Abstract
Smith-Lemli-Opitz syndrome (SLOS) is caused by an inherited defect in the last step in cholesterol (Chol) biosynthesis, leading to abnormal accumulation of 7-dehydrocholesterol and decreased Chol levels. Progressive retinal degeneration occurs in an animal model of SLOS, induced by treating rats with AY9944, a selective inhibitor of the enzyme affected in SLOS. Here we evaluated alterations in the biochemical and physical properties of retinal rod outer segment (ROS) membranes in this animal model. At 1 month of AY9944 treatment, there were modest alterations in fatty acid composition, but no significant differences in cis-parinaric acid (cPA) spectroscopic parameters in ROS membranes from treated versus control rats. However, at 3 months, ROS docosahexaenoic acid (DHA) content was dramatically reduced, and cPA fluorescence anisotropy values were decreased, relative to controls. Also, 1,6-diphenyl-1,3,5-hexatriene exhibited decreased rotational motion and increased orientational order in ROS membranes from 3 month-old AY9944-treated rats, relative to controls. No significant changes in protein:lipid ratios were observed; however, rhodopsin regenerability was compromised by 3 months of treatment. These findings are consistent with reduced ROS membrane fluidity in the SLOS rat model, relative to controls, primarily due to the dramatic reduction in membrane DHA levels, rather than altered sterol composition.
Highlights
Smith-Lemli-Opitz syndrome (SLOS) is caused by an inherited defect in the last step in cholesterol (Chol) biosynthesis, leading to abnormal accumulation of 7-dehydrocholesterol and decreased Chol levels
We previously described a progressive retinal degeneration in such an Abbreviations: 7DHC, 7-dehydrochosterol; BRD, Brownian rotational diffusion; Chol, cholesterol; cPA, cis-parinaric acid; DHA, docosahexaenoic acid; DPH, 1,6-diphenyl-1,3,5-hexatriene; mAb, monoclonal antibody; ROS, rod outer segment; SLOS, Smith-Lemli-Opitz syndrome
We confirmed that AY9944 had produced the desired and expected effect on sterol metabolism in the rat under the given treatment conditions, which would be reflected in the abnormal steady-state accumulation of 7DHC and reduction of both Chol levels as well as total sterol levels in serum (Table 1)
Summary
AY9944 (trans-1,4-bis(2-chlorobenzylamino-methyl)cyclohexanedihydrochloride) was custom synthesized, and matched the spectroscopic and physical properties of an authentic sample of AY9944 (kindly provided by Wyeth-Ayerst Laboratories, Monmouth, NJ). Detergent-solubilized ROS membranes from control and AY9944-treated rats were prepared as described previously [19]; proteins were separated on 12% SDS-PAGE under reducing conditions and immunoblotted essentially per the method of Towbin, Staehelin, and Gordon [20], normalized to total lipid phosphorus load (1 mg) per lane. Quantitative analysis of sterols was performed in comparison with authentic standards of pure 7DHC and Chol; integrated peak areas were analyzed with respect to empirically determined response factors for each sterol, with calculated masses corrected for recovery efficiency of the internal [3H]cholesterol standard. This system permits baseline separation of 7DHC and Chol, with a lower detection limit of ?10 pmol
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