DHCEO accumulation is a critical mediator of pathophysiology in a Smith-Lemli-Opitz syndrome model

Abstract

Smith-Lemli-Opitz syndrome (SLOS) is an inborn error of metabolism caused by defective cholesterol biosynthesis. Mutations within the gene encoding 7-dehydrocholesterol reductase (DHCR7), the last enzyme in the pathway, lead to the accumulation of 7-dehydrocholesterol (7-DHC) in the brain tissue and blood of the SLOS patients. The objective of this study was to determine the consequences of the accumulation of an immediate cholesterol precursor, 7-DHC and its oxysterol metabolite, 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), in the brain tissue of Dhcr7-KO mouse, a model for SLOS. We found that cholesterol, 7-DHC and DHCEO show region-specific distribution, suggesting that the midbrain and the cortex are the primary sites of vulnerability. We also report that neurons are ten fold more susceptible to a 7-DHC-derived oxysterol mixture than glial cells, and that DHCEO accelerates differentiation and arborization of cortical neurons. The overall results suggest that 7-DHC oxidative metabolites are critical contributors to altered neural development in SLOS. The future studies will test if antioxidant supplementation will ameliorate some of the clinical symptoms associated with this devastating disease.

Figure 1. Structure and expression of cholesterol, 7-dehydrocholesterol and DHCEO in WT and Dhcr7-KO mouse brain tissue

A) Chemical structure of cholesterol, immediate cholesterol precursor 7-DHC, and DHCEO, which is derived from 7-DHC by oxidation. B) Expression of cholesterol, 7-DHC, and DHCEO levels in cortex, midbrain, hippocampus and cerebellum in E20 mouse brain tissue. 7-DHC levels < 0.2 μg/mg in WT are consistent with previously published measurements of 7-DHC in the mouse brain tissue^, . DHCEO level in WT tissue was below the limit of detection for LC/MS (less than 2 ng). Note the high, but variable levels of 7-DHC and DHCEO across the various brain regions of the Dhcr7-KO mice.

Figure 2. 7-DHC and DHCEO accumulation vary across brain regions in Dhcr7-KO mice

A) Normalized cholesterol levels are highest in the cortex and cerebellum in the WT mouse brain. B) The highest amount of 7-DHC is found in midbrain and cortex in Dhcr7-KO mice, with strongly diminished and uniform levels of cholesterol across all the brain regions. C) The highest levels of DHCEO are formed in cortex and midbrain. Note that cholesterol and/or 7-DHC are not predictive of DHCEO levels across the brain regions.

Figure 3. 7-DHC-derived oxysterols are highly neurotoxic

X-axis denotes concentrations of oxysterols while y-axis shows the percentage of live cells compared to untreated controls. Primary cortical neurons and astrocytes were treated with different concentrations of 7-DHC oxysterol mixture for 72 hrs. Cell survival was measured using CellTiterProliferation Assay (Promega). Note that oxysterols, in concentrations comparable to those found in the brain of SLOS mice, show approximately 10-fold higher cytotoxicity to neocortical neurons than astrocytes.

Figure 4. Differentiation of neuronal processes in vitro is accelerated in the presence of DHCEO

Primary cortical neurons were grown for 3 days and then treated with 7-DHC-derived oxysterol DHCEO for 48 hrs. A-B) Example of control and DHCEO-treated cultures stained with MAP2 antibody (cal bar = 50 μm). Unaltered images were acquired at the same exposure times showing more pronounced intensity of staining in DHCEO-treated cultures, suggesting relative immaturity of control cultures compared to DHCEO-treated ones. C-H) Examples of different neuronal types in cultures exposed to sham- and DHCEO treatment (cal bar = 100 μm). Note that in all figures DHCEO-treated neurons show more extensive neuronal arborization.

Figure 5. DHCEO-treated neurons show a statistically significant increase in process arborization across the various morphological classes of neurons

A) Pyramidal-like, multipolar and bipolar neurons all report a statistically significant increase in total process length after 72 hours of DHCEO exposure (10-14 neurons per group, *p<0.01; **p<0.001). B) Typical examples of neurolucida-traced cortical neurons exposed to sham or DHCEO treatment. C) Graphical representation of process arborization as determined by Sholl Analysis, performed on neurolucida-traced neurons. Each bar represents a different neuron.