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Defective cholesterol clearance limits remyelination in the aged central nervous system

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Science  09 Feb 2018:
Vol. 359, Issue 6376, pp. 684-688
DOI: 10.1126/science.aan4183
  • Fig. 1 Defective cholesterol clearance limits lesion regeneration in aged mice.

    (A) Images of corpus callosum lesions stained with fluoromyelin (green) and IBA1 (red) in 3-month-old (3M), 12-month-old (12M), and 12-month-old mice treated with GW3965 (12M+GW3965) at 14 dpi. Scale bar, 100 μm. (B and C) Quantification of lesion area in square millimeters, determined by luxol fast blue (LFB) staining, and in cubic millimeters, determined by fluoromyelin staining, at 14 dpi. (D) Confocal images of 3M, 12M, and 12M+GW3965 corpus callosum lesions showing IBA1 (red), fluoromyelin (green), and LAMP1 (blue). Scale bars, 40 μm. For 12M animals, the panels at right are magnified images of the boxed areas at left. (E) Change in number of IBA1+ cells in corpus callosum lesions compared with the contralateral unlesioned side. (F) Number of IBA1+, fluoromyelin+, and LAMP1+ cells in lesioned corpus callosum tissue at 14 dpi. (G) Semithin sections and (H) quantification of foam cells of 3M, 12M, and 12M+GW3965 corpus callosum lesions. Scale bars, 10 μm. (I) Reflection microscopy images of spinal cord lesions, showing crystals (white) in lectin+ phagocytes (red), and (J) relative quantification of the fluorescence intensity of the reflected light. Scale bar, 25 μm. DAPI, 4′,6-diamidino-2-phenylindole; a.u., arbitrary units. (K to M) Quantitative PCR analysis of Apoe, Abca1, and Abcg1 in 4-dpi lesions of 3M and 12M mice. All data are mean ± SEM (error bars); *P < 0.05, **P < 0.01, ***P < 0.001 by one-way analysis of variance (ANOVA) test, with Tukey’s multiple comparison test.

  • Fig. 2 APOE is required for cholesterol clearance in demyelinating lesions.

    (A and B) Images of corpus callosum lesions in WT and APOE−/− mice at 21 dpi showing fluoromyelin staining (green), IBA1 (white), DAPI (blue), and LAMP1 (red). (C and D) Quantification of lesion area in square millimeters at 21 dpi, as determined by fluoromyelin and LFB staining. (E) Lesion volume in cubic millimeters, as measured by fluoromyelin staining in consecutive sections. (F) Number of IBA1+, fluoromyelin+, and LAMP1+ cells per square millimeters in lesions at 21 dpi. (G and H) Change in the number of IBA1+ and MHC II+ cells in the lesioned corpus callosum as compared with the contralateral unlesioned side. (I) Representative images (boxed area of bottom left panel shown at larger magnification in right corner) and (K) quantification of foam cells in spinal cord lesions in WT and APOE−/− mice at 21 dpi. (J) Reflection microscopy images of 21-dpi lysolecithin lesions, showing crystals (white) in lectin+ phagocytes (red), and (L) relative quantification of the fluorescence intensity of the reflected light. (M) Transmission electron microscopy images of foam cells in 21-dpi lesions of APOE−/− mice (the boxed area is shown below at higher magnification), showing needle-like cholesterol crystals (black arrows) and lipid droplets (white arrowheads), and (N) relative quantification of crystals. All data are mean ± SEM (error bars); *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student’s t test. Scale bars in (A), (B), (I), and (J), 25 μm; scale bar in (K), 2.5 μm.

  • Fig. 3 Enhancing cholesterol clearance prevents lysosomal storage of myelin debris and crystal formation.

    (A) Representative images with crystals (white) and lectin+ phagocytes (red), and (B) quantification of cholesterol crystals in lysolecithin lesions of WT, APOE−/−, and APOE−/− mice treated with HβCD or phosphate-buffered saline (PBS). Scale bar, 25 μm. (C) Representative images of methylenblue-azur II staining and (D) quantification of remyelination in lysolecithin lesions at 21 dpi. In (C), the edges of the lesions are identified by dashed lines. Scale bar, 25 μm. (E) Confocal images of primary microglial cell cultures prepared from WT or APOE−/− mice, treated with myelin debris and stained for LAMP1 (red), fluoromyelin (white), and DAPI (blue) at 24 hours posttreatment. Microglia from WT or APOE−/− mice were treated with myelin debris and subsequently transferred in media conditioned either by WT or APOE−/− astrocytes. APOE−/− microglia cells in APOE−/− conditioned media (CM) were additionally treated with APOE mimetic peptide (ATI). Scale bar, 25 μm. (F) Change in the area of myelin particles per cell as compared with WT cells in WT conditioned media. All data are mean ± SEM (error bars); **P < 0.01, ***P < 0.001 by two-way ANOVA, with Tukey’s multiple comparison test.

  • Fig. 4 Defective myelin debris clearance activates the NLRP3 inflammasome.

    (A) Immunoblot of cytosol and membrane fractions of primary BMDMs 12 hours after treatment with myelin debris for cathepsin B and γ-tubulin. (B) Cathepsin B activity assay of the cytosolic fraction of control macrophages 12 hours after treatment with myelin debris in the presence or absence of the cathepsin B inhibitor CA074me (10 μM). AMC, 7-amino-4-methylcoumarin. (C and D) Immunoblot and quantification of the active subunit of caspase 1 (p20) after myelin debris treatment of WT or NLRP3−/− BMDMs. The intensity of the p20 band was normalized to γ-tubulin. (E) Caspase 1 activity in lysates from lysolecithin lesions of WT and APOE−/− mice at 21 dpi. (F) Enzyme-linked immunosorbent assay for IL-1β release in WT BMDMs, after treatment with myelin debris with or without YVAD. (G) Quantification of the percentage of dead cells [propidium iodide–positive (PI+)] after myelin debris treatment (12 hours) in the presence or absence of a caspase-1 inhibitor (YVAD). (H) Quantification of PI+ cells after treatment of WT and NLRP3−/− BMDMs with myelin debris for 12 hours. (I) Methylenblue-azur II staining of remyelinating lesions in the spinal cord of 3M, 12M, GW3965-treated 12M, and 12M NLRP3−/− mice and (J) relative quantification of myelinated fibers. All data are mean ± SEM (error bars); *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA test, with Tukey’s multiple comparison test and two-tailed Student’s t test (E). Scale bar in (I), 25 μm.

Supplementary Materials

  • Defective cholesterol clearance limits remyelination in the aged central nervous system

    Ludovico Cantuti-Castelvetri, Dirk Fitzner, Mar Bosch-Queralt, Marie-Theres Weil, Minhui Su, Paromita Sen, Torben Ruhwedel, Miso Mitkovski, George Trendelenburg, Dieter Lütjohann, Wiebke Möbius, Mikael Simons

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S10
    • References

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