ROLE OF Ca2+ AND SODIUM-CALCIUM EXCHANGER (NCX) ON CELLULAR / MITOCHONDRIAL UPTAKE AND TOXICITY OF THE PARKINSONIAN TOXIN, MPP+ A Dissertation by Mapa Mudiyanselage Sumudu Tharangani Mapa Master of Science, Wichita State University, 2016 Master of Philosophy, University of Kelaniya, Sri Lanka, 2009 Bachelor of Science, University of Kelaniya, Sri Lanka, 2007 Submitted to the Department of Chemistry and the faculty of the Graduate School of Wichita State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy December 2016 i © Copyright 2016 by Mapa S. T. Mapa All Rights Reserved ii ROLE OF Ca2+ AND SODIUM-CALCIUM EXCHANGER (NCX) ON THE CELLULAR / MITOCHONDRIAL UPTAKE AND TOXICITY OF PARKINSONIAN TOXIN, MPP+ The following faculty members have examined the final copy of this dissertation for form and content, and recommend that it be accepted in partial fulfillment of the requirement for the degree of Doctor of Philosophy with a major in Chemistry. _____________________________________________________ Kandatege Wimalasena, Committee Chair _____________________________________________________ David Eichhorn, Committee Member _____________________________________________________ James G. Bann, Committee Member _____________________________________________________ Moriah Beck, Committee Member _____________________________________________________ George Bousfield, Committee Member Accepted for the College of Liberal Arts and Sciences _____________________________________________ Ron Matson, Dean Accepted for the Graduate School _____________________________________________ Dennis Livesay, Dean iii DEDICATION To my loving Parents & Husband iv ACKNOWLEDGEMENTS I would like to offer my sincerest gratitude to my supervisor Prof. K. Wimalasena, for his guidance and support throughout my research work in the graduate school. He encouraged me to develop my independent and critical thinking and research skills and greatly assisted me with scientific writing. I am grateful for the opportunity to work under his supervision and without his enthusiasm and persistent help this dissertation would not have been possible. I am also very grateful to express my sincere appreciation to the members of dissertation committee, Profs. David Eichhorn, James Bann, Moriah Beck and George Bousfield for serving in my committee and for their support and advice. My deepest gratitude goes to Dr. Shyamali Wimalasena for continuous support throughout my graduate studies and exhaustively proofreading of the manuscripts and this dissertation. My sincere thanks go to Dr. Yamuna Kollalpitiya, Dr. Viet Le, and Dr. Chamila Kadigamuwa for their help and support in my research works. I would like to thank my dad, as well as my brothers for their encouragement during this period. Specially, I want to express my loving gratitude to my husband, Chamila for his endless support, patience and love during this time of endeavor and throughout my life. Finally I would like to thank to the faculty and staff of the WSU Chemistry department and friends for assisting me in numerous ways. v ABSTRACT Although the exact cause(s) of dopaminergic cell death in Parkinson's disease (PD) is not fully understood, the discovery that 1-methyl-4-phenylpyridinium (MPP+) selectively destroys dopaminergic neurons and causes PD