Abstract
Purpose: :
We previously found that photoreceptor cytochrome c (cyto c) was nitrated in the early phase of experimental autoimmune uveoretinitis (EAU), on day 5 postimmunization (p.i.). Although nitrated cyto c is apoptogenic, no apoptosis was observed in the photoreceptors. At this stage of EAU, we also detected upregulation of alphaA crystallin in the retina. In this study, we determine whether the crystallin prevents the apoptotic cascade induced by the nitrated cyto c.
Methods: :
Experimental uveitis was induced in Lewis rats by bovine S–antigen. The superoxide scavenging capacity of alphaA crystallin was estimated spectrophotometrically using hypoxanthine and xanthine oxidase to generate superoxide in vitro. Retinal cytosolic and mitochondrial fractions were separated, and the release of nitrated cyto c into the cytosol was detected by UV/VIS absorption and immunoblot. The interaction of nitrated cyto c with alphaA crystallin was demonstrated by co–immunoprecipitation with anti–alphaA crysallin. The binding of alphaA crystallin to the procaspase –3 subunit p24 was detected using retinal cell–free systems, and apoptosis was initiated by the addition of dATP.
Results: :
AlphaA crystallin displayed no superoxide scavenging properties. Nitrated cyto c was released into retinal cytosol, but no apoptosis was detected until day 12 p.i. when the presence of inflammatory cells is apparent. Immunoblot detected co–precipitation of alphaA crystallin with cyto c (probed by anti–cyto c), and the cyto c was also nitrated (probed by anti–nitrotyrosine). In a cell–free system from the control retina, alphaA crystallin intercepted the apoptotic pathway triggered by nitrated cyto c/dATP by binding to the procaspase–3 intermediate subunit p24, preventing further generation of the active subunits p20 and p17.
Conclusions: :
These results indicated that alphaA crystallin suppresses nitrated cyto c–mediated apoptosis through direct interactions with key apoptogenic components, including the upstream molecule, nitrated cyto c, and the downstream subunits p24, a partially processed procaspase–3. These processes effectively abolished the subsequent generation of the executioner caspase–3 subunits, p20/p17, thereby protecting the photoreceptors from the oxidative stress in the early EAU.
Keywords: apoptosis/cell death • oxidation/oxidative or free radical damage • uveitis-clinical/animal model