molecule (Lipman, 1941). Along with the discovery of ATP’s role in intracellular energetics, Alan Drury and Albert Szent-Györgyi von Nagyrápolt (Drury & Szent-Györgyi, 1929) described the extracellular effects of the purine’s counterparts. They observed that crude extracts from various tissues of sheep exerted intense pharmacological effects, like cardiac arrest and arrhythmia, when injected intravenously into other species. Later, other researchers found that ATP could also induce a pronounced heart block (Drury, 1936). In the 1940s, the war conflicts were intensified leading to increased visibility of traumatic shock’s studies. The purinergic field proposed that the injury caused by trauma would release augmented amounts of ATP and other adenine molecules contributing to vasodilatation (Bielschowsky & Green, 1944). The study with injured patients led to findings described in the book Biological Actions of Adenine Nucleotides (Green & Stoner, 1950). The book detailed the correlation between the length of the phosphate chain and biological activity in the heart. These findings were later corroborated by many researchers in different tissues, such as muscles, intestine, uterus, etc. (reviewed in Burnstock, 2012). In the 1960s, the studies of electrophysiology had significantly improved showing membrane hyperpolarization upon adenosine application and depolarization upon ATP  application in neuromuscular junctions and arteries (Axelsson & Holmberg, 1969). Studies of the neuromuscular junction were tremendously important for the elucidation of the purinergic signaling. Firstly, Buchthal and Folkow (1948) reported potentiation of acetylcholine-evoked contraction of skeletal muscle fibers upon ATP exposure. Then, in the 1950s, Pamela Holton reported the transient elevations of extracellular ATP in electric-stimulated auricular nerves. She detected ATP through the firefly luminescence method (Strehler & Totter, 1952, 1954) and proposed that noradrenaline liberation from sympathetic nerve endings would stimulate ATP

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liberation as well (Holton, 1959). These findings are extremely important and the basis of the concept of purinergic co-transmission, described by Geoffrey Burnstock in 1976 (Burnstock, 1976). Corroborating this concept, Zimmerman and Pull and McIlwain identified electrical stimulation with ATP release (Pull et  al., 1972; Zimmermann, 1978), Dowdall detected ATP  storage in synaptosomes (Dowdall et  al., 1974), and another study detected ATP-induced depolarization through a reduction in K+ conductance (Akasu et al., 1983). The identification of ATP’s role in the neurotransmission by Geoffrey Burnstock started as an experimental surprise (Burnstock et al., 1970). At the time, Geoffrey and his students, Max Bennett and Graham Campbell, stimulated the nerves of the smooth muscle from Taenia coli with atropine in the presence of bretylium which is a blocker of noradrenaline’s release from nerve terminals. They expected to report depolarization and contraction upon direct muscle stimulation; instead, they observed hyperpolarization and muscle relaxation (Burnstock et al., 1963). Their data were discussed worldwide recognizing the non-adrenergic, non-cholinergic (NANC) neurotransmission. During the 1960s and 1970s, various research groups accumulated evidence for NANC neurotransmission in different systems and models, like respiratory, cardiovascular, urogenital, and gastrointestinal tracts (Axelsson & Holmberg, 1969). Aiming on the identification of the neurotransmitter of NANC, Geoffrey Burnstock intensified the studies in gut and bladder NANC. At that time, some criteria characterizing neurotransmitter were already established in the literature, such as the synthesis and storage in nerve terminals, intracellular Ca2+ signaling dependence, inactivation by reuptake or by degradation through ectoenzymes, and blockage or potentiation due to nerve stimulation or exogenous application (reviewed by Burnstock, 2006). After some unsuccessful trials with the known neurotransmitter candidates including monoamines, neuropeptides, amino acids, etc., Geoffrey Burnstock decided to have a try on ATP, based on the early findings of ATP role in the cardiovascular system. Surprisingly, ATP worked beautifully in NANC, while fulfilling all the demands of a neurotransmitter (Burnstock et al., 1970, 1978). Thus, in 1972, Geoffrey published an article in Pharmacological Reviews journal, proposing the purinergic neurotransmission (Burnstock, 1972). This proposition encountered extensive resistance from academics and was even ridiculed at some symposia. The main reason for such denial was the well-described role of ATP as the energetic molecule in biochemical pathways. In the mid-1970s, some researchers detected the release of purines in the synapses of the central nervous system and even observed the release from synaptosomes obtained from the whole brain, cortex, and striatum. The release was triggered by high K+ concentrations  or veratridine (White, 1978, 1984; Pamela Potter & White, 1980). In the 1990s and early 2000s, electrophysiological recordings proved the existence of ATP-evoked depolarization, induced currents, and synaptic transmission in diverse neurons, including celiac ganglion, spinal cord, hippocampus, locus coeruleus, and cortex (Pankratov et al., 2002, 2006; Messemer et al., 2013). The extensive role of ATP and adenosine in the brain, from trophic factors to neurotransmission and inflammation roles, has been widely studied as we shall detail in the following chapters. Figure 1.1 summarizes the main discoveries of the field.

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Fig. 1.1  Timeline highlighting the main discoveries of the purinergic signaling field

Nomenclature and Components The purinergic signaling system mainly comprises the extracellular receptors that are activated by ATP/ADP or adenosine and ectoenzymes that convert ATP into adenosine by sequential hydrolysis of the phosphate groups. The receptors and enzymes are ubiquitously expressed throughout the organism; the tissues vary on the levels and subtypes of each component. Geoffrey Burnstock initially proposed two different types of receptors based on the ligands, P1 and P2, for adenosine and ATP/ADP, respectively (Burnstock, 1976; Burnstock et  al., 1978). In 1985, after cloning of some receptors, an additional division was created, separating P2X and P2Y subtypes (Burnstock & Kennedy, 1985). P1 receptors are composed of four different seven-transmembrane G-coupled receptor subtypes, named A1, A2A, A2B, and A3. They are activated by adenosine with distinct  binding  affinities: A1 (1–10  nM), A2A (30  nM), A2B (1000  nM), A3 (100 nM) (Ciruela et al., 2010). The type of coupled G-protein also varies according to each receptor, diversifying intracellular signals mediated by adenosine. The A1 subtype is coupled to Gi/o protein, leading to inhibition of adenylyl cyclase and decreased levels of cAMP, while also activating phospholipase C and triggering Ca2+ signaling. The A2A receptor is coupled to Gs proteins promoting adenylyl cyclase activity. A2B and A3 subtypes  are coupled to Gs or Gq/11  proteins trigerring both adenylyl cyclase and phospholipase C  activities (Fredholm et  al., 2011). Caffeine and theophyline are antagonists of every adenosine receptor (Rivera-Oliver & Díaz-Ríos, 2014). The NH2 terminus of adenosine receptors is located extracellularly and possesses some glycosylation sites that can afect the activation of the receptor, while the COOH terminus shows sites for both phosphorylation and palmitoylation, which interfere in the desensitization and internalization mechanisms (Sun et al., 2017). Thereby, longer COOH terminus tail provides different functions and intracellular transmission, where A2A receptor presents a 122-amino acid-long tail while others vary between 30 and 40 amino acids (Tong, 2017).

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P2X receptors have two putative transmembrane domains and are trimeric ligand-gated ion channels that can assemble as hetero- or homomeric; the seven cloned subunits are named P2X(1-7) in humans (ref). They are non-selective cation channels triggered by ATP only. Its activation causes membrane depolarization and intracellular Ca2+ influx (Burnstock, 2007). The P2Y receptors are also seven-transmembrane G-coupled receptors, named P2Y (1,2,4,6,10,11–14), encoded by distinct genes (Burnstock, 2013). Most of P2Y receptors are coupled to Gq protein, triggering Ca2+ release from intracellular stores. Recombinant P2Y11 receptor can also modify intracellular cAMP levels, while P2Y12, P2Y13, P2Y14 are coupled to Gi/o (Erb & Weisman, 2012). The first references for each receptor is as follows: P2Y1 (Webb et al., 1993), P2Y2 (Lustig et al., 1993), P2Y4 (Communi et  al., 1995), P2Y6 (Communi et  al., 1996), P2Y11 (Communi et al., 1997), P2Y12 (Hollopeter et al., 2001), P2Y13 (Communi et al., 2001), and P2Y14 (Chambers et al., 2000). Some non-mammalian orthologues or receptors with sequence homology that are not sensitive to purines or pyrimidines represent the missing numbers (p2y3, p2y5, p2y8-10) and are known as orphan receptors. The p2y7 receptor is a leukotriene B4 (LTB4) receptor (Tong, 2017), while further receptors were cloned in Xenopus laevis (p2y8) and chick (p2y3 and p2y5). P and Y upper case letters can only be applied in for mammalian receptors, following the current NC-IUPHAR policy nomenclature. Human P2Y receptor types differ in their sensitivity to ligands. For instance, P2Y1, P2Y12, P2Y13 subtypes  are ADP receptors, while the P2Y11 subtype  is mainly ATP-sensitive, and P2Y4 or P2Y6 receptors are mainly activated by pyrimidines, such as UTP and UDP, respectively (Schachter et al., 1996; Fumagalli et al., 2004; Gachet & Hechler, 2005). Both ATP and UTP activate the P2Y2 receptor (Nicholas et al., 1996). Interestingly, the P2Y14 receptor is activated by UDP and nucleotide sugars such as UDP-glucose and UDP-galactose (Chambers et al., 2000). Extracellular purine and pyrimidine concentration levels vary according to their transportation balance across the membrane and the activity of extracellular ectoenzymes called ectonucleotidases (Cappellari et al., 2012). These enzymes hydrolyze nucleoside tri-, di-, and monophosphates and dinucleoside polyphosphates and produce nucleoside diphosphates, nucleoside monophosphates, nucleosides, phosphate, and inorganic pyrophosphate (PPi) (Zimmermann et  al., 2012). The final products of the hydrolysis are salvaged by reuptake through specific transporters and rephosphorylation inside the cell (Zimmermann et al., 2012). Ectonucleotidases are divided into four major groups: ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases), ecto-5′-nucleotidase (eN), ecto-­ nucleotide pyrophosphatase/phosphodiesterases (E-NPPs), and alkaline phosphatases (APs) (Zimmermann et al., 2012). E-NTPDases are the main nucleotide-hydrolyzing enzymes in purinergic signaling, by hydrolyzing nucleoside tri- or diphosphates to nucleoside monophosphates (Zimmermann et al., 2012). The ecto-5′-nucleotidase hydrolyzes nucleoside monophosphate producing extracellular adenosine from AMP (Yegutkin, 2014). E-NPPs do not hydrolyze AMP; instead, they hydrolyze nucleoside tri- and diphosphates, dinucleoside polyphosphates, ADP ribose, and NAD+ (Zimmermann et al., 2012).

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APs hydrolyze nucleoside tri-, di-, and monophosphates, pyrophosphate, and a variety of monoesters of phosphoric acid (Zimmermann et al., 2012). There are eight NTDPases in mammalian tissues, including the extracellular NTPDase1, NTPDase2, NTPDase3, and NTPDase8. NTPDase1 is also known as CD39, while eN is known as CD73 (Zimmermann et al., 2012). NTPDase1 to NTPDase3 enzymes were found as dimers to tetramers. There are seven different E-NPPs (NPP1-7) and four APs (Tissue nonspecific AP, Placental AP, Germ cell AP, and Intestinal AP) (Zimmermann et al., 2012). Some ecto enzymes,  such as ecto-nucleoside diphosphate kinase and ecto-­ adenylate kinase, may act in opposite ways by interconverting extracellular nucleotides, such as ecto-nucleoside diphosphate kinase and ecto-adenylate kinase. Adenosine deaminase deaminates extracellular adenosine to inosine, controlling the activity of P1 receptors (Goueli & Hsiao, 2019). Figure  1.2 shows a simplified scheme with the connections and components of purinergic signaling.

Purinergic Signaling Perspectives Purinergic signaling research has vastly increased in the past decades, since its discovery. Diverse pharmacological and genetic engineering  tools have been developed to modulate purinergic  receptors and ectoenzyme activities and expression levels for the understanding of purinergic signaling mechanisms. Corroborating the importance of the field in pathophysiological processes, some of purinergic signaling activity  modulators have been already tested in clinical trials. In addition, diverse diseases have been implicated with altered purinergic signaling. The

Fig. 1.2  Scheme at cellular membrane level showing the P1 (A1, A2A, A2B, A3) and P2 (P2X and P2Y) receptors interacting  with their natural ligands. Extracellular enzymes convert adenosine 5`-triphosphate (ATP) into adenosine (ADO) through consecutive hydrolysis. Adenosine (ADO) can be converted into inosine or recycled by reentering the cell through equilibrative nucleoside transporters (ENT). Further abbreviations: ENTDPase ecto-nucleoside triphosphate diphosphohydrolase, CD73 ecto-5’-nucleotidase, ENPP ectonucleotide pyrophosphatase/phosphodiesterase

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following chapters of this book show the promising evidences of the field of purinergic signaling in neurodevelopment, neuroinflammation and neurodegeneration, thus highlighting the recent advances.

References Abbracchio, M.  P., & Williams, M. (2001). Purinergic neurotransmission: An historical background. In Purinergic and pyrimidinergic signalling I., (Newby 1984) (pp. 1–16). https://doi. org/10.1007/978-­3-­662-­09604-­8_1 Akasu, T., Hirai, K., & Koketsu, K. (1983). Modulatory actions of ATP on membrane potentials of bullfrog sympathetic ganglion cells. Brain Research, 258(2), 313–317. https://doi. org/10.1016/0006-­8993(83)91157-­5 Axelsson, J., & Holmberg, B. (1969). The effects of extracellularly applied ATP and related compounds on electrical and mechanical activity of the smooth muscle Taenia coli from the Guinea-pig. Acta Physiologica Scandinavica, 75(1–2), 149–156. https://doi.org/10.1111/j.1748­1716.1969.tb04366.x Bielschowsky, M., & Green, H. (1944). Organic and inorganic pyrophosphates as shock-inducing agents. Nature, 153, 524–525. Buchthal, F., & Folkow, B. (1948). Interaction between acetylcholine and adenosine triphosphate in normal, curarised and Denervated muscle. Acta Physiologica Scandinavica, 15(2), 150–160. https://doi.org/10.1111/j.1748-­1716.1948.tb00492.x Burnstock, G. (1972). Purinergic nerves. Pharmacological Reviews, 24, 509–581. Burnstock, G. (1976). Do some nerve cells release more than one transmitter? Neuroscience, 1(4), 239–248. Burnstock G. (2006). Historical review: ATP as a neurotransmitter. Trends in Pharmacological Sciences, 27, 166–176. Burnstock, G. (2007). Purine and pyrimidine receptors. Cellular and Molecular Life Sciences, 64(12), 1471–1483. https://doi.org/10.1007/s00018-­007-­6497-­0 Burnstock, G. (2012). Purinergic signalling: Its unpopular beginning, its acceptance and its exciting future. BioEssays, 34(3), 218–225. https://doi.org/10.1002/bies.201100130 Burnstock, G. (2013). Purinergic signalling: Pathophysiology and therapeutic potential. The Keio Journal of Medicine, 62(3), 63–73. Burnstock, G., & Kennedy, C. (1985). Is there a basis for distinguishing two types of P2-purinoceptor? General Pharmacology, 16(5), 433–440. Burnstock, B. Y. G., Dewhurst, D. J., & Simon, S. E. (1963). Sodium exchange in smooth muscle distribution of ions in smooth muscle may differ considerably from that of observed by Goodford & Hermansen (1961). They found that over 95% of. Journal of Physiology, 167, 210–228. Burnstock, G., et al. (1970). Evidence that adenosine triphosphate or a related nucleotide is the transmitter substance released by non-adrenergic inhibitory nerves in the gut. British Journal of Pharmacology, 40(4), 668–688. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcg i?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=4322041 Burnstock, G., Cocks, T., Crowe, R., & Kasakov, L. (1978). Purinergic innervation of the Guinea-­ pig urinary bladder. British Journal of Pharmacology, 63, 125–138. Cappellari, A. R., et al. (2012). Characterization of Ectonucleotidases in human Medulloblastoma cell lines: Ecto-5′NT/CD73  in metastasis as potential prognostic factor. PLoS One, 7(10), e47468. Chambers, J.  K., et  al. (2000). A G protein-coupled receptor for UDP-glucose. The Journal of Biological Chemistry, 275(15), 10767–10771. https://doi.org/10.1074/jbc.275.15.10767

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Ciruela, F., et al. (2010). Adenosine receptors interacting proteins (ARIPs): Behind the biology of adenosine signaling. Biochimica et Biophysica Acta, 1798(1), 9–20. S0005-2736(09)00369-1 [pii]. https://doi.org/10.1016/j.bbamem.2009.10.016 Communi, D., et  al. (1995). Cloning and functional expression of a human uridine nucleotide receptor. Journal of Biological Chemistry, 270(52), 30849–30852. https://doi.org/10.1074/ jbc.270.52.30849 Communi, D., Parmentier, M., & Boeynaems, J.-M. (1996). Cloning, functional expression and tissue distribution of the human P2Y6Receptor. Biochemical and Biophysical Research Communications, 222(2), 303–308. https://doi.org/10.1006/bbrc.1996.0739 Communi, D., et al. (1997). Cloning of a human purinergic P2Y receptor coupled to phospholipase C and adenylyl cyclase. Journal of Biological Chemistry, 272(51), 31969–31973. https://doi. org/10.1074/jbc.272.51.31969 Communi, D., et al. (2001). Identification of a novel human ADP receptor coupled to Gi. Journal of Biological Chemistry, 276(44), 41479–41485. https://doi.org/10.1074/jbc.M105912200 Dowdall, M. J., Boyne, A. F., & Whittaker, V. P. (1974). Adenosine triphosphate. A constituent of cholinergic synaptic vesicles. The Biochemical Journal, 140(1), 1–12. Drury, A. N. (1936). The effective refractory period, full recovery time, and premature response interval of ventricular muscle in the intact unanæsthetised cat and rabbit. Quarterly Journal of Experimental Physiology, 26(2), 181–200. Drury, A. N., & Szent-Györgyi, A. (1929). The physiological activity of adenine compounds with especial reference to their action upon the mammalian heart. The Journal of Physiology, 68(3), 213–237. https://doi.org/10.1113/jphysiol.1929.sp002608 Erb, L., & Weisman, G.  A. (2012). Coupling of P2Y receptors to G proteins and other signaling pathways. Wiley Interdisciplinary Reviews. Membrane Transport and Signaling, 1(6), 789–803. https://doi.org/10.1002/wmts.62 Fiske, C.  H., & Subbarow, Y. (1929). Phosphorus compounds of muscle and liver. Science, 70(1816), 381–382. Franzen, R., & Tois, J. (2012). Purine and sugar chemistry on solid phase - 100 years after the Emil Fischers Chemistry Nobel Prize 1902. Combinatorial Chemistry & High Throughput Screening, 6(5), 433–444. https://doi.org/10.2174/138620703106298617 Fredholm, B.  B., et  al. (2011). International union of basic and clinical pharmacology. LXXXI. Nomenclature and classification of adenosine receptors - an update. Pharmacological Reviews, 63(1), 1–34. https://doi.org/10.1124/pr.110.003285 Fumagalli, M., et al. (2004). Cloning, pharmacological characterisation and distribution of the rat G-protein-coupled P2Y13 receptor. Biochemical Pharmacology, 68(1), 113–124. https://doi. org/10.1016/j.bcp.2004.02.038 Gachet, C., & Hechler, B. (2005). The platelet P2 receptors in thrombosis. Seminars in Thrombosis and Hemostasis, 31(02), 162–167. Goueli, S. A., & Hsiao, K. (2019). Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39. PLoS One, 14(10), 1–19. https://doi.org/10.1371/journal. pone.0220094 Green, H. N., & Stoner, H. B. (1950). Biological actions of the adenine nucleotides. H.K. Lewis. Hollopeter, G., et al. (2001). Identification of the platelet ADP receptor targeted by antithrombotic drugs. Nature, 409(6817), 202–207. https://doi.org/10.1038/35051599 Holton, P. (1959). The liberation of adenosine triphosphate on antidromic stimulation of sensory nerves. The Journal of Physiology, 145, 494–504. Levene, P.  A., & Jacobs, W.  A. (1908). On glycothionic acid. The Journal of Experimental Medicine, 8(10), 557–558. Levene, P. A., & Tipson, R. S. (1931). The ring structure of adenosine. Science, 74, 521. Lipman, F. (1941). Metabolic generation and utilization of phosphate bond energy. In Advances in enzymology and related subjects of biochemistry (1st ed., pp. 99–162). Lohmann. (1929). Über die Pyrophosphatfraktion im Muskel. Naturwissenschaften, 17, 624–625. Lustig, K. D., et al. (1993). Expression cloning of an ATP receptor from mouse neuroblastoma cells. Proceedings of the National Academy of Sciences of the United States of America, 90(11), 5113–5117.

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Messemer, N., et  al. (2013). P2X7 receptors at adult neural progenitor cells of the mouse subventricular zone. Neuropharmacology, 73, 122–137. S0028-3908(13)00228-1 [pii]. https://doi. org/10.1016/j.neuropharm.2013.05.017 Miescher, F. (1874). Die Spermatozoen einiger Wirbelthiere. Ein Beitrag zur Histochemie. Verha. Naturforsch. Ges. Basel 6, 38–208. Nicholas, R. A., et al. (1996). Uridine nucleotide selectivity of three phospholipase C-activating P2 receptors: Identification of a UDP-selective, a UTP-selective, and an ATP- and UTP-specific receptor. Molecular Pharmacology, 50(2), 224–229. Available at: https://molpharm.aspetjournals.org/content/50/2/224 Pamela Potter, T. D., & White. (1980). Release of adenosine 5′-triphosphate from synaptosomes from different regions of rat brain. Neuroscience, 5(7), 1351–1356. Pankratov, Y. V., Lalo, U. V., & Krishtal, O. A. (2002). Role for P2X receptors in long-term potentiation. Journal of Neuroscience, 22(19), 8363–8369. Pankratov, Y., et al. (2006). Vesicular release of ATP at central synapses. Pflugers Archiv European Journal of Physiology. [Preprint]. https://doi.org/10.1007/s00424-­006-­0061-­x Pull, I., Jones, D.  A., & Mcilwain, H. (1972). Superfused cerebral tissues in hypoxia: Neurotransmitter and amino acid retention; labile constituents and response to excitation. Hypoxic conditions have been imposed on incubating superfused neocortical tissues. Their lactate production increased and K and phosphocreatine content fell moderately; under these conditions the release of amino acid and putative neurotransmitters into the superfusing medium was studied. During the hypoxia, total free amino acids in. Journal of Neurobiology, 3(4), 311–323. Rivera-Oliver, M., & Díaz-Ríos, M. (2014). Using caffeine and other adenosine receptor antagonists and agonists as therapeutic tools against neurodegenerative diseases: A review. Life Sciences, 101(1–2), 1–9. Schachter, J. B., et al. (1996). Second messenger cascade specificity and pharmacological selectivity of the human P2Y1-purinoceptor. British Journal of Pharmacology, 118(1), 167–173. https://doi.org/10.1111/j.1476-­5381.1996.tb15381.x Strehler, B. L., & Totter, J. R. (1952). Firefly luminescence in the study of energy transfer mechanisms. I.  Substrate and enzyme determination. Archives of Biochemistry and Biophysics, 1(40), 28–41. Strehler, B. L., & Totter, J. R. (1954). Determination of ATP and related compounds: Firefly luminescence and other methods. Methods of Biochemical Analysis, 1, 341–356. Sun, B., et  al. (2017). Crystal structure of the adenosine A2A receptor bound to an antagonist reveals a potential allosteric pocket. Proceedings of the National Academy of Sciences of the United States of America, 114(8), 2066–2071. https://doi.org/10.1073/pnas.1621423114 Tong, B.  C.-K. (2017). Adenosine A2a receptors form distinct oligomers in protein detergent complexes Nicole. Physiology & Behavior, 176(5), 139–148. https://doi.org/10.1002/1873­3468.12367.Adenosine Webb, T. E., et al. (1993). Cloning and functional expression of a brain G-protein-coupled ATP receptor. FEBS Letters, 324(2), 219–225. White, T. D. (1978). Release of ATP from a synaptosomal preparation by elevated extracellular K+ and by veratridine. Journal of Neurochemistry, 30(2), 329–336. White, T.  D. (1984). Characteristics of neuronal release of ATP. Progress in Neuro-­ Psychopharmacology & Biological Psychiatry, 8(4–6), 487–493. Yegutkin, G. G. (2014). Enzymes involved in metabolism of extracellular nucleotides and nucleosides: Functional implications and measurement of activities. Critical Reviews in Biochemistry and Molecular Biology. [Preprint]. https://doi.org/10.3109/10409238.2014.953627 Zimmermann, H. (1978). Turnover of adenine nucleotides in cholinergic synaptic vesicles of the Torpedo electric organ. Neuroscience, 3(9), 827–836. https://doi. org/10.1016/0306-­4522(78)90035-­0 Zimmermann, H., Zebisch, M., & Sträter, N. (2012). Cellular function and molecular structure of ecto-nucleotidases. Purinergic Signalling. [Preprint]. https://doi.org/10.1007/ s11302-­012-­9309-­4

Chapter 2

Adenosine A2A Receptor-Containing Heteromers and Neuroprotection Rafael Franco, Rafael Rivas-Santisteban, Alejandro Lillo, Jaume Lillo, Iu Raïch, Catalina Pérez-Olives, Claudia Llinas del Torrent, Gemma Navarro, and Irene Reyes-Resina Abstract  Adenosine is a nucleoside with key functions in cell metabolism and in the regulation of several physiological processes in mammals. Its actions are mediated by cell surface adenosine receptors, which belong to class A rhodopsin-like G protein-coupled receptors (GPCRs). Four have been so far identified: A1, A2A, A2B, and A3. The potential of these receptors as therapeutic targets has culminated in the relatively recent approval of istradefylline in patients with Parkinson’s disease. Istradefylline is a selective antagonist of the A2A receptor (A2AR), which is considered a target for neurodegeneration. The A2AR is among the GPCRs for which more receptor-receptor interactions have been identified, thus opening the possibility that the real therapeutic target is the A2AR in a receptor heteromer context. This chapter aims at presenting the partners of the A2AR with a special focus on the potential of their ligands, agonists, antagonists and allosteric modulators, in affording neuroprotection. The neuroprotective potential of A2AR-containing heteromers expressed in microglia is also presented. Keywords  Microglia · Receptor heteromers · Neurodegenerative diseases · Neuroprotection

