Exploring the role of C5a-C5aR1 signalling in development through pluripotent stem cell modelling Owen Hawksworth BSc (Hons) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2017 Faculty of Medicine Abstract The complement system has been traditionally described as a powerful controller of innate immunity. With activation through the recognition of pathogenic surfaces, spontaneous hydrolysis, or extrinsic cleavage, a cleavage cascade is initiated culminating in the formation of the active complement fragments C3a, C3b, C5a, and C5b. These fragments direct immune cells to sites of inflammation, as well as tagging pathogens for destruction and directly lysing foreign cells. It is now clear however, that this complex family of proteins possesses a wide range of functions outside of immune regulation. From fertilisation and morphogenesis, to the control of foetal and adult stem cell populations, studies have described novel actions of complement factors. This thesis is focussed on the central complement receptor, C5aR1. Traditionally described as a potent activating and chemotactic receptor for immune cells, C5aR1 has been shown to function in a number of non- immune cell populations. Of note, our laboratory has previously described a role for C5aR1 in neural tube closure, with loss of C5aR1 signalling associated with increased neural tube defects under folate deficient conditions. However, a mechanistic role of C5aR1 at this stage of development was not described. In this thesis, pluripotent stem cells were utilised as an in vitro model of human development to interrogate the expression and function of C5aR1 at a number of developmental stages. Specifically, in pluripotent stem cells representative of the blastocyst inner cell mass, in neural rosettes representative of the developing ventricular zone, and in matured post-mitotic cortical neurons. This builds on previous work by our laboratory, which had identified C5aR1 actions in the mouse ventricular zone, analogous to late neural rosette cultures, and had shown a neurotoxic effect of C5aR1 in post-mitotic mouse neurons. C5aR1 was found to be expressed in pluripotent stem cells, with a role in promoting maintenance of pluripotency in the absence of FGF2 signalling. Additionally, C5aR1 was found to be apically expressed in human neural rosettes, with signalling promoting proliferation and maintenance of cell polarity. This work correlated well with mouse studies performed outside of this thesis, to show that in vivo, loss of C5aR1 in this neural progenitor population resulted in behavioural deficits and microstructural brain changes. Lastly, we determined the mRNA expression of C5aR1 in human post- mitotic cortical neurons. However, in contrast to previous mouse studies, exogenous C5a, alone or in the presence of secondary stressors, had little effect on the survival of these cells. Overall, the results presented in this thesis have further expanded our knowledge of C5a-C5aR1 signalling in development, describing novel roles for this signalling pathway. The use of pluripotent stem cells allowed for the exploration of C5aR1 actions in human development that would otherwise be limited to animal models. Additionally, it allowed for the correlation of previous animal results to ii a human cell type, with both inter-species conservation of function, and discordance, identified. Additionally, the use of pluripotent stem cell modelling has provided a useful platform for the future study of complement in both development and disease. This work has direct implications to the clinical environment, highlighting the important developmental roles of C5aR1, and how modulation of C5aR1 signalling to target complement driven disease could have detrimental effects on foetal development. iii Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. iv Publications During Candidature Journal Articles Hawksworth OA, Coulthard LG, Woodruff TM (2016) Complement in the fundamental processes of the cell Molecular Immunology 84; 17-25 Hawksworth OA, Xiang L, Coulthard LG, Wolvetang EJ & Woodruff TM (2017) New concepts on the therapeutic control of terminal complement receptors Molecular Immunology Epub ahead of print Hawksworth OA, Coulthard LG, Taylor SM, Wolvetang EJ & Woodruff TM (2014) Complement C5a promotes human embryonic stem cell pluripotency in the absence of FGF2 Stem Cells 32 (12); 3278-3284 Coulthard LG, Hawksworth OA, Li R, Balachandran A, Lee JD, Sepehrband F, Kurniawan N, Jeanes A, Simmons DG, Wolvetang EJ & Woodruff TM (2017) Complement C5aR1 Signaling Promotes Polarization and Proliferation of Embryonic Neural Progenitor Cells through PKCζ Journal of Neuroscience 37 (22); 5395-5407 Bellows-Peterson ML, Fung HK, Floudas CA, Kieslich CA, Zhang L, Morikis D, Wareham KJ, Monk PN, Hawksworth OA, Woodruff TM (2012) De novo peptide design with C3a receptor agonist and antagonist activities: theoretical predictions and experimental validation Journal of Medicinal Chemistry 55 (9); 4159-68 Reports Hawksworth OA, Coulthard LG & Woodruff TM (2013) Complement peptide receptors: C3a receptor. International Union of Basic and Clinical Pharmacology Database. http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=31 Coulthard LG, Hawksworth OA & Woodruff TM (2013) Complement peptide receptors: C5a receptor. International Union of Basic and Clinical Pharmacology Database. http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=32 Hawksworth OA, Woodruff TM (2016) Flash News: Complement and microglia mediate early synapse loss in Alzheimer mouse models Focus in Complement 43; 2 v Book Chapters Coulhard LG, Hawksworth OA, Woodruff TM “Chapters: C3aR, C5aR1, and C5aR2” The Complement FactsBook 2nd edition, S. Barnum & T. Schein (Ed.) Academic Press, October 2017 International Conference Abstracts Hawksworth OA, Coulthard LGJ, Taylor SM, Wolvetang EJ & Woodruff TM (2012) Expression of complement factors and functional C5a receptors in human embryonic stem cells and induced pluripotent stem cells Immunobiology 217 (11), 1169 Presented at the 24th International Complement Workshop, Chania, Crete, Greece Winner Aegean Conference Trainee Award (2012) vi Publications Included in this Thesis Chapter 1 Hawksworth OA, Coulthard LG, Woodruff TM (2016) Complement in the fundamental processes of the cell Molecular Immunology 84; 17-25 Contributor Statement of Contribution Hawksworth OA Researched the paper (100%) Wrote the paper (95%) Coulthard LG Wrote and edited the paper (5%) Woodruff TM Edited the paper (100%) Hawksworth OA, Xiang L, Coulthard LG, Wolvetang EJ & Woodruff TM (2017) New concepts on the therapeutic control of terminal complement receptors Molecular Immunology Epub ahead of print Contributor Statement of Contribution Hawksworth OA Researched the paper (95%) Wrote the paper (95%) Xiang L Researched and wrote the paper (5%) Coulthard LG Edited the paper (25%) Wolvetang EJ Edited the paper (25%) Woodruff TM Edited the paper (50%) Chapter 2 Hawksworth OA, Coulthard LG, Taylor SM, Wolvetang EJ & Woodruff TM (2014) Complement C5a promotes human embryonic stem cell pluripotency in the absence of FGF2 Stem Cells 32 (12); 3278-3284 Contributor Statement of Contribution Hawksworth OA Performed the experiments (100%) Analysed the data (95%) Wrote the paper (80%) Coulthard LG Analysed the data (5%) Edited the paper Taylor SM Edited the paper and intellectual input (20%) Wolvetang EJ Edited the paper and intellectual input (40%) Woodruff TM Edited the paper and intellectual input (40%) vii Chapter 3 Coulthard LG*, Hawksworth OA*, Li R, Balachandran A, Lee JD, Sepehrband F, Kurniawan N, Jeanes A, Simmons DG, Wolvetang EJ & Woodruff TM (2017) Complement C5aR1 Signaling Promotes Polarization and Proliferation of Embryonic Neural Progenitor Cells through PKCζ Journal of Neuroscience 37 (22); 5395-5407 *These authors contributed equally Contributor Statement of Contribution Hawksworth OA Performed and analysed the human experiments (100%) Wrote the