symptoms in humans and other mammals has strengthened the environmental hypothesis of PD. The current model for the toxicity of MPP+ is centered on its specific uptake into dopaminergic cells through the dopamine transporter (DAT), electrogenic accumulation into the mitochondria, inhibition of the mitochondrial complex I, leading to ATP depletion, increased reactive oxygen species (ROS) production, and apoptotic cell death. However, MPP+ is taken up into many cell types through a number of other transporters, challenging the major hypothesis of this model, that the specific dopaminergic toxicity of MPP+ is due to the specific uptake through DAT. In order to address this discrepancy, we have characterized a series of 4'-substituted MPP+ derivatives with varying hydrophilicities. The photophysical studies of these derivatives show that 4'I-MPP+ is fluorescent and can be used to localize the intracellular distribution of MPP+. Using these novel probes, we have shown that both cellular and mitochondrial uptake of MPP+ are modulated by the cell and mitochondrial membrane potentials and found that Ca2+ play a key role in these MPP+ uptakes and toxicities. Furthermore, the effects of Ca2+ on MPP+ uptake and toxicity are mediated through mitochondrial and plasma membrane sodium calcium-exchangers (NCX) and the mitochondrial permeability transition pore (PTP). For the first time, we show that the specific mitochondrial NCX (mNCX) inhibitor, CGP37157 inhibits mitochondrial uptake of MPP+ and protects dopaminergic MN9D cells from MPP+ toxicity. Thus, these findings could be further exploited for development of pharmacological agents to protect central nervous system (CNS) dopaminergic neurons from PD-causing environmental toxins. vi TABLE OF CONTENTS Chapter Page 1 INTRODUCTION . 1 2 BACKGROUND AND SIGNIFICANCE . 5 2.1 Neurologic Disorders . 5 2.2 Neurodegenerative Diseases . 5 2.3 Parkinson's Disease (PD). 6 2.3.1 Genetic Factors of PD. 7 2.3.2 Environmental Factors of PD . 8 2.3.2.1 MPTP/MPP+ Model . 8 2.3.2.2 Rotenone . 11 2.4 MPP+ Uptake Mechanism(s) and Transporters . 12 2.4.1 Dopamine Transporter (DAT). 12 2.4.2 Norepinephrine Transporter (NET) . 13 2.4.3 Organic Cation Transporter (OCT) . 14 2.4.4 Plasma Monoamine Transporter (PMAT) . 14 2.4.5 Vesicular Monoamine Transporter (VMAT) . 15 2.5 Ca2+ Homeostasis . 16 2.5.1 Intracellular Ca2+ Stores . 17 2.5.2 Voltage Gated Ca2+ Channels (VGCC) . 19 2.5.3 Ligand Gated Ca2+ Channels (LGCC) . 19 2.5.4 Na+/Ca2+ Exchanger (NCX) . 20 2.5.5 Mitochondrial Ca2+ Uniporter (MCU). 22 2.5.6 Mitochondrial Permeability Transition Pore (PTP) . 22 2.5.7 Transient Receptor Potential (TRP) Channels. 23 2.5.8 Calcium Channel Inhibitors . 23 2.6 Mechanism(s) of MPP+ Toxicity . 24 2.6.1 Mitochondrial Membrane Depolarization . 25 2.6.2 Mitochondrial Complex I Inhibition . 25 2.6.3 Reactive Oxygen Species (ROS) Production . 26 2.6.4 Apoptosis . 28 2.7 Cell Culture . 29 2.7.1 MN9D Cells . 30 2.7.2 SH-SY5Y Cells . 30 2.7.3 HEK293 Cells . 31 2.7.4 HepG2 Cells . 31 vii TABLE OF CONTENTS (continued) Chapter Page 3 EXPERIMENTAL METHODS . 32 3.1 Materials . 32 3.2 Instrumentation . 33 3.3 Methods . 34 3.3.1 Synthesis of 3'Hydroxy MPP+ Derivative and 4'Halogenated MPP+ Derivatives. 34 3.3.2 Cell Culture . 35 3.3.3 Differentiation of MN9D Cells . 