R. Franco (*) Department of Biochemistry and Molecular Biomedicine, School of Biology, University of Barcelona, Barcelona, Spain Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas (CiberNed), Spanish National Institute of Health, Carlos III, Madrid, Spain School of Chemistry, University of Barcelona, Barcelona, Spain e-mail: [email protected] R. Rivas-Santisteban · J. Lillo · C. Pérez-Olives Department of Biochemistry and Molecular Biomedicine, School of Biology, University of Barcelona, Barcelona, Spain © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 H. Ulrich et al. (eds.), Purinergic Signaling in Neurodevelopment, Neuroinflammation and Neurodegeneration, https://doi.org/10.1007/978-3-031-26945-5_2

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Introduction Adenosine, a purine nucleoside, is an autocoid, that is, a molecule that participates in events that occur in virtually all living cells on Earth. Among others, adenosine is a sneak of local metabolic status; its concentration increases when ATP is used during a physiological event, for example in a muscle in action, or as a consequence of a pathological event such as a stroke. Consequently, the modulatory role of adenosine is variable and depends on the type of cell and whether it is a physiological or a pathological situation. Adenosine regulates cell events via four receptors, A1,  A2A, A2B, and A3, that belong to rhodopsin-like class A G-protein-coupled receptors (GPCRs). The first discovered adenosine receptor, the A1, as well as the A3, are coupled to Gi proteins and, therefore, activation by agonists leads to the inactivation of adenylate cyclase and a decrease in the level of cytosolic cAMP. In contrast, Gs is the cognate protein of A2A and A2B receptors, whose activation leads to activation of the adenylate cyclase and increases of cAMP levels. Activation of adenosine receptors may regulate ion channel activity, mitogen-activated protein kinase (MAPK) signaling pathway activation, etc. (see Alexander et al. (2021) for review). GPCRs are able to form heteromers. Probably the first heteromers reported were for taste receptors but, also, heteromerization of class C GPCRs was discovered decades ago (Chandrashekar et al., 2006; Kniazeff et al., 2011). The formation of heteromers for class A receptors was proposed several decades ago, but it was only Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas (CiberNed), Spanish National Institute of Health, Carlos III, Madrid, Spain A. Lillo Department of Biochemistry and Physiology, Faculty of Pharmacy and Food Science, University of Barcelona, Barcelona, Spain I. Raïch Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas (CiberNed), Spanish National Institute of Health, Carlos III, Madrid, Spain Department of Biochemistry and Physiology, Faculty of Pharmacy and Food Science, University of Barcelona, Barcelona, Spain C. Llinas del Torrent Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, Campus Universitari, Bellaterra (Barcelona), Spain G. Navarro Department of Biochemistry and Physiology, Faculty of Pharmacy and Food Science, University of Barcelona, Barcelona, Spain Institut de Neurociències de la Universitat de Barcelona, Barcelona, Spain I. Reyes-Resina Department of Biochemistry and Molecular Biomedicine, School of Biology, University of Barcelona, Barcelona, Spain Department of Biochemistry and Physiology, Faculty of Pharmacy and Food Science, University of Barcelona, Barcelona, Spain

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in the late twentieth century that experimental evidence was provided. Despite residual reluctance in the field, there is consensus in that class A GPCRs may form homodimers, heterodimers, and even higher-order structures via direct receptor-­ receptor interactions (Fuxe & Ungerstedt, 1974; Agnati et al., 1981, 1982; Franco et al., 2007; Ferré et al., 2009; Cordomí et al., 2015). The main difference between class A and class C receptor dimers is that intramembrane helices participate in class A dimer formation, whereas the enormous extracellular N-terminal domain of class C receptors is the main driver of receptor-receptor interactions in such a subfamily of GPCRs. In that sense, there are crystal structures of class C receptors formed using only the extracellular domains, that is, lacking transmembrane domains (Felder et  al., 1999; Kunishima et  al., 2000; Reyes-Cruz et  al., 2001; Tsuchiya et al., 2002; Muto et al., 2007). There are currently more than 600 heteromers described in the scientific literature. An ad hoc website (http://www.gpcr-­hetnet.com/) informs about GPCRs that do interact (or that do not interact) and provides the appropriate reference (for interaction or lack thereof) (Borroto-Escuela et  al., 2014). A quick inspection of the network in the “gpcr-hetnet” homepage reveals the highly populated “self” interactions between taste receptors, the less populated “self” interactions between class C metabotropic glutamate receptors, and about a dozen interactions involving class B GPCRs. The vast majority (hundreds) of identified heteromers are found for class A receptors. Interestingly, the adenosine A1 (A1R) and A2A (A2AR) receptors are among the few that show more than five possible interactions. Checking http://www.gpcr-­ hetnet.com/ (accessed on Nov 17, 2021), and just taking into account the four adenosine receptor types, the A1 receptor interacts with 1 class C and 10 class A GPCRs and the A2AR interacts with 1 class C and 11 class A GPCRs. So far, the interactions identified for the A3 are with the A1R (Hill et al., 2014) and the A2AR, (Lillo et al., 2020) and the single interaction identified for the A2BR is with the A2AR (Hinz et al., 2018; Gnad et al., 2020). For comparison, the ß1-adrenoceptor interacts (so far) with five class A GPCRs, one of them being the A1R (Mercier et al., 2002; Xu et al., 2003; Zhu et al., 2005; Somvanshi et al., 2011; González et al., 2012; Chandrasekera et al., 2013).

Adenosine and Neuroprotection Adenosine is a key neuromodulator whose role changes depending on the physiological situation. In addition, in any neurological alteration adenosine receptors are involved, turning them into therapeutic targets. A main issue is identifying the best receptor to target and whether the optimal drug is an agonist, an antagonist, or an allosteric modulator. For example, generally speaking, A1R agonism is neuroprotective but can cause side effects, whereas A2AR antagonism is both neuroprotective and devoid of serious side effects. In fact, istradefylline, sold as NouriastR in Japan and NourianzR in the USA, has been approved for adjuvant anti-parkinsonian therapy (Mizuno & Kondo, 2013; Saki et al., 2013; Kondo et al., 2015; Berger et al., 2020).

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The compound addresses symptoms, although it may delay disease progression, i.e., it may prevent the death of dopaminergic neurons. Unfortunately, regulatory bodies do not have any protocol to address neuroprotection in humans. Therefore, the neuroprotective potential of ligands of adenosine receptors can only be addressed in cell and animal models. Informative reviews of the neuroprotective potential of A2AR as target for neuroprotection are found elsewhere (Ongini et al., 1997; Schwarzschild et al., 2002, 2006; Popoli et al., 2004; Stone, 2005; Simola et al., 2008; Johnson et al., 2008; Sebastião & Ribeiro, 2009; Jenner et al., 2009; Armentero et al., 2011; Chen, 2014; Jenner, 2014; Kondo et al., 2015; Navarro et al., 2015; Cunha, 2016; Chen & Cunha, 2020). This chapter is devoted to the potential of the A2AR but from the point of view of A2AR-containing heteromers. A summary of mediators and mode of action of targeting the A2AR to afford neuroprotection is provided in Fig. 2.1.

Heteromers of A2AR with Class C Metabotropic Glutamate Receptors The number of interactions reported for class A and class C receptors is, so far, limited. In what concerns the A2AR, only the interaction with the class C metabotropic glutamate receptor 5 has been, to our knowledge, reported (Nishi et al., 2003; Cabello et al., 2009). The interaction is framed in what is known, at the rodent level, as the “indirect pathway” of the SNC motor control circuit. Macromolecular complexes formed by the A2AR, the metabotropic glutamate-5 receptor, and the dopamine D2 receptor are expressed in striatal medium spiny GABAergic neurons of this “indirect” pathway. The macromolecular complex has a relevant role in integrating the adenosine, dopamine, and glutamate inputs although its role in neuroprotection is uncertain (Ferré et al., 2007; Borroto-Escuela et al., 2018). In other words, the neuroprotective role attributed to A2AR antagonists may be mediated by complexes in the striatum made up of the adenosine receptor and other GPCRs.

Heteromers of A2AR with Other Adenosine Receptor Subtypes Fifteen years ago, we reported the interaction between two adenosine receptors: the A1R and the A2AR. It was soon noticed that it participated in the regulation of neurotransmitter release/uptake by neurons or glia (Ciruela et  al., 2006; Cristóvão-­ Ferreira et  al., 2013). Remarkably, the A1R/A2AR heteromer is a sensor of the concentration of extracellular adenosine. The heteromer allows both positive and negative regulation of glutamate release (in synapses from cortical neurons) (Ciruela et  al., 2006), something that individually expressed receptors could  not achieve. Mechanistically there is a Gi-mediated signaling at low adenosine concentrations and a Gs-mediated signaling at high adenosine concentrations. Heteromer allows

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Fig. 2.1  Mediators and potential mechanisms of neuroprotection via A2AR-containing macromolecular complexes

alternative signaling, via A1R or via A2AR; individual receptors would not be able to get alternative Gi or Gs engagement. A structural model consisting of a heterotetramer formed by two homodimers coupled to one Gs and one Gi protein has been constructed (Navarro et al., 2016, 2018). At low adenosine levels, the A1R-Gi unit is engaged (due to higher affinity of the agonist/receptor interaction), and at higher concentrations of the nucleoside, the A2AR-Gs unit is engaged while A1R-Gi operation is blocked; the positioning of the long C-terminal domain of the activated A2AR would impede A1R activation and Gi engagement. As indicated in the paper we identified a new mechanism of signal transduction that implies a cross-communication between Gi and Gs proteins guided by the C-terminal tail of the A2AR.  This mechanism provides the molecular basis for the operation of the A1-A2AHet as an adenosine concentration-­sensing device that modulates the signals originating at both A1R and A2AR (Navarro et al., 2018). Based on these results, it is tempting to speculate that the A1R/A2AR heteromer should be considered as potential therapeutic target in pathological situations where glutamate excitotoxicity contributes to neurodegeneration. A2A and A2B receptors are coupled to Gs proteins  and, therefore, it was not expected that these receptor types would interact. However, recent data from different laboratories have demonstrated such an interaction (Hinz et  al., 2018; Gnad et al., 2020), thus suggesting that all four adenosine receptor types may interact. The benefit of the occurrence of A2A/A2B receptor heteromers is still an open question. On the one hand, the higher the expression of the A2BR and the higher the expression of the heteromer, the lower the functional activity of the A2AR. Apart from the possible conformational limitations in the activation mechanisms, the affinity of the selective A2AR ligands is markedly reduced in the heteromeric context. Future

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research should identify the circumstances that require A2AR inactivation through upregulation of A2BR cell surface expression and heteromerization (Hinz et  al., 2018). On the other hand, it has been proved a prominent role of the heteromer in brown adipose tissue in the sense that the key regulatory role of adenosine in this tissue is mainly mediated by the A2AR/A2BR heteromer. In fact, some properties of adenosine receptor-mediated regulation of cell metabolism in brown adipose tissues are lost in animals KO for the A2AR or for the A2BR (Gnad et al., 2020). A relevant finding that has been silent for years may shed light on the potential of the heteromer in neuroprotection, namely that A2BR is not only an adenosine but also a netrin receptor (Corset et  al., 2000). Apart from participating in axon guidance, netrin is involved in, among other, neuroprotective events, neurological recovery after spinal cord injury (Gao et al., 2020) and neuroprotection of nigral dopaminergic neurons in Parkinson’s disease animal models (Jasmin et al., 2021). Research is required on the integration of adenosine receptor-mediated functionality with the actions of netrin. We have recently demonstrated the occurrence of A2AR/A3R heteromer in heterologous expression systems but also in primary neurons and microglia. At first sight, it may seem that the heteromer would behave as the A1R/A2AR heteromer because one receptor is coupled to Gi and another to Gs. However, one property of the heteromer is a reduction of Gi-mediated signaling even in the absence of selective A2AR agonists. Interestingly, the selective A2AR antagonists reverses the blockade of A3R function (Lillo et al., 2020). In terms of neuroprotection, these findings open the possibility of increasing the neuroprotective effect of A2AR antagonists also because they allow A3R-mediated signaling; does neuroprotection via A2AR antagonists require the activation (by endogenous adenosine) of the A3R?

Heteromers of A2AR with Other Class A GPCRs Considering the data and references in http://www.gpcr-­hetnet.com/, the A2AR interacts with dopamine D2, D3, and D4, with GPR37, with μ-opioid, with cannabinoid CB1 and CB2, and with purinergic P2Y1, P2Y2, P2Y12, and P2Y13 receptors (Yoshioka et al., 2002; Hillion et al., 2002; Canals et al., 2003; Woods et al., 2005; Fuxe et al., 2005; Carriba et al., 2007, 2008; Navarro et al., 2008; Schicker et al., 2009; Dunham et  al., 2009; Borroto-Escuela et  al., 2010; Nakata et  al., 2010; Marcellino et al., 2010; Suzuki et al., 2013; Bonaventura et al., 2014; Franco et al., 2019; Torvinen et  al., 2005; Borroto-Escuela et  al., 2021; Beggiato et  al., 2014, 2016; Chiodi et al., 2016). In addition, it is reported that the A2AR may interact with angiotensin II AT1 (De Oliveira et al., 2017) and histamine H3 (Márquez-Gómez et al., 2018) receptors. Almost any of those receptors that are capable of interacting with the A2AR have been linked to  neurodegeneration or to  neuroprotection. Accordingly, it would be good to consider the A2AR when addressing the neuroprotective potential of targeting these class A receptors (dopamine, histamine, angiotensin) that are able to interact with the A2AR.

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A2AR-Containing Heteromers, Microglia, and Neuroprotection Neurons targeted to die progressively in a neurological disease, such as Parkinson’s or Alzheimer’s, are already committed, and there are few options to extend their lifetime by directly targeting them. Probably the best approach would be to boost, via brain-penetrant molecules arriving via systemic circulation, the antioxidant mechanisms that would decrease oxidative stress occurring in a suffering neuron. Targeting neuronal receptors may in  vitro increase pro-survival factors, but it is doubtful that the approach may be relevant in real neuropathological situations related to neurodegeneration. Targeting glia is a very promising approach as glial cells are more resistant to adverse conditions. In addition, it is often found that microglial cells surrounding neurodegenerative structures are activated; therefore, activated microglia appear as attractive targets. In fact, pharmacological manipulation of microglia can lead to phenotypically different cells. Despite recent controversy of the several phenotypes that microglial cells may display, for operative purposes there are two basic phenotypes among activated microglia  – the proinflammatory (or M1 according to the nomenclature used for macrophages) and the neuroprotective (or M2 according to the nomenclature used for macrophages) (see Franco and Fernández-Suárez (2015) for review). Can adenosine receptors in microglia be targeted to skew microglia toward the M2 phenotype? Indeed, this is a possibility, as there are adenosine receptors expressed in activated microglia and it is a matter of finding the right receptor(s) to be targeted and the right compound (agonist, antagonist, inverse agonist, allosteric modulator) or compound combinations. Several years ago, we found that microglia surrounding pathological structures in the brain of patients having Alzheimer’s disease expressed the A2AR, whereas the expression of the receptor in the microglia in control brains (supposedly non activated resting microglia) was negligible (Angulo et al., 2003). Recent studies in our laboratory confirm A2AR occurrence in microglia from animal models of Alzheimer’s disease and even upregulation of the A2AR-A3R heteromer. Besides, RNAseq data indicate a marked  expression of the A1R in primary microglia obtained from the brains of transgenic and control mice with Alzheimer’s disease (data in preparation). In summary, the A2AR and the A2AR-containing heteromers in microglia deserve attention to manage the skewing of microglia toward phenotypes that lead to increased neuronal survival in neurodegenerative diseases.

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Chapter 3

Purinergic Signaling in Brain Physiology Talita Glaser and Henning Ulrich

Abstract  Since the discovery of purinergic signaling in the 1960s, many researchers focused on the central nervous system neurotransmission. Some neurons present high permeability to Ca2+ upon ATP challenge. Here, in this chapter, we highlight some of the most relevant findings regarding communication in the brain through P1 adenosine receptors or P2 purine receptors. Glial cells may regulate synaptic transmission and modulate neuronal activity by releasing neuroactive substances including ATP. Conversely, ATP may act at glial P2 receptors influencing glial functions. The role of ATP as gliotransmitter involved in the control of neuronal or neuroglial circuits will be discussed in this chapter. Keywords  Adenosine · Extracellular ATP · Neuron · Astrocytes · Microglia · Neural communication

Adenosine Receptors Adenosine has been extensively studied regarding its functions in the modulation of synaptic plasticity through the activation of presynaptic G-protein coupled receptors in the presynaptic membrane. The class of G-protein adenosine receptors comprises four subtypes (A1, A2A, A2B, and A3 receptors). Adenosine receptors are classified according to the G protein with which they interact. While A1 and A3 receptors bind to Gi/o proteins, the A2A and A2B receptor subtypes interact with Gs/ olf proteins. A1 and A2A receptors bind with high affinities to their agonist T. Glaser Department of Biochemistry, Universidade de São Paulo, São Paulo, Brazil H. Ulrich (*) Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, Brazil e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 H. Ulrich et al. (eds.), Purinergic Signaling in Neurodevelopment, Neuroinflammation and Neurodegeneration, https://doi.org/10.1007/978-3-031-26945-5_3

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adenosine, while A2B and A3 receptors reveal less binding affinity for adenosine (Borea et al., 2018). Adenosine receptors are differently expressed in the brain, suggesting that they exert distinct functions in respective brain regions. A1 subtypes are expressed by neurons, astrocytes, oligodendrocytes, and microglial cells in the cortex, hippocampus, cerebellum, basal cortex, and olfactory bulb, among other brain regions (Liu et  al., 2019). A2A receptors are also expressed in the cerebral cortex, amygdala, hypothalamus, hippocampus, thalamus, and cerebellum, while A2B and A3 receptors are found at reduced expression rates in the brain and are mainly in the hippocampus and cerebellum (Stockwell et al., 2017; Liu et al., 2019). Intracellular and extracellular adenosine formation occurs by degradation of ATP into AMP which then is further hydrolyzed into adenosine. Adenosine transport through the plasma membrane occurs through the actions of equilibrative nucleoside transporters (ENT 1 and 2) (Ho et al., 2020), which are highly expressed in the brain. Adenosine may be converted into AMP or degraded into inosine and hypoxanthine or uric acid by adenosine deaminase or xanthine oxidase, respectively. As reviewed by Sebastião et al. (2018), adenosine kinase is highly expressed by astrocytes, and there is a close interaction between neurons and glial cells in regards of the control of synaptic activity and plasticity. Importantly, different from the liberation of neurotransmitters by exocytosis triggered by membrane depolarization, adenosine is released into the extracellular space by nucleoside transporters and then exerts its effects on synaptic activity. The inhibitory action of the presynaptic A1 receptor on synaptic activity results from its reduction of intracellular cAMP accumulation, due to the activation of Gi/o protein, and reduction of protein kinase A phosphorylation cascade and inhibition of calcium channels, resulting in a reduction of exocytotic neurotransmitter release (Liu et  al., 2019). On the other hand, upon stimulation by adenosine, A1 receptors in the postsynaptic membrane activate potassium channels, and the resulting K+ outflow hyperpolarizes the membrane potential. The ATP-sensitive potassium channel is activated in the substantia nigra with subsequent membrane potential hyperpolarization (Liu et al., 2019). Different from the A1 receptor reducing synaptic activity, the A2A receptor augments the synaptic potential and induces the release of the excitatory neurotransmitter glutamate by activating Gs protein-mediated cAMP and protein kinase A signaling (Liu et  al., 2019). The A2A receptor  mediates desensitization of the A1 receptor, whereby removing the A1 receptor-mediated blockade (Lopes et al., 2002) by protein kinase A mediated facilitation of synaptic transmission (Rebola et al., 2003). In view of that, the balance between inhibitory A1 and excitatory A2A receptor activities is extremely important for physiological conditions in the brain. Co-localization of presynaptic A1 and A2A receptors has been found at the presynaptic membranes of cortico-thalamic glutamatergic terminals (Ciruela et al., 2006a, 2006b; Fernández-Dueñas et al., 2017). While low adenosine concentrations mostly stimulate the A1 receptor, maintaining synaptic inhibition, higher adenosine concentrations result in A2A activation inducing glutamate release (Fernández-Dueñas et al., 2017). Interactions between A2A receptors with NMDA and mGlu5 glutamate receptors have been demonstrated (Fig. 3.1). NMDA receptors may enhance long-­ term potentiation by evoking excitatory postsynaptic currents, while A2A-mGlu5

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Fig. 3.1  Adenosine receptor roles at pre- and post-synapses. When located at the pre-synapses, the A1 receptor inhibits adenylate cyclase, while the A2A receptor activates adenylate cyclase and parallelly inhibits A1 receptor action, therefore modulating glutamate release. Postsynaptic A1 receptors modulate K+ channel activity and increases the permeability for this ion, causing hyperpolarization. The A2A receptor dimerizes with glutamate receptors, such as NMDA or mGlu5 receptors, and enhances their responses upon activation by their agonists

receptor interactions may result in the facilitation of glutamate release (reviewed by (Glaser et al., 2020)). Disturbances in A1/A2A receptor signaling is observed in neurodegenerative diseases. Excessive A2A receptor activity occurs in neurodegenerative disorders, such as in Parkinson’s disease. Therefore, A2A receptor antagonism is a therapeutic approach for Parkinson’s disease (Hinz et al., 2018). Besides affecting synaptic potential and thereby the exocytosis of glutamate-­ induced excitatory neurotransmission, adenosine acts in the modulation of synapses by interacting with other receptors as reviewed by Sebastião et  al. (2018). Such modulatory interactions occur by desensitization of autofacilitatory nicotinic acetylcholine receptors. The desensitization of autofacilitatory presynaptic nicotinic receptors is enhanced in conditions of A2A receptor activation and diminished in the presence of A2A receptor antagonists. On the other hand, A1 receptor inhibition augments presynaptic nicotinic receptor desensitization. It can be concluded that A2A and A1 receptors have opposite functions, such as also in the regulation of excitatory glutamate liberation (Ribeiro & Sebastião, 2010). A2A receptor-mediated adenosinergic regulation of autofacilitation of acetylcholine release by desensitization of presynaptic facilitatory nicotinic receptors was shown in the hippocampus (Cunha & Ribeiro, 2000). Autofacilitatory regulation of acetylcholine release by adenosine

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receptors is also important for neuromuscular transmission, as demonstrated by Nascimento et al. (2014). Direct interactions were also described between D2 dopamine and A2A receptors, which are highly expressed in the striatum, where A2A receptors exert antagonistic actions to those of D2 receptors. A2A receptor stimulation promoted inhibition of D2  receptor-mediated locomotor activation, while A2A receptor antagonism had opposite effects (Prasad et al., 2021). A further target for direct adenosine action is the calcitonin gene-related peptide (CGRP) receptor, which participates in vascular nociception and pain sensation (reviewed by Ribeiro & Sebastião, 2010). In view of that, adenosinergic signaling has been studied for the management of neuropathic pain (Shaw et al., 2020). The A2A receptor is important for the liberation of neurotrophic factors and facilitating of synaptic actions of these factors, such as by BDNF (Tebano et al., 2008) and GDNF (Gomes et al., 2006), being in line with promotion of neuronal survival. Expression of the A2B receptor is upregulated in conditions of metabolic cell death, occurring during stroke and other types of brain injury (Long et al., 2013). Selective agonism of this receptor has shown to be neuroprotective in conditions of transient focal brain ischemia in a rat model (Dettori et al., 2021). In the same line, agonism of the A3 receptor reduced brain injury subjected to subarachnoid hemorrhage (Li et al., 2020). Neuroprotective activities exerted by A2B and A3 receptor activation involves anti-inflammatory features. Overall, adenosinergic signaling has crucial functions in the regulation of excitatory neurotransmission as well as in cell survival pathways, which become important following brain injury and during neurodegenerative diseases.

P2 Receptors The roles of ATP as a neurotransmitter in the CNS were postulated by Geoffrey Burnstock in 1972 (Burnstock, 1972). In this seminal paper, he describes extracellular ATP as a non-adrenergic and non-cholinergic transmitter in the gastrointestinal smooth muscle (reviewed by Verkhratsky et al., 2020). Later, this hypothesis was extended by the co-transmission of ATP with acetylcholine and noradrenaline (Burnstock, 1976). P2 receptors were then grouped into P2X and P2Y receptor classes (Burnstock & Kennedy, 1985). The physiological roles of ATP secretion into the extracellular space and signal transduction are mediated by ionotropic P2X receptors, which are ATP-gated ion channels, and P2Y receptors, which couple to G-proteins. P2 receptors are widely expressed in various tissues, and their actions are not limited to the nervous system. In the CNS, postsynaptic actions of P2 receptors modulate the effects of neurotransmitters (Illes & Alexandre Ribeiro, 2004; Hussl & Boehm, 2006; Fischer & Krügel, 2007) (see Figure 3.2. for brain area-specific P2 receptor expression). While at presynaptic sites and at the synapses as well, purinergic signaling through P2X and P2Y receptor subtypes control the release of the major neurotransmitters (ACh,

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Fig. 3.2  Relative expression levels of the different P2 receptors in the brain regions of the mouse. Darker shades represent higher detection of transcripts and proteins

Fig. 3.3  Highlights of P2X and P2Y receptors in neurons, astrocytes, and microglia

norepinephrine, dopamine, serotonin, glutamate, GABA) (Yang et al., 1994; Cunha & Ribeiro, 2000; Papp et al., 2004; Sperlágh et al., 2007; von Kügelgen & Hoffmann, 2016), among other functions, which are discussed in this chapter. Besides their roles in neurotransmission and maintaining homeostasis in cellular, tissue, and body functions, these receptors also act in diverse developmental and tissue regeneration related processes, such as brain development (reviewed by (Huang et al., 2019)), bone development (Lenertz et  al., 2015) and repair (Jørgensen, 2019), cardiac development (Burnstock, 2017), differentiation of hematopoietic stem cells (Filippin et al., 2020), immune response regulation (Sévigny et al., 2015), and neuroprotection (Miras-Portugal et  al., 2016) (Fig.  3.3). P2 receptors are intensively investigated based on their roles in pathophysiological conditions, including inflammation, cardiovascular disease, neurodegeneration, and cancer, as well as in therapy of these conditions (Burnstock 2006).

