Florey Department of Neuroscience and Mental Health - Theses

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Author: Walsh, Alexander Dominic × Start date: 2020 × Subject: Neurodegeneration ×

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    Gene Regulatory Networks and MicroRNAs in Microglia
    Walsh, Alexander Dominic ( 2022)
    Microglia are important regulators of central nervous system (CNS) development, homeostasis and immunity. Microglia exhibit a significant level of cellular plasticity which is facilitated by a dynamic transcriptional landscape that is modulated by genetic and environmental factors. However, our understanding of these mechanisms and the complexity of microglial transcription remains incomplete. Dysregulation of microglial gene expression and cellular function is implicated in numerous neurodevelopmental and neurodegenerative diseases, including the autoimmune demyelinating disease multiple sclerosis (MS). Therefore, understanding the transcriptional control of microglia provides invaluable knowledge of these cells and their unique roles in the CNS. The central aim of this thesis was to further characterise microglial transcription by investigating two genetic mechanisms that are strongly implicated in microglial biology. The first genetic mechanism that was investigated was the influence of small non-coding miRNAs on microglial gene expression. Here, I have comprehensively sequenced and characterised miRNA expression in microglia derived from a developmental mouse cohort and human resected brain tissue, identifying distinct subsets of known and novel miRNAs that regulate key microglial processes. Integrated miRNA-mRNA expression network analysis revealed miRNAs involved in microglial developmental processes including neurogenesis, myelination, endocytosis as well as key immune signalling pathways. Evidence from both species indicated a significant influence of age, but not sex on miRNA expression in these cells. The second genetic mechanism that was investigated was the transcriptional regulation of MERTK. MERTK has been shown to regulate microglial/myeloid cell-mediated apoptotic clearance and the resolution of inflammation in the CNS. More specifically, genetic variation in MERTK is associated with MS susceptibility. In this study, I investigated the single nucleotide polymorphism (SNP), rs7422195, which is associated with both changing MERTK expression in CD14+ve monocytes and modifying MS risk, hypothesising that this SNP is a causal disease modifying variant. While in vitro evidence suggested that variation at this genomic site drives differential protein binding, I was not able to experimentally validate specific protein interactions occurring at this region in vivo in primary monocytes. Furthermore, to increase our understanding of the transcriptional regulation of MERTK, I characterised the promoter region of MERTK gene, identifying distinct regulatory elements that may be responsible for driving gene expression in microglial/myeloid cells. Overall, this thesis has contributed to our knowledge of two separate, but important mechanisms that regulate microglial phenotype with important consequences for their function in the healthy and diseased CNS.