Abstract
Purpose :
Increasing evidence has linked the methylation states of histone and DNA to the growth or regenerative ability of the optic nerve. We propose that by modulating the methyltransferases of histone (HMTs) or DNA (DNMTs), we may be able to reset the methylation pattern in mature neurons to rejuvenate their axon growth ability. We aimed to identify histone and DNA methyltransferase changes that are associated with retinal ganglion cell (RGC) development in mouse and investigate their potential in promoting RGC axon regeneration following optic nerve injury.
Methods :
RGCs from mice at developmental age embryonic day 16, postnatal day 0, and 10 were collected for RNA profiling of HMT and DNMT expression by quantitative PCR. Inhibitors of HMTs or DNMTs were added to the cultured primary mouse RGCs or retinal explants, and neurite outgrowth was quantified. Mice with conditional knockout of candidate HMT and DNMT specifically in RGCs driven under the VGLUT2 promoter were generated. Retinal explants collected from the conditional knockout mice were cultured for 7-days and evaluated for neurite outgrowth. Moreover, optic nerve crush injury was carried out in mutant mice, and nerve regeneration was quantified at 14 days after injury.
Results :
By screening methyltransferase changes associated with RGC development in mouse, we detected dynamic expression of a major HMT, enhancer of zeste homolog 1 (Ezh1) and key members of DNMTs, DNMT1 and DNMT3a, that correlates with the period of retinal neuritogenesis or retaining of optic nerve growth/regenerative capacity in developing RGCs. Small molecule pan inhibitors of Ezh1 and DNMTs increased neurite outgrowth in primary RGC or retinal explant cultures, with DNMT inhibition showing higher axon growth potential. We then generated mice carrying RGC-specific knockout of DNMT1 or DNMT3a and found that DNMT3a deficiency induced robust axon re-growth in retinal explant cultures as well as in the optic nerve crush injury model in mice.
Conclusions :
Collectively, we identified DNMT3a as the methyltransferase with a critical modulatory role of RGC axon growth or regeneration. Strategic inhibition of DNMT3a may open a potential therapeutic paradigm for neuronal degenerative diseases and to utilizing epigenomic modification as a mean of neurogenesis modulation.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.