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Wayne State University Wayne State University Dissertations 1-1-2013 Progress Towards Understanding Of Mechanisms Of Action Of Potent Multifunctional Disease Modifying Therapeutics For Parkinson's Disease & Investigating The ethM amphetamine-Induced Striatal Microglia Activation. Mrudang M. Shah Wayne State University, Follow this and additional works at: http://digitalcommons.wayne.edu/oa_dissertations Part of the Medicinal Chemistry and Pharmaceutics Commons, Neurosciences Commons, and the Pharmacology Commons Recommended Citation Shah, Mrudang M., "Progress Towards Understanding Of Mechanisms Of Action Of Potent Multifunctional Disease Modifying Therapeutics For Parkinson's Disease & Investigating The eM thamphetamine-Induced Striatal Microglia Activation." (2013). Wayne State University Dissertations. Paper 916. This Open Access Dissertation is brought to you for free and open access by DigitalCommons@WayneState. It has been accepted for inclusion in Wayne State University Dissertations by an authorized administrator of DigitalCommons@WayneState. PROGRESS TOWARDS UNDERSTANDING OF MECHANISMS OF ACTION OF POTENT MULTIFUNCTIONAL DISEASE MODIFYING THERAPEUTICS FOR PARKINSON’S DISEASE & INVESTIGATING THE METHAMPHETAMINE-INDUCED STRIATAL MICROGLIA ACTIVATION by MRUDANG MANOJKUMAR SHAH DISSERTATION Submitted to the Graduate School Of Wayne State University, Detroit, Michigan in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY 2014 MAJOR: PHARMACEUTICAL SCIENCES Approved by: _____________________________________ Advisor Date _____________________________________ _____________________________________ _____________________________________ _____________________________________ DEDICATION This dissertation is dedicated to two most important persons in my life, my wife, Nirali Shah and my sister, Shivani Shah. It would not have been possible to reach this milestone of my career without proper guidance, support and motivation from my parents, Manoj Shah and Dharini Shah. ii ACKNOWLEDGEMENTS First of all, I would like to acknowledge my previous mentor Dr. David Thomas, who provided me an opportunity to delve into the field of neuroscience research. The independence provided by him was phenomenal and the most important factor that helped me develop broad knowledge of analytical skills during my tenure as a graduate student. I can’t thank my current mentor Dr. Aloke Dutta enough for all his support during critical stage of my graduate studies. Dr. Aloke Dutta has been the most important person for providing me a meaningful insight to pursue research with maximum accuracy, efficiency and enthusiasm. Dr. Aloke Dutta will always be my inspiration for approaching difficult problems with critical thinking and unique approach. I would also like to extend my appreciation for all the help and guidance provided by Dr. Donald Kuhn. He has been always very inspirational and encouraging throughout my journey as a graduate student. I would like to thank all my committee members, Dr. Randall Commissaris, Dr. Anna Moszczynska, and Dr. Paul Stemmer for their continuous support and availability to discuss critical issues related to my research project. I would especially like to thank my colleagues Ms. Liping Xu and Dr. Chandra Shekhar Voshavar for their unconditional support and guidance throughout my tenure in Dr. Aloke Dutta’s lab. The relationship established by them has made me believe them a part of my academic family. I would also like to thank my other colleagues Dr. Bhaskar Gopishetty, Dr. Mark Johnson, Mr. Gyan Prakash Modi, Dr. Horrick Sharma, Dr. Banibrata Das, Dr. Seenuvasan Vedachalam, Ms. Dan Liu, Ms. Asma Elmabruk, Mr. Fahd Dholkawala from Dr. Aloke Dutta’s iii lab, Dr. Abiy Moussa Mohammad from Dr. David Thomas’s lab, Dr. Mariana Angoa-Pérez and Ms. Dina Francescutti-Verbeem from Dr. Donald Kuhn’s lab. I am also grateful to all the collaborators who provided me broader perspective of neuroscience research. I would really like to thank Dr. Deepak Bhalla and Dr. Shane Perrine for providing me an additional angle to view neuroscience research. I really appreciate the help and extra-ordinary efforts of Wayne State Proteomics Core and Wayne State Flow Cytometry Core, who helped me carry out many important experiments smoothly. I would particularly like to thank Ms. Namhee Shin, Dr. Paul Stemmer, Dr. Jessica Back, and Mr. Eric Van Buren for making my proteomics based and flow cytometry related experiments possible. I am really appreciative of Dr. Timothy Stemmler for providing us an ability to avail FPLC system for protein purification experiments. I would like to especially thank Ms. Lindsey Nico for her help with FPLC troubleshooting. My journey would not have been enjoyable and exciting without