Abstract
Smith-Lemli-Opitz syndrome (SLOS) is a metabolic and developmental disorder caused by mutations in the gene encoding the enzyme 7-dehydrocholesterol reductase (Dhcr7). This reductase catalyzes the last step in cholesterol biosynthesis, and levels of 7-dehydrocholesterol (7-DHC), the substrate for this enzyme, are elevated in SLOS patients as a result of this defect. Our group has previously shown that 7-DHC is extremely prone to free radical autoxidation, and we identified about a dozen different oxysterols formed from oxidation of 7-DHC. We report here that 7-DHC-derived oxysterols reduce cell viability in a dose- and time-dependent manner, some of the compounds showing activity at sub-micromolar concentrations. The reduction of cell survival is caused by a combination of reduced proliferation and induced differentiation of the Neuro2a cells. The complex 7-DHC oxysterol mixture added to control Neuro2a cells also triggers the gene expression changes that were previously identified in Dhcr7-deficient Neuro2a cells. Based on the identification of overlapping gene expression changes in Dhcr7-deficient and 7-DHC oxysterol-treated Neuro2a cells, we hypothesize that some of the pathophysiological findings in the mouse SLOS model and SLOS patients might be due to accumulated 7-DHC oxysterols.
Highlights
Smith-Lemli-Opitz syndrome (SLOS) is a metabolic and developmental disorder caused by mutations in the gene encoding the enzyme 7-dehydrocholesterol reductase (Dhcr7)
The concentration of the 7-DHC oxysterol mixture that led to a 50% reduction in cell survival was used to treat Neuro2a cultures for 24–72 h, and the morphological changes of the cells were analyzed by the use of Tu20 and p75 antibodies
Based on the studies reported here, we conclude that the majority of the oxysterols formed from peroxidation of 7-DHC are bioactive and strongly affect cell viability
Summary
The initiator 2,2’-azobis(4-methoxy-2,4-dimethylvaleronitrile) was purchased from Wako Chemicals, dried under vacuum, and stored at Ϫ40°C. 7-DHC (>98%) and Rose Bengal (95%) were purchased from Sigma-Aldrich Co. and were used without further purification. Cells were cultured with medium containing 10% cholesterol-deficient serum (Thermo Scientific HyClone Lipid Reduced FBS). Because the 7-DHC oxysterol mixture contains over a dozen compounds, we designed experiments to determine the activity of individual fractions collected from HPLC, aiming to find the most potent peaks. The primary 7-DHC-oxidation mixture (0.11 M, 100 l) was subject to separation by normal phase HPLC-UV (25 cm × 10 mm, 5 Si; elution solvent: 10% 2-propanol in hexanes, flow rate: 4.0 ml/min, monitoring wavelengths: 210 and 246 nm) over a 40 min period. A parallel HPLC-UV separation of 7-DHC oxidation products reduced with P(OMe) was carried out to collect peaks eluting at 6.9, 7.9, 8.9, 9.2, 10.9, 11.9, 12.5, 13.0, and 15.5 min. Cell viability tests on purified compounds are consistent with this observation
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