Research ArticleMultiple Sclerosis

BCAS1 expression defines a population of early myelinating oligodendrocytes in multiple sclerosis lesions

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Science Translational Medicine  06 Dec 2017:
Vol. 9, Issue 419, eaam7816
DOI: 10.1126/scitranslmed.aam7816
  • Fig. 1. BCAS1 expression identifies newly generated oligodendrocytes.

    (A) Confocal images of breast carcinoma amplified sequence 1 (BCAS1) (pink) and oligodendrocyte transcription factor 2 (OLIG2) (white) in the corpus callosum (CC) of postnatal day 40 (P40) mice. Scale bars, 5 μm. (B) Bar graph shows the percentage of BCAS1+ cells expressing sex-determining region Y box 10 protein (SOX10), OLIG1, or OLIG2. (C) Bar graph shows the percentage of BCAS1+ cells expressing neural/glial antigen 2 (NG2), CC1, or myelin-associated glycoprotein (MAG) in the corpus callosum of P40 mice. (D) Left (experimental plan): NG2-CreERT2:R26R-tdTomato-mEGFP mice were used to induce the expression of mEGFP by tamoxifen (TMX) at P30 as illustrated. Right: Quantification of the percentage of double-positive [GFP+ (green fluorescent protein–positive) and BCAS1+] cells over time. One-way analysis of variance (ANOVA), followed by Bonferroni’s post hoc test, *P < 0.05; n = 3, three sections per animal. Quantitative data are means ± SEM.

  • Fig. 2. BCAS1+ premyelinating and myelinating oligodendrocytes are present during development and in adult mice.

    (A) Immunohistochemical analysis of transgenic mice in which the proteolipid protein (PLP) promoter drives the expression of GFP (PLP-GFP mice); representative confocal images show BCAS1 (pink) and GFP (green) expression in premyelinating oligodendrocytes (pre-mOLGs) in the developing and adult somatosensory cortex (CX) of P9, P14, and 4-month-old mice. Blue arrows indicate BCAS1+/GFP, and yellow arrows indicate BCAS1+/GFP+ pre-mOLGs. Scale bars, 10 μm. (B) Quantification of the percentage of pre-mOLGs positive for GFP, BCAS1, or both in the somatosensory cortex of P9, P14, and 4-month-old mice. Student’s two-tailed t test, unpaired,*P < 0.05, ***P < 0.001. (C) Representative confocal image of BCAS1+ segments (pink) aligned along neurofilament-positive (white) axons in the somatosensory cortex of P15 mice. Scale bar, 4 μm. (D) Left: Representative confocal image of BCAS1+ segments (pink) flanked by contactin-associated protein 1 (CASPR) clusters (white) on both sides of a node in the somatosensory cortex of P15 mice. Scale bar, 3 μm. Right: Quantification of the percentage of nodes in the somatosensory cortex of P15 mice showing BCAS1+ segments and CASPR expression on one side (one BCAS1+ paranode), both sides (two BCAS1+ paranodes), or without BCAS1+ segments (zero BCAS1+ paranode). (E) Representative confocal images showing BCAS1 (pink) and GFP (green; PLP-GFP mice) expression in myelinating (mOLGs) in the developing and adult somatosensory cortex of P9, P14, and 4-month-old mice. Blue, yellow, and white arrows show BCAS1+/GFP, BCAS1+/GFP+, and BCAS1/GFP+ mOLGs, respectively. Scale bars, 10 μm. (F) Quantification of the percentage of mOLG positive for GFP, BCAS1, or both in the somatosensory cortex of P9, P14, and 4-month-old mice. Quantitative data are means ± SEM; n = 3, three sections per animal [only n = 2 for P9 in (F)]; one-way ANOVA, followed by Bonferroni’s post hoc test, ***P < 0.001. (G) Schematic illustration of the phases of OLG maturation and the relative expression of NG2, CC1, and BCAS1. OPCs, oligodendrocyte progenitor cells. (H) Schematic illustration representing the pattern of the expression of BCAS1 and PLP (green) during development and in adulthood.

  • Fig. 3. BCAS1+ oligodendrocytes are a transient cell population.

