ABSTRACT KHAREL, PRAKASH, Ph.D., December 2018 Chemistry & Biochemistry RNA OXIDATION IN MULTIPLE SCLEROSIS NEURODEGENERATION (204 PP.) Dissertation advisor: Soumitra Basu An increase in the production of reactive oxygen species (ROS) and the inability of cellular machinery to adequately neutralize the ROS thus produced, lead the cells towards oxidative stress. ROS can damage all major classes of biomolecules including proteins, DNA and RNA. Recent findings show the evidence of high level of RNA oxidation in the neuronal cells of many neurological disorders suggesting a link between RNA oxidation and neurodegeneration. We have recently discovered the presence of extensive oxidative damage in the RNA molecules of neuronal cells in postmortem brains of multiple sclerosis (MS) patients. Working on the MS model system, we have established the identity of selectively oxidized mRNA molecules under MS microenvironment in human neuronal cells. Our study reveals that many of the mRNAs linked to various neurological pathways are selectively oxidized under oxidative stress. We have demonstrated the functional consequences of mRNA oxidation in two neuropathology related mRNAs (namely Nat8l and Nlrp3 mRNA). N-acetyl aspartate transferase 8 like protein (NAT8L) is a key trans-mitochondrial membrane protein that catalyzes the transfer of acetyl group from acetyl-CoA to aspartate to form N-acetyl aspartate (NAA) and transports NAA to neuronal cytoplasm. We have discovered that the oxidation in Nat8l mRNA molecule i results in the reduced expression of NAT8L enzyme. We also observed a reduced level of NAA present in the neuronal cells under oxidative stress. Using the neuronal tissue culture and MS animal model (cuprizone mouse model), we have established that a higher mRNA oxidation results in a reduced expression of NAT8L protein, which presumably contributes to the MS disease progression by weakening the myelin production machinery. In a different context, we have also observed a unique situation where an oxidation in the Nlrp3 mRNA could lead towards the activation of alternative inflammasome pathway in human neurons under MS microenvironment. To the best of our knowledge, our study is the first to establish the direct connection between mRNA oxidation and MS neurodegeneration. In another study, we defined the impact of base oxidation in the structure and function of RNA molecules. We worked on an engineered version of the Tetrahymena group 1 intron (a ribozyme) to evaluate its enzymatic activity under oxidative stress. Our in vitro findings suggest the progressive loss of ribozyme function with increasing oxidation. Additionally, our investigation revealed that RNA oxidation is detrimental in RNA folding. We have also synthesized a novel 8- OHG analog (phosphorothioate) molecule with a potential to map the interference in the RNA structure due to the presence of oxidatively modified nucleosides. ii RNA OXIDATION IN MULTIPLE SCLEROSIS NEURODEGENERATION A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Prakash Kharel December 2018 © Copyright All rights reserved Except for the previously published materials iii Dissertation written by Prakash Kharel B.S., Tribhuvan University, 2006 M.S., Tribhuvan University, 2010 Ph.D., Kent State University, 2018 Approved by _____________________________, Chair, Doctoral Dissertation Committee Soumitra Basu, Ph.D., MBA. _____________________________, Members, Doctoral Dissertation Committee Sanjaya Abeysirigunawardena, Ph.D. _____________________________ Jacob Shelley, PhD. _____________________________ Jennifer McDonough, Ph.D. _____________________________ Ernest Freeman, Ph.D. Accepted by ____________________________, Chair, Department of Chemistry and Biochemistry Soumitra Basu, Ph.D., MBA. _____________________________, Dean, College of Arts and Sciences James L. Blank, Ph.D. i TABLE OF CONTENTS Page TABLE OF CONTENTS ………………………………………………………………...v LIST OF FIGURES ……………………………………………………..….…………....x LIST OF TABLES ..……………………………………………….………......................xv LIST OF ABBREVIATIONS…..……………………………………………………....xvii DEDICATION…………………………………………………………………………..xix ACKNOWLEDGEMENTS………..………………………………….……..……....…..xx CHAPTERS CHAPTER 1. Introduction ……………………………………………….……………….1 1.1. Multiple sclerosis……………………………………………………………..1 1.2. Multiple sclerosis genetics…………………………………………………....4 1.3. Autoimmune attacks in multiple sclerosis…………………………………....5 1.4. Mitochondrial dysfunction in multiple sclerosis……………………………..6 1.5. Oxidative stress in multiple sclerosis and other neurological disorders ………………………………………………………………………………....8 1.6. Metal dysregulation in multiple sclerosis brain………………………….….12 1.7. ROS in CNS inflammation……………………………………………….