No description available.

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.

oligodendrocytes migrate from the corpus callosum into the overlying cortex. The incidence of cilia did not change markedly across age groups, and did not vary consistently with the number of processes per cell, which was used as an indication of the maturation stage of OPCs and young OLs. The mean percent of Olig1 immunopositive (Olig1+) cells having cilia across ages was 33.1% + 16.5%, with all ages combined. In O4+ cells of these mice, 56.7 + 3.6% had primary cilia. If it is the case that adult OLs do not have cilia, the point in the lineage when primary cilia are lost is still unknown. Adult mice that had been injected with cyclopamine to block cilia-dependent Shh signaling were examined to determine whether the rate of generating new OPCs was influenced. In the CC of control mice, the numerical density of Olig1+/BrdU+ cells was 1.29 + 0.07/mm2 was reduced to 0.68 + 0.38/mm2 in the cyclopamine-injected group, and the numerical density of all BrdU+ cells (including both Olig1+ and Olig1- cells) of 4.55 + 1.50/mm2 in the control group was reduced to 3.14 + 1.27/mm2 in the cyclopamine-injected group. However, there were only 2 mice in each group and the differences were not statistically significant.

Major depressive disorder is a common severe psychiatric disorder with unknown etiology. Recent studies show that the loss and malfunction of oligodendrocytes are closely related to the neuropathological changes in depression, which can be reversed by antidepressant treatment. St. Johns wort is an effective and safe herbal treatment for depression in several clinical trials. However, the underlying mechanism of its therapeutic effects is unclear. In this study, we evaluated the effects of hyperforin, a major active component of this herb, on the proliferation, mitochondrial function and development of oligodendrocytes. We have demonstrated that hyperforin increases mitochondrial function and prevents mitochondrial toxin-induced cytotoxicity in oligodendrocyte lineage cells. Hyperforin promotes the maturation of oligodendrocytes but does not increase the proliferation of oligodendrocyte progenitor cell line and neural stem/progenitor cells. Our findings suggest that chronic hyperforin treatment may stimulate the development and function of oligodendrocytes. These results suggest a new mechanism of hyperforin in depression treatment. Future in vitro and in vivo studies are required to further characterize the mechanisms of hyperforin.

The Quaking (qk) gene has been implicated in the development of oligodendroglial cells which are the primary source of myelin in the mammalian central nervous system (CNS). Qk encodes three alternatively spliced variants, QKI-5, QKI-6 and QKI-7, all of which are RNA binding proteins. Loss of QKI-6 and QKI-7 results in a dysmyelination phenotype that is present shortly after birth while loss of QKI-5 results in embryonic lethality. CG4 oligodendroglial cells were transfected with either pIRES2-QKI5 to up regulate QKI-5 expression or a QKI-5 specific siRNA to down regulate QKI-5. Cells were cultured for 6d in differentiation medium (DM) following which total RNA and protein was collected from the cell cultures, and coverslips with attached cells were processed for immunofluorescence. Increased QKI-5 expression following transfection with pIRES2-QKI5 resulted in increased Sirt2 and Plp mRNA expression, but did not affect SIRT2 and PLP protein expression. Down regulation of QKI-5 expression had no significant effect on mRNA or protein levels for QKI-6, QKI-7, Plp or Sirt2. Immunocytochemistry revealed that up regulation of QKI-5 resulted in significantly higher percentage of A2B5+ cells and a lower percentage of GalC+ cells, whereas siRNA treatment resulted in an increase in the percentage of GalC+ cells. Our results suggest QKI-5 regulates oligodendrocyte differentiation and modulates the transcription and availability of target mRNAs, such as Sirt2 and Plp, for translation. In order to gain a more complete understanding of the relationship between qk and both Sirt2 and Plp, future studies would include RNA coimmunoprecipitation, miRNA studies, and expanding the list of target genes to include various cell cycle components.

