Abstract
Purpose :
Among the seven known ATP-activated P2X receptor subtypes, only P2X7 has been extensively studied in purinergic signaling and inflammation associated with age-related macular degeneration (AMD). This study tested the hypothesis that the less-explored P2X4 may also play a functional role in AMD pathogenesis.
Methods :
Human post-mortem eye sections were obtained from five different donor groups: Young (age ≤ 55 years; n = 5), Old (age ≥ 70 years; n = 8), Geographic Atrophy (GA; n = 5), Choroidal Neovascularization (CNV; n = 5), and Soft Drusen (SD; n = 4). ARPE-19 cells were cultured in DMEM with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin. P2X4 and P2X7 mRNA and protein expressions were detected in cultured ARPE-19 cells and human post-mortem eyes by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunofluorescence staining, respectively. Data was analyzed using a two-tailed Student's t-test and the Mann-Whitney U test.
Results :
RT-qPCR revealed a significantly higher mRNA expression level for P2X4 over P2X7 in ARPE-19 cells (p = 0.00011). Immunocytochemistry indicated greater expression of P2X4 than P2X7 in ARPE-19 cells, particularly when permeabilized with Triton X-100 (p < 0.0001). Immunohistochemistry on human post-mortem eyes showed that P2X4 in the retinal pigment epithelium (RPE) was most expressed in the SD group, with a 57% and 88% increase compared to the least expressed GA and CNV donors (late-stage AMD), respectively. In the choroid, P2X4 was also the most expressed in the SD group, with a 75% and 99% increase compared to the GA and Old donors, respectively. In contrast, P2X7 in the RPE was lowest in the SD group, with a 60% decrease compared to both highest expressed donor groups (Old and GA). P2X7 in the choroid was most expressed in the GA group, with a 115% increase compared to the least expressed Young donor group.
Conclusions :
P2X4 and P2X7 receptors are expressed in the human retina, but their expression levels vary with age and retinal degeneration. P2X4 receptors may potentially play a role in RPE cells, particularly in patients with soft drusen, warranting further investigation into their function and interaction with P2X7 receptors in AMD pathogenesis.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.