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Master Document Template Copyright by Dana Most 2016 THE DISSERTATION COMMITTEE FOR DANA MOST CERTIFIES THAT THIS IS THE APPROVED VERSION OF THE FOLLOWING DISSERTATION: THE ROLE OF SYNAPTIC MICRORNA IN CHRONIC ALCOHOL CONSUMPTION AND ITS EFFECTS ON SYNAPTIC COMPOSITION Committee: R. Adron Harris, Supervisor Roy Dayne Mayfield, Co-Supervisor Robert Messing Kimberly Raab-Graham Jonathan Pierce-Shimomura Rajesh Miranda THE ROLE OF SYNAPTIC MICRORNA IN CHRONIC ALCOHOL CONSUMPTION AND ITS EFFECTS ON SYNAPTIC COMPOSITION BY DANA MOST, B.SC. DISSERTATION Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY THE UNIVERSITY OF TEXAS AT AUSTIN AUGUST 2016 I DEDICATE THIS TO MY PARENTS PNINA YOUNG, BRAD YOUNG AND MIKE MOST. I DERIVE MY INSPIRATION FROM YOU. ACKNOWLEDGEMENTS This dissertation is the culmination of fascinating scientific projects and amazing life experiences of the past six years. These would never have been possible without the support of my two wonderful advisors and mentors, Adron Harris and Dayne Mayfield. It has been my privilege to work for them and with them. For this, I am forever grateful. I thank Adron for his unmatched inspiring mentorship and for his enduring friendship. He has given me the freedom to explore a wide range of ideas and scientific paths, all while supporting, encouraging and guiding me through the challenges. I thank Dayne for his continuous support and guidance, technical advice, and dedication to scientific excellence, without which I would have not been where I am today. Most students have one extraordinary advisor. I was fortunate to have had two. Thank you both. Special thanks to Dr. Yuri Blednov, who contributed significantly to my ability to execute these experiments, both technically and conceptually, and added to my education and training. I extended a warm appreciation to my committee members, Drs. Robert Messing, Kimberly Raab-Graham, Jonathan Pierce-Shimomura and Rajesh Miranda, for their guidance, feedback, scientific inspiration, expertise, and thoughtful questions which greatly enhanced my understanding of the topic and helped refine my project. I would like to thank the scientific community at the University of Texas in Austin, particularly the Waggoner Center for Alcohol and Addiction Research, the Institute of Neuroscience and the Graduate School. These centers cultivated a fostering environment for me to thrive in, as well as helped fund my research project. A big thanks to Krystal Phu, Jayna Dickson, and Jody Mayfield for their continuous assistance and input throughout grad school paper work, deadlines, grant and fellowship applications, and v scientific publications. I am appreciative of my lab mates, old and new, for innumerable discussions, both scientific and not, and for assistance with experiment execution, especially Olga Ponomarev, Gayatri Tiwari, Kelly Jameson and Jason Wang. I would like to thank the organizations that provided funding opportunities without which this science would never have seen the light of day. These include the F31 award from NIAAA; the Bruce-Jones Fellowship in Addiction Biology; the Graduate School prestigious award; the NIH Training Grant; and the Graduate School Recruitment Fellowship. I have worked beside many wonderful scientific peers who are both colleagues and friends. I thank Shlomi Yeshurun, John Valenta, Ashley Vena, Rick Gray, Laura Ferguson and Claire Stelly for their scientific input, encouragement, and friendship throughout graduate school. I have had the great good fortune of having wonderful friends around the world. Thank you Shlomi Yeshurun, Anat Tal-Gagnon, Daren Goldin, Tamara Spach and Leor Katz, for your support, inspiration and long-lasting friendship. I could always rely on you and you have always been there for me in any capacity. I would like to thank my three parents and brother for their decades of love, support and encouragement. Without them, I would never have made it this far. From a young age they challenged me in every way possible, always pushed me to reach my full potential, and believed in me even when I didn’t believe in myself. They have built my ability to dream big and shoot for the stars, even if it means I may land on the moon: My mom, for her everlasting belief in me, for always pushing me forward, for being my best friend and for the many food packages she has mailed me throughout the years, which sustained my body and soul during long work days in the lab; vi My step dad, for always supporting my many (crazy) scientific endeavors, for reviewing, editing and challenging me in my publications and grant applications, and for doing so happily during many sleepless nights. None of this could have been done without you; My dad, for being the happiest