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A Role for the P75 Neurotrophin Receptor in Axonal Degeneration and Apoptosis Induced by Oxidative Stress

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A Role for the P75 Neurotrophin Receptor in Axonal Degeneration and Apoptosis Induced by Oxidative Stress A Role for the p75 Neurotrophin Receptor in Axonal Degeneration and Apoptosis Induced by Oxidative Stress By Bradley Kraemer Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY In Neuroscience December, 2014 Nashville, Tennessee Approved: Bruce D. Carter, Ph.D. Aaron B. Bowman, Ph.D. Christine L. Konradi, Ph.D. Scott W. Hiebert, Ph.D. William H. Valentine, DVM, Ph.D. To my best friend and beautiful wife Maria, who has always been there to comfort me in times of frustration and to celebrate with me in times of joy; and to my wonderful parents, whose loving support and ever-optimistic counsel continuously helped me to achieve this goal. ii ACKNOWLEDGEMENTS This work would not have been possible without the support of the Vanderbilt Interdisciplinary Graduate Program, NIMH Training in Fundamental Neuroscience Training Program T32 MH064913, and NIH grant R01NS038220. I am sincerely grateful to my mentor, Dr. Bruce Carter, for the tremendous amount of time and effort dedicated to my training over the last five years. From the hours upon hours spent in your office asking questions while studying for my qualifying exam, to the countless discussions in our weekly meetings, you have always had an open door and have been willing to listen to my questions or concerns. Your unique perspective and scientific curiosity has been a great example for me, and your guidance during my time in your lab has greatly shaped my development into an independent scientist. Thank you also for always promoting such a positive and supportive lab environment. The many lab trips, social events, and meetings to help others with preparations for qualifying exams, conference presentations, or other events have all had a tremendous effect on lab comradery and have made the lab a supportive and often fun place to work. Lastly, thank you for helping me to develop a taste for higher-quality beer, and to recognize the difference between a lager and an ale. The Carter lab has been much more than a workplace for me. From playing tennis with Bruce, to whitewater rafting and canoeing with my friends in the lab, to meeting my wife, the Carter lab has been a significant component of my social and personal life. I am deeply grateful to all of the members of the lab who have helped me in so many ways. Thank you to the former lab members Allison Limpert, Rajappa Kenchappa, Ana Perdigoto, and Jami Scheib, who were so instrumental in my early lab training, as well as iii to Uzma Saeed, who helped me to optimize neuronal cultures of superior cervical ganglia, and Malathi Naryan, who assisted with experiments designed to analyze tyrosine hydroxylase staining in the irides of mice. I am also grateful to current lab member Amrita Pathak, who contributed valuable control data indicating that BDNF-induced apoptosis is blocked by the antibody 9650. I would also like to thank all of the other current lab members: Bret Mobley, Emily Stanley, Chelsea Sullivan, Alex Trevisan, Eddie Hickman, and Rose Follis. All of you have been invaluable to the progress of my research. Your ideas in lab meeting, technical advice, and overall support have been incredibly helpful. I will never forget how genuinely happy everyone in lab was for me when I received positive reviews for my manuscript. Thank you so much for all of your encouragement. I would like to express my gratitude to the members of my committee. I am greatly appreciative of the time, resources, and valuable insight that you devoted to my research project. Thank you to Dr. Aaron Bowman, the chair of my committee, for all of your time and suggestions. Your advice regarding optimization of our p75 NTR cleavage assays and your shared protocols related to antibody purification greatly helped to move the project forward, as well as assisted several other members of the Carter lab. I am also appreciative of all of the advice and suggestions of Dr. Christine Konradi. Thank you for your constant enthusiasm and especially for your technical advice, which again and again was instrumental to the success of my experiments. I am grateful for the time and assistance of Dr. Scott Hiebert, who consistently during committee meetings would ask the type of practical and big-picture questions that kept us focused on investigating that which truly matters. Finally, I am appreciative of Dr. William Valentine, who has been a wonderful adviser and collaborator. Thank you so much for providing me with 4-hydroxy- iv 2-nonenal, meeting with me on numerous occasions to discuss the direction of my project, and offering insightful ideas and advice related to oxidative stress and neurodegeneration. Your expertise and guidance was a critical part of my success. I am greatly appreciative to all of my collaborators. Thank you to my first undergraduate trainee, John Snow, who first began work with me in the early stages of this dissertation project. Though the experiments were challenging, your hard work helped us tremendously. Together, we were able to develop our cell culture assays, determine the appropriate treatment conditions to expose cultured neurons to 4-hydroxy- 2-nonenal or 6-hydroxydopamine, and optimize experimental conditions for detecting NTR NTR p75 cleavage. In addition to other results, you contributed to data revealing that p75 is cleaved in response to HNE, and that such cleavage requires γ-secretase. These findings were a major component of our recently published manuscript, and I am grateful for all of your hard work and help to progress the project. I am also appreciative to Dr. Ariel Deutch, who collaborated with us to investigate the effects of intraperitoneal administration of 6-OHDA to wildtype and p75 NTR -/- mice, as well as to Deutch lab members Pete Vollbrecht and Lauren Herrera, who performed cardiac perfusions, tissue collection, and tyrosine hydroxylase staining in the spleen and heart. I benefitted greatly from your advice and research contribution. I would not be where I am today without the love and support of my family. To my parents, thank you so much for always encouraging me to ask questions and take an interest in the unknown. Your enthusiasm for science, as well as your loving encouragement, has kept me strong during the most difficult times of my dissertation research. Thank you to my brothers, Ryan and Kyle, who have always been there for me v in so many ways. To my newest parents, Cindy and Joe, thank you for your loving support and for your interest in my research. Lastly, to my wife Maria, who understands perhaps better than anyone the obstacles and challenges of my dissertation research, thank you for always being there for me. You enrich my life, and I am grateful for you every day. vi TABLE OF CONTENTS Page DEDICATION .................................................................................................................. ii ACKNOWLEDGEMENTS .............................................................................................. iii LIST OF FIGURES......................................................................................................... ix LIST OF ABBREVIATIONS ............................................................................................ x Chapter I. Introduction Neurotrophins and Neurotrophin Receptors ............................................................. 1 The p75 Neurotrophin Receptor ............................................................................... 3 Structure ............................................................................................................ 4 Apoptotic Signaling ............................................................................................ 7 Activation of the Mitochondrial Cascade ............................................................ 8 Cytosolic Factors Linking p75 NTR to JNK .......................................................... 10 Other Factors Involved in p75 NTR -mediated Apoptosis..................................... 14 Regulated Intramembrane Proteolysis of p75 NTR ............................................. 16 Proneurotrophins and Sortilin........................................................................... 20 Apoptotic Role of p75 NTR in Pathology ............................................................. 22 Promotion of Cell Survival ................................................................................ 28 P75 NTR Forms a High-Affinity Complex with the Trks ....................................... 29 P75 NTR and Trk Receptors: Mechanisms of Association .................................. 31 Downstream Pro-survival Signals Induced by p75 NTR ..................................... 33 Determinants of Survival Outcome After p75 NTR Activation .............................. 36 Summary.......................................................................................................... 37 Oxidative Stress Sources of Reactive Oxygen Species .............................................................. 37 Antioxidants ..................................................................................................... 38 Oxidative Stress-induced Apoptotic Signaling .................................................. 39 Lipid Peroxidation ............................................................................................ 40 Dissertation Goals: Exploring a Hypothesis about Oxidative Stress and p75 NTR ...................................................................
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