Major depressive disorder is a common severe psychiatric disorder with unknown etiology. Recent studies show that the loss and malfunction of oligodendrocytes are closely related to the neuropathological changes in depression, which can be reversed by antidepressant treatment. St. Johns wort is an effective and safe herbal treatment for depression in several clinical trials. However, the underlying mechanism of its therapeutic effects is unclear. In this study, we evaluated the effects of hyperforin, a major active component of this herb, on the proliferation, mitochondrial function and development of oligodendrocytes. We have demonstrated that hyperforin increases mitochondrial function and prevents mitochondrial toxin-induced cytotoxicity in oligodendrocyte lineage cells. Hyperforin promotes the maturation of oligodendrocytes but does not increase the proliferation of oligodendrocyte progenitor cell line and neural stem/progenitor cells. Our findings suggest that chronic hyperforin treatment may stimulate the development and function of oligodendrocytes. These results suggest a new mechanism of hyperforin in depression treatment. Future in vitro and in vivo studies are required to further characterize the mechanisms of hyperforin.

No description available.

Oligodendrocytes (OL) are the myelinating cells of the central nervous system (CNS). A series of complex cell signaling events in the CNS ensures successful myelination. Various molecular cues including growth factors, transcription factors and cytokines regulate myelination by inducing OL migration, proliferation and differentiation. Plateletderived growth factor A (PDGF-A) and fibroblast growth factor 2 (FGF2) are two of the most well characterized regulators of OP migration. The current study hypothesizes that PDGF-A and FGF2 regulate the migration of OP through transient activation of the extracellular signal-regulated protein kinase (ERK) signaling pathway. The results show that activation of ERK is required for OP migration. It also demonstrates the significance of threshold levels of growth factors and temporal regulation for OP migration. Furthermore, the chemokine CXCL1 has been shown to play a critical role in regulating the dispersal of OP during development, although the mechanisms underlying this regulation are unknown. Previous studies have shown that calcium flux is required for OP migration. CXCL1 induces calcium flux in cells; therefore we hypothesized that CXCL1 inhibition of OP migration was regulated via changes in intracellular calcium flux. However, our results show that CXCL1 inhibition of OP migration is independent of calcium signaling. In addition, we show that CXCL1 inhibition of OP migration is specific to PDGF-A induced migration. Lastly, the current study identifies a transcriptional regulator, methyl-CpG-binding protein 2 (MeCP2) as regulating the expression of myelin specific genes in a transgenic mouse. Interestingly, gene expression of myelin associated proteins myelin basic protein (MBP), myelin associated glycoprotein (MAG)and proteolipid protein (PLP), which play an important role in regulation of OL differentiation and subsequent formation of myelin of the myelin sheath, where found to be dysregulated. Overall, these findings reveal previously unknown roles of various intrinsic factors in successive phases of OL development. It aims to provide a better understanding of complexity to myelin development, function and disease.

Oligodendrocytes (OL) are the myelinating cells of the central nervous system (CNS). A series of complex cell signaling events in the CNS ensures successful myelination. Various molecular cues including growth factors, transcription factors and cytokines regulate myelination by inducing OL migration, proliferation and differentiation. Plateletderived growth factor A (PDGF-A) and fibroblast growth factor 2 (FGF2) are two of the most well characterized regulators of OP migration. The current study hypothesizes that PDGF-A and FGF2 regulate the migration of OP through transient activation of the extracellular signal-regulated protein kinase (ERK) signaling pathway. The results show that activation of ERK is required for OP migration. It also demonstrates the significance of threshold levels of growth factors and temporal regulation for OP migration. Furthermore, the chemokine CXCL1 has been shown to play a critical role in regulating the dispersal of OP during development, although the mechanisms underlying this regulation are unknown. Previous studies have shown that calcium flux is required for OP migration. CXCL1 induces calcium flux in cells; therefore we hypothesized that CXCL1 inhibition of OP migration was regulated via changes in intracellular calcium flux. However, our results show that CXCL1 inhibition of OP migration is independent of calcium signaling. In addition, we show that CXCL1 inhibition of OP migration is specific to PDGF-A induced migration. Lastly, the current study identifies a transcriptional regulator, methyl-CpG-binding protein 2 (MeCP2) as regulating the expression of myelin specific genes in a transgenic mouse. Interestingly, gene expression of myelin associated proteins myelin basic protein (MBP), myelin associated glycoprotein (MAG)and proteolipid protein (PLP), which play an important role in regulation of OL differentiation and subsequent formation of myelin of the myelin sheath, where found to be dysregulated. Overall, these findings reveal previously unknown roles of various intrinsic factors in successive phases of OL development. It aims to provide a better understanding of complexity to myelin development, function and disease.

