Regulation of Glutathione Peroxidase 4 in Diabetic Retinopathy

Oxidative stress-mitochondrial dysfunction plays a central role in diabetic retinopathy. Glutathione peroxidase 4 (GPx4), a key antioxidant enzyme that detoxifies lipid peroxides, is inhibited in diabetes, but the mechanism underlying its inhibition is unclear. Here, we have investigated the role of ubiquitination in retinal GPx4 inhibition in diabetes. Primary human retinal endothelial cells and Müller cells, incubated in 20 mM (high) D-glucose, were analyzed for GPx4 activity and its ubiquitination. GPx4 interaction with ubiquitin ligase TRIM46 and with mitochondrial membrane transporters was analyzed by immunofluorescence and by co-IP. The role of TRIM46 in mediating GPx4 ubiquitination and mitochondrial membrane damage was confirmed using TRIM46-siRNA. Results were confirmed in the retina from streptozotocin-induced diabetic mice and from human donors with documented diabetic retinopathy. Compared to cells in normal glucose, high glucose significantly increased GPx4 ubiquitination in both retinal endothelial and Müller cells, inhibited cytosolic and mitochondrial GPx4 activity by >30%, and upregulated TRIM46 expression and TRIM46-GPx4 interactions. High glucose had no effect on TRIM46 mitochondrial import, but GPx4 import was decreased, and GPx4 in the mitochondria was more ubiquitinated; TRIM46-siRNA prevented the decrease in cytosolic and mtGPx4 activities. Similarly, retina from diabetic mice and human donors with diabetic retinopathy had increased TRIM46 and ubiquitinated GPx4 and decreased GPx4 activity. Diabetes induces TRIM46-mediated ubiquitination of GPx4 in the cytosol and impairs its mitochondrial import, leading to mitochondrial damage and cell death.