Abstract
BackgroundDJ-1 is found in many tissues, including the brain, where it has been extensively studied due to its association with Parkinson’s disease. DJ-1 functions as a redox-sensitive molecular chaperone and transcription regulator that robustly protects cells from oxidative stress.MethodologyRetinal pigment epithelial (RPE) cultures were treated with H2O2 for various times followed by biochemical and immunohistological analysis. Cells were transfected with adenoviruses carrying the full-length human DJ-1 cDNA and a mutant construct, which has the cysteine residues at amino acid 46, 53 and 106 mutated to serine (C to S) prior to stress experiments. DJ-1 localization, levels of expression and reactive oxygen species (ROS) generation were also analyzed in cells expressing exogenous DJ-1 under baseline and oxidative stress conditions. The presence of DJ-1 and oxidized DJ-1 was evaluated in human RPE total lysates. The distribution of DJ-1 was assessed in AMD and non-AMD cryosectionss and in isolated human Bruch’s membrane (BM)/choroid from AMD eyes.Principal FindingsDJ-1 in RPE cells under baseline conditions, displays a diffuse cytoplasmic and nuclear staining. After oxidative challenge, more DJ-1 was associated with mitochondria. Increasing concentrations of H2O2 resulted in a dose-dependent increase in DJ-1. Overexpression of DJ-1 but not the C to S mutant prior to exposure to oxidative stress led to significant decrease in the generation of ROS. DJ-1 and oxDJ-1 intensity of immunoreactivity was significantly higher in the RPE lysates from AMD eyes. More DJ-1 was localized to RPE cells from AMD donors with geographic atrophy and DJ-1 was also present in isolated human BM/choroid from AMD eyes.Conclusions/SignificanceDJ-1 regulates RPE responses to oxidative stress. Most importantly, increased DJ-1 expression prior to oxidative stress leads to decreased generation of ROS, which will be relevant for future studies of AMD since oxidative stress is a known factor affecting this disease.
Highlights
The retinal pigment epithelium (RPE) constitutes a monolayer of cuboidal cells
Immunoblots of RPE lysates obtained from all cell cultures demonstrated heterogeneity in the levels of expression of DJ-1 when compared to the expression of the loading control protein GAPDH (Fig. 1B)
Immunoblots of lysates revealed that ARPE-19 cultures transduced with the hDJ-1 (Fig. 5A, lane 3) and with the C to S Ad constructs (Fig. 5, lane 2) displayed significant increased immunoreactivity of DJ-1 when compared to ARPE-19 control cultures (Fig. 5A, lane 1) and normalized to the levels of GAPDH (Fig. 5B) under baseline conditions
Summary
The retinal pigment epithelium (RPE) constitutes a monolayer of cuboidal cells. Its apical surface faces a very complex extracellular matrix called the interphotoreceptor matrix (IPM) that surrounds the photoreceptor cells projecting from the outer retina. It was suggested that oxidative stress affecting the physiological function and leading to focal loss of the RPE cells is a major factor contributing to geographic atrophy, and vision loss in the elderly blinding disease age-related macular degeneration (AMD) [6,7]. It remains to be determined why the initial retinal degeneration occurs and how degeneration processes progress as a result of continued oxidative insults. DJ-1 functions as a redox-sensitive molecular chaperone and transcription regulator that robustly protects cells from oxidative stress
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