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The Role of Purinergic Receptor A1 in Neurogenesis Modulation from Subventricular Zone

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The Role of Purinergic Receptor A1 in Neurogenesis Modulation from Subventricular Zone Departamento de Neurociencias Neurozientziak Saila The role of purinergic receptor A1 in neurogenesis modulation from subventricular zone Mónica Benito Muñoz Leioa, June 2016 (c)2016 MONICA BENITO MUÑOZ Index INTRODUCTION 1. ADULT NEUROGENESIS…………………………………………………………………………………..……..3 2. THE SUBVENTRICULAR ZONE…………………………………………………………………………………5 3. THE SUBVENTRICULAR ZONE AND NEUROGENESIS IN HUMAN BRAIN………………....8 4. NEUROGENESIS MODULATION………………………………………………………………………………9 5. MODULATION IN PHYSIOLOGICAL CONDITIONS…………………………………………………..10 6. MODULATION IN PATHOLOGICAL CONDITIONS……………………………………………………14 7. MODULATION OF NEUROGENESIS IN ISCHEMIC CONDITIONS HUMAN BRAIN ISCHEMIA...........................................................................19 8. EXPERIMENTAL IN VIVO MODELS…………………………………………………………………………21 9. PATHOPHYSIOLOGY OF ISCHEMIA-REPERFUSION INJURY…………………………………….24 10. NEUROGENESIS IN ISCHEMIC CONDITIONS………………………………………………………….28 11. PURINERGIC SYSTEM……………………………………………………………………………………………29 12. EXTRACELLULAR RECEPTORS……………………………………………………………….………………30 13. IONOTROPIC RECEPTORS……………………………………………………………………………………..31 14. METABOTROPIC RECEPTORS………………………………………………………………………………..33 P2Y Receptors…………………………………………………………………………………………33 P1 Receptors…………………………………………………………………………………………..34 15. NUCLEOTIDE METABOLISM………………………………………………………………………………….36 16. FUNCTIONS OF PURINERGIC SYSTEM……………………………………………………………..……38 OBJECTIVES.................................................................................................................................43 MATERIAL AND METHODS…………………………………………………………………………………………………….47 1. IN VITRO CULTURES………………………………………………………………………………..…………..47 1.1. Neurosphere culture………………………………………………………………………..47 1.2. Neuronal differentiation…………………………………………………………………..47 1.3. Astrogliogenesis in vitro……………………………………………………………………48 1.4. Organotypic culture………………………………………………………………………….48 2. CELL VIABILITY……………………………………………………………………………………………………..49 3. Ca2+ RECORDINGS………………………………………………………………………………………………..49 4. IMMUNOLABELING………………………………………………………………………………………………50 4.1. Immunofluorescence……………………………………………………………………….50 4.2. Cytofluorimetry assay……………………………………………………………………….52 5. GENE EXPRESSION ANALYSIS……………………………………………………………………………….53 5.1. RNA extraction and quantification……………………………………………………53 5.2. Retrotranscription……………………………………………………………………………53 5.3. Quantitative real time polymerase chain reaction (qRT-PCR)…………..53 5.4. PCR gene expression array……………………………………………………………….54 5.5. Adora1 silencing……………………………………………………………………………...54 6. PROTEIN EXPRESION……………………………………………………………………………………………55 6.1. Protein extraction and Western blot…………………………………………..……55 6.2. Synaptosome preparation………………………………………………………………..56 7. LUMINISCENCE ASSAYS………………………………………………………………………………………..57 7.1. ELISA assay……………………………………………………………………………………….57 7.2. Luciferin/ Luciferase assay………………………………………………………………..57 8. IN VIVO EXPERIMENTS…………………………………………………………………………………………58 8.1. Osmotic pumps releasing CPA………………………………..