- Anatomy and Neuroscience - Theses
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ItemDevelopment of in vitro and in vivo models for the study of myelin plasticityBujalka, Helena ( 2019)The central nervous system (CNS) constantly responds to changes in environmental stimuli by undergoing structural and functional modifications. Some stimuli induce persistent CNS changes which in turn underpin adaptive behaviours that enable individual animals to function in their unique environmental circumstances. This phenomenon, referred to as neuroplasticity, has been studied predominantly with respect to adaptive neuronal changes, and has focused primarily on synaptic changes and the molecular transduction mechanisms that mediate them. It is increasingly recognised, however, that glial cells can also be modified by external stimuli. Oligodendrocytes – the myelinating glia of the CNS which facilitate efficient nerve impulse conduction and support axonal metabolism – have also been demonstrated to undergo long term changes in response to environmental stimuli. Experience-dependent changes in oligodendrocyte number or myelination could underpin adaptive behaviours via modifications to neuronal metabolism and nerve impulse conduction. The emerging consensus is that stimulation – whether indirectly through modulating sensory, motor, or social experience, or directly through modulating neuronal activity – increases oligodendroglial lineage progression and myelin production. It has further been demonstrated that myelin plasticity is an axon-specific phenomenon whereby, when given the choice, myelin segments preferentially form on axons that are more highly active relative to those that are nearby but less active. The molecular mechanisms that mediate myelin plasticity are not well understood, and studies addressing this question have predominantly focused on the role of extracellular, pro-myelinating signals released by neurons in an activity-dependent manner. Comparatively little is known about the oligodendroglial intrinsic molecular transduction mechanisms that mediate myelin plasticity. This thesis aimed to develop a model system for studying myelin plasticity, including in particular to investigate the molecular transduction mechanisms that are triggered within oligodendroglia to mediate myelin plasticity. In developing such a model, two approaches were employed. First, an in vitro myelinating co-culture model was developed. A standard co-culture protocol was adopted and refined to produce robustly myelinating co-cultures of dorsal root ganglion (DRG) neurons and oligodendrocyte precursor cells (OPCs). To stimulate neuronal activity, both the hM3Dq pharmacogenetic and the channelrhodopsin-2 (ChR2) optogenetic techniques were explored. The pharmacogenetic stimulation technique was ineffective at driving DRG neurons to the levels of activity reportedly required for inducing myelin plasticity. In contrast, the optogenetic stimulation technique reliably drove DRG neurons to fire at the required frequency. Contrary to expectations, optogenetic stimulation did not increase myelin production in co-cultures, nor did it increase the propensity of myelin segments to preferentially form on optogenetically stimulated relative to control axons. The reasons for this are unclear, but are unlikely to be related to phototoxicity and are more likely to be explained by a negative effect of high ChR2 expression on myelination in these co-cultures. Second, an in vivo pharmacogenetic model was employed to drive activity of cortical neurons in juvenile hM3Dq transgenic mice. Contrary to expectations, there was no evidence for an activity-dependent increase in oligodendroglial lineage progression. The reasons for this are unclear, however they could relate to the young age of the animals in this relative to other studies of myelin plasticity or to the large population of neurons undergoing activity manipulation in this relative to other studies of myelin plasticity. The implications for glial plasticity, and for how it is studied, are discussed.
