In vivo epigenetic reprogramming of retinal ganglion cells in aging mice with glaucoma - Glaucoma is a major cause of irreversible blindness worldwide that specifically targets retinal ganglion cells (RGCs) and their axons, causing loss of function, and ultimately cell death. Currently approved therapies for glaucoma patients lower intraocular pressure (IOP), which can slow but not stop disease progression. Importantly, these treatments only indirectly affect the retina and do not provide any direct effect on RGC survival. Therefore, an important unmet need is to identify new therapeutic targets for glaucoma patients that protect and/or rejuvenate RGCs. Although aging is a highly significant risk factor for glaucoma, very little is known about how and why aging causes patients to become more susceptible. Recent studies by colleagues demonstrated that RGC susceptibility to IOP-induced stress increases with age and coincides with histone and chromatin changes, indicating an epigenetic mediated mechanism. Moreover, IOP-induced-injury accelerated RGC aging, directly connecting injury with the aging process. Thus, there are at least two important components of glaucoma (i) stress induced RGC injury by elevated IOP, and (ii) the negative effects of aging that increases RGC susceptibility to stress. Therefore, the only way to completely understand the pathobiology of glaucoma is to use model systems that include the effects of both aging, and stress. Another pressing reason to understand how aging contributes to glaucoma is that we, along with our collaborators, showed that aging can be reversed. We were the first to reverse in vivo physiological aging of any tissues within the mouse. This was achieved using a gene therapy that triggered “transient epigenetic reprogramming” of RGCs via AAV2 delivery of three of the four Yamanaka factors OSK (Oct4, Sox2, Klf4) which rejuvenated RGCs and restored visual function lost due to aging. The experiments described herein will extend our previous research to (i) investigate the combined effects of aging and elevated IOP on the development of glaucoma and the RGC transcriptome, (ii) identify the age- and glaucoma-specific transcriptomic changes, and (iii) determine how these transcriptomic changes are altered by OSK epigenetic reprogramming. We hypothesize the rejuvenating effects of OSK mediated epigenetic reprogramming in aging mice with glaucoma are due to the reversal of both age- and IOP-induced changes in the RGC transcriptome, which are epigenetically dependent upon the Tet 1,2 mediated CpG methyl transferase enzymes. Three specific aims are proposed: (Aim 1) demonstrate elevated IOP induced glaucoma in aging mice is more severe and in vivo transient AAV2-OSK mediated epigenetic reprogramming restores visual function, rejuvenates RGCs, and prevents loss of RGCs and axons, (Aim 2) determine the age-associated changes that occur in the RGC transcriptome using scRNAseq and how AAV2-OSK epigenetic reprogramming reverses these changes and restores function, and (Aim 3) determine the elevated IOP induced changes in the RGC transcriptome of aging mice and how AAV2-OSK epigenetic reprogramming reverses these changes and restores function.
