1. Biochemistry and Chemical Biology
  2. Cell Biology
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The Ormdl genes regulate the sphingolipid synthesis pathway to ensure proper myelination and neurologic function in mice

  1. Benjamin A Clarke
  2. Saurav Majumder
  3. Hongling Zhu
  4. Y Terry Lee
  5. Mari Kono
  6. Cuiling Li
  7. Caroline Khanna
  8. Hailey Blain
  9. Ronit Schwartz
  10. Vienna L Huso
  11. Colleen Byrnes
  12. Galina Tuymetova
  13. Teresa M Dunn
  14. Maria L Allende  Is a corresponding author
  15. Richard L Proia  Is a corresponding author
  1. National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, United States
  2. Uniformed Services University of the Health Sciences, United States
Research Article
Cite this article as: eLife 2019;8:e51067 doi: 10.7554/eLife.51067
4 figures, 2 tables and 1 additional file

Figures

Figure 1 with 2 supplements
Ormdl3, but not Ormdl1 or Ormdl2, single knockout (KO) mice exhibit significantly increased levels of sphingolipids in the brain.

(A) Schematic of the de novo sphingolipid biosynthetic pathway and its feedback inhibition by ORMDLs through the sensing of ceramide levels. SPT, serine palmitoyltransferase; 3KDHSph, 3-keto-dihydrosphingosine; DHSph, dihydrosphingosine; DHCer, dihydroceramide; Cer, ceramide; Sph, sphingosine; S1P, sphingosine-1-phosphate. (B–D) Generation of Ormdl KO mice. Panels show the intron-exon organizations of the Ormdl genes and the protein coding regions (white). (B, C) Ormdl1 and Ormdl2 KO mice were produced by CRISPR/Cas9-induced mutations, resulting in frameshifts and premature stop codons. The locations of sgRNA sequences (red), PAM sites (green), as well as the changes in DNA and protein are indicated. The base insertion in the CRISPR/Cas9 modified Ormdl2 gene is underlined. (D) Ormdl3 KO mice were generated by germline Cre-LoxP recombination to excise exons 2, 3, and part of exon 4, resulting in the deletion of the entire protein-coding sequence. (E) RT-qPCR of Ormdl WT RNA in brain of Ormdl KO mice relative to that in WT mice. The mice were 8 weeks old. Probes detect the WT Ormdl sequences. Data are expressed as means ± SD. Unpaired Student’s t test; ***p<0.001. nd, not detectable. n = 4 for all genotypes. (F) Levels of dihydrosphingosine, total dihydroceramide, total ceramide, and sphingosine were determined by HPLC-tandem MS on lipid extracts of whole brains harvested from 8-week-old WT, Ormdl1 KO, Ormdl2 KO, Ormdl3 KO, Ormdl1/2 double KO, Ormdl1/3 double KO, and Ormdl2/3 double KO mice (Figure 1—source data 1). Data are expressed as means ± SD. One-way ANOVA with Bonferroni correction; *p<0.05, ***p<0.001. n = 8 for all genotypes. DKO, double knockout.

Figure 1—source data 1

Levels of dihydrosphingosine, total dihydroceramide, total ceramide, and sphingosine from brains of WT, Ormdl1 KO, Ormdl2 KO, Ormdl3 KO, Ormdl1/2 double KO, Ormdl1/3 double KO, and Ormdl2/3 double KO mice.

https://cdn.elifesciences.org/articles/51067/elife-51067-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Generation of floxed Ormdl3 mice.

(A) The Ormdl3 gene contains four exons. The protein-coding region (white) extends from exon 2 to exon 4. (B) A gene targeting vector was designed with a 2.4 kb 5′ homology arm (green) and a 5.9 kb 3′ homology arm (red). A LoxP/FRT-flanked neomycin expression cassette was inserted 179 bp upstream of exon 2 in a transcriptional orientation opposite from Ormdl3. A single LoxP site was inserted into the 3′ UTR of exon 4. The genomic region flanked by the LoxP sites was 1.9 kb and included exons 2 and 3, as well as 428 bp of exon 4. (C) Structure of the targeted allele. (D) The neomycin (Neo) cassette was removed by FRT-mediated recombination, resulting in the floxed Ormdl3 -Neo allele.

Figure 1—figure supplement 2
Levels of brain ceramide and dihydroceramide subspecies in Ormdl KO mice.