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P2X Receptors The CNS show the expression of all the seven subunits (P2X1–P2X7) of ionotropic P2 receptors (Soto & Rubio, 2001). These are trimeric assembled channels that allow fast entrance of cations (Na+, K+, Ca2+) across the cell membrane (Ralevic & Burnstock, 1998; Burnstock, 2007). Cation flux through P2X receptor channels leads to membrane depolarization and opening of voltage-gated calcium channels. Ca2+ inflow activates down-stream signaling cascades, such as protein kinase C, PI3 kinase/Akt, GSK3, Ca2+/calmodulin-dependent protein Kinases II (CAMKII), or mitogen-activated protein kinases (MAPKs) (Khakh & Egan, 2005; Miras-Portugal et al., 2016; Kopp et al., 2019). P2X receptors assemble from three homo- or heteromeric subunits with distinct pharmacological properties. An exception from the P2X receptor cation channels is the P2X5 subtype that allows Cl− inflow (Bo et al., 2003). P2X2, P2X2/3, P2X4, and P2X7 receptors open large pores allowing the conductance of permeable cations and fluorescent dyes, upon long agonist exposure (Lin et al., 2016). In the CNS, P2X receptor signaling can cross talk to some neurotransmitter receptors, such as nicotinic acetylcholine (nACh), GABAA, and 5-HT3 receptor channels (Fig. 3.2). P2X1 receptor detection in cerebellar astrocytes (Burnstock & Loesch, 2017) mainly assemble with P2X5 receptor subunits forming a functional heteromer. P2X2 receptors are abundantly expressed in the CNS, such as cerebral and cerebellar cortex, hippocampus, habenula, nigrostriatal system, basal ganglia, ventrolateral medulla, area postrema, nucleus of solitary tract, and spinal cord (Nörenberg & Illes, 2000; Pankratov et al., 2009). The P2X2 receptor subtype may be co-­assembled with P2X6 or P2X3 receptor subunits. These receptors control presynaptic neurotransmitter release (Nörenberg & Illes, 2000; Roberts et al., 2006). However, the broadly expressed P2X4 receptor overlaps with P2X2/3 receptor expression  and controls neurotransmitter release (Nörenberg & Illes, 2000; Soto & Rubio, 2001). The P2X5 receptor is less present in the CNS, its detection is mainly in astrocytes as P2X1/5 receptor heteromer (Nörenberg & Illes, 2000) while the P2X6 receptor assembles with P2X2 and P2X4 subunits. The P2X7 receptor is the most studied receptor in the field, although, its presence in neurons is controversial  discussed (Illes et al., 2017). The expression of the P2X7 receptor in neurons has been questioned (Sim, 2004), even though some researchers detected this receptor in neuronal preparations of cultured cerebrocortical neurons (Wirkner et  al., 2005; Fischer et al., 2009), in presynaptic nerve terminals of the CNS (Deuchars et al., 2001) and synaptosomal preparations of cerebellar granule cells (Sánchez-Nogueiro et  al., 2005), hippocampal neurons (Rodrigues et al., 2005), and cerebrocortical neurons (Alloisio et  al., 2008). Responsive P2X7 receptors  were reported in presynaptic neuron and synaptosomes even in the samples of P2X7 receptor knockout mice (Nörenberg & Illes, 2000; Sánchez-Nogueiro et  al., 2005; Sperlágh et  al., 2006; Carrasquero et al., 2009; Oliveira et al., 2010). Later, various splice variants and small nucleotide polymorphisms of mammalian P2X7 receptors were described

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(Sperlágh & Illes, 2014). Interestingly, this receptor is broadly distributed in the CNS and may escape inactivation by genetic deletion (Sperlágh & Illes, 2014). This discussion between experts led to the establishment of three criteria in order to classify the purinergic receptor as the P2X7 receptor subtype: (1) low divalent cation concentration bathing medium potentiating ATP/Bz-ATP response (Yan et  al., 2011); (2) Bz-ATP potency higher than that of ATP; and (3) effective blockage by low concentrations of selective P2X7 receptor antagonists. Microglia cells are the immune cells of the brain and show intense P2X4/P2X7 receptor expression, whose expression levels increase following nerve injury (Tsuda et  al., 2003; Guo et  al., 2007). Neuronal and astroglial P2X7 receptors regulate neuro- and gliotransmitter release, connecting the responses between microglia to those of neurons/astrocytes (Ferrari et al., 2006; Sperlágh et al., 2006). Activation of P2X7 receptors also triggers several signaling pathways, such as interleukin-1β secretion, phospholipase D and nuclear factor k-light-chain-enhancer of activated B cell  (NF-kB)  activation, apoptosis, production of reactive oxygen species, rearrangement of skeleton, and membrane blebbing (Ferrari et  al., 2006; Sperlágh et al., 2006). P2X receptors show widespread expression throughout the CNS. When expressed on neurons, they support the role for extracellular ATP acting as a fast neurotransmitter. Postsynaptic P2X receptor-mediated fast synaptic currents were  first described in the medial habenula (Edwards et al., 1997). Then, ATP-induced currents were registered in various CNS regions, including the spinal cord (Bardoni et al., 1997), locus coeruleus (Nieber et al., 1997), hypothalamic arcuate nucleus (Wakamori & Sorimachi, 2004), hippocampal CA1 (Pankratov et al., 1998), CA3 regions (Mori et al., 2001), and the somatosensory cortex (Pankratov et al., 2003). A typical situation is that ATP-mediated synaptic currents account for 5–15% of total excitatory postsynaptic currents (EPSCs), which are largely mediated by glutamate (Pankratov et al., 2007; Abbracchio et al., 2009). ATP-induced currents in neurons of medial habenula and somatosensory cortex show high Ca2+ permeability. In addition, when NMDA receptors are blocked by Mg2+, P2X receptors are an important route for Ca2+ influx during resting membrane potentials (Abbracchio et al., 2009). The Ca2+ entry may lead to the assembly of either P2X or AMPA receptors in the plasma membrane and the consequences are strong synaptic connections over a longer period (long-term potentiation, LTP or long-term depression, LTD) (Pankratov et al., 2007). As a matter of fact, P2XR are involved in regulation of synaptic plasticity by controlling LTP and LTD in different brain regions (Pankratov et al., 2009). Interestingly, P2X receptors may interact with diverse ionotropic receptors, such as nicotinic acetylcholine receptors, GABAA, and 5-HT3 receptor channels (Nakazawa et al., 1994). The interaction occurs at the intracellular domains, between the cytoplasmic loop of the Cys-loop channels and the C-terminus of the P2X receptors (Boué-Grabot et al., 2003; Khakh & Egan, 2005).

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P2Y Receptors P2Y receptors are coupled to G-proteins, which α and βγ subunits are tightly associated. Binding of a ligand to the P2Y receptor triggers the dissociation of the α subunit from the βγ dimer, allowing the Gα subunit to initiate the further downstream events. Interestingly, the βγ subunits are also active participants in P2Y receptor signaling, such as by regulation of ion channel activities. Each P2Y receptor subtypes may be linked to one or more of the four main heterotrimeric G protein subfamilies (Gs, Gi/o, Gq/11, and G12/13) (Abbracchio et al., 2006; Erb & Weisman, 2012). For example, receptor-induced activation of Gq/11 proteins stimulates phospholipase C (PLC)-β that then cleaves phosphatidylinositol-bisphosphate (PIP2) in the membrane into inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 binds to its receptor on the endoplasmic reticulum, mobilizing intracellular calcium, while DAG activates protein kinase C (PKC) leading to an intracellular phosphorylation cascade. In addition, once complexed to Ca2+, calmodulin activates the calmodulin kinase (CaMK) inducing diverse intracellular events. For instance, Gq/11 coupling was observed for P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors (Abbracchio et  al., 2006; Erb & Weisman, 2012; Volonte et  al., 2012). Unexpectedly, P2Y2, P2Y4, P2Y13, and P2Y14 receptors via Gi/o-protein activation can also stimulate the PLC–IP3–DAG–Ca2+ release pathway  and PKC and/or CaMK signaling (Abbracchio et al., 2006, 2009; Erb & Weisman, 2012). P2Y12, P2Y13, and P2Y14 receptors preferentially bind to Gi/o-proteins. Moreover, Go was also reported for P2Y2, P2Y4, and P2Y11 receptors. Gi-protein activation is classically associated with the inhibition of adenylate cyclase and decreased cyclic AMP (cAMP) levels; however, the activation of the Gi/o-proteins may also have other consequences. Hence, differential activation of G protein subtypes can also be agonist-specific for P2Y receptors. In this case, ATP binding to the P2Y11 receptor stimulates the Gs protein activating adenylate cyclase, while UTP stimulation leads to Gq protein stimulation and PLC activation (Communi & Boeynaems, 1997; White et al., 2003). Some P2Y receptors may combine with other monomeric G proteins (Erb & Weisman, 2012), such as Gα12/13 subunits for P2Y2 or P2Y12 receptor activation. Moreover, P2Y receptors can couple to the MAPK/extracellular signal-regulated kinase (ERK) pathway. P2Y receptor signaling is known for interacting with integrins and cross talking with tyrosine kinase receptors (Fig. 3.2). P2Y receptors are expressed in both neurons and glial cell types in the CNS (Fischer & Krügel, 2007; Abbracchio et al., 2009). P2Y1 receptors are the dominant P2Y receptors in neurons, while they also participate in astrocyte functions (Illes & Alexandre Ribeiro, 2004; Verkhratsky, 2010). The distribution of P2Y1 receptors in the brain is broad and includes cerebral and cerebellar cortex, hippocampus, caudate nucleus, nucleus accumbens, the basal ganglia, subthalamic nucleus, and the midbrain (Burnstock & Knight, 2004), while the P2Y2 receptor involved in ATP-­ induced signaling is expressed at lower levels in all regions of the human brain, including neurons and astrocytes. Similarly, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14 receptors were detected in both neuronal and glial cell types of the CNS

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(Verkhratsky, 2010). Microglia also expresses multiple P2Y receptor subtypes (P2Y1, P2Y2, P2Y2/4, P2Y6, and P2Y12), Especially, P2Y6 and P2Y12 receptors appear to be functionally important (Boucsein et  al., 2003; Sasaki et  al., 2003; Inoue, 2008).

P2 Receptors in Neurons and Glia The main mediators connecting neurons and glial cells are glutamate and ATP (Koizumi et al., 2003). Activation of P2YR or P2X7R via ATP triggers release of gliotransmitter by astrocytes. In this regard, diverse reports observed that the P2X7  receptor induced the release of glutamate, GABA, or ATP from astrocytes (Duan et al., 2003; Sperlágh et al., 2006; Khakpay et al., 2010). Likewise, astrocytes of the brainstem release ATP under low blood pH (Gourine et al., 2010) and induce P2Y1 receptor activity in respiratory neurons to indicate and elevate partial pressure of pCO2.

Presynaptic Receptors P2X receptors  show high Ca2+ permeability, thus facilitating the Ca2+-dependent neurotransmitter release. Likely, ATP action via P2X receptor stimulation increases acetylcholine release at peripheral neurons or neuromuscular junctions (Sperlágh et al., 2007). The presynaptic facilitator action of ATP on noradrenergic transmission is also described in the locus coeruleus (Fröhlich et al., 1996) and the hippocampus (Papp et al., 2004). Moreover, P2X receptor activation influences glutamate release in central synapses, including the spinal cord (Nakatsuka & Gu, 2001), brain stem nuclei such as nucleus tractus solitarii, nucleus ambiguous (Inoue et al., 1992; Bowser & Khakh, 2004; Jin et al., 2004; Jameson et al., 2008), hippocampus (Sperlágh et al., 2002; Rodrigues et al., 2005), and cortical synaptosomes (Patti et al., 2006). Likewise, P2X receptor facilitates the release of the inhibitory transmitter GABA in the brain stem, midbrain, and hippocampus (Aihara et al., 2007; Calovi et al., 2018). Glycine may also be released upon presynaptic P2X receptor activation in the spinal cord (Rhee et al., 2000) and the trigeminal nucleus (Wang et al., 2001). Metabotropic receptors located at the pre-synapse are usually inhibitory modulators. In contrast, some P2Y receptor subtypes induce dopamine release in the striatum (Navarro et al., 2015), orin the nucleus accumbens (Krügel, 2016), as well as glutamate release potentiation in the medial habenula nucleus (Price et al., 2003). In addition to P2X receptors, the P2Y1 receptor also increases glutamate release in hippocampal slices (Kawamura et al., 2004). ATP was also reported to inhibit the release of serotonin and dopamine through P2Y receptor  activation (von Kügelgen & Hoffmann, 2016). ATP and its  stable

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analogs inhibit the depolarization-evoked glutamate release in rat brain cortical slices (Bennett & Boarder, 2000) likely through P2Y1, P2Y2, and P2Y4 receptor activation, as demonstrated by Rodrigues et al. (2005). Similarly, in hippocampal slides, P2Y1  receptors  excited interneurons and inhibited hippocampal circuits (Bowser & Khakh, 2004).

Postsynaptic P2 Receptors Postsynaptic P2 receptors can modulate activities or responses mediated by other receptors. For instance, the P2Y1 receptor  in cerebellar Purkinje cells increased GABAA receptor sensitivity through G protein-coupled free intracellular Ca2+ elevation (Saitow et al., 2005). P2Y receptors can also complex with ionotropic glutamate NMDA receptors in the prefrontal cortex. In this case, P2Y1 receptor activation inhibit NMDA receptor function through a physical crosstalk (Luthardt et al., 2003). Opposingly, P2Y4 receptor  activation facilitates NMDA receptor conductance in pyramidal neurons (Wirkner et al., 2007).

P2 Receptors in Glia Communication Astrocytes are important for information flow circuitry in the CNS. Astrocytes may release neuroactive substances that in turn modulate neuronal synapses (Newman, 2003). Thus, ATP plays an important role in glial signaling. ATP transport occurs by exocytosis or diffusion through hemichannels, pannexins, volume-regulated channels, or even through P2X7 receptor channels  (Guthrie et  al., 1999; Bowser & Khakh, 2004). In addition, ATP release from astrocytes induces propagation of Ca2+ waves within the astroglia syncytium (Abbracchio et al., 2009; Verkhratsky, 2010). Diverse P2R subtypes have been detected in astrocytes: all P2X receptor subunits except the P2X6 subunit were identified in rat astrocyte cultures (Fumagalli et al., 2003); P2Y1 and P2Y2 receptors, P2Y4, P2Y6 and P2Y12, and P2Y13 and P2Y14 receptors are as well expressed in astrocytes (Fumagalli et al., 2003; Fischer et al., 2009). During physiological rest state, low and transient increases in extracellular levels of ATP reinforce glial receptors for coordination of glial and neuronal functions. However, after injury or during inflammatory conditions, immense amounts of ATP are released triggering the amplification of the inflammatory responses and contributing to neural damage (James & Butt, 2002; Burnstock & Verkhratsky, 2009), such as in response to IL-1β treatment or inhibition of connexin 43 synthesis (Verkhratsky et al., 2009). P2Y receptors  in astrocytes trigger PLC-dependent Ca2+ mobilization (Pearce et  al., 1989), which can propagate to neighboring astrocytes, contributing to the mechanism of propagation of glial Ca2+ waves (Guthrie et al., 1999). Both P2Y1 and

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P2Y2 receptors  are involved in ATP-mediated Ca2+ wave propagation (Salter & Hicks, 1995; Verkhrasky et al., 2009). P2Y2 receptor-triggered waves travel faster and further than those propagated via P2Y1 receptors (Gallagher & Salter, 2003). These Ca2+ waves can be propagated via inositol triphosphate diffusion through gap junctions or by frequent ATP release (Verkhratsky et al., 2009). Regarding P2X  receptors, cortical astrocytes present a P2X1/5 heteromeric receptor, which is sensitive to ATP at nanomolar concentrations. Its desensitization is slow (Pankratov et al., 2009). Another peculiar P2X receptor type of the cortical astrocytes is the P2X7 receptor (Sperlágh et al., 2006) that participates in interleukin maturation during inflammatory processes along with microglial cells.

P2 Receptors in Microglia Microglia cells are the immune cells in the CNS that play crucial roles during infection. Therefore, their overstimulation leads to pathological conditions. Microglial activation usually occurs during brain damage or neurodegenerative diseases, and includes cell migration to the site of injury, release of proinflammatory molecules, and phagocytosis of damaged cells (Tsuda et al., 2003; Inoue, 2008). Microglial cells express both P2X and P2Y receptors  (Fröhlich et  al., 1996), while they are the source of extracellular ATP. Interestingly, the functional expression profile of the different P2 receptor subtypes changes upon stimulation. Under resting conditions, P2X7, P2Y1, P2Y2, and P2Y4  receptors  are prevalent, while P2Y6, P2Y12, P2Y13, and P2Y14 receptors are upregulated in the activated phenotype (Bianco et al., 2005). Extracellular ATP act as a chemo-attractant for microglia cells via P2Y12 receptor activation (Haynes et al., 2006). Neuronal injury leads to increased release or leakage of ATP and UTP, which also triggers P2Y6 receptor activation and microglia signaling for motility and engulfment (Inoue, 2008; Inoue et al., 2009). The P2X7 receptor is strongly involved in excessive inflammation responses by microglial cells (Parvathenani et al., 2003; McLarnon et al., 2006). This receptor controls the production of cytokines and expression of inflammation-related genes, such as NF-kB (Ferrari et al., 2006), IL-1α and IL-1β (Di Virgilio et al., 2017; Illes et al., 2020), TNF-α (Hide et al., 2000), and IL-18 (Rampe et al., 2004). Both P2X7 and P2X4 receptors have also been implicated in brain-blood barrier permeability  regulation (Aslam et  al., 2021), which is an important feature  for brain  inflammation. In addition, hyperactive microglia strikingly increases the expression of P2X4 receptors (Tsuda et al., 2003). Taken together, these findings suggest that microglial P2 receptors are novel and promising therapeutic targets for neuroinflammatory disorders caused by abnormal microglial response.

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Conclusion P1 adenosine and P2 receptors for extracellular nucleotides are widely expressed in the CNS. Their functions range from neurotransmission to the control of pre- and postsynaptic activity. The receptors are expressed by neurons, astrocytes, oligodendrocytes as well as by brain immune cells and have lately been shown to regulate brain-blood barrier function. As discussed throughout this book, correct functions and dysfunctions of these receptors define brain physiology and pathophysiology, such as neuroinflammation and neurodegeneration, and are important for neurogenesis and repair of brain functions.

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Chapter 4

Neurotrophic Actions of Adenosine and Guanosine: Implications for Neural Development and Regeneration? Filipa F. Ribeiro, Joaquim A. Ribeiro, and Ana M. Sebastião

Abstract  Purinergic nucleosides are for a long time known as neuromodulators, controlling the release and action of neurotransmitters. More recently, evidence emerged for adenosine and guanosine to have trophic actions. In this chapter, we address evidence for neurotrophic actions mediated by adenosine and guanosine in cell proliferation, cell migration, cell differentiation, neurite outgrowth, and synaptogenesis. Alterations in any of these processes may compromise brain function, leading to cognitive impairment and neurodegenerative diseases. Therefore, understanding the molecular and cellular mechanisms that modulate these trophic actions is the center of attention of regenerative research. Purine nucleosides could certainly play a role there. Keywords  Adenosine · Guanosine · Cellular proliferation · Neurite outgrowth · Neuronal differentiation Purine nucleosides are present in all cells and organ systems where they have both intracellular and extracellular roles. Purinergic nucleosides result from the reaction of a purine derived nucleobase, namely adenine or guanine, with a ribose, giving rise to the purine nucleosides adenosine and guanosine. Adenosine plays important roles in many biochemical processes, such as in energy transfer, in the synthesis of adenine nucleotides, in signaling transduction, in the formation of cyclic adenosine monophosphate (cAMP), and in the integration of the structure of nucleic acids. In addition to these widely known functions, adenosine acts as a neuromodulator, not only by regulating neurotransmitter release, synaptic transmission, and plasticity, but also by the action of other receptors and neuromodulators, such as neurotrophins (Sebastião & Ribeiro, 2015). Similarly, guanosine is a naturally occurring F. F. Ribeiro · J. A. Ribeiro · A. M. Sebastião (*) Instituto de Farmacologia e Neurociências, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 H. Ulrich et al. (eds.), Purinergic Signaling in Neurodevelopment, Neuroinflammation and Neurodegeneration, https://doi.org/10.1007/978-3-031-26945-5_4

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nucleoside that, besides integrating nucleic acids, also acts in signaling transduction, in the form of cyclic guanosine monophosphate (cGMP). Guanosine has been shown to have a neuromodulatory role in brain cells regulating, among different functions, neuroprotection and several cellular processes such as growth, differentiation, and survival (Di Liberto et al., 2016; Tasca et al., 2018). In fact, both adenosine and guanosine exert trophic effects on neuronal and glial cells (e.g., Di Liberto et al., 2016; Rathbone et al., 1999).

Purine Nucleoside Regulation Under physiological conditions, the extracellular concentration of purine nucleosides is controlled by the amount of released purines, their re-uptake, and by the activity of extracellular metabolizing enzymes (Fig.  4.1). Purine nucleosides are produced and released by different cells of the central nervous system (CNS), including neurons and glia (Rathbone et al., 1999), with the source of extracellular guanosine being mainly glial cells (Ciccarelli et  al., 1999). Intracellularly, these nucleosides are produced from dephosphorylation of nucleoside monophosphate (NMP) by the activity of cytosolic 5′-nucleotidases (cN), in particular the cN-I and cN-II, which play a role respectively in AMP breakdown to adenosine and in GMP breakdown to guanosine (Sala-Newby et  al., 2000; Hunsucker et  al., 2005). Adenosine can also be generated from the hydrolysis of S-adenosylhomocysteine (SAH) by S-adenosylhomocysteine hydrolase (Broch & Ueland, 1980). Moreover, the presence of unidirectional and bidirectional nucleoside transporters in the plasma membrane allows adenosine and guanosine to diffuse between the intracellular and extracellular compartments, thus regulating the inner and outer levels of these nucleosides (Pastor-Anglada et  al., 2001; Fredholm et  al., 2001; Schmidt et al., 2007; Parkinson et al., 2011). Adenosine and guanosine can also be produced extracellularly from nucleotides, namely ATP, ADP and AMP, and GTP, GDP, and GMP, respectively. When released to the extracellular space from presynaptic nerve terminals and glial cells (Wagner et al., 1978; Santos et al., 2006), ATP and GTP are catabolized by a cascade of ectonucleotidases present both on the cell surface of the extracellular space or soluble on the extracellular milieu, thus producing multiple breakdown products (Zimmermann, 1996; Zimmermann & Braun, 1996; Todorov et al., 1997; Fredholm et al., 2001; Yegutkin, 2008). A family of ectoenzymes is the ecto-nucleotide triphosphatases (ecto-NTPase) which comprises ecto-ATPase and ecto-ATP- diphosphohydrolase or apyrase (ecto-NTPDase). Ecto-ATPase is responsible for hydrolyzing ATP to ADP and GTP to GDP, whereas ecto-NTPDase is responsible for producing AMP from either ATP or ADP, and GMP from GTP or GDP (Yegutkin, 2008; Tasca et al., 2018). Another family of ectoenzymes is represented by ecto-5′nucleotidase (or CD73), which is responsible for the final step of the extracellular nucleotide catabolism of AMP to adenosine, GMP to guanosine, and IMP to inosine (Zimmermann & Braun, 1996; Zimmermann, 2000; Fredholm et  al., 2001;

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Fig. 4.1  Schematic representation of purinergic nucleoside metabolism. Adenosine and guanosine can be produced intra- and extracellularly and cross the plasma membrane through bidirectional specific transporters, namely concentrative nucleoside transporters and equilibrative nucleoside transporters. ATP: adenosine triphosphate; GTP: guanosine triphosphate; ADP: adenosine diphosphate; GDP: guanosine diphosphate; AMP: adenosine monophosphate; GMP: guanosine monophosphate; AMPD: AMP deaminase; ADA: adenosine deaminase; AK: adenosine kinase; IMP: inosine monophosphate; SAH: S-adenosylhomocysteine; XO: xanthine oxidase; PNP: purine nucleoside phosphorylase; GDA: guanine deaminase; cN-I and cN-II: cytosolic 5′-nucleotidases; CD73: ecto-5′-nucleotidase; ENT: equilibrative nucleoside transporter; NT: neurotransmitter; TNAP: tissue non-specific alkaline phosphatase; A1R, A2AR, A2BR and A3R: adenosine receptors; GR: putative guanosine receptor. (Adapted from Ribeiro (2017))

Hunsucker et  al., 2005). Moreover, the tissue non-specific alkaline phosphatase (TNAP) is the most studied alkaline phosphatase and is responsible for generating nucleosides from the hydrolysis of extracellular nucleotides (ATP, GTP, ADP, GDP, AMP, and GMP) (Zimmermann, 2000; Langer et al., 2007; Yegutkin, 2014). The action of either adenosine or guanosine can be limited by converting them to other products of the purine catabolism or using it to re-synthesize adenine nucleotides.

