Validation of Grx2 Gene Knockout Mice as a New Model for Age-Related Retinal Degeneration

Abstract

Purpose : Age-related macular degeneration (AMD) is a leading cause of blindness worldwide. The poorly understood pathogenesis has greatly hindered our progress in therapeutic development. To address this shortcoming, this project was designed to examine how retinal redox dysregulation leads to AMD and characterize glutaredoxin 2 (Grx2), a mitochondrial thiol redox regulating enzyme, knockout mice as a potential new animal model for AMD.

Methods : The retinal pigment epithelium (RPE) layers were isolated from healthy (n=5) and AMD donor eyes (n=5). Grx2 levels were mesured using western blot. Primary RPE cells were isolated from wild-type (WT) and Grx2 knockout (KO) mice for the in vitro study. The visual functions of WT and Grx2 KO mice were examined by fundus photography and scotopic electroretinography (ERG). H&E staining was used for histological exams. RPE structural changes were assessed by immunostaining of tight junction protein ZO-1. Lipofuscin autofluorescence was examined on cryostat sections. The level of protein glutathionylation (PSSG) was measured by immunoblotting using anti-PSSG antibody.

Results : Grx2 protein levels and enzyme activities were decreased by approximately 30% in AMD donor eyes. Primary RPE cells isolated from Grx2 KO mice were more sensitive to oxidative damage. Grx2 KO mice developed age-dependent retinal degenerative pathology. By 12 months of age, Grx2 KO mice (n=8) showed ~50% decrease in a-wave and ~30% decline in b-wave amplitudes (n=8, P<0.001). Yellowish retinal deposits with features of drusen were also identified by fundus photography. Histological analysis and ZO-1 immunostaining revealed extensive RPE lesions, including RPE atrophy, vacuolation, hyper- and hypo-pigmentation, sub-RPE deposits, and loss of tight junction integrity. Age-dependent lipofuscin accumulation was observed in Grx2 KO mice. Furthermore, Grx2 KO mice demonstrated increased markers of mitochondrial oxidative damage including PSSG accumulation.

Conclusions : Grx2 plays a critical role in maintaining the mitochondrial redox homeostasis in the aging retina. Grx2 deficiency causes PSSG accumulation and sensitizes RPE cells to age-related oxidative damage, leading to RPE degeneration and photoreceptor damage. As a new animal model for AMD, Grx2 KO mice will provide new insights into the pathogenesis and potential therapeutic targets of AMD.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.