    (A) Experimental design: 5-Ethynyl-2′-deoxyuridine (EdU) was administered to a 4-month-old mice as shown in the scheme. Analyses were performed 10 (+0 days), 17 (+7 days), 28 (+18 days), and 50 (+40 days) days after injection. (B and C) Quantification of the percentage of EdU+/BCAS1+ cells directly (0 days) and 40 days after the end of the treatment in the corpus callosum (B) and somatosensory cortex (C). Student’s two-tailed t test, unpaired, *P < 0.05, ***P < 0.001. (D) Line graph showing the time-course expression analysis of the fraction of CC1+ oligodendrocytes that were EdU+ and either BCAS1+ or BCAS1 plotted versus the time after the end of the EdU exposure in the corpus callosum. One-way ANOVA, followed by Newman-Keuls’ post hoc test, *P < 0.05, **P < 0.01. (E) Experimental design: Proliferating OPCs were ablated by tamoxifen injection in inducible Sox10-iCreERT2 × Esco2fl/fl × CAG-EGFP mice (FL) and compared to control animals Sox10-iCreERT2 × Esco2wt/wt × CAG-EGFP (WT). Esco2, establishment of sister chromatid cohesion N-acetyltransferase 2. Tamoxifen was injected in 8- to 10-week-old animals, and analyses were performed 6 and 16 weeks after injection. Recombined cells are marked by EGFP. (F and G) Representative images of BCAS1 and GFP expression in the somatosensory cortex of WT and FL mice 16 weeks after tamoxifen injection (F) and quantification of the percentage of BCAS1+ mOLGs that are positive for GFP at 6 and 16 weeks postinduction (wpi) in the somatosensory cortex (G). Scale bars, 15 μm. Student’s two-tailed t test, unpaired, **P < 0.01, ***P < 0.001. 6 wpi/WT versus 16 wpi/KO, P = 0.0028; 6 wpi/KO versus 16wpi/KO, P = 0.0028; 16 wpi/WT versus 16 wpi/KO, P = 0.0004. KO, knockout. For all quantifications, n = 3 (only n = 2 for the +6 wpi group), three sections per animal. Quantitative data are means ± SEM.

  • Fig. 4. Myelination continues into adulthood but decreases in aged mice.

    (A) Cell density quantification (left) and immunohistochemistry (right) of BCAS1+ pre-mOLGs and mOLGs in the somatosensory cortex in P40 and 4-, 7-, and 21-month-old mice. Scale bars, 30 μm. White and blue arrows indicate BCAS1+ pre-mOLGs and BCAS1+ mOLGs, respectively. One-way ANOVA, followed by Newman-Keuls’ post hoc test, *P < 0.05, **P < 0.01. Student’s two-tailed t test, unpaired, for intergroup analysis, *P < 0.05, ***P < 0.001. n = 3, three sections per animal. Quantitative data are means ± SEM. (B) Left (experimental design): Mass spectrometry analysis was performed in 1-, 4-, 18-, and 26-month-old mice fed for 30 or 60 days with a diet containing lysine labeled with 13C (13C-isotope). Right: The 13C isotope incorporation rates (13C-lysines/12C-lysines ratio) are shown for three axonal proteins—neurofilament light (NFL), neurofilament medium (NFM), and neurofilament heavy (NFH) chains—and for the three major myelin proteins—PLP, myelin basic protein (MBP), and CLD11; four animals per group, 4-month-old groups versus 18- and 26-month-old groups, one-way ANOVA, followed by Bonferroni’s post hoc test. 30 days feeding: P < 0.0001, PLP; P = 0.0226, MBP; P < 0.0001, CLD11; P = 0.0800, NFH; P = 0.2355, NFM; P = 0.2700, NFL. 60 days feeding: P < 0.0001, PLP; P = 0.0016, MBP; P < 0.0001, CLD11; P = 0.1338, NFH; P = 0.0671, NFM; NFL, P = 0.0229. n = 4, three technical replicates. Error bars indicate SD.

  • Fig. 5. BCAS1+ cells represent a transient oligodendroglial subpopulation enriched during active myelination in the human brain.

