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S. J. Fliesler, R. J. Kapphahn, D. A. Ferrington; Proteome Alteration and Enhanced Oxidative Modification of Retinal Proteins in a Rat Model of Smith-Lemli-Opitz Syndrome. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4477.
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Oxidative modification of proteins often disrupts their structure/function and can compromise cellular viability. Such modifications include, among others, 4-hydroxynonenal (HNE) and carboxyethylpyrrole (CEP), as well as nitrotyrosine (NTyr). We compared the retinal proteome and levels of such modifications as a function of age in a rat model of Smith-Lemli-Opitz syndrome (SLOS), in comparison to age-matched controls. Some SLOS rats also were fed a high-cholesterol (HC) diet.
Sprague-Dawley rats were treated with AY9944 to produce the SLOS model (see Fliesler et al., Arch. Ophthalmol. 122:1190, 2004); untreated age-matched rats served as controls. Retinas were harvested at 1, 2, and 3 mo of age, flash frozen in liquid nitrogen, and subsequently were either subjected to proteomic analysis (2D IEF/SDS-PAGE, silver staining/ quantitative densitometry, and MS analysis) or analyzed by immuno-slot blot, probing with antibodies raised against HNE, CEP, and NTyr, followed by quantitative densitometry.
Of 139 2D-gel spots analyzed, 16 were significantly (p≥0.05) increased in staining intensity, while 5 were minimal or absent, in retinas from SLOS rats, compared to controls (1-mo). By MS analysis, 3 of the enhanced spots were revealed to be crystallins (alpha-A and alpha-B) and peroxiredoxin 6, which are known oxidative stress markers. HNE modification of retinal proteins was >15-fold higher at 3 mo in SLOS rats vs. controls (p=0.04), but showed no differences at 1 or 2 mo; the HC diet significantly reduced HNE modification of retinal proteins at 3 mo (by 3.8-fold; p=0.04). CEP modification was greater at 1, 2, and 3 mo in SLOS rats vs. controls (p=0.02), but showed no age dependence; the HC diet did not alter CEP levels. In contrast, both SLOS and control rats exhibited an age-dependent increase in NTyr modification of retinal proteins (p=0.001), but were not different from one another at the same age; the HC diet reduced NTyr levels by 2-fold (p=0.02).
The retinal proteome is dramatically altered in the SLOS rat model compared to untreated controls, including up-regulation of oxidative stress markers. Oxidative modification of retinal proteins is also enhanced in SLOS rats; some of these (HNE, NTyr) show age-dependence, while CEP is elevated at all ages in SLOS rats. A HC diet significantly reduced the levels of HNE and NTyr, but not CEP. Hence, cholesterol supplementation (the current standard of care for SLOS patients) may have a biologically relevant (and efficacious) impact beyond normalizing cholesterol levels in SLOS.
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