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Chengjian Tu, Xiaosheng Jiang, Jun Qu, Lowell G. Sheflin, Joyce E. Young, Bruce A. Pfeffer, Steven J. Fliesler; Retina Proteome Profiling in a Rat Model of Smith-Lemli-Opitz Syndrome Using an Ion-Current-Based Method. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3332.
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© ARVO (1962-2015); The Authors (2016-present)
Rats treated with AY9944, a selective inhibitor of DHCR7 (3β-hydroxy-Δ7-dehydrocholesterol reductase), undergo progressive retinal degeneration and provide an animal model of Smith-Lemli-Opitz syndrome (SLOS). Conventional proteomic methods suffer from poor proteomic coverage and difficulties in analyzing membrane proteins. Here, we examined proteomic changes in retinas from AY9944-treated and age-matched control rats using a gel-free, comprehensive proteomics approach.
Sprague-Dawley rats were treated with AY9944 as previously described (Arch. Ophthalmol. 122:1190, 2004); untreated age-matched rats served as controls. Retinas (N=5 per group) were harvested at ca. 2 mo of age, and subjected to nano-LC/Oribtrap analysis (J. Proteome Res. 8:2838, 2009). Paraffin-embedded eyes from companion animals were subjected to conventional histological and correlative TUNEL analysis.
>3500 unique proteins were analyzed, with a false-discovery rate (FDR) of 0.1%; the false-positive rate for quantification was ≤2%, indicating accurate profiling. Levels of ≥132 proteins were significantly (p<0.05) altered by AY9944 treatment, representing a diversity of biological processes, e.g., apoptosis, visual transduction, lipid metabolism, vesicular transport, oxidative/stress response, and ion channel activity. ROM1, rhodopsin, and rhodopsin kinase levels were decreased, relative to controls, whereas GFAP levels were increased, consistent with retinal degeneration. Pyknosis and TUNEL-positive staining in treated, but not control, retinas was observed almost exclusively in the outer nuclear layer, indicating photoreceptor apoptosis.
Multiple proteomic changes in the retina occur in the SLOS rat model during the normal-to-pathological transition period, consistent with previous histological observations as well as transcriptome perturbations in this model (Siddiqui et al.; ARVO 2008, 2009). Thus, beyond the initial insult to cholesterol biosynthesis, proteomic alterations are implicated in the etiology of retinal degeneration in this model, and may have broader relevance to the SLOS disease mechanism.
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