Abstract
Purpose: :
DJ-1 is a protein ubiquitously expressed in many tissues including the brain where it has been shown to function as antioxidant, redox-sensitive molecular chaperone and transcription regulator, which robustly protect cells from oxidative stress. DJ-1 peptides were detected in rat retinal pigment epithelium (RPE) cell fractions subjected to proteomic analysis. The present study was conducted to define the precise molecular mechanisms regulating the expression and function of DJ-1 in oxidative stress response in RPE and retina.
Methods: :
To decipher the DJ-1 function, RPE cultures were treated with H2O2 (100 to 800uM) and 4-HNE (5 to 100uM) for various times followed by biochemical and immunohistological analysis. In addition, cells were infected with a replication-deficient adenovirus carrying the full-length human DJ-1 cDNA and a mutant construct, which has the cysteine (C) residues at amino acid 46, 53 and 106 mutated to serine (S)(CtoS construct) prior to stress experiments. Results were analyzed by immunofluorescence, and Westerns. In addition, the effects of DJ-1 deletion were examined in knockout (KO) mice through non-invasive, fundus imaging in vivo using SLO and OCT, histological and immunohistological evaluation of the retinas of young adult and aged DJ-1 KO and control mice.
Results: :
In RPE cells under baseline conditions, DJ-1 displays a diffuse cytoplasmic and nuclear staining. Upon oxidative injury, a large portion of DJ-1 redistributed to mitochondria. Increase in DJ-1 expression was observed when cells were exposed to oxidative stress as analyzed by immunocytochemical and biochemical assays. In addition, cells exposed to oxidative stress generated oxidized DJ-1. Overexpression of full-length DJ-1 prior to exposure to oxidative stress led to significant decrease in the generation of reactive oxygen species (ROS) stress species and oxidative stress-related cellular damage. In contrast, the overexpression of CtoS DJ-1 prior to oxidative stress experiments did not affect ROS generation by cells subjected to oxidative stress. Overexpressed CtoS DJ-1 also failed to change its cytoplasmic distribution to mitochondria in response to oxidative stress. SLO and OCT imaging did not reveal any obvious, qualitative differences between the retinal morphology of control and DJ-1 KO mice. However, histology revealed altered inner and outer nuclei layer and RPE thinning in young adult mice.
Conclusions: :
DJ-1 expression is increased upon exposure to oxidative stress. DJ-1 overexpression promotes protection of RPE cells to oxidative stress. The three DJ-1 cysteine residues are involved in the oxidative stress function of DJ-1 in RPE cells. DJ-1 KO mice display early signs of retinal degeneration.
Keywords: retinal pigment epithelium • oxidation/oxidative or free radical damage • immunohistochemistry