MOLECULAR AND GENETIC ANALYSIS OF PARKIN IN MICROGLIAL ACTIVATION AND INFLAMMATION-RELATED NEURODEGENERATION APPROVED BY SUPERVISORY COMMITTEE Malú Tansey, Ph.D. Matthew S. Goldberg, Ph.D. Zhijian Chen, Ph.D. David Farrar, Ph.D. Gang Yu, Ph.D. DEDICATION This is dedicated to my family for their love and support and to my husband (to be) Andy. MOLECULAR AND GENETIC ANALYSIS OF PARKIN IN MICROGLIAL ACTIVATION AND INFLAMMATION-RELATED NEURODEGENERATION by THI ANH TRAN DISSERTATION Presented to the Faculty of the Graduate School of Biomedical Sciences The University of Texas Southwestern Medical Center at Dallas In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY The University of Texas Southwestern Medical Center at Dallas Dallas, Texas March, 2010 Copyright by THI ANH TRAN, 2010 All Rights Reserved MOLECULAR AND GENETIC ANALYSIS OF PARKIN IN MICROGLIAL ACTIVATION AND INFLAMMATION-RELATED NEURODEGENERATION THI ANH TRAN The University of Texas Southwestern Medical Center at Dallas, 2010 MALU TANSEY, Ph.D. Parkinson’s disease (PD) is a progressive, neurodegenerative disease characterized by the loss of dopaminergic (DA) neurons in the substantia nigra (SN). Genetic mutations account for only 5-10% of PD cases. Oxidative stress and inflammation have both been linked to sporadic PD. Inflammation-induced injury to dopaminergic neurons can be significantly attenuated by impairment of microglial activation. In addition, previous studies from our lab reported that parkin-/- mice are more susceptible to inflammation- induced degeneration of nigral DA neurons. Therefore, inflammatory responses are a critical determinant of DA neuronal survival. v Microglia support neuronal survival by providing trophic factors and phagocytosing debris. However, with chronic inflammation glia release chemical mediators which are toxic to surrounding neurons. Our data provide evidence that Parkin is a negative regulator of microglial activation. parkin-/- mice display increased cytokine expression in the midbrain and increased cytokines in the serum suggesting parkin-/- mice are basally inflamed. Parkin loss-of-function mutations are linked to autosomal recessive PD. The parkin gene encodes an E3 ubiquitin ligase linked to mitochondrial dysfunction. Most studies on Parkin concentrate on its role in neurons, however, we hypothesize that Parkin function in microglial activation and inflammatory signaling also affect DA neuron survival. Our biochemical analyses of primary wild type microglia show Parkin expression is negatively regulated by inflammatory stimuli. Pharmacological or genetic inhibition of NF-κB, a transcription factor activated by inflammatory stimulation, blocks the inflammation-induced decrease in Parkin levels. Additionally, our data suggests that NF-κB may bind the parkin promoter, further implicating Parkin function in the inflammatory activation pathway. These novel findings suggest that in a normal cell experiencing inflammation, the decreased expression of Parkin, which has been shown to antagonize apoptotic signaling cascades, may render the cell more susceptible to death. Additionally, sources of inflammation including environmental triggers, infection, or traumatic injury could cause a normal individual to have the same susceptibility to PD as an individual with an inherited mutation, because inflammation leads to Parkin loss of function. vi ACKNOWLEDGEMENTS During these brief years at UT Southwestern, I have learned and grown much as a scientist and as a person. I am indebted to family, friends, and peers for their support, patience and stimulating conversations about biology and life in general. First, I would like thank my mentor Malú Tansey for finding a place for me in her lab when I was struggling, and for believing in my potential. I am grateful that she provided an environment to arm me with the technical skills, critical eye and the confidence that I would need to succeed in this field. I would not have finished with a smile still on my face without her encouragement when the science was not working quite right, her beatings with the wet noodle to remind me of the caliber of excellence that was expected, and her overall enthusiasm. I would also like to thank my committee members Dr. David Farrar, Dr. James Chen, Dr. Gang Yu, and my chair Dr. Matt Goldberg for taking the time to discuss projects and share their expertise, as well as offer support and reagents to see those projects to completion. I would also like to thank Dr. Melanie Cobb, Dean of the Graduate School, and Nancy McKinney for keeping me on track, and for their sincere concern in my development. Special thanks go out to Tansey lab members past and present for being helpful and making the lab environment so enjoyable. I am grateful for Dr. Jae Lee and Dr. Laura Alto for their patience in teaching me techniques, their valuable input on my projects, and advice on how to survive graduate school. I would also like to thank the previous graduate students Dr. Phil Wrage, Dr. Melissa McCoy and Dr. Fiona McAlpine for vii paving the way in the lab, and especially Terina Martinez and Ashley Harms for their friendship and support as we journeyed together, and their willingness to make chocolate runs when the road was rough. Special thanks to our lab manager Kelly Ruhn for not letting me crowd the freezer, and teaching me about mice, soccer and how not to be so type A. I am grateful for my friends who helped me balance work and play by sprinkling in art and music. Thank you to my former roommates for leaving the light on when I would work late, and sharing cold pizza and warm cups of coffee. Grad school would not have been the same without my graduate school classmates and peers, so thanks for all the camaraderie and karaoke. Thank you most especially to my grad school peer turned significant other, Andy, for inspiring me not only by his incredible work ethic and intellect, but by his infinite patience through this writing process. Finally, I am here because of the unwavering love of my parents Tu and Anne, my brothers Kiet and Hieu, and my incredible aunts, uncles and cousins. Thanks Mom for packing me dinners and sending me sunshine in all the ways that you did. Thanks Dad for cultivating my curiosity in life and fostering an attitude in which I need not fear the unknown. viii TABLE OF CONTENTS DEDICATION .................................................................................................................. ii ABSTRACT ...................................................................................................................... v ACKNOWLEDGEMENTS ............................................................................................ vii TABLE OF CONTENTS ................................................................................................. ix PRIOR PUBLICATIONS .............................................................................................. xiii LIST OF FIGURES ....................................................................................................... xiv LIST OF APPENDICES ............................................................................................... xvii LIST OF DEFINITIONS ............................................................................................. xviii CHAPTER I: GENERAL INTRODUCTION .................................................................. 1 A. Parkinson’s Disease ....................................................................................... 1 a. Clinical Features and Pathophysiology ................................................. 1 b. Etiology – Genetics ................................................................................ 2 c. Etiology – Infection ................................................................................ 3 d. Etiology – Mitochondrial Toxins ............................................................ 5 e. PD-Associated Genes and Biological Function .................................. 6 f. Protein Aggregation and Degradation................................................. 6 g. Protein Oxidation.................................................................................. 7 h. PD-Associated Genes and Microglia Function................................... 8 B. Microglia ....................................................................................................... 10 a. The Brain-Resident Macrophage ....................................................... 10 b. Biological Function............................................................................. 11 ix c. Microglial activation........................................................................... 13 C. Neuroinflammation ....................................................................................... 14 a. Microglia Role in Neuroinflammation............................................... 14 b. Chronic Neuroinflammation............................................................... 14 c. Neuroinflammation in PD................................................................... 15 D. Parkin ............................................................................................................ 17 a. Historical Perspective......................................................................... 16 b. Parkin Structure and Function........................................................... 17 c. Post-Translational Modification ........................................................ 18 d. Putative Substrates.............................................................................