Sphingolipid concentrations were determined by HPLC-tandem MS on lipid extracts of whole brains harvested from 8-week-old WT, Ormdl1 KO, Ormdl2 KO, Ormdl3 KO, Ormdl1/2 double KO, Ormdl1/3 DKO, and Ormdl2/3 double KO mice (Figure 1—figure supplement 2—source data 1 file 1). (A) Individual ceramide subspecies with different fatty-acid chain lengths and C18 sphingoid bases. (B) Individual dihydroceramide subspecies with different fatty-acid chain lengths. Data are expressed as means ± SD. One-way ANOVA with Bonferroni correction; *p<0.05, ***p<0.001 versus WT mice. n = 8 for all genotypes. DKO, double knockout.

Figure 1—figure supplement 2—source data 1

Levels of individual ceramide and dihydroceramide subspecies with different fatty-acid chain lengths from brains of WT, Ormdl1 KO, Ormdl2 KO, Ormdl3 KO, Ormdl1/2 double KO, Ormdl1/3 double KO, and Ormdl2/3 double KO mice.

https://cdn.elifesciences.org/articles/51067/elife-51067-fig1-figsupp2-data1-v1.xlsx
Elevated sphingolipids, neurologic phenotype and reduced viability when multiple Ormdls are deleted.

(A) Body weight of 8-week-old male (n = 7–14) and female mice (n = 7–10) of the indicated genotypes. Each circle represents the weight of an individual mouse. Unpaired Student’s t test; ***p<0.001 versus WT mice. (B) Litter size was determined at weaning for offspring of the indicated matings. Each circle represents the number of weanlings from an individual litter (n = 19–25). bnd, breeding not done. One-way ANOVA with Bonferroni correction. (C) Image of an 8-week-old Ormdl1/3 double KO mouse (right) showing characteristic clasping of the hindlimbs upon tail suspension, a sign of neurodegeneration. An Ormdl1–/– Ormdl3+/– littermate is shown (left). (D) Wire hang behavioral test. Eight-week-old mice were allowed to hang from a suspended wire using the forelimbs and the latency time to fall was recorded. The maximum hanging time was 180 s. Circles represent the mean of three determinations for each mouse (n = 16–29 mice for each genotype). One-way ANOVA with Bonferroni correction; ***p<0.001 versus WT mice. DKO, double knockout.

Figure 3 with 1 supplement
Myelination is disrupted in Ormdl1/3 double KO mice.

(A) Representative transmission EM images of sciatic nerve of 6-week-old WT, Ormdl1 KO, Ormdl3 KO, and Ormdl1/3 double KO mice. Scale bars, 10 μm. (B) Axon density in sciatic nerve of 6-week-old WT, Ormdl1 KO, Ormdl3 KO, and Ormdl1/3 double KO mice was determined by quantifying the number of myelinated axons in 7–12 EM fields per genotype. Data are expressed as means ± SD. One-way ANOVA with Bonferroni correction; ***p<0.001. n = 3 for WT, n = 2 for Ormdl1 KO, n = 2 for Ormdl3 KO, n = 3 for Ormdl1/3 double KO mice. (C) Example images of redundant myelin figures in sciatic nerve axons of 6-week-old Ormdl1/3 double KO mice. Scale bars, 5 μm. (D) Percentage of myelinated axons in sciatic nerve of 6-week-old WT, Ormdl1 KO, Ormdl3 KO, and Ormdl1/3 double KO mice showing redundant myelination was quantified in 7–12 EM fields per genotype. Data are expressed as means ± SD. One-way ANOVA with Bonferroni correction; ***p<0.001. n = 3 for WT, n = 2 for Ormdl1 KO, n = 2 for Ormdl3 KO, n = 3 for Ormdl1/3 double KO mice. (E) Levels of dihydrosphingosine, total dihydroceramide, total ceramide, sphingosine and hexosylceramide were determined by HPLC-tandem MS on lipid extracts of sciatic nerve from 8-week-old WT, Ormdl1 KO, Ormdl3 KO, and Ormdl1/3 double KO mice (Figure 3—source data 1). Data are expressed as means ± SD. One-way ANOVA with Bonferroni correction; ***p<0.001. n = 8 for all genotypes. DKO, double knockout.

Figure 3—source data 1

Levels of dihydrosphingosine, total dihydroceramide, total ceramide, sphingosine and total hexosylceramide from sciatic nerves of WT, Ormdl1 KO, Ormdl3 KO, and Ormdl1/3 double KO mice.

https://cdn.elifesciences.org/articles/51067/elife-51067-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Levels of sciatic nerve ceramide, dihydroceramide and hexosylceramide subspecies in Ormdl1 KO, Ormdl3 KO, and Ormdl1/3 double KO mice.

