Anatomy and Neuroscience - Theses
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ItemDevelopment of in vitro and in vivo models for the study of myelin plasticity( 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.
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ItemUsing induced pluripotent stem cells to model primary open-angle glaucoma( 2019)Glaucoma is a group of optic neuropathies that may be characterized by gradual degeneration of retinal ganglion cells (RGCs) and their axons leading to irreversible vision loss [1, 2]. Glaucoma is the second leading cause of blindness worldwide [3-5] and it is estimated that the number of people affected by the disease will reach 80 million by 2020, while more than 11 millions will be bilaterally blind [6]. In this project I focus on primary open-angle glaucoma (POAG), as it accounts for the majority of glaucoma cases worldwide. So far, multiple risk factors for glaucoma have been identified; however, the exact mechanism causing RGC loss in patients remains elusive. Furthermore, examination of RGCs affected in POAG is difficult pre-mortem due to their anatomical location. To overcome this problem, somatic cells can be reprogrammed into patient-specific induced pluripotent stem cells (iPSCs), which can be then differentiated into cell type of interest, i.e. RGCs. This PhD project consisted of several steps. First, I assessed the feasibility of transferring the iPSC culture into the automated platform. Using automation was essential to generate large number of samples required for analysis. The transition to automation was successful, as evidenced by maintenance of iPSC morphology, expression of pluripotency markers and ability to differentiate into derivatives of three germ layers. I also demonstrated that incorporating automation into human (h) iPSC culture allows standardization of maintenance and passaging procedures reducing inter-sample variability and human error. I subsequently used the platform to generate over 300 hiPSC lines for POAG modelling. In parallel, I optimized RGC differentiation protocol to obtain sufficient number of cells for their examination with single cell RNA sequencing (scRNA-seq). Next, iPSC-derived RGCs were subjected to scRNA-seq to gain in-depth information about transcriptomic differences between healthy controls and POAG patients. Understanding mechanisms underlying RGC function, maintenance of homeostasis and those conferring susceptibility to POAG is crucial to discover new therapeutic targets and commence the process of drug discovery.