Abstract
Purpose :
Aging is the primary risk factor for the onset of several age-related retinal diseases, including age-related macular degeneration (AMD). Recent studies show the retinal pigment epithelium (RPE) is more susceptible to dysfunction with age than other retinal layers. In this study, we report on RNA-seq analyses of age-related transcriptional changes in the mouse RPE.
Methods :
Total RNA was isolated from RPE/choroid of young (1-2 months, n=3) and aged (20-24 months, n=3) C57BL/6J mice. Six poly(A) enriched sequencing libraries were prepared and sequenced on the Illumina HiSeq 2500 platform. Differential expression testing was done using the R package, DESeq2, and genes were considered differentially expressed (DEGs) using the cutoff criteria of the adjusted p-value (< 0.05) and log2 fold change (≥ 1.0 or ≤ −1.0). Gene expression data was used for bioinformatic analysis. Principal component analysis (PCA) and volcano plots were used to capture age-related gene expression changes. Enrichment analysis of DEGs was performed using gene ontology (GO), and Reactome pathway analysis tools. qPCR was used to validate the expression of selected genes.
Results :
PCA of expression data revealed age was the main factor clustering the young and aged RPE/choroid. RNA-seq analysis identified 595 and 723 upregulated genes in young and aged mice RPE/choroid, respectively. GO analysis showed enrichment of the immune response, regulation of the immune system, and leukocyte activation in aging RPE. Consistent with the GO results, Reactome pathways demonstrated the involvement of regulation of signaling in natural killer and myeloid cells, secondary messengers of immune pathways, cell differentiation, and survival. GO-enriched terms associated with DEGs downregulated in aged RPE/choroid were mainly related to extracellular matrix organization, and organ and tissue development, indicative of depleting regenerative ability of aging RPE. Heatmaps of annotated immune and inflammation genes revealed increased expression in aged RPE/choroid compared to young mice.
Conclusions :
Differential gene expression analyses in aged mouse RPE/choroid demonstrated a pro-inflammatory and immunogenic transcriptomic profile. These findings may aid in understanding the molecular mechanisms of age-related loss of RPE cell viability and retinal degenerations such as AMD.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.