Validate Grx2 gene knockout mice as a new model for age-related retinal degeneration

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 new animal model for AMD. Methods: The retinal pigment epithelium (RPE) layers were isolated from healthy and AMD donor eyes. Grx2 protein levels were measured by Western blot analysis. Primary RPE cells were isolated from wild-type (WT) and Grx2 knockout (KO) mice for the in vitro study. The visual function 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 cryostatsections. The level of protein glutathionylation (PSSG) was measured by immunoblotting using anti-PSSG antibody. Results: Grx2 protein level and enzyme activities were decreased by approximately 30% in AMD donor eyes. Primary RPE cells isolated from Grx2 KO mice were more sensitive to H2O2-induced oxidative damage than WT RPE cells. Grx2 KO mice developed age-dependent retinal degenerative pathology. By 12 months of age, Grx2 null mice showed ~50% decrease in a-wave and ~30% decline in b-wave amplitudes (n=8, P 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 therapeutics of AMD.