Reduced cholesterol levels impair Smoothened activation in Smith–Lemli–Opitz syndrome

Abstract

Smith–Lemli–Opitz syndrome (SLOS) is a common autosomal-recessive disorder that results from mutations in the gene encoding the cholesterol biosynthetic enzyme 7-dehydrocholesterol reductase (DHCR7). Impaired DHCR7 function is associated with a spectrum of congenital malformations, intellectual impairment, epileptiform activity and autism spectrum disorder. Biochemically, there is a deficit in cholesterol and an accumulation of its metabolic precursor 7-dehydrocholesterol (7DHC) in developing tissues. Morphological abnormalities in SLOS resemble those seen in congenital Sonic Hedgehog (SHH)-deficient conditions, leading to the proposal that the pathogenesis of SLOS is mediated by aberrant SHH signalling. SHH signalling is transduced through the transmembrane protein Smoothened (SMO), which localizes to the primary cilium of a cell on activation and is both positively and negatively regulated by sterol molecules derived from cholesterol biosynthesis. One proposed mechanism of SLOS involves SMO dysregulation by altered sterol levels, but the salient sterol species has not been identified. Here, we clarify the relationship between disrupted cholesterol metabolism and reduced SHH signalling in SLOS by modelling the disorder in vitro. Our results indicate that a deficit in cholesterol, as opposed to an accumulation of 7DHC, impairs SMO activation and its localization to the primary cilium.

Cholesterol activates the G-protein coupled receptor Smoothened to promote Hedgehog signaling

“…Cholesterol has been shown to be necessary for SMO activation, based on experiments using inhibitors of cholesterol biosynthesis and high concentrations of naked MβCD to strip the plasma membrane of cholesterol (Cooper et al, 2003). Impaired SMO activation caused by cholesterol deficiency has also been noted in Smith-Lemli-Opitz syndrome (SLOS), a congenital malformation syndrome caused by defects in the enzyme that converts 7-dehydrocholesterol to cholesterol (Blassberg et al, 2016; Cooper et al, 2003). In contrast to our results, the SMO CRD is dispensable for this permissive role of cholesterol.…”

“…In contrast to our results, the SMO CRD is dispensable for this permissive role of cholesterol. The depletion of cholesterol reduces signaling by SMO mutants lacking the entire CRD (Myers et al, 2013) or carrying mutations in the CRD binding-groove (Blassberg et al, 2016). By analogy with other GPCRs, these permissive effects are likely to be mediated by the SMO 7TMD.…”

“…While the best-defined role for cholesterol is in the biogenesis of Hh ligands (Porter et al, 1996), it also plays an independent role in the reception of Hh signals. Pharmacological or genetic depletion of cholesterol reduces cellular responses to Hh ligands, which has led to the view that cholesterol is permissive for Hh signaling (Blassberg et al, 2016; Cooper et al, 1998; Cooper et al, 2003; Incardona et al, 1998; Incardona and Roelink, 2000). Distinct from these previous observations, we find that an acute increase in plasma membrane cholesterol is sufficient to activate Hh signaling.…”

Endogenous B-ring oxysterols inhibit the Hedgehog component Smoothened in a manner distinct from cyclopamine or side-chain oxysterols

“…As a result, cholesterol is depleted, whereas 7-DHC accumulates and is converted into abnormal derivatives. One of the underlying reasons for the malformations in Smith-Lemli-Opitz syndrome (SLOS) (DHCR7 deficiency) is thought to be cholesterol deficiency, and pharmacologic depletion of cellular sterols by methyl-β-cyclodextrin (MCD) inhibits Smoothened activity in wild-type cells (9,10). It is not clear whether the sterols depleted by MCD directly activate Smoothened or act as permissive factors for Smoothened activation, as noted for sterol regulation of many other GPCR superfamily members (11).…”

A new trick for an old lipid

“…Unexpectedly, they found a cholesterol molecule occupied a hydrophobic (water-fearing) pocket in the cysteine-rich domain. Since disrupting cholesterol production in humans and mice affects Smoothened activity (Blassberg et al, 2016; Cooper et al, 2003), this raised the possibility that cholesterol might directly bind to and regulate Smoothened.…”