Concentrations of sphingolipids with C18 sphingoid bases were determined by HPLC-tandem MS on lipid extracts of sciatic nerves harvested from 8-week-old WT, Ormdl1 KO, Ormdl3 KO, and Ormdl1/3 double KO mice (Figure 3—figure supplement 1—source data 1 file 1). (A) Individual ceramide subspecies with different fatty-acid chain lengths. (B) Individual dihydroceramide subspecies with different fatty-acid chain lengths. (C) Individual hexosylceramide subspecies with different fatty-acid chain lengths. Data are expressed as means ± SD. One-way ANOVA with Bonferroni correction; *p<0.05, **p<0.01, ***p<0.001 versus WT mice. n = 8 for all genotypes. DKO, double knockout.

Figure 3—figure supplement 1—source data 1

Levels of individual ceramide, dihydroceramide and hexosylceramide subspecies with different fatty-acid chain lengths from sciatic nerves of WT, Ormdl1 KO, Ormdl3 KO, and Ormdl1/3 double KO mice.

https://cdn.elifesciences.org/articles/51067/elife-51067-fig3-figsupp1-data1-v1.xlsx
Figure 4 with 2 supplements
Increased de novo sphingolipid biosynthesis in myelin-producing cells mimicked the phenotype of Ormdl1/3 KO mice.

(A) Six-week-old fSPT/iPlp1-Cre (fSPT) mouse after treatment with tamoxifen for 2 weeks. (B) Western blot of fSPT expression in sciatic nerve of 6-week-old mice carrying Stop-fSPT without and with iPlp1-Cre (fSPT) after treatment with tamoxifen for 2 weeks. Bottom panel represents the same blot reprobed with an antibody against β-actin as a loading control. n = 3 for both genotypes. (C) Representative transmission EM images of sciatic nerve of 6-week-old Stop-fSPT and Stop-fSPT/iPlp1-Cre (fSPT)mice after treatment with tamoxifen for 2 weeks. Scale bars, 5 μm. (D) Axon density in sciatic nerve of 6-week-old Stop-fSPT and fSPT/iPlp1-Cre (fSPT) mice after treatment with tamoxifen for 2 weeks was determined by quantifying the number of myelinated axons in five and six EM fields for Stop-fSPT and fSPT/iPlp1-Cre mice (fSPT), respectively. Data are expressed as means ± SD. One-way ANOVA with Bonferroni correction; ***p<0.001. n = 2 for both genotypes. (E) Example images of redundant myelin figures in sciatic nerve of 6-week-old fSPT/iPlp1-Cre (fSPT) mice after treatment with tamoxifen for 2 weeks. Scale bars, 2 μm. (F) Percentage of myelinated axons showing redundant myelination in sciatic nerve of 6-week-old Stop-fSPT and fSPT/iPlp1-Cre (fSPT) mice after treatment with tamoxifen for 2 weeks, quantified in five and six EM fields for Stop-fSPT and fSPT mice, respectively. Data are expressed as means ± SD. One-way ANOVA with Bonferroni correction; ***p<0.001. n = 2 for both genotypes. (G) Levels of dihydrosphingosine, total dihydroceramide, total ceramide, sphingosine and hexosylceramide were determined by HPLC-tandem MS on lipid extracts of sciatic nerve from 6-week-old Stop-fSPT and fSPT/iPlp1-Cre (fSPT) mice after treatment with tamoxifen for 2 weeks (Figure 4—source data 1). Data are expressed as means ± SD. One-way ANOVA with Bonferroni correction; ***p<0.001. n = 5 for Stop-fSPT, n = 7 for fSPT/iPlp1-Cre (fSPT) mice after treatment with tamoxifen for 2 weeks.

Figure 4—source data 1

Levels of dihydrosphingosine, total dihydroceramide, total ceramide, sphingosine and hexosylceramide from sciatic nerves of mice carrying Stop-fSPT, without or with iPlp1-Cre (fSPT), after treatment with tamoxifen.

https://cdn.elifesciences.org/articles/51067/elife-51067-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Generation and characterization of fSPT conditional mutant mice.

