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Role of ABCA5 in the Pathogenesis of Parkinson's Disease

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Role of ABCA5 in the Pathogenesis of Parkinson's Disease Role of ABCA5 in the Pathogenesis of Parkinson’s disease Lisa Mak A thesis in total fulfilment of the requirements for the degree of Masters by Research School of Medical Science Faculty of Medicine August 2013 ORIGINALITY STATEMENT „I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.‟ Signed …………………………………………….............. Date …………………………………………….............. TABLE OF CONTENT PAGE Originality statement I Acknowledgement II Abbreviations III Abstract V 1. INTRODUCTION 1 1.1 Parkinson‟s disease 1 1.2 Brain Lipids 3 1.2.1 Importance of lipids in the brain 3 1.2.2 Lipids and Parkinson‟s disease 4 1.3 ATP-binding cassette (ABC) transporters 5 1.3.1 ABCA subfamily 6 1.3.2 ABCA transporter and neurodegeneration 12 1.4 ABCA5 12 1.4.1 ABCA5 expression 13 1.4.2 Function of ABCA5 13 1.4.3 Regulation of ABCA5 15 1.5 Hypothesis and aims of project 16 2. MATERIALS AND METHODS 17 2.1 Materials 18 2.2 Cell culture methods 20 2.2.1 Thawing cell lines from liquid nitrogen stock 20 2.2.2 Maintaining and culturing cells 20 2.2.3 Transfection 20 2.3 Genetic techniques 21 2.3.1 RNA extraction from cells and brain tissues 21 2.3.1.1 RNA extraction from cells 21 2.3.1.2 RNA extraction from brain tissues 22 2.3.2 Reverse transcription 22 2.3.3 Quantitative real-time polymerase chain reaction 23 2.4 Protein methods 25 2.4.1 Cell lysates (RIPA) 25 2.4.2 Western blotting 25 2.5 BODIPY-cholesterol efflux assay 26 2.6 Statistical analyses 28 3. RESULTS 29 3.1 Expression of ABCA5 in the human brain 30 3.2 Expression of α-synuclein in the human brain 33 3.2.1 Impact of ABCA5 expression on α-synuclein in SK-N-SH cells 34 3.3 Function and regulation of ABCA5 35 3.3.1 Development of protocol for measuring cholesterol efflux 35 3.3.2 ABCA5 as a cholesterol transporter 36 3.3.3 PPAR-activator induces ABCA5 expression in SK-N-SH cells 39 4. DISCUSSION 43 5. REFERENCES 48 COPYRIGHT STATEMENT ‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' Signed ……………………………………………........................... Date ……………………………………………........................... AUTHENTICITY STATEMENT ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ Signed ……………………………………………........................... Date ……………………………………………........................... I ACKNOWLEDGEMENTS Firstly, I would like to give thanks to my supervisor Dr. Scott Kim for his much appreciated support and his continuous advices throughout my candidature. I am also very grateful to my co-supervisor Prof. Glenda Halliday for all her support and encouragement. I would also like to extend my appreciation to the rest of my colleagues within the Halliday Group and within Neuroscience Research Australia for their support and assistance and friendship throughout the years. I am also grateful to Mrs. Elizabeth Gilbert for her generous Gilbert scholarship during my candidature. Special thanks to my close friend and sister in Christ Puika for sharing all the joy and tears and always being there for me. I am deeply grateful to my family and my fiancé, Victor, for their tremendous amount of love, support and encouragement they have given to me throughout my candidature. Last but not least, I would like to give all the glory to my lord Jesus and am extremely grateful for giving me such a valuable experience to learn and grow. II LIST OF ABBREVIATIONS ABC transporter ATP-binding cassette transporter ABCA1 ATP-binding cassette subfamily A1 ABCA2 ATP-binding cassette subfamily A2 ABCA3 ATP-binding cassette subfamily A3 ABCA4 ATP-binding cassette subfamily A4 ABCA5 ATP-binding cassette subfamily A5 ABCA7 ATP-binding cassette subfamily A7 ABCA8 ATP-binding cassette subfamily A8 AD Alzheimer‟s disease ApoA-I Apolipoprotein A-I ApoE Apolipoprotein E APP Amyloid precursor protein Aβ Amyloid-β BODIPY Dipyrromethene boron difluoride BSA Bovine serum albumin CD Cyclodextrin cDNA Complementary deoxyribonucleic acid CNS Central nervous system Ct Comparative threshold cycle dNTP Deoxyribonucleotide triphosphate EDTA Ethylenediaminetetraacetic