the support, inspiration and encouragement from my friends and colleagues. I would really like to thank Dr. Rohan Uttarwar, Dr. Amit Wani, Mr. Hardik Doshi, Mr. Anand Gupta, Mr. Manit Shah, Dr. Venkat Nadithe, Dr. Roberta Traini, Dr. Bhavaani Jayaram, Ms. Sunitha Ravichandran, Ms. Anuja Vedapathak, Mrs. Sahana Nagaraja, Mrs. Kena Shah, and Ms. Heli Chauhan for all their support and love throughout my graduate studies. Last but not the least; I would like to thank the department of Pharmaceutical Sciences for providing me an excellent opportunity to blossom into an experienced researcher from a novice student. I would like to extend special thanks to the departmental chair, Dr. George Corcoran for helping me out throughout my tenure as a graduate student. It would not have been iv possible to develop into researcher without appropriate guidance from graduate student directors Dr. David Oupický and Dr. Steven Firestine. v TABLE OF CONTENTS Dedication……………………………………………………………………………………….. ii Acknowledgements………………………………………………………………………….…...iii List of Tables……………………………………………………………………………………viii List of Figures…………..………………………………………………………………………...ix List of Abbreviations…………………………………………………………………………....xiv PROGRESS TOWARDS UNDERSTANDING OF MECHANISMS OF ACTION OF POTENT MULTIFUNCTIONAL DISEASE MODIFYING THERAPEUTICS FOR PARKINSON’S DISEASE Chapter 1: Introduction…………………………………………………………………………....1 Chapter 2: Hypotheses, rationale and study design……………………………………………...35 Chapter 3: Materials and Methods……………………………………………………………….47 Chapter 4: D-512 confers significant neuroprotection in PC12 and MN9D cell-lines………………………………………………………..70 Chapter 5: D-240, D-436, and D-520 significantly modify aggregation kinetics of α-synuclein………………………………………………….95 INVESTIGATING THE METHAMPHETAMINE-INDUCED STRIATAL MICROGLIA ACTIVATION Chapter 6: Introduction…………………………………………………………………………128 Chapter 7: Hypotheses, rationale and study design…………………………………………….157 Chapter 8: Materials and Methods…...…………………………………………………………163 vi Chapter 9: Significant protein expression changes are induced by Methamphetamine in cell-culture and microglia isolated from animals……………………………………………………………….173 References………………………………………………………………………………………202 Abstract…………………………………………………………………………………………230 Autobiographical Statement…………………………………………………………………….234 vii LIST OF TABLES Table 1-1: Evidence of oxidative stress in Parkinson’s disease…………………………………16 Table 1-2: Loci, genes and susceptibility factors involved in Parkinson’s disease……………...25 Table 1-3: Current Parkinson’s disease therapy……………………………………………........32 Table 3-1: Gradient elution protocol for purification of α-synuclein using Q-sepharose HP column…………..…………………………………………………69 viii LIST OF FIGURES Figure 1-1: Neuropathology of Parkinson’s disease….…………………………………………...4 Figure 1-2: Immunostained Lewy bodies in SNPc dopaminergic neurons of Parkinson’s disease…………………………………………………………………...6 Figure 1-3: Substantia nigra from Parkinson’s disease patient stained for α-synuclein………………………………………………………………………..7 Figure 1-4: Spread of idiopathic Parkinson’s disease pathology………………………………….8 Figure 1-5: Dopamine metabolism by enzymatic reaction and autooxidation…………………..10 Figure 1-6: Iron(III) catalyzed reactions…………………………………………………...........11 Figure 1-7: Mitochondrial Electron Transport System (ETS)…………………………………...13 Figure 1-8: Lipid peroxidation process in presence of free radicals……………………………..15 Figure 1-9: Products of guanine oxidative damage via the intermediate C8-OH adduct radical ……………………………………………………………...16 Figure 1-10: Carbonylation of protein amino acid side-chain…………………………………...17 Figure 1-11: Glutathione synthesis and metabolism in the central nervous system……………..20 Figure 1-12: Representation of intracellular pathways of MPP+ toxicity……………………….22 Figure 1-13: Structural similarity between MPP+ and Paraquat………………………………...23 Figure 1-14: Aggregation and functional domains of α-synuclein………………………………27 Figure 1-15: Prion-like mechanism of α-synuclein transfer…………………………………….28 Figure 1-16: Mode of action of current Parkinson’s disease therapeutics………………………31 Figure 2-1: Design of potential multifunctional compounds for Parkinson’s disease…………..36 Figure 2-2: Structures of known α-synuclein aggregation inhibitors and some lead compounds from our lab…………………………………………………………….38 Figure 2-3: Imbalance in reactive oxygen species generation and elimination………………….39 Figure 4-1: MTT assay 6-OHDA dose dependence…………………………………………….70 ix Figure 4-2: MTT assay D-512 dose dependence……………….………………………………..71 Figure 4-3: Neuroprotective effect of D-512 pre-treatment and co-treatment on 6-OHDA induced cell death in PC12 cells………………………………………72 Figure 4-4: Neuroprotective effect of D-512 pre-treatment on 6-OHDA induced cell death in PC12 cells……….…………………………………72