Discovery of Molecular Mechanisms Underlying Lysosomal and Mitochondrial Defects in Parkinson’S Disease
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Discovery of Molecular Mechanisms Underlying Lysosomal and Mitochondrial Defects In Parkinson’s Disease Brigitte Phillips Supervisor: Associate Professor Antony Cooper A thesis in fulfillment of the requirements for the degree of Doctor of Philosophy St Vincent’s Clinical School, Faculty of Medicine The University of New South Wales & The Garvan Institute of Medical Research April, 2018 THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: Phillips First name: Brigitte Other names/s: Radinovic Abbreviation for degree as given in the University calendar: PhD School: St Vincent’s Clinical School Title: The emerging contributions of the lysosome and mitochondria to Parkinson’s diseases Abstract 350 words maximum Parkinson’s disease (PD) is a common, debilitating neurodegenerative disease yet the causes of cell dysfunction in PD remain unclear. By integrating available patient data with data from an unbiased assessment of proteomic changes in multiple cellular PD models, this study has identified new aspects of mitochondrial and lysosomal dysfunction that likely contribute to PD. Two areas were investigated in detail; the mitochondrial protein CHCHD2 and the V-ATPase complex, which acidifies endolysosomal compartments. CHCHD2 had not been well characterized or associated with PD when identified in this study, however PD-causative variants have since been described and CHCHD2 has recently been proposed to regulate mitochondrial cristae structure and interact with cytochrome c. Data from this thesis extends CHCHD2 dysfunction to sporadic PD patients, where its reduced expression was identified in the brain. In exploring the potential function of CHCHD2, mitochondrial impairment resulted in rapid translational up- regulation of CHCHD2 and its specific accumulation in depolarised mitochondria, suggesting that CHCHD2 plays a targeted role in aiding mitochondrial recovery or quarantining cytochrome c in response to mitochondrial damage. A recently identified PINK1/PUF protein-mediated translational control mechanism is proposed to regulate this response and is under investigation. This thesis also identified and explored the contributions of V-ATPase dysfunction to PD. Protein and mRNA levels of multiple V-ATPase subunits were down-regulated in patients’ brain regions and these reduced V-ATPase protein levels inversely correlated with increased levels of αSynuclein, the toxic protein that accumulates in PD. Depletion of V-ATPase subunits in vitro increased αSynuclein protein levels and produced many PD-associated lysosomal and mitochondrial dysfunctions, including increased mitochondrial Ca2 and reactive oxygen species. V-ATPase depletion also impaired lysosomal clustering in the cell perinuclear region, aligning with previous indications that the V-ATPase facilitates microtubule- mediated lysosomal transport and suggesting that lysosomal transport and maturation may be impaired in PD, a dysfunction evident in similar neurodegenerative diseases but not yet explored in PD. These results characterise new consequences of V-ATPase dysfunction and implicate V-ATPase depletion as a contributing factor to αSynuclein accumulation, lysosome and mitochondrial dysfunction in PD. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in 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 property 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 Abstracts International (this is applicable to doctoral theses only). …………………………… …………………………… ……………………………23/10/2017 Signature Witness Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. FOR OFFICE USE ONLY Date of completion of requirements for Award: i 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..........................................23/10/17 ii 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 ..............................................................................23/10/17 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 ..............................................................................23/10/17 iii ABSTRACT Parkinson’s disease (PD) is a common, debilitating neurodegenerative disease yet the causes of cell dysfunction in PD remain unclear. By integrating available patient data with data from an unbiased assessment of proteomic changes in multiple cellular PD models, this study has identified new aspects of mitochondrial and lysosomal dysfunction that likely contribute to PD. Two areas were investigated in detail; the mitochondrial protein CHCHD2 and the V-ATPase complex, which acidifies endolysosomal compartments. CHCHD2 had not been well characterized or associated with PD when identified in this study, however PD-causative variants have since been described and CHCHD2 has recently been proposed to regulate mitochondrial cristae structure and interact with cytochrome c. Data from this thesis extends CHCHD2 dysfunction to sporadic PD patients, where its reduced expression was identified in the brain. In exploring the potential function of CHCHD2, mitochondrial impairment resulted in rapid translational up-regulation of CHCHD2 and its specific accumulation in depolarised mitochondria, suggesting that CHCHD2 plays a targeted role in aiding mitochondrial recovery or quarantining cytochrome c in response to mitochondrial damage. A recently identified PINK1/PUF protein-mediated translational control mechanism is proposed to regulate this response and is under investigation. This thesis also identified and explored the contributions of V-ATPase dysfunction to PD. Protein and mRNA levels of multiple V-ATPase subunits were down-regulated in patients’ brain regions and these reduced V-ATPase protein levels inversely correlated with increased levels of αSynuclein, the toxic protein that accumulates in PD. Depletion of V-ATPase subunits in vitro increased αSynuclein protein levels and produced many PD-associated lysosomal and mitochondrial dysfunctions, including increased mitochondrial Ca2 and reactive oxygen species. V-ATPase depletion also impaired lysosomal clustering in the cell perinuclear region, aligning with previous indications that the V-ATPase facilitates microtubule-mediated lysosomal transport iv and suggesting that lysosomal transport and maturation may be impaired in PD, a dysfunction evident in similar neurodegenerative diseases but not yet explored in PD. These results characterise new consequences of V-ATPase dysfunction and implicate V-ATPase depletion as a contributing factor to αSynuclein accumulation, lysosome and mitochondrial dysfunction in PD. v PUBLICATIONS FROM THIS THESIS Oral Presentations Live Cell Imaging of mitochondrial function in Parkinson’s disease-like cells, EMBL Australia PhD Symposium, Sydney, Nov 2014 Poster Presentations The Role