Myelin is a protective layer wrapped around axons which helps them conduct electrical signals rapidly, and provides them with metabolic support. In the central nervous system (CNS), myelin is produced by specialised glial cells called oligodendrocytes. Loss of myelin (demyelination) is associated with degeneration of axons and many neurodegenerative disorders, including multiple sclerosis (MS). The restoration of myelin sheaths by remyelination may protect axons and help functional recovery of patients, but achieving this requires better understanding of how the process unfolds at the cellular level. To investigate the processes of de- and remyelination in vivo, I have characterised a transgenic zebrafish line in which expression of the bacterial enzyme nitroreductase (NTR) is driven under the myelin basic protein promoter, thus in myelinating glia. I treat larvae with the NTR substrate metronidazole (Mtz). The reaction between NTR and Mtz results in a toxic metabolite which selectively kills NTR-expressing cells. The treatment with Mtz consistently ablates two-thirds of oligodendrocytes while not harming the animals otherwise. Myelin sheaths continue to deteriorate after the end of the treatment, such that seven days later, extensive demyelination is observed by electron microscopy. By 16 days after Mtz-treatment, robust recovery has occurred, with no discernible axon loss and myelin thickness restored to control levels. At this time point, oligodendrocyte numbers have also returned to control levels. During the demyelinated phase, I observe a striking increase in microglia and macrophages in the spinal cord. In order to study the role of the innate immune system in recovery, I used a mutant line, irf8-/- which lacks a transcription factor essential for development of microglia and macrophages. I am in the process of determining the ability of these mutants to regenerate their oligodendrocytes and myelin; preliminary results suggest that they are able to restore their myelin sheaths fully, but seem to have a delay in regenerating their oligodendrocytes compared to wild-types. The model I have established can be used in the future to better understand the consequences of demyelination to axon health, as well as chemical screening to identify compounds that could accelerate the remyelination process or enhance the thickness of myelin generated during remyelination. Insights arising from such studies will be useful in designing strategies to reduce axon loss and improve myelin regeneration in demyelinating diseases.

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Remyelination is a spontaneous regenerative process whereby myelin sheaths are restored to demyelinated axons. Key players in this process are oligodendrocyte progenitor cells (OPCs), a widespread population of CNS progenitor cells which persist into adulthood. Remyelination is impaired in patients with chronic demyelinating conditions such as Multiple Sclerosis, and as with other regenerative processes, its efficiency declines with increasing age. Hence, there is a need for the development of therapeutic interventions that will aid in promoting endogenous remyelination when the endogenous regenerative potential is compromised. The nuclear receptor RXR$\gamma$ is an important positive regulator of OPC differentiation and an accelerator of endogenous remyelination in aged rats. RXR$\gamma$ functions as a ligand-induced transcription factor and is able to regulate gene transcription. It does so by heterodimerising with other nuclear receptors and recruiting co-regulators involved in chromatin remodelling. However, we lack understanding on the specific mechanism by which RXR$\gamma$ promotes OPC differentiation. With the work presented in this thesis I demonstrate that RXR$\gamma$ function is regulated at multiple signalling levels. Proximity ligation assays revealed that RXR$\gamma$ remains consistently bound to its partners throughout the oligodendrocyte lineage, and the biological relevance of each heterodimer is determined by the dynamic association of co-regulators. This is in turn influenced by ligand presence and subcellular receptor localisation. To identify the genes controlled by RXR$\gamma$ in OPCs I carried out ChIP sequencing, which revealed genes involved in proliferation and cell cycle control. Further functional assessments aided me in the development of a hypothesis whereby RXR$\gamma$ activation does not directly influence oligodendrocyte formation, but rather promotes cell cycle exit thereby accelerating and facilitating OPC differentiation. Altered nuclear receptor expression and ligand presence in ageing OPCs may consequently impair this process. My thesis provides an alternative hypothesis to how RXR$\gamma$ regulates lineage cell progression, highlighting a new avenue in the development of therapeutic interventions targeting generic stem cell functions for which drugs are already FDA approved, rather than oligodendrocyte-specific pathways.

Multiple Sclerosis (MS) is an autoimmune demyelination disease of the central nervous system (CNS). The traditional immunotherapies so far have not been able to stop the progression of the disease. Galanin (GAL) is an inducible neuropeptide. It has diverse regulatory effects in the nervous. The expression of galanin is induced by the administration of estrogen and is increased during pregnancy in rodents. In women with MS, pregnancy has a protective effect associated with significant remission of MS symptoms during the second and third trimester. In this study, using the cuprizone (CPZ) demyelination model of MS I demonstrated that GAL has a pronounced neuroprotective effect on demyelination and remyelination, with estrogen the protection was further enhanced. Moreover, I also found differential activation of GAL receptors in the demyelination and remyelination processes. These results suggest GAL alone or combined with estrogen might introduce next generation strategies for the treatment of MS.

