Ganglioside lipidomics of CNS myelination using direct infusion shotgun mass spectrometry

Figure 1. Ganglioside synthesis pathway Synthesis of gangliosides of the asialo, a-, b-, and c-series is shown. All gangliosides are synthesized from ceramide by the addition of sugar groups and sialic acid. Gangliosides that are not synthesized anymore in St3gal5/B4galnt1-deficient mice are highlighted in red. Major brain gangliosides are encircled in green.

Figure 2. Developmental change of ganglioside content in the brain of wild-type mice at P12, P21, and 6 months (adult) (A) PCA shows clustering of biological replicates for every age. The lipid species’ percentage of moles per age was used as input data. (B) Ganglioside composition. Intensities are normalized to GM1 standard and then to total lipid amounts. Graphs represent mean values. n = 4 for every age. All values are represented as mean ± SD. Abbreviations: Lipid classes as defined by Lipid Maps Classification. See also Tables S1, S2, and S3; Figures S1 and S2.

Figure 3. Developmental change of ganglioside content in the optic nerve of wild-type mice at P12, P21, and 6 months (adult) (A) PCA shows clustering of biological replicates for every age. The lipid species’ percentage of moles per age was used as input data. (B) Ganglioside composition. Intensities are normalized to GM1 standard and then to total lipid amounts. Graphs represent mean values. n = 4 for every age. All values are represented as mean ± SD. Abbreviations: Lipid classes as defined by Lipid Maps Classification. See also Tables S1 and S3; Figure S3.

Figure 4. Comparison of the gangliosides distribution in the brain of P21 St3gal5/B4galnt1-deficient mice with St3gal5/B4galnt1 double heterozygous and wild-type animals (A) PCA shows clustering of wild-type and St3gal5+//B4galnt1+/− samples, whereas the samples of St3gal5−/−/B4galnt1−/− mice spread in both dimensions. The lipid species’ percentage of moles per genotype was used as input data. (B) Ganglioside composition. Intensities for each genotype are normalized to GM1 standard and then to total lipid amounts. Graphs represent mean values. n = 3 for St3gal5+//B4galnt1+/− and St3gal5−/−/B4galnt1−/− samples and n = 4 for wild-type samples. All values are represented as mean ± SD. Abbreviations: Lipid classes as defined by Lipid Maps Classification. See also Tables S1 and S3; Figure S4.

Figure 5. Comparison of the gangliosides and lipid class distribution in the optic nerve of P21 St3gal5/B4galnt1-deficient mice with double heterozygous and wild-type (A) PCA shows clustering of each, wild-type and St3gal5+//B4galnt1+/− samples, whereas the samples of St3gal5−/−/B4galnt1−/− mice spread in both dimensions. The lipid species’ percentage of moles per genotype was used as input data. (B) Ganglioside composition. Intensities for each genotype are normalized to GM1 and then to total lipid amounts. Graphs represent mean values. n = 3 for St3gal5+//B4galnt1+/− and St3gal5−/−/B4galnt1−/− samples and n = 4 for wild-type samples. All values are represented as mean ± SD. Abbreviations: Lipid classes as defined by Lipid Maps Classification. See also Tables S1 and S3; Figure S5.

Figure 6. Ultrastructural analysis of mouse optic nerves reveals hypomyelination in St3gal5/B4galnt1-deficient mice (A) Representative TEM images at p21 show less myelinated axons and thinner myelin sheath in St3gal5−/−/B4galnt1−/− mice. Asterisks mark unmyelinated axons. Arrows show myelinated axons. Scale bar 5μm. (B) Graph displays the percentage of myelinated axons over the total number of axons at p12 and p21. We did not detect differences at p12 (p = 0.279). At p21 St3gal5−/−/B4galnt1−/− mice displayed significantly less myelinated axons (p = 0.045). Differences were tested with an unpaired t-test, two-tailed. (C) G ratios are calculated by dividing the axonal diameter with the myelinated fiber diameter. (D) Scatterplot displaying the g ratios of individual myelinated axons as a function of the respective axon caliber with simple linear regression at p21. St3gal+//B4galnt+/− slope = 0.15, St3gal5−/−/B4galnt1−/− slope = 0.13. All values are represented as mean ± SD. Figure related to Figure S6.