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J. D. Ribich, C. J. Lieven, A. L. Kloosterboer, L. A. Levin; Identification of Retinal Proteins Undergoing Sulfhydryl Oxidation After Optic Nerve Crush. Invest. Ophthalmol. Vis. Sci. 2009;50(13):141.
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Axonal injury leads to retinal ganglion cell (RGC) death via apoptosis. We previously showed that there is a rise in intracellular superoxide after axotomy (Lieven CJ et al, IOVS 47:1477, 2006), signaling RGC apoptosis. To further understand how a superoxide burst could transduce this signal transduction pathway, we isolated rat retinal proteins undergoing sulfhydryl oxidation following surgically-induced axonal injury.
Optic nerve crush was performed in one eye of Long Evans rats. Retinas were removed 5 days after the surgery and prepped for gel electrophoresis. Following a first dimension run, gel lanes were excised and incubated in dithiothreitol (DTT) to reduce disulfide bonds and then in iodoacetamide to alkylate free sulfhydryls. The gel lanes were then rotated 90 degrees and run in the second dimension, followed by staining with SYPRO Ruby.
The vast majority of proteins were on a 45 degree diagonal, indicating that they did not contain disulfide bonds when first isolated. A number of protein spots were visible below the main diagonal, consistent with the presence of disulfides that were reduced during the incubation with DTT. Protein spots that were present in the axonal injury retinas and not in the control retinas, or vice versa, were excised and analyzed by mass spectrometry.
Proteins that change sulfhydryl redox status after axotomy are potential signaling molecules for the transduction of superoxide-induced apoptosis. Further work should clarify whether these redox changes are necessary and sufficient for RGC death after axonal injury.
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