35 3.3.4 Measurement of Cell Viability . 35 3.3.5 Measurement of Cellular Uptake of MPP+ or MPP+ Derivatives . 36 3.3.6 MPP+ or MPP+ Derivative Uptake Into MN9D Mitochondria . 37 3.3.7 Measurement of the Mitochondrial Membrane Potential . 38 3.3.8 Mitochondrial Localization of 4'I-MPP+ . 38 3.3.9 Effect of FCCP and 85 mM K+ Ions on the Cellular Uptake of MPP+ and MPP+ Derivatives . 38 3.3.10 Measurement of the Mitochondrial Ca2+ Level . 39 3.3.11 NCX Transfection . 40 3.3.11.1 Determination of the NCX Expression Levels in Transfected Cells Using Western Blot . 41 3.3.12 Rat Brain Mitochondria Isolation . 42 3.3.13 Mitochondrial Complex I inhibition Studies . 42 3.3.14 Measurement of Intracellular ATP Levels . 43 3.3.15 Measurement of Reactive Oxygen Species (ROS) . 43 3.3.16 Chromatin Condensation Test for Apoptosis . 44 3.3.17 Protein Determination . 44 3.3.18 Data Analyses . 45 3.3.19 Technical Statement . 45 4 RESULTS . 46 4.1 Photophysical Properties of MPP+ Derivatives and Intracellular Accumulation of 4'I-MPP+ . 46 4.2 Relative Dopaminergic Cell Toxicities of 4'-Substituted MPP+ Derivatives . 49 4.3 Characteristics of the Cellular Uptake of 4'-Substituted MPP+ Derivatives . 52 4.4 Mitochondrial Uptake of 4'-Substituted MPP+ Derivatives . 55 4.5 Intracellular 4'I-MPP+ Localized into the Mitochondria . 57 4.6 Membrane Potentials Drive Active Cellular and Mitochondrial Accumulation of MPP+ and its 4'-Substituted Derivatives . 58 viii TABLE OF CONTENTS (continued) Chapter Page 4.7 The Effect of Extracellular Ca2+ on Cellular Uptake of MPP+ . 61 4.8 The Effect of Ca2+ on Mitochondrial Accumulation of MPP+ . 63 4.8.1 The Effect of Extra-Mitochondrial (Cytosolic) Ca2+ and Na+ on Mitochondrial Accumulation of MPP+ . 64 4.8.2 The Effect of Ca2+ Channel Blockers on Mitochondrial Accumulation of MPP+ . 65 4.8.2.1 The Effect of CGP37157 and KBR7943 on Mitochondrial Membrane Potential With 4'I-MPP+ Uptake . 67 4.8.2.2 CGP37157 Protect MN9D Cells From MPP+ and 4'I-MPP+ Toxicity . 69 4.9 Does mNCX Play a Role in Mitochondrial Accumulation and Toxicity of MPP+? . 69 4.9.1 NCX Transfection into HEK293 Cells Increases 4'I-MPP+ Uptake and Toxicity . 70 4.9.2 NCX Transfection into HEK293 Cells Depolarizes Mitochondrial Membrane Potential by 4'I-MPP+ Uptake . 72 4.10 The Effect of Mitochondrial PTP Inhibitor on MPP+ Cellular Uptake . 72 4.11 The Effect of MPP+ on Mitochondrial Complex I . 74 4.12 The Effect of MPP+ on ATP Depletion . 76 4.13 MPP+ and Its 4'-Substituted Derivatives Increase Intracellular ROS Production Specially in MN9D Cells . 77 4.14 MPP+ Cause Specific Apoptotic MN9D Cell Death . 79 5 DISCUSSION . 81 6 CONCLUSIONS . 89 7 REFERENCES . 91 ix LIST OF TABLES Table Page 1 Major Types of Neurological Disorders . 5 2+ 2 Ca Channel Inhibitors and Functions . 24 3 Comparison of Relative Antioxidant Enzyme Levels in Various Cell Types . 28 + 4 Retention Times For MPP Derivatives . 46 + 5 UV-Vis Analysis of MPP Derivatives . 47 + 6 IC50 Values of MPP Derivatives in MN9D Cells . 52 7 FCCP and 85 mM K+ Percent Inhibition of MPP+ Derivatives in the Presence and Absence of Extracellular Ca2+ in MN9D Cells . 59 x LIST OF FIGURES Figure Page 1 Proposed Mechanisms of MPTP/MPP+ Uptake and Toxicity . 11 2 Calcium Uptake and Extrusion Mechanisms in Mitochondria .