Abstract
Purpose :
AMD manifests as a progressive degeneration of structures in and around the macula that ultimately leads to photoreceptor (PR) cell death and consequent vision impairment. The missense variant, rs10490924 (G>T: A69S) is a frequently observed ARMS2 AMD risk allele in the human population. We have previously determined ARMS2 expression in human RPE. Little is known about the impact of this variant and in particular how this variant affects the neuroretina. This study investigates the effects of the ARMS2 AMD high-risk A69S variant on the retina in a hybrid co-culture model composed of human RPE cells and porcine retinal explants.
Methods :
hTERT-RPE1 (RPE) cells were seeded on transwell inserts and transfected with expression plasmids for ARMS2 non-risk and ARMS2 A69S on the next day. Transfection with the empty expression vector served as a negative control. After 24h, the RPE cells were co-cultured with retinal explants, obtained from the visual streak of the porcine retina, rich in cone PRs. Co-cultures were maintained for 3 days, then fixed and sectioned for imaging (mean±SEM, n=6). PR cell survival was assessed by counting DAPI-stained cell rows and measuring the percentage of TUNEL-positive cells in the outer nuclear layer (ONL). Cone opsin distribution and microglial activation were also analyzed using cell-specific antibodies.
Results :
Retinae co-cultured with ARMS2 A69S RPE cells displayed enhanced retinal degeneration compared to ARMS2 non-risk RPE. In particular, we observed that ARMS2 A69S RPE significantly induced the number of dying cells in the ONL (%TUNEL: %4,02 ± 0,62 vs %2,5 ± 0,42). The number of PR cell rows in the ONL was decreased in retinae co-cultured with ARMS2 high-risk RPE cells (6,19 ± 0,2 vs 7,02 ± 0,19). Notably, those retinae also had a lower number of cones (6,28 ± 0,43 vs 10,54 ± 0,74) and displayed more microglia activation (Iba-1 positive cells in the ONL: 2,3 ± 0,29 vs 1,18 ± 0,17) when co-cultured with ARMS2 A69S RPE compared to ARMS2 non-risk RPE.
Conclusions :
Our data show that RPE cells overexpressing the ARMS2 AMD high-risk variant enhance retinal degeneration in the porcine neuroretina compared to ARMS2 non-risk cells. Our results suggest a link between the ARMS2 A69S polymorphism in RPE cells and loss of PR as seen in AMD patients. This encourages us to further investigate the effects of ARMS2 A69S on RPE-retina interactions in AMD pathology.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.