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On the one hand, adenosine deaminase (ADA) converts adenosine into the nucleoside inosine, which in turn, through the action of the enzyme purine nucleoside phosphorylase (PNP), is then phosphorylated into the nitrogenous base hypoxanthine and then degraded to the stable end product uric acid (Yegutkin, 2008; Ipata et al., 2011). PNP can also convert guanosine into guanine, which, through guanine deaminase (GDA) or cypin, is deaminated to produce xanthine (Tasca et al., 2018). On the other hand, adenosine can also be used to regenerate adenine nucleotides through the action of the enzyme adenylate kinase mainly localized in the cytosol (Yegutkin, 2008, 2014). The actions of these enzymes are responsible for controlling the levels of the different purines. Adenosine concentration is maintained in the range of 25–250 nM in basal conditions, which is sufficient to tonically activate a significant portion of adenosine receptors (Dunwiddie & Masino, 2001) and mediate the regulation of several physiological processes such as sleeping and local neuronal excitability. Guanosine levels are about two to three-fold higher than adenosine levels in the culture medium (Ciccarelli et al., 1999). However, in pathological conditions, such as hypoxia and ischemia, there is overexpression of 5′-nucleotidase and a simultaneous blockade of the PNP activity. The consequence is accelerated hydrolysis of the nucleotides to nucleosides, and prevented conversion of nucleosides into the correspondent nucleobases. Thus, accumulating intra- and extracellular nucleosides, with adenosine and guanosine levels drastically increased (Ciccarelli et al., 1999; Sebastião & Ribeiro, 2009). In particular, after an ischemia or trauma, large quantities of purines are released to the extracellular space with extracellular levels of guanosine becoming particularly elevated (Rathbone et al., 1999), both in cultured astrocyte (Ciccarelli et al., 1999) and in an in vivo model of focal cerebral ischemia (Uemura et al., 1991).

Purine Nucleoside Receptors Adenosine receptors (ARs) belong to a family of receptors called P1 receptor family. The P1 receptor family includes four specific metabotropic G-protein coupled receptors (GPCRs), namely the A1, A2A, A2B, and A3 subtypes. GPCRs trigger the activation of a variety of transduction mechanisms, with the main intracellular signaling pathway involving the formation of cAMP. On the one hand, A1Rs and A3Rs are mainly coupled to adenylyl cyclase inhibitory G-proteins (Gi or Go), which are responsible for inhibiting adenylyl cyclase, although A3Rs are also coupled to PLC excitatory G-protein (Gq). On the other hand, A2ARs and A2BRs are coupled to adenylyl cyclase stimulatory G-proteins (Gs), which, in turn, are responsible for stimulating adenylyl cyclase to produce cAMP, though coupling to PLC excitatory G-protein (Gq) can also occur (Fredholm et al., 2001; Burnstock, 2007). Adenosine receptors are widely distributed through the brain and are expressed by different neural cells, namely neurons, astrocytes, oligodendrocytes, and microglia (Burnstock, 2007; Daré et al., 2007; Zimmermann, 2011; Del Puerto et al., 2013),

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being involved in neuron–glia communication, consequently displaying different roles such as in cell proliferation, migration, and differentiation (Neary & Zimmermann, 2009). Contrary to adenosine, guanosine is considered an orphan neuromodulator, given its putative receptor has not been identified yet. Nevertheless, the possible existence of a single high affinity binding site for [3H]-guanosine has been suggested in rat brain membranes. Guanosine has been described to activate a putative GPCR that is distinct from the well-characterized purinergic adenosine receptors, since adenosine receptor agonists are unable to stimulate this binding site (Traversa et  al., 2002, 2003; Volpini et al., 2011). In fact, there are effects of guanosine that are independent of adenosine receptors, persisting in the presence of P1 receptor antagonists (Gysbers & Rathbone, 1992). On the other hand, there is also evidence indicating that guanosine may signal through complexes of adenosine receptors, namely A1R and/or A2AR (Ciruela, 2013; Lanznaster et al., 2019), as the blockade of these receptors affects the action of guanosine, such as in neuroprotection of hippocampal cells against oxidative and inflammatory processes, as well as in stimulating glutamate uptake (Ciccarelli et al., 2000; Dal-Cim et al., 2013, 2019). Also, in astrocytes, the protective effects of guanosine against ischemic-like situations were abolished by A1R antagonist and A2AR agonist (Dal-Cim et al., 2019). Guanosine may, therefore, act through different mechanisms, either dependent, or independent of adenosine receptors. It is important to refer that adenosine-receptor mechanisms may not imply the need for guanosine binding to adenosine receptors, but interactions between signaling cascades.

Trophic Actions of Purine Nucleosides Whereas neuromodulatory actions of nucleosides are known for more than 50 years, attention for their neurotrophic actions started to emerge only in the last 20–25 years, but it is now clear that they regulate neuronal cells proliferation, migration, differentiation, and synaptogenesis, besides promoting synthesis and release of trophic factors, which in turn further affect those functions.

Cell Proliferation It is nowadays accepted that neural cell proliferation occurs not only during embryogenesis but also in adulthood, even in non-pathologic conditions. It takes place in neurogenic niches where neural stem cells (NSCs) and cell progenitors or glial cells divide, giving rise to two new-born cells, which after a cascade of other processes eventually give rise to functional newborn differentiated cells. Importantly, cell proliferation may also spread all over the brain giving rise to growing brain cancers. Neural cell proliferation from neurogenic niches can also be boosted by

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neurodegenerative conditions, eventually due to an intrinsic effort to compensate for neural cell loss (Beckervordersandforth & Rolando, 2020). NSCs are integrated in a highly complex neurogenic niche, which is surrounded by astrocytes, endothelial cells, and mature neurons to provide structural support and regulation of the NSCs. NSCs are tightly regulated by several factors. Purine nucleosides have been recently recognized as modulators of NSCs. Different cells from the niche express ectoenzymes that allow the degradation of released nucleotides into nucleosides. The ecto-NTPDase2 is expressed in the subventricular zone (SVZ) and the rostral migratory stream of the adult rat brain, particularly bound to the membrane of type B cells, as well as transiently expressed in radial glial cells of the hippocampal dentate gyrus (DG) (Braun et al., 2003; Shukla et al., 2005). TNAP, the enzyme responsible for generating nucleosides from the hydrolysis of extracellular nucleotides, was detected in NSCs and neural progenitors during embryonic brain development at E14, becoming more restricted to the ventricular and SVZ regions during further embryonic development (Narisawa et al., 1994; Langer et al., 2007; Delic & Zimmermann, 2010). In the adult brain, TNAP was expressed in NSCs (type B cells), neuroblasts (type A cells), and in some subsets of transient amplifying precursor cells (type C cells) in the SVZ. Moreover, TNAP was expressed throughout the rostral migratory stream (RMS) in astrocytes that form the glial tubes around clusters of migrating neuroblasts (Langer et al., 2007). Additionally, neurospheres derived from the adult SVZ expressed ecto-nucleotidases NTPDase2 and TNAP (Mishra et al., 2006). Therefore, regulating the enzymes that participate in the metabolism of nucleosides will have an impact in nucleosides concentration and consequently an impact in NSC regulation. By knocking down TNAP, a reduction in proliferation was observed in NSCs isolated from adult mouse SVZ (Kermer et al., 2010). Adenosine A1R and A2AR have been detected during brain development in several brain regions, in particular in hippocampus, cerebral cortex, thalamus, midbrain, and cerebellum for A1R, and in striatum for A2AR, although with transient expression in other regions such as the cerebral cortex (Mateus et  al., 2019). However, very few reports have detected the presence of adenosine receptors, namely A1Rs, A2ARs, A2BRs, and A3Rs, in NSCs isolated from the adult mice SVZ (Stafford et al., 2007; Migita et al., 2008; Benito-Muñoz et al., 2016). Regarding the hippocampus, much less information exists. A2ARs were detected in neurospheres and cultured cells isolated from the DG (Ribeiro et al., 2021) and mouse single-cell gene sequencing identified neuronal progenitors and immature neurons as the A2AR-­ expressing cells (Shin et  al., 2015; Hochgerner et  al., 2018). Despite the lack of information concerning adenosine receptor expression, there are several studies revealing significant effects when activating or blocking these receptors in NSCs. Adenosine and selective A1R activation was shown to promote SVZ-derived NSC proliferation via MEK/ERK and Akt signaling pathways (Migita et al., 2008; Benito-Muñoz et al., 2016). However, A1R activation inhibited proliferation of cultured rat astrocytes (Ciccarelli et al., 1994), and, in turn, A2AR activation promoted astrogliosis, both in rat primary cultures and in vivo (Hindley et al., 1994; Brambilla et al., 2003). Moreover, the number and size of primary 6-day-old neurospheres was

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reduced in the presence of extracellular adenosine through a mechanism based on A2AR antagonist response, although A2AR agonists were devoid of effect (Stafford et al., 2007). Conversely, an opposite effect of A2AR activation was observed in the hippocampus. Pharmacological blockade of A2AR protects precursor subgranular zone (SGZ) cells from decreased proliferation induced by oxygen and glucose deprivation (Maraula et  al., 2013), and potentiates neuroblast proliferation in the dorsal hippocampus (Oliveros et al., 2017). In the same line, A2AR knockout mice have decreased proliferation of newborn hippocampal cells, which is associated to cognitive impairment (Moscoso-Castro et al., 2017). In fact, a gene-based association analysis associated ADORA2A, the adenosine A2AR gene, with a larger hippocampal volume and better memory (Horgusluoglu-Moloch et  al., 2017), reinforcing a role of A2AR in promoting adult neurogenesis. More recently, intraperitoneal administration of an A2AR selective agonist in rats was shown to prevent the reduction in Ki67 and doublecortin (DCX) positive cells, as well as spatial memory impairment as a result of noise-induced hearing loss exposure (Shukla et al., 2019). We have also observed that A2AR activation promotes cell proliferation of type 2b and type 3 cells from cultures derived from DG, but not of type 1 nor type 2a cells (Ribeiro et al., 2021), suggesting that A2AR activation preferentially acts on cells from the middle to later stages of neurogenesis where these receptors are more expressed. On the other hand, caffeine, the nonselective antagonist of adenosine receptors, affects proliferation of precursor cells in the adult hippocampus in a time- and dose-­ dependent manner (Han et al., 2007; Wentz & Magavi, 2009). Long-term administration (2–4  weeks) of low doses of caffeine reduced cell proliferation and hippocampus-dependent learning and memory in rats (Han et al., 2007). In turn, a 7-day administration of caffeine differently affected the proliferation of adult hippocampal neuronal precursors in mice accordingly to the dose: with moderate doses decreasing proliferation whereas supra-physiological doses enhancing proliferation of neural precursors (Wentz & Magavi, 2009). Interestingly, consumption of acute moderate dose of caffeine has a different impact on rats that are sleeping or in active periods. Though caffeine consumption during sleep period reduces DG cell proliferation, during the active period the effect of caffeine is greatly weakened or even absent, which suggests that the acute action during the sleeping period results from caffeine-mediated sleep deprivation (Kochman et al., 2009). During sleep deprivation, extracellular adenosine levels are increased in the basal forebrain, neocortex, and hippocampus (Porkka-Heiskanen et  al., 1997, 2000; Basheer et  al., 1999; Schmitt et  al., 2012; Sahu et  al., 2013), and when sleep-deprivation turns into a pathological condition, caffeine has a protective role. After 48 h of sleep deprivation, caffeine treatment in rats prevented the reduction of cell proliferation and neuronal differentiation, also attenuating the loss of brain-derived neurotrophic factor (BDNF) in the DG (Sahu et al., 2013). It is possible that these opposite actions of caffeine are related to differential blockade of adenosine receptors. A1R activation contributes to the deleterious effects of sleep deprivation on hippocampal synaptic plasticity and hippocampus-dependent memory (Halassa et al., 2009; Florian et al., 2011; Schmitt et  al., 2012). Selective blockade of A1R mitigates 48-h sleep

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deprivation decline in spatial reference memory in rats and the reduction in the number of neural progenitor cells (Chauhan et al., 2016). This action of A1R blockade could be possibly due to upregulation of BDNF levels in DG and CA1 regions, given that BDNF expression levels in the DG and CA1 region was observed to decline after sleep deprivation and be rescued by A1R antagonist treatment (Chauhan et al., 2016). Lower attention has been given to the actions of A2BRs and A3Rs. On the one hand, A2BRs were described to be involved in the recruitment of proliferating cells during adult fin regeneration in zebrafish (Rampon et  al., 2014), and to inhibit human progenitor smooth muscle cell growth (Dubey et  al., 2020). On the other hand, in  vitro stress stimulation induces the increase of adenosine in fibroblasts through A3R and mitogen-activated protein kinase (MAPK) signaling pathway, leading to fibroblast proliferation (Qu et al., 2020). In recent years, these adenosine receptors have been shown to play an important role in cancer development. Purine nucleosides, namely adenosine, regulate cell proliferation in glioblastoma, which is the most common and devastating form of brain primary tumor, as they are characterized by malignant proliferation and recurrence. Hypoxia regions in glioblastoma are associated with tumor growth, with adenosine accumulating in those regions. In fact, either adenosine or inhibiting ADK with 5-iodotubercidin (ITU) caused a reduction of cell proliferation/viability in human astrocytes and glioblastoma (Marcelino et  al., 2020, 2021). Stimulating A1R and A2BR resulted in an anti-­ proliferative/pro-apoptotic effect on cancer stem cells (Daniele et  al., 2014). Conversely, the enzyme CD73, a protein involved in extracellular adenosine production, has been implicated in cancer pathogenesis, promoting glioblastoma growth, angiogenesis, and invasiveness. Inhibition of CD73 impairs cell proliferation. When administered to a rat preclinical glioblastoma model, via nasal route, a cationic nanoemulsion to deliver CD73siRNA (NE-siRNA CD73R), there was a reduction of 60% of tumor growth (Azambuja et  al., 2020). A2BRs are highly expressed in glioblastoma (Yan et al., 2019) and play an important role in tumor cell proliferation, angiogenesis, metastasis, and immune suppression, with A2BR antagonists and CD73 inhibitors constituting potentially attractive anticancer agents (Yan et al., 2019; Gao & Jacobson, 2019). In fact, contrary to an anti-proliferative/pro-­ apoptotic effect reported by Daniele et  al., hRNA-mediated knockdown of ADORA2B expression decreases proliferation (Yi et al., 2020; Wilkat et al., 2020), and A2BR selective antagonism with MRS1754 reduces in  vitro proliferation and migration and in vivo tumor growth of renal cell carcinoma, via the MAPK/JNK pathway (Yi et al., 2020). Similarly, A3R and its ligands are important participants in cancer onset and development, being overexpressed in a variety of cancer types. In some cases, A3R activation leads to tumor growth, cell proliferation, and survival, whereas in other cases, A3R triggers cytostatic and apoptotic pathways (Mazziotta et al., 2022). It is important to highlight that the action of adenosine in cell proliferation in non-tumor and in tumor cells differs. Indeed, there is a strong attenuation of adenosine anti-proliferative effect in glioblastoma cell lines, as compared with human astrocytes, probably resulting from increased adenosine elimination due to ADK upregulation, suggesting a proliferative-prone adaptation of tumor cells to

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increased adenosine levels (Marcelino et al., 2021). Importantly also, the inhibitory action of adenosine on astrocytic proliferation may occur through receptor-­ independent mechanisms (Marcelino et al., 2020; Gebril et al., 2021). Reversion of the reaction catalyzed by S-adenosylhomocysteine (SAH) hydrolase, leading to SAH accumulation, as well as inhibition of S-adenosylmethionine (SAM)dependent methyltransferases (Marcelino et al., 2020), or even adenosine-induced alterations in DNA methylation (Losenkova et  al., 2020; Wahba et  al., 2021; Murugan et al., 2021), all have been reported to affect proliferation with impact in cancer, including in glioblastoma cell proliferation. Another nucleoside with impact in cell proliferation is guanosine, although it is not yet certain how guanosine regulates progenitor cells. It has been described that intraperitoneal treatment with guanosine (8 mg/kg) for 2–8 weeks days boosts proliferating BrdU+ neuroprogenitor cells in the adult mouse hippocampal DG, rat spinal cord, and in the SVZ of a Parkinson’s disease mouse model (Jiang et  al., 2008; Su et al., 2009; Piermartiri et al., 2020). Furthermore, chronic treatment with guanosine increases remyelination after spinal cord injury, by increasing proliferation and differentiation of endogenous adult oligodendroglial progenitor cells (Jiang et al., 2008). In contrast, guanosine-treated mice (5 mg/kg/day) for 3 weeks did not affect the number of proliferating cells positive for the proliferating markers cell nuclear antigen (PCNA) and Ki-67  in the entire hippocampal DG (Bettio et  al., 2016). This difference may be due to the use of Ki-67 for proliferation, on the one hand, that reflects the amount of proliferation at the time of tissue collection and, on the other hand, to the repeated administration of BrdU that allows labeling dividing cells from a larger time period and thus, at the moment of tissue collection, some of the cells may be no longer dividing. Therefore, the number of BrdU+ cells depends not only on cell proliferation but also on cell survival and differentiation capacity. To study the effect of guanosine on proliferation of NSCs and neural progenitor cells, Piermartiri and colleagues used a neurosphere assay, where neurospheres derived from SVZ and DG NSCs of adult mice were grown in suspension in the presence of the growth factors (human basic fibroblast growth factor, bFGF; epidermal growth factor, EGF). Continuous treatment with guanosine increased the number of neurospheres and proliferation of neural stem and progenitor cells. Interestingly, this effect of guanosine in cell proliferation was reduced by removing adenosine from the culture medium (Piermartiri et al., 2020). In similar proliferative conditions, in the presence of growth factors, we have recently shown that selective activation of A2ARs increases the number of secondary neurospheres (Ribeiro et al., 2021). Moreover, guanosine was found to stimulate proliferation of in vitro NSCs through a mechanism that involves cAMP accumulation and CREB phosphorylation (Su et al., 2013), and not through guanosine metabolism since the product of guanosine cleavage, guanine, has no stimulating effect on NSC proliferation (Su et al., 2013). Nevertheless, it is not clear whether guanosine acts through increasing adenosine levels or through direct interaction with adenosine receptors. Guanosine also mediates astrocyte proliferation. This action of guanosine has been associated with adenosine receptor activation (Rathbone et al., 1991; Ciccarelli et al., 2000). The mitogenic activity of guanosine on astrocytes was shown to be

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inhibited by antagonists of A2ARs (Rathbone et al., 1991), or partly inhibited by A1R and A2BR antagonists (Ciccarelli et al., 2000). On the other hand, exogenous guanosine treatment has been also shown to promote astrocyte proliferation and extracellular accumulation of adenosine and inosine, which may be directly released from astrocytes or derived from the extracellular breakdown of ATP (Kim et al., 1991; Rathbone et al., 1991, 1998; Ciccarelli et al., 2000, 2001). The enzyme that converts adenosine into inosine, ADA, reduced but did not abolish guanosine-induced astrocyte proliferation, whereas ADA inhibition amplified guanosine effect (Ciccarelli et al., 2000). In fact, both extracellular guanosine and adenosine stimulated astrocyte proliferation in vitro, with increased intracellular cAMP being involved in this process. The effects of both guanosine and adenosine on cell proliferation and cAMP levels were inhibited by antagonists of A2ARs and enhanced by A1Rs antagonists, suggesting that guanosine effects are indirectly exerted by increasing the endogenous extracellular adenosine concentration (Rathbone et  al., 1991). Moreover, guanosine has been shown to stimulate the production of large amounts of neuroprotective factors (Rathbone et al., 1999; Ciccarelli et al., 2001), and the mitogenic activity of guanosine on rat astrocytes was enhanced by the co-presence in the culture of microglial cells releasing soluble factors (Ciccarelli et al., 2000), thus suggesting that guanosine actions may be also dependent on the release of trophic factors. In summary, the actions of purine nucleosides on neural cell proliferation may occur through a diversity of mechanisms that may synergize or oppose to each other, depending on the cell metabolism, physiological versus pathological cell state, or even tumor adaptative responses. Further research on this topic is indeed needed for better understanding of the role of purines and their impact in brain tumors.

Cell Migration Cell migration plays a critical role in different cell types, being essential for organogenesis, tissue regeneration, immune surveillance, and tumor metastases formation. Neurons migrate from their neurogenic regions to the regions where they are needed to form neural circuits. Neuronal migration is regulated by a complex variety of chemical guides and signals. A defect in neuronal migration may result in a neurological disorder (Rahimi-Balaei et al., 2018). The role of purine nucleosides in cell migration is understudied. The few reports relate adenosine receptors but not guanosine. Caffeine intake during pregnancy affects the development of the fetal brain (Weaver, 1996). The addition of caffeine to pregnant mouse water supply delays embryonic migration of some neurons during the maturation period, resulting in the offspring being susceptible to seizures and showing impaired memory (Weaver, 1996). Moreover, exposure to A2AR antagonists, including caffeine, during the period of pregnancy and lactation in mice leads to delayed migration and insertion of γ-aminobutyric acid (GABA) neurons in the developing hippocampus, clearly detected during the first postnatal week (Silva

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et al., 2013). Importantly, these mice exhibited a loss of hippocampal GABAergic neurons in adulthood and revealed some cognitive deficits (Silva et al., 2013). A1R stimulated OPC migration, consequently inducing myelin protection and remyelination in a rat optic chiasm demyelination experimental model (Othman et al., 2003). In contrast A2AR may inhibit OPC migration since their inhibition during development causes ectopic OPC migration out of the spinal cord, from motor exit point in transition zones in zebrafish larvae (Fontenas et al., 2019). Another adenosine receptor with a role in migration is A3R, which, when activated by released adenosine under hypoxic conditions, promotes cell migration/ invasion of glioblastoma stem-like cells (Torres et al., 2019). A3R signaling has also been shown to facilitate P2Y12-stimulated microglia process extension and migration (Ohsawa et al., 2012).

Cell Differentiation NSCs are multipotent, having the capacity to differentiation into cells from the different neural lineages, namely astrocytes, oligodendrocytes, and neurons, through processes that are called astrogliogenesis, oligodendrogenesis, and neurogenesis. These processes of neural genesis start during brain development and continue throughout adulthood, not only in the neurogenic niches, namely the SVZ lining the lateral ventricles and the DG of the hippocampus, where NSCs are present, but also from glial precursor cells in the brain parenchyma. These processes of the formation of new cells are tightly regulated by several factors. Purine nucleosides, such as adenosine, have been described to regulate neurogenesis and oligodendrogenesis, while guanosine has been only described to regulate neurogenesis. In fact, the enzyme TNAP that generates nucleosides has been reported to regulate in vitro NSC differentiation, with TNAP knockdown in NSCs being responsible for reducing differentiation of these cells into neurons and oligodendrocytes (Kermer et al., 2010). A1R activation induces downregulation of proneurogenic genes as demonstrated by gene expression analysis. Furthermore, adenosine, via A1R activation, reduces neuronal differentiation of NSCs and stimulates astrogliogenesis, an action that has been shown to occur in neurosphere cultures generated from postnatal SVZ, as well as in vivo in the olfactory bulb (Benito-Muñoz et al., 2016). Corroborating these data, sleep deprivation increased extracellular adenosine levels and impaired adult neurogenesis, an action that may eventually exacerbate cognitive impairments due to sleep deprivation. A selective blockade of A1R mitigates the decline in spatial reference memory and doublecortin (DCX) positive cells in 48-h sleep-deprived rats, although no changes were observed in NeuN expression in DG (Chauhan et al., 2016). Accordingly, the non-selective adenosine receptor antagonist caffeine prevents the decline in cognition, as well as the decline in neuronal differentiation by increasing post-mitotic DCX positive cells during sleep deprivation (Sahu et  al., 2013, p. 20). Caffeine can also have a positive regulatory action upon adult neurogenesis after hyperoxia, as caffeine administration was reported to protect neural

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cells by preventing the loss of immature and mature neurons caused by high oxygen exposure (Endesfelder et al., 2014). Sleep deprivation and hypoxia surely affect a broad range of processes, besides neurogenesis, but their impact in neurogenesis may contribute to the negative outcome of those conditions. Regarding A2AR, studies indicate that stimulation of these receptors enhances neurogenesis and reduces neuronal damage in an animal model of spinal cord injury (Irrera et al., 2018). Corroborating this data, inhibition of A2ARs induced impulsive behavior accompanied by increased immature neuroblast proliferation in the hippocampus (Oliveros et al., 2017). Recently, we demonstrated that A2AR activation promotes neuronal differentiation in the DG, by increasing the number of total immature (DCX+ cells) and newborn mature neurons (NeuN+ cells) both in vivo and in vitro, favoring glutamatergic neurons. Interestingly, this action of A2ARs in neuronal differentiation was shown to be dependent on the presence of extracellular BDNF (Ribeiro et al., 2021). We could not observe, however, any influence on the regulation of BrdU+GFAP+ glial cells. In particular, A2AR selective activation enhanced neurogenesis in the dorsal hippocampus, by increasing the number of adult-born neurons (Ribeiro et al., 2021). Accordingly, intraperitoneal administration of an A2AR agonist in rats prevented the reduction of DCX+ cells as a consequence of noise-induced hearing loss (Shukla et  al., 2019). For the best of our knowledge, no information so far exists on the role of A2BRs and A3Rs in the regulation of neuronal differentiation. Concerning the actions of guanosine, it also affects neuronal differentiation. In a rat model of Parkinson disease, chronic systemic administration of guanosine increased the number of tyrosine hydroxylase-positive dopaminergic neurons in the substantia nigra pars compacta while inducing a significant and stable recovery of locomotor function (Su et  al., 2009). Accordingly, chronic oral administration of guanosine to adult mice promoted an increase in the number of immature neurons in the ventral hippocampal DG, known to regulate emotional and motivational behaviors, and resulted in antidepressant-like effects following 3 weeks of treatment (Bettio et al., 2016). In cultured neurospheres derived from DG, guanosine increased the percentage of β-tubulin III+ neurons, indicating a facilitation of neuronal differentiation; this was further confirmed in  vivo, where intraperitoneal chronic administration of guanosine to adult C57BL/6 mice increased hippocampal DG ratio of BrdU+/DCX+ to BrdU+ cells, which was accompanied by antidepressant-­ like effects (Piermartiri et  al., 2020). Guanosine also induced an increase in the number of cultured cerebellar neurons, indicating that this nucleoside promotes the survival or maturation of primary cerebellar neurons; interestingly, this effect was prevented by the blockade of A2ARs, thus suggesting an interplay between A2ARs and guanosine actions (Decker et al., 2019). Oligodendrocytes are responsible for forming myelin sheaths, which allow fast neuronal communication. Oligodendrocyte degeneration leads to demyelinating diseases such as multiple sclerosis. Remyelination requires the differentiation of oligodendrocyte progenitor cells (OPCs) into mature oligodendrocytes. Adenosine and its receptors are essential mediators in remyelination processes (Cherchi et al.,

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2021). OPCs express all four subtypes of adenosine receptors (Stevens et al., 2002), with oligodendrocytes also expressing the equilibrative nucleoside transporters ENT1 and ENT2, as well as adenosine degrading enzymes, such as adenosine deaminase and adenosine kinase (González-Fernández et  al., 2014). TNAP is expressed in the nodes of Ranvier. Ablation of TNAP function compromises myelination in the mouse brain, resulting in a reduction of the white matter of the spinal cord accompanied by cellular degradation around the paranodal regions and a decreased ratio and diameter of the myelinated axons, as well as resulting in the virtual absence of myelinated axons in the cerebral cortex (Hanics et al., 2012). On the other hand, in the presence of adenosine, OPCs differentiate into myelinating OLs, consequently contributing to axon myelination (Stevens et al., 2002). As evaluated by studies on cultured OPCs, adenosine acts as a dual modulator of OPC development. Selective activation of A1Rs protects myelin and induces remyelination in an experimental model of rat optic chiasm demyelination (Asghari et al., 2013). In accordance, through A1R activation, adenosine increases voltage-­ dependent outward K+ currents and facilitates OPC maturation (Stevens et  al., 2002) and stimulates OPC migration (Othman et al., 2003). In turn, selective activation of either A2AR and A2BR inhibits OPC differentiation and maturation by reducing voltage-dependent K+ currents (Coppi et al., 2013, 2020). The possible opposite effects mediated by A1Rs and A2ARs on oligodendrocyte differentiation and myelination have been attributed, at least in part, to their different coupling to the adenylate cyclase/cAMP levels and their different ability to modify K+ conductance. Interestingly, however, under certain pathological conditions A2AR activation may promote myelination. This has been shown in both genetic (Ferrante et al., 2016) and pharmacological models (De Nuccio et al., 2019) of Niemann Pick type C disease, a rare devastating lysosomal-lipid storage disorder, which among other dysfunctions leads to oligodendrocyte maturation arrest. In these models, A2AR activation attenuated dysfunction of oligodendrocyte-like cultured cells (Ferrante et  al., 2016), rescued oligodendrocyte progenitors from maturational arrest, and promoted their differentiation to mature oligodendrocytes (De Nuccio et al., 2019). Together these data suggest that drugs that may promote A2ARs activation may prove therapeutically relevant to correct demyelination in Niemann Pick type C disease. Concerning A3Rs, the information so far available concerns viability of oligodendrocytes, since their activation in cultured oligodendrocytes causes oxidative stress and mitochondrial membrane depolarization, leading to apoptotic and necrotic cell death (González-Fernández et  al., 2014); accordingly incubation of optic nerves ex  vivo with an A3R agonist induced oligodendrocyte damage, and myelin loss (González-Fernández et al., 2014). The role of guanosine on oligodendrocyte differentiation is almost unknown. As far as we know, there is only one study showing that systemic administration of guanosine to rats with spinal cord injuries improves locomotor function by stimulating maturation of endogenous oligodendrocyte progenitors and remyelination of axons at the injury site (Jiang et al., 2008).