person I know, for his persistence that I should also be that way and for proof-reading this thesis; And my brother, the shining light in my sometimes dark tunnel, for supporting me throughout my roughest life moments and for being unapologetically himself; Finally, I would like to thank my beautiful cat, Sammy, for his unconditional love throughout the long work days and nights. vii THE ROLE OF SYNAPTIC MICRORNAS IN CHRONIC ALCOHOL CONSUMPTION AND ITS EFFECTS ON SYNAPTIC COMPOSITION Dana Most, Ph.D. The University of Texas at Austin, 2016 Supervisor: R. Adron Harris Co-supervisor: Roy Dayne Mayfield Drug dependence is the process by which the brain learns to depend on a drug and crave for it in its absence. This learning takes place through modifications of synaptic connections between neurons, changing synaptic structure and function, and causing long-lasting neuroadaptations. Over the past decade, several microRNAs have been proposed as playing a key role in regulating local mRNA translation into protein, specifically in the synaptic compartments of the cell (pre-synaptic terminals and post- synaptic densities). There is limited evidence, however, regarding how synaptic microRNAs control local mRNA translation during chronic drug exposure and how this contributes to the development of dependence. Can alcohol-responsive synaptic mRNAs and microRNA be identified? Do synaptic microRNAs regulate the synaptic mRNA expression changes in response to alcohol? Can changes in synaptic microRNA composition affect alcohol consumption? In this thesis, I present research supporting microRNA regulation of local mRNA translation and how drugs of abuse target this process. In the first section, I focus on the identification of alcohol-responsive synaptic mRNAs. In the second section, I focus on identifying alcohol-responsive synaptic microRNAs and predict key mRNA-microRNA viii interactions. Identifying the primary regulatory elements in the synapse is imperative for an accurate model of the synaptic interactions that lead to mRNA translation underlying plasticity. Since the neuroadaptations associated with response to alcohol rely on many mRNAs, therapy with microRNAs provides a potential treatment for alcohol dependence. In the third section, I focus on manipulating key microRNAs in-vivo to reverse and prevent alcohol consumption and alcohol-induced-neuroadaptations. The ability of synaptic microRNAs to rapidly regulate mRNAs provides a discrete, localized system that could potentially be used as diagnostic and treatment tools for alcohol and other addiction disorders. ix TABLE OF CONTENTS Acknowledgements .................................................................................................. v List of Tables ......................................................................................................... xiv List of Figures ........................................................................................................ xvi Chapter 1: Introduction .......................................................................................... 1 Overview ........................................................................................................ 1 The Effects of Alcohol on the Nervous System .............................................. 1 Primary Targets ..................................................................................... 4 The Effects on DNA ...................................................................... 8 The Effects on Neurotransmitter Systems ................................. 13 The Effects on the Neuroimmune System ................................. 29 The Effects of Alcohol on Synaptic Elements ............................................... 34 Local Translation and mRNAs ............................................................. 35 Local Translation and microRNA Regulation ...................................... 38 MicroRNAs Regulation Following Exposure to Stimulants ................. 41 MicroRNA Regulation Following Alcohol Exposure ............................ 46 Summary ...................................................................................................... 49 Chapter 2: The Synaptoneurosome Transcriptome: A Model for Profiling the Molecular Effects of Alcohol.................................................................. 53 Abstract ........................................................................................................ 53 Introduction ................................................................................................. 53 Materials and Methods ................................................................................ 55 Animal Housing and Alcohol Self-Administration
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