    (A) Immunohistochemistry showing colocalization of BCAS1 with OLIG2, NG2, CC1, and tubulin polymerization promoting protein (TPPP/p25). (B) Quantification of the percentage of BCAS1+ cells positive for SOX10, OLIG2, NG2, TPPP/p25, and CC1 in newborns (n = 3 to 4). (C) Quantification of the density of BCAS1+ and NG2+ cells in the white matter throughout development and adult life (BCAS1: 17 to 27 weeks, n = 2; all other time points, n = 3 to 5; NG2, n = 2 to 5 per time point; Mann-Whitney test,*P < 0.05, **P < 0.01; BCAS1 0 year versus BCAS1 2 to 8 years, P = 0.0357; BCAS1 2 to 8 years versus NG2 2 to 8 years, P = 0.0079; BCAS1 11 to 16 years versus NG2 11 to 16 years, P = 0.0159; BCAS1 18 to 26 years versus NG2 18 to 26 years, P = 0.0179; BCAS1 38 to 46 years versus NG2 38 to 46 years, P = 0.0357; BCAS1 83 to 90 years versus NG2 83 to 90 years, P = 0.0286). (D) BCAS1 expression in the white matter around birth (0 year) and at 1 year of age (1 year). Inset and image in the center show magnification of the BCAS1+ cells in a newborn human, as indicated by the arrowhead. (E) Quantification of BCAS1+ cell density in the frontal cortex (n = 3 to 6). (F) Representative images of BCAS1 expression in the frontal cortex in humans around birth (0 year) and at 2 to 8, 38 to 46, and 57 to 65 years of age (as indicated). The insets show magnifications of BCAS1+ cells, as indicated by arrowheads, with few or no segments (left) and with several segments (right). (G) Percentage of BCAS1+ cells with a myelinating morphology in the frontal cortex in humans at different ages; n = 3 to 5. Kruskal-Wallis test with Dunn’s post hoc test, *P < 0.05. Quantitative data are means ± SEM. Scale bars, 50 μm.

  • Fig. 6. BCAS1+ oligodendrocytes are reformed during remyelination in rodents.

    (A) Representative images of BCAS1 expression of lysolecithin-induced lesions in the rat corpus callosum on days 3 and 6 after injection. BCAS1+ cells are shown in brown, and cell nuclei are shown in blue. (B) Quantification of BCAS1+ cell density in lysolecithin-induced lesions 3, 6, 12, and 20 days after injection (n = 3; Kruskal-Wallis test with Dunn’s post hoc test, *P < 0.05). (C) Representative images of BCAS1 expression in the corpus callosum 1 and 4 weeks after the start of cuprizone treatment in mice. BCAS1+ cells are shown in brown, and cell nuclei are shown in blue. (D) Quantification of BCAS1+ cell density in the corpus callosum 1 week after the start of cuprizone treatment and 2 days remyelination (2d RM) and 3 weeks remyelination (3wks RM) after the treatment was discontinued; n = 4 to 14 animals. Kruskal-Wallis test with Dunn’s post hoc test, *P < 0.05, **P < 0.01. Quantitative data are means ± SEM. Scale bars, 50 μm.

  • Fig. 7. BCAS1+ oligodendrocytes identify areas of ongoing remyelination in chronic multiple sclerosis.

    (A) Quantification of BCAS1+ cell density in the normal-appearing white matter (NAWM), the immediate periplaque white matter (PPWM), the lesion center (LC), and remyelinated lesion areas (RM), if present, of chronic demyelinated lesions (n = 10), chronic lesions with areas of remyelination at the lesion edge (n = 18; ***P < 0.001), and fully remyelinated shadow plaques (n = 7). Kruskal-Wallis test with Dunn’s post hoc test was used for statistical analysis. (B) Luxol fast blue/periodic acid–Schiff (LFB/PAS) histochemistry depicts the frontal subcortical white matter containing a chronic demyelinated multiple sclerosis lesion with two adjoining sharply delineated lesion areas of pale LFB staining (RM1 and RM2) and a neighboring lesion area with more intense but still visibly reduced LFB staining [shadow plaque (SP)]. (C) BCAS1 immunohistochemistry on a serial section of (B). Frames in (C) and (D) indicate lesion areas depicted at a higher magnification below. LB, lesion border. (D to J) Multiple sclerosis lesion areas as indicated in (C) immunostained for BCAS1. Arrowheads indicate BCAS1+ cells. Error bars indicate SEM. Scale bars, 2 mm (B and C) and 50 μm (all other images).

  • Fig. 8. Ongoing remyelination occurs independently from multiple sclerosis lesion activity.