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Neurite Outgrowth There are several evidence that relate purine nucleosides with neurite outgrowth or axon growth and guidance. Adenosine is responsible for decreasing neurite total length in adult dorsal root ganglion (DRG) neurons, an action that is mediated by A1Rs (Shaban et  al., 1998). Moreover, A1R activation has been shown to inhibit neurite growth in neuronal pheochromocytoma (PC12) cells and in primary cultures of cortical and hippocampal neurons. These inhibitory effects of A1R are dependent on Rho kinase pathways. Particularly, in PC12 cells, A1R activation prevented nerve growth factor (NGF)-induced neurite growth and induced stress fiber formation, an action that was independent from p44/42 MAP kinase activity (Thevananther et al., 2001). Engrailed is a transcription factor, with a key role in axon guidance. It stimulates rapid ATP synthesis and secretion from growth cones which is then hydrolyzed to adenosine. A tight interaction between Engrailed, ephrin A5, and adenosine A1R activation had been shown to control retinal ganglion cell (RGC) axonal guidance, likely leading to increased precision of the retinal projection map (Stettler et al., 2012). There are several studies, using different models, relating A2ARs with neurite outgrowth. Although the A2AR agonist, CGS 21680, was shown to reduce neurite total length in adult dorsal root ganglion (DRG) neurons, this effect was seen at high micromolar concentrations and was blocked by A1R but not A2R antagonists, so it has to be considered as an A1R-mediated effect (Shaban et  al., 1998). In A2AR-­ expressing PC12 cells, the inhibition of ecto-5′-nucleotidase, which is expected to reduce extracellular adenosine levels, impairs neurite outgrowth (Heilbronn & Zimmermann, 1995). Moreover, A2AR activation has been shown to enhance NGF-­ induced neuritogenesis (Gysbers & Rathbone, 1992), and to rescue the blockade of NGF-mediated neurite outgrowth as a result of the inhibition of the MAPK cascade (Cheng et  al., 2002). The A2AR-mediated rescuing effect was shown to involve cAMP/PKA and cAMP response element-binding protein (CREB) activation (Gysbers & Rathbone, 1996a; Cheng et al., 2002). In neuronal PC12 cells whose neurite outgrowth was impaired by chemical hypoxia with rotenone, Tomaselli and colleagues showed that, in combination with NGF, adenosine can partially rescue neurite outgrowth through a mechanism dependent on MAPK pathway (Tomaselli et  al., 2005). Furthermore, the bacterial nucleoside N6-methyldeoxyadenosine induces an A2AR-dependent process outgrowth in PC12 cells, together with stimulation of adenylate cyclase, and the activation of the MAPK cascade (Charles et al., 2003). In fact, these data suggest that both MAPK and PKA are important for A2AR-­ mediated neurite outgrowth. In addition, Canals and colleagues (Canals et al., 2005) explored the effect of A1R and A2AR activation not only in human neuroblastoma cell line SH-SY5Y but also in primary cultures of striatal neurons. They showed that either A1R and A2AR activation leads to neuritogenesis, through a mechanism dependent on MAPK and protein kinase C (PKC) signaling pathways, with A2AR being also dependent on PKA pathway (Canals et al., 2005). Also, co-stimulation of A1R

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and A2AR was shown to not lead to synergistic effects, indicating that the neuritogenic effects of adenosine are dependent on the concentration of this purine nucleoside that will differently activate separately each adenosine receptor (Canals et al., 2005). Furthermore, A2AR activation rescued neuritogenesis in primary hippocampal neurons and PC12 cells impaired by p53 blockage, in a PKA- and PKC-­ independent mechanism (Sun et al., 2006, 2010). On the other hand, in rat cortical neurons, methyl 3,4-dihydroxybenzoate was shown to promote neurite outgrowth through the adenosine A2AR/PI3K/Akt signaling pathway (Zhang et  al., 2015). More recently, we have shown that A2AR activation enhances axonal elongation and dendritic branching in primary cultures of cortical neurons (Ribeiro et al., 2016a). While axonal elongation was shown to be dependent on PI3K/Akt and MAPK signaling pathways, and independent of the presence of endogenous BDNF, A2AR-­ mediated dendritic branching was dependent of endogenous BDNF (Ribeiro et al., 2016a). The implications, but also the complexity, of some of the actions of adenosine upon neuronal outgrowth were reported by Alves and colleagues (Alves et al., 2020) using an attention deficit and hyperactivity disorder (ADHD) model, where neurons display less neurite branching, shorter maximal neurite length, and decreased axonal outgrowth. While caffeine recovered neurite branching and elongation from ADHD neurons via both PKA and PI3K signaling, the A2AR selective agonist CGS 21680 promoted more neurite branching via PKA signaling, which suggests that the action of caffeine upon neurite branching does not result from A2AR blockade. However, the selective A2AR antagonist Scheme 58261 recovered axonal outgrowth from ADHD neurons through PI3K and not PKA signaling (Alves et  al., 2020). These findings point toward different mechanisms involved in axonal outgrowth and neurite branching. Accordingly, in  vivo intracerebroventricular diffusion of CGS21680 for 4 weeks contributed to a higher DG suprapyramidal granule neuronal dendritic complexity, without affecting total dendritic length or volume (Ribeiro et al., 2021). It is important to note that some actions of adenosine may be exerted through the modulation of the action of neurotrophins, well-known regulators of neuronal survival, cell proliferation, neurite extension, and synaptogenesis of developing neurons. In fact, adenosine, acting through A2ARs, can transactivate TrkB receptors, i.e., promote receptor autophosphorylation in the absence of neurotrophin binding (Lee & Chao, 2001; Wiese et al., 2007). Furthermore, it can potentiate the action of BDNF upon TrkB receptors (Diógenes et al., 2004; Ribeiro et al., 2016b). A2AR also regulate BDNF expression and release (Tebano et al., 2008; Jeon et al., 2011). Moreover, NGF expression has been described to be increased by the activation of A2AR in microglia (Heese et al., 1997) and by A1R in astrocytes (Ciccarelli et al., 1999). Netrins are a family of laminin-related secreted proteins and are important in controlling axon elongation and pathfinding. Netrin-1-dependent outgrowth of dorsal spinal cord axons not only requires a receptor complex containing DCC (deleted in colorectal cancer) protein but also A2BR (Corset et al., 2000). Activation of A2BR by adenosine analogues results in PKCα-dependent removal of UNC5A from the cell surface, which results in a reduction in the number of growth cones that

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collapse in response to netrin-1, thus converting netrin-1-mediated repulsion to attraction (McKenna et al., 2008). A2BRs were also shown to be involved in nerve growth during adult regeneration in zebrafish (Rampon et  al., 2014). Regarding A3R, less is known. A3R agonist 2-Cl-IB-MECA significantly promoted in vitro and in vivo neurite outgrowth during the regeneration of rat RGCs, an action that was caused by the activation of an Akt-dependent signaling pathway (Nakashima et al., 2018). Inosine has been shown to support neurite outgrowth of primary cultured neocortical mouse neurons, an action that was suppressed by the A1R and A2AR antagonists 8-cyclopentyl theophylline (CPT) and 8-(3-chlorostyryl) caffeine, thus likely involving direct or indirect interaction between inosine and adenosine receptors. Moreover, oral administration of inosine produced antidepressant-like effects in chronically stressed mice, enhancing cell proliferation in the DG and an increase in transcript level of BDNF (Muto et al., 2014). After chemical hypoxia of primary rat cerebellar granule cells induced with rotenone, an inhibitor of mitochondrial complex I, inosine has been shown to partially restore neurite outgrowth (Böcklinger et al., 2004). This rescuing action of inosine also occurred in PC12 cells in combination with NGF, where MAPK pathway plays a vital role (Tomaselli et al., 2005). Apart from that, inosine was also shown to stimulate axon growth in the adult CNS. In fact, unlike mammals, goldfish retinal ganglion cells can regenerate their axons. In response to the purine nucleoside inosine, extensive axon growth occurs in the mature rat corticospinal tract, which is also associated to changes in gene expression that underlie regenerative growth. Following unilateral transection of the corticospinal tract, inosine stimulated layer 5 pyramidal cells of the intact sensorimotor cortex to upregulate GAP-43 expression and to sprout axon collaterals (Benowitz et al., 2002). The purine nucleoside guanosine also has a role in neurite outgrowth. Extracellular guanosine stimulates the de novo extension of neurites from rat PC12 cells (Gysbers & Rathbone, 1992, 1996a). This action of guanosine occurred through a cAMP-­ dependent and -independent mechanism, as it was partially blocked by the adenylate cyclase inhibitor SQ22536 (Gysbers & Rathbone, 1996a). In human SH-SY5Y neuroblastoma cells, guanosine increases the number and the length of neurites (Guarnieri et al., 2009). Guanosine also acts in a synergistic way with saturating concentrations of NGF to enhance neuritogenesis (Gysbers & Rathbone, 1992). Moreover, when the A1/A2R agonist NECA was added together with guanosine in the presence of NGF, these compounds elicited a greatly enhanced neuritogenic response (Gysbers & Rathbone, 1992). On the other hand, guanosine-induced neurite outgrowth seems to be independent of A1Rs and A2Rs, as A1R and A2Rs antagonists, DPMX, CGS15943, or PACPX, did not affect this action of guanosine (Gysbers & Rathbone, 1996b). This suggests that the mechanisms through which NECA modulates the neuritogenic effects may be different from those of guanosine and NGF (Gysbers & Rathbone, 1992). These effects of guanosine on neurites can also be associated to the regulation of other factors expression and release. In fact, guanosine has been shown to enhance the synthesis and release of growth factors, among which is NGF, as well as of adenine-based purines, from cultured astrocytes (Middlemiss et al., 1995; Rathbone et al., 1999; Ciccarelli et al., 2001).

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Synaptogenesis The formation of synapses, a process called synaptogenesis, is essential to establish neuronal communication and is regulated by several factors, including purine nucleosides and the enzymes involved in their metabolism. The nucleoside-generating ectoenzymes TNAP and ecto-5′-nucleotidase (CD73) have been proposed as essential during the formation of synapses (Hanics et  al., 2012; Gomez-Castro et al., 2021). In fact, TNAP is normally expressed in the synaptic cleft, and animals with ablation of TNAP function display a higher percentage of immature cortical synapses (Hanics et  al., 2012). In the case of ecto-5′nucleotidase, it has a transient increase in expression at rat excitatory cerebellar synapses during brain development and during lesion-induced synaptogenesis (Schoen et al., 1991). Additionally, increased 5′-nucleotidase activity of axon terminals has been observed in adult rats during reactive synaptogenesis of the hippocampal DG, which had been deprived of its entorhinal afferents (Schoen & Kreutzberg, 1994). Rat models of epilepsy have increased 5′-nucleotidase activity in the CA3 region and in the inner molecular layer of the DG. In both amygdala-­ kindled and kainate-treated rats, 5′-nucleotidase was clearly detected in terminals within the inner molecular layer with plastic sprouting response characteristics, leading to the proposal that the activity of this enzyme would contribute to attenuate the glutamatergic transmission of the aberrant mossy fibers in epileptic rats, by producing adenosine (Schoen et al., 1999). These data, together with an increase of both ATP and adenosine, as well as a peak of expression ecto-5′-nucleotidase and of peri/post-synaptic A2AR, during the period of synaptogenesis in the developing hippocampus (Gomez-Castro et  al., 2021), suggest that this purine nucleoside has a role in synaptogenesis. Although little is known about the role of A2AR signaling in synaptogenesis, a recent work (Gomez-Castro et al., 2021) elegantly demonstrated a role of adenosine, through A2ARs, on stabilization of hippocampal GABAergic synapses during a critical post-natal period. Suppression of A2AR activity with shRNA or pharmacological approaches led to a loss of GABAergic synapses in cultured hippocampal neurons. Importantly also, A2AR activation proved sufficient to stabilize GABAergic synapses even in the absence of GABAA receptor signaling (Gomez-Castro et al., 2021). A2AR-mediated GABAergic synapse stabilization is dependent on adenylyl cyclase/cAMP/PKA signaling cascade and the postsynaptic scaffolding molecule gephyrin (Gomez-Castro et al., 2021), which is a central element that anchors, clusters, and stabilizes glycine and GABAA receptors at inhibitory synapses (Choii & Ko, 2015; Pizzarelli et al., 2020). ARs, specially A1R and A2AR subtypes, also play important roles during neuromuscular junction (NMJ) synaptogenesis (reviewed in Tomàs et al., 2018). During nervous system development, there is an overwhelming production of synapses, which is followed by their activity-dependent reduction. ARs contribute to the developmental synapse elimination process during NMJ synaptogenesis, helping to select axon terminals through Hebbian competition. For that, ARs establish several

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synergistic and antagonistic relations with mAChR subtypes and TrkB receptors. Moreover, in the adult, ARs help to modulate ACh transmitter release and preserve synaptic function by having a protective role against synaptic depression during repetitive activity (Tomàs et al., 2018). Concerning guanosine, it has been observed that in vivo 7-day administration of guanosine using an osmotic pump to the rat visual cortex promotes an increase in the number and size of synaptic buttons along the axonal branches projecting from the rat visual cortex to other cortical areas and to subcortical structures (Gerrikagoitia & Martínez-Millán, 2009). Astrocyte-derived cholesterol promotes the formation and function of synapses in RGC cultures from postnatal rats. In fact, dendrite differentiation is the rate-limiting step for glia-induced synaptogenesis in RGCs, with this process requiring cholesterol. Moreover, cholesterol enhances directly presynaptic differentiation, supporting continuous synaptogenesis and the stability of synaptic transmission (Goritz et  al., 2005). Interestingly, guanosine can increase cholesterol efflux from astrocytes, through phosphoinositide 3 kinase/extracellular signal-regulated kinase 1/2 (PI3K/ERK1/2) (Ballerini et  al., 2006). As a whole, these data suggest a relevant role for guanosine in regulating synaptogenesis.

Conclusion Over the last years, purinergic nucleosides, such as adenosine and guanosine, have emerged as molecules that exert trophic actions, regulating the processes of cell proliferation, migration, differentiation, neurite outgrowth, and synaptogenesis. However, these different trophic actions involve quite distinct molecular processes and depend on the presence and expression of receptor subtypes, interaction with other molecules, and activation of different signaling pathways. While adenosine can exert its actions through differential activation of four distinct adenosine receptors, a receptor for guanosine was not yet identified. Some of the guanosine action require co-activation of adenosine receptors but other seem to be adenosine-­receptor independent. Moreover, some actions of these nucleosides might be dependent on the regulation of trophic factors expression, such as NGF and BDNF.  A deeper understanding of the relative hierarchy established between these modulatory molecules and their receptors, and of the intrinsic mechanisms involved both in physiological and pathological conditions, will certainly result in a better understanding of the nervous system maturation but also likely in the development of therapeutic strategies for brain regeneration. Acknowledgments  The authors research is supported by Fundação para a Ciência e a Tecnologia (FCT) (PTDC/MED-FAR/4834/2021 and PTDC/MED-FAR/30933/2017) and by European Union H2020-WIDESPREAD-05-2017-Twinning (EpiEpinet) (grant agreement No. 952455). F.F.R. received a fellowship from FCT (IMM/CT/76-2021).

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Chapter 5

Purinergic Signaling in Neurogenesis and Neural Fate Determination: Current Knowledge and Future Challenges Roberta Andrejew, Natalia Turrini, Qing Ye, Yong Tang, Peter Illes, and Henning Ulrich

Abstract  Neurogenesis is responsible for the generation of the neuronal network during the development of the central nervous system and remains active with reduced activity in the postnatal and adult brain. Purinergic signaling participates in these processes. Here, we discuss the participation of purinergic P1 and P2 as well as of ectonucleotidases converting ATP into ADP and adenosine in the regulation of

Roberta Andrejew and Natalia Turrini contributed equally with all other contributors.

R. Andrejew · N. Turrini Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, SP, Brazil Q. Ye Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, SP, Brazil Key Laboratory of Sichuan Province for Acupuncture and Chronobiology, Chengdu, China International Joint Research Centre on Purinergic Signalling, Chengdu, China Y. Tang Key Laboratory of Sichuan Province for Acupuncture and Chronobiology, Chengdu, China International Joint Research Centre on Purinergic Signalling, Chengdu, China P. Illes International Joint Research Centre on Purinergic Signalling, Chengdu, China Pharmacology and Toxicology, Leipzig University, Leipzig, Sachsen, Germany H. Ulrich (*) Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, Brazil e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 H. Ulrich et al. (eds.), Purinergic Signaling in Neurodevelopment, Neuroinflammation and Neurodegeneration, https://doi.org/10.1007/978-3-031-26945-5_5

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neural stem and progenitor cell proliferation, migration, and differentiation-based phenotype determination. Major regulatory functions have been suggested for P2X7, P2Y1, P2Y13, and A2A receptors together with connexins for the synchronization of migration and differentiation. These receptors are also active during adult neurogenesis occurring in the subventricular zone and subgranular zone of the dentate gyrus by stimulating the liberation of growth factors enhancing neuronal survival and differentiation. The elucidation of purinergic signaling mechanisms is essential for understanding neurodevelopmental diseases and endogenous repair mechanisms of the brain. Keywords  Neural precursor cells · Dentate gyrus · ATP · Adenosine · P2X7 · P2Y1 · P2Y13 · A2A

Neurogenesis Neurogenesis is a fundamental process in which stem and progenitor cells self-­ renew, migrate, and generate new functional neurons. It is currently well established that this process occurs in the embryonic and adult central nervous system (CNS). Most neurons of the nervous system are born during embryonic development, which is characterized by an intense proliferative activity of stem cell populations. During this process, there are two essential types of cells: the primary and multipotent neural stem cells (NSCs) that generate the second type, the neural precursor cells (NPCs), which generate neurons, astrocytes, and oligodendrocytes (Urbán & Guillemot, 2014). Both cell types share some essential characteristics such as self-­ renewal and capacity for differentiation (Bjornson et al., 1999; Urbán & Guillemot, 2014). These two essentialities allow the formation of the entire CNS, and neurons can originate from three types of NSCs and NPCs: basal progenitors, neuroepithelial cells, and radial glial cells (RGC) (Götz & Huttner, 2005). At first, new neurons are generated in neurogenic complexes considered functional units of cells. The fate of these cells is managed by the extracellular matrix and the secreted molecules of this environment (Alvarez-Buylla & García-Verdugo, 2002). Cell maturation is complex and involves expansion, differentiation, migration, and final integration in the synaptic network. Each step is modulated by transcriptional and epigenetic signals triggered by cell communication, internal signaling cascades, ligand-induced signaling, and a mass of different molecules participating in this immense and incredible process called neurogenesis (Götz & Huttner, 2005). However, once the differentiation is complete and the mature neuronal cells have been formed, these lose their ability of self-renewal and are not capable of differentiating into another cell type. Neurogenesis and embryonic nervous system development need to take place correctly, as any dysfunction during development may result in severe congenital neuropathies. Adult neurogenesis is a recent discovery. Compared to embryonic neurogenesis, it happens at lower levels. NPCs are present in the adult CNS in small quantities and

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in rodents  is resctricted mostly in two specific regions, the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG). Progenitor cells present in the SVZ are divided into three types: Type B, a quiescent radial glia-like cell that is slowly proliferative cells and expresses nestin, vimentin, and glial fibrillary acidic protein (GFAP); Type C, transit-­ amplifying progenitor cells that are proliferative and express mainly nestin and achaete-scute complex homolog-1 (Mash1) proteins; and Type A cells, proliferative neuroblasts cells that express doublecortin (DCX) and distal-less homeobox 1 (Dlx1) proteins. These cells are committed to the neuronal fate and migrate through the rostral migratory stream (RMS) to the olfactory bulb where they mature locally as interneurons (Fiorelli et al., 2015; Ming & Song, 2011). Additionally, ependymal cells, astrocytes, and microglia are glial cells also present in the adult SVZ neurogenic niche. In the SGZ, three types of progenitor cells are important: type I, highly proliferative NPCs (radial glia-like cells) that express nestin, SRY-Box Transcription Factor 2 (Sox2), GFAP, and brain lipid-binding protein (BLBP) and generate type II cells; type II (IIa and IIb), transit amplifying progenitor cells that are more proliferative and express T-box brain gene 2 (Trb2) and minichromosome maintenance protein 2 (MCM2); and neuroblasts that are migrating immature neurons expressing Trb2, MCM2, and DCX.  These neuroblasts can generate immature neurons and integrate into the microenvironment. For generating new granule neurons, cells must migrate to the DG, elongate their dendrites and axons, and integrate into the complex DG circuitry (Nicola et al., 2015). The angiogenic environment interferes in these neurogenic areas (Palmer, 2002). The contact of the neurogenic cells with endothelial and adjacent cells modulates the differentiation process. Hormones, trophic factors, neurotransmitters, and further bioactive molecules are secreted into the microenvironment. The existence of adult neurogenesis in humans has been questioned for many years (Snyder, 2018; Sorrells et al., 2018). Nethertheless evidence accumulated to date strongly indicates its existence, even though it is probably more limited in humans than in rodents (Ernst et al., 2014; Kuhn et al., 2018).

 xpression of Purinergic Signaling Elements E During Embryogenesis Purinergic receptors have been identified at all stages of brain embryonic development indicating their importance in this process. ATP and its metabolites through purinergic receptors mediate the release of growth factors and chemokines and subsequent intracellular signaling pathways that are fundamental for stem cell proliferation and differentiation (Burnstock & Ulrich, 2011; Fields & Burnstock, 2006). Here, we shall discuss the implication of purinergic signaling in brain development focusing on NSCs, which give rise to the three major cell lineages of the nervous system: neurons, oligodendrocytes, and astrocytes.

