THE CONTRIBUTIONS OF MICROGLIAL VPS35 TO ADULT HIPPOCAMPAL NEUROGENESIS AND NEURODEGENERATIVE DISORDERS By Joanna Ruth Erion Submitted to the Faculty of the Graduate School of Augusta University in partial fulfillment of the Requirements of the Degree of Doctor of Philosophy April 2017 COPYRIGHT© 2017 by Joanna Ruth Erion THE CONTRIBUTIONS OF MICROGLIAL VPS35 TO ADULT HIPPOCAMPAL NEUROGENESIS AND NEURODEGENERATIVE DISORDERS This thesis/dissertation is submitted by Joanna Ruth Erion and has been examined and approved by an appointed committee of the faculty of the Graduate School of Augusta University. The signatures which appear below verify the fact that all required changes have been incorporated and that the thesis/dissertation has received final approval with reference to content, form and accuracy of presentation. This thesis/dissertation is therefore in partial fulfillment of the requirements for the degree of Doctor of Philosophy). ___________________ __________________________________ Date Major Advisor __________________________________ Departmental Chairperson __________________________________ Dean, Graduate School ACKNOWLEDGEMENTS I will be forever grateful to my mentor, Dr. Wen-Cheng Xiong, for welcoming me into her laboratory and providing me with the honor and opportunity to work under her astute leadership. I have learned many valuable lessons under her guidance, all of which have enabled me to grow as both a student and a scientist. It was with her erudite guidance and suggestions that I was able to examine aspects of my investigations that I had yet to consider and ask questions that would not have otherwise occurred to me. I would also like to thank Dr. Lin Mei for contributing not only his laboratory and resources to my endeavors, but also his guidance and input into my investigations, providing me with additional avenues for directing my efforts. The collaborative effort of the Mei-Xiong lab provided me with a rich atmosphere for my learning and growth. I wish to extend thanks to my many coworkers and fellow collaborators throughout the years, not only to members of the Mei-Xiong lab family, but to every individual who has provided me with input, guidance, education, and experience. There are too many to list, but I wish to point out the following for their assistance and support during my studies: Fulei Tang who taught me not only the basic techniques essential to my experiments, but who also helped me learn to troubleshoot and self-teach - skills that I have learned are invaluable to one's success in science; Dong Sun for his assistance with animal treatments; Fang Lui for always being willing to help and assist; Hongsheng Zhang for contributing the retroviral vector applied in my studies; Thiri Lin for providing so much support in all things lab-related; and very importantly, Shiyang Ye for the much needed support and assistance in making my way to the finish line. With his help, I was able to pursue additional experiments that I may not otherwise have had the time to investigate. I wish to extend my gratitude to The Graduate School (TGS) at Augusta University – the TGS administration has been infinitely supportive of my efforts, beginning with my very first communications during the application process and ongoing throughout the course of my studies. Not only have TGS faculty supported me through all of my academic and personal endeavors, but they have also provided an atmosphere rich with opportunity for exploring future career options and acquiring the skills and knowledge necessary to succeed professionally. Additionally, I am grateful to the Department of Neuroscience and Regenerative Medicine and the Molecular Medicine program for providing me with a supportive environment for my education. Dr. Lynette McCluskey has gone above and beyond her role as program director by providing mentorship as well as friendship to all students within the Molecular Medicine program. I also wish to extend my gratitude to my committee members - Dr. Krishnan Dhandapani, Dr. Amyn Rojiani, Dr. Nilkantha Sen, and Dr. Bobby Thomas - for contributing their time, guidance, and support towards my studies. The direction of my thesis work has depended upon each and every individual on my committee and the valuable input and feedback they have provided along the way as they have each provided their own unique perspective into my research. I also wish to thank my thesis reader Dr. Sangeetha Sukumari-Ramesh for providing her time and feedback to better the overall content of this thesis. In a short time, she has already provided valuable guidance and direction. Finally, I am most thankful to my family and friends who have shared this journey with me: my mother Debra Barlow who has always supported my dreams and ambitions, no matter how