The Ormdl genes regulate the sphingolipid synthesis pathway to ensure proper myelination and neurologic function in mice
- National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, United States
- Uniformed Services University of the Health Sciences, United States
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, …
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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 …
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 Or…
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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
Figure 2
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 …
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, …
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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 …
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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 …
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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 …
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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-C…
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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 …
| 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 # | 16 | 20 | 9 | 33 | 62 | 0 | 11 | 5 | 0 |
| Observed % | 10.26 | 12.82 | 5.77 | 21.15 | 39.74 | 0 | 7.05 | 3.21 | 0 |
| Predicted # | 9.75 | 19.5 | 9.75 | 19.5 | 39 | 19.5 | 9.75 | 19.5 | 9.75 |
| Predicted % | 6.25 | 12.5 | 6.25 | 12.5 | 25 | 12.5 | 6.25 | 12.5 | 6.25 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (M. musculus) | Ormdl1 KO mice | This paper | RRID: IMSR_JAX:034646 | See 'Materials and methods' |
| Genetic reagent (M. musculus) | Ormdl2 KO mice | This paper | RRID: IMSR_JAX:034645 | See 'Materials and methods' |
| Genetic reagent (M. musculus) | Ormdl3 Floxed mice | This paper | RRID: IMSR_JAX:034644 | See 'Materials and methods' |
| Genetic reagent (M. musculus) | EIIA-Cre mice | Jackson Laboratory | RRID:IMSR_JAX:003724 | Mice expressing Cre transgene under the control EIIa promoter |
| Genetic reagent (M. musculus) | STOP-fSPT transgenic mice | This paper | See 'Materials and methods' | |
| Genetic reagent (M. musculus) | PLP/creERT mice | Jackson Laboratory | RRID:IMSR_JAX:005975 | Mice expressing tamoxifen inducible-mouse Plp1 promoter |
| Genetic reagent (M. musculus) | Mx-Cre mice | Jackson Laboratory | RRID:IMSR_JAX:003556 | Mice expressing Cre transgene under the control of the mouse Mx1 promoter |
| Antibody | Monoclonal anti-human SPTLC1 | Santa Cruz Bio-technology | RRID:AB_10917035; clone H-1, cat. # sc-374143 | For western blot (1:1000 dilution) |
| Antibody | Anti-mouse IgG-HRP conjugated | Millipore | RRID:AB_9045; Cat. # AP-124P | For western blot (1:1000 dilution) |
| Antibody | Anti-mouse β-actin-HRP conjugated | Abcam | RRID:AB_8674; AC-15, cat # ab49900 | For western blot (1:100,000 dilution) |
| Sequence-based reagent | Ormdl1For | PCR primer | Genotyping Ormdl1 KO mice: 5′-TGTAATGAACAGCCGTGGTAT | |
| Sequence-based reagent | Ormdl1Rev | PCR primer | Genotyping Ormdl1 KO mice: 5′-GCAGAAGGGGATGCTGAGTAATA | |
| Sequence-based reagent | Ormdl2For | PCR primer | Genotyping Ormdl2 KO mice: 5′-CACATGCAGCAGTCCTACCA | |
| Sequence-based reagent | Ormdl2Rev | PCR primer | Genotyping Ormdl2 KO mice: 5′-GTTGGACTCCTGCCTGATCC | |
| Sequence-based reagent | NDEL1 | PCR primer | Genotyping Ormdl3 KO mice: 5′-GCAGGAGGAAGAGGCCCTCAG | |
| Sequence-based reagent | NDEL2 | PCR primer | Genotyping Ormdl3 KO mice: 5′-CTCTTGACTGCCGCTCTGCAAAAGAG | |
| Sequence-based reagent | SIAE4 | PCR primer | Genotyping Ormdl3 KO mice: 5′-CACGGCGCAGGGTTCTAATACATAC | |
| Sequence-based reagent | ForSPT | PCR primer | Genotyping fSPT mice: 5′-CATCGAGCTGAAGGGCATCG | |
| Sequence-based reagent | RevSPT | PCR primer | Genotyping fSPT mice: 5′-GTTATCTAGAATTATAGACGCGCTAG | |
| Sequence-based reagent | ForR26 | PCR primer | Genotyping fSPT mice: 5′-AGTTCTCTGCTGCCTCCTGGCTTCT | |
| Sequence-based reagent | CreFor | PCR primer | Genotyping fSPT mice: 5′-GCCTGCATTACCGGTCGATGC | |
| Sequence-based reagent | CreRev | PCR primer | Genotyping: 5′-CAGGGTGTTATAAGCAATCCC | |
| Sequence-based reagent | TaqmanFWD | Ormdl1 taqman assay primer | Ormdl1 taqman assay primer: 5′-ATGAATGTTGGAGTTGCCCAC | |
| Sequence-based reagent | TaqmanREV | Ormdl1 taqman assay primer | Ormdl1 taqman assay primer: 5′-GAACACTGCAGAAGGGGATG | |
| Sequence-based reagent | Ormdl1 probe | Applied Biosystems | Custom | Ormdl1 taqman assay probe: 5′-TGTAGATGACCCAAAATGGT |
| Sequence-based reagent | Ormdl2 assay-on-demand | Applied Biosystems | Mm00452481_g1 | |
| Sequence-based reagent | Ormdl3 assay-on-demand | Applied Biosystems | Mm00787910_sH |