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The importance of the P2X3 receptor in CNS development was shown in vitro and in vivo. Norton and colleagues showed the expression of the P2X3 subunit in vitro by using in situ hybridization. Gene expression data were confirmed in vivo in rat CNS from embryonic day 11 (E11) onward, the exact time when gangliogenesis starts (Cheung & Burnstock, 2002; Norton et al., 2000). Additionally, P2X3 receptor expression pattern follows the development of the regulatory parts of the autonomic nervous system such as the medulla oblongata and spinal cord, which generate sensory nerves and craniofacial motoneurons (Massé et al., 2007; Massé & Dale, 2012). Gene knockdown of the P2X3 receptor zebrafish paralog, p2xr3.1 caused defects in craniofacial and sensory circuit formation (Kucenas et al., 2009), reinforcing the capability of the P2X3 receptor of orchestrating CNS development of different species. P2X2 and P2X7 receptors start their expression on E14 (Cheung et al., 2005), while the P2X5 receptor was detected beginning from E8 in the neural tube with expression gradually increasing until E9-E13 and declining after this day, being detected again just after the completion of the CNS development (Guo et al., 2013) (Fig. 5.1a). P2Y1 and P2Y4 receptors have been expressed since E11, while the P2Y2 receptor gradually increased its expression from E14 (Cheung et  al., 2003) (Fig.  5.1a). The P2Y1 receptor is predominantly  localized in proliferative regions, such as the ventricular zone (VZ) and the SVZ; and is also expressed by RGC, which are NSCs; and intermediate precursor cells (IPCs), such as intermediate neuronal progenitors (INPs) (Liu et al., 2008; Weissman et al., 2004; Zeng et al., 2008). In agreement with purinergic receptor implication in neurogenesis, in vitro studies showed that NSCs and NPCs, express several P2 purinergic receptors, such as P2X7, P2X4, P2Y1, P2Y2, P2Y4, P2Y6, P2Y12, and P2Y14 subtypes, and respond to ATP. Moreover, the neurospheres in the mitotic process secrete ATP and, due to purinergic receptors’ action, cause intracellular calcium ([Ca2+i]) mobilization, which suggests that purinergic receptors have a role in NPC expansion (Lin et al., 2007). Connexins are involved in the physiological release of ATP to extracellular environment, the major endogenous agonist of P2X receptors (Taruno, 2018) with crucial roles during embryonic neurogenesis. Neuron-specific connexin36 is dynamically expressed throughout embryonic neurogenesis (Gulisano et al., 2000; Söhl et al., 1998). Along with connexin26, they are localized in the VZ at least during the first wave of neurogenesis, suggesting their  participation in the cellular expansion as well (Cina et al., 2007). Together, these data show the importance of P2 purinergic receptors and connexins in a period of intense neurogenesis in proliferative regions at the same time (Delarasse et al., 2009; Lin et al., 2007; Maric et al., 2000; Oliveira et al., 2016; Scemes et al., 2003). Ectoenzymes are essential for purinergic modulation since they fine-regulate the concentration of nucleotides and nucleosides in the extracellular space. Ecto-­ nucleotide pyrophosphatases/phosphodiesterases (E-NPPs) are a family of ectonucleotidases that hydrolyze ATP directly to AMP, releasing pyrophosphate. NPP expression patterns change with ongoing neuronal development (de Cognato et al., 2008). NPP1 gradually increases in expression during the development of different structures, such as the olfactory bulb, cerebral cortex, striatum, and cerebellum

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Fig. 5.1  Purinergic signaling  roles in embryonic neurogenesis. (a) Purinergic receptors and ectonucleotidases are gradually expressed during embryo development. (b) Purinergic signaling regulates the proliferation, migration, and differentiation of NSCs and NPCs. The P2Y1 receptor is abundantly expressed in RCGs. This receptor and TNAP positively modulate the proliferation of NPCs. Connexin43 releases ATP that can be hydrolyzed into ADP, which activates P2Y1 receptors and consequently promote the migration of NPCs. During differentiation, TNAP, connexin36, and UTP promote neuronal fate determination, whereas TNAP can also induce oligodendrocyte differentiation. The receptor, on which UTP acts, is still unknown. (c) Purinergic signaling can also synchronize the cell cycle which impacts proliferation and migration processes. Proliferative RGCs express P2Y1 receptors and TNAP, which can act as a source of ADP production. P2Y1 receptor activation increases the free intracellular Ca2+ concentration ([Ca2+]i) causing growth factor, ATP, and neurotransmitter release. Consequently, extracellular ATP activates P2Y1 receptors in neighbor cells triggering the same signaling. Connexin channels form connexons that propagate the signal. These mechanisms can induce the entrance  into the cell cycle, or exit  from the cell cycle, resulting in synchronicity in the S phase of the neural progenitor cell cycle, with consequently increased proliferation and migration rates. ADP adenosine 5'-diphosphate, ATP adenosine 5'-triphosphate, NTPDase2 ectonucleoside triphosphate diphosphohydrolase 2, TNAP tissue nonspecific alkaline phosphatase, UTP Uridine 5′-triphosphate

(de Cognato et al., 2008). Meanwhile, NPP2 was detected at E8.5 in the neural tube and posterior region of the midbrain (Bächner et al., 1999). From E13.5 until birth, NPP2 expression is restricted to mesenchymal tissues and choroid plexus epithelium (Bächner et al., 1999). After birth, NPP2 expression remained constant during

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the process of brain maturation (de Cognato et al., 2008), especially in oligodendrocyte differentiation and myelin formation (Fuss et  al., 1997). NPP3 expression decreases during the development process, especially in the cerebellum, hippocampus, and olfactory bulb. Few precursor cells transiently express NPP3, mainly in a specific germinal layer of the VZ of the immature rat brain, as shown in in vitro experiments (Blass-Kampmann et al., 1997). Tissue nonspecific alkaline phosphatase (TNAP) expression starts to appear at E8.5 in the neuroepithelium, and at E14, the majority of the telencephalic vesicle has TNAP activity, including DCX+ neuroblasts. This enzyme equally hydrolyzes ATP, ADP, or AMP generating adenosine. During further embryonic development, enhanced TNAP activity became restricted to cells of the VZ  and SVZ (Langer et  al., 2007). Ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) is an enzyme that preferentially hydrolyzes ATP over ADP and consequently produces ADP. Its expression becomes evident during late embryonic development on E17, is prominent during postnatal periods (Shukla et  al., 2005) (Fig.  5.1a). Few functions were attributed until now to this enzyme family during embryogenesis, but its function appears to be mainly associated with migration, further discussed below. A study showed that mouse fetal ventral midbrain-derived cells encode almost all P2X and P2Y receptors as well as NTPDase2 and TNAP (Delic & Zimmermann, 2010). Essentially, all components of the purinergic signaling were also expressed by the cultured cells derived from E10 and E13.5 (Delic & Zimmermann, 2010) (Fig. 5.1a). Besides that, this study demonstrates the presence of functional nucleotide and adenosine receptors in progenitors cultured from mouse fetal midbrain (Delic & Zimmermann, 2010). In fact, all these purinergic components possess functionalities during embryogenesis and modulate proliferation, migration, and differentiation of progenitor cells that will be discussed below.

 he Role of Purinergic Signaling in the Proliferation of Stem T and Progenitor Cells ATP has been described as an inductor of mitosis in cultured immortalized NPCs (Ryu et al., 2003), and sustains the proliferation of NPCs isolated from E16 mouse embryos. Inhibition of P2Y receptors prevented this effect. Real-time bioluminescence imaging of extracellular ATP revealed that the source of extracellular nucleotides is the progenitor cells themselves, which release ATP in episodic burst events (Lin et al., 2007) (Fig. 5.1b). Extracellular ATP can regulate embryonic neurogenesis by activating the P2Y1 receptor in RGCs. Transient spikes of [Ca2+]i in RGCs cause the release of growth factors, ATP, and other neurotransmitters. In a feedback loop, ATP can activate the P2Y1 receptor of the neighboring cells triggering [Ca2+]i

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mobilization and consequent release of growth factors, ATP, and neurotransmitters as well in the neighbor cells (Wiencken-Barger et al., 2007) (Fig. 5.1c). The passage of second messengers through connexin channels in non-junctional membranes propagates signals in sequence and creates calcium waves in the neighborhood for a long distance (Weissman et al., 2004). Coupling or uncoupling of these hemichannels that connect adjacent cells induces entrance or exit from the cell cycle. ATP and purinergic receptors have been recognized as triggers of these events. This phenomenon can promote synchronization in the S phase of the cell cycle of neuronal progenitor  cells, resulting in proliferation and neurogenesis (Weissman et  al., 2004) (Fig. 5.1c). In fact, treatment with the P2 receptor pan-antagonist, suramin, or specific P2Y1 receptor antagonist, MRS2179, or apyrase, which promotes ATP degradation, caused a reduction in [Ca2+]i waves and consequently  the absence of cell cycle synchronism, reducing cell proliferation and migration in the cortex. The P2Y1 receptor proved to be fundamental for the maintenance and increase of proliferation in embryonic neurogenesis (Weissman et al., 2004). Lin and colleagues showed that the neuronal differentiation process occurs associated with decreasing in ATP release and loss of P2Y receptors, which cause downregulation of purinergic signaling (Lin et  al., 2007). In fact, acute P2X7 receptor stimulation with BzATP induced proliferative behavior in E14Tg2A mouse embryonic stem cells (Glaser et  al., 2014). However, prolonged activation of the P2X7 receptor with extracellular ATP or BzATP leads to lysis/necrosis of NPCs (E14.5) accompanied by a loss of mitochondrial membrane potential. Surprisingly, NPC death mediated by P2X7 receptor stimulation is not associated with the opening of a non-selective pore, which is assumed to be responsible for ionic imbalances leading to cell death. Corroborating these findings, these effects were not observed in NPCs derived from P2X7 receptor knockout (KO) mice (Delarasse et  al., 2009). These initial data instigate the investigation of purines in the CNS development. Further investigation found that during human embryonic development, NPCs express P2X7 receptors and when activated, P2X7 receptors promote phagocytosis of apoptotic immortalized human neural progenitor cells (ReNcell), apoptotic neuroblasts, and latex beads in both neural progenitors and neuroblasts (Lovelace et  al., 2015). P2X7 receptor antagonists or siRNA knockdown inhibited this phenomenon, suggesting that the P2X7 receptor may act as a scavenger receptor on NPCs within the developing human CNS (Lovelace et al., 2015). Alkaline phosphatases are also important for the proliferation process since they regulate nucleotide levels, releasing inorganic phosphate. So far, it is known that TNAP is the most expressed ectoenzyme during embryogenesis in the mammalian CNS. In vitro and in vivo studies mapped that TNAP expression is enriched in neurogenic niches being the main regulator of purinergic signaling during development, as well as can modulate the proliferation of embryonic stem cells (Braun et al., 2003; Heo & Han, 2006; Langer et al., 2007; Sebastián-Serrano et al., 2015) (Fig. 5.1b).

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The Role of Purinergic Signaling in Progenitor Cell Migration Migration is part of embryonic neurogenesis since the neurons are born in the neural tube and must arrive at distant regions of the CNS to populate them. It has been proven that ATP and P2Y1 receptors are involved in modulating the migration of NPCs to distant regions, especially in the neocortical SVZ. An in vivo study using shRNA for downregulation of P2Y1 receptor expression  during development showed alterations of [Ca2+]i waves in NPCs and migratory mechanisms (Scemes et al., 2003) (Fig. 5.1b). Correspondingly, the P2Y1 receptor antagonist MRS2179 decreased proliferation and migration and blocked [Ca2+]i transients induced by 2-MeSATP, a P2Y receptor agonist, in embryonic mouse neurospheres. Connexins mediate protein-protein interactions and affect behavior and cell functions, such as migration. Connexin43, for example,  may allow non-permanent cell-cell interactions that are responsible for orienting the cell without the formation of any functional channel in the embryonic brain (Elias et al., 2007, 2010; Qi et al., 2015). It was observed in vitro that a specific part of connexin43 is responsible for the prevention of premature differentiation by interacting with signaling proteins, such as Src kinase. The deletion of this site of interaction caused impairment of [Ca2+]i transients induced by purinergic receptors (Santiago et  al., 2010; Sorgen et  al., 2004). Experiments using connexin43 KO mice showed impaired neurogenesis and cortical organization (Wiencken-Barger et al., 2007) (Fig. 5.1b). The impaired neurogenesis could be explained given that connexin43 genetic deletion induced reduction of P2Y1 receptor expression and neurosphere migration and [Ca2+]i transients, which were reverted when P2Y1 receptor was exogenously expressed (Scemes et al., 2003). These data show that connexin channels are important not only for intracellular signaling synchronization of the cell cycle but also for migration and differentiation of RGCs. Ectonucleotidases are also involved in migration. An in vivo study showed that NTPDase2 is associated with the migration of NPCs in the developing DG during embryogenesis (Shukla et al., 2005) (Fig. 5.1b). Moreover, during the development, strong expression of TNAP was observed in neuroendothelial cells of the neural tube at embryonic day E8.5 and in migrating germ cells (Langer et  al., 2007; Narisawa, 2015).

The Role of Purinergic Signaling in Cell Fate Destination Firstly, the gap junctions mediated by connexins between NPCs form a cluster of cells that respond coordinately to extrinsic signals; thus, connexins can synchronize cell aggregates  (Peinado, 2001; Russo et  al., 2008). Connexins can also mediate intracellular communication between NPCs through small signaling molecules. It was possible to identify this in vitro because NPCs can follow different cell fate destinations depending on the treatment and feeder layers, on which they are

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cultured (Song & Ghosh, 2004). As an example, connexin36 is a strong positive regulator of neuronal differentiation for striatal NPCs in mouse embryonic cells (Hartfield et al., 2011) (Fig. 5.1b). After it migrates correctly, the neuronal precursor must grow and contact other cells. Several molecules that can guide the cells in this process. During development, ATP can be released by different cell  types under diverse circumstances and generate a plethora of degradation products under ectonucleotidase action. One of them is the TNAP, which can regulate differentiation processes into neurons and oligodendrocytes in vitro during the embryonic stage (Kermer et al., 2010) (Fig. 5.1b). Interesting studies showed that hippocampal neurons from E17 stage onward express P2X7, P2Y1, and P2Y13 receptors in the distal domain of axons and ATP inhibits axonal elongation (del Puerto et al., 2012). An in vitro study conducted with a hippocampal neuronal culture from rat E17 showed that the decreased axon length was reverted following pharmacological treatment with the P2X7 receptor antagonist Brilliant Blue G (BBG) or suppression of P2X7 receptor gene expression using shRNA (Díaz-Hernandez et al., 2008). Contrarily, ADP caused a significant increase in axon lengths through the activation of P2Y1 receptors (del Puerto et al., 2012). Opposite effects were observed in conditions of P2Y13 receptor activation by ADP. These three receptors, P2X7, P2Y1, and P2Y13, are connected by a signaling pathway that controls cAMP levels, an important second messenger for pathways of axon elongation and formation. In contrast, they differ on how to produce the cAMP, controlled by adenylyl cyclase 5 (AC5). First, AC5 is inhibited by submicromolar concentrations of [Ca2+]i, which explains why the P2X7 receptor blocks axon elongation, whereas it was increased by the P2Y1 receptor through activation of a Gq protein and AC5, resulting in increased cAMP levels. On the other hand, the P2Y13 receptor, also activated by ADP, stimulates a Gi protein that inhibits AC5, abolishing axon elongation. This is just a part of the axon and dendrite elongation processes which are morphologically important, given the fact that these also control neuronal polarity. Activity modulation of purinergic receptors consequently affects other pathways, such as PI3K-Akt-GSK3α/β signaling, being equally important for axon growth (del Puerto et al., 2012). Besides axonal growth, P2 receptors also regulate proliferation, migration, and differentiation of oligodendrocyte precursors (Agresti et al., 2005). Gliogenesis is not within the scope of the present work, but it is noteworthy that P2Y1 receptors also play an important role in oligodendrocyte differentiation (for an in-depth review, see Agresti et  al. (2005) and Welsh and Kucenas (2018)). Roles for the P2X7 receptor in embryogenesis are contradictory. In a study conducted by Tsao and co-workers, PKC-dependent extracellular signal-regulated kinase pathway (ERK) phosphorylation was essential for P2X7 receptor-mediated neuronal differentiation of NPCs. The stimulation of P2X7 receptors in an NPC culture from E15.5 induced Ca2+ influx inhibited proliferation, modified the cell cycle, increased the expression of neuronal markers, such as βIII-tubulin and microtubule-­associated protein 2 (MAP2), and induced activation of ERK1/2. All these effects were abolished by the removal of extracellular Ca2+ and by the treatment with blockers of P2X7 receptor and PKC activities (Tsao et  al., 2013).

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Knockdown of P2X7 receptor  expression by shRNA extinguished the agonist-­ stimulated [Ca2+]i increase and the expression of βIII-tubulin and NeuN. The authors concluded that the activation of P2X7 receptors in NPCs induced neuronal differentiation through a PKC-ERK1/2 signaling pathway (Tsao et al., 2013). On the contrary, in the E14Tg2A embryonic stem cell line, P2X7 receptor expression decreased gradually according to the progress of neuronal differentiation. P2X7 receptor stimulation promoted proliferation and inhibited the expression of genes related to the differentiation of embryonic stem cells (Glaser et al., 2014). There are many interesting facts about the effects of extracellular nucleotides on the proliferation and differentiation of human mesencephalic neural stem/precursor cells (hmNPCs). When combined with the mitogens epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2), UTP increased expression of the proliferative marker in hmNPCs and this effect was prevented by the P2 receptor blocker PPADS.  Besides proliferation, UTP also stimulated dopaminergic differentiation (Fig.  5.1b). Treatment with UTP significantly augmented the number of tyrosine hydroxylase (TH)+- cells as well as TH protein concentration, and stimulated the ERK pathway. As in the proliferation assay, the differentiation-inducing effect of UTP was abolished by P2 receptor antagonists. The administration of other P2 receptor agonists (ATP, ADP, and the more stable and putatively selective P2Y1 receptor agonist, ADPβS) did not cause the same effects as UTP did (Milosevic et al., 2006). Concerning P1 receptors, the A2A receptor agonist, CGS21680, increased brain-­ derived neurotrophic factor (BDNF) production, whereas its antagonist ZM241385 prevented this effect in rat primary cortical neuron culture. A2A receptor stimulation induced BDNF production that depended on Akt-GSK3β signaling. The A2A receptor-mediated BDNF production had a role in neuronal protection against excitotoxicity, in neurite extension,  and synapse development (Jeon et  al., 2011). Similar experiments showed that the treatment of rat primary cultures of cortical neurons from E18 with the A2A receptor agonist CGS21680 enhanced axonal elongation and dendritic branching. This effect was blocked by inhibitors of PI3K, mitogen-­ activated protein kinase (MAPK), and phospholipase C (PLC) (Ribeiro et al., 2016). In summary, purinergic signaling affects differentiating NSCs and NPCs, thereby regulating the progress of differentiation and contributing to cell fate determination. The balance of these processes and how purines and their receptors together with many further signaling cues reach out to these cells determine which specific type of CNS cell each precursor cell will be transformed into.

Neurogenesis in the Postnatal Brain Purinergic signaling-mediated effects on neurogenesis are strictly necessary not only during embryonic period but during postnatal brain development as well, including the adult brain. NPCs express mostly P1 and P2 receptors and through extracellular nucleotides and nucleotides actions, they control proliferation,

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migration, and cell fate destination. As detailed below, purinergic signaling modulates chemokines and cytokines release, MAPK pathway, and acts synergically with growth factors resulting in regulation of neurogenesis directly or indirectly by glial modulation. In this section, we will address the two main neurogenic niches in adults: the SVZ of lateral ventricle (Fig. 5.2a) and SGZ of the hippocampal DG.

 urinergic Signaling Role in Neurogenesis P of the Subventricular Zone NPCs of the SVZ express virtually all P2X, P2Y, and P1 receptors, although the expression of some of them is contradictorily discussed (Benito-Muñoz et al., 2016; Grimm et al., 2009; Messemer et al., 2013a, 2013b; Mishra et al., 2005; Stafford et  al., 2007; Suyama et  al., 2012). Regarding nucleotide metabolism, these cells express NTPDase2, hydrolyzing ATP to ADP, and TNAP, an enzyme that hydrolyzes nucleotides and generates adenosine (Grimm et al., 2009; Mishra et al., 2005). TNAP is highly expressed during embryogenesis and in the ventricular and SVZ areas of the early postnatal brain. From postnatal day 21 until the adult stage, TNAP expression is gradually reduced but it remains present in SVZ and RMS. In the adult SVZ, TNAP is expressed mostly by proliferative cells, mainly Type B, A, and C cells (Langer et al., 2007). On the contrary, NTPDase2 expression is scarcely detectable during embryogenesis and it  increases during postnatal brain development, reaching adult levels at postnatal day 21, which is confined to SVZ (Langer et al., 2007). NTPDase2 expression is high in adult SVZ, mainly in Type B cells, and RMS (Braun et  al., 2003; Gampe et  al., 2015), as well as is the main NTPDase expressed in astrocytes (Robson et al., 2006) (Fig. 5.2b, c). Beyond gene and protein expression, neurospheres from adult SVZ exhibit functional ectonucleotidases and P2 receptors, being capable of hydrolyzing nucleotides and responding to ATP, ADP, and UTP stimuli (and its stable analogs) with [Ca2+]i transients, mainly through P2Y1 and P2Y2 receptors (Mishra et  al., 2005). Additionally, ADPβS, UTP, and low concentrations of adenosine augmented EGF and FGF-2-induced NPC proliferation in neurospheres, suggesting an overlap between downstream signaling of purinergic and growth factors for cell cycle control (Mishra et al., 2005). Further experiments demonstrated that P2Y1 receptors and possibly P2Y2 receptors promote growth factor signaling (Mishra et al., 2005). Grimm and coworkers showed afterward that ADPβS and UTP induced ERK1/2 and CREB phosphorylation in undifferentiated NPCs neurospheres were partially mediated by P2Y1, P2Y2, and P2Y13 receptors (Grimm et al., 2009). In addition, ATP, ADPβS, and UTP also augmented in  vitro migration of NPCs from SVZ (Grimm et  al., 2010). Corroborating these findings, mice with global KO for NTPDase2 presented increased BrdU+ (proliferation marker) cells in adult SVZ in vivo (Gampe et al., 2015). These data indicate that extracellular nucleotides and ADP- and UTP-responsive P2Y receptors are involved with signaling related to the

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Fig. 5.2  Purinergic signaling modulates adult neurogenesis in the SVZ and the migratory process in the RMS. (a) The SVZ region in the lateral ventricle is an adult neurogenic niche that have NPCs and glial cells, responsible to maintain homeostasis and fine regulate the appearance of newborn neurons in the adult stage. The NPCs in SVZ are classified as (b) Type B cells, Type C cells, and Type A cells, whereas glial cells with relevant purinergic function are astrocytes and ependymal cells. (b-I) Type B cells express connexin45 and release ATP, which is hydrolyzed into ADP by NTPDase2 or metabolized to AMP by TNAP action. Both ADP, produced by NTPDase2, and guanosine promote the self-renewal of Type B cells. However, neither ADP nor guanosine receptors involved with the proliferative process are described in Type B cells. P2X7 receptor seems to exert a countervailing effect, as its activation may promote apoptosis of Type B cells. (b-II) Type C cells express connexin45 which releases ATP and TNAP, which converts ATP into ADP, AMP, or adenosine. The ADP-sensitive P2Y1 receptor is abundantly expressed in Type C cells. Connexin45, extracellular ATP, and P2Y1 receptor activation were found to have a prominent role in cell  self-renewal. In addition, ATP, through P2X7 receptor  stimulation, promotes phagocytic activity and transmembrane pore formation in Type C cells. Adenosine, through A1 receptor activation, inhibits Type C cell differentiation into Type A/neuroblasts. (b-III) The adenosine A1 receptor also inhibits the differentiation of Type A/neuroblast into mature neurons. These cells abundantly express P2Y1 receptors, and activation of these receptors, which occurs endogenously since cells express connexin45 and TNAP, stimulates self-renewal and migration. (b-IV) Glial cells, especially  astrocytes, modulate NPC differentiation. Astrocytes of the SVZ express connexin45 and P2Y1 and A1 receptors. NTPDase2 is the main NTPDase in astrocytes. P2Y1 and A1 receptors induce astrocyte reactivity that may contribute to the neurogenic niche. P2Y1 receptor activation also induces the release of chemoattractant chemokines that positively modulate NPC proliferation and migration. Ependymal cells exhibit functional P2X7 receptors, but their role remains unknown. (c) Progenitor cells of the RMS express connexin45 for ATP release, which is metabolized into adenosine by TNAP enzymes that are also present. Further studies are necessary to establish the role of purinergic signaling in migratory cells in the RMS.  ADP adenosine 5'-diphosphate, AMP adenosine 5'-monophosphate, ATP adenosine 5'-triphosphate, LV lateral ventricle, NPCs neural progenitor cells, NTPDase2 ectonucleoside triphosphate diphosphohydrolase 2, RMS rostral migratory stream, SVZ subventricular zone, TNAP tissue nonspecific alkaline phosphatase

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proliferation, growth, and survival of NPCs. Contradictory results were reported by Stafford and colleagues who showed that ATPγS and ADPβS decreased primary neurosphere frequency and proliferation, whereas the P2Y1 and P2Y12 specific antagonists, 30  μM MRS2179 and 10  μM MRS2395, respectively, reverted this inhibition. However, after replating, secondary  formed neurospheres treated with ADPβS presented increased cell numbers, whereas ATPγS continued to inhibit neurosphere formation (Stafford et al., 2007), partially agreeing with the literature data. These controversies about nucleotide-induced proliferative effects may be related to NPC cultures performed as monolayer or neurospheres, as well as to growth factor supplementation. Despite this uncertainty regarding the effects of nucleotides on proliferation, additional studies have supported this positive modulation and the importance of the P2Y1 receptor in regulating this process. The P2Y1 receptor is abundantly expressed in NPCs and is mostly present in Mash1+/Type C and DCX+/Type A cells (Suyama et al., 2012). ADPβS, via P2Y1 receptor stimulation, increased neurosphere numbers and proliferation rates, while P2Y1 receptor activation reduced the size of neurospheres, as well as their differentiation rates  into neurons (βIII-tubulin+ cells) (Boccazzi et al., 2013). ADPβS induced astrocyte reactivity and increased expression of CXCL1, CCL3, and CCL20 chemoattractant chemokines, which were also involved in cell migration and proliferation of replated neurospheres. In vivo analysis corroborated these findings once 100 μM ADPβS infusion into the left ventricle for 7 days induced reactive astrogliosis and increased the number of BrdU+ cells co-stained for Mash1, GLAST, or DCX in the SVZ (Boccazzi et al., 2013). The role of the P2Y1 receptor was further scrutinized, since 2 μg/kg/h ATP infusions into the lateral ventricle for 3 days induced proliferation of Mash1+/Type C cells in the SVZ of adult mice. P2Y1 receptor inhibition, using 6 μg/kg/h MRS2179, or P2Y1 receptor KO mice revealed reduced proliferation of Mash1+/Type C cells. Noteworthy, P2Y1 receptor KO completely blocked the effect of ATP-induced proliferation in mice (Suyama et al., 2012) (Fig. 5.2b). These results indicate that although P2Y12 and P2Y13 receptors may participate in this effect, as ADP is their agonist, the P2Y1 receptor appears to have a prominent effect on the proliferation of neural progenitors Type C cells and Type A/migrating immature neurons. Recent evidence points to P2Y2 receptor participation in neurogenesis control since P2Y2 KO mice presented a reduction in the total BrdU+ population in the SVZ without affecting the RMS population or the migration to the olfactory bulb (Ali et al., 2021). Besides P2Y receptor functions in neurogenesis, connexins release ATP into the extracellular environment as above-mentioned. Connexin45 is expressed in neurogenic areas during postnatal brain development, including Mash1+/Type C, DCX+/ Type A, and GFAP+ cells, which might be Type B cells and mature astrocytes of SVZ/RMS (Imbeault et al., 2009; Khodosevich et al., 2012). Genetic deletion of connexin45 reduced the proliferation of Mash1+/Type C cells, whereas its genetic overexpression increased the proliferation of these cells. This mechanism depended on ATP release and Ca2+ signaling, probably via P2X receptors (Khodosevich et al.,

