Texas Medical Center Library DigitalCommons@TMC UT GSBS Dissertations and Theses (Open Access) Graduate School of Biomedical Sciences 12-2016 Characterization of Vesicular Monoamine Transporter 2 and its role in Parkinson's Disease Pathogenesis using Drosophila Antonio Joel Tito Jr. Sheng Zhang Follow this and additional works at: http://digitalcommons.library.tmc.edu/utgsbs_dissertations Part of the Cell Biology Commons, Medicine and Health Sciences Commons, Molecular and Cellular Neuroscience Commons, Molecular Biology Commons, and the Molecular Genetics Commons Recommended Citation Tito, Antonio Joel Jr. and Zhang, Sheng, "Characterization of Vesicular Monoamine Transporter 2 and its role in Parkinson's Disease Pathogenesis using Drosophila" (2016). UT GSBS Dissertations and Theses (Open Access). 719. http://digitalcommons.library.tmc.edu/utgsbs_dissertations/719 This Dissertation (PhD) is brought to you for free and open access by the Graduate School of Biomedical Sciences at DigitalCommons@TMC. It has been accepted for inclusion in UT GSBS Dissertations and Theses (Open Access) by an authorized administrator of DigitalCommons@TMC. For more information, please contact [email protected]. CHARACTERIZATION OF VESICULAR MONOAMINE TRANSPORTER 2 AND ITS ROLE IN PARKINSON’S DISEASE PATHOGENESIS USING DROSOPHILA By Antonio Joel Tito Jr., M.S. APPROVED: _______________________ Sheng Zhang, Ph.D. _______________________ Hugo Bellen, Ph.D. _______________________ Richard Behringer, Ph.D. _______________________ Kartik Venkatachalam, Ph.D. _______________________ Momiao Xiong, Ph.D. _______________________ Nick Justice, Ph.D. _______________________ Andrew Bean, Ph.D. APPROVED: _______________________ Dean, UTHealth Graduate School of Biomedical Sciences at Houston CHARACTERIZATION OF VESICULAR MONOAMINE TRANSPORTER 2 AND ITS ROLE IN PARKINSON’S DISEASE PATHOGENESIS USING DROSOPHILA A DISSERTATION Presented to the Faculty of The University of Texas Health Science Center at Houston And The University of Texas M.D. Anderson Cancer Center Graduate School of Biomedical Sciences In partial fulfillment of the requirements For the degree of DOCTOR OF PHILOSOPHY By Antonio Joel Tito Jr., M.S. Houston, Texas December, 2016 II DEDICATION I dedicate this dissertation to God, my father, Jorge Tito-Izquierdo, Ph.D. P.E., my mother, Elizabeth Tito, my brothers, Juan Pablo Tito and Jorge Francisco Tito for their patience and devotion to growing in faith, wisdom, knowledge, and love. “The aim of natural science is not simply to accept the statements of others, but to investigate the causes that are at work in nature.” By St. Albertus Magnus, Doctor Universalis of the Church III ACKNOWLEDGEMENTS I am particularly grateful to the Holy Trinity and His mother for without their continuous inspiration and loving graces, this dissertation would not have been completed. I would like to thank the selfless dedication of my PhD advisor, Dr. Sheng Zhang, and to all my friends, colleagues, and professors from the Center for Metabolic and Degenerative Diseases (CMD) at the Institute of Molecular Medicine at UT-Health; the students and faculty at the Human Molecular Genetics Program (HMG) at the Graduate School of Biomedical Sciences (GSBS) at UT-Health; and the staff and administrators at the GSBS. I am deeply thankful for Sheng’s commitment to scientific rigor, as well as for his patience, guidance, words of encouragement, and for all the times he has selflessly offered the most needed personal, scientific, and academic support. The way he communicates his deep knowledge in the sciences with so much passion and care has made me into a better scientist and future mentor. I am very thankful and quite honored and privileged to have such a talented, passionate, and caring advisor as Sheng! I am also in great debt for the intellectual contribution and moral support by all my colleagues, fellow students, and professors at the CMD and HMG. Our conversations have led to great scientific insights, which will serve as the foundation for future discoveries. In addition, a warm thank you to the members of my committee: Dr. Hugo Bellen, Dr. Nicholas Justice, Dr. Andrew Bean, Dr. Richard Behringer, Dr. Momiao Xiong, and Dr. Kartik Venkatachalam. I am particularly grateful for convincingly conveying a great excitement in regard to critically thinking about the future implications of my experiments. Likewise, I would like to express my sincere appreciation to our collaborators and their laboratory personnel and students who offered their selfless support and care for the success of my project: Dr. Hugo Bellen and Dr. Sonal Nagarkar-Jaiswal at Baylor College of Medicine, Dr. Jay Hirsh at the University of Virginia, Dr. Kristen Eckel-Mahan and Dr. Alex Ribas at