Developing novel therapeutics for excitotoxic neuronal death following stroke

ii PREFACE . iii TABLE OF CONTENTS . vi LIST OF TABLES . ix LIST OF FIGURES . x LIST OF ABBREVIATIONS . xiii LIST OF PLASMIDS . xv ACKNOWLEDGEMENTS . xvii DEDICATION . xix CHAPTER 1: INTRODUCTION . 1 1.1 OVERVIEW . 1 1.2 STROKE AND NMDA RECEPTORS . 2 1.2.1 Glutamate mediates excitotoxicity . 3 1.2.2 The calcium hypothesis . 4 1.2.3 Modifications to the calcium hypothesis . 6 1.2.4 Excitotoxicity following stroke . 8 1.3 DUAL ROLES OF NMDARS IN DEATH AND SURVIVAL . 8 1.3.1 The NMDAR location hypothesis . 9 1.3.2 The NMDAR subtype hypothesis . 11 1.3.2.1 Subtype hypothesis Early work . 12 1.3.2.2 Subtype hypothesis Later work . 13 1.3. 3 A unified hypothesis . 15 1.4 DEATH SIGNALING PROTEINS IN THE NDC . 16 1.4. 1 The NR2B-PSD95-nNOS signaling complex . 17 1.4. 2 The NR2BR-PTEN signaling complex . 19 1.4. 3 The NR2B-DAPK1 signaling complex . 20 1.5 BEYOND THE NDC . 22 1.5. 1 The calpains family of death-signaling proteases . 22 1.5. 2 AMPAR trafficking as an excitotoxic process . 24 1.5. 3 MAPKs in neuronal survival and death . 25 1.5. 3.1 Early death-signaling by p38 kinase . 26 1.5. 3.2 Delayed death-signaling by JNK . 27 1.5. 4 SREBP1 as a pro-death transcription factor . 28 1.6 NON-NEURONAL NEUROTOXIC MECHANISMS . 29 1.7 THERAPEUTIC STRATEGIES FOR STROKE INTERVENTION . 30 1.7. 1 tPA and its limitations . 31 1.7. 2 NMDAR blockers and their limitations . 31 1.7. 2.1 Clinical failure of NMDAR blockers . 32 1.7. 2.2 NR2BR-specific blockers and limitations . 33 1.7. 3 Interference of NMDAR signaling by synthetic peptides . 34 #""! 1.8 RATIONALE AND HYPOTHESES . 35 1.8. 1 Objective 1: Synaptic/NR2A NMDAR stimulation . 37 1.8. 2 Objective 2: Inhibiting SREBP1 death signaling . 38 1.8. 3 Objective 3: Novel method for protein knockdown . 39 CHAPTER 2: MATERIALS AND METHODS . 45 2.1 ANIMAL MODEL OF FOCAL ISCHEMIC STROKE . 45 2.1.1 Experimental animals . 45 2.1.2 Middle Cerebral Arterial Occlusion (MCAO) . 45 2.1.3 Neurological score . 47 2.1.4 Histology and infarct measurements . 48 2.1.5 Criteria for exclusion . 49 2.2 ANIMAL MODEL OF GLOBAL ISCHEMIA . 49 2.2.1 Experimental animals . 49 2.2.2 Global ischemia . 49 2.2.3 Electrophysiology in vivo . 51 2.2.4 Perfusion and fixation . 52 2.3 HUMAN EMBRYONIC KIDNEY CELLS MODEL . 54 2.3.1 Maintenance . 54 2.3.2 Transfection and drug treatment . 54 2.3. 3 Storage of HEK cells . 55 2.4 TISSUE COLLECTION AND WESTERN BLOT ANALYSIS . 55 2.4. 1 Animal tissue collection whole cell lysate . 55 2.4. 2 Animal tissue collection nuclear fraction . 56 2.4. 3 HEK 293 cell tissue collection whole cell lysate . 57 2.4. 4 Electrophoresis and Western blot analysis . 58 2.5 HISTOLOGY, IMMUNOHISTOCHEMISTRY, AND IMMUNOFLUORESCENCE . 59 2.5. 1 Cresyl violet staining . 59 2.5. 2 Fluoro-jade staining . 60 2.5. 3 Immunohistochemistry NeuN . 61 2.5. 4 Immunofluorescence NR1 and SREBP1 . 62 2.6 MOLECULAR BIOLOGY AND GENETIC ENGINEERING . 63 2.6. 1 Plasmid source . 63 2.6. 2 DNA amplification, purification, and storage . 63 2.6. 3 Site directed mutagenesis . 64 2.6. 4 Restriction and ligation . 65 2.6. 5 Plasmid production . 66 CHAPTER 3: SYNAPTIC/NR2A NMDAR STIMULATION . 77 3.1 SPECIFIC BACKGROUND . 77 3.1.1 NMDAR electrophysiology and pharmacology . 77 3.1.2 Stimulating pro-survival NMDARs . 78 3.2 RESULTS . 79 3.2.1 NMDAR subtypes on stroke damage . 79 3.2.2 Glycine treatment: neuroprotection against focal ischemic stroke . 79 3.2.3 Glycine treatment: neuroprotection against global ischemia . 82