RPE-specific knockout of REV-ERBα induces age-dependent RPE degeneration in mice

Abstract

Purpose : Retinal pigment epithelium (RPE) dysfunction and atrophy occur in dry age-related macular degeneration (AMD) and are associated with chronic oxidative stress. Previously, we found that systemic deficiency of nuclear receptor REV-ERBα, a redox-sensitive transcription factor, results in RPE dysfunction and degeneration in mice due to dysregulation of the oxidative stress response in RPE. Here we investigated if RPE-specific knockout of REV-ERBα is sufficient to induce RPE degeneration in mice.

Methods : Mice with RPE-specific deletion of REV-ERBα, BEST1-cre:Rev-erbα^fl/fl (RPE-KO), was generated by breeding Rev-erbα^flox/flox (fl/fl) mice with BEST1-cre (cre) mice, with both fl/fl and cre mice as controls. Knockout efficiency and specificity of REV-ERBα in RPE were measured by RT-qPCR, Western blot, and immunostaining of RPE/choroid flat mounts and cross-sections. RPE degeneration of fl/fl (n=4), cre (n=5) and RPE-KO (n=7) mice were monitored by fundus imaging at 3, 6 and 9 months old, and sub-retinal lesions were quantified. RPE atrophy was analyzed by immunostaining of RPE/choroid flat-mounts. RNA and protein expression of REV-ERBα target genes including antioxidant regulators and enzymes was examined in fl/fl and RPE-KO RPE. Oxidative DNA damage was evaluated by immunostaining of 8-hydroxy-2'deoxyguanosine (8-OHdG).

Results : REV-ERBα knockout was specific to RPE cells in RPE-KO mice with up to 80% efficiency in cell counting and mRNA and protein levels. Fundus imaging of RPE-KO mice showed significantly greater number of whitish yellow lesions at both 6- and 9-month-old, compared to both fl/fl and cre controls. In addition, RPE-KO mice showed more severely distorted RPE patches in flat-mounts by immunostaining of β-Catenin, compared with cre controls. Significant downregulation of NRF2 and its target genes Sod1, Sod2 and Catalase was found in RPE-KO vs. fl/fl RPE at both mRNA and protein levels. Furthermore, RPE-KO mice exhibit elevated oxidative stress with 8-OHdG staining, compared to cre RPE at 6 months old.

Conclusions : These findings suggest that RPE specific depletion of REV-ERBα leads to fundus lesions and RPE degeneration similar to those in systemic REV-ERBα knockout mice, demonstrating an RPE cell-autonomous role of REV-ERBα in regulating RPE function and health, potentially through modulating NRF2 transcription and antioxidant self-defense.

This is a 2021 ARVO Annual Meeting abstract.