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N. A. Rao, S. Saraswathy; In Experimental Autoimmune Uveitis the Mitochondrial Proteomics Reveal Mitochondrial Specific Oxidative Stress in the Retina. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1352.
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Recent advances in proteomics have made it possible to determine the precise changes in protein expression in the mitochondrial compartment in various pathologic conditions. We investigated alterations in retinal mitochondrial protein levels in response to oxidative stress during the early phase of experimental autoimmune uveitis (EAU).
EAU was induced in 18 B10RIII mice with 25 µg of interphotoreceptor retinoid-binding protein emulsified with complete Freund’s adjuvant (CFA); 18 mice injected with CFA alone served as controls. The retinas were dissected out from 12 animals each from experimental and control groups on postimmunization day 7; mitochondrial fractions were prepared and submitted to a novel fluorescence technique, two-dimensional difference gel electrophoresis (2D - DIGE). The protein spots showing differential expression were excised and subjected to MALDI-TOF for peptide identification. Another group of six experimental and six controls were killed on postimmunization days 1, 3, 5, and 7; iNOS translocation to the mitochondria from the cytosol was studied using Western blot analysis of mitochondrial and cytosolic retinal components. mRNA expression levels of TNF-α, IL1ß, IL-12, IL-17, IL-18, IL-21 and IL-27 were quantitated by real-time PCR
2D - DIGE analysis revealed differential expression of 18 proteins: 13 were overexpresed (including aconitase, stress protein 70, αA crystallin, ßB2 crystallin, manganese-SOD, fructose bisphosphate aldolase) and 5 were downregulated (including. ATP synthase, malate dehydrogenase, guanine nucleotide binding protein and calretinin). The above cytokines including iNOS were upregulated significantly beginning on day 3, and iNOS protein was translocated to the mitochondria on postimmunization day 4
The presence of mitochondrial specific oxidative stress related proteins in the early EAU retina along with downregulation of ATP synthase provides early evidence of the stress-related retinal damage. The mitochondrial stress appears to be mediated by T-cell cytokines and mitochondrial translocation of iNOS.
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