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Vera Bonilha, Mary Rayborn, Xiaoping Yang, Karen Shadrach, Joe Hollyfield; Increased Levels of DJ-1 are present in cells exposed to carboxyethylpyrrole (CEP), and in Retina, RPE, Bruch’s Membrane and Drusen from AMD donor eyes. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6102.
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DJ-1 is a protein known to function as an antioxidant, redox-sensitive molecular chaperone and transcription regulator, which robustly protect cells from oxidative stress. To analyze the distribution of DJ-1 in cultured cells incubated with human serum albumin (HSA) and HSA adducted to carboxyethylpyrrole (CEP), and retinas from AMD donors the following studies were performed.
ARPE-19 cell cultures were incubated with HSA and HSA-CEP, the oxidation fragment of docosahexaenoic acid previously found in eye tissue and plasma from AMD patients. RPE cultures were treated with HAS and HAS-CEP (100 to 600ug/ml) for various times followed by biochemical and immunohistological analysis. Generation of reactive oxygen species (ROS) was analyzed by incubation of RPE cultures with 10uM of CM-H2DCFDA followed by confocal microsocopy. Viability/Cytotoxicity of RPE cultures incubated with HAS and HSA-CEP was assayed using the LIVE/DEAD Viability/Cytotoxicity Kit for mammalian cells. Cryosections of isolated human Bruch’s membrane/choroid (with drusen) in the perimacular area were processed for DAB reaction with an antibody that recognizes DJ-1. The presence of DJ-1 and oxDJ-1 (DJ-1 oxidized at residue 106) was also analyzed in retina and RPE total lysates.
Incubation of RPE cultures with HSA-CEP but not HSA led to the generation of ROS. However, cell viability was not significantly affected and remained >90%. Increasing concentrations of HSA-CEP resulted in a dose-dependent increase in DJ-1 expression as analyzed by immunocytochemical and biochemical assays. Both DJ-1 and oxDJ-1 were detected by western blot in human retina and RPE lysates. However, the intensity of expression was significantly higher in the AMD eyes. Isolated human Bruch’s membrane/choroid (with drusen) from AMD eyes displayed DJ-1 immune reaction.
DJ-1 expression is increased upon exposure to CEP. DJ-1 is expressed in the human retina of normal and AMD eyes. The expression of DJ-1 in AMD eyes is significantly increased. Anti-DJ-1 immunoreactivity is present in Bruch’s membrane and drusen isolated from AMD eyes.
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