The Effects of Clusterin on the Aggregation and Pathogenicity of TDP-43, a Protein Implicated in Amyotrophic Lateral Sclerosis

University of Wollongong Research Online University of Wollongong Thesis Collection 1954-2016 University of Wollongong Thesis Collections 2015 The effects of clusterin on the aggregation and pathogenicity of TDP-43, a protein implicated in amyotrophic lateral sclerosis Rebecca Anne Brown Follow this and additional works at: https://ro.uow.edu.au/theses University of Wollongong Copyright Warning You may print or download ONE copy of this document for the purpose of your own research or study. The University does not authorise you to copy, communicate or otherwise make available electronically to any other person any copyright material contained on this site. You are reminded of the following: This work is copyright. Apart from any use permitted under the Copyright Act 1968, no part of this work may be reproduced by any process, nor may any other exclusive right be exercised, without the permission of the author. 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Recommended Citation Brown, Rebecca Anne, The effects of clusterin on the aggregation and pathogenicity of TDP-43, a protein implicated in amyotrophic lateral sclerosis, Doctor of Philosophy thesis, School of Biological Sciences, University of Wollongong, 2015. https://ro.uow.edu.au/theses/4664 Research Online is the open access institutional repository for the University of Wollongong. For further information contact the UOW Library: [email protected] The effects of clusterin on the aggregation and pathogenicity of TDP-43, a protein implicated in Amyotrophic Lateral Sclerosis By Rebecca Anne Brown Bachelor of Science (Biological Sciences) Honours 1 This thesis is presented as part of the requirements for Degree of Doctor of Philosophy School of Biological Sciences University of Wollongong Wollongong, Australia November 2015 i DECLARATION OF AUTHENTICITY This thesis is submitted in accordance with the regulations of the University of Wollongong in fulfilment of the Degree of Doctor of Philosophy. It does not include any material published by another person except where due reference is made in the text. The experimental work described in this thesis is original and has not been submitted for a degree to any other university. Rebecca Anne Brown ii ACKNOWLEDGEMENTS The work presented in this thesis would not be possible without Australian Rotary Health, who awarded me the Lloyd Binet PhD Scholarship which was co-funded by the Rotary Club of Hunters Hill. Thank you for this great privilege, it was a pleasure meeting members of the club and sharing my research with you. I would like to thank my supervisor Senior Professor Mark Wilson for all his advice, knowledge and guidance throughout my PhD. Thank you for giving me the opportunity to be part of such a wonderful lab group, and extra thanks for putting up with my endless stressing! I left every meeting feeling better about my work than when I walked in, for which I am very grateful. Everyone in lab 210 deserves a big thank you, as the encouragement and support I received from all of you is a big part of what made my PhD enjoyable. Special thanks to Nat, Jen, Dan, Pat, Sharon, Luke and BJ for all the lab help you have given me, but mostly for making Uni fun! Thank you also to my co-supervisor Dr Justin Yerbury for all your help and expertise over the past few years. I was lucky enough to spend a significant part of my PhD at the University of Cambridge and I would like to thank Professor Christopher Dobson for his help in making this possible. Thank you especially to Dr Leila Luheshi for teaching me everything about flies and making me feel so welcome from day one. I am also very grateful to Jenna and Teresa for all the help they gave me in the fly lab. Thank you also to the basement lab and lab 290 for all your help, but mostly for making sure I had such a great time in Cambridge. Particular thanks goes to Sharon, Maya and Janice. Thank you especially to James for always believing in me and putting up with my thesis induced hissy fits. Even when you were on the other side of the world you have always been there for me. Lastly, I want to thank my family for their endless support. Dad, thank you for driving me to Uni all these years and for providing me with wine which also helped a lot. I really did enjoy our car chats each day. Mum thank you for always listening to me whenever I was overly dramatic about my PhD, and for always managing to make me feel better every time. Also all the crazy pyjamas you have bought me made great thesis writing clothes. Sammy thanks for being the most supportive sister ever, and also for teaching me how to