Tocopherol derivative TFA-12 promotes myelin repair in experimental models of multiple sclerosis

Abstract

Multiple sclerosis (MS) is an inflammatory disease of the CNS that is associated with demyelination and axonal loss, resulting in severe neurological handicap. Current MS therapies mostly target neuroinflammation but have only a little impact on CNS myelin repair. Progress toward treatments that enhance remyelination would therefore represent major advances in MS treatment. Here, we examined the ability of TFA-12, a new synthetic compound belonging to tocopherol long-chain fatty alcohols, to promote oligodendrocyte regeneration and remyelination in experimental models of MS. We showed that TFA-12 significantly ameliorates neurological deficit and severity of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE) in mice. Histological evaluation of mouse EAE spinal cords showed that TFA-12 treatment reduces inflammation, astrogliosis, and myelin loss. Additionally, we demonstrated that TFA-12 accelerates remyelination of focal demyelinated lesions induced by lysolecithin injections. We also found that this compound induces the differentiation of oligodendrocyte precursor cells into mature oligodendrocytes through the inhibition of the Notch/Jagged1 signaling pathway. Altogether, our data provide important proof of principle indicating that TFA-12 could be a potential therapeutic compound for myelin repair in MS.

Figure 1.

TFA-12 inhibits astroglial and microglial activation in vitro. A, Molecular structure of TFA-12. B, RT-PCR analysis of gfap, vimentin, N-cadherin, NosII, and Tnf-α gene expression in astrocyte primary cultures, treated or untreated with TFA-12 (10⁻⁶ m) and with or without LPS (1 μg/ml). TFA-12 inhibits both astrogliosis and inflammatory gene response. C, RT-PCR gene expression analysis in MMGT12 microglial cells. RNA extraction was performed after 6 h of incubation with TFA-12 (10⁻⁵ m), with or without LPS activation (0.01 μg/ml). Expression of NosII, Il-1β, and Tnf-α transcripts in TFA-12-treated MMGT12 cells are significantly decreased. β-Actin was used as an internal control. D, E, ELISA quantification of TNFα (D) and IL1β (E) protein levels in LPS-stimulated MMGT12 microglial cultures treated and untreated with TFA-12 for 24 h. TFA-12 inhibits both TNF-α and IL-1β secretion. Data are mean ± SEM values obtained from three experiments, each conducted in independent cultures (n = 3). *p < 0.01, Student's t test. Ctr, Control.

Figure 2.

TFA-12 improves the clinical course of MOG-induced EAE. A, EAE was induced in C57BL/6 mice by immunization with MOG_35–55 peptides, and clinical scores were evaluated daily. TFA-12 (0.39 mg/kg) or saline solution alone was injected daily from disease onset at 12 dpi. TFA-12 injections improves significantly (*p < 0.05) the mean clinical scores in EAE mice compared with vehicle treatment (n = 20 for each group) from 20 to 25 dpi. B, C, LFB-cresyl violet staining of spinal cord sections from vehicle-treated (B) and TFA-12-treated (C) EAE mice at 24 dpi. Vehicle-treated mice show large inflammatory foci (arrows) in the spinal cord white matter (B), whereas few very cellular infiltrates are detected in spinal cords of TFA-12-treated mice (C). Scale bar, 100 μm.

Figure 3.

Inflammation, astrogliosis, and demyelination are reduced by TFA-12 treatment. Thoracic spinal cord sections from vehicle-treated (B, E, H, K) and TFA-12-treated (A, D, G, J) mice were analyzed at 24 dpi. A, B, CD45 immunohistochemistry on spinal cord sections of vehicle-treated (A) and TFA-12-treated (B) EAE mice reveals immune foci (dashed lines) in the dorsal funiculus. C, The number of CD45+ cells is decreased by 2.8-fold in TFA-12-treated mice compared with controls. D, E, In vehicle-treated mice, demyelinated lesions are characterized by the lack of MBP staining and myelin debris (D), whereas demyelination is barely visible in TFA-12-treated groups (E). F, The extent of demyelination is 4.2-fold lower in TFA-12-treated mice than in controls. G–I, GFAP+ astrogliosis is also drastically reduced by TFA-12 treatment. J–L, ORO staining (red) reveals intense macrophage activity in EAE lesions from vehicle-treated mice (J), whereas macrophage activity is significantly reduced under TFA-12 conditions (K, L). All quantifications were made on three independent experiments. *p < 0.05, Student's t test. Scale bar, 50 μm.

Figure 4.

TFA-12 treatment reduced microglial activation in MOG-induced EAE. Coronal spinal cord sections from vehicle-treated (A, B) and TFA-12-treated (D–F) EAE mice stained for MOG (A, D) and F4/80 (B, E) at 24 dpi. EAE lesions are delimited by dashed lines. A, B, D, E, In vehicle-treated spinal cords, lesions lacking MOG labeling (A) were filled with numerous activated F4/80+ microglia (B), whereas in TFA-12-treated mice (D), F4/80+ microglial cells are strongly reduced (E). C, F, Merge fields are shown. Scale bar, 50 μm.

Figure 5.