(A) fSPT was linked to EGFP by a T2A sequence and inserted into a vector containing a CAG promoter, a transcriptional stop sequence flanked by LoxP sites, and a polyA signal. This transcriptional cassette was flanked by attB sites. The plasmid along with phiC31 integrase protein was microinjected into zygotes from FVB Rosa26-3attP mice, which contained three tandem attP sites (3attP) in the Rosa26 locus, resulting in integration into the Rosa26 locus as indicated. The integrase catalyzes recombination between the attP and attB sites, resulting in two new hybrid sites, attL and attR. Expression of Cre excises the Stop sequence, enabling expression of the fSPT. Primers for genotyping (ForSPT, RevSPT, ForR26, RevR26) are shown and described in the 'Materials and methods' section. (B) Western blot of fSPT expression in liver of 12-week-old mice carrying Stop-fSPT, without (left) and with (right) Mx1-Cre (fSPT),after treatment with pIpC for 4 weeks. The bottom panel presents the same blot stripped and reprobed with an antibody against β-actin as a loading control. n = 3 for both genotypes. (C) SPT enzyme activity in liver of 12-week-old mice carrying Stop-fSPT without and with Mx1-Cre (fSPT) after treatment with pIpC for 4 weeks. Data are expressed as mean SPT activity (pmol of [3H] serine incorporated into sphingoid bases/mg protein/min) ± SD. Unpaired Student’s t test; **p<0.01. n = 3 for both genotypes. (D) Levels of individual ceramide subspecies with different fatty-acid chain lengths and C18 sphingoid bases were determined by HPLC-tandem MS on lipid extracts of liver harvested from 12-week-old mice carrying Stop-fSPT, without and with Mx1-Cre (fSPT), after treatment with pIpC for 4 weeks (Figure 4—figure supplement 1—source data 1 file 1). DHC16, C16-dihydroceramide. Data are expressed as means ± SD. Unpaired Student’s t test; *p<0.05, **p<0.01, ***p<0.001. n = 5 for Stop-fSPT, n = 8 for fSPT/Mx1-Cre (fSPT), after treatment with pIpC.

Figure 4—figure supplement 1—source data 1

Levels of individual ceramide subspecies with different fatty-acid chain lengths, C16-dihydroceramide, dihydrosphingosine, total ceramide, and sphingosine from liver of mice carrying Stop-fSPT, without and with Mx1-Cre (fSPT), after treatment with pIpC.

DHC16, C16-dihydroceramide.

https://cdn.elifesciences.org/articles/51067/elife-51067-fig4-figsupp1-data1-v1.xlsx
Figure 4—figure supplement 2
Levels of sciatic nerve ceramide and dihydroceramide subspecies in mice overexpressing fSPT.

Concentrations of sphingolipid with C18 sphingoid bases were determined by HPLC-tandem MS on lipid extracts of sciatic nerve harvested from 6-week-old mice carrying Stop-fSPT, without or with iPlp1-Cre (fSPT), after treatment with tamoxifen for 2 weeks (Figure 4—figure supplement 1—source data 1 file 2). (A) Individual ceramide subspecies with different fatty-acid chain lengths. Data are expressed as means ± SD. One-way ANOVA with Bonferroni correction; *p<0.05, ***p<0.001. n = 8 for both genotypes. (B) Individual dihydroceramide subspecies with different fatty-acid chain lengths. (C) Individual hexosylceramide subspecies with different fatty-acid chain lengths. Data are expressed as means ± SD. One-way ANOVA with Bonferroni correction; *p<0.05, **p<0.01, ***p<0.001. n = 5 for Stop-fSPT, n = 7 for fSPT/iPlp1-Cre (fSPT) mice after treatment with tamoxifen for 2 weeks.

Figure 4—figure supplement 2—source data 1

Levels of individual subspecies of ceramide, dihydroceramide and hexosylceramide with different fatty-acid chain lengths from sciatic nerves of mice carrying Stop-fSPT, without or with iPlp1-Cre (fSPT), after treatment with tamoxifen.

https://cdn.elifesciences.org/articles/51067/elife-51067-fig4-figsupp2-data1-v1.xlsx

Tables

Table 1
Analysis of offspring from Ormdl1–/– Ormdl2+/– Ormdl3+/– intercrosses.

Mouse genotypes were determined by PCR of tail-snip DNA of 156 pups at weaning from 38 litters derived from Ormdl1–/– Ormdl2+/– Ormdl3+/– intercrosses. The genotype distribution frequency of offspring, predicted by Mendelian considerations and actually obtained, is shown. The Chi-square test was used to determine whether the obtained distribution of genotypes was statistically different from the predicted Mendelian ratios; p<0.0001.

