Oxidation of DJ-1 Cysteines in Retinal Pigment Epithelium Function.

Abstract

The retina and RPE cells are regularly exposed to chronic oxidative stress as a tissue with high metabolic demand and ROS generation. DJ-1 is a multifunctional protein in the retina and RPE that has been shown to protect cells from oxidative stress in several cell types robustly. Oxidation of DJ-1 cysteine (C) residues is important for its function under oxidative conditions. The present study was conducted to analyze the impact of DJ-1 expression changes and oxidation of its C residues on RPE function. Monolayers of the ARPE-19 cell line and primary human fetal RPE (hfRPE) cultures were infected with replication-deficient adenoviruses to investigate the effects of increased levels of DJ-1 in these monolayers. Adenoviruses carried the full-length human DJ-1 cDNA (hDJ) and mutant constructs of DJ-1, which had all or each of its three C residues individually mutated to serine (S). Alternatively, endogenous DJ-1 levels were decreased by transfection and transduction with shPARK7 lentivirus. These monolayers were then assayed under baseline and low oxidative stress conditions. The results were analyzed by immunofluorescence, Western blot, RT-PCR, mitochondrial membrane potential, and viability assays. We determined that decreased levels of endogenous DJ-1 levels resulted in increased levels of ROS. Furthermore, we observed morphological changes in the mitochondria structure of all the RPE monolayers transduced with all the DJ-1 constructs. The mitochondrial membrane potential of ARPE-19 monolayers overexpressing all DJ-1 constructs displayed a significant decrease, while hfRPE monolayers only displayed a significant decrease in their ΔΨm when overexpressing the C2S mutation. Viability significantly decreased in ARPE-19 cells transduced with the C53S construct. Our data suggest that the oxidation of C53 is crucial for regulating endogenous levels of ROS and viability in RPE cells.