……………………….58 8.1.1. Sacrifice and tissue processing…………………………………………………59 8.2. Ischemic animals treated with DPCPX……………………………….……….……59 8.2.1. Parameters………………………………………………………………………………61 8.2.2. Experimental protocol and drug treatment……………………………..62 8.2.3. Tissue fixation, dissection and vibratome………………………………..63 8.2.4. Measure of infarct volume………………………………………………………63 8.2.5. Immunohistochemistry: Double fluorescence immunolabeling………………………………………………………………………………………64 8.2.6. Image acquisition and analysis…………………………………………………65 8.3. Positron tomography emission (PET)………………………………………………..65 8.3.1. Radiochemistry………………………………………………………………………..66 8.3.2. PET scans and data acquisition………………………………………………..66 8.3.3. PET Image analysis…………………………………………………………………..66 9. STATISTICAL ANALYSIS…………………………………………………………………………………………67 RESULTS…………………………………………………………………………………………………………………………………71 1. CHARACTERIZATION ON NEUROSPHERE CULTURES……………………………………………..71 2. EXTRACELULAR ADENOSINE NEGATIVELY MODULATES NEURONAL DIFFERENTIATION………………………………………………………………………………………………..75 3. STIMULATION OF ADENOSINE RECEPTORS DURING DIFFERENTIATION INHIBITS THE TRANSPORT OF SYNAPTIC VESICLE.…………………………………………………………………..…77 4. ADENOSINE INHIBITS NEURONAL DIFFERENTIATION THROUGH THE ACTIVATION OF A1 RECEPTOR………………………………………………………………………………………………….81 5. A1 RECEPTORS INHIBIT DIFFERENTIATION FROM TRANSIT AMPLIFYING PRECURSORS TO NEUROBLATS…………………………………………………………………………….86 6. A1 RECEPTORS INHIBIT DIFFERENTIATION FROM TRANSIT AMPLIFYING PRECURSORS TO NEUROBLASTS 6.1. Activation of A1 receptor modulates IL-10 release……………………………88 6.2. A1 stimulation activates the Bmp2/Smad pathway…………………………..92 7. IN VIVO INHIBITION OF NEUROGENESIS BY SELECTIVE ACTIVATION OF A1 RECEPTOR……………………………………………………………………………………………………………94 8. ACTIVATION OF A1 RECEPTOR DURING BRAIN ISCHEMIA ACTIVATES ASTROGLIOGENESIS TO THE DETRIMENT OF NEUROGENESIS………………………………98 8.1. Body weight, neurological score, and survival after DPCPX treatment in tMCAO…………………………………………………………………………………………………….98 8.2. Infarct size and brain volume…………………………….……………………………100 Index 8.3. Blocking A1 receptor during tMCAO improves mobility shortly after ischemia………………………………………………………………………………………………..102 8.4. Positron Tomography Emission (PET) analysis of cell proliferation….103 8.5. Activation of A1 receptor modulates neurogenesis and astrogliogenesis during brain ischemia………………………………………………….105 DISCUSSION………………………………………………………………………………………………………………………….111 1. ADENOSINE AND NEUROGENESIS………………………………………………………………………111 2. A1 RECEPTOR-MEDIATED MECHANISMS OF NEUROGENESIS INHIBITION………….112 3. CHARACTERIZATION OF A1 RECEPTOR MECHANISMS…………………………….………….113 4. A1 RECEPTOR-MEDIATED STIMULATION OF NEUROGENESIS IN ISCHEMIC MICE………………………………………………………………………………………………………………….116 CONCLUSIONS………………………………………………………………………………………………………………………121 BIBLIOGRAPHY..........................................................................................................................125 Abrevations AAV Adeno-associated virus Ache Acetyl cholinesterase AD Alzhemier disease aNSC Activated neural stem cell ADA Adenosine deaminase ADK Adenosine kinase ADP Adenosine diphosphate ADPβS Adenosine-5′-O-(2-thiodiphosphate) Ado Adenosine AMP Adenosine monophosphate AP Alkaline phosphatases AR Adenosine receptor ATP Adenosine triphosphate BBB Blood