The Effects of Exposure to the Environmental Neurotoxicant Manganese on Α-Synuclein and Its Cell-To-Cell Transmission Via Exoso
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Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2016 The effects of exposure to the environmental neurotoxicant manganese on α-synuclein and its cell-to-cell transmission via exosomes Dilshan Shanaka Gurunnaselage Don Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Neuroscience and Neurobiology Commons, and the Toxicology Commons Recommended Citation Gurunnaselage Don, Dilshan Shanaka, "The effects of exposure to the environmental neurotoxicant manganese on α-synuclein and its cell-to-cell transmission via exosomes" (2016). Graduate Theses and Dissertations. 15009. https://lib.dr.iastate.edu/etd/15009 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. The effects of exposure to the environmental neurotoxicant manganese on α-synuclein and its cell-to-cell transmission via exosomes by Dilshan Gurunnanselage Don A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Toxicology Program of Study Committee: Dr. Anumantha Kanthasamy, Major Professor Dr. Arthi Kanthasamy Dr. Wilson Rumbeiha Dr. Stephanie Hansen Dr. Sanjeevi Sivasankar Iowa State University Ames, Iowa 2016 Copyright © Dilshan Gurunnanselage Don, 2016. All rights reserved. ii To my father G.D. Harischandra and mother Priyani Jayamanna, thank you for allowing me to follow my ambitions throughout my childhood iii TABLE OF CONTENTS ABSTRACT v CHAPTER I. GENERAL INTRODUCTION 1 Dissertation Organization 1 Introduction 3 Background and Literature Review I 8 Manganese; its role and significance in Parkinsonism Spectrum Disorders 8 Background and Literature Review II 64 Exosomes: Nanoscale dumpsters or delivery trucks? 64 CHAPTER II α-SYNUCLEIN PROTECTS AGAINST MANGANESE NEUROTOXIC INSULT DURING THE EARLY STAGES OF EXPOSURE IN A DOPAMINERGIC CELL MODEL OF PARKINSON’S DISEASE 81 Abstract 82 Introduction 83 Materials and Methods 86 Results 96 Discussion 104 References 110 Figures 115 CHAPTER III. THE ENVIRONMENTAL NEUROTOXICANT MANGANESE PROMOTES PRION-LIKE CELL-TO-CELL TRANSMISSION OF α- SYNUCLEIN VIA EXOSOMES IN CELL CULTURE AND ANIMAL MODELS OF PARKINSONISM 126 Abstract 127 Introduction 128 Materials and Methods 131 iv Results 144 Discussion 163 References 172 Figures 182 CHAPTER IV. ENVIRONMENTAL NEUROTOXICANT MANGANESE INCREASES EXOSOME-MEDIATED MIRNA DELIVARY AND AUTOPHAGIC REGULATION IN CELL CULTURE MODEL OF PARKINSON’S DISEASE. 196 Abstract 197 Introduction 198 Materials and Methods 202 Results 208 Discussion 211 References 214 Figures 218 CHAPTER V. GENERAL CONCLUSIONS 221 References 230 APPENDIX I. MANGANESE AND PRION DISEASE 231 APPENDIX II. ROLE OF PROTEOLYTIC ACTIVATION OF PROTEIN KINASE Cδ IN THE PATHOGENESIS OF PRION DISEASE 280 APPENDIX III. ANTIOXIDANTS AND REDOX-BASED THERAPEUTICS IN PARKINSON’S DISEASE 327 ACKNOWLEDGMENTS 350 v ABSTRACT Parkinson’s disease (PD) is a chronic multifaceted neurodegenerative condition caused by a complex interplay of genetic and environmental factors that affects about 1% of people over the age of 60. Although the progressive loss of dopaminergic neurons and accumulation of aggregated α-Synuclein (αSyn) protein in Lewy bodies are considered key pathophysiological features of the disease, the physiological function of αSyn and the molecular mechanisms leading to protein aggregation and propagation remain unknown. Manganese (Mn) is considered a key inhaled pollutant implicated in environmentally- linked PD as evidenced by epidemiological studies done on humans exposed to Mn during mining, welding metals, and dry battery manufacturing. However, the exact molecular mechanisms underlying Mn-induced protein aggregation are not well understood. Considering the role of the divalent metal Mn in PD-like neurological disorders, we conducted a comprehensive characterization of the role of αSyn in Mn- induced dopaminergic neurotoxicity, cell-to-cell spreading of αSyn protein aggregates and aberrant miRNA delivery via exosomes. Using an αSyn-expressing dopaminergic cell model, we show that wildtype αSyn significantly attenuates Mn-induced neurotoxicity during the early stages of exposure while prolonged Mn exposure promotes αSyn aggregation and dampens its neuroprotective effect. Our subsequent studies show that upon Mn exposure, misfolded αSyn-containing exosomes are released to its extracellular milieu, which may in turn induce neuroinflammatory, and neurodegenerative responses in cell culture and animal models of PD. We also found enhanced accumulation of misfolded αSyn species in serum exosomes of welders exposed to Mn indicating the possibility of using exosomes as biomarkers of Mn- vi neurotoxicity. To further elucidate the regulatory role of exosomes in Mn-induced miRNA dysregulation, we performed next-generation miRNA sequencing and identified multiple differentially expressed miRNAs in Mn-stimulated exosomes in contrast to control exosomes. Herein, our results suggest that Mn induces a novel mechanism of cell injury through modulating the protein and miRNA cargo in exosomes and altering gene expression. This may contribute to the cell-to-cell transmission of the aggregated αSyn protein and progression on neurodegeneration. 