acid GWAS Genome-wide association study HCl Hydrogen chloride III HDL High density lipoprotein LB Lewy bodies LDL Low-density lipoprotein LXR Liver-X receptor MES cells Mesencephalic neuronal cells M-MLV Moloney Murine Leukemia Virus mRNA Messenger RNA NaCl Sodium chloride NBDs Nucleotide-binding domains Opti-MEM Opti-minimal essential medium PBS Phosphate buffered saline PD Parkinson‟s disease PMI Post-mortem interval PPAR Peroxisome proliferator-activated receptor PUFAs Poly-unsaturated fatty acids qRT-PCR Quantitative real-time polymerase chain reaction RIPA Radioimmunoprecipitation RXR Retinoid X receptor SDS-PAGE SDS polyacrylamide gels SE Standard error SNPC Substantianigra pars compacta SP cells Side population cells TMDs Transmembrane domains α-syn α-synuclein IV ABSTRACT Parkinson‟s disease (PD) is one of the most common neurological movement disorders in humans. Its pathological hallmark is the deposition of intracellular aggregates of the neuronal protein α-synuclein. Increasing evidence indicates that α-synuclein binds and interacts with lipids, which are transported in the central nervous system (CNS) by a group of proteins called ATP-Binding Cassette subfamily A (ABCA) transporters. A genome-wide association study reported that ABCA5 is associated with PD. However, very little is known about the function of ABCA5 in the brain or PD. The aim of this project is to investigate the potential function of ABCA5 in the brain and in the disease process of PD. This was achieved by analysing changes in ABCA5 expression in samples of human post-mortem PD brain compared with age- and gender- matched disease-free controls, andby assessing PD relevant molecular changes inin vitro models transiently expressing ABCA5. In the anterior cingulate cortex, ABCA5 gene expression, but not the expression of other ABCA transporters, was significantly reduced in PD compared to controls. The levels of α-synuclein expression were also significantly decreased in the same region as ABCA5. Moreover, SK-N-SH neuronal cells transiently transfected with ABCA5 demonstrated significantly up-regulated α- synuclein mRNA expression with no increase in α-synuclein protein levels. Transiently expressed ABCA5 was also shown to stimulate the efflux of cholesterol from neurons to discsmade from apolipoproteinE. Upregulation of ABCA5 gene expression could be stimulated by peroxisome proliferator-activated receptor (PPAR)in a time - and dose- dependent manner. These findings suggest that ABCA5 may be involved in regulation of lipid homeostasis in the pathological process of PD. V Chapter 1: Introduction 1 1.1.PARKINSON’S DISEASE Parkinson‟s disease (PD) was first described in “An essay on the shaking palsy” by James Parkinson in 1817 (Parkinson, 2002) where he detailed his observations of six individuals; one followed in detail over a long period of time, and the other five observed at a distance. Despite the small number of patients, Parkinson was able to provide a very accurate account of the symptoms of the disease as: “Involuntary tremulous motion, with lessened muscular power, in parts not in action and even when supported; with a propensity to bend the trunk forward, and to pass from a walking to a running pace”. Motor features remain the main criteria for the clinical diagnosis of PD, but it is now known that PD patients may also experience a number of non-motor features such as neuropsychiatric symptoms (hallucinations, depression and dementia) (Sanchez-Ramos et al., 1996), sleep disturbances (Stacy, 2002), constipation (Sadjadpour, 1983), hypotension (Goldstein, 2006), fatigue (van Hilten et al., 1993), olfactory deficits and autonomic dysfunctions. PD is one of the most prevalent neurodegenerative diseases in humans. It affects approximately 1% of people over the age of 65, and the prevalence rises to 5% of people over the age of 85 (Van Den Eeden et al., 2003). It can be characterized as early onset or late onset. Early onset PD, which is estimated at 3% of PD cases, occurs before the age of 50 years and is usually due to genetic mutations. The majority of the PD cases are late onset and mostly sporadic.
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