Cell Biology

Loss of SPNS1, a lysosomal transporter, in the nervous system causes dysmyelination and white matter dysplasia

Yoshinobu Ichimura
Yuki Sugiura
Yoshinori Katsuragi
Yu-Shin Sou
Takefumi Uemura
Naoki Tamura
Satoko Komatsu-Hirota
Takashi Ueno
Masato Koike
Satoshi Waguri
Masaaki Komatsu author has email address

Department of Physiology, Juntendo University Graduate School of Medicine, Bunkyo-ku, Tokyo, Japan
Center for Cancer Immunotherapy and Immunobiology, Kyoto University, Kyoto, Japan
Department of Nursing, Niigata College of Nursing, Joetsu, Japan
Department of Cell Biology and Neuroscience, Juntendo University Graduate School of Medicine, Bunkyo-ku, Tokyo, Japan
Laboratory of Proteomics and Biomolecular Science, Biomedical Research Core Facilities, Juntendo University Graduate School of Medicine, Bunkyo-ku, Tokyo, Japan; Department of Anatomy and Histology, Fukushima Medical University School of Medicine, Fukushima, Japan
Autophagy Research Center, Juntendo University Graduate School of Medicine, Bunkyo-ku, Tokyo, Japan

https://doi.org/10.7554/eLife.99913.1

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Figures and data

Impairment of lysosomal integrity by the loss of SPNS1.
(A) Immunofluorescence analysis. Parental, SPNS1^-/-HeLa cells and SPNS1^-/- HeLa cells expressing SPNS1-FLAG were immunostained with the LAMP-1 antibody. The left scatter plot shows the results of quantitative analysis of the size of LAMP-1-positive structures per cell in parental (WT) (n = 34401), SPNS1^-/- HeLa (n = 40011), and SPNS1^-/-HeLa expressing SPNS1-FL (n = 34812). The right scatter plot shows the number of LAMP-1-positive structures per area (μm²) in WT (n = 663), SPNS1^-/- HeLa (n = 740), and SPNS1^-/- HeLa expressing SPNS1-FL (n = 463). Blue indicates nuclei stained with Hoechst 33342. Each dot corresponds to individual data points, and the horizontal line indicates the mean. Statistical analysis was performed using the Šidák’s test after one-way ANOVA. Scale bars, 20 μm (main panels), 2 μm (inset panels). (B) Electron micrographs of cells indicated in (A). Boxed regions are enlarged and shown below. The number of lysosomes per area (μm²) of WT (n = 11), SPNS1^-/- HeLa (n = 11), and SPNS1^-/-HeLa expressing SPNS1-FL (n = 11) were quantified using the Image J software and presented in scatter plots. Each dot corresponds to individual data points, and the horizontal line indicates the mean. Statistical analysis was performed using the Šidák’s test after one-way ANOVA. Arrowheads indicate lysosomes. Bars, top panels: 5 μm, bottom panels: 500 nm. (C) Immunofluorescence microscopy. Liver sections from Spns1^flox/flox and Spns1^flox/flox;albumin-Cre mice at 3 months were stained with SPNS1 (green) and LAMP-1 (red) antibodies, and nuclei was stained with Hoechst 33342 (blue) as indicated. Boxed regions are enlarged and shown in the insets. Bar, 20 μm. (D) Electron micrographs of mouse liver sections, as indicated. Boxed regions are enlarged and shown on the right. Arrows indicate lysosome-like structures with undigested materials. Bars, 10 μm (left) and 1 μm (right). (E) Immunoblot analysis. Homogenates and lysosomal fractions prepared from the liver of Spns1^flox/flox (n = 5) and Spns1^flox/flox;albumin-Cre mice (n = 3) at 6 weeks of age were subjected to SDS-PAGE followed by immunoblotting with Cathepsin B and Actin antibodies. Scatter plots show the results of densitometric analysis of the intensity of the Cathepsin B band relative to that of the Actin band. Each dot corresponds to individual data points, and the horizontal line represents the mean. Statistical analysis was performed using the Welch’s t-test. (F) Cathepsin activity. The activity of Cathepsin B + L and that of Cathepsin B in lysosomal fractions shown in (E) was measured. Each dot corresponds to individual data points, and the horizontal line represents the mean. Statistical analysis was performed using the Welch’s t-test.

Growth and mortality of Spns1^flox/flox;nestin-Cre mice.
(A) Immunoblot analysis. Brain homogenates prepared from the indicated genotype mice were subjected to immunoblot analysis with the SPNS1 antibody and Ponceau-S staining of the gel after electrophoresis. Data shown are representative of three separate experiments. (B) Growth curve of Spns1^flox/flox (n = 43 at 2 weeks of age, n = 41 at 3 weeks of age, n = 65 at 4 weeks of age, and n = 5 at 5 weeks of age) and Spns1^flox/flox;nestin-Cre (n = 17 at 2 weeks of age, n = 18 at 3 weeks of age, n = 22 at 4 weeks of age, and n = 27 at 5 weeks of age) mice. Data are means ± s.e.m. Statistical analysis was performed using the Welch’s t-test. (C) Kaplan–Meier analysis of of Spns1^flox/flox (n = 15) and Spns1^flox/flox;nestin-Cre (n = 22) mice.

