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The Parkinson's Disease-Associated Protein DJ-1 Protects Dictyostelium Cells from AMPK-Dependent Outcomes of Oxidative Stress.

ABSTRACT: Mitochondrial dysfunction has been implicated in the pathology of Parkinson's disease (PD). In Dictyostelium discoideum, strains with mitochondrial dysfunction present consistent, AMPK-dependent phenotypes. This provides an opportunity to investigate if the loss of function of specific PD-associated genes produces cellular pathology by causing mitochondrial dysfunction with AMPK-mediated consequences. DJ-1 is a PD-associated, cytosolic protein with a conserved oxidizable cysteine residue that is important for the protein's ability to protect cells from the pathological consequences of oxidative stress. Dictyostelium DJ-1 (encoded by the gene deeJ) is located in the cytosol from where it indirectly inhibits mitochondrial respiration and also exerts a positive, nonmitochondrial role in endocytosis (particularly phagocytosis). Its loss in unstressed cells impairs endocytosis and causes correspondingly slower growth, while also stimulating mitochondrial respiration. We report here that oxidative stress in Dictyostelium cells inhibits mitochondrial respiration and impairs phagocytosis in an AMPK-dependent manner. This adds to the separate impairment of phagocytosis caused by DJ-1 knockdown. Oxidative stress also combines with DJ-1 loss in an AMPK-dependent manner to impair or exacerbate defects in phototaxis, morphogenesis and growth. It thereby phenocopies mitochondrial dysfunction. These results support a model in which the oxidized but not the reduced form of DJ-1 inhibits AMPK in the cytosol, thereby protecting cells from the adverse consequences of oxidative stress, mitochondrial dysfunction and the resulting AMPK hyperactivity.

SUBMITTER: Chen S

PROVIDER: S-EPMC8392454 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:The loss of function of DJ-1 caused by mutations in DJ1 causes a form of familial Parkinson's disease (PD). However, the role of DJ-1 in healthy and in PD cells is poorly understood. Even its subcellular localization in mammalian cells is uncertain, with both cytosolic and mitochondrial locations having been reported. We show here that DJ-1 is normally located in the cytoplasm in healthy Dictyostelium discoideum cells. With its unique life cycle, straightforward genotype-phenotype relationships, experimental accessibility and genetic tractability, D.discoideum offers an attractive model to investigate the roles of PD-associated genes. Furthermore, the study of mitochondrial biology, mitochondrial genome transcription and AMP-activated protein kinase-mediated cytopathologies in mitochondrial dysfunction have been well developed in this organism. Unlike mammalian systems, Dictyostelium mitochondrial dysfunction causes a reproducible and readily assayed array of aberrant phenotypes: defective phototaxis, impaired growth, normal rates of endocytosis and characteristic defects in multicellular morphogenesis. This makes it possible to study whether the underlying cytopathological mechanisms of familial PD involve mitochondrial dysfunction. DJ-1 has a single homologue in the Dictyostelium genome. By regulating the expression level of DJ-1 in D. discoideum, we show here that in unstressed cells, DJ-1 is required for normal rates of endocytic nutrient uptake (phagocytosis and, to a lesser extent, pinocytosis) and thus growth. Reduced expression of DJ-1 had no effect on phototaxis in the multicellular migratory 'slug' stage of the life cycle, but resulted in thickened stalks in the final fruiting bodies. This pattern of phenotypes is distinct from that observed in Dictyostelium to result from mitochondrial dyfunction. Direct measurement of mitochondrial respiratory function in intact cells revealed that DJ-1 knockdown stimulates whereas DJ-1 overexpression inhibits mitochondrial activity. Together, our results suggest positive roles for DJ-1 in endocytic pathways and loss-of-function cytopathologies that are not associated with impaired mitochondrial function.

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The Parkinson's Disease-Associated Protein DJ-1 Protects Dictyostelium Cells from AMPK-Dependent Outcomes of Oxidative Stress.

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