UT-Health at Houston, and Dr. Mian Jiang at the University of Houston-Downtown. I am truly grateful for their unwavering support and scientific rigor; especially to Dr. Bellen who kindly offered to serve as co-PI for the NINDS Diversity RO1 Supplement Award and offer an open-door policy to his Howards Hughes Laboratory at BCM. I am also highly appreciative and in deep gratitude to my parents, brothers, brothers in Christ at Church, friends, and Shebna Cheema for all their support and care. Immeasurable appreciation and thanks goes to Pilar Rodriguez for her detailed critique of the dissertation as well as for her emotional support in my moments of hardship. Last but not least, special thanks goes to all my students and friends: Shebna Cheema, Enes Mehmet-Inam, Kiara Arroyo-Andrade, Pilar Rodriguez, Sam Vallagomesa, Wenting Li, Linden Shih, Kira Wegner-Clemens, Zoe Tao, Daniel Colchado, Mohammed Alsheikh-Kassim, Jiang Ge, Rachel Chyau-wen Lin, Jehanne Hou, Marc Rinosa, and Hongyue Jiang. They have one way or another contributed this and many other investigations in Dr. Zhang’s lab, under my tutelage. Most importantly, they have taught me the art of teaching, patience, and imparted a mission onto me to mentor more students in the near future. IV Characterization of Drosophila homologue of vmat2 and its role in a genetic model of Parkinson’s disease Antonio J. Tito Jr., M.Sc. Supervisory Professor: Sheng Zhang, Ph.D. Parkinson’s disease (PD) is a progressive neurodegenerative disorder caused by the selective loss of the dopaminergic neurons (DN) in the substantia nigra pars compacta region of the brain. PD is also the most common neurodegenerative disorder and the second most common movement disorder. PD patients exhibit the cardinal symptoms, including tremor of the extremities, rigidity, slowness of movement, and postural instability, after 70-80% of DN degenerate. It is, therefore, imperative to elucidate the underlying mechanisms involved in the selective degeneration of DN. Although increasing numbers of PD genes have been identified, why these largely widely expressed genes induce selective loss of DN is still not known. Notably, dopamine (DA) itself is a chemically labile molecule and can become oxidized to toxic byproducts while induce the accumulation of harmful molecules such as Reactive Oxygen Species (ROS). Accordingly, DA toxicity has long been suspected to play a role in selective neuronal loss in PD. Vesicular monoamine transporter (VMAT) is essential for proper vesicular storage of monoamines such as DA and their regulated release. Increasing evidence have linked VMAT dysfunction with Parkinson’s disease. In this study, we reexamine the gain- and loss-of-function phenotypes of the sole VMAT homolog in Drosophila. Our results suggest that the C-terminal sequences in the two encoded VMAT isoforms not only determine their differential subcellular localizations, but also their activities in content release. In particular, VMAT2 orthologue potentially poses a unique, previously unexplored activity in promoting DA release. On the other hand, by examining DA distribution in wildtype and VMAT mutant animals, we find that there exists intrinsic difference in the dynamics of intracellular DA handling among DN clustered in different brain regions. Furthermore, loss of VMAT causes severe loss of total DA levels and a redistribution of DA in Drosophila brain. Lastly, removal of both VMAT and another PD gene parkin, which is also conserved in Drosophila, results in the selective loss of DN, primarily in the protocerebral anterior medial (PAM) clusters of the brain. Our results suggest a potential involvement of cytoplasmic DA in selective degeneration of DN and also implicating a role of differential intracellular DA handling in the regional specificity of neuronal loss in PD. V TABLE OF CONTENTS ACCEPTANCE PAGE .……………………………………………………………………………………………………………...I TITLE PAGE ..…………………………………………………………………………………………………………………………II DEDICATION ………………………………………………………………………………………………………………………..III ACKNOWLEDGMENTS …………………………………………………………………………………………………………IV ABSTRACT …………………………………………………………………………………………………………………………...V TABLE OF CONTENTS …………………………………………………………………………………………………………..VI LIST OF FIGURES …………………………………………………………………………………………………………….....VII LIST OF ABBREVIATIONS AND ACRONYMS …………………………………………………………….………...VIII LIST OF APPENDICES ………………………………………………………………………………………………..………….IX CHAPTER 1: LITERATURE REVIEW AND INTRODUCTION……………………………………………….…………1 1.1 Pathological Hallmark and PD Symptoms ………………………………………….………………………………….1 1.2 Clinical Manifestations of the Disease …………………………………………..………………………………………2 1.3 PD Prevalence …………………………..…………………………………………………………………………………...……3 1.4 A Bio-Molecular Snapshot of PD Treatments………………………………………………..…………………..…..3