CERT1 mutations perturb human development by disrupting sphingolipid homeostasis

Abstract

Neural differentiation, synaptic transmission, and action potential propagation depend on membrane sphingolipids, whose metabolism is tightly regulated. Mutations in the ceramide transporter CERT (CERT1), which is involved in sphingolipid biosynthesis, are associated with intellectual disability, but the pathogenic mechanism remains obscure. Here, we characterize 31 individuals with de novo missense variants in CERT1. Several variants fall into a previously uncharacterized dimeric helical domain that enables CERT homeostatic inactivation, without which sphingolipid production goes unchecked. The clinical severity reflects the degree to which CERT autoregulation is disrupted, and inhibiting CERT pharmacologically corrects morphological and motor abnormalities in a Drosophila model of the disease, which we call ceramide transporter (CerTra) syndrome. These findings uncover a central role for CERT autoregulation in the control of sphingolipid biosynthetic flux, provide unexpected insight into the structural organization of CERT, and suggest a possible therapeutic approach for patients with CerTra syndrome.

Keywords: Cell Biology; Genetics; Lipid rafts; Neurodevelopment.

Figure 1. Mutations in CERT1 lead to a neurodevelopmental syndrome.

(A) Schematic representation of functional domains in CERT. The N-terminal PH domain interacts with phosphoinositide phosphatidylinositol-4-phosphate [PtdIns(4)P] (25) on the trans Golgi. The SRR is the target of protein kinase D (PKD) and casein kinase 1γ2 (CSNK1G2) phosphorylation. The FFAT (2 phenylalanines in an acidic tract) motif interacts with the integral membrane proteins VAP-A and VAP-B on the ER (70), and a C-terminal START-related domain extracts Cer from the ER membrane and delivers it to the trans Golgi (7). The schematic shows coding variants in CERT1 (NP_005704) in our cohort of 31 individuals above the gene diagram and other individuals identified from clinical databases (DECIPHER, version 9.31, ClinVar, and VKGL) below it (Supplemental Table 1). Colors indicate the age of onset; gray indicates that no information is available. The distribution of gnomAD singleton missense variants for healthy individuals is plotted below. (B) Range of severity in motor delays compared with the 75th percentile (light gray) and 25th percentile (dark gray), adapted from values published by the Denver Developmental Screening Test II. White and black circles indicate delayed sitting and walking ages, respectively; asterisks indicate that the individual needs sitting or walking support. White circles with an arrow indicate individuals who are currently immobile or have not yet developed independent walking. (C) Heatmap shows the degree of intellectual disability (ID), speech delay (SD), and motor delay (MD) of the patients bearing frequent CERT1 mutations. See Supplemental Table 2 for scores. C1, cluster 1; C2, cluster 2; C3, cluster 3; C4, cluster 4.

Figure 2. Disease-causing variants result in CERT misregulation.

(A) CERT-GFP WT and mutant localization in HeLa cells analyzed by confocal microscopy. Scale bar: 20 μm. (B) Percentage of CERT-GFP WT and mutants associated with the Golgi complex in HeLa cells. Cells were stained with Hoechst and anti-GM130 antibody and analyzed by automated fluorescence microscopy (n >1,000 cells per condition; ****P < 0.0001, 1-way ANOVA, effect size >15%). WT CERT is shown in gray, and CerTra mutants are shown in green. Data are shown as box-and-whisker plots. Bars represent the median value of each data set. (C) Western blot of HeLa cells expressing CERT-GFP WT or mutants (n = 3). Hyperphosphorylated (Hyper p-) and de-/hypophosphorylated (D/hypo-) bands are indicated by arrowheads. The clusters, as in Figure 1, are indicated throughout A–C.

Figure 3. Disease-causing CERT variants are susceptible to phosphoregulation.

(A) Percentage of CERT-GFP WT and mutants from clusters 1, 2, and 3 associated with the Golgi complex in HeLa cells overexpressing CSNK1G2-HA (k) or PP2Cε-HA (p). Cells were stained with Hoechst, anti-GM130 antibody, and anti-HA antibody and analyzed by automated fluorescence microscopy (n >500 cells per condition; **P < 0.01, ***P < 0.001, and ****P < 0.0001, 1-way ANOVA). Bars represent the median value of each data set. (B) Subcellular localization of CERT-GFP WT, p.S132L, p.T166A, and p.G243R in HeLa cells expressing PP2Cε-HA or CSNK1G-HA. Cells were stained with DAPI (blue) and anti-HA antibody and analyzed by confocal microscopy. Asterisks indicate cotransfected cells. Scale bar: 20 μm. (C) Western blot analysis of HeLa cells coexpressing CERT-GFP WT or mutants with CSNK1G2-HA or PP2Cε-HA (n = 3). C, cluster (as represented in Figure 1).

