Quantitative Proteomics Analysis of Inborn Errors of Cholesterol Synthesis: IDENTIFICATION OF ALTERED METABOLIC PATHWAYS IN DHCR7 and SC5D DEFICIENCY*

Smith-Lemli-Opitz syndrome (SLOS) and lathosterolosis are malformation syndromes with cognitive deficits caused by mutations of 7-dehydrocholesterol reductase (DHCR7) and lathosterol 5-desaturase (SC5D), respectively. DHCR7 encodes the last enzyme in the Kandutsch-Russel cholesterol biosynthetic pathway, and impaired DHCR7 activity leads to a deficiency of cholesterol and an accumulation of 7-dehydrocholesterol. SC5D catalyzes the synthesis of 7-dehydrocholesterol from lathosterol. Impaired SC5D activity leads to a similar deficiency of cholesterol but an accumulation of lathosterol. Although the genetic and biochemical causes underlying both syndromes are known, the pathophysiological processes leading to the developmental defects remain unclear. To study the pathophysiological mechanisms underlying SLOS and lathosterolosis neurological symptoms, we performed quantitative proteomics analysis of SLOS and lathosterolosis mouse brain tissue and identified multiple biological pathways affected in Dhcr7^{Δ3–5/Δ3–5} and Sc5d^−/− E18.5 embryos. These include alterations in mevalonate metabolism, apoptosis, glycolysis, oxidative stress, protein biosynthesis, intracellular trafficking, and cytoskeleton. Comparison of proteome alterations in both Dhcr7^{Δ3–5/Δ3–5} and Sc5d^−/− brain tissues helps elucidate whether perturbed protein expression was due to decreased cholesterol or a toxic effect of sterol precursors. Validation of the proteomics results confirmed increased expression of isoprenoid and cholesterol synthetic enzymes. This alteration of isoprenoid synthesis may underlie the altered posttranslational modification of Rab7, a small GTPase that is functionally dependent on prenylation with geranylgeranyl, that we identified and validated in this study. These data suggested that although cholesterol synthesis is impaired in both Dhcr7^{Δ3–5/Δ3–5} and Sc5d^−/− embryonic brain tissues the synthesis of nonsterol isoprenoids may be increased and thus contribute to SLOS and lathosterolosis pathology. This proteomics study has provided insight into the pathophysiological mechanisms of SLOS and lathosterolosis, and understanding these pathophysiological changes will help guide clinical therapy for SLOS and lathosterolosis.Smith-Lemli-Opitz syndrome (SLOS¹; Online Mendelian Inheritance in Man 270400) is a multiple malformation syndrome with cognitive and behavioral deficiencies due to an inborn error of cholesterol synthesis. Typical findings in SLOS include dysmorphic facial features, limb defects, genital anomalies, growth retardation, cognitive disabilities, behavioral problems, and autistic features (for a review, see Ref. 1). The incidence of SLOS has been estimated to be on the order of 1/20,000–1/70,000 (1). SLOS is an autosomal recessive disorder caused by mutation of the 7-dehydrocholesterol reductase gene (DHCR7) (2–4). DHCR7 catalyzes the final step in the Kandutsch-Russel cholesterol biosynthetic pathway. Impaired DHCR7 activity results in increased 7-dehydrocholesterol (7DHC) and decreased cholesterol levels (Fig. 1A). Lathosterolosis is a rare “SLOS-like” malformation syndrome due to mutations of lathosterol 5-desaturase (SC5D) (5–7). SC5D catalyzes the conversion of lathosterol to 7DHC. Thus, in lathosterolosis, like SLOS, there is a deficiency of cholesterol. However, the accumulating precursor sterol is lathosterol rather than 7DHC (Fig. 1A). Because of its rarity and the fact that all known cases of lathosterolosis were ascertained due to similarity with SLOS, the phenotypic spectrum of lathosterolosis has not been defined.Open in a separate windowFig. 1.Representative 2-DE maps of SLOS and lathosterolosis mouse brain proteins. A, SLOS and lathosterolosis are inborn errors of cholesterol synthesis. SLOS is caused by mutations in the DHCR7 gene. DHCR7 catalyzes the final step in cholesterol synthesis. Lathosterolosis is caused by mutations of the SC5D gene. Cholesterol levels are decreased in both SLOS and lathosterolosis, but the accumulating precursor sterol differs. In SLOS, 7DHC accumulates, whereas in lathosterolosis, the accumulating sterol is lathosterol. B, representative 2-DE maps of control (Dhcr7^+/+ and Sc5d^+/+), Dhcr7^{Δ3–5/Δ3–5}, and Sc5d^−/− mouse brain proteins. Eighty micrograms of the pooled protein sample from Dhcr7^+/+, Dhcr7^{Δ3–5/Δ3–5}, Sc5d^+/+, and Sc5d^−/− embryonic mouse brain tissues were separated on a pH 3–10 nonlinear IPG strip followed by electrophoretic separation on a 12% SDS-polyacrylamide gel. Acidic pH is to the left, and increased molecular mass is at the top. Compared with Dhcr7^+/+ mouse brains, the protein spots with significantly decreased or increased expression in Dhcr7^{Δ3–5/Δ3–5} mouse brains are marked in Dhcr7^+/+ and Dhcr7^{Δ3–5/Δ3–5} mouse brain 