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2012). Ependymal cells of the SVZ also participate in neurogenesis control (Lim et al., 2000). As these cells express functional P2X7 receptors (Genzen et al., 2009), these receptors may indirectly or directly modulate adult neurogenesis. P2X7 receptors are also  abundantly expressed and functional in ex  vivo slices and cultured NPCs from the adult SVZ (Messemer et al., 2013a), except in astrocytes or neuroblasts (Genzen et  al., 2009). P2X7 receptor activation in undifferentiated neurospheres reduced NPCs viability, increased active caspase-3 expression in nestin+ cells, and induced transmembrane pore formation (Messemer et  al., 2013a). A recent study found that P2X7 receptors are expressed in in vitro primary culture and also in vivo in NPCs of the adult SVZ, mainly in Type C cells (Leeson et al., 2018). P2X7 receptor activation by ATP or BzATP induced Ca2+ influx and transmembrane pore formation in NPCs that were blocked with specific antagonists (Leeson et al., 2018). Interestingly, ATP treatment reduced NPC proliferation which did not occur in P2X7 receptor KO cells. Additionally, in agreement with the effects of the P2X7 receptor in embryogenesis, it acted as a scavenger receptor and promoted prominent phagocytic functions of NPCs from the SVZ (Leeson et al., 2018) (Fig. 5.2b). The phagocytic activity of P2X7 receptors in innate immune responses in the brain has been debated (Gu & Wiley, 2018). The first report demonstrating NPC phagocytic activity through P2X7 receptors (Leeson et al., 2018) has repercussions for brain and adult neurogenesis homeostasis. Intriguingly, a specialized microglia phenotype in SVZ/RMS controls neurogenic processes. These cells have low responsiveness to ATP stimulus and express low levels of P2Y12 and P2Y6 receptors, suggesting that purinergic signaling is downregulated in these phagocytic cells to avoid unwanted activation/phagocytosis (Ribeiro Xavier et al., 2015). We speculate that P2X7 receptor-induced phagocytosis in NPCs may be compensating or replacing specialized microglial cell functions in circumstances of high extracellular ATP concentrations, which act as a danger signal. These data indicate a new opportunity in the field for studying either direct control or glial control of neurogenesis by P2X7 receptors. Actions of adenosine on NPC proliferation has not been completely established. When NPCs had been cultured as primary or secondary neurospheres supplemented with FGF and EGF, adenosine treatment (10, 20, 30, or 50 μM) decreased neurosphere proliferation (Mishra et al., 2005; Stafford et al., 2007), whereas A2A and A3 receptor antagonists, 5 μM SCH58261 and 5 μM MRS1523, respectively, reverted this scenario (Stafford et al., 2007). Interestingly, under the same conditions, Mishra and coworkers found that low doses of adenosine (1 μM) stimulated neurosphere proliferation (Mishra et al., 2005), whereas Stafford and coworkers found no differences in neurosphere proliferation (Stafford et al., 2007). Accordingly, under similar culture conditions, 100 μM adenosine decreased the cell viability of neurospheres (Benito-Muñoz et al., 2016). However, when NPCs had been cultured as monolayers with several supplements (human transferrin, progesterone, putrescine, sodium selenite, insulin, bFGF, and EGF), adenosine treatment (10 or 30 μM) stimulated proliferation, while a lower adenosine concentration (3  μM) did not cause any

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effects (Migita et al., 2008). Further experiments revealed that A1 receptors, but not A2A or A3 receptors, were exclusively involved in the proliferation stimulation via MEK/ERK and the Akt pathway (Migita et al., 2008). These controversies indicate that cell culture conditions, such as monolayer/neurospheres and medium supplementation components, may determine proliferation and cell viability processes, and these concerns must be considered when interpreting the data. Adenosinergic signaling during neuronal differentiation was assessed as well. Initial experiments in neurospheres indicated that adenosine treatment (1, 10, and 100 μM) reduced the number of βIII-tubulin+ cells. These results were later confirmed with the specific 100 μM A1 receptor agonist CPA treatment at postnatal and adult stages. In fact, further experiments showed that the A1 receptor blocks neuronal differentiation by inhibiting differentiation of Type C cells into neuroblasts, inducing IL-10 release, and stimulating the STAT3/Bmp2/SMAD pathway. In vivo experiments corroborated in vitro findings given that 500 μM CPA infusion into the lateral ventricles was associated with neurogenesis inhibition and astrogliogenesis promotion in the SVZ and olfactory bulb (Benito-Muñoz et al., 2016) (Fig. 5.2b). Guanosine, a purine nucleoside with known neuroprotective effects (Bettio et al., 2016), is being investigated in the context of neurogenesis. Initial findings show that 8 mg/kg guanosine treatment for 8 weeks resulted in increased BrdU+ and nestin+ populations in SVZ in the rat with parkinsonism-like behavior induced by a proteasome inhibitor (PSI) (Su et al., 2009). Recent results suggested neurogenic effects of guanosine given that 100 μM guanosine augmented the number and density of adult SVZ neurospheres (Piermartiri et al., 2020) (Fig. 5.2b). In summary, the above-mentioned findings show that all neural progenitors and astrocytes express connexin45. Thus, all these cells are sources of extracellular ATP, indicating that purinergic signaling is fundamental for adult SVZ neurogenesis. In addition, the P2Y1 receptor has a major role in the proliferation of Type C and Type A cells, as well as in the migratory functions of neuroblasts. NTPDase2 is abundantly expressed in Type B cells; thus, it is likely that the proliferation of the Type B population is mediated by NTPDase2. Purinergic receptor expression has been shown for Type B cells, although it is apparent that ADP generated by NTPDase2, guanosine, and ATP potentially regulate Type B cell function. Initial reports indicate that P2X7 receptor-mediated apoptosis in Type B cells. P2X7 receptor has a prominent role in Type C cells, regulating phagocytic activity and transmembrane pore formation, which are innovative functions in NPCs. A1 receptors possess inhibitory activities on SVZ neurogenesis. Novel results regarding guanosine effects on neurogenesis indicate its role in neurogenesis promotion through stimulation of Type B cell proliferation. Finally, purinergic signaling in migrating progenitor cells in RMS lacks knowledge, which will be necessary for the complete understanding of this pathway in migratory behavior. These overall findings indicate several new goals that could be further explored in future studies.

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 urinergic Signaling Role in Neurogenesis of the Dentate P Gyrus Subgranular Zone Neurogenesis in the SGZ of the hippocampal DG (Fig. 5.3) has been studied for many years and the purinergic system has been increasingly shown to perform a crucial function in its regulation. Initial studies showed that NTPDase2 is also expressed in proliferative NPCs from the DG, mainly in Type I and neuroblast cells, whereas is absent in mature granule cells (Shukla et al., 2005), indicating that extracellular nucleotides, mostly ATP and ADP (its levels are controlled by NTPDase2), may regulate progenitor cell proliferation and migration. This modulation may be direct or indirect: regarding direct regulation, it was shown that ATP, ATPγS, and ADP treatment induced the proliferation of NPCs from adult DG, whereas PPADs, suramin, and MRS2179 completely blocked this effect. Neither adenosine nor the selective A1 antagonist, DCPCX, had any effect on proliferation, indicating that P2X and P2Y1 receptors are involved in the proliferative process of hippocampal NPCs (Cao et al., 2013). Glial control of neurogenesis is an important indirect mechanism (Falk & Götz, 2017) that can be modulated by extracellular ATP levels. ATP released by astrocytes was shown to be sufficient to induce proliferation of NPCs from the adult hippocampus. This mechanism was controlled by P2Y1 receptors expressed in NPCs (Cao et al., 2013). Moreover, P2Y1 receptors also promoted axonal outgrowth, via PI3K-Akt and MAPK-ERK pathways, in ex  vivo slices of the hippocampus and entorhinal cortex during the postnatal period (Heine et al., 2015) (Fig. 5.3b). P2Y12 and P2Y13 subtypes are also ADP-sensitive receptors and are prominently expressed in microglial cells. Microglial cells also contribute to the neurogenic niche homeostasis (Araki et al., 2021), and through these cells, both receptors exert antagonistic effects in neurogenesis. The P2Y12 receptor is currently considered a marker of healthy microglia that modulates extension processes and chemotaxis (Koizumi et al., 2013). P2Y12 KO mice presented the impaired phagocytic activity of microglia with a consequent reduction of the number of total BrdU+ cells and DCX+/neuroblasts in the adult DG.  In addition, diminished proliferative DCX+  neuroblast and BrdU+/NeuN+ newborn neurons were identified (Diaz-­ Aparicio et  al., 2020). These findings indicate that P2Y12 receptors regulate the phagocytic process of microglia that promotes the presence of self-renewing, migratory, and differentiated cells. Regarding P2Y13 receptor function in hippocampal neurogenesis, a report showed this receptor was not expressed by NPCs from DG, but was expressed by microglial cells of the hippocampus (Stefani et  al., 2018). Accordingly, P2Y13 KO increased BrdU+ cell numbers in the DG of young adult and aged mice. In P2Y13 receptor KO young adult  mice, increased proliferative Tbr2+/Type IIa and DCX+ neuroblast cells, but not nestin+ cells, were found in SGZ of DG. However, P2Y13 receptor KO mice also presented an increase in caspase-3 apoptosis marker expression and disruption of microglial complexity and process extension, indicating that the P2Y13 receptor is essential for microglial and hippocampal neurogenesis homeostasis (Stefani et al., 2018) (Fig. 5.3b). Recent findings

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Fig. 5.3  Purinergic signaling modulates adult neurogenesis in the SGZ of hippocampal DG. (a) General overview of hippocampus and DG location in the rodent adult brain. (b) Type II cells and astrocytes are the main source of extracellular ATP in SGZ neurogenic niche. ATP can be hydrolyzed by NTPDase2 expressed on Type I cells and neuroblasts that generate ADP. ADP activates P2Y12 and P2Y13 receptors expressed in microglial cells. Inhibition of microglial P2Y12 receptors blocks neuroblast self-renewal and newborn neuron appearance. On the contrary, inhibition of microglial P2Y13 receptor positively modulate Type II cell and neuroblast self-renewal. A1 receptors are expressed in Type I cells, neuroblasts, and immature and mature granule neurons, and their inhibition in neuroblasts promotes self-renewal. In contrast, A2A receptor activation stimulates Type II cell, neuroblast, and immature granule neuron self-renewal, as well as maturation of granule neurons. Guanosine, through an unknown receptor, also promotes neuroblast self-renewal. The P2X7 receptor is mainly expressed in Type II cells and its activation inhibits NPC proliferation. ADP adenosine 5'-diphosphate, ATP adenosine 5'-triphosphate, DG dentate gyrus, GCL granular cell layer, NPCs neural progenitor cells, NTPDase2 ectonucleoside triphosphate diphosphohydrolase 2, SGZ subgranular zone

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suggest the participation of the UTP-sensitive P2Y2 receptor in the proliferation and migration of hippocampal NPCs. P2Y2 receptor KO mice exhibited reductions in the total BrdU+ and DCX+ cell populations in the DG but did not reveal any alterations in neuronal fate determination, as measured by anti-NeuN immunostaining (Ali et al., 2021). As mentioned above, extracellular ATP signaling directly or indirectly regulates neurogenesis. Accordingly, connexin45, one of the ATP release pathways, is expressed by Type IIa cells of the DG during early postnatal brain development (Imbeault et al., 2009). Functional P2X7 receptor expression was demonstrated in adult hippocampal NPCs in their undifferentiated state and following differentiation (Hogg et al., 2004). Recently, it was corroborated that functional P2X7 receptors are expressed in the adult DG, mainly in Type II cells in both in vitro and in vivo conditions. Upon stimulation by ATP or BzATP, Ca2+ influx, and permeable transmembrane pore were induced in NPCs derived from the hippocampus, whereas the P2X7 receptor-specific antagonists AZ10606120 and A438079 inhibited these effects (Leeson et al., 2018) (Fig. 5.3b). Moreover, ATP treatment also promoted the phagocytic behavior of NPCs and decreased their proliferation through P2X7 receptor signaling (Leeson et  al., 2018), unlike what was found in previous studies (Cao et al., 2013). Besides  the here discussed effects of di- and triphosphate nucleotides, the nucleoside adenosine has been demonstrated to exert a relevant role in hippocampal neurogenesis. Single-cell gene expression analysis revealed that the A1 adenosine receptor is expressed in Type I NPCs, neuroblasts, and immature and mature granule neurons, whereas the adenosine A2A receptor is expressed in neuroblasts and immature neurons in mouse DG (Hochgerner et al., 2018, online browsable resource is available on http://linnarssonlab.org/dentate/). Treatment with the pan-antagonist of adenosine receptors, caffeine (60 mg/kg/day), reverted the total decrease in the numbers of proliferative BrdU+ cells and proliferative BrdU+/DCX+ neuroblast cells in the DG induced by 48 h of sleep deprivation (Sahu et al., 2013). Later on it was shown that the A1 receptor was responsible for the prevention of neurogenesis inhibition after sleep deprivation. Moreover, specific A1 receptor inhibition by 8-CPT at a concentration of 20 mg/kg/day restored the number of DCX+ cells at proliferative and intermediate stages and the BDNF-induced decreased expression by 48 h of sleep deprivation (Chauhan et al., 2016). Antagonistic effects were found for the A2A receptor since deletion of its gene resulted in a reduction of BrdU+ cells that was accompanied by increased expression of synaptophysin and PSD95 in the hippocampus of adult and aged A2A receptor KO mice. In addition, SNAP25 expression in the hippocampus was decreased in adult and increased in aged A2A KO mice (Moscoso-Castro et al., 2017). A recent study demonstrated that A2A receptor activation, with 100 nM CGS21680 for 28 days, increased DCX+ population and newborn mature neurons BrdU+/NeuN+ in DG of adult rats. Accordingly, in DG neurospheres obtained from early postnatal (P1-3) rats, 30 nM CGS21680 increased the frequencies of Type IIb cells (Nestin+/SOX2+/DCX+), neuroblasts (DCX+), and immature neurons (Nestin−/SOX2−/DCX+) cell populations and reduced neuroblast death. Remarkably, A2A receptor activation was essential for BDNF-mediated proliferation

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and differentiation into mature neurons (Ribeiro et al., 2021). However, in a pathogenic context, such as the fat mass and obesity-associated gene (Fto) deficiency model, which results in adipogenesis and energy homeostasis dysfunction, inhibition of adult neurogenesis in the DG and consequent increase of adenosine levels in the hippocampus were found (Gao et al., 2020). Adenosine treatment of in vitro differentiated NPCs increased caspase-3 apoptotic marker expression, possibly through A2A receptor activation (Gao et al., 2020) (Fig. 5.3b). These findings indicate that adenosine through A2A receptors may act differently according to the physiological or disease state: physiologically, the A2A receptor may promote the proliferation of NPCs and neuronal differentiation, whereas pathologically it may induce apoptosis of newborn neurons. In addition to adenosine, guanosine treatment increased the number, density, and proliferation of nestin+ and β-III tubulin+ cells of neurospheres from DG (Piermartiri et al., 2020). These data were further corroborated in vivo since adult mice treated with 8 mg/kg of guanosine for 26 days exhibited increased total BrdU+ cells and proliferative BrdU+/DCX+ cells (Piermartiri et al., 2020) (Fig. 5.3b), indicating the neurogenic role of guanosine and cell fate destination with neuronal commitment in the hippocampus. Altogether, we conclude that purinergic signaling has an important regulatory role in adult DG neurogenesis. Purinergic signaling may regulate neural progenitor functions directly, by expressing P2 and P1 receptors in NPCs and granule neurons; and indirectly, mainly by microglial function. Further studies are needed to establish which ectonucleotidases are expressed in the DG neurogenic niche. However, it is known that the ADP-generating enzyme, NTPDase2, is expressed in this niche. Remarkably, ADP acts on P2Y12 and P2Y13 receptors that  exist on microglial cells, and although both are coupled to an inhibitory G protein, they exert antagonistic modulation in NPCs from DG. While P2Y12 receptor inhibition blocks neuroblast self-renewal and newborn neurons appearance, P2Y13 receptor inhibition promotes Type II cell and neuroblast self-renewal. Likewise, A1 and A2A receptors also exert antagonistic roles. A1 receptor inhibition in neuroblasts promotes selfrenewal. In contrast, A2A receptor activation stimulates Type II cell, neuroblast, and immature granule neuron self-renewal, as well as the maturation of granule neurons. P2X7 receptor activation inhibits NPC proliferation. Initial studies concerning guanosine effects indicate that guanosine treatment promote neuroblast self-renewal, although the mechanisms remain unclear.

 ovel Strategies to Study Purinergic Role in Neurogenesis N and Concluding Remarks We postulate that purinergic signaling is fundamental for correct embryogenesis, brain development, and maintenance of adult neurogenic niches. Besides extensive literature regarding purinergic signaling regulation in different stages of neurogenesis gathered here, we noticed that there are still several gaps in the literature to have

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full knowledge of the field. In fact, in addition to the two classic neurogenic niches brought up in this review, evidence has accumulated that hypothalamus also presents neurogenesis (Yoo & Blackshaw, 2018). Tanycytes from the hypothalamus, radial glia-like cells, also express pluripotency markers such as Rax, LHX2, Sox2, Sox9, nestin, vimentin and GFAP, and Notch proteins (Chauvet et al., 1998; Lee et al., 2012). These cells are located close to the third ventricle, and their cell body contacts the cerebrospinal fluid (Yoo & Blackshaw, 2018). Due to their location at the interface between cerebrospinal fluid and brain parenchyma, tanycytes’ function has been studied regarding feeding regulation and energy balance (Yoo & Blackshaw, 2018). Remarkably, initial studies found that tanycytes are sensitive to glucose and extracellular ATP, showing [Ca2+]i wave responses (Frayling et al., 2011). In the following, 2MeSADP-induced [Ca2+]i  transients were  shown to be completely, but reversibly, blocked by the P2Y1 receptor antagonist MRS2500 indicating that ATP responses were mediated by P2Y1 receptors in tanycytes. Further experiments showed that tanycytes released ATP after glucose stimulus and glucose-evoked Ca2+ wave depended on P2Y1 receptor function (Frayling et al., 2011). Tanycytes also express NTPDase2 (Braun et al., 2003) and connexin43 that are involved in glucose-­ evoked [Ca2+]i  transients. These events are mediated by ATP release that consequently stimulates tanycytes proliferation via P2Y1 receptors during the early postnatal period (Orellana et al., 2012; Recabal et al., 2021). Corresponding with the findings about SGZ and SVZ neurogenesis, the initial results on tanycytes’ neurogenesis suggest that similar purinergic regulation may occur, supporting the relevance of purinergic signaling in neurogenesis. Most purinergic receptors are expressed in embryonic NPCs. TNAP is the prominent ectoenzyme present in NSCs and NPCs. NTPDase2 expression appears later in the embryo at E17. The P2Y1 receptor is the main purinergic receptor involved in NSCs and NPCs proliferation and migration, whereas the P2X7 receptor’s role in proliferation and differentiation is contradictory and more studies are needed to confirm those findings. Remarkably, TNAP, connexin36, and UTP promote neuronal fate determination. However, whether UTP receptors act on neuronal differentiation during the embryonic stage is still unknown. Remarkably, connexin45 seems to be the most relevant connexin in adult neurogenesis. Through its action, all progenitor cells and astrocytes are sources of ATP in the  SVZ, whereas in the  DG, astrocytes and Type II cells are the major sources of extracellular ATP. There is no information available about ATP-degrading enzymes,  besides NTPDase2, in the  DG.  Accordingly, the P2Y1 receptor has  a major role  in modulating the proliferation of Type C cells and neuroblasts in the SVZ. In addition, it is known that adenosine has a role in neurogenesis as well. A1 and A2A receptors exhibit antagonistic roles in controlling DG neurogenesis. The A2A receptor favors proliferation and neuronal fate determination in the DG. In both DG and SVZ, the A1 receptor seems to exert an inhibitory function on proliferation and differentiation, respectively, indicating an overall neurogenesis inhibition. In both neurogenic niches, P2X7 receptor stimulation induced phagocytic activity and transmembrane pore formation in NPCs, indicating novel functions of NPCs that could be further explored. Additionally, glial control of neurogenesis is also an

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important regulatory mechanism. The P2X7 receptor is expressed and functional in ependymal cells of the SVZ; however, its effects are unknown. Microglia also possesses a fundamental role in neurogenesis control, and recent findings have shown that P2Y12 and P2Y13 receptors have antagonistic roles in the DG that should be further explored. Regarding Type B cells of the  SVZ, there is little information about purinergic signaling, mainly limited to purinergic receptors, although this cell type expresses NTPDase2 and TNAP enzymes that are a great source of extracellular nucleotides and nucleosides. Pioneering and recent evidence regarding nucleoside guanosine promoting neurogenesis in both SVZ and DG has been collected. However, it remains unclear through which receptor guanosine acts as well as the intracellular signaling mechanisms induced by guanosine. In conclusion, there are several goals to achieve in deciphering purinergic control of neurogenesis. Noteworthy more information is needed regarding  intracellular mechanisms, glial modulation, and several other purinergic receptors, which have been poorly investigated. The elucidation of purinergic signaling mechanisms is essential for understanding neurodevelopmental diseases and endogenous repair mechanisms of the brain.

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Chapter 6

Purinergic Signaling in Autism Spectrum Disorder Iohanna Deckmann, Júlio Santos-Terra, and Carmem Gottfried

Abstract  Autism spectrum disorder (ASD) is a highly prevalent neurodevelopmental disorder estimated to affect 1:36 individuals, characterized by impairments in the social approach and stereotyped behavior patterns. The etiology of this disorder is still unclear; however, disturbances in the regulation of purinergic signaling contribute to establishing the ASD phenotype. Purinergic signaling is a system that involves second extracellular messengers able to trigger responses in several biological processes, activating the immune system, modulating bioenergetics, and promoting neuromodulation, by activation of specific receptors subtypes: metabotropic P1, ionotropic P2X, and metabotropic P2Y. In addition, several psychiatric disorders, including epilepsy, schizophrenia, Alzheimer’s disease, and, in recent

Iohanna Deckmann and Júlio Santos-Terra contributed equally with all other contributors. I. Deckmann · J. Santos-Terra Translational Research Group in Autism Spectrum Disorder - GETTEA, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil National Institute of Science and Technology in Neuroimmunomodulation - INCT-NIM, Rio de Janeiro, Brazil Autism Wellbeing and Research Development - AWARD - Initiative BR-UK-CA, Porto Alegre, Brazil C. Gottfried (*) Translational Research Group in Autism Spectrum Disorder - GETTEA, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil National Institute of Science and Technology in Neuroimmunomodulation - INCT-NIM, Rio de Janeiro, Brazil Autism Wellbeing and Research Development - AWARD - Initiative BR-UK-CA, Porto Alegre, Brazil Departamento de Bioquímica, ICBS, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 H. Ulrich et al. (eds.), Purinergic Signaling in Neurodevelopment, Neuroinflammation and Neurodegeneration, https://doi.org/10.1007/978-3-031-26945-5_6

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years, ASD, present altered components associated with the purinergic system, such as mitochondrial dysfunction, polymorphisms in genes that encode purinergic receptors, abnormalities in intermediaries of purine metabolism, among others. This chapter summarizes the contribution of purinergic signaling in ASD phenotype and hypothesizes how this intricate puzzle involves extracellular messengers and ASD pathophysiology, focusing on pathways associated with metabolism, neuroimmune modulation, and neurodevelopment. Keywords  Autism spectrum disorder · Purinergic signaling · Immunomodulation · Neurodevelopment · MicroRNA · Second messengers · Metabolism · Cell signaling

Introduction Autism spectrum disorder (ASD) is one of the neuropsychiatric disorders with the highest incidence nowadays. As it is a condition that originates during early neurodevelopment, involving epigenetic changes, several experimental approaches shed light on possible mechanisms involved in ASD triggering. In this context, the purinergic system emerges as a strong candidate for its phenotype modulation.

ASD ASD is a neurodevelopmental disorder characterized by two sets of characteristic behavioral alterations: communication/social interaction impairments and stereotyped/repetitive behaviors (American Psychiatric Association, 2013). Although the ASD etiology remains unclear, it is already known that both genetic and environmental risk factors can contribute to the onset (Gottfried et al., 2015). Furthermore, the most recent prevalence data of ASD from the USA shows that 1:36 8-year-old children are affected, and that the ratio of males/females is 4:1 (Maenner et  al., 2023), demonstrating a high prevalence that has been rising in the last decades. Besides that, the absence of biomarkers and the heterogeneity of the disorder challenge the diagnosis and, consequently, the implementation of adequate therapeutic strategies (Masi et al., 2017). Beyond the core behavioral dyad, ASD individuals may also experience a series of comorbidities, including epilepsy, anxiety, perception alterations, gastrointestinal disturbance, sleep impairments, and many others (Doshi-Velez et  al., 2014). The high incidences of electrophysiological (8–30% of the individuals with ASD) (Spence & Schneider, 2009; Bolton et al., 2011; Lukmanji et al., 2019) and sensory alterations (more than 90% of the individuals) (Chang et al., 2014) support the connectivity theory in ASD: the brain, in this case, presents an excitatory/inhibitory imbalance added to impaired connectivity of different brain regions, leading to local hyper processing, resulting in the impaired interpretation of the different stimuli.