Myelin is essential for normal nervous system conduction as well as providing metabolic support for the ensheathed axon and has been implicated to influence axon calibre (diameter of the axon body) growth. In demyelinating diseases, the disruption of these functions causes axon degeneration resulting in neurological impairment. The neurons that are myelinated in the CNS and the axon-oligodendrocyte (axon- OL) interactions that might regulate axon calibre and myelination during myelinated axon formation are still mostly unknown, preventing a deeper understanding of CNS development and repair. This doctoral thesis identifies a specific subset of interneurons that are myelinated and investigates the axon-oligodendrocyte interactions during axon calibre growth and initial myelination. In the zebrafish spinal cord, Commisural Primary Ascending interneurons (CoPA), Circumferential Descending interneurons (CiD) and reticulospinal neurons are amongst the first to be myelinated, whereas Commisural Bifurcating Longitudinal interneurons (CoBL) and Circumferential Ascending interneuron (CiA) are not myelinated during early developmental stages. Of the myelinated neurons, axon calibre of reticulo spinal neurons is increased in time with myelin ensheathment, while the axon calibre of CoPA and CiD interneurons is not increased with the onset of myelination. In order to investigate whether there might be a causative relationship between axon calibre increase and myelin ensheathment, the majority of oligodendrocytes were eliminated by olig2 morpholino knockdown. In the absence of oligodendrocytes, the axon calibre of reticulospinal neurons was normal, demonstrating that axon calibre growth is independent of axon-OL interactions and myelin ensheathment. In order to further investigate which aspects of myelinated axon formation might be regulated by axon-OL interactions, axonal activity was reduced through inhibition of synaptic vesicle release by global expression of Tetanus-toxin (TetTx). TetTx treated zebrafish showed a 40% decrease of myelinated axons in the spinal cord. Interestingly, only 10% of this reduction was caused by a decrease in oligodendrocyte number in the spinal cord. Single cell analysis of individual oligodendrocytes revealed a 30% reduction of myelin sheaths per oligodendrocyte in TetTx treated animals, indicating a positive correlation between synaptic vesicle release and the extent of myelination. Timelapse analysis of the myelinating behaviour of individual oligodendrocytes revealed that the decrease in myelin sheaths per cell in the absence of synaptic vesicle release results from a reduction in the initial formation of sheaths rather than an increased retraction of myelin sheaths. Furthermore, individual myelin sheaths formed by the same oligodendrocyte exhibit a dynamic range of different growth rates in control animals, which was reduced to a more uniform, slow growth of myelin sheaths in the absence of synaptic vesicle release. This suggests that local axon-OL interactions can regulate the dynamic myelin sheath growth through synaptic vesicle release. The analyses in this doctoral thesis identifies a subset of the neurons that are myelinated during the onset of myelination in the zebrafish spinal cord, demonstrates that axon caliber growth of these neurons is independent of myelin ensheathment and that axon-OL interactions mediated by synaptic vesicle release can regulate the extent of myelination and influence the dynamic myelinating behavior of oligodendrocytes in vivo. These findings begin to elucidate the axon-OL interactions underlying myelinated axon formation during CNS development, from which future studies might derive neuro-regenerative treatments for demyelinating diseases.