University of Kentucky UKnowledge Theses and Dissertations--Chemistry Chemistry 2017 EPIGENETIC MODIFICATIONS TO CYTOSINE AND ALZHEIMER’S DISEASE: A QUANTITATIVE ANALYSIS OF POST-MORTEM TISSUE Elizabeth M. Ellison University of Kentucky, [email protected] Digital Object Identifier: https://doi.org/10.13023/ETD.2017.398 Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Ellison, Elizabeth M., "EPIGENETIC MODIFICATIONS TO CYTOSINE AND ALZHEIMER’S DISEASE: A QUANTITATIVE ANALYSIS OF POST-MORTEM TISSUE" (2017). Theses and Dissertations--Chemistry. 86. https://uknowledge.uky.edu/chemistry_etds/86 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. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless an embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. 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 thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Elizabeth M. Ellison, Student Dr. Mark A. Lovell, Major Professor Dr. Mark A. Lovell, Director of Graduate Studies EPIGENETIC MODIFICATIONS TO CYTOSINE AND ALZHEIMER’S DISEASE: A QUANTITATIVE ANALYSIS OF POST-MORTEM TISSUE 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 Elizabeth Marie Ellison Lexington, Kentucky Director: Dr. Mark A. Lovell, Professor of Chemistry Lexington, Kentucky 2017 Copyright © Elizabeth Marie Ellison 2017 ABSTRACT OF DISSERTATION EPIGENETIC MODIFICATIONS TO CYTOSINE AND ALZHEIMER’S DISEASE: A QUANTITATIVE ANALYSIS OF POST-MORTEM TISSUE Alzheimer’s disease (AD) is the most common form of dementia and the sixth leading cause of death in the United States, with no therapeutic option to slow or halt disease progression. Development of two characteristic pathologic lesions, amyloid beta plaques and neurofibrillary tangles, in the brain are associated with synaptic dysfunction and neuron loss leading to memory impairment and cognitive decline. Although mutations in genes involved in amyloid beta processing are linked to increased plaque formation in the inherited familial form of AD, the more common idiopathic form, termed sporadic AD, develops in the absence of gene mutations. In contrast, alterations in gene expression and transcription occur in plaque and tangle susceptible brain regions of sporadic AD subjects, even in the earliest stages of development of pathologic burden, and may give insight into the pathogenesis of AD. Epigenetic modifications to cytosine are known to alter transcriptional states and gene expression in embryonic development as well as in cancer studies. With the discovery of enzymatically oxidized derivatives of 5-methylcytosine (5- mC), the most common epigenetic cytosine modification, a probable demethylation pathway has been suggested to alter transcriptional states of DNA. The most abundant 5- mC derivative, 5-hydroxymethylcytosine (5-hmC), while expressed at low concentrations throughout the body, is expressed at high concentrations in brain cells. To determine the role cytosine modifications play in AD, this study was directed at the quantification of epigenetic modifications to cytosine in several stages of AD progression using global, genome-wide, and gene-specific studies. To determine global levels of each cytosine derivative in brain regions relevant to AD progression, a gas chromatography/mass spectrometry quantitative analysis was utilized to analyze cytosine, 5-mC, and 5-hmC in tissue specimens from multiple brain regions of AD subjects, including early and late stages of AD progression. To determine the genome-wide impact of 5-hmC on biologically relevant pathways in AD, a single-base resolution sequencing analysis was used to map hydroxymethylation throughout the hippocampus of late stage AD subjects. Finally, to determine gene-specific levels of cytosine, 5-mC, and 5-hmC, a quantitative polymerase chain reaction (qPCR) protocol was paired with specific restriction enzyme digestion to analyze target sequences within exons of genes related to sporadic AD. Results from these studies show epigenetic modifications to cytosine are altered on the global, genome-wide, and gene-specific levels in AD subjects compared to normal aging, particularly in early stages of AD progression, suggesting alterations to the epigenetic landscape may play a role in the dysregulation of transcription and the pathogenesis of AD. KEYWORDS: cytosine, 5-hydroxymethylcytosine, 5-methylcytosine, Alzheimer’s disease, epigenetic modifications Elizabeth Marie Ellison _ _________July 7, 2017__________ EPIGENETIC MODIFICATIONS TO CYTOSINE AND ALZHEIMER’S DISEASE: A QUANTITATIVE ANALYSIS OF POST-MORTEM TISSUE By Elizabeth Marie Ellison ____Dr. Mark A Lovell__ __ Director of Dissertation ____Dr. Mark A Lovell__ __ Director of Graduate Studies ______July 7, 2017__ _____ Dedicated to my dad, James F. Ellison For your unwavering belief in me ACKNOWLEDGMENTS There are many people I need to thank, but none more so than my research advisor, Dr. Mark A. Lovell. My graduate career and this dissertation would not have been possible without the guidance, patience, and mentorship of Dr. Lovell. Countless questions, meetings, seminar practices, edits of manuscripts, advice, encouragement, as well as new artist and album suggestions, have prepared me for my future as a bioanalytical chemist (and music enthusiast). You are a wonderful educator and a great mentor. Thank you so much boss. In addition to my research advisor, my advisory committee members, Dr. Bert Lynn, Dr. Chris Richards, and Dr. Paul Murphy, have gone above and beyond, providing insight and scope to my research projects, and encouraged me to excel. Thank you all. I would also like to mention two other fellow scientists, Dr. R. Daniel Johnson and Coach Chad Hodge. My undergraduate research advisor, Dr. RDJ, who introduced me to chemistry research by initiated my career as a chemistry research assistant. Thank you for your encouragement to pursue a chemistry doctoral degree and for believing in me. I treasure those years working in the lab and the fellow members of the lab group. In addition to RDJ, I’d also like to thank Coach Hodge, my high school AP chemistry teacher, for introducing me to chemistry and making high school chemistry class captivating. Thank you both so much for your passion for science and passion for teaching. My fellow lab mates, Shuling Fister, Dr. Melissa Bradley-Whitman, and Dr. Sony Soman, thank you all so much for your patience with me. Thank you for your guidance and for troubleshooting road-blocks with me these past five years. Just about every technique I have learned came from one of you, and I could not be more grateful. Thank you for all the birthday lunches, Christmas presents, and all the laughs we’ve shared over the years. Thank you to all the faculty and staff of Sanders-Brown Center on Aging, for allowing me to use instrumentation and lab space, offered guidance, and aided me in obtaining samples for iii my research projects. Specifically, I would like to thank Ms. Paula Thomason. Thank you for editing my manuscripts, arranging potlucks and get-togethers, and laughing and sharing your life with me and our lab. You’ve been so kind and a warm presence in Sanders-Brown, and I feel blessed to know you. In addition, special thanks to Dr. Erin Abner for her biostatistics expertise. Thank you so much for all of your help and for explaining (and re-explaining) all the complicated statistics I needed for this dissertation. Finally, and most importantly, thank you to the brave men and women who volunteer to be enrolled in the aging and dementia studies at Sanders-Brown. Your sacrifice and selflessness will be the reason that Alzheimer’s disease can be cured. To my fellow chemistry graduate students, you keep me sane and lift my spirits when science is impossible. Thank you for all the coffee trips, bagels, lunches, and bowling nights to ease the stress and frustration of graduate school. To my closest friends, I am so blessed to know each of you and can’t thank you enough for all the support and laughter you’ve given me. To my family, thank you Mom, Dad, Rick and Rob, for loving me unconditionally and believing, without any doubt, that I could and would do this. Thank you for expecting me to succeed and not letting me give up when I thought it was too difficult. Thank you for being my support system, my biggest cheerleaders, and my favorite people.