Research ArticleNEUROSCIENCE

Inhibition of neutral sphingomyelinase 2 promotes remyelination

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Science Advances  02 Oct 2020:
Vol. 6, no. 40, eaba5210
DOI: 10.1126/sciadv.aba5210
  • Fig. 1 Modifications in the lipid content of remyelinated axons.

    (A) Black gold staining in pCC of mice fed a normal diet (ND), a diet containing CPZ (4 weeks), and a CPZ diet (4 weeks), followed by a return to ND (CPZ + ND) (4 weeks). Images show extensive demyelination in the CPZ group and a partial restoration of myelin structure in CPZ + ND group. Scale bar, 200 μm. (B) Quantitation of black gold staining in pCC of mice fed ND, CPZ, and CPZ + ND (n = 5). (C) Representative electron microscope images of the pCC in mice fed a ND, a CPZ diet, and a CPZ + ND diet. Images show extensive demyelination in the CPZ group and partial restoration of myelin ultrastructure in the CPZ + ND group with many thinly myelinated axons with disorganized structure. Bottom images are magnifications of the corresponding top images. Scale bars, 2 μm (top) and 500 nm (bottom). (D) Quantitation of the percentage of myelinated axons and g-ratio (E) in pCC after the indicated treatment conditions (100 to 250 axons; n = 3). (F) nSMase2 activity in pCC after the indicated treatment conditions (n = 5). (G) Heatmaps (left) and quantitative comparisons (right) of the indicated sphingolipids (n = 5). Data show increases in multiple ceramides with decreases in SMs and sulfatides in the CPZ group, with time-dependent reductions of ceramides and increases in SMs in the CPZ + ND groups. Sulfatides do not recover in the CPZ + ND groups and remain depleted. Data are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.05, and ###P < 0.001 as indicated. Analysis of variance (ANOVA) with Tukey post hoc comparisons. cps, counts per second.

  • Fig. 2 TNFα and IL-1β promote the survival and migration of oligodendrocyte progenitor cells but promote death during the differentiation of progenitors to mature oligodendrocytes.

    (A) Quantitation of oligodendrocyte progenitor cell (OPCs) proliferation following a dose response of TNFα (0 to 100 ng/ml) or IL-1β (0 to 100 ng/ml) as determined by incorporation of bromodeoxyuridine (BrdU; 5 μg/ml) (n = 3). (B) Quantitation of cell death in OPCs by treatment with a dose response of TNFα (0 to 100 ng/ml) or IL-1β (0 to 100 ng/ml) for four consecutive days as determined by the number of active caspase3 (Cas3)–immunopositive cells (n = 3). (C) Quantitation of OPCs migration over a 12-hour time frame showing increased cell migration following treatment with TNFα (25 or 100 ng/ml) (n = 6). (D) Representative images of OPCs induced to differentiate by the removal of platelet-derived growth factor (PDGF). Cultures showed a reduction in the number of MBP+ cells after treatment with TNFα or IL-1β (100 ng/ml) every other day for 7 days. Scale bars, 50 μm. (E) Quantitation of OPC differentiation (n = 3). Stage of differentiation was determined by quantitative analysis of MBP-immunopositive (red) cells, expressed as a percentage of total cell numbers [4′,6-diamidino-2-phenylindole (DAPI); blue]. (F) Images of caspase3 activation during differentiation of OPCs to GalC+ immature oligodendrocytes. Most caspase3-immunopositive cells were also GalC-immunopositive (green) (merge is yellow), demonstrating that caspase3 was activated during cell differentiation in response to TNFα or IL-1β. Scale bar, 50 μm. (G) Quantitation of caspase3 activation during differentiation of OPCs to GalC+ immature oligodendrocytes. Cell death was quantified as the percentage of caspase3-immunopositive (red) cells (n = 6). Data are presented as means ± SD. *P < 0.05, ***P < 0.001, #P < 0.05, ##P < 0.01, and ###P < 0.001 compared to untreated control cultures. ANOVA with Tukey post hoc comparisons. N.S., not significant.