….13 1.8. Damage in the biomolecules due to ROS…………………………………….15 1.8.1. Lipid oxidation…………………………………………………...15 v 1.8.2. Protein oxidation…….…………………………………...............16 1.8.3. DNA oxidation…………………………………………………...17 1.8.4. RNA oxidation in MS and other neurological disorders………....17 1.8.5. Coping with RNA oxidative damage………………………….….24 1.9. Hypotheses of the study……………………………………………………...27 1.10. References………………………………………………………………..28 CHAPTER 2. Evidence of extensive RNA oxidation in the neurons of normal appearing cortex of Multiple Sclerosis brain…..…………………………………………………………...41 2.1. Introduction………………………………………………………………….41 2.2. Materials and methods………………….……...….........................................43 2.3. Results………………………………………………...……………………..49 2.4. Discussion……………………………………………….…………………..59 2.5. Conclusion…………………………………………………………………...62 2.6. References…………………………………………………………………...62 CHAPTER 3. Investigation of mRNA oxidation in human neuronal cells reveals selective oxidation of mRNAs could be linked to MS neuropathology………................................65 3.1. Introduction…………………………………………………..……………...65 3.2. Materials and methods……………………………………....……………….68 3.3. Results………………………………………………….…….……………...73 3.4. Discussion………………………………………………………..………….88 3.5. Conclusion…………………………………………………………………...92 vi 3.6. References…………………………………………………………...........92 CHAPTER 4. Oxidative damage to Nat8l mRNA in human neurons is linked to multiple sclerosis pathogenesis ……………………………………………………………...….95 4.1. Introduction………………………………………………………………..95 4.2. Materials and methods………………….……...…...................................100 4.3. Results ……………………………………………...…………………....105 4.4. Discussion …………………………………………….…………............120 4.5. Conclusion ……………………………………………………………….125 4.6. References………………………………………………………………..126 CHAPTER 5. Evidence of existence of NLRP3 inflammasome pathway in human neuronal cells which could be altered under SNP mediated oxidative stress ...………………..130 5.1. Introduction ..…………………………………………………………….130 5.2. Materials and methods………………….……...…...................................134 5.3. Results ……………………………………………...…………………....136 5.4. Discussion …………………………………………….………………….143 5.5. Conclusion ……………………………………………………………….145 5.6. References………………………………………………………………..145 CHAPTER 6. RNA oxidation impairs RNA function: Synthesis of a novel 8- hydroxyguanosine phosphorothioate analog with a potential to probe the interference of RNA oxidation in structure and function ……………….…………………………...148 vii 6.1. Introduction ..……………………………………………………………148 6.2. Materials and methods………………….……...…..................................153 6.3. Results ……………………………………………...…………………...160 6.4. Discussion …………………………………………….………………...169 6.5. Conclusion ………………………………………………………………171 6.6. References……………………………………………………………….172 CHAPTER 7. Concluding Remarks………………..………………..…….………….176 CHAPTER 8. Future Perspectives…………………………………………………….178 APPENDIX..………………………………………………………………………….181 viii LIST OF FIGURES Page Chapter 1. Introduction Figure 1.1. Overview of MS symptoms, neurons & non-neuronal brain cells and neurodegeneration…………………………………………….…………………...........3 Figure 1.2. Players in MS pathogenesis………………………………………………15 Figure 1.3. Common oxidative base modifications in DNA and RNA……………….20 Figure 1.4. Mutagenic nature of 8-OHG …………………………….………………..21 Figure 1.5. Proposed role of RNA oxidation in the pathogenesis of neurological disorders and cancer……………………………………………………………………………....23 Figure 1.6. A hypothesis to decipher the role of selective RNA oxidation in MS neurodegeneration…………………………………………………................................28 Chapter 2. Evidence of extensive RNA oxidation in the neurons of normal appearing cortex of multiple sclerosis brain Figure 2.1. PLP staining to distinguish between lesion and non-lesion areas of MS brain…………………………………...…………………………..…...........................50 Figure 2.2. Immunohistochemical analyses of RNA oxidation in MS brain.................51 Figure 2.3. Comparative analysis of RNA and DNA oxidation in MS and non-MS brains………..………….................................................................................................53 ix Figure 2.4. RNA oxidation is predominant in neuronal cells……………………..…..54 Figure 2.5. RNA oxidation is less prominent in oligodendrocyte cells……………….55 Figure 2.6. HPLC analyses of 8-OHG in MS vs non-MS RNA and DNA…………...57 Figure 2.7. Comparison between RNA oxidation in lesion vs non-lesion areas of MS brain ……………………………………………………………………………………...….58 Figure 2.8. mRNA molecules are highly oxidized in MS brain…………...………….59 Chapter 3. Investigation of mRNA oxidation in human neuronal cells reveals selective oxidation of mRNAs could be linked to MS neuropathology Figure 3.1. A schematic of