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G.-S. Wu, Y.-H. Liu, S. Saraswathy, A. Rodriquez, N. A. Rao; EAU in iNOS-Deficient Mice Reveals Role of iNOS and Uncoupling Protein 3 in Photoreceptor Mitochondrial Oxidative Stress. Invest. Ophthalmol. Vis. Sci. 2007;48(13):583.
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We previously found that 3 major tyrosine-nitrated retinal proteins in the early phase of EAU were mitochondria-related proteins (IOVS 2005;46:2271). The prominent mediator for this process before the entry of inflammatory cells appears to be inducible nitric oxide synthase (iNOS). In this study, we investigated iNOS-deficient mice, uncoupling protein 3 (UCP 3) and the site of mitochondrial oxidative stress.
Retinas of iNOS-deficient mice (KO; C57BL/6NTac-Nos2tm1N12) were screened against wild-type mice (WT; C57BL/6NTac) using 96-well PCR array preassembled for NO· signaling pathway genes. EAU was induced and retinas were collected on day (D) 10 postimmunization (p.i.). Peak inflammation occurs on D21. The lipophilic cationic dye, JC-1 was used to probe mitochondrial membrane potential at D0, D6, D7, D10 and D11 p.i. Retinal explants were incubated with JC-1 and frozen sections were cut immediately for confocal measurement of red (585 nm, hyperpolarized) and green (520 nm, depolarized) fluorescence. Dichlorofluorescein (DCF) and mitotracker red were used to localize site of oxidative stress by confocal microscopy.
PCR array of WT retinas revealed a 71% increase in UPC 3 compared with that in iNOS KO mice. Other oxidative stress genes, such as NADPH oxidases and their activators, hepsin, glutathione peroxidase and eosinophil peroxidase, were also upregulated in WT mice. JC-1 staining was seen exclusively in the photoreceptor layer. The ratio of intact mitochondria vs. constant JC-1 monomer declined from 0.88±0.12 on D0 to 0.32±0.03 on D11. At D7, DCF (green) and mitotracker (red) intensely co-localized to the photoreceptor inner segments, whereas only minimal DCF staining was seen for controls.
Our results indicate that mitochondrial oxidative stress is primarily localized in photoreceptors and is mediated by the upregulated iNOS in the early phase of EAU. Mitochondrial stress is also revealed by the UPC 3 upregulation. UCP 3 protects cells from oxidative stress by limiting production of reactive oxygen species. Suppression of superoxide, however, delivers two deleterious consequences: reduced mitochondrial respiration; and decreased membrane potential. This initial loss of membrane potential increases mitochondrial membrane permeability and releases the apoptogenic molecule, cytochrome c to start apoptotic cascade.
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