  • Fig. 3 Manipulation of nSMase2 expression regulates cell survival in response to TNFα.

    (A) Images of nSMase2 expression (red) in mature oligodendrocytes (MBP, green) and a lack of nSMase2 expression in oligodendrocyte progenitor cells (OPCs; NG2, green) (colocalization appears as yellow). Scale bars, 20 μm. (B) Analysis of sphingolipids in oligodendrocytes following treatment with TNFα (100 ng/ml) showing increases in ceramide and decreases in sphingosine (n = 3). (C) TNFα (100 ng/ml) decreased ceramide and increased sphingosine 1-phosphate (sphingosine 1-P) in OPCs (n = 3). (D) Immunoblots of nSMase2 overexpression in OPCs transfected with empty vector (Mock) or pcDNA-nSMase2. (E) Fluorescent images of NG2+ OPCs (green) transfected with an empty vector (Mock) or a vector expressing nSMase2 (pcDNA-nSMase2, red). Nuclei were stained with DAPI (blue). Scale bars, 50 μm. (F) Quantitation of cell death (pyknotic nuclei expressed as the percentage of EGFP-immunopositive cells) in OPCs transfected with empty vector or pcDNA-nSMase2 (cotransfected with EGFP-C2 as transfection indicator), followed by treatment with TNFα (100 ng/ml) (n = 3). Inhibition of nSMase2 with altenusin (25 μM) confirmed that nSMase2 expression in OPCs regulated TNFα-induced cell death of pcDNA-nSMase2 cells. (G) Knockdown of nSMase2 in oligodendrocytes transduced with a control lentivirus expressing scrambled RNA (scrambled shRNA-GFP) or shRNA directed against nSMase2 (sh-nSMase2-GFP). (H) Fluorescent images of oligodendrocytes transduced with a control lentivirus expressing scrambled RNA (scrambled shRNA-GFP) or shRNA directed against nSMase2 (sh-nSMase2-GFP). Scale bars, 50 μm. (I) Quantitation of cell death in oligodendrocytes transduced with the indicated vectors and treated with vehicle, TNFα (100 ng/ml), TNFα + altenusin (25 μM), or TNFα + cambinol (10 μM) for 24 hours (n = 3). Pyknotic nuclei were expressed as the percentage of GFP+ cells. Data are presented as means ± SD. *P < 0.05, **P < 0.01, #P < 0.05, and ##P < 0.01 as indicated. ANOVA with Tukey post hoc comparisons.

  • Fig. 4 Inhibition of nSMase2 blocked CPZ-induced demyelination.

    (A) Schematic illustration showing the timing of cambinol infusion during CPZ-induced demyelination. Cambinol was unilaterally infused into lateral ventricle 2 days before CPZ feeding for 28 days at the rate of 0.31 μg/kg per day. (B) Quantitation of nSMase2 activity in the cortex of mice exposed to CPZ following vehicle or cambinol infusion, showing that cambinol reduced CPZ-induced nSMase2 activity (n = 5 in each group). (C) Representative images of black gold–stained myelin in the pCC of mice from the indicated treatment groups. Arrowheads show the region of cambinol-mediated protection against CPZ-induced demyelination. Scale bar, 200 μm. (D) Quantification of black gold–stained area in the pCC showing protection from demyelination in the CPZ + cambinol group (n = 5 in each group). (E) Heatmaps and quantitative analysis of the indicated class and species of sphingolipids in the pCC of mice fed a standard diet (normal, n = 6), mice fed CPZ with vehicle infusion (n = 6), and mice fed CPZ + cambinol (n = 5). CPZ-induced increases in ceramide were reduced by cambinol infusion. Data are presented as means ± SD *P < 0.05, ***P < 0.001, #P < 0.05, ##P < 0.01, and ###P < 0.001 as indicated. ANOVA with Tukey post hoc comparisons.

  • Fig. 5 Pharmacological inhibition of nSMase2 enhanced remyelination and improved myelin compaction.

