TOWARDS RETINA REGENERATION: THE EPIGENETIC BASIS OF NEURONAL AND AXON GUIDANCE GENE REGULATION

Degenerative disorders, such as glaucoma are among the leading causes of irreversible blindness worldwide. With these disorders affecting a larger fraction of the population yearly, the need for regenerative retina and optic nerve therapy is self-evident. Increasing evidence suggests that Müller glia are potential stem cells in the adult mammalian retina but have a limited potential to differentiate into retinal ganglion cell (RGC). Understanding the mechanisms regulating expression of RGC specific and axon-guidance genes during development and in retinal stem cells is key for successful optic nerve regeneration. I proposed that dedifferentation of Müller glia to RGCs is restricted by silencing of RGC specific and axon guidance genes by chromatin remodeling mechanisms such as DNA methylation. The DNA methylation pattern of RGC determining gene Atoh7 and axon guidance genes EphA5 and EphB1 in Müller glia derived spheres were investigated prior to and following demethylation. Bisulfite sequencing (BS) showed that in ImM10 spheres, the promoters of these genes exhibited a high frequency of methylation and quantitative RT-PCR showed that the genes are not transcribed. Demethylation with 5-azadeoxycytidine (AzadC) resulted in a significant decrease of methylation at the gene promoters and expression of their mRNA. Priming ImM10 derived spheres in the presence of EGF and differentiating the cells in the presence of BDNF resulted in increased expression of pluripotent, RGC developmental and retinal pigment epithelium specific genes. These gene expression changes were associated with cell morphology changes consistent with that of cells of epithelial identity. Furthermore, I showed that DNA methylation is required for this BDNF driven morphological change. Additional evidence for the role of DNA methylation in the regulation of the retinal temporal-nasal gradient of EphA5 was found in BS analysis of the P0 mouse retina. In the nasal retina a modest, but statistically significant increase in methylation was correlated with lower levels of receptor mRNA expression compared to the temporal retina. The inverse relationship between EphA5 promoter methylation and mRNA expression is consistent with a role for DNA methylation in modulating the spatial patterns of EphA5 gene expression in the retina and in silencing EphA5 expression in ImM10 cells. In addition to regulation by DNA methylation, the EphA5 proximal promoter contains four predicted TCF/LEF binding sites, suggesting a potential role for WNT signaling in the transcriptional regulation of EphA5. In luciferase assays, activation of the canonical WNT signaling pathway increased the activity of mouse EphA5 promoter constructs in HEK293. WNT signaling activation increased expression of the endogenous EphA5 gene in retinal progenitor cells in vitro but failed to upregulate expression of the gene in retinal explants ex vivo, possibly due to the lack of one or several WNT signaling components in the P7 retina. Taken together these data provide the first evidence for a direct role of DNA methylation in the regulation of RGC specific genes. The combinated effects of BDNF treatment and DNA demethylation are consistent with epigenetic limitations on the proliferative and neurogenic potential of Müller glia in vitro. Reversing epigenetic silencing of neuronal genes combined with growth factor treatments to enhance neuronal survival offer a novel strategy for increasing neurogenesis from Müller-derived stem cells.