Abstract
Oligodendrocytes insulate axons in the central nervous system (CNS) with myelin sheaths, providing metabolic support and improving saltatory conduction. In multiple sclerosis (MS), myelin sheaths are stripped from axons in a process termed demyelination. Oligodendrocyte progenitor cells (OPCs) migrate to the site of injury in response to this damage, proliferate, differentiate into mature oligodendrocytes, and carry out the myelin regenerative process known as remyelination. Unfortunately, this is not always successful, resulting in a lack of functional recovery and subsequent disease progression. Research has shown that immune signalling is important in facilitating effective remyelination, and our group has shown that regulatory T cells (Treg) are required for successful myelin regeneration. However, the molecular mechanisms within OPCs underlying this effect of Treg are unknown. To address this gap in knowledge I screened, validated, and assessed for functional significance, a candidate identified from RNA sequencing (RNAseq) of OPCs stimulated by Treg-conditioned medium.Treg-conditioned media generated for this study were confirmed to be bioactive and validated through an assay confirming that media drove a significant increase in oligodendrocyte differentiation. Ribonucleic acid (RNA) was collected from OPC cultures treated with Treg-conditioned media for RNAseq and selected candidates were validated through quantitative polymerase chain reaction (qPCR). Antagonism of one such candidate, transient receptor potential channel 4 (TRPC4), resulted in a significant abrogation of the Treg-mediated effect on oligodendrocyte differentiation, as measured by myelin basic protein (MBP) area. I observed that TRPC4 is expressed in demyelinated spinal cord lesions. Cellular profiling identified that in this setting, TRPC4 is expressed in oligodendrocytes, neurons, and astrocytes, but not in microglia or macrophages. A downward but not significant trend was observed for the number of TRPC4+ oligodendrocytes, neurons, and astrocytes observed in mice lacking Treg. Antagonism of TRPC4 had no effect on myelination in brain slice cultures, but decreased Treg-mediated remyelination of brain slices in a dose dependent manner. Brain slices generated from TRPC4 knockout (KO) mice had a significant increase in axonal density during myelination, and a significant reduction in remyelination at 14 days. Loss of TRPC4 through antagonism or genetic deficiency had no effect on OPC proliferation or oligodendrocyte differentiation in vivo.
The results from this thesis identified candidates involved in the molecular mechanisms of immune-mediated oligodendrocyte differentiation, and identified potential novel therapeutic targets for myelin repair in MS and other demyelinating diseases that warrant further investigation.
Thesis is embargoed until 31 December 2028.
| Date of Award | Dec 2023 |
|---|---|
| Original language | English |
| Awarding Institution |
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| Sponsors | Northern Ireland Department for the Economy & Biotechnology & Biological Sciences Research Council |
| Supervisor | Denise Fitzgerald (Supervisor) & Beckie Ingram (Supervisor) |