Translational alterations of retinoid receptors, their binding partners and the retinoid signalling pathway in schizophrenia brain and blood Shan-Yuan Tsai Submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy School of Psychiatry Faculty of Medicine August 2018 Thesis/Dissertation Sheet Australia's Global University Surname/Family Name Tsai Given Name/s Shan-Yuan Abbreviation for degree as give in the University calendar PhD Faculty Faulty of Medicine School School of Psychiatry Translational alt�ations of retinoid receptors,their binding partners and the Thesis Tille retinoid signalling pathway in schizophrenia brain and blood Abstract350 words maximum: (PLEASE TYPE) Schizophrenia is a debilitating mentalillness characterised by positive, negative and cognitive symptoms. Epidemiological studies implicate vitamin D in the aetiologyof schizophrenia and protein studies show altered circulating levels of the ligand vitamin D and implicate relinoids in schizophrenia. However, the gene expressions of vitamin D and rebnoid receptors have not beenext ensively studied. In parallel, there is increasing interest in the role ofneuroinflammation in the aetiology and progressionof schizophrenia and both vitamin D and retJnoids (vitamin A andits derivatives) have known anti-inflammatory actions. My thesis aims to measure expressionof molecules involved in vitamin D andretino id signalling and determine 1heinfluence of elevated inflammatory markers on these molecules. My findings are obtained from two cohoris: a post moriern braincohort comprisingtissue samples from the frontal cortex fromt hirty-seven individualswith schizophrenia and thirty-seven matched controls; and a clinical cohort of eighty-six patients with schizophrenia and seventy-sevencon trols who provided peripheral bloodsamp les. Firstly, I used next generation sequencing data and real lime polymerasechain reaction in the postmortem brain cohortto identify decreased mRNA expression of one retinoid X receptor subtype (RXRB) and of two of its binding partners, the NR4A1 and NR4A2 receptors, in schizophrenia. Secondly, using previously identified subgroups of people with neuroinflammation in the poot mortem cohort. I found that expression of two receptors, RXRB andret inoic acid receptor gamma (RARG). were increased in schizophrenia with neuroinflammation. whereas twoorphan nuclear receptors, NR4A1 and NR4A3, were decreased in the same group. Finally, I analysed mRNA transcript levels in blood, and identified dysregulation in the retinoid signalling pathwayin schizophrenia, principally involving the disruption of the conversion of retinol to retinoic acid (the bioactive form of vitamin A). I also found evidence of crosstalk between retinoid signalling molecules and pro inflammatory cytokines. The dysregulalion in gene transcriptsof the retinoid signalling pathway also affected cognitive perfonnance on tasks involving the prefrontal cortexand hippocampus. My studies suggest retinoIds contribute to the aetiology of schizophrenia and identify a direct relationshipv.ith pro inflammatorycytol< ines in both brain and blood. 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Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. OR 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 institute, 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………………………………………………………………27/04/2019 I II Acknowledgements There are a number of people I would like to thank with whom this work would not have been possible. My supervisor, Professor Cynthia Shannon Weickert, for your support and opportunity to pursue a PhD with the Schizophrenia Research Laboratory. Your vision and passion to cure schizophrenia is inspiring. My co-supervisors, Dr Vibeke Catts, and Dr Janice Fullerton, thank you both for your guidance, expertise and invaluable feedback in thesis writing. Vibeke, thank you for your selfless time, care, and patience. Jan, thank you for your insight into genetics in schizophrenia. A huge thank you to Mrs Laurie Cowled for financial support. Your generosity in enriching and aiding women students across many disciplines to further their education is amazing. I would also like to thank everyone from the Schizophrenia Research Laboratory. Debora for laboratory logistical help and moral support, Inara for administrative help, humour and proof- reading my thesis and Tertia for answering my random lab related questions. Thank you also to all the previous and current research assistants for technical help, with special mentions to Danny, Alice R, Isabella, Alice Z and Sam O. Thank you to Cam and DD for statistics help and Wai-Kit for IT help. Thank you for the moral support from fellow students, especially Dr Yiru Zhang for the chats, Christin and Hayley for making sure I was not financially stranded overseas. To my friends, thank you for your patience and understanding, particularly towards the end of my PhD journey. Christine, thank you for being my rock, and Fiona, thank you for keeping me sane. A huge thank you to my family. Mum and Dad, thank you for all your encouragement and offering unlimited babysitting. To my sisters, Belinda and Joanne, thank you for your support and keeping me grounded with your banter. To my son, Aiden, thank you for making bringing so much joy to my life. I’ve enjoyed and look forward to each milestone and our nightly bedtime stories. Every day with you is a wonder. II Thesis Abstract Schizophrenia is a debilitating mental illness characterised by positive, negative and cognitive symptoms. Epidemiological studies implicate vitamin D in the aetiology of schizophrenia and protein studies show altered circulating levels of the ligand vitamin D and implicate retinoids in schizophrenia. However, the gene expressions of vitamin D and retinoid receptors have not been extensively studied. In parallel, there is increasing interest in the role of neuroinflammation in the aetiology and progression of schizophrenia and both vitamin D and retinoids (vitamin A and its derivatives) have known anti-inflammatory actions. My thesis aims to measure expression of molecules involved in vitamin D and retinoid signalling and determine the influence of elevated inflammatory markers on these molecules. My findings are obtained from two cohorts: a post-mortem brain cohort comprising tissue samples from the frontal cortex from thirty-seven individuals with schizophrenia and thirty-seven matched controls; and a clinical cohort of eighty-six patients with schizophrenia and seventy-seven controls who provided peripheral blood samples. Firstly, I used next generation sequencing data and real-time polymerase chain reaction in the post-mortem brain cohort to identify decreased mRNA expression of one retinoid X receptor subtype (RXRB) and of two of its binding partners, the NR4A1 and NR4A2 receptors, in schizophrenia. Secondly, using previously identified subgroups of people with neuroinflammation in the post-mortem cohort, I found that expression of two receptors, RXRB and retinoic acid receptor gamma (RARG), were increased in schizophrenia with neuroinflammation, whereas two orphan nuclear receptors, NR4A1 and NR4A3, were decreased in the same group. Finally, I analysed mRNA transcript levels in blood, and identified dysregulation in the retinoid signalling pathway in schizophrenia, principally involving the disruption of the conversion of retinol to retinoic acid (the bioactive form of vitamin A). I also found evidence of crosstalk between retinoid signalling molecules and pro-inflammatory cytokines. The dysregulation in gene transcripts of the retinoid signalling pathway also affected cognitive performance on tasks involving the prefrontal cortex and hippocampus. My studies suggest retinoids contribute to the aetiology of schizophrenia and identify a direct relationship