Anatomy and Neuroscience - Theses
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ItemThe role of neuronal TrkB in central nervous system myelination( 2019)Myelin, the specialised membrane surrounding many axons in the nervous system, is vital for the normal sensory and motor function as well as high order function such as learning and memory. Despite the importance of myelin in neural development and functions, little is known whether neuronally expressed molecules can promote the central nervous system (CNS) myelinating process. Through selectively deleting the expression of neurotrophin receptor TrkB in neurons in vivo (TrKB cKO), I have found that neuronal TrkB is essential for oligodendroglial cell production and lineage progression in multiple CNS regions including the lumbar spinal cord white matter tracts, cerebral cortex and corpus callosum and importantly de novo myelination, independent of axonal number and calibre in vivo. I have performed ultrastructural analysis of myelinated axons and found that TrkB cKO mice have significantly fewer myelinated axons compared to control mice during early postnatal development, indicative of delayed initiation of myelination. The analysis of myelin thickness via G ratio has revealed thinner myelin membranes in TrkB cKO mice compared to littermate control mice during development, persisting to late adulthood (n=3 mice/genotype/timepoint). This hypomyelinating phenotype has resulted in impaired myelin function, as evident by reduced optomoter responses in TrkB cKO mice compared to control mice. Moreover, I have found that TrkB cKO mice have significantly fewer oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes in both white and grey matter regions throughout postnatal development and into adulthood compared to littermate controls. A 24-hour EdU labelling experiment has demonstrated that neuronal TrkB expression is required for oligodendroglial production. Interestingly, the overall number of astrocytes and microglia has remained unchanged after neuronal TrkB deletion, suggesting a specific effect upon oligodendroglial lineage cells and myelination. In addition, assessment of neuronal morphology revealed no significant difference in overall dendritic branching including the number of primary and secondary processes. Taken together, results of this PhD study identify that TrkB is a novel neuronal signal that instructs oligodendroglial lineage development and de novo myelination within different neural circuits, indicating a new mechanism that underpins nervous system plasticity.
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ItemInvestigating the role of Gpr62 in oligodendrocyte development and central nervous system myelination( 2015)Myelination is a highly regulated process in the vertebrate nervous system whereby glial cells wraps axons with insulating myelin in order to allow rapid electrical conduction and metabolic support, a process which is fundamental for proper axon function and survival. Current research in the central nervous system (CNS) has focused on factors affecting oligodendroglial differentiation, yet the molecular interactions that occur post-differentiation between the oligodendrocyte and other CNS cell types during developmental myelination and myelin maintenance are still unclear. This thesis investigates the role the orphan G-protein coupled receptor (GPCR) Gpr62, as it relates to oligodendrocyte development and myelination. First, in silico analysis in conjunction with in situ hybridization showed that Gpr62 is specific to and highly expressed in the CNS, specifically within myelinating oligodendrocytes. Moreover, Gpr62 expression was shown to be regulated by myelin regulatory factor (MyRF), a key transcription factor regulating expression of major myelin genes. Secondly, a germline knockout mouse strain of Gpr62 was investigated. Gpr62 knockout mice showed no significant affect on myelin thickness, myelin gene or protein expression, initiation of myelination, or oligodendrocyte formation relative to their wildtype littermates, all of which were assessed in the developing, adult, and aging brain. An RNA-sequencing analysis revealed few differentially expressed genes between the knockout and wildtype controls, which were not validated in independent cohorts. Interestingly, the RNA- sequencing revealed several differentially expressed genes in close proximity to the Gpr62 locus. Further analysis indicated that these differentially regulated genes proximal to the Gpr62 locus were the result of Sv129-inherited DNA from the ES cells used for targeting, a result with significant implications for other germline knockout lines generated in the same fashion. Finally, forced viral expression of a tagged version of Gpr62 using an AAV virus demonstrated for the first time the cellular localization of the Gpr62 protein, showing expression within the oligodendrocyte extensions and myelin sheath, expressed along the adaxonal space. Collectively, these results indicated that Gpr62 is a novel oligodendrocyte-specific GPCR located along the myelin sheath, yet it is dispensable for myelin development and maintenance. Thus, failure to identify a role for Gpr62 may be the results Gpr62 having a function in the oligodendrocyte outside of developmental myelination. Indeed, the specificity and regulation of Gpr62 expression in oligodendrocytes by MyRF, and its localization within the myelin sheath, suggests Gpr62 may play a role in the oligodendrocyte that has yet to be explored.