ItemMolecular mechanisms of BDNF signalling in central nervous system myelinationPECKHAM, HALEY ( 2016)Myelination within the CNS allows the fast and efficient transmission of action potentials along axons. Both myelin and the oligodendrocytes that generate myelin and wrap the axons are crucial for the development and maintenance of an adaptive and healthy nervous system. Interestingly, the process of myelination is responsive to neural activity so myelination also contributes to a form of neuroplasticity called white matter plasticity. Disorders as varied as multiple sclerosis and schizophrenia result when myelination is compromised, and experiences in the world, from childhood neglect and maltreatment to learning a second language, leave their trace in the white matter of our brains. Much remains to be discovered about the factors that regulate myelination and the signalling networks these factors utilise. Our laboratory has taken an interest in a growth factor that has a recognised role in synaptic plasticity and is epigenetically regulated. This molecule is brain-derived neurotrophic factor (BDNF). Our studies reveal that BDNF exerts a pro-myelinating effect in vitro and utilises Erk1/2 to mediate this effect. Specifically, BDNF acts on oligodendroglial TrkB receptors to myelinate the axons of DRG neurons in co-culture with oligodendrocytes. Studies in vivo confirm a role for BDNF, TrkB and Erk1/2 in myelination. Previous work from our laboratory indicates that loss of TrkB from myelinating oligodendrocytes leads to a phenotype of thinner myelin in the CNS, and my results reveal that when TrkB is deleted from the time of oligodendrocyte specification, this phenotype is restricted to thinner myelin only around large diameter axons. This suggests that there compensation occurs for the loss of TrkB but the factors that contribute to this putative mechanism of compensation are not yet known. Surprisingly, despite TrkB null oligodendrocytes only yielding a relatively mild hypomyelinating phenotype in vivo, I discovered that in the context of a myelinating co-culture, TrkB null oligodendrocytes exhibit a severely reduced capacity to myelinate. This led me to pose the question: how were oligodendrocytes that had a very limited capacity to myelinate in vitro able to myelinate the majority of axons normally in vivo? The result suggested that a cell or factor that was present in vivo but absent in the myelinating co-culture system was enhancing the oligodendrocytes capacity to myelinate. Interestingly, in vitro studies demonstrate that Fyn kinase associates with and is activated by TrkB, and that Fyn translocates neuronal TrkB receptors to lipid rafts in response to BDNF treatment. Similarly to BDNF, in vivo studies on Fyn kinase reveal that it too has a role in synaptic plasticity and myelination and this potentially suggests they utilise a common signalling pathway. In this work I interrogated BDNF signalling in oligodendrocytes and identified that BDNF and Fyn kinase do share a common signalling pathway: Fyn kinase is a downstream mediator of the pro-myelinating effect of BDNF. I then hypothesised that Fyn kinase could form part of a mechanism that compensates for the loss of oligodendroglial TrkB in myelination in vivo. I demonstrated that forced over-expression of Fyn kinase by TrkB null oligodendrocytes enhanced myelination in vitro. However, I also found that in isolated, cultured TrkB null oligodendrocytes the expression of Fyn kinase protein was lower than control oligodendrocytes although proportionally more of it was active. Taken together, these results suggest that Fyn kinase could, in principle, contribute towards redundancy in myelination but requires factors or cells, absent from the in vitro myelination assay, but present in vivo that increase its activation or expression level. Further studies are required to identify these factors and to determine if this putative compensation mechanism occurs in vivo. BDNF and Fyn kinase both have roles in neural plasticity and it is consistent with a maxim of biological economy that they would have complementary roles in white matter plasticity. BDNF switches oligodendrocytes into an activity dependent mode of myelination, and the findings of this study indicate that Fyn kinase mediates BDNF driven myelination, suggesting a role for Fyn kinase in white matter plasticity as well. Due to the burgeoning burden of neurodegeneration and the recognition that white matter plasticity is affected in mental illness and by experiences of trauma, it is imperative that we interrogate the molecular mechanisms of white matter plasticity to better target treatments to alleviate the distress caused by these challenges.
ItemInvestigating the role of Gpr62 in oligodendrocyte development and central nervous system myelinationHay, 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.
ItemInvestigation of the mechanisms BDNF utilises to promote central nervous system myelinationFerner, Anita Hilja ( 2014)Brain-derived neurotrophic factor (BDNF) promotes CNS myelination, which is crucial for normal CNS function. This thesis investigates the influence BDNF and its signalling exerts upon myelination. Surprisingly, I found that conditional deletion of TrkB in oligodendroglia exerted no effect on myelination in vivo. However, over-expression of Erk2, a molecule activated by BDNF signalling, promotes myelination in vitro. I found that Erk1/2 interacts with and phosphorylates the oligodendroglial transcription factors Olig1 and Olig2, which may contribute to Erk1/2’s promyelinating effects.