The Retinal Pigment Epithelium has increased oxidative stress and is more susceptible to oxidative stress-induced degeneration in Sorsby Fundus Dystrophy

Abstract

Purpose : Sorsby Fundus Dystrophy (SFD) is an autosomal dominant retinal dystrophy that shares a clinical phenotype with Age-related Macular Degeneration (AMD), including the development of sub-retinal deposits, RPE degeneration, and choroidal neovascularization. SFD is caused by mutations in TIMP3 and the Ser179Cys-TIMP3 mutation is associated with severe pathology in patients. We wanted to evaluate if mice carrying the S179C-TIMP3 mutation were more susceptible to oxidative stress-induced RPE degeneration.

Methods : To test oxidative stress, 4-month-old mice (S179C-TIMP3 and age-matched wild-type (WT) littermates) received a single tail vein injection of 10 and 20 mg/kg body weight of NaIO₃; parallel groups of mice were injected with PBS. RPE degeneration was evaluated by histology: staining of plastic sections with Toluidene Blue and whole mount immunohistochemistry of RPE/Choroid with phalloidin-TRITC. Quantitative PCR was performed on RPE and retina for antioxidant genes at baseline and under experimental conditions. Reactive oxygen species (ROS) staining was performed on cultured ARPE-19 cells expressing S179C-TIMP3, WT-TIMP3, or vector only control incubated with CM-H₂DCFDA, a reagent that is only fluorescent upon oxidation by ROS.

Results : Both WT and S179C-TIMP3 mice developed severe RPE degeneration at 20 mg/kg of NaIO₃. However, at the lower dose of NaIO₃ (10 mg/kg), only the S179C-TIMP3 mice showed RPE degeneration. DJ-1, Nrf2, Sod1, and Sod2 expression was significantly upregulated in the RPE at baseline S179C-TIMP3 mice compared to WT controls. When S179C-TIMP3 and WT mice were subjected to 10 mg/kg NaIO₃, S179C-TIMP3 mice show a trend of downregulation of these same genes while the WT have no change. There was no change in antioxidant gene expression in the retina. Cultured S179C-TIMP3 RPE cells showed increased ROS compared to WT-TIMP3 or vector only cells.

Conclusions : The pathogenic S179C-TIMP3 mutation results in increased ROS in the RPE at baseline, upregulation of antioxidant genes, and increased sucetibility to increased oxidative stress induced by NaIO₃. Increased oxidative stress affects mainly the RPE cells in our SFD model.

This is a 2020 ARVO Annual Meeting abstract.