Alteration of retinal rod outer segment membrane fluidity in a rat model of Smith-Lemli-Opitz syndrome

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.

Fig. 1.

Fatty acid profiles of retinal rod outer segment (ROS) membranes from AY9944-treated (filled bars) and control (open bars) rats at (A) 1 and (B) 3 postnatal months of age. Values are mean ± SD (N = 3), expressed as relative mole percent. * Statistical significance (P < 0.05; multivariant ANOVA with post-hoc Scheffe test) of differences between treated and control values for a given fatty acid species.

Fig. 2.

Opsin protein levels in control (C) and AY9944-treated (AY) rat ROS membranes. A: Western blot probed with monoclonal anti-opsin antibodies (mAb 4D2). Samples were adjusted in order to load an equivalent amount (1 mg total) of lipid phosphorus per lane. Immunoreactivity was visualized using HRP-conjugated secondary antibodies and enhanced chemifluorescence detection. Migration positions of protein molecular weight markers (M_r in kDa) are indicated at left. B: Quantitative densitometric analysis of Western blots. Graph represents measurements (mean ± SD) obtained from four independent blots per treatment and time point (1 and 3 months). C: Quantitative RT-PCR analysis of opsin expression in retinas isolated from control and AY9944-treated rats. Graph shows values (mean ± SD, N = 4) expressed in arbitrary units.

Fig. 3.

Steady-state anisotropy measurements of cis-parinaric acid (cPA) in purified ROS membranes from 1 and 3 month-old rats. Open bars, controls; filled bars, AY9944-treated. Values are mean ± SD (N = 4), measured at 37°C. * Statistically significant difference (P = 0.0068) between treated versus control samples at 3 months of age.

Fig. 4.

Measures of acyl chain ensemble order (membrane fluidity) obtained from analysis of 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence anisotropy decays. A: The free volume (or disorder parameter), f_v, from analysis via the Brownian rotational diffusion (BRD) model. The smaller value for the membranes from AY9944-treated animals, compared with controls, indicates decreased acyl chain packing disorder (P < 0.001, N = 6). B: The order parameter, S, calculated from the model-independent analysis of the anisotropy decays in terms of the sum of three exponential decays. The higher S value for the membranes from AY9944-treated animals, compared with controls, indicates increased orientational order of DPH (P < 0.001, N = 6). Error bars indicate ± SD.

Fig. 5.

Measures of DPH rotational motion obtained from analysis of DPH fluorescence anisotropy decays. A: The diffusion constant for DPH rotation about its long axis, D_perp, from the BRD model analysis. The smaller value in ROS membranes from the AY9944-treated animals, compared with controls, indicates slower DPH rotation (P = 0.006, N = 6). B: The weighted average of the three empirical rotational time constants, <ϕ>, from the model-independent analysis. The higher value for membranes from AY9944-treated animals, compared with controls, indicates slower DPH rotation (P = 0.006, N = 6). Error bars indicate ± SD.

Fig. 6.

Rhodopsin regeneration in ROS membranes from control and AY9944-treated rats. Values expressed as percent regeneration, shown as a function of time ROS membranes were incubated with excess 11-cis retinal at 37°C. Comparison of values from 1 month-old control (open circles) versus AY9944-treated rats (filled circles) shows no statistically significant difference (N = 3). In contrast, comparison of values from 3 month-old control (open squares) and AY9944-treated rats (filled squares) showed a statistically significant difference (P = 0.0006, N = 3, at 24 h). Error bars indicate ± SD.