Oxidative stress-induced RPE and retinal cell death involves multiple regulated cell death pathways.

Abstract

Purpose : To dissect oxidative stress-related cell death pathways in RPE and retina using the NaIO₃ mouse degeneration model.

Methods : Three-month-old C57BL/6J mice received a single tail vein injection of 10 mg/Kg NaIO₃; the DJ-1 KO mice were also analyzed as a model of increased sensitivity to oxidative stress. This concentration of NaIO₃ induces degeneration only in the DJ-1 KO mice. Parallel groups of mice were injected with PBS. Retina and RPE were isolated, lysed, and analyzed by LC-MS/MS. Panther analysis tool and Ingenuity Pathway Analysis were used to analyze the proteomics data. Main observations were further assayed by Western blotting, immunohistochemistry, confocal microscopy, and histology in mice injected with increasing concentrations of NaIO₃ (10-30 mg/Kg).

Results : Proteomics data analyses suggested cell death and survival as one of the top modulated molecular and cellular functions in RPE and retina of C57BL/6J and DJ-1 KO mice injected with 10 mg/Kg NaIO₃ compared to PBS injected equivalent. Apoptosis and necroptosis pathways were analyzed using active caspase-3 (acas3) and p-MLKL immunostaining. In PBS-injected mice, we observed increased acas3 reactivity in the DJ-1 KO mice compared to C57BL/6J mice; p-MLKL was observed in both mice strains in the photoreceptor inner segments and GCL. With 10 mg/Kg NaIO₃, acas3 was observed only in the photoreceptor inner segment of C57BL/6J retinas, whereas it was observed in RPE and photoreceptors with 20 mg/Kg NaIO₃. In DJ-1 KO mice, acas3 was expressed in RPE and photoreceptor degenerating cells and OPL and INL at both concentrations of NaIO₃. In C57BL/6J retinas, p-MLKL staining was observed only in GCL with 10 mg/Kg NaIO₃, whereas its expression was observed in RPE, ONL, and GCL with 20 mg/Kg NaIO₃. In DJ-1 KO mice, p-MLKL was localized in ONL and GCL with 10 and 20mg/Kg NaIO₃. Injection of the pan-caspase inhibitor, Z-VAD-Fmk, after 10 mg/Kg NaIO₃ injection in DJ-1 KO mice completely inhibited RPE degeneration in DJ-1 KO, thereby, confirming the role of caspase-mediated apoptosis of RPE in the initiation of the degeneration in this mouse model.

Conclusions : Oxidative stress-induced RPE and retinal cell death involve activation of both apoptosis and necroptosis.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.