    (A) Representative images showing the expression of the pan-macrophage/activated microglia marker KiM1P (brown) and MBP (blue) at the rim of multiple sclerosis lesions. Rows represent different inflammatory and demyelinating lesion activities according to Kuhlmann et al. (33). Insets in (A) highlight KiM1P+ phagocytes containing MBP+ particles. (B) Representative images showing BCAS1 expression at the rim of multiple sclerosis lesions of varying inflammatory and demyelinating activity; BCAS1+ oligodendrocytes are indicated by arrowheads. BCAS1+ oligodendrocytes extending multiple processes are observed at the lesion rim in all stages of lesion activity. (C) Percentage of lesions with different inflammatory and demyelinating activities containing areas of ongoing remyelination (ongoing RM) with BCAS1+ cells (n = 15). (D) Quantification of BCAS1+ cell density in areas with ongoing remyelination of multiple sclerosis lesions of different lesion activities (n = 15). Quantitative data are means ± SEM. Scale bars, 500 μm (A, left column) and 50 μm (all other images).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/419/eaam7816/DC1

    Materials and Methods

    Fig. S1. Validation of antiserum raised against BCAS1.

    Fig. S2. BCAS1 specifically labels cells of oligodendrocyte lineage.

    Fig. S3. Characterization of BCAS1+ pre-mOLGs and mOLGs.

    Fig. S4. Characterization of ENPP6+ oligodendrocytes in mice.

    Fig. S5. BCAS1 is down-regulated in fully matured oligodendrocytes prepared from mice.

    Fig. S6. Comparison of BCAS1 with O1 and O4 immunolabeling.

    Fig. S7. BCAS1 is down-regulated in fully matured oligodendrocytes prepared from human iPSCs.

    Fig. S8. Dynamics of BCAS1+ cells generation in adult mouse cortex.

    Fig. S9. Quantification of the number of BCAS1+ cells in the adult mouse brain.

    Fig. S10. Developmentally born mOLGs continue myelination into late adulthood.

    Fig. S11. BCAS1+ oligodendrocytes are distinct from PLP+ and ENPP6+ oligodendrocytes in the human brain.

    Fig. S12. Characterization of BCAS1+ oligodendrocytes in the human brain.

    Fig. S13. BCAS1+ oligodendroglia are rare in and around chronic demyelinated MS lesions and fully remyelinated shadow plaques.

    Table S1. Clinical characteristics of patients to study the developmental expression of BCAS1.

    Table S2. Clinical characteristics of MS patients.

    Table S3. Primary data.

    References (4648)

  • Supplementary Material for:

    BCAS1 expression defines a population of early myelinating oligodendrocytes in multiple sclerosis lesions

    Maryam K. Fard,* Franziska van der Meer, Paula Sánchez, Ludovico Cantuti-Castelvetri, Sunit Mandad, Sarah Jäkel, Eugenio F. Fornasiero, Sebastian Schmitt, Marc Ehrlich, Laura Starost, Tanja Kuhlmann, Christina Sergiou, Verena Schultz, Claudia Wrzos, Wolfgang Brück, Henning Urlaub, Leda Dimou, Christine Stadelmann,* Mikael Simons*

    *Corresponding author. Email: cstadelmann{at}med.uni-goettingen.de (C.S.); khojasteh{at}em.mpg.de (M.K.F.); msimons{at}gwdg.de (M.S.)

    Published 6 December 2017, Sci. Transl. Med. 9, eaam7816 (2017)
    DOI: 10.1126/scitranslmed.aam7816

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Validation of antiserum raised against BCAS1.
    • Fig. S2. BCAS1 specifically labels cells of oligodendrocyte lineage.
    • Fig. S3. Characterization of BCAS1+ pre-mOLGs and mOLGs.
    • Fig. S4. Characterization of ENPP6+ oligodendrocytes in mice.
    • Fig. S5. BCAS1 is down-regulated in fully matured oligodendrocytes prepared from mice.
    • Fig. S6. Comparison of BCAS1 with O1 and O4 immunolabeling.
    • Fig. S7. BCAS1 is down-regulated in fully matured oligodendrocytes prepared from human iPSCs.
    • Fig. S8. Dynamics of BCAS1+ cells generation in adult mouse cortex.
    • Fig. S9. Quantification of the number of BCAS1+ cells in the adult mouse brain.
    • Fig. S10. Developmentally born mOLGs continue myelination into late adulthood.
    • Fig. S11. BCAS1+ oligodendrocytes are distinct from PLP+ and ENPP6+ oligodendrocytes in the human brain.
    • Fig. S12. Characterization of BCAS1+ oligodendrocytes in the human brain.
    • Fig. S13. BCAS1+ oligodendroglia are rare in and around chronic demyelinated MS lesions and fully remyelinated shadow plaques.
    • Table S1. Clinical characteristics of patients to study the developmental expression of BCAS1.
    • Table S2. Clinical characteristics of MS patients.
    • References (46–48)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S3. (Microsoft Excel format). Primary data.

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