Single-nucleus analysis of the aging mouse retina reveals transcriptional and regulatory differences in rod and cone response to aging.

Abstract

Purpose : To identify age-related changes in gene regulatory networks in retinal photoreceptors during aging and their potential role in age-related diseases

Methods : Retinal tissue was isolated from 28 days (n=3), 2.5 month (n=3), 5 month (n=3), and 21 month-old (n=3) mice and analyzed via single-nucleus RNA-seq and ATAC-seq. Retinal cell types were identified by cluster analysis of the snRNA-seq data (~62,000 nuclei) and snATAC-seq (113,487 nuclei). Differentially expressed genes (DEGs) were identified by comparing expression levels between different age points. Select genes were validated by qRT-PCR and immunohistochemistry.

Results : Cluster analysis revealed 18 clusters [AR1] of known retinal cell types, the two largest clusters were rods and cones with 20,399 and 6,196 nuclei, respectively. 457 DEGs were identified in rods and 180 in cones between 28 days and 21 months. Among these, 65 genes were changing with age in rods and cones. These genes are involved in diverse pathways such as neuron adhesion and axon development were decreasing, while genes in basement membrane organization increasing. Across all age comparisons, rods showed more DEGs than cones. Significantly more DEGs were detected in cones between 28 days and 21 months compared to 2.5 months and 21 months. Additional subclustering of rods and cones of all ages identified 3 cell subtypes each for both rods and cones. These subtypes had almost equal contributions from all ages. Intriguingly, One of these subpopulations, cluster 3 of rods, showed 28 genes upregulated only in this cluster. These genes are involved in cytochrome-c oxidase activity (mt-CO2/mt-CO3/Ndufa4), lipid metabolism (Apoe), and PI3K/AKT/mTOR signaling (GNGT1), pathways known to be associated with AMD. Motif analysis using ATAC-seq discovered that Mef2d motif was enriched in older age rods. It has been shown that insufficient Mef2d can lead to greater vulnerability to oxidative stress in photoreceptors.

Conclusions : More genes were differentially expressed with age in rods than cones. Clustering of all photoreceptors at all ages revealed 6 distinct subclusters. One rod cluster showed a unique transcriptional profile, with unique genes upregulated that are involved in several AMD related pathways. Taken together, we hypothesize that this cluster of rods is more vulnerable to age-related pathology and transcriptional changes.

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