Cellular mechanisms affecting Alzheimer’s amyloid-beta aggregation in Saccharomyces cerevisiae A thesis presented for the degree of Doctor of Philosophy by Suresh Nair School of Biotechnology and Biomolecular Sciences University of New South Wales August, 2012 THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: NAIR First name: SURESH Other name/s: Abbreviation for degree as given in the University calendar: PhD School: Biological Sciences Faculty: SCIENCE Title: Cellular mechanisms affecting Alzheimer’s amyloid-beta aggregation in Saccharomyces cerevisiae Abstract (350 words maximum): Amyloid-beta (Aβ) plaques are a major neuropathological feature of Alzheimer’s disease (AD). These plaques are primarily composed of aggregates of Aβ peptides generated via the amyloidogenic processing of the amyloid precursor protein. The two major isoforms of Aβ peptide are Aβ40 and Aβ42, of which the latter is highly prone to aggregation. Increased presence and aggregation of intracellular Aβ42 peptides is an early event in the disease progression of AD. Improved understanding of cellular processes involved in Aβ42 aggregation may have implications for understanding AD progression and development of therapeutic strategies. Here Aβ42 fused to green fluorescent protein (Aβ42GFP) was expressed in each mutant of the Saccharomyces cerevisiae genome-wide deletion library to identify proteins and cellular processes that affect intracellular Aβ42 aggregation by assessing the fluorescence associated with expression of the Aβ42GFP fusion protein. This screening identified 110 mutants exhibiting intense Aβ42GFP-associated fluorescence. Four major cellular processes were over- represented in the data set, including phospholipid homeostasis and mitochondrial function. Analysis of the S. cerevisiae lipidome by quantitative mass spectrometry led to the discovery that disruption of phosphatidylcholine, phosphatidylserine and/or phosphatidylinositol metabolism had a major effect on intracellular Aβ42 aggregation and localisation. Distinctive subcellular localisation of Aβ42GFP in the phospholipid mutants was observed. Confocal microscopy indicated that Aβ42GFP in the phospholipid mutants was juxtaposed to the nucleus of the cell, associated with the endoplasmic reticulum. A novel outcome of the Aβ42GFP fusion protein has been to also identify putative roles for two proteins of previously uncharacterised function. This study further exploited the powerful and flexible platform of applying GFP-derived fluorescence-based assay for the screening of the Library of Pharmacologically Active Compounds (LOPAC) and the SPECTRUM Collection chemical/drug libraries for compounds with amyloidogenic and anti-amyloidogenic properties. These libraries include FDA-approved drugs and identifying such drugs as affecting intracellular Aβ42 aggregation may significantly reduce costs and the exhaustive process of introducing new drugs onto the market. These data provide genome-wide evidence of cellular processes and chemical compounds that affect intracellular Aβ42GFP aggregation and may have important implications for understanding cellular mechanisms that affect intracellular Aβ42 aggregation and ultimately AD progression. 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). 20 – 12 – 2012 …………………………………………… ……………………………………………..……..……… ………..…..…….… 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: 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 acknowledged. Signed ……………………….. Suresh Nair Date: 20 – 12 – 2012 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 ……………………….. Suresh Nair Date: 20 – 12 – 2012 AUTHENICITY 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 ……………………….. Suresh Nair Date: 20 – 12 – 2012 Preface ABSTRACT Amyloid-beta (Aβ) plaques are a major neuropathological feature of Alzheimer’s disease (AD). These plaques are primarily composed of aggregates of Aβ peptides generated via the amyloidogenic processing of the amyloid precursor protein. The two major isoforms of Aβ peptide are Aβ40 and Aβ42, of which the latter is highly prone to aggregation. Increased presence and aggregation of intracellular Aβ42 peptides is an early event in the disease progression of AD. Improved understanding of cellular processes involved in Aβ42 aggregation may have implications for understanding AD progression and development of therapeutic strategies. Here Aβ42 fused to green fluorescent protein (Aβ42GFP) was expressed in each mutant of the homozygous diploid Saccharomyces cerevisiae genome-wide deletion library (Winzeler et al., 1999) to identify proteins and cellular processes that affect intracellular Aβ42 aggregation by assessing the fluorescence associated with expression of the Aβ42GFP fusion protein. This screening identified 110 mutants exhibiting intense Aβ42GFP-associated fluorescence. Four major cellular processes were over-represented in the data set, including phospholipid homeostasis and mitochondrial function. Global analysis of the S. cerevisiae lipidome by quantitative shotgun mass spectrometry led to the discovery that disruption of phosphatidylcholine, phosphatidylserine and/or phosphatidylinositol metabolism had a major effect on intracellular Aβ42 aggregation and localisation. Distinctive subcellular localisation of Aβ42GFP in the phospholipid mutants was observed. Confocal microscopy indicated that Aβ42GFP in the phospholipid mutants was juxtaposed to the nucleus of the cell, associated with the endoplasmic reticulum. Another novel outcome of the Aβ42GFP fusion protein has been to identify putative roles for two proteins of previously uncharacterised function. This study further exploited the powerful and flexible platform of applying GFP-derived fluorescence- based assay for the screening of the Library of Pharmacologically Active Compounds (LOPAC) and the SPECTRUM chemical/drug library for compounds with amyloidogenic and anti-amyloidogenic properties. These libraries include FDA- approved drugs and identifying such drugs as affecting intracellular Aβ42 aggregation may significantly reduce costs and the exhaustive process of introducing new drugs onto I Preface the market. Compounds identified through this approach may potentially change the way these drugs are currently administered to patients. These data provide the first genome-wide evidence of cellular processes and chemical compounds that affect intracellular Aβ42GFP aggregation and may have important implications for understanding cellular mechanisms that affect intracellular Aβ42 aggregation and ultimately disease progression of AD. II Preface ACKNOWLEDGEMENTS I thank Ian, my supervisor, for his guidance and encouragement throughout my candidature. Your passion in