Ormdl1–/–
Ormdl2+/+
Ormdl3+/–
Ormdl1–/–
Ormdl2+/+
Ormdl3+/+
Ormdl1–/–
Ormdl2+/+
Ormdl3–/–
Ormdl1–/–
Ormdl2+/–
Ormdl3+/–
Ormdl1–/–
Ormdl2+/–
Ormdl3+/+
Ormdl1–/–
Ormdl2+/–
Ormdl3–/–
Ormdl1–/–
Ormdl2–/–
Ormdl3+/+
Ormdl1–/–
Ormdl2–/–
Ormdl3+/–
Ormdl1–/–
Ormdl2–/–
Ormdl3–/–
Observed #16209336201150
Observed %10.2612.825.7721.1539.7407.053.210
Predicted #9.7519.59.7519.53919.59.7519.59.75
Predicted %6.2512.56.2512.52512.56.2512.56.25
Key resources table
Reagent type
(species) or
resource
DesignationSource or referenceIdentifiersAdditional
information
Genetic reagent
(M. musculus)
Ormdl1 KO miceThis paperRRID:
IMSR_JAX:034646
See 'Materials and methods'
Genetic reagent
(M. musculus)
Ormdl2 KO miceThis paperRRID:
IMSR_JAX:034645
See 'Materials and methods'
Genetic reagent
(M. musculus)
Ormdl3 Floxed miceThis paperRRID:
IMSR_JAX:034644
See 'Materials and methods'
Genetic reagent
(M. musculus)
EIIA-Cre miceJackson LaboratoryRRID:IMSR_JAX:003724Mice expressing Cre transgene under the control EIIa promoter
Genetic reagent
(M. musculus)
STOP-fSPT transgenic miceThis paperSee 'Materials and methods'
Genetic reagent
(M. musculus)
PLP/creERT miceJackson LaboratoryRRID:IMSR_JAX:005975Mice expressing tamoxifen inducible-mouse Plp1 promoter
Genetic reagent
(M. musculus)
Mx-Cre miceJackson LaboratoryRRID:IMSR_JAX:003556Mice expressing Cre transgene under the control of the mouse Mx1 promoter
AntibodyMonoclonal anti-human SPTLC1Santa Cruz Bio-technologyRRID:AB_10917035; clone H-1, cat. # sc-374143For western blot (1:1000 dilution)
AntibodyAnti-mouse IgG-HRP conjugatedMilliporeRRID:AB_9045; Cat. # AP-124PFor western blot (1:1000 dilution)
AntibodyAnti-mouse β-actin-HRP conjugatedAbcamRRID:AB_8674; AC-15, cat # ab49900For western blot (1:100,000 dilution)
Sequence-based reagentOrmdl1ForPCR primerGenotyping Ormdl1 KO mice: 5′-TGTAATGAACAGCCGTGGTAT
Sequence-based reagentOrmdl1RevPCR primerGenotyping Ormdl1 KO mice: 5′-GCAGAAGGGGATGCTGAGTAATA
Sequence-based reagentOrmdl2ForPCR primerGenotyping Ormdl2 KO mice: 5′-CACATGCAGCAGTCCTACCA
Sequence-based reagentOrmdl2RevPCR primerGenotyping Ormdl2 KO mice: 5′-GTTGGACTCCTGCCTGATCC
Sequence-based reagentNDEL1PCR primerGenotyping Ormdl3 KO mice: 5′-GCAGGAGGAAGAGGCCCTCAG
Sequence-based reagentNDEL2PCR primerGenotyping Ormdl3 KO mice: 5′-CTCTTGACTGCCGCTCTGCAAAAGAG
Sequence-based reagentSIAE4PCR primerGenotyping Ormdl3 KO mice: 5′-CACGGCGCAGGGTTCTAATACATAC
Sequence-based reagentForSPTPCR primerGenotyping fSPT mice: 5′-CATCGAGCTGAAGGGCATCG
Sequence-based reagentRevSPTPCR primerGenotyping fSPT mice: 5′-GTTATCTAGAATTATAGACGCGCTAG
Sequence-based reagentForR26PCR primerGenotyping fSPT mice: 5′-AGTTCTCTGCTGCCTCCTGGCTTCT
Sequence-based reagentCreForPCR primerGenotyping fSPT mice: 5′-GCCTGCATTACCGGTCGATGC
Sequence-based reagentCreRevPCR primerGenotyping: 5′-CAGGGTGTTATAAGCAATCCC
Sequence-based reagentTaqmanFWDOrmdl1 taqman assay primerOrmdl1 taqman assay primer: 5′-ATGAATGTTGGAGTTGCCCAC
Sequence-based reagentTaqmanREVOrmdl1 taqman assay primerOrmdl1 taqman assay primer: 5′-GAACACTGCAGAAGGGGATG
Sequence-based reagentOrmdl1 probeApplied BiosystemsCustomOrmdl1 taqman assay probe: 5′-TGTAGATGACCCAAAATGGT
Sequence-based reagentOrmdl2 assay-on-demandApplied BiosystemsMm00452481_g1
Sequence-based reagentOrmdl3 assay-on-demandApplied
Biosystems
Mm00787910_sH

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