Myelination in Multiple Sclerosis Lesions Is Associated with Regulation of Bone Morphogenetic Protein 4 and Its Antagonist Noggin

Abstract

Remyelination is a central aspect of new multiple sclerosis (MS) therapies, in which one aims to alleviate disease symptoms by improving axonal protection. However, a central problem is mediators expressed in MS lesions that prevent effective remyelination. Bone morphogenetic protein4 (BMP4) inhibits the development of mature oligodendrocytes in cell culture and also blocks the expression of myelin proteins. Additionally, numerous studies have shown that Noggin (SYM1)-among other physiological antagonists of BMP4-plays a prominent role in myelin formation in the developing but also the adult central nervous system. Nonetheless, neither BMP4 nor Noggin have been systematically studied in human MS lesions. In this study, we demonstrated by transcript analysis and immunohistochemistry that BMP4 is expressed by astrocytes and microglia/macrophages in association with inflammatory infiltrates in MS lesions, and that astrocytes also express BMP4 in chronic inactive lesions that failed to remyelinate. Furthermore, the demonstration of an increased expression of Noggin in so-called shadow plaques (i.e., remyelinated lesions with thinner myelin sheaths) in comparison to chronically inactive demyelinated lesions implies that antagonizing BMP4 is associated with successful remyelination in MS plaques in humans. However, although BMP4 is strongly overexpressed in inflammatory lesion areas, its levels are also elevated in remyelinated lesion areas, which raises the possibility that BMP4 signaling itself may be required for remyelination. Therefore, remyelination might be influenced by a small number of key factors. Manipulating these molecules, i.e., BMP4 and Noggin, could be a promising therapeutic approach for effective remyelination.

Keywords: BMP4; Noggin; bone morphogenetic protein 4; multiple sclerosis; remyelination; remyelination block.

Figure 1

Bone morphogenetic protein4 (BMP4) expression and Noggin expression in multiple sclerosis lesions. (A) BMP4 is upregulated in chronic white matter lesions. The values were obtained by qPCR from frozen tissue RNA from four different multiple sclerosis (MS) patients and four controls. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene. The Mann-Whitney rank sum test was applied for statistical analysis. (* p < 0.05). (B,C) Upregulation of BMP4 in chronic white matter MS lesions and upregulation of BMP4 antagonist Noggin in remyelinated lesion areas. The results were obtained using nanostring technology from RNA obtained from macrodissected lesion areas of formalin-fixed and paraffin-embedded autopsy (FFPE) tissue. GAPDH and beta-2 microglobulin (B2M) served as housekeeping genes for comparison. The point of reference for the statistical analysis (Mann-Whitney rank sum test) was always the corresponding control tissue of the white or grey substance (* p < 0.05 (B, control white matter: n = 8, remyelinated lesion areas: n = 5, MS NAWM: n = 7, chronic white matter lesions: n = 9, subpial controls: n = 8, subpial lesions: n = 11, leukocortical controls: n = 8, leukocortical lesions: n = 9) and ** p < 0.01 (C, control white matter: n = 8, remyelinated lesion areas: n = 5, MS NAWM: n = 7, chronic white matter lesions: n = 9, subpial controls: n = 8, subpial lesions: n = 11, leukocortical controls: n = 8, leukocortical lesions: n = 9), mean + SEM).