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Several pathways have already been described as altered in ASD, both in the brain and in peripheral tissues. Immune system alterations stand out in ASD, including descriptions of increased content of pro-inflammatory cytokines, altered lymphocyte profile, and high levels of autoimmune diseases (Deckmann et al., 2018). In the brain, the highlights are for routes associated with glutamate and GABA neurotransmission (Horder et al., 2018), as well as pathways involved in synaptic plasticity (Bourgeron, 2015) and the activation and reactivity of glial cells such as astrocytes and microglia (Petrelli et al., 2016). Purinergic signaling studies in ASD are emerging in recent years, demonstrating several roles that will be discussed in this chapter. In an overview, this system can be the link that unites immune and brain dysfunctions, helping to understand the pathophysiology of autism.

Purinergic System and Autism-Like Features As reviewed by Ulrich et al. (2012) and Fumagalli et al. (2017), brain development in embryonic life is finely regulated by a range of biological processes. It has been demonstrated that purinergic signaling plays an essential role in organizing embryonic and fetal development and organogenesis in a time-dependent manner, controlling purinergic signaling molecules, such as adenosine (ADO) triphosphate (ATP), the Ca2+ release from radial glia, differential receptor subtypes expression, among others (Ulrich et al., 2012; Fumagalli et al., 2017). Here, we present an overview of the contribution of the dysfunction of purines and pyrimidines metabolism in ASD. A summary of the main findings can be seen in Fig. 6.1.

Humans The neurobiological bases of ASD-like features (social impairments and stereotyped behavior) are still a challenge for science due to the dynamics and crosslinking of distinct biological pathways, including purinergic signaling throughout development. Single nucleotide polymorphisms (SNP) in the ADO A2A receptor gene (ADORA2A) were already associated with ASD (rs2236624-CC and rs2298383) and phenotypic variability, including impaired scores in behavioral assessments (rs3761422, rs5751876, and rs35320474) (Freitag et  al., 2009). Interestingly, ASD patients also demonstrated mutations in the ADO A3 receptor gene (ADORA3), identified as rs77883500 and rs139935750, and, in an in  vitro assay, the presence of the first SNP induced enhanced levels of cGMP, resulting in increased activity of the serotonin transporter (Campbell et al., 2013). A postmortem analysis of cerebellum from idiopathic ASD patients demonstrated that the cluster of genes associated with impaired social behavior had gene

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Fig. 6.1  Main findings on purinergic system dysfunctions and autism-like features. Here we provide a clear overview of changes described in cell culture, in knockout animals for constituents of the purinergic system, and in animal models of ASD, as well as in humans diagnosed with ASD, emphasizing the important contribution of P2X4, P2X7, P2Y1, and A1 receptors in the pathophysiology of ASD. (1) Freitag et al. (2009), (2) Campbell et al. (2013), (3) Luo et al. (2018), (4) Takahashi et al. (2020), (5) Braun et al. (2007), (6) Stubbs et al. (1982), (7) Persico et al. (2000), (8) Bottini et al. (2001), (9) Avendaño et al. (2015), (10) Chávez et al. (2019), (11) Garré et al. (2020), (12) Horváth et al. (2019), (13) Trang et al. (2009), (14) Montilla et al. (2020), (15) Wyatt et al. (2013), (16) Mastrangelo et al. (2012), (17) Naviaux et al. (2013), (18) Hirsch et al. (2020), (19) Squillace et al. (2014), (20) Zimmermann et al. (2017), (21) López-Cruz et al. (2017)

ontology enrichment for purinergic-signaling genes, demonstrating an important overlap (Ginsberg et al., 2012). In a recent study, gene set enrichment analysis of a polygenic risk score in ASD individuals demonstrated an association (R2 = 0.064; β, −5.30; SE 1.30; P  A2B (0.03) > A3 (0.1) >> A2A (1) P2X1 (0.05–1) P2Y1 (8) P2X2 (1–30) P2Y2 (0.1) P2X3 (0.3–1) P2Y4 (2.5) P2X4 (1–10) P2Y11 (17) P2X5 (1–10) P2Y13 (0.26) P2X6 (1–12) P2X7 P2Y12 (0.07) ≈ P2Y13 (0.06) P2Y2 (0.2) > P2Y6 (6) P2Y6 (0.3) P2Y14 (0.1–0.5)

References Borea et al. (2018) Vitiello et al. (2012)

ATP adenosine triphosphate, ADP adenosine-5′-diphosphate, UTP uridine-5′-triphosphate, UDP uridine-5′-diphosphate

adenosine can be directly released outside the cell through nucleoside transporters (Garcia-Gil et al., 2021). Adenosine activates P1 receptors while purine/pyrimidine nucleotides stimulate P2 receptors with different affinities (Table 7.2). Based on molecular cloning and pharmacological differences, P2 receptors can be further divided into P2Y (metabotropic) and P2X (ligand-gated cation channels) receptors (Kennedy, 2021). Currently, eight subtypes of metabotropic P2Y receptors (P2Y1, 2, 4, 6, 11, 12, 13, 14) and seven subtypes of ionotropic P2X receptors (P2X1–7) have been described (Burnstock, 2018; Kennedy, 2021). The role of purinergic signaling in stress and mood disorders has been largely discussed in the last decades (Illes et  al., 2019; Ribeiro et al., 2019b, d). In this chapter, we will revisit the main signaling mechanisms conveyed by adenosine and ATP and their possible involvement in stress and depression, with new perspectives for novel mechanisms and treatment options.

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Adenosine Signaling in Mood Regulation and Mood Disorders Overview of the Adenosinergic System Adenosine is a ubiquitous molecule in all living cells. Aside from constituting the backbone of nucleic acids and being a central molecule in cellular metabolism, adenosine can be released in an activity-dependent manner to modulate cellular metabolism in conditions where energy charge is compromised. Despite its actions as a homeostatic modulator, adenosine exerts another important extracellular function as a neuromodulator even in the absence of metabolic imbalance. The neuromodulatory role of adenosine occurs mainly at the synaptic level and involves the control of neurotransmitter release, post-synaptic responsiveness, and interactions between different receptor systems (Ribeiro et al., 2003). Extracellular adenosine is involved in multiple physiological processes of the central nervous system regulating sleep, cognition, memory, and different behavioral and emotional states (Gomes et al., 2011). Adenosine is mainly formed by the breakdown of intra- or extracellular adenine nucleotides. There are two main mechanisms involved in intracellular adenosine synthesis. The first occurs through the dephosphorylation of AMP by the cytosolic enzyme 5′-nucleotidase (Latini & Pedata, 2001). The second source of intercellular adenosine is the hydrolysis of S-adenosyl-homocysteine (SAH) by the enzyme SAH hydrolase, formed from transmethylase activity on S-adenosylmethionine (SAM) (Ueland, 1982; Pak et al., 1994). In the extracellular compartment, adenosine can be formed by the breakdown of released adenine nucleotides, especially ATP. Intracellular ATP can be released by exocytosis by neurons or glial cells or after transient or permanent damage of cell membranes during trauma (Dunwiddie et  al., 1997). ATP undergoes rapid enzymatic catabolism originating ADP, AMP, and adenosine in both situations. This catabolic process involves the coordinated action of the ectoenzymes nucleoside triphosphate diphosphohydrolases and ecto-­5-­ nucleotidases (Zimmermann, 2021). Adenosine can be transported across the membrane by four subtypes of bidirectional equilibrative nucleoside transporters (ENT1–4). These transporters are responsible for the release or uptake of purine and pyrimidine nucleosides in a concentration-­dependent manner (Parkinson et al., 2011; Pastor-Anglada & Perez-­ Torras, 2018). On the other hand, the CNS also possesses concentrative nucleoside transporters (CNT1–3) involved in the uptake of extracellular nucleosides dependent on the transmembrane sodium (Na+) gradient (Pastor-Anglada & Perez-Torras, 2018). The catabolism of adenosine can occur intra- and extracellularly through a conversion to inosine catalyzed by the enzyme ADA. However, extracellular ADA expression is usually relatively low, and most extracellular adenosine is taken up by the transporters and then catabolized. Intracellular adenosine can also be converted to AMP by ADK (Lloyd & Fredholm, 1995). The mechanisms underlying adenosinergic control of emotional behavior involve modulation of synaptic transmission and strength, neuroplasticity, glial cell

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reactivity, neurogenesis, glial-neuron communication, and others (Gomes et  al., 2011). Most of these functions are attributed to adenosine actions on metabotropic A1, A2A, A2B, and A3 receptors (P1 receptors) (Fredholm et al., 2001). These receptors are composed of seven transmembrane glycoproteins coupled with G proteins. A2A and A2B receptors are coupled to Gs proteins, being linked to adenylyl cyclase activation and protein kinase A (PKA)-dependent signaling. Alternatively, A1 and A3 receptors are functionally coupled to members of the pertussis-toxin-sensitive family of G proteins (Gi/o), and their activation inhibits adenylyl cyclase and raises intracellular concentration of calcium ([Ca2+]i) through a pathway involving phospholipase C (PLC) activation. A2A, A2B, and A3 receptors can also couple to Gq/11 proteins and striatal A2A receptors are mainly coupled to Golf, which also activates adenylyl cyclase (Fredholm et al., 2001; Sebastiao & Ribeiro, 2009). The neuromodulatory role of adenosine and its ability to normalize synaptic transmission is mediated through concerted action on inhibitory A1 and facilitatory A2A receptors. Adenosine in concentrations present under basal conditions is sufficient to activate A1, A2A, and A3 receptors. By contrast, adenosine A2B receptors require higher concentrations of adenosine to be significantly activated (Ribeiro et al., 2002). However, adenosine receptors have been shown to form homodimers, which are believed to be the functional receptor species placed at the cell surface, and also heterodimers with other receptors including dopaminergic and glutamatergic receptors (Canals et al., 2004; Ginés et al., 2000; Ciruela et al., 2001). Adenosine has the ability to control and integrate the activity of neurotransmitter systems, including dopaminergic, serotonergic, GABAergic, and glutamatergic systems, which regulate positive and negative emotional states and are altered in mood- and anxiety-­related disorders, including MDD, bipolar disorder (BD), and post-traumatic stress disorder, and others (Gomes et al., 2011, 2021; van Calker & Biber, 2005). The widespread expression of adenosine receptors and their implication in numerous physiological and pathophysiological conditions had made them pivotal targets for developing clinically effective agents for different conditions.

Preclinical Evidence Over the past decades, several pieces of evidence have reinforced the existence of a relevant role for the adenosinergic system in mood regulation and mood disorders. Initially, research was focused on two main topics: the impact of antidepressant treatments on the adenosine release, metabolism, and receptors, as well as the behavioral changes induced by the pharmacological modulation of the adenosinergic system in animal models. Both topics were recently revised by J. I. Gomes et al. (2021) and van Calker and Biber (2005). Here, we present some of the major findings that lead to our current understanding of the potential of adenosine receptors for mood regulation. Some of the initial studies showed that tricyclic antidepressants such as nortriptyline, clomipramine, and desipramine increased the release of adenosine in cortical

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neurons (Phillis, 1984). Moreover, the activity and expression of different proteins involved in adenosine metabolism and transport, including the enzymes ectonucleotidases (Pedrazza et al., 2007, 2008), ADA (Herken et al., 2007), and the CNT2, were sensitive to the action of different classes of antidepressants (Nagai & Konishi, 2014; Sawynok et al., 2005). The effects of some mood stabilizers, such as carbamazepine, also appear to involve the modulation of the adenosinergic system, especially A1 receptors (Biber et  al., 1999). Despite differences in the classes of the compounds evaluated, concentrations, and cell types investigated in different experiments, the results initially suggested that the modulation of the adenosinergic system might be a common event involved in the pharmacological compounds used to treat mood alterations. These observations were further reinforced by studies showing that non-pharmacological therapies for mood disorders also impact the adenosinergic system. Sleep deprivation, electroconvulsive therapy, and deep brain stimulation were associated with an increase in the release of ATP and adenosine, increase in the expression of A1 receptors, and decrease in A2 receptor expression (Bekar et al., 2008; Gleiter et al., 1989; van Calker & Biber, 2005). Despite these initial observations, causality was not established and the normalization of the adenosinergic system might be a secondary effect in mood regulation. In the early 2000s, a second line of evidence emerged to explore the relationship between the adenosinergic system and mood regulation, based on different behavioral data using rodent models. These studies included models such as the forced swim test (FST), tail suspension test (TST), the learned helplessness (LH) paradigm, olfactory bulbectomy, chronic unpredictable stress (CUS), social defeat stress, and others. In these models, genetic activation/inactivation of adenosine receptors as well as non-selective or selective ligands was explored. It is important to highlight that these studies are heterogeneous and strongly impacted by differences in the models and the intrinsic limitations of mimicking psychiatry disorders in rodents (Planchez et  al., 2019). However, they provided initial evidence and potential mechanisms associated with adenosine receptors and emotional regulation. It was demonstrated that acute central or systemic adenosine administration elicited antidepressant-like effects in mice submitted to the FST and TST (Kaster et al., 2004). However, it must be noted that in rats submitted to the bilateral olfactory bulbectomy model of depression associated with hyperlocomotion, chronic adenosine treatment had no effect on the FST or sucrose preference test (SPT) (Padilla et al., 2018). The effects of adenosine administration are hard to explain, in part due to its short half-life but also by its ability to interact with multiple targets both in the periphery and in the CNS. Moreover, inosine, the main adenosine metabolite, also presented antidepressant-like effects after acute and chronic systemic administration in mice, and prevented the hyperlocomotion in a model of manic behavior in rats (Camerini et  al., 2020; Goncalves et  al., 2017a, b; Muto et  al., 2014; Yuan et al., 2018). Some of the strongest evidence pointing to the potential of adenosine receptors on mood modulation came from studies with A2A receptors. These receptors, initially identified in the striatum, are present in the nerve terminals of important brain regions regulating emotional behavior such as the HIPP, PFC, and amygdala (AMY)

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(Rebola et  al., 2005; Simões et  al., 2016). The administration of adenosine A2A receptor antagonists and A2A receptor deficiency induced active escape behaviors interpreted as antidepressant-like effects (El Yacoubi et  al., 2001, 2003). These results were further reinforced by different A2A receptor antagonists with selective (SCH58261, SCH412348, istradefylline, KD66, KD167, and KD206) and non-­ selective properties (caffeine, theophylline) in predictive rodent models such as the FST, TST, reserpine-induced depressive behavior, or LH (Dziubina et  al., 2016; Hodgson et al., 2009; Minor et al., 2003; Turgeon et al., 2020; Yamada et al., 2013, 2014). Caffeine was also shown to enhance the antidepressant-like activity of classical antidepressant drugs in the FST in mice (Szopa et al., 2016; Poleszak et al., 2016). Accordingly, A2A receptors overexpression in forebrain neurons increased depression-like behavior and anhedonia (Coelho et al., 2014). The use of different animal models with more substantial validity was an important step to increase the reliability of the previous findings and reinforce the role of the adenosinergic system in mood regulation. Chronic unpredictable mild stress (CUMS) leads to depression-like and anhedonic behavior and is associated with a decrease in synaptic plasticity and an increase of A2A receptors in the striatum and in glutamatergic terminals in the HIPP (Crema et  al., 2013; Kaster et  al., 2015). Both caffeine and the selective A2A antagonist istradefylline prevented these effects (Kaster et  al., 2015). Chronic caffeine treatment also enhanced the resilience to social defeat stress in mice (Ibrahim et al., 2020; Yin et al., 2015). Furthermore, A2A receptor blockade also prevented stress-induced hippocampal-related deficits induced by subchronic restraint (Cunha et al., 2006), maternal separation (Batalha et  al., 2016), and olfactory bulbectomy (Padilla et  al., 2018). There are multiple mechanisms associated with the potential antidepressant effects of A2A receptor antagonists, including modulation of neuroplasticity (Kaster et al., 2015), microglial reactivity, and neuroinflammation (Dias et al., 2021), as well as their ability to modulate the dopaminergic and glutamatergic systems (Boison, 2008) and hypothalamic-­pituitary-adrenal (HPA) axis (Batalha et al., 2016). However, since A2A receptors are often found to inhibit the actions of A1 receptors (Stockwell et al., 2017), one possible explanation for the antidepressant-like effects of A2A receptor antagonists might involve the facilitation of A1 receptors signaling (Hines et al., 2013). The activation of A1 receptors can decrease neuronal excitability and has well-documented neuroprotective effects (Sebastiao & Ribeiro, 2009). Antidepressant effects of A1 receptor activation were first suggested by van Calker and Biber (2005), based on the effects of electroconvulsive therapy. These findings were further reinforced by studies showing that sleep deprivation, another highly effective treatment for depression, requires A1 receptor activation (Hines et  al., 2013). The central administration of an A1 receptor agonist led to antidepressant and anti-anhedonia effects (Hines et al., 2013). Finally, Serchov et al. using transgenic mice overexpressing adenosine A1 receptors confirmed its antidepressant potential in animal models. The activation of neuronal A1 receptors produced antidepressant-­ like effects in different paradigms, while A1 receptor knockout (KO) mice displayed depressive-like behavior (Serchov et al., 2015).

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The involvement of A2B and A3 receptors in mood modulation and in mood disorders is not well established so far and just a few studies evaluated this possibility. It was reported that fluoxetine-mediated increase in BDNF in astrocytes involved astroglial A2B receptors activation (Kinoshita et al., 2018). However, the selective A2B receptor antagonist alloxazine was tested in the reserpine-induced pharmacological model but failed to revert the increased immobility in the FST (Minor & Hanff, 2015). For A3 receptors, behavioral phenotyping of KO mice revealed increased locomotor activity but a decrease in active escape behaviors in the FST compared to wild-type mice, suggesting a depression-like phenotype (Fedorova et al., 2003). The role of A2B and A3 receptors in mood modulation needs to be further investigated, especially using more robust models and in patients with mood disorders.

Human Studies In 1999, Elgun et  al. demonstrated that ADA activity is reduced in the serum of patients with MDD and reported the existence of an inverse correlation with the severity of depression (Elgun et al., 1999). Additionally, a blunted adenosine A2A receptor response was found in platelets of depressed patients (Berk et al., 2001). In patients with MDD, chronic treatment with citalopram enhanced adenosine plasma levels to a similar extent and profile as serotonin (Blardi et al., 2005). Almost at the same time, the adenosinergic system was investigated in patients with BD. It was demonstrated that during the manic phase, the excretion of the adenosine metabolite uric acid was increased, suggesting a dysfunction in the adenosinergic system (De Berardis et al., 2008; Machado-Vieira et al., 2002; Salvadore et al., 2010). Besides that, serum adenosine levels were decreased in euthymic bipolar patients compared to controls, and a negative association was found between adenosine levels, depression scores, and functional impairment (Gubert et al., 2016). Despite these results, it is still unclear whether the peripheral findings are associated with changes in brain function or can predict the disease’s course. Another strong group of evidence pointing to an adenosinergic role in mood regulation and mood disorders came from studies with caffeine in humans. Caffeine is the most consumed psychoactive substance, and in the concentrations reached by the average consumption, it acts primarily as a non-selective adenosine receptor antagonist (Fredholm et al., 1999). From the epidemiological perspective, caffeine consumption is heterogeneous, and genetic factors can impact its metabolization and pharmacodynamics. Thus, the impact of caffeine in psychiatric symptoms has both positive (Amendola et  al., 1998; Fine et  al., 1994; Lieberman et  al., 1987; Lucas et al., 2011; Ritchie et al., 2007) and negative (Gilliland & Andress, 1981; Greden et al., 1978; James & Crosbie, 1987; Bertasi et al., 2021) results reported by the literature. High doses of caffeine are associated with anxiety, depressive symptoms, psychosis, and exacerbation of manic symptoms in patients with BD (Broderick & Benjamin, 2004; Tondo & Rudas, 1991). On the other hand, the

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Chapter 8

Roles of Purinergic Receptors in Alzheimer’s Disease Cécile Delarasse and David Blum

Abstract  Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by major memory impairments. Neuropathologically, AD is defined by the presence of neurofibrillary tangles (NFT) made up of intraneuronal fibrillar aggregates of hyperphosphorylated and abnormally phosphorylated Tau proteins and the extracellular accumulation of Aβ peptides into amyloid plaques. Both lesions are mainly observed at the cortical level, with a vulnerability of the internal temporal lobe. In the present chapter, we particularly focus on the role of purinergic signaling mediated by A2A and P2X7 receptors in the pathophysiology of AD and their involvement in the neuroglial control of plasticity and neurodegeneration. Keywords A2A receptor · P2X7 receptor · Aβ peptides · Fibrillary aggregates

Introduction Alzheimer’s disease (AD) is an age-related neurodegenerative disease characterized by the loss of cognitive functions, with memory impairment in the foreground. The definitive diagnosis is based on the neuropathological observation of amyloid plaques and neurofibrillary lesions, leading to synaptic deficits, driving cognitive decline. Amyloid plaques consist of extracellular deposits of aggregated β-amyloid (Aβ) peptides, resulting from the sequential proteolysis of the precursor of the C. Delarasse (*) Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France e-mail: [email protected] D. Blum (*) University of Lille, Inserm, CHU Lille, U1172 – LilNCog – Lille Neuroscience & Cognition, Lille, France Alzheimer & Tauopathies, LabEx DISTALZ, Lille, France e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 H. Ulrich et al. (eds.), Purinergic Signaling in Neurodevelopment, Neuroinflammation and Neurodegeneration, https://doi.org/10.1007/978-3-031-26945-5_8

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amyloid protein (APP) by β and γ secretases. Neurofibrillary lesions consist of intraneuronal fibrillar aggregates of hyperphosphorylated and abnormally phosphorylated Tau proteins. The AD brain is also characterized by microglial and astrocytic reactivities as well as the development of neuro-inflammation, which instrumentally participate to the lesion development and synapse loss.

Purinergic Signalization in AD ATP and its hydrolysis pathways are involved in the delicate balances between excitation and inhibition which shapes neuronal circuits but also non-neuronal functions, astrocyte communication, and microglial activation. The ultrafine tuning of this cellular signalization is ensured by the difference between intracellular (3–10 mM) and extracellular (20–50 nm) concentration of ATP. A very low fluctuation will have major consequences on this ratio allowing extraordinary sensitivity of purinergic receptors to ATP and its catabolites. In case of cell damage, high amount of ATP is released, which acts as a danger signal. The P2X7 receptor has a reduced sensitivity to ATP compared to the other purinergic receptors (EC50: 0.1–1 mM) and will be activated by high local concentration of ATP at sites of tissue injury. The purinergic P2X7 receptor is a key element in the activation of the inflammasome NLRP3 (NOD-like receptor family pyrin domain-containing 3) that leads to the release of pro-inflammatory cytokines such as interleukin-1β (IL-1β) and IL-18 (Kanellopoulos & Delarasse, 2019). Brief stimulation of P2X7 receptors  also induces the release of other pro-inflammatory mediators such as TNFα and production of reactive oxygen species (ROS). ATP is subject to fast catabolism by ectoenzymes, CD39 phosphohydrolyze ATP into ADP and AMP, and then AMP will be rapidly converted into adenosine by CD73. In the brain, adenosine effects essentially rely on two G-protein-coupled receptors: A1 and A2A subtypes (Cunha, 2016). In homeostatic conditions, adenosine, by regulating both receptors, particularly fine tunes synaptic plasticity with a particular importance of the A1 presynaptic and postsynaptic signaling onto glutamate release and regulatory effects. In pathological conditions, enhanced extracellular concentrations of ATP – but also intraneuronal ATP breakdown – promote a rise in the extracellular levels of adenosine which then favors the activation of A2A over A1 receptors  (Cunha, 2016), dysregulating synaptic activity in a neuron-autonomous and nonautonomous manner, favoring synaptic deficits and memory impairments. Moreover, both P2X7 and A2A receptors have been physiopathologically linked to Alzheimer’s disease. Indeed, expression of P2X7 receptors is upregulated in rat’s brain following intra-hippocampal Aβ peptide injection (McLarnon et al., 2006) and around Aβ plaques in the hippocampus of transgenic mice models of AD (Tg2576 and APPPS1) as well as in mouse models of Tau lesion (THY-Tau22, P301S) (Parvathenani et al., 2003; Martin et al., 2019; Carvalho et al., 2021; Di Lauro et al., 2021). A marked upregulation of P2X7 receptor expression  was also reported in the brain of AD patients, notably in

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microglia and astrocytes surrounding Aβ plaques (McLarnon et al., 2006; Martin et al., 2019). Regarding A2A receptors, early studies on cortical plasma membranes revealed that cortex of AD patients exhibit enhanced A2A binding and response (Albasanz et al., 2008). More recent data emphasized that A2A expression is abnormally increased not only in brain cortical parenchyma of AD patients (Orr et al., 2015; Temido-Ferreira et al., 2020) but also on transgenic mouse models (Viana da Silva et al., 2016; Faivre et al., 2018; Lee et al., 2018; Orr et al., 2018; Silva et al., 2018). A2A receptor expression changes have been shown to occur at both neuronal/ synaptic (Viana da Silva et al., 2016; Temido-Ferreira et al., 2020) and astroglial (Orr et al., 2015, 2018; Lee et al., 2018) levels.

The P2X7 Purinergic Receptor and AD Increased expression of P2X7 receptors in models of AD and in the brain of AD patients and its role in inflammatory processes suggest that P2X7 receptors may be involved in the development and progression of AD. In vitro, we showed that brief stimulation (