TFA-12 impacts on oligodendroglial cells in EAE lesions. A, B, Immunolabeling for NG2+ OPCs in EAE lesions of vehicle-treated (A) and TFA12-treated (B) mouse spinal cords. C, Cell counts in EAE lesions reveal a significant reduction in the number of NG2+ cells (arrows), under TFA-12 conditions. D, E, CC1 immunolabeling (arrows) in spinal cord EAE lesions from vehicle- and TFA-12-treated mice. F, Quantification of CC1+ cells shows a 1.6-fold increase of the number CC1+ oligodendrocytes in TFA-12-treated groups. The dashed lines outline the border of EAE, as defined by CD45 immunolabeling on adjacent sections. **p < 0.01, Student's t test. Scale bar, 50 μm.

Figure 6.

TFA-12 treatment enhances amount of remyelinated axons in LPC-induced lesions. A, Schematic representation of LPC-induced demyelination in the mouse spinal cord dorsal funiculus. B, Toluidine blue semithin section illustrating LPC-induced demyelination at 2 dpi. C, D, Ultrathin micrographs of the lesions in vehicle-treated (C) and TFA-12-treated (D) mice at 15 dpi. TFA-12 treatment enhances the number of remyelinated axons, identified by their thin myelin sheaths (D, asterisks), whereas vehicle-treated mice had significantly fewer remyelinated axons (C). E, Quantification of remyelinated axons over the total number of axons within the lesion shows a significant increase under TFA-12 treatment. ***p < 0.001, Student's t test. Scale bars: B, 100 μm; C, D, 5 μm.

Figure 7.

TFA-12 promotes CG4 cell differentiation. CG4 oligodendroglial cells were differentiated in N1 medium (A, D, G, J) or in N1 supplemented with 5 × 10⁻⁷ m TFA-12 (B, E, H, K) for 48 h for O4 and Ki67 and 5 d for GalC staining. A, B, The number of O4+ oligodendrocytes (arrows) is increased by TFA-12 treatment (B) compared with untreated cultures (A). C, The percentage of O4+ oligodendrocytes is 2.5-fold higher in TFA-12-treated than untreated cultures. D, E, Morphology of untreated (D) and TFA-12-treated (E) O4+ oligodendrocytes. F, Quantification of O4+ oligodendrocytes, according to their number of processes, shows that TFA-12 promotes OPC differentiation. G, H, Proliferating Ki67+ cells (arrows) are reduced in N1 medium plus TFA-12 (H) compared with N1 alone (G). I, Quantification of the percentage of Ki67+ cells shows an 11-fold reduction of cell proliferation. J, K, GalC immunostaining of CG4 culture after 5 d of differentiation in N1 medium alone (J) or N1 supplemented with 5 × 10⁻⁷ m TFA-12 (K). L, The number of GalC+ differentiated oligodendrocytes was significantly enhanced by TFA-12 treatment. *p < 0.05; **p < 0.01, Student's test. Scale bars: A, B, G, H, J, K, 50 μm; D, E, 25 μm.

Figure 8.

TFA-12 enhances oligodendrocyte differentiation in primary OPC cultures. Primary OPCs, prepared from CAG-GFP transgenic newborn rat brains, were cultured 5 d in N medium alone or in N + 5 × 10⁻⁷ m TFA-12. A, B, Ki67 immunocytochemistry in vehicle-treated (A) and TFA-12 treated (B) cultures. C, The number of Ki67+ cells is significantly reduced in TFA-12-treated cultures. D–F, GalC and GFP coimmunostaining in vehicle-treated (D) and TFA-12-treated (E) OPC cultures. Cells counts confirm the significant increase in the number of GalC+ differentiated oligodendrocytes with complex processes (D–F). G, H, MBP and GFP coimmunolabeling in control (G) and TFA-12-treated (H) cultures reveals a significant increase of MBP+ mature oligodendrocytes bearing myelin-like sheaths (G, H, arrowheads; I). **p ≤ 0.01; ***p ≤ 0.001, Student's t test. Scale bar, 100 μm.

Figure 9.

TFA-12 acts as an antagonist of the Notch signaling pathway. A, Real-time RT-PCR of Hes1, Hes5, and Mash1 gene expression levels in CG4 cells untreated and treated with TFA-12 for 48 h. The expression of Notch downstream effectors Hes1 and Hes5 is reduced by fourfold under TFA-12 treatment, whereas Mash1 expression is increased by fourfold, indicating that this compound acts as potent inhibitor of Notch signaling. The Wilcoxon test performed on ΔCt was used to confirm significance between TFA-12 and control groups (*p < 0.05). B, Quantification of O4+ cells after 3 d of differentiation in N1, N1+Jagged1/Fc, and N1+Jagged1/Fc+TFA-12. As expected, Jagged-mediated Notch activation prevents CG4 cell differentiation into O4+ oligodendrocytes. This effect was significantly reversed by TFA-12 at 5 × 10⁻⁷ m. *p < 0.05 for N1+Jagged1/Fc versus N1+Jagged1/Fc+ TFA-12, Student's t test. Data are mean ± SEM of three independent experiments.