Figure 2

Comprehensive immunohistochemical investigation of the white matter pathology of BMP4 and its antagonist Noggin (representative results). (A–E) inflammatory (early active*) lesion: BMP4 (A) is strongly expressed in foamy macrophages (left) in the lesion. In the lesion rim (right), astrocytic cells with clear overexpression of BMP4 are frequently seen. Increased expression of the BMP4 antagonist Noggin in active lesions (B). The inflammatory infiltrate was shown here with the T cell marker CD8 (C), which detects more than 50% of the T-cells in the infiltrate (Supplementary Figure S1). The foamy macrophages in the lesion are marked positively in the staining against CD68 (KiM1P) (D). In the marginal area of the lesion, the staining against the myelin protein MBP (E) shows intact myelin sheaths (lower area). In addition, some macrophages are present in the infiltrate (middle region) in whose cytoplasm myelin degradation products are recognizable (in this case MBP; CNP also contained in macrophages, not shown). (F–J) chronic inactive lesion: BMP4 (F) is expressed in some astrocytes in the lesion center. Noggin is hardly expressed (G). In the chronically inactive lesions, only a few T-cells (CD8, H) and slightly activated microglia/residual macrophages (CD68 (KiM1P), (I) are still present. Myelin sheath are not detectable any more in the lesion centre (MBP, J). (K–O) remyelinated lesion area: BMP4 (K), like Noggin (L), is expressed in some cell elements in remyelinated lesion areas. Also in these lesion areas there are only few inflammatory infiltrates (CD8, M) and only solitary activated microglial cells (CD68 (KiM1P), N). In the MBP staining some thin (newly formed) myelin sheaths are stained (O). (P–T) white matter control tissue: No cells are marked positively in staining against BMP4 (P). Some cells express Noggin (Q). No T-lymphocytes are marked in the parenchyma (R). Only solitary microglial cells are detected in staining against CD68 (KiM1P) (S). The myelin sheaths are intact (MBP, T). (* early active in this context means that, in addition to inflammatory infiltrate in macrophages, small phagocytosed myelin protein fragments (CNP, not shown) could be detected in the lesion [17]); (scale bar = 50 µm).

Figure 3

Software-assisted immunohistochemical quantification of BMP4-positive cells associated with the white matter pathology of multiple sclerosis: (A) Quantification of immunopositive cells in inflammatory i.e., active lesions, the lesion margin of chronically inactive lesions, the lesion center of chronically inactive lesions, remyelinated lesion sites and normal appearing white matter (NAWM) of MS cases with respect to control tissue (white matter of cases without apparent neurological disease). A significantly increased number of BMP4-positive cells is found in active lesions. A larger number of BMP4-positive cells are also seen in the periphery and center of chronically inactive lesions (** p < 0.01, * p < 0.05, n = 6–18, mean + SEM, ns = not significant). (B,C) Quantification of CD8-positive T-cells and the microglial/macrophagocytic infiltrate (CD68 (KiM1P) area fraction) in corresponding lesion areas as described under A. Inflammatory T-cellular infiltrate and microglial/macrophagocytic infiltrate are particularly present in active lesions and in the margins of chronically inactive lesions (** p < 0.01, n = 6–18, mean + SEM, ns = not significant). (D,E) Quantification of the abundance of myelin sheaths (detected by staining for the myelin protein MBP; MBP area fraction) and of mature oligodendrocytes (NogoA-staining). Compared to control tissue, myelin sheaths (D) are reduced particularly in the chronically inactive lesion centers and also in remyelinated lesion sites (** p < 0.01, n = 6–18, mean + SEM, ns = not significant). The number of mature oligodendrocytes (NogoA-staining, E) is significantly reduced only in the lesion centers of chronically inactive lesions. In the remyelinated lesion areas, their number has increased again and there is no significant difference compared to control tissue. (** p < 0.01, n = 6–18, mean + SEM, ns = not significant). (F) Quantification of the BMP4 antagonist Noggin (area fraction). Increased expression is observed in inflammatory lesions. Especially in lesion centers of chronically inactive lesions, one observes a considerably reduced expression of Noggin. There is a marked difference between the lesion centers of chronically inactive lesions and remyelinated lesion sites in which Noggin expression is restored. Both the inflammatory T-cellular infiltrate and the microglial infiltrate correlate with the number of BMP4-positive cells (** p < 0.01, r = 0.85 (G), and r = 0.89 (H)). A negative correlation between BMP4 expression and myelin sheathing is observed (** p < 0.01, r = −0.46 (I).

Figure 4

Chronic white matter lesions (WML) or grey matter lesions (GML) with BMP4 expressing cells. The double fluorescence staining of BMP4 (A–D, green) with GFAP (A, red), CD68 (Kim1P) (B, red), and NeuN (C, red) shows that BMP4 is expressed by astrocytes (A), microglia/macrophages (B), and neurons (C). The NogoA-positive adult oligodendrocytes (D, red) do not express BMP4; (scale bar = 25 µm).