2-DE maps, respectively. Compared with Sc5d^+/+ mouse brains, the protein spots with significantly decreased or increased expression in Sc5d^−/− mouse brains are marked in Sc5d^+/+ and Sc5d^−/− mouse brain 2-DE maps, respectively. Supplemental Table 2 provides detailed information on the differentially expressed protein spots.Although the genetic and biochemical causes of SLOS are defined, the pathophysiological mechanisms contributing to specific malformations have not been delineated. The classic paradigm for the pathogenesis of an inborn error of metabolism includes the accumulation of a toxic precursor and/or deficiency of an essential product. In the case of SLOS, the observed defects are postulated to be caused, either singly or in combination, by cholesterol deficiency or the accumulation of 7DHC (8, 9).Cholesterol is an essential lipid with multiple critical functions. In addition to being a structural lipid in membranes and myelin, cholesterol is the precursor for bile acid, steroid hormone, neuroactive steroid, and oxysterol synthesis. In cellular membranes, cholesterol rafts are microdomains that function in receptor-mediated signal transduction. Functional defects in IgE receptor-mediated mast cell degranulation and cytokine production (10), N-methyl-d-aspartate receptor function (11), and serotonin 1A receptor ligand binding (12, 13) have been reported in SLOS. The altered sterol composition in SLOS affects the physiochemical properties and function of lipid rafts. Membrane domains incorporating 7DHC differ from those containing only cholesterol in protein composition (14), packing (15), and stability (16–18). Substitution of 7DHC for cholesterol also decreases membrane bending rigidity (19). In addition, model membranes mimicking SLOS membranes have been reported to exhibit atypical membrane organization (20) and curvature (19). These alterations may have functional consequences. Depletion of cholesterol from hippocampal membranes and replenishment with 7-dehydrocholesterol does not restore ligand binding activity of the serotonin 1A receptor despite the recovery of the overall membrane order (12). Cholesterol is also necessary for maturation and function of the hedgehog family of morphogens during embryonic development, and several mechanisms by which sonic hedgehog signaling might be impaired in SLOS have been proposed (21–23).To understand the pathophysiological processes underlying cognitive defects found in SLOS, we need to consider the potential detrimental effects of decreased cholesterol/functional sterol levels versus the potential toxic effects of increased 7DHC. To give insight into pathological effects due to cholesterol deficiency and precursor accumulation, we have produced mouse models deficient in either 7-dehydrocholesterol reductase (11) or lathosterol reductase (6) activity (Dhcr7^{Δ3–5/Δ3–5} and Sc5d^−/−, respectively). Although the two models are similar in many respects, significant differences exist. Dhcr7 pups have relatively few physical malformations other than a low frequency of cleft palate but die during the 1st day of life due to failure to feed (11). In contrast Sc5d mutant embryos are stillborn and have multiple developmental malformations (6). In addition, although secretory granule formation is altered in both models, consistent with differing physiochemical properties of the two precursor sterols, the specific changes differ between the two models (19). For these reasons, a comparison of the two models will provide insight into common mechanisms that are likely due to cholesterol/sterol deficiency and syndrome-specific mechanisms that are due to specific effects of one of the two precursors.We now report the use of two-dimensional electrophoresis (2-DE) mass spectrometry proteomics analysis to identify proteins with altered expression in brain tissue from both Dhcr7 and Sc5d mutants with the goal of identifying novel pathophysiological mechanisms contributing to the neurological deficits in these two inborn errors of cholesterol synthesis. Because our focus was on identifying processes that could contribute to abnormal neurological development, our analysis was focused on brain tissue from E18.5 embryos. This embryonic age was selected because the biochemical defect increases with embryonic age (6, 11), and it is the latest time point for which we could obtain viable tissue for both mutants. Western blot analysis was used to validate selected individual proteins and pathways. Functional annotation suggested that alterations in mevalonate metabolism, glycolysis, oxidative stress, apoptosis, protein biosynthesis, intracellular trafficking, and cytoskeleton may contribute to the pathology of inborn errors of cholesterol synthesis. In addition, our data are consistent with the hypothesis that both cholesterol deficiency and increased precursor sterol levels contribute to SLOS and lathosterolosis pathology.