Anatomy and Neuroscience - Theses

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    The role of the TAM receptors in CNS myelination and demyelination
    Akkermann, Rainer ( 2015)
    Multiple Sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS). The TAM family of receptor-tyrosine kinases, comprising Tyro3, Axl and Mertk, is widely expressed in the CNS and has been shown to be critical in the outcome of both toxin-induced, as well as inflammatory demyelination. In addition, previous work has demonstrated a direct impact of Gas6-mediated TAM signalling in the regulation of myelination. However, our understanding of the contributions of each individual receptor in these processes remains poor. In order to potentially utilise TAM receptor functions in the development of new therapeutics, it is of crucial importance to dissect these contributions as pan activation of all three receptors may have undesired off-target effects. The aim of this thesis was therefore to provide further insight into which TAM receptor transduces the pro-myelinating effects of Gas6 and which receptors may be important in limiting demyelination. Using electron microscopy, I found that while deletion of Gas6 only results in a mild, non-statistically significant reduction in developmental myelination, Tyro3 deficiency significantly impairs initiation of this process. In vitro data suggest that Tyro3 expressed on oligodendrocytes is required for normal myelination and that this receptor is required for Gas6-mediated enhancement of myelination. Oligodendrocytes deficient in Tyro3 display a reduction in the activation of Erk1, a signalling molecule involved in the induction of myelin gene expression, suggesting that the effects of Tyro3 upon myelination may be mediated at least in part by Erk1. I also could demonstrate that Tyro3 deficiency alone is not sufficient to significantly alter cuprizone-induced demyelination. This is also true for heterozygous microglia-specific Mertk deletion, indicating that homozygous deletion may be required to unravel potential effects of this receptor in experimental demyelination. Finally, injection of Axl or Mertk activating antibodies did not alter EAE disease course which may have been due to detrimental effects probably caused by antibody-mediated hyperactivation of the immune system. In summary, the data presented in this thesis describe for the first time that Tyro3 is a regulator of CNS myelination and that this is regulated by its expression on oligodendrocytes, possibly to an extent through Erk1 activation. Neither Tyro3 deletion nor partial deletion of Mertk in microglia alone affected cuprizone-induced demyelination. Finally, my findings suggest that TAM activating antibodies may not be ideal for therapeutic activation of these receptors in inflammatory conditions.
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    Investigating the role of Gpr62 in oligodendrocyte development and central nervous system myelination
    Hay, Curtis Mackenzie ( 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.