Wallis, Marcus
(2020)
Investigating the potential role of DNA (hydroxy)methylation in the control of post-stroke axon growth.
PhD thesis, University of Nottingham.
Abstract
An important mechanistic factor of functional recovery following ischemic stroke is the sprouting of new axons and repair of damaged axons in the region around the infarct. To induce this axonal growth, regulation of gene expression is required. Whilst some previous research has characterised a role of DNA methylation (5-methylcytosine, 5mC) in this regulation, less is known about what role, if any, oxidised forms of 5mC perform. 5mC is oxidised by Tet enzymes to form 5-hydroxymethylcytosine (5hmC), which acts mainly as an intermediate of demethylation and is therefore associated with increased gene expression. It has been previously shown that global 5hmC levels increase following ischemic stroke, although no determination has been made as to how this increase influences post-stroke axon growth.
The use of an in vivo model of ischemic stroke helped to determine that post-stroke increases in global 5hmC are delayed, peaking at 2-7 days post-stroke, and return to baseline after 1 month. The temporal peak in 5hmC coincides with previously determined peak expression of axon growth-related genes, suggesting a potential role of DNA hydroxymethylation in the regulation of post-stroke axon growth via altered gene expression. Furthermore, isolation of specific cell types from the post-ischemic brain revealed that post-stroke increases in 5hmC are observed not only in the total cell population, but also in microglial cells specifically. These post-stroke increases in 5hmC are accompanied by significantly increased expression of microglial activation markers. This therefore suggests that DNA hydroxymethylation is involved in the regulation of the brains innate immune response to ischemic stroke, which in itself has implications on post-stroke axon growth.
An in vitro model of ischemic stroke and reperfusion was also used to determine a direct effect of Tet enzyme expression on axon growth, with decreased expression of Tet2 and Tet3 each leading to significantly reduced axon growth. Additionally, it was seen that global 5hmC levels and Tet3 expression are significantly increased following hypoxic insult. Taken together, this suggests that ischemia may act to induce Tet3 expression in the brain, which subsequently acts to promote axonal growth as part of the central nervous system’s endogenous repair response.
Overall, the body of work detailed in this thesis has revealed a potential role of 5hmC, and the Tet family of enzymes that catalyse its formation, in the regulation of post-stroke axonal growth. Though the novelty of these findings necessitates that further research be performed to consolidate this effect, it is hoped that this methylomic response to ischemic stroke can be used to develop delayed therapeutic treatments aimed at promoting post-stroke axonal growth and functional recovery.
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