Dysmyelination due to the lack of oligodendrocytes in Spns1^flox/flox;nestin-Cre mice.
(A) Gross anatomy of whole brain (left) and sagittal sections (right) of brain from Spns1^flox/flox and Spns1^flox/flox;nestin-Cre mice at P26. Arrowheads indicate corpus callosum and arrows indicate cerebellar medulla. Bar, 1 cm. (B) Hematoxylin and eosin staining of the corpus callosum (CC) of brains from Spns1^flox/flox and Spns1^flox/flox;nestin-Cre mice at P24. Bars, 1 mm (left) and 100 μm (right). (C) Immunofluorescence images of sagittal sections of the brain from Spns1^flox/flox and Spns1^flox/flox;nestin-Cre mice at P26, stained with the MBP antibody (red) and Hoechst 33342 (blue). CC: corpus callosum; Cx: cerebral cortex; hip: hippocampus. Bar, 100 μm. (D) Immunoblot analysis. Homogenates of the brain tissue from Spns1^flox/flox, Spns1^flox/+;nestin-Cre and Spns1^flox/flox;nestin-Cre mice at P12 were subjected to SDS-PAGE followed by immunoblotting with MBP and Ponceau-S staining of the gel. Scatter plots show the results of densitometric analysis of the MBP band relative to the whole protein content estimated using Ponceau-S staining (n = 3 each group). The dots represent individual data points, and the horizontal lines indicate the means. Statistical analysis was performed using the Šidák’s test after one-way ANOVA. (E) Electron micrographs of the corpus callosum (CC) of Spns1^flox/flox and Spns1^flox/flox;nestin-Cre mice at P14. Bar, 2 μm. (F) Immunofluorescence images of the sagittal sections of the brain from Spns1^flox/flox and Spns1^flox/flox;nestin-Cre mice at P0 (left) and P18 (right) stained with the OLIG2 antibody and Hoechst 33342. CC; corpus callosum. Bars, 100 μm. Scatter plots show the results of quantitative analysis of the number of OLIG2-positive cells. Spns1^flox/flox(n = 4 at P0 and 1, n = 3 at P14 and 18, and Spns1^flox/flox;nestin-Cre mice (n = 3 at P0 and 1, n = 3 at P14 and 18). The dots correspond to individual data points, and the horizontal lines represent the means. Statistical analysis was performed using the Welch’s t-test.

Quantification and visualization of lysophospholipids in Spns1^flox/flox;nestin-Cre mice.
(A) Lipidome analysis of the brain of Spns1^flox/flox or Spns1^flox/+ (n = 10), Spns1^flox/+;nestin-Cre (n = 4), and Spns1^flox/flox;nestin-Cre (n = 3) mice at P14 was overviewed using hierarchical cluster analysis, which led to the identification of a cluster of accumulation of metabolites, including lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), and lysophosphatidylinositol (LPI), in the brain of Spns1^flox/flox;nestin-Cre mice. (B) Lipidome of the brain of Spns1^flox/flox or Spns1^flox/+ (n = 10) and Spns1^flox/flox;nestin-Cre (n = 3) mice at P14 was compared using the volcano plot analysis; among the molecular species with significant accumulation in Spns1-deficient brain, lysophospholipids were annotated. (C) Results of quantitative analysis of lysophospholipids, LPC, LPI, and LPE in the mouse brain described in (A); PUFA-containing LPs are highlighted with red dashed lines. The dots correspond to individual data points, and the horizontal lines represent the means. (D) Imaging mass spectrometry to visualize the pattern of lysophospholipid accumulation in sagittal sections of the brain of Spns1^flox/flox and Spns1^flox/flox;nestin-Cre mice at P14. Arrows indicate hippocampus. Bars, 3 mm.

Spns1-deficient brain shows accumulation of sphingosine and decrease in myelin glycolipids.
(A) Quantification of sphingosine and major sphingoglycolipids in the brain of Spns1^flox/flox (n = 10), Spns1^flox/+;nestin-Cre (n = 4), and Spns1^flox/flox;nestin-Cre (n = 3) mice at P14. The dots correspond to individual data points, and the horizontal lines represent the means. A schematic illustration of sphingosine metabolic pathway. Sphingosine (Sph) are synthesized de novo synthesis in the Endoplasmic Reticulum (ER) or salvage pathway of the lysosome. Cer: ceramide; SM: sphingomyeline; S1P: sphingosine-1-phosphate; CoA: palmitoyl coenzyme A. (B) Imaging mass spectrometry to visualize the localization pattern of major sphingoglycolipid molecular species in the sagittal section of the brain of Spns1^flox/floxand Spns1^flox/flox;nestin-Cre mice at P14. Bars, 3 mm. (C) Results of the enrichment analysis of the dataset obtained from the water-soluble metabolome analysis of the brain comparing the brain of Spns1^flox/floxand Sns1^flox/flox;nestin-Cre mice at P14. (D) Quantification results of a sialic acid (N-acetyl neuraminic acid) and a corresponding sialic acid nucleotide (CMP-N-acetyl neuraminic acid) in the brain of Spns1^flox/flox(n = 12) and Spns1^flox/flox;nestin-Cre (n = 6) mice at P14. The dots correspond to individual data points, and the horizontal lines represent the means. (E) Imaging mass spectrometry to visualize the localization pattern of gangliosides in the sagittal sections of the brain of Spns1^flox/flox and Spns1^flox/flox;nestin-Cre mice at P14. Arrowheads indicate hippocampus. Bar, 3 mm.

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