Abstract
Purpose :
Genome-wide association studies have identified a myriad of genetic loci and variants linked to age-related macular degeneration (AMD), yet our understanding of how these loci contribute to AMD is lacking. One such locus contains the coding TNFRSF10A and noncoding AC100861.1 genes. Within AC100861.1 lies a SNP whose minor variant is associated with AMD, but the function of the transcribed long noncoding RNA (lncRNA) and the effect of the SNP are not known. Considering that retinal pigmented epithelium (RPE) cell stress is a major AMD risk factor and that TNFRSF10A is involved in death receptor signaling, we investigated AC100861.1 function through the lens of apoptosis to understand how disruption of this locus may contribute to the RPE death associated with AMD.
Methods :
TNFRSF10A and AC100861.1 RNA levels were quantified in donor macular RPE samples and cell line isolates via qPCR. Production of TNFRSF10A and AC100861.1 was manipulated within ARPE-19 cells via transfection of siRNAs or expression vectors. The extent of apoptosis was measured through TUNEL staining followed by flow cytometry. Protein levels were measured through western blot analysis. Transcript localization was assessed in ARPE-19 cells through RNA fluorescent in situ hybridization (RNA-FISH).
Results :
Expression from TNFRSF10A and AC100861.1 were found to be downregulated in early-stage AMD, yet in more advanced AMD, expression from both genes was upregulated. TUNEL analysis indicated that knockdown of the AC100861.1 reduced susceptibility to apoptosis in ARPE-19 cells in response to cell stress, while AC100861.1 overexpression had the opposite effect. Conversely, knockdown and overexpression of TNFRSF10A elicited minimal changes in the apoptotic susceptibility. Neither knockdown nor overexpression of AC100861.1 affected TNFRSF10A protein levels. RNA-FISH data identified the AC100861.1 transcript as localized to the cytoplasm.
Conclusions :
Our findings reveal an altered expression of TNFRSF10A and AC100861.1 in AMD-affected macular RPE, reflecting a dysregulation of this locus in the disease state. AC100861.1 appears to increase susceptibility to apoptosis, although this does not appear to be mediated through an alteration of TNFRSF10A protein levels. Since the AC100861.1 transcript is primarily localized to the cytoplasm it is more likely to influence processes there, yet the mechanism is not yet known.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.