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Protein Kinase C Signaling in Neurodegeneration A

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Protein Kinase C Signaling in Neurodegeneration A PROTEIN KINASE C SIGNALING IN NEURODEGENERATION A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy By Varun Kumar May, 2016 © Copyright All rights reserved Except for previously published materials Dissertation written by Varun Kumar B.Sc., University of Delhi, 2008 M.Sc., Devi Ahilya University, 2010 Ph.D., Kent State University, 2016 Approved by Wen-Hai Chou , Chair, Doctoral Dissertation Committee Srinivasan Vijayaraghavan , Members of Doctoral Dissertation Committee Alexander L. Mdzinarishvili , Werner J. Geldenhuys , Gail C. Fraizer , Accepted by Ernest J. Freeman , Director, School of Biomedical Sciences James L. Blank , Dean, College of Arts and Sciences ii TABLE OF CONTENTS……………………………………………………………………iii LIST OF FIGURES………………………………………………………………………….vi LIST OF ABBREVIATIONS……………………………………………………………….viii ACKNOWLEDGEMENTS………………………………………………………………….x CHAPTERS 1. Introduction……………………………………………………………………………...1 1.1 Protein Kinase C………………………………………………………………….1 1.2 Mode of PKC regulation………………………………………………………….2 1.3 Molecular targets in preconditioning……………………………………………..2 1. 4 PKC in ischemia………………………………………………………………...5 1.5 Activating Transcriptional factor 2………………………………………….…....6 1.6 Regulation of ATF2……………………………………………………………….7 1.7 Role of ATF2 in diseases…………………………………………………………10 1.8 The chemical-genetics approach…………………………………………….……11 1.9 Overview…………………………………………………………..…………….. 14 2. Determine the effect of PKCε deficiency following global cerebral ischemia………16 2.1 Introduction and rational……………………………………………………….....16 2.2 Material and methods……………………………………………………………..20 2.3 Results…………………………………………………………………………….25 2.3.1 Determine hippocampal neuronal degeneration in PKCε WT and KO mice after global cerebral ischemia…………………………………………………25 2.3.2 Determine hippocampal neuronal cell death in PKCε WT and KO mice after iii global cerebral ischemia………………………………………………………27 2.3.3 Determine the cerebrovascular anatomy in PKCε WT and KO mice………...29 2.3.4 Determine the regional cerebral blood flow before and after global cerebral ischemia in PKCε WT and KO mice..………………………………….……. 31 2.3.5 Determine the physiological parameters for PKCε WT and KO mice………...33 2.4 Conclusion……………………………………………………………………..….36 3. Determine the molecular mechanisms of cell death after global cerebral ischemia..37 3.1 Introduction and rational..…………………………………………………………....37 3.2 Material and methods………………………………………………………………...40 3.3 Results………………………………………………………………………………..42 3.3.1 Determine the phosphorylation of ATF2 at Thr52 in mouse hippocampus……42 3.3.2 Determine temporal expression profile of PKCε in mouse hippocampus before and after global cerebral ischemia……………………………..…………..……44 3.3.3 Determine temporal expression profile of ATF2 in mouse hippocampus before and after global cerebral ischemia…………………………………..……….…46 3.3.4 Determine cytochrome c expression in mouse hippocampus after global cerebral ischemia……………………………………………………………..………….48 3.4 Conclusion………………………………………………………………..………….50 4. Generation and characterization of ATP analog-specific PKCδ homology model in silico…………………………………………………………………………………….51 4.1 Introduction and rational..…………………………………………………….……51 4.2 Material and methods………………………………………………………………52 iv 4.3 Results………………………………………………………………………….......54 4.3.1 Generate human PKCδ homology model using MOE software……………...54 4.3.2 Perform docking studies for substrates (N6-(benzyl)-ADP) and PP1 derived inhibitors in the PKC homology model……………………………………..57 4.4 Conclusion…………………………………………………………………………65 5. Discussion and future directions……………………………………………………66 5.1 Discussion………………………………………………………………………….66 5.2 Future directions…………………………………………………………………...72 6. Bibliography………………………………………………………………………….74 v LIST OF FIGURES Figure 1. Schematic representation of regulation of ATF2 via PKC Figure 2. The chemical-genetics approach………………………………………………...........13 Figure 3. Hippocampal neurodegeneration in PKCε+/+ and PKCε-/- mice after global cerebral ischemia……………………………………………………………………………….26 Figure 4. Hippocampal neuronal cell death in PKCε+/+ and PKCε-/- ischemia………………………………………………………………………………28 Figure 5. Assessment of cerebrovascular anatomy……………………………………………..30 Figure 6. Determination of regional cerebral blood flow (rCBF)………………………….........32 Figure 7. ATF2 (Thr52) phosphorylation by PKC in vivo……………………………………..43 Figure 8. Cytosolic PKC decreased in mouse hippocampus after global cerebral ischemia…..45 Figure 9. Temporal expression of ATF2 in mitochondria for PKCε+/+ and PKCε-/- mice after global cerebral ischemia……………………………………………………………...47 Figure 10. Decreased release of cytochrome c in the cytosol for PKCε-/- mice after global cerebral ischemia ………………………………………………………..