1 CHAPTER I: GENERAL INTRODUCTION Dissertation Organization The alternative format was chosen for this thesis and consists of manuscripts that have been published, or are being prepared for submission. The dissertation contains a general introduction, two book chapters, three research papers and a conclusions/future directions section that briefly discusses the overall findings from all chapters. The references for each manuscript chapter are listed at the end of that specific section. References pertaining to the background and literature review as well as those used in general conclusion section are listed at the end of the dissertation. The introduction section under Chapter 1 provides a brief background and overview of Parkinson’s disease (PD), manganese (Mn) neurotoxicity and exosomes which this thesis is heavily based upon. The Background and Literature Review-I section covers in depth review on the recent advances on elucidating cellular and molecular mechanisms of Mn-induced protein misfolding, mitochondrial dysfunction and consequent neuroinflammation and neurodegeneration; this section will be submitted to Pharmacology and Therapeutics for publication. In the background and literature review II section, authors discuss and summarize current literature on exosome composition, biogenesis and their potential function as intercellular messengers in cell-to-cell transmission of proteins and small- RNA species. The manuscript from Chapter 2 was recently published in the Toxicological Science. It demonstrates a potential neuroprotective role of wild-type α-Syn against Mn- induced neurotoxicity during the early stages of exposure in a dopaminergic neuronal 2 model of PD. Chapter 3 explores how aggregated -synuclein, the major component of PD-associated Lewy bodies and a gene linked to the development of familial PD, can transfer from one to another through exosomes leading to inflammation and neurodegeneration in experimental models on Mn-toxicity. Chapter 4 studies how exosomes contributes to cell-to-cell transmission of miRNAs and thereby manipulate recipient cell gene expression. Chapters 3 and 4 are in the process of being submitted for publication. This dissertation also contains three appendix sections. Appendix I is a book chapter that was recently published in Manganese in Health and Disease summerizeing role of manganese in Prion disease. Appendix II is published in the Jornal Prion evaluating the role of oxidative stress-sensitive, pro-apoptotic protein kinase Cδ (PKCδ) in prion-induced neuronal cell death using cerebellar organotypic slice cultures (COSC) and mouse models of prion diseases. Appendix III is a book chapter that was recently published in the Inflammation, Oxidative Stress and Age-Related Disease summarizing the recent discoveries of antioxidant therapeutics, including mitochondria-targeted antioxidants for modulating oxidative damage in PD and its potential as a possible treatment for PD. This dissertation contains the experimental data and results obtained by the author during his Ph.D. study under the supervision of his major professor Dr. Anumantha G. Kanthasamy at Iowa State University, Department of Biomedical Sciences. 3 Introduction Parkinson’s disease Parkinson’s disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s disease, and is the most common movement disorder in people over the age of 65. PD also recognized as one of the most common neurologic disorders, affecting approximately 1% of individuals older than 60 years causing progressive disability characterized by severe motor symptoms including uncontrollable tremor, postural imbalance, slowness of movement and rigidity (Lotharius and Brundin, 2002). Pathologically, this disease is characterized by a progressive degeneration of dopaminergic neurons projecting from the substantia nigra pars compacta (SNpc) to the striatum resulting in pronounced loss of neurotransmitter dopamine resulting above