ucf-101 and Retinal-Degeneration

To investigate the role of HtrA2/Omi, a nuclear-encoded mitochondrial serine protease with a proapoptosis function, under H(2)O(2)-induced oxidative stress in human RPE, in the Ccl2(-)(/)(-)Cx3cr1(-)(/)(-) double-knockout (DKO) mouse retina, and the HtrA2/Omi-deficient mice.. Oxidative stress was induced in ARPE-19 cells by 1 mM H(2)O(2) for 2 hours. HtrA2/Omi and caspase-3 expression was evaluated using RQ-PCR, immunohistochemistry, or Western blot. Cell viability was detected by MTT assay. HtrA2/Omi expression in the subcellular components and activated caspase-3 were measured. These processes were also evaluated in cells treated with UCF-101, an HtrA2/Omi inhibitor or in cells subjected to RNAi against HtrA2/Omi. Oxidative stress was assayed and compared in retinas of DKO and wild-type (WT) mice by determining serum NADPH oxidase subunits and nitrite levels. Transmission electron microscopy was used to view the retinal ultrastructure of the HtrA2/Omi-deficient mice.. H(2)O(2)-induced oxidative damage resulted in HtrA2/Omi translocation from mitochondria to cytosol, leading to RPE cell apoptosis via a caspase-mediated pathway. Treatment of RPE cells with UCF-101 reduced the cytosolic translocation of HtrA2/Omi, attenuated caspase-3 activation, and decreased apoptosis. After specific HtrA2 downregulation, increased cell viability was measured in H(2)O(2)-treated ARPE-19 cells. Retina of DKO mice exhibit increased oxidative stress and upregulation of HtrA2/Omi. Fewer and abnormal mitochondria were found in HtrA2/Omi(-)(/)(-) photoreceptors and RPE.. These findings suggest that HtrA2/Omi is related to RPE apoptosis due to oxidative stress, which may play an important role in the integrity of mitochondria and the pathogenesis of AMD.

Topics: Animals; Apoptosis; Blotting, Western; Caspase 3; Cell Line; Cell Survival; Chemokine CCL2; CX3C Chemokine Receptor 1; Cytosol; Disease Models, Animal; Enzyme Inhibitors; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation, Enzymologic; High-Temperature Requirement A Serine Peptidase 2; Humans; Hydrogen Peroxide; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Mitochondrial Proteins; Oxidative Stress; Protein Transport; Pyrimidinones; Receptors, Chemokine; Retinal Degeneration; Retinal Pigment Epithelium; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Serine Endopeptidases; Thiones; X-Linked Inhibitor of Apoptosis Protein