Multiple Sclerosis (MS) is an autoimmune demyelination disease of the central nervous system (CNS). The traditional immunotherapies so far have not been able to stop the progression of the disease. Galanin (GAL) is an inducible neuropeptide. It has diverse regulatory effects in the nervous. The expression of galanin is induced by the administration of estrogen and is increased during pregnancy in rodents. In women with MS, pregnancy has a protective effect associated with significant remission of MS symptoms during the second and third trimester. In this study, using the cuprizone (CPZ) demyelination model of MS I demonstrated that GAL has a pronounced neuroprotective effect on demyelination and remyelination, with estrogen the protection was further enhanced. Moreover, I also found differential activation of GAL receptors in the demyelination and remyelination processes. These results suggest GAL alone or combined with estrogen might introduce next generation strategies for the treatment of MS.

Multiple sclerosis is thought to be an autoimmune disease that causes lesions and demyelination of axons in the central nervous system. A reduction in relapses is seen in the third trimester of pregnancy when estrogen levels are highest, followed by an increase in relapses in the first three months post-partum when estrogen levels drop. This thesis focuses on the effect of 17beta- and 17alpha-estradiol on myelination and remyelination in cultured rat cerebellar slices. No reproducible effect of 17beta-estradiol on myelination was detected. However, during lysolecithin-induced demyelination it had a possible protective effect in males and females, although it was not statistically significant. During myelination, 100 nM 17alpha-estradiol caused a small but significant decrease in MBP expression in females. During lysolecithin-induced demyelination, it had a possible, but not statistically significant, protective effect in males. Neither 17beta- nor 17alpha-estradiol had a reproducible or significant effect on remyelination in males or females.

Multiple sclerosis is thought to be an autoimmune disease that causes lesions and demyelination of axons in the central nervous system. A reduction in relapses is seen in the third trimester of pregnancy when estrogen levels are highest, followed by an increase in relapses in the first three months post-partum when estrogen levels drop. This thesis focuses on the effect of 17beta- and 17alpha-estradiol on myelination and remyelination in cultured rat cerebellar slices. No reproducible effect of 17beta-estradiol on myelination was detected. However, during lysolecithin-induced demyelination it had a possible protective effect in males and females, although it was not statistically significant. During myelination, 100 nM 17alpha-estradiol caused a small but significant decrease in MBP expression in females. During lysolecithin-induced demyelination, it had a possible, but not statistically significant, protective effect in males. Neither 17beta- nor 17alpha-estradiol had a reproducible or significant effect on remyelination in males or females.

Cortical injuries, such as those caused by stroke and other insults, are the leading cause of death and disability worldwide. While thrombolytics can be used to restore blood flow immediately following the onset of symptoms of an ischemic stroke, there are currently no neurorestorative therapeutics that can enhance long-term recovery of function following injury. Neuregulins are a family of growth factors involved with the survival and function of neurons and glia. Glial Growth Factor 2 (GGF2) is an isoform of neuregulin-1 that has demonstrated significant effects in the recovery of function in rodent models of stroke. Histological analyses suggest GGF2 promotes recovery by enhancing endogenous mechanisms to reduce inflammation and promote plasticity. To further explore the efficacy of GGF2, we used our rhesus monkey model of cortical injury and fine motor impairment to compare the rate and pattern of recovery in monkeys treated with GGF2. Twenty-four young adult male monkeys (ages 4-10 years old) were pre-trained on our task of fine motor function of the hand before undergoing surgery to produce a cortical lesion limited to the hand representation of the primary motor cortex on one side. Intravenous (IV) administration of GGF2 (0.5 mg/kg) began 24 hours after surgery and continued daily for 7 days. This was followed by 21 days of sub-cutaneous administration of GGF2 at two different dose levels (0.1 mg/kg or 0.3 mg/kg). Post-operative testing began two weeks after the lesion and continued for 12 weeks. All trials were video recorded and latency to retrieve a reward was quantitatively measured to assess the trajectory of post-operative response latency and grasp pattern compared to pre-operative levels. The results showed no significant differences between the groups in the recovery of fine motor function. Moreover, all vehicle control monkeys returned to their pre-operative levels of latency and grasp pattern despite no significant differences in lesion volume from the experimental groups. In addition to measures of behavioral recovery, we processed the brain tissue with immunohistochemistry to investigate the role of GGF2 treatment in reducing the pro-inflammatory response of microglia and enhancing axonal sprouting and synaptogenesis following injury. All groups had a greater density of Iba1+ microglia in the perilesional grey matter and sublesional white matter, but there were no significant differences in the numerical density or phenotypes of microglia between the groups. We also found no significant differences in axonal sprouting between the groups. However, GGF2 treatment did enhance expression of synaptophysin in the contralesional hemisphere of monkeys that received subcutaneous doses of GGF2 following the initial 7 days of intravenous GGF2 treatment. This suggests that high dose GGF2 treatment may enhance plasticity of compensatory circuits involving the intact hemisphere and that this effect is dose dependent. In addition, we followed up these analyses using a subset of monkeys from the larger GGF2 study to optimize and validate a method that labels newly synthesized myelin. This is accomplished by in vivo administration of a choline analog, propargylcholine (P-Cho) that labels newly synthesized myelin and can be visualized post-mortem. Our results demonstrate effective and stable incorporation of P-Cho with post injection survival of 1 to 6 weeks. Using this method to quantify new myelin after cortical injury to the primary motor cortex, showed significantly greater P-Cho labeling and co-localization with myelin basic protein (MBP) in the white matter underlying the ipsilesional hemisphere when compared with the contralesional hemisphere. This validates P-Cho for assessing myelin plasticity in a nonhuman primate brain and how it might be used to assess therapeutics aimed at inducing remyelination and enhancing myelin synthesis. Finally, this dissertation also includes the comparison of sex differences in recovery of motor function after cortical injury. In a cohort of aged male and female monkeys, postmortem analysis showed no differences in lesion volume between the males and females. However, behaviorally, the females returned to their pre-operative latency and grasp patterns significantly faster and more completely than the males. These findings demonstrate the need for additional studies to further investigate the role of estrogens and other sex hormones that may differentially affect recovery outcomes in the primate brain. Collectively, the results presented in this dissertation highlight the complexity of evaluating treatments and mechanisms underlying recovery of function by enhancing neuroplasticity. Specifically, we were unable to effectively evaluate GGF2 as a treatment due to the behavioral recovery of all control monkeys. Follow up studies should investigate treatment with GGF2 in aging monkeys and compare the results with our findings. Additionally, it is necessary to further explore the recovery of fine motor function in young monkeys. Finally, our study showing sex differences in recovery of function provides evidence that sex hormones may play a significant role in providing neuroprotection in the aging brain following cortical injury. Future studies should measure post-operative estrogen levels and evaluate supplementation as a potential treatment option.

