Role of the Prolyl Isomerase Pin1 in the Pathogenesis of Parkinson's
Total Page：16
File Type：pdf, Size：1020Kb
Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2012 Role of the prolyl isomerase Pin1 in the pathogenesis of Parkinson's disease and neuroprotection by novel targeted compounds in pre-clinical animal models of the disease Anamitra Ghosh Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Immunology and Infectious Disease Commons, Neuroscience and Neurobiology Commons, and the Toxicology Commons Recommended Citation Ghosh, Anamitra, "Role of the prolyl isomerase Pin1 in the pathogenesis of Parkinson's disease and neuroprotection by novel targeted compounds in pre-clinical animal models of the disease" (2012). Graduate Theses and Dissertations. 12327. https://lib.dr.iastate.edu/etd/12327 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]. Role of the prolyl isomerase Pin1 in the pathogenesis of Parkinson’s disease and neuroprotection by novel targeted compounds in pre-clinical animal models of the disease by Anamitra Ghosh A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Neuroscience Program of Study Committee: Anumantha G. Kanthasamy, Major Professor Ann Smiley-Oyen Arthi Kanthasamy Marian L. Kohut N. Matthew Ellinwood Iowa State University Ames, Iowa 2012 Copyright© Anamitra Ghosh, 2012. All rights reserved. ii TABLE OF CONTENTS ABSTRACT iv CHAPTER I. GENERAL INTRODUCTION Dissertation Organization 01 Introduction 01 Background and Literature Review I: Pin1- a new physiological substrate in human Diseases including cancers and neurodegenerative disorders 05 Background and Literature Review II: Neuroinflammation and oxidative stress in Parkinson’s disease 36 CHAPTER II. ACTIVATION OF NOVEL PROAPOPTOTIC PROTEIN PIN1 (PEPTIDYL-PROLYL CIS/TRANS ISOMERASE) IN CELL CULTURE AND ANIMAL MODEL OF PARKINSON’S DISEASE 58 Abstract 58 Introduction 59 Materials and Methods 61 Results 71 Discussion 82 References 87 CHAPTER III. PIN1 MODULATES ACTIVATION OF PROINFLAMMATORY TRANSCRIPTION FACTOR NF-ΚB IN NEUROINFLAMMATORY REACTIONS IN PARKINSON’S DISEASE 115 Abstract 115 Introduction 116 Materials and Methods 119 Results 125 Discussion 133 References 138 iii CHAPTER IV. ANTI-INFLAMMATORY AND NEUROPROTECTIVE EFFECTS OF A NOVEL COMPOUND DIAPOCYNIN IN A MURINE MODEL OF PARKINSON’S DISEASE 161 Abstract 161 Introduction 162 Materials and Methods 164 Results 169 Discussion 175 References 181 CHAPTER V. NOVEL COMPOUND MITO-APOCYNIN PROTECTS DOPAMINERGIC NEURONS VIA ATTENUATING OXIDATIVE STRESS AND INFLAMMATION IN ANIMAL MODEL OF PARKINSON’S DISEASE 205 Abstract 205 Introduction 206 Materials and Methods 208 Results 215 Discussion 224 References 229 CHAPTERVI. GENERAL CONCLUSION 255 References 261 ACKNOWLEDGEMENT 304 iv ABSTRACT Parkinson’s disease (PD) is a chronic and progressive neurodegenerative disorder named by the French neurologist Jean-Martin Charot, after the British physician James Parkinson who first described the disease as “Shaking Palsy”. Pathologically, PD is characterized by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc), a marked reduction of dopamine in the striatum, the presence of ubiquitin and α-synuclein positive cytoplasmic inclusions known as Lewy bodies, and depigmentation of the locus cereleus. The prevailing theory regarding processes that are likely to account for progressive degeneration of the dopaminergic neurons in the nigrostriatal axis involves on mitochondrial dysfunction, oxidative stress, excitotoxicity and neuroinflammation. Of those, neuroinflammation and oxidative stress have gained the most focus recently. Research over the last decade has provided extensive evidence that the sustained microglial and astroglial neuroinflammatory responses cause progressive and delayed dopaminergic neurodegeneration. However, the mechanisms by which glial cells activation and subsequent inflammation lead to dopaminergic neurodegeneration remain poorly understood. Development of effective therapeutic approaches to halt the disease progression of PD is of paramount importance. My objective in this Ph.D. thesis work was to characterize important signaling molecules activated in neuroinflammation mediated neurodegenerative pathways as well as characterze of novel compounds in a pre-clinical mouse model of PD. Protein interacting with never in mitosis – A (Pin1) is a peptidyl-prolyl isomerase, that specifically recognizes phosphorylated serine or threonine residues immediately preceding