    (A) Timing of cambinol treatment following CPZ-induced demyelination. After 4 weeks of a CPZ-containing diet, cambinol was unilaterally infused for 28 days into the lateral ventricle during return to an ND. (B) Quantitation of nSMase2 activity in the cortex of mice fed a ND, a CPZ-containing diet (4 weeks), after return to a ND (4 weeks), and after return to a ND with cambinol infusion (4 weeks), showing that cambinol infusion blocks CPZ-induced up-regulation of nSMase2 activity (n = 5). (C) Cambinol infusion blocks CPZ-induced up-regulation of multiple ceramides but does not modify increases in SMs or decreases in the sulfatide content of pCC (n = 5). (D) Representative images and (E) quantitation of black gold staining in pCC following the indicated treatments (n = 5). Scale bar, 200 μm. (F) Electron microscopy images of axons in pCC following the indicated treatment conditions. Scale bars, 2 μm (top) and 500 nm (bottom). (G) Quantitation of myelinated axons in pCC showing a larger percentage of myelinated axons in CPZ + ND + cambinol mice compared to CPZ + ND + vehicle (100 to 250 axons; n = 3). (H) g-ratios in pCC showing thicker compact myelin structures in CPZ + ND + cambinol mice compared to CPZ + ND + vehicle (100 to 250 axons; n = 3). (I) Staining and (J) quantitation of APP in the pCC of mice following the indicted treatments (n = 5 in each group). Scale bar, 200 μm. (K) Heatmaps and quantitation of CEs and cholesterol in pCC from the indicated treatment groups (n = 5). Data are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.01, and ###P < 0.001 as indicated. ANOVA with Tukey post hoc comparisons.

  • Fig. 6 Genetic deletion of nSMase2 during remyelination improves myelin compaction.

    (A) Schematic illustration for creating PDGFRα-CreER-smpd3fl/fl mice. IRES, internal ribosomal entry site. (B) Schematic illustration showing the timing of CPZ feeding and tamoxifen treatment (1 mg/day, over 5 days) during the return to ND. (C) Images of MBP (green) and nSMase2 (red) fluorescence in the pCC of mice fed a ND, a CPZ-containing diet (4 weeks), a CPZ diet and return to ND (CPZ + ND) (4 weeks), and CPZ with tamoxifen induction of nSMase2 deletion during the first 5 days of return to ND (CPZ + ND + Tam). Increased expression of nSMase2 in the CPZ + ND group is absent in the CPZ + ND + Tam group, and MBP staining is more robust in the CPZ + ND + Tam group compared to the CPZ + ND group. Scale bar, 100 μm. (D) Representative electron microscopy images of pCC in mice treated with indicated conditions. Scale bar, 500 nm. (E) Quantitation of myelinated fibers in pCC showing a greater percentage of myelinated fibers in the CPZ + ND + Tam mice compared with the CPZ + ND mice (100 to 250 axons; n = 3). (F) Analysis of g-ratios for the pCC of mice showing thicker and compact myelin structures in the CPZ + ND + Tam mice compared with the CPZ + ND mice (100 to 250 axons; n = 3). (G) Heatmaps and quantitative analysis of the indicated class and species of sphingolipids in pCC of mice from the indicated treatment groups (n = 4 to 5). Ceramide content of the pCC was reduced, but SMs remained elevated and sulfatides reduced in the CPZ + ND + Tam mice compared with the CPZ + ND mice. Data are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.01, and ###P < 0.001 as indicated. ANOVA with Tukey post hoc comparisons.

Supplementary Materials

  • Supplementary Materials

    Inhibition of neutral sphingomyelinase 2 promotes remyelination

    Seung-Wan Yoo, Amit Agarwal, Matthew D. Smith, Saja S. Khuder, Emily G. Baxi, Ajit G. Thomas, Camilo Rojas, Mohammed Moniruzzaman, Barbara S. Slusher, Dwight E. Bergles, Peter A. Calabresi, Norman J. Haughey

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