brain barrier BDNF Brain-derived neurotrophic factor bFGF Fibroblast growth factor bHLH Basic helix-loop-helix BMP Bone morphogenetic protein BrdU 5-bromo-2´-deoxyuridine BfdA Brefeldin A 2´,3´-BzATP 2'(3')-O-(4-Benzoylbenzoyl)adenosine-5'-triphosphate Caff Caffeine cAMP Cyclic adenosine monophosphate CBDN Calbindin CCA Common carotid artery CNS Central nervous system CNT Concentrative nucleoside transporter CNTF Ciliary neurotrophic factor Abrevations CR Calretinin CBF Cerebral blood flow CPA N6-Cyclopentyladenosine CRYAB Crystallin alpha B CSF Cerebrospinal fluid CTP Cytidine-5´-triphosphate DAG Dyacilglycerol DAI Days after ischemia DARP Dopamine-releasing protein DCX Doublecourtin DIV Days in vitro Dlg4 Discs, large homolog 4 (Drosophila) DM Differentiation medium DNA Deoxyribonucleic acid dNTP Dinucleotidetriphosphate DP Dipyridamol DPCPX 1, 3-dipropyl-8-cyclopentylxanthine DTT Dithiothreitol ECA External carotid artery EGF Epidermal growth factor ELISA Enzyme-linked immunosorbent assay EMC Extracellular matrix components E-NPP Ectonucleotide pyrophosphate/phosphodiesterase ENT Equilibrative nucleoside transporter Epo Erythropoietin ET-1 Endothelin-1 FGF Fibroblast growth factor [18F] FLT 3'-deoxy-3'-[18F]-fluorothymidine Abrevations GABA Gamma-amino butyric acid GAPDH Glyceraldehyde-3-phosphate dehydrogenase GC Granule cell GDNF Glial cell -derived neurotrophic factor GFAP Glial fibrillary acidic protein GL Glomerular layer GPCR G protein couple receptor Gpi Glucose-6-phosphate-isomerase GrL Granular layer GSK3β Glycogen synthase kinase 3 beta GTP Guanosine-5´-triphosphate 5-HT 5-hydroxytryptamine HPRT1 Hypoxanthine phosphoribosyl-transferase 1 ICA Internal carotid artery IdU 5-Iodo-2´-deoxyuridine IFN-γ Interferon IGF-I Insulin-likeγ growth factor I IL Interleukin IMP Inosine monophosphate I.P. Intraperitoneal Ip5I Diinosine pentaphosphate InsP3 Inositol triphosphate JAK Janus kinase JGN Juxtaglomerular neuron LIF Leukemia inhibitory factor lncRNA long non-coding ribonucleic acid LV Lateral ventricule MAPK Mitogen activated protein kinase Abrevations MCAO Middle cerebral artery occlusion MCA Middle cerebral artery MCP-1 Monocyte chemoattractant protein 1 α,β-meATP α,β-methyleneadenosine 5′-triphosphate 2-MeSATP 2-(Methylthio)adenosine 5′-triphosphate mRNA Messenger ribonucleic acid miRNA Micro ribonucleic acid MS Multiple sclerosis N Neurosphere NAD+ Nicotinamide adenine dinucleotide NECA 5´-N-ethylcarboxamido-adenosine NFM Medium neurofilaments NGF Nerve growth factor NINDS National Institute of Neurological Disorders and Stroke NMDA N-methyl-D-aspartate NO Nitric oxide NOS Nitric oxide synthase NSC Neural stem cell NT-3 Neurotrophin-3 NTP Nucleoside triphosphate NTPDase Ectonucleoside triphosphate diphosphohydrolase OB Olfactory bulb PBS Phosphate-buffered saline PD Parkinson disease PEDF Pigment epithelium-derived factor PET Positron tomography emission PFA Paraformaldehyde PGE2 Prostaglandin E2 Abrevations PGC Periglomerular cell PKC Protein kinase C PPADS Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate PSA-NCAM Polysialylated-neural cell adhesion molecule Ptn Pleiotrophin qNSC Quiescent neural stem cell qRT-PCR Quantitative real-time polymerase chain reaction RMS Rostral migratory
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