proline (pSer/Thr-Pro) in a subset of proteins in which it isomerizes the cis/trans conformation of the v peptide bond. High expression levels of Pin1 in terminally differentiated and post mitotic neurons suggest that it plays an important role in neurons, except in cell cycle regulation and proliferation. Recently, Pin1 overexpression was shown to facilitate formation of -synuclein inclusions in a cellular model of -synuclein aggregation. Pin1 was also localized in Lewy bodies in PD patients. But the level, activity, and role of Pin1 in the pathogenesis of PD are incompletely known. We hypothesized that Pin1 is differentially activated in neuronal and glial cells of the nigral dopaminergic system, and that it regulates NF-κB-mediated sustained neuroinflammatory processes in cell culture and in an animal model of Parkinson’s disease. Herein, we demonstrate for the first time that there is a dopaminergic neuron specific upregulation of Pin1 in human postmortem PD brain sections as well as in cell culture and animal models. We observed a rapid increase in Pin1 expression in both the 1-methyl- 4phenyl pyridinium (MPP+)-treated cell culture and in 1-methyl-4-phenyl-1-2-3-6- tetrahydroptridine MPTP treated mice. Also, Pin1 acts as an important pro-apoptotic factor in the selective degeneration of dopaminergic neurons. Importantly, pharmacological inhibition of Pin1 attenuates MPTP-induced Pin1 expression in vitro and in vivo and protects against MPTP-induced neurodegeneration in the nigrostriatal axis. We also demonstrate for the first time that, microglia and astrocytes express Pin1 and that there is a strong association between Pin1 and NF-κB p65 in BV2 microglial cells. Recent studies demonstrated that the promoter regions of proinflammatory molecules contain the DNA binding site for NF-κB. We have shown that Pin1 and NF-κB p65 inhibition by Juglone leads to attenuation of glial cells activation and subsequent reduction of proinflammatory reactions in cell culture and in an animal model of PD. While characterizing the role of Pin1 in the pathogenesis of PD, we also tested the efficacy of novel compounds for protection of dopaminergic neurons in a mouse vi model of PD. We demonstrate that the novel compound diapocynin, a metabolite of apocynin, blocks MPTP-induced activation of microglial and astroglial cells, thus inhibiting the inflammatory and oxidative stress processes in MPTP-treated mice. Diapocynin also protects the nigrostriatum against MPTP toxicity. The final chapter of this work characterizes the anti-inflammatory and neuroprotective properties of mito-apocynin, a mitochondria targeted compound in the MPTP mouse model of PD. Mitoapocynin prevents the behavioral imapariments and dopamine loss caused by MPTP-induced toxicity. We have shown that mito-apocynin protects the nigrostriatum by attenuating glial cell mediated neuroinflammation and oxidative stress. Collectively, the research described herein characterizes the novel and important roles of Pin1 in the neuroinflammation and pathophysiology of Parkinson’s disease, as well as establishes the efficacy of novel compounds in protection of the nigrostriatum in a pre-clinical mouse model of Parkinson’s disease. 1 CHAPTER I GENERAL INTRODUCTION Thesis Layout and Organization The alternative format was chosen for writing this dissertation as it consists of manuscripts that are either under peer review or being prepared for submission. The dissertation contains a general introduction, four research papers, a general conclusion that discusses the overall findings from all the chapters and acknowledgement. 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 General Introduction (Chapter I) briefly provides the current knowledge on Parkinson’s disease and describes an overview of the research objective. The Background and Literature Review I provide background information on Parkinson’s disease (PD), pathological features of PD, physiology and chemistry of dopamine, genetics of PD and apoptosis in PD, MPTP mouse models of PD and role transcription factor NF-κB in neurodegeneration. It also has the current knowledge of peptidyl prolyl cis/trans isomerase, Pin1 in cancers and neurodegenerative disorders, and provides an overview of the research objectives pertaining to chapters II and III. Background and Literature Review II summarize the current evidences implicating a pathogenic role for reactive gliosis in dopaminergic neurodegeneration, role of oxidative stress