University of Kentucky UKnowledge Theses and Dissertations--Chemistry Chemistry 2013 OXIDATIVE DAMAGE TO DNA IN ALZHEIMER'S DISEASE Sony Soman University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Soman, Sony, "OXIDATIVE DAMAGE TO DNA IN ALZHEIMER'S DISEASE" (2013). Theses and Dissertations--Chemistry. 28. https://uknowledge.uky.edu/chemistry_etds/28 This Doctoral Dissertation is brought to you for free and open access by the Chemistry at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Chemistry by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. 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REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s dissertation including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Sony Soman, Student Dr. Mark A. Lovell, Major Professor Dr. Dong-Sheng Yang, Director of Graduate Studies OXIDATIVE DAMAGE TO DNA IN ALZHEIMERS DISEASE ——————————————— ABSTRACT OF DISSERTATION ______________________________ A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Arts and Sciences at the University of Kentucky By Sony Soman Lexington, Kentucky Director: Dr. Mark A. Lovell, Professor of Chemistry Lexington, Kentucky 2013 Copyright © Sony Soman 2013 ABSTRACT OF DISSERTATION OXIDATIVE DAMAGE TO DNA IN ALZHEIMERS DISEASE Previous studies from our laboratory and others show a significant increase in levels of both nuclear and mitochondrial DNA and RNA oxidation in vulnerable brain regions in the progression of Alzheimer’s disease (AD). Although total DNA oxidation is increased in AD it remains unclear whether oxidative damage is widespread throughout the genome or is concentrated to specific genes. To test the hypothesis that specific genes are more highly oxidized in the progression of AD, we propose to quantify the percent oxidative damage in genes coding for proteins shown to be altered in the progression of AD using quantitative/real-time polymerase chain reaction (qPCR/ RT-PCR). To further test the hypothesis that diminished DNA repair capacity in the progression of AD contributes to increased DNA oxidation we will use custom PCR arrays and qPCR, Western blot analysis and activity assays to quantify changes in enzymes involved in base excision repair (BER). In order to carry out these studies tissue specimens from superior and middle temporal gyri (SMTG) and inferior parietal lobe (IP), as well as, a non-vulnerable region, the cerebellum (CER) will be analyzed from normal control (NC) subjects and subjects throughout the progression of AD including those with preclinical AD (PCAD), mild cognitive impairment (MCI), and late stage AD (LAD). We will also analyze specimens from diseased control subjects (DC; Frontotemporal dementia (FTD) and dementia with Lewy bodies (DLB)) to determine if the changes we observe in AD are specific. KEYWORDS: Alzheimer’s disease (AD), oxidative stress, base excision repair, DNA oxidation OXIDATIVE DAMAGE TO DNA IN ALZHEIMERS DISEASE By Sony Soman Dr. Mark A. Lovell Director of Dissertation Dr. Dong-Sheng Yang Director of Graduate Study October 8, 2013 ACKNOWLEDGMENTS I must start my acknowledgements by thanking Dr. Lovell for being the best advisor, a graduate student could ask for and for creating a wonderful learning environment in the lab. “Boss”, you’ve taught me an incredible amount; self-reliance in research, encouragement to pursue ideas that piqued my interest and most of all to just keep moving on even when you are completely discouraged. I’ve always appreciated your indefatigable determination and the freedoms allowed to me while in your group. You have inspired me for a lifetime. I would not have been able to even start my research without the help of my wonderful colleagues, Shuling Xiong-Fister, Melissa Bradley and Ganna Lyubartseva. Without their help in training on various instruments and techniques and their constructive critiques of experimental methods and results, my research efforts would not even have taken off the ground in the first place. Shuling and Melissa, I