Figure 4. Several CERT1 mutations increase sphingolipid levels.

(A) Schematic representation of the de novo sphingolipid biosynthetic pathway with the main enzymes involved (shading indicates the prevalent intracellular localization of synthetic reactions). (B) Mass spectrometry profile of sphingolipids in HeLa CERT1-KO cells overexpressing selected CERT1 mutants from clusters 1, 2, and 3. Values are total levels across the major fatty acid chain lengths of the indicated sphingolipids. The levels of individual species are reported in Supplemental Figure 4A. n = 3. Data are the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA. (C) Effect on the LCB of CERT mutations in HeLa cells. LCB profiles were evaluated by incorporation of an isotope labeled (2,3,3-d³,¹⁵N)-l-serine. n = 3. Data are the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA. (D) The de novo sphingolipid biosynthetic pathway as modified by CERT1 mutations in clusters 1, 2, and 3. CerS, ceramide synthases; DES, dihydroceramide desaturase; SMS1, sphingomyelin synthase 1; UGCG, glucosylceramide synthase; ST3Gal5, GM3 synthase; A4GalT, Gb3 synthase; Sph, sphingolipid.

Figure 5. Disease-causing CERT1 mutations affect the central core structure of CERT.

(A) Domain organization of CERT with its CCD and the predicted H1 and H2 helices. The global percentage of HDX is shown for all peptides graphed according to their central residue number. The mean of 3 experiments is shown. (B) Deuterium incorporation over time of 4 selected peptides (highlighted with arrows on the HDX profile). Data are from Supplemental Tables 5 and 6. (C) Molecular model of CERT’s CCD based on contact prediction. Helix H1 is shown in yellow and H2 in red. (D) Thermostability of intervening regions showing a difference in HDX by circular dichroism: the samples were heated from 4°C to 94°C; the percentage indicates the helicity of each construct at 20°C. (E) Deconvoluted mass spectrum of purified recombinant CERT 151-309 WT. The value 18.5 kDa represents the molecular weight of the monomer, 37 kDa a dimer, and 71.5 kDa a tetramer. (F) Molecular model of CERT 151-309 WT as an antiparallel dimer. (G) Model showing the location of aa mutated in CerTra syndrome. The areas differentially exposed to deuterium exchange are indicated according to the color scale. (H) Molecular model of CERT WT as an antiparallel dimer. (I) Molecular model of CERT WT at the ER-TGN membrane contact site in its active and inactive conformations. (J) Thermostability of CERT 154-187 WT and p.T166A and CERT 151-309 WT and p.G243R by circular dichroism. (K) Peptide in the CCD displaying decreases in exchange in the p.G243R mutant compared with WT. These changes are mapped on the hypothetical CCD structure.

Figure 6. CERT gain of function causes neurological defects in D. melanogaster.

(A) Schematic of transgenic dCERT flies on the w¹¹¹⁸ background. dCERT^WT, +WT; dCERT^p.S149L, +SL; Chr, chromosome. (B) Quantification of endogenous or exogenous dCERT mRNA levels in flies on the w¹¹¹⁸ background (Ctrl) in dCERT-transgenic flies. Data indicate the log₂ fold change over Ctrl or +WT (n = 6; data are the mean ± SD). (C) Representative specimens of Ctrl, +WT, and +SL adult flies. Head length (HL), abdomen length (AL), and wing length (WL) were measured. Scale bars: 1 mm. (D) 3D rendering of a micro-CT scan of the heads from Ctrl, +WT, and +SL adults (frontal view). Brain volume is highlighted in pink. Body axes are dorsal (D) and left (L). Scale bars: 100 μm. (E) Z-projections of confocal stacks of whole-mount adult Ctrl, +WT, and +SL adult brains (frontal view) immunostained with anti-nc82. Scale bars: 100 μm. (F) HL/AL ratio of flies reared on DMSO or 10 μM HPA-12 (HPA) (Ctrl, n = 26 or 30, +WT n = 31 or 31, and +SL, n = 25 or 38). (G) Brain volume for Ctrl, +WT, and +SL flies as determined by confocal microscopy (n = 3–4; data are the mean ± SEM). (H) Mass spectrometry profile of sphingolipids in Ctrl, +WT, and +SL adult heads (n = 8). (I) Locomotion of Ctrl, +WT, and +SL flies plotted as total counts per 30 minutes over time (n = 16). (J) Locomotion of flies pretreated with 0 or 10 μM HPA-12 (n = 12). (K) Climbing ability of flies at 2, 9, 16, and 23 days post eclosion (dpe) after vigorous mechanical stress (n = 9). Data shown are the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 1-way ANOVA.