Abstract/Summary Aging is the principal risk factor for age-related macular degeneration (AMD), a neurodegenerative disease characterized by the irreversible loss of vision. Clinical and mouse studies indicate that consumption of diets with higher dietary glycemic indices increase AMD risk. Atrophy of the retinal pigmented epithelium (RPE) layer is an AMD hallmark that precedes photoreceptor cell loss. However, the mechanisms underlying RPE impairment with aging and exacerbation by poor diet are unclear. Thus, therapeutic approaches to maintain RPE function with aging and prevent AMD are yet to be developed. Epigenetic processes (DNA modifications and chromatin accessibility) in the RPE may play a central mechanistic role in the pathogenesis and progression of AMD. DNA modifications, [cytosine base methylation and hydroxymethylation (mC and hmC respectively)], are fundamental regulators of DNA accessibility and gene regulation/expression. A barrier to progress in understanding the role of epigenetic mechanisms in RPE aging and DNA modifications in particular, has been the lack of quantitatively accurate, genome-wide data in this specific cell type. Without the knowledge of the specific genomic locations of altered modifications/accessibility with aging it is impossible to design mechanistic studies that unravel the functional effects of epigenetic reconfiguration. Therefore, the critical next step for the field is to generate these genome-wide maps of mC and hmC in CG and CH contexts and genomic accessibility in the primary cellular site of AMD pathogenesis, the RPE, from both sexes across the lifespan. To address this critical issue, we have developed a cell-type specific, tamoxifen-inducible Cre, transgenic NuTRAP model to allow isolation of nucleic acids (DNA & RNA), specifically from RPE cells. In Aim 1, changes in mC/hmC and chromatin accessibility patterns with aging will be examined by whole genome oxidative bisulfite sequencing (WGoxBS) and ATAC-seq in RPE. In Aim 2, the RPE-specific differential changes in the translatome will be identified as a function of aging. In prior studies we have determined that age-related DNA modification changes can be prevented by caloric restriction. In aim 3, we will interrogate the potential of Western and ketogenic dietary patterns, in combination with impaired oxidative stress resolution pathways, to exacerbate or ameliorate changes in the RPE epigenome and gene expression profiles. Paired epigenomic and transcriptomic data from the same animals will be used to: 1) assess aging with ‘epigenetic clocks’ in RPE, 2) determine the role of altered modification patterns in age- and dietary/oxidative stress- related changes in gene expression, 3) determine enrichment of differential modifications/accessibility in regulatory regions of the genome, and 4) identify and refine genomic loci for epigenome editing. These studies will determine critical genomic regions with altered DNA modification patterns that can be manipulated in future interventional studies. The ultimate goal of the research is to develop clinical interventions that target the RPE epigenome to maintain visual function with aging and prevent AMD.

Project Narrative Our scientific premise is that maladaptive changes in DNA modifications and chromatin accessibility accumulate in the retinal pigmented epithelium (RPE) with aging leading to RPE dysfunction and contributing to age-related macular degeneration (AMD) pathogenesis. Whether RPE epigenetic aging is exacerbated by poor diet and can be slowed by ketogenic diet will also be tested. These studies will provide fundamental knowledge to the field and new biological/functional insights into RPE aging as well as identify RPE-specific epigenomic targets for manipulation to maintain RPE function with aging and prevent AMD pathogenesis.