Central nervous system (CNS) myelination is important for proper nervous system function in vertebrates. In demyelinating diseases such as multiple sclerosis, autoimmune-mediated myelin destruction results in neurological impairment; and although remyelination does occur spontaneously, it is poorly understood and insufficient in humans. Zebrafish (Danio rerio) are known to harbour tremendous regenerative capacity of various CNS tissues; however, there is presently only little knowledge of their myelin repair efficiency. An experimental model of myelin injury in zebrafish would permit study of the mechanisms involved in successful remyelination and could potentially guide the development of novel therapeutic agents for mammalian remyelination. This doctoral thesis describes the characterisation of the novel myelin protein Claudin k in zebrafish, demonstrates the establishment of adult zebrafish as an experimental model for CNS de- and remyelination and explores some mechanisms underlying myelin repair. A variety of myelin markers have previously been investigated in zebrafish, including myelin basic protein and myelin protein zero. However, the use of these is limited by either late developmental expression or presence in compact myelin only. Claudin k is a novel tight junction protein specific to zebrafish CNS and PNS, which can be observed early in development and throughout nervous system regeneration. Utilising specific antibodies and a novel transgenic zebrafish line, in which the claudin k promoter drives the expression of green fluorescent protein in myelinating cells, the studies herein characterise the expression of Claudin k, demonstrate the fidelity of the transgenic construct, and investigate the relationship of Claudin k with established myelin and CNS inflammation markers. Data demonstrate that Claudin k expression closely resembles expression patterns of the endogenous gene, and as such provides a key tool for examining CNS myelination in zebrafish. For the study of de- and remyelination in the zebrafish, the experiments herein describe the use of lysophosphatidylcholine (LPC), a detergent-like myelin toxin, which is used widely in rodent models to demyelinate axons. Its application to the adult zebrafish optic nerve induced focal demyelinating lesions, critically without detectable axonal injury, and permitted the study of time course and efficiency of remyelination. Myelin in the lesion area was reduced as detected by both immunohistochemistry and electron microscopy at 8 days post lesion (dpl), and return of the markers to control levels suggested regeneration by 28 dpl. In addition microglial activation was observed along the optic pathway, which also returned to levels compared to unlesioned control by 28 dpl. In young zebrafish (aged 4-6 months), the myelin thickness of remyelinated fibres showed no difference to the pre-lesion state, which is different to mammals, where the myelin thickness is reduced. However, in old fish (aged 18+ months), remyelinated fibres presented with thinner myelin, suggesting that the regenerative capacity of zebrafish declines with age. While the zebrafish as an experimental system has tremendous benefits, such as potential for drug screens using the transparent larvae, capacity for transgenesis and live imaging, experimental models in zebrafish potentially bear several limitations, in particular their distant relationship to humans. To determine whether zebrafish remyelination involves homologous signalling mechanisms to mammals, demyelinated zebrafish optic nerves were treated with human recombinant Semaphorin 3A, an axonal guidance molecule which is well known to inhibit oligodendrocyte precursor cell (OPC) recruitment and remyelination in mammals. Results demonstrated fewer oligodendroglial cells at 14 dpl and less myelinated fibres at 28 dpl in the optic nerve lesion area compared to control treated animals, supporting the hypothesis that zebrafish remyelination may indeed respond to human signalling molecules. Taken together, the findings in this doctoral thesis suggest that this new experimental zebrafish-based model of CNS remyelination can be added to the suite of current models to better understand the remyelination process and that some signalling mechanisms observed in mammals around myelination and OPC recruitment are likely conserved in the zebrafish. In addition, it could potentially be used to discover novel therapeutic targets that promote myelination in injury.