ambitious they may seem; my Father, Charles Erion, who served as my steadfast source of sage advice, inspiring in me my dedication to seek and uphold truth and knowledge; my husband, Brian Appel, who I met over the course of my journey as a graduate student and who has become my ultimate cornerstone, providing his faith in my abilities and his support through all the ups and downs, and guiding my success in life; my bonus daughter, Avery Appel, who inspired in me a new outlook on life and provided me a future to aspire for; and my youngest daughter, Victoria Appel, who accompanied me for the final stages of my journey as a graduate student and who has become my utmost inspiration and motivation for purpose and success. I have become the person I am because of the support and encouragement of each and every individual acknowledged here, and I will be forever grateful to everyone who has invested their time, resources, and encouragement in their support of my success. ABSTRACT JOANNA RUTH ERION The contributions of microglial VPS35 to adult hippocampal neurogenesis and neurodegenerative disorders (Under the direction of WEN-CHENG XIONG) The retromer complex is a multimeric protein complex which facilitates intracellular trafficking of a variety of transmembrane proteins. Vacuolar protein sorting- associated protein 35 (VPS35) is a critical component of retromer’s cargo recognition subunit and has been implicated in neurodegenerative disease pathology, including Alzheimer’s disease (AD). Without functional VPS35, trafficking of retromer cargo is often impaired, compromising cargo function and/or downstream signaling events. VPS35 expression is ubiquitous and can be found throughout the central nervous system (CNS). Microglia express VPS35, and microglia from AD patients exhibit reduced VPS35 expression. We sought to determine how microglial VPS35 loss-of-function might contribute to neurodegenerative disease pathology. We examined the CNS of a mouse model of heterozygous VPS35 deletion and found evidence suggesting upregulated microglial activity. To specifically examine the effects of microglial VPS35 loss-of-function, we developed VPS35CX3CR1 mice, a model which induces microglial-specific VPS35 depletion. Microglial VPS35 loss-of-function upregulated microglial density in a region-specific manner, which we determined to be an effect of increased microglial differentiation/survival. Upon further inspection, we found evidence of upregulated microglial inflammatory activity in the hippocampus, including increased levels of proinflammatory cytokine interleukin-6. Microglial density was increased within the subgranular zone (SGZ) of VPS35CX3CR1 mice, so we sought to determine if microglial VPS35 loss-of-function has any effect on hippocampal neurogenesis. While we found decreased doublecortin+ (DCX) immature neurons, an increase was observed in the differentiation/survival of neural progenitor cells (NPCs). Further analysis suggested the cell cycle exit of VPS35CX3CR1 hippocampal NPCs may be compromised. To examine morphology of newborn neurons, hippocampi were labeled with a retroviral vector, revealing impaired dendritic development in newborn hippocampal VPS35CX3CR1 neurons. Aberrant regulation of hippocampal neurogenesis in VPS35CX3CR1 mice was associated with a depressive behavioral phenotype and long-term memory impairment. These findings implicate a novel microglial-specific role of VPS35 that might contribute to neurodegenerative disease pathogenesis. The full extent of this role remains to be determined, as well as the mechanisms underlying our observations. It also remains to be determined what role microglial VPS35 might play in other brain regions, and how microglial VPS35 depletion might contribute to other aspects of neurodegenerative disease pathology. KEY WORDS: Retromer, VPS35, Neurodegeneration, Alzheimer's Disease, Parkinson's Disease, Microglia, Hippocampus, Adult Neurogenesis, Subgranular Zone Table of Contents Chapter Page 1 INTRODUCTION ...........................................................................................#1 2 MATERIALS AND METHODS ...................................................................#27 3 RESULTS ......................................................................................................#37 4 DISCUSSION ................................................................................................#65 5 SUMMARY ...................................................................................................#75 References ........................................................................................................................#76 Appendix A ....................................................................................................................#100