Characterization of Mice with Altered Dopamine Transporter and Vesicular Monoamine Transporter 2 Levels by Shababa Tanzeel Masoud A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Pharmacology and Toxicology University of Toronto © Copyright by Shababa Tanzeel Masoud 2017 Characterization of Mice with Altered Dopamine Transporter and Vesicular Monoamine Transporter 2 Levels Shababa Tanzeel Masoud Doctor of Philosophy Department of Pharmacology and Toxicology University of Toronto 2017 Abstract Dopamine is a key neurotransmitter that regulates motor coordination and dysfunction of the dopamine system gives rise to Parkinson’s disease. Nigrostriatal dopamine neurons are vulnerable to various genetic and environmental insults, suggesting that these cells are inherently at-risk. A cell-specific risk factor for these neurons is the neurotransmitter, dopamine itself. If intracellular dopamine is not appropriately sequestered into vesicles, it can accumulate in the cytosol. Cytosolic dopamine is highly reactive and can trigger oxidative stress, leading to cellular toxicity. Cytosolic dopamine levels are modulated by the plasma membrane dopamine transporter (DAT) that takes up dopamine from the extracellular space, and the vesicular monoamine transporter 2 (VMAT2) that stores dopamine into vesicles. In this thesis, we altered DAT and VMAT2 levels to investigate the detrimental consequences of potentially amplifying cytosolic dopamine in transgenic mice. Project 1 focused on selective over-expression of DAT in dopaminergic cells of transgenic mice (DAT-tg). DAT-tg mice displayed phenotypes of dopaminergic damage: increased dopamine-specific oxidative stress, L-DOPA-reversible fine motor deficits and enhanced sensitivity to toxicant insult, suggesting that increasing DAT- mediated dopamine uptake is detrimental for dopamine cells. As an extension of Project 1, ii Project 2 focused on mice that simultaneously over-express DAT and under-express VMAT2 (DAT-tg/VMAT2-kd mice). These animals were hypothesized to demonstrate exacerbated dopaminergic toxicity due to buildup of cytosolic dopamine caused by increased uptake and decreased packaging. While DAT-tg/VMAT2-kd mice displayed detrimental phenotypes (poor survival, decreased body weight, reduced dopamine tissue content and release) and compensatory changes (increased dopamine receptors and metabolism), they did not show dopamine cell loss. This is due to unexpected loss of phenotypes in DAT-tg mice from a new colony that no longer displayed dopaminergic neurodegeneration. Thus, instead of Parkinsonian behavior, DAT-tg/VMAT2-kd mice showed novel phenotypes such as hyperactivity and improved fine-motor and cognitive skills compared to other genotypes. DAT-tg/VMAT2-kd mice were also highly sensitive to amphetamine-induced locomotion. Hence, in the absence of neurodegeneration, altered DAT and VMAT2 levels produced unique behavioral changes in DAT-tg/VMAT2-kd mice, shedding light on the complex function of the dopamine system. Collectively, these studies demonstrate how perturbations in dopamine compartmentalization can impact dopamine homeostasis and behavior. iii Acknowledgments First, I would like to thank my supervisor, Dr. Ali Salahpour, for his immense guidance and mentorship during my Ph.D. As one of his first students, I have had the privilege of learning from him directly and seeing the lab grow over the years. His enthusiasm for science, positive outlook, understanding nature and approachability make him a truly unique supervisor. To my committee members, Drs. Peter G. Wells, José Nobrega, W. M. Burnham and David S. Riddick: you have been my guiding light throughout this Ph.D. You have challenged, supported and encouraged me. I am eternally grateful for having the best Ph.D. supervisory committee I could ever hope for. A special thank you to Dr. David S. Riddick for playing the dual role of my co-supervisor and thesis reader. You have always had the time to check up on me, provide constructive criticism and guide me in the right direction. Also, a special thank you to Dr. W. M Burnham – I started my scientific journey in your lab as a 4th year project student and since then, I have shared a great working relationship with you as the TA for PCL475. Thank you for your kindness and for always having my best interest in mind. To Dr. Amy Ramsey, thank you for offering your expertise and advice throughout my Ph.D. To our collaborators: Drs. Gary W. Miller, Jason Richardson, Jonathan Brotchie and Andrei Starostin – I truly appreciate your invaluable technical help with my projects. I would like to gratefully acknowledge Dr. Salah El Mestikawy for being my external examiner. A special mention for Lien Nguyen, my undergraduate project student, for her useful contribution to these experiments. I am also grateful for my sources of funding from Parkinson Society of Canada, Canadian Institutes of Health Research and the University of Toronto. Wendy Horsfall, you are the backbone of our lab – I cannot thank you enough for sharing your knowledge and being so patient with us. Marija Milenkovic and Dr. Laura Vecchio, thank you for helping me every day and being my voice of reason. To all members of the Salahpour and Ramsey labs, I am grateful to have shared this journey with you. Finally, I would like to extend my deepest gratitude to my family. To my parents, Chowdhury A. Masud and Shabina M. Masud - you never doubted me even for a moment. You stood by me as pillars of strength throughout all my struggles and I will forever remain grateful. To Nafees, you supported me in every way imaginable. Thank you for being my teammate. iv Table of Contents Acknowledgments.......................................................................................................................... iv Table of Contents .............................................................................................................................v List of Figures ..................................................................................................................................x List of Tables ............................................................................................................................... xiii List of Appendices ....................................................................................................................... xiv List of Publications ........................................................................................................................xv List of Abbreviations ................................................................................................................... xvi Chapter 1 Introduction .....................................................................................................................1 Introduction .................................................................................................................................1 1.1 Statement of Research Problem ...........................................................................................1 1.2 Literature Review.................................................................................................................2 1.2.1 Dopamine function in the brain ................................................................................2 1.2.1.1 Nigrostriatal pathway and movement ........................................................3 1.2.1.2 Other dopaminergic pathways ...................................................................8 1.2.2 Dopamine homeostasis .............................................................................................9 1.2.2.1 Synthesis ....................................................................................................9 1.2.2.2 Release .....................................................................................................11 1.2.2.3 Degradation .............................................................................................12 1.2.3 Dopamine transport ................................................................................................14 1.2.3.1 Plasma membrane transport ....................................................................14 1.2.3.2 Vesicular membrane transport .................................................................15 1.2.4 Dopamine compartmentalization and its effects ....................................................17 1.2.4.1 Extracellular dopamine ............................................................................17 1.2.4.1.1 Dopamine Receptors............................................................... 17 v 1.2.4.2 Intracellular dopamine .............................................................................20 1.2.4.2.1 Cytosolic dopamine ................................................................ 20 Reactivity .................................................................. 21 Toxicity .................................................................... 24 1.2.5 Classical drugs that interact with the dopamine system .........................................28 1.2.5.1 Enzyme ligands .......................................................................................29 1.2.5.2 DAT ligands ............................................................................................30 1.2.5.3 VMAT2 ligands .......................................................................................33 1.2.5.4 Dopamine receptor ligands ......................................................................35