…………..49 Figure 11. ATP representation in the nucleotide-binding pocket of WT and AS-PKCδ…..........55 Figure 12. ATP interactions in the nucleotide-binding pocket of WT and AS-PKCδ…………..56 vi Figure 13. Comparison of N6-(benzyl)-ADP (BZ-ADP) interactions in the nucleotide-binding pocket of AS and WT-PKCδ………………………………………………………..59 Figure 14. Comparison of 1 NA-PP1 interactions in the nucleotide-binding pocket of WT and AS-PKCδ…………………………………………………………………………….60 Figure 15. Interactions of 1NM-PP1 and 2NM-PP1 with AS-PKCδ…………………………...61 Figure 16. Interactions of 1NA-PP1 and 1NM-PP1 with AS-PKA…………………………….62 Figure 17. Interactions of 2MB-PP1 with AS-PKCδ…………………………………………...63 Figure 18. Interactions of N6-(benzyl)-ATP and 1NA-PP1 with mAC…………………………64 Figure 19. Model for apoptosis after global cerebral ischemia………………………………….71 vii LIST OF ABBREVIATIONS ACA anterior cerebral artery AP1 activator protein 1 AS analog-specific ATF2 activating transcription factor 2 ATP adenosine triphosphate BCCAO bilateral common carotid artery occlusion bZIP basic leucine zipper domain CREB c-AMP response element-binding protein DAG diacylglycerol ERK extracellular signal regulated kinase FJC fluoro-jade c HK1 hexokinase-1 HSP heat shock proteins IPC ischemic preconditioning IP3 inositol triphosphates JNK c-Jun N-terminal kinase mAC mammalian adenylyl cyclase MANT 2’(3’)-O-(N-methylanthraniloyl) MAPK mitogen-activated protein kinase MCA middle cerebral artery 2MB-PP1 1-(tert-butyl)-3-(2-methylbenzyl)-1H-pyrazolo[3,4-d]- pyrimidin-4-amine 1NA-PP1 1-(tert-butyl)-3-(1-naphthyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine viii 1NM-PP1 1-tert-butyl-3-(1-naphthalenylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 2NM-PP1 1-tert-butyl-3-(2- naphthalenylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine NES nuclear export signal OGD oxygen glucose deprivation PcomA post-communicating artery PDB protein data bank PFA paraformaldehyde PIP2 phosphatidylinositol 4,5-bisphosphate PMA phorbol 12-myristate 13-acetate PNBM para-nitrobenzyl mesylate PP1 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine RACK receptor for activated C kinase rCBF regional cerebral blood flow ROS reactive oxygen species TNF tumor necrosis factor VDAC1 voltage-dependent anion-selective channel protein 1 ix ACKNOWLEDGEMENTS First of all, I would like to express my sincere gratitude to my advisor, Dr. Wen-Hai Chou, for his continuous guidance and encouragement during my time in the lab. He was always there for me when I needed help in my work. He constantly motivated me to be inquisitive and think independently in all my experiments. I am deeply indebted to his endeavors for providing a great research environment in the laboratory. I also appreciate his patience for other matters not related to research. It has really been a great time working in his laboratory. I would also like to thank all my committee members: Dr. Alexander Mdzinarishvili, Dr. Srinivasan Vijayaraghavan, Dr. Werner J. Geldenhuys and Dr. Gail C Fraizer for continuous support, constructive comments and encouragement during my PhD. I really appreciate their time and patience when needed during my stay at Kent State University. I also express my sincere gratitude to Dr. Tibor Kristian for his help in establishing mouse model of global cerebral ischemia. My sincere thanks to Dr. Yi-Chinn Weng, who provided constructive insights into my experiments. She took care of everything when needed during my PhD. I doubt that I would be able to convey my appreciation fully, but I owe her my eternal gratitude. I would also like to thank my fellow lab mates, Guona Wang, Xiqian Han, JD, Isabella, Vivek, and Supreet for their support in my experiments. Last but not the least, I would like to thank my parents, sisters, brother-in-law and all my cousins for their continuous support for what I do. I would like to express my special gratitude to my father, who always encourages and appreciates my work better than me. Words are not enough to express my gratitude fully to all my family members, who were always there for me. x CHAPTER 1 Introduction 1.1 Protein Kinase C Protein Kinase C (PKC) is a family of 10 serine-threonine kinases that regulate a broad spectrum of cellular functions (Newton and Messing, 2010; Steinberg, 2008). They are lipid-sensitive enzymes that are activated by growth factor receptors. These growth factor receptors stimulate phospholipase C (PLC), the enzyme that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to generate membrane-bound diacylglycerol (DAG) and inositol trisphosphate (IP3). DAG activates PKC whereas, IP3 mobilizes intracellular calcium. Tumor-promoting phorbol esters such as phorbol 12-myristate 13-acetate (PMA) also activate PKCs. All PKC family proteins contain a diverse regulatory domain
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