appreciate your constant encouragement and friendship during my stay within the group. I have never felt the absence of my family here because of these two truly awesome friends and their families. I would also like to thank my doctoral committee for their support during this process. Dr. Lynn, it has been a pleasure sparring with you in my committee meetings. Your comments got me thinking about my research from a different perspective many times. Dr. Levine, I appreciate your support, your professionalism and your kind words of encouragement whenever we talked about my research. Dr. Wei, thank you for your support and guidance throughout this process. I must extend thanks to Dr. Peter Nelson, Dr. Donna Wilcock and their lab members for their help and support. I am also grateful for the funding that supported my research including my RCTF fellowship, the iii University of Kentucky Graduate College, and the University of Kentucky Chemistry department. Last but not the least I must thank my husband Partha for being my pillar of strength through so many difficult times. I would like to express my love and gratitude to my parents C.G. Soman Pillai and Thankam S. Pillai and and my sister Swapna Soman for all the love and encouragement that they have given me all my life. I would also like to mention here the love of my life, my son Rishi Soman Jana who has made my life so beautiful. A special thanks to my friends from the Sanders Brown Center on Aging for making my stay in UK so agreeable. iv TABLE OF CONTENTS ACKNOWLEDGMENTS …………………………………………...….……..……...... iii TABLE OF CONTENTS ………………………….….…..…………………..….……….v LIST OF TABLES ……………………………………..……….…………………..…... ix LIST OF FIGURES ……………………….…….…..……...…………………...………..x CHAPTER ONE: Introduction and Background ………….…………………........…1 1.1. Alzheimer’s disease (AD) ……………………………………………..….…...….. 1 1.1.1. Discovery and incidence.…………………………………………....……...... 1 1.1.2. Clinical characteristics.…………………………………..…………...……… 2 1.1.3. Pathological features ……………………………………….……….…….......3 1.1.4. Risk factors ….…………………………………….……….…………...…… 9 1.1.5. Early stages ……….………………………………..…………………...…….10 1.1.6. Etiology ……….…………………………………………………....…….…..11 1.2. Oxidative stress and AD ………………………………………...…………..…....12 1.2.1.Reactive Oxygen Species (ROS)...……….…………………………..……...12 1.2.2.Oxidative Stress…………...………………………………………...…..….. 13 1.2.3.Lipid peroxidation and protein oxidation……………...………..…..…...… 16 1.2.4.Nucleic acid oxidation …………….……………….…………………….… 17 1.3. DNA repair pathways …………………………………..…………..…………….25 1.3.1.Base excision repair pathway ..………………………...……....…………. ..25 1.3.2.Nucleotide excision repair pathway .…………………...……………….…..30 v 1.3.3.Double strand break repair pathway…………………………………..….....30 1.3.4.Mismatch repair pathway ……….…………….……………………...……...31 1.3.5.Other genes involved in DNA repair………………………………………....31 1.4. Additional neurodegenerative diseases……………………………………….......32 1.4.1.Dementia with lewy bodies……………………………….……………….…32 1.4.2.Frontotemporal dementia (FTD)………………………………………..……33 1.4.2.1. Behavioral variant FTD………………………..……………........33 1.4.2.2. Primary progressive aphasia……………………….…………..…33 1.4.2.3. FTD movement disorders………………………….………..........34 1.5. Statements of research projects ………………………………..…….……...….. .34 1.5.1.Oxidative DNA damage in genes of proteins modified during AD ………….34 1.5.1.1. Polymerase chain reaction………………….………….......…......36 1.5.1.2. Real time quantitative PCR (qPCR)…………………….....……...45 1.5.1.3. qPCR data analysis………………………………………..……....46 1.5.1.3.1. Absolute quantification/ Standard curve method ....…….....48 −ΔΔ Ct 1.5.1.3.2. Relative quantification/ comapartive Ct/ 2 method …………………………………………………….………........48 1.5.1.4. Voltage Dependent Anion Channels (VDACs)……………....….49 1.5.2.Changes in DNA repair enzymes of BER pathway in AD subjects…….…..50 CHAPTER TWO: Materials and methods ……………………………..…...……...50 2.1. Materials …………………….…………………………………………...…….....50 2.1.1.Brain specimen sampling ………………………………………………...….50