The Retinal Pigmented Epithelium translatomic responses to aging are sexually divergent

Abstract

Purpose : Aging is the main risk factor for age-related macular degeneration (AMD). Retinal pigmented epithelium (RPE) atrophy is an AMD hallmark that precedes photoreceptor loss. Genome-wide chromatin accessibility, DNA methylation, and gene expression studies of AMD and control RPE suggest epigenomic/transcriptomic changes play a critical role in AMD onset and progression. However, mechanisms by which normal aging impairs RPE function and contributes to AMD pathogenesis are unclear. The aim of this study is to define the normative cell-specific translatomic profile of the RPE in both sexes and with advanced age.

Methods : We generated Aldh1l1-cre/ERT2^+/wt; NuTRAP^flox/wt (Aldh1l1-cre/ERT2⁺; NuTRAP⁺) mice which express EGFP and mCherry tags allowing for affinity isolation of nuclei and ribosomes after tamoxifen induction. After induction, female and male mice were euthanized at 4 or 24 months of age and their eyes harvested and processed for immunohistochemistry/confocal microscopy of RPE-eyecup flatmount preparations; RPE ribosome bound RNA isolation via TRAP; qPCR; and stranded RNAseq profiling.

Results : Co-localization of EGFP and mCherry was observed in the RPE of Aldh1l1-cre/ERT2⁺; NuTRAP⁺ samples. RNA-seq bioinformatic analysis revealed enrichment of RPE genes by TRAP relative to input lysates. Comparing TRAP-isolated RPE- and published human RPE- transcriptomes, we found a strong correlation not only in what genes are expressed in the mouse and human RPE but also their relative expression. Pathway analysis showed that RPE-specific transcriptomic changes with aging were associated with activation of neuroinflammation, pattern recognition receptor signaling, and phagosome formation, and predicted activation of upstream regulators Irf7 and Stat3 in aged RPE vs young RPE. Principal component analysis of TRAP-RPE translatomes showed separation of samples by age in the first component and sex in the second component. Two-way-ANOVA revealed 1,001 differentially expressed genes (DEGs) with age, 305 DEGs with sex, and 167 DEGs with age and sex main effects.

Conclusions : The Aldh1l1-NuTRAP model is ideal for studying the RPE translatomic profile with aging. Differential age- and sex- associated transcript expression with overrepresentation of pathways related to inflammation were found. Our dataset provides a resource for vision researchers seeking to evaluate sex and age as factors in their preclinical studies.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.