make potato bake. That doesn’t have anything to do with my PhD but it’s really delicious. I couldn’t have done this without you guys. iii ABSTRACT Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and fatal neurodegenerative disease with an international prevalence of approximately 3-7 people per 100,000. The disease is characterised by the selective degeneration of both upper and lower motor neurons, and the presence of insoluble protein aggregates within degenerating neurons and surrounding cells. Typically, pathological changes begin at a focal point of onset and spread radially outwards over time. The pathogenic mechanisms that mediate neurodegeneration in ALS are multifactorial and involve interactions between genetic, cellular and molecular pathways including protein aggregation and disruption to proteostasis, glutamate induced excitotoxicity and the activation of non-neuronal cells. Transactive response DNA binding protein (TDP-43) is a major component of ubiquitinated inclusions in ALS and mutations in the aggregation prone C-terminal region are associated with disease pathogenesis. Normally a nuclear protein involved in RNA processing and exon splicing, in ALS TDP-43 is depleted from the nucleus and accumulates in the cytosol of motor neurons in insoluble aggregates. The human body has evolved complex protein quality control systems to prevent protein misfolding and limit the accumulation of misfolded or aggregated proteins. Chaperones are the key components of these systems in the extracellular and intracellular spaces, and work by binding to the exposed regions of hydrophobicity on non-native proteins to either assist in folding/refolding or target them for degradation. Studies have reported interactions between proteins involved in ALS and various intracellular chaperones including αB-crystallin, Hsp70 and Hsp40, and Hsp27/25, suggesting that chaperones may influence ALS onset and progression. However the effects of extracellular chaperone action on the disease still remain to be thoroughly elucidated. Clusterin is a potent extracellular chaperone with broad substrate specificity and can be retrotranslocated from the endoplasmic reticulum (ER) to the cytoplasm under conditions of ER stress. It was previously unknown whether clusterin could inhibit the aggregation of TDP-43, or if it was a constituent of TDP-43 inclusions. Cell-to-cell spread is a “prion-like” (refer to section 1.1 and 1.7) mechanism of protein propagation and studies have shown that TDP-43 and Cu/Zn superoxide dismutase (SOD1) are both capable of such spread in cultured cells. Prion-like mechanisms of protein propagation could explain the outward spread of symptoms observed in ALS. SOD1 has been shown to spread from cell-to-cell in vivo, but no published reports describe if TDP-43 is also able to do so. Understanding whether TDP-43 is able to do so would lead to an increased understanding of the mechanisms of protein propagation in ALS, mechanisms which could be targeted in the development of new therapeutic strategies. This study aimed to examine whether TDP-43 was capable of cell-to-cell spread in cell culture and in vivo using a Drosophila model, and if so whether the extracellular chaperone clusterin could prevent iv this spread. Additionally it was determined whether the expression of TDP-43 in a subset of Drosophila glial cells was sufficient to induce motor neuron defects and mortality, and whether the co-expression of clusterin was able to reduce this. The ability of clusterin to inhibit the aggregation of TDP-43 in vitro was also tested. Finally, thoracic spinal cord sections from ALS patients were examined via immunohistochemistry to determine if clusterin was a constituent of TDP-43 inclusions, which would be consistent with the two proteins interacting during ALS progression. Results from both cell-culture and in vivo experiments demonstrated that TDP-43 was able to spread from the glial cells where it was originally expressed, to surrounding cells which included motor neurons. Taken together these results suggest that like SOD1, TDP-43 may have prion-like modes of transmission. In Drosophila larvae the co-expression of clusterin was able to reduce this spread. Expression of TDP-43 in the glial cells of Drosophila caused a significant reduction of lifespan and locomotor defects. While the co-expression of clusterin was not able to extend lifespan, it did result in the preservation of locomotor ability. This effect may have resulted from clusterin inhibiting TDP-43 aggregation, as it was found that clusterin could potently inhibit the aggregation of TDP-43286-331, a synthetic peptide corresponding to residues 286-331 of TDP-43.

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