title: DJ-1-dependent regulation of oxidative stress in the retinal pigment epithelium (RPE).
abstract: BACKGROUND
  DJ-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.
  
  
  METHODS
  Retinal 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.
  
  
  RESULTS
  DJ-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
  DJ-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.
authors.affiliation: Department of Ophthalmic Research, The Cole Eye Institute, Cleveland Clinic Lerner College of Medicine, Cleveland, Ohio, United States of America.
authors.forename: Karen G
authors.initials: KG
authors.lastname: Shadrach
authors.forename: Mary E
authors.initials: ME
authors.lastname: Rayborn
authors.forename: Joe G
authors.initials: JG
authors.lastname: Hollyfield
authors.forename: Vera L
authors.initials: VL
authors.lastname: Bonilha
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007150:Immunohistochemistry; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008268:Macular Degeneration / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism; D009154:Mutation; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D000071617:Protein Deglycase DJ-1; D021381:Protein Transport / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D015513:Oncogene Proteins; D016877:Oxidants; D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide; C105131:PARK7 protein, human; D000071617:Protein Deglycase DJ-1