Teriflunomide Promotes Oligodendroglial 8,9-Unsaturated Sterol Accumulation and CNS Remyelination

(A and B) Newborn-derived OPCs cultures fixed at DIV 7. Coimmunolabeling for Olig2 and MOG in control (Ctrl)- (A) and TF-treated conditions (B). White arrowheads indicate cells coexpressing Olig2 and MOG. (C) A higher proportion of Olig2+ cells were coexpressing MOG when treated with TF (10 nM–1 µM) compared with the control condition. (D and E) Adult OPCs were isolated by fluorescence-activated cell sorting from the CNS of 2 months old PDGFRa:GFP mice and immunostained for Olig2 and MOG in control (Ctrl)- (D) and TF-treated conditions (E). (F) The percentage of Olig2⁺ cells coexpressing MOG is significantly increased in cultures treated with TF compared to control. Data shown are mean ± SEM from 3 to 5 individual experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (1-way ANOVA, Dunn post hoc test (C); Student 2-tailed unpaired t test [F]). Scale bar in A–F = 10 µm. MOG = myelin oligodendrocyte glycoprotein; OPC = oligodendrocyte precursor cell; TF = teriflunomide.

(A) OPCs treated with unconditioned (Ctrl) or TF and/or Ro 48-8071 were immunostained against Olig2 and MOG at 96 hours following treatment. The percentage of Olig2⁺ cells coexpressing MOG was significantly increased in cultures treated with TF compared with control, an effect blocked by the Ro 48-8071 treatment. (B) Quantitation of sterol levels in OPCs treated with TF and/or Ro 48-8071. In OPC cultures treated for 24 hours with TF, the level of zymosterol was increased (results are expressed as % of controls), whereas lanosterol and cholesterol levels were unchanged. (C and D) EBP and DHODH messenger RNA levels measured by reverse transcriptase-quantitative polymerase chain reaction in OPCs, Br, and SC. The Li and Sp were used as positive control for EBP and DHODH enzymatic gene expression, respectively. Glyceraldehyde 3-phosphate dehydrogenase was used as the reference gene. (E) The percentage of MOG⁺/Olig2⁺ oligodendrocytes at 96 hours was significantly increased in OPCs treated with TF compared with control, an effect reversed by the uridine treatment. (F) Uridine blocked the accumulation of zymosterol induced by teriflunomide. (G) The percentage of MOG⁺/Olig2⁺ oligodendrocytes at 96 hours following treatment with TF and/or IFNγ/IL-17 was significantly decreased in OPCs treated with TF and exposed to IFNγ/IL-17 cytokines compared with TF. Data are shown as mean ± SEM from 3 individual experiments. *p < 0.05; ***p < 0.001 (1-way ANOVA, Newman-Keuls post hoc test). Br = brain; DHODH =dihydroorotate dehydrogenase; EBP = emopamil binding protein; Li = liver; OPC = oligodendrocyte precursor cell; SC = spinal cord; Sp = spleen; TF = teriflunomide.

Dose response of remyelination potency of TF. (A and B) Detection of GFP⁺ oligodendroglial cells in optic nerves of stage53 Mbp:GFP-NTR tadpoles. Demyelination of stage53 Mbp:GFP-NTR tadpoles was achieved by 10 days exposure to metronidazole (10 mM) in the swimming water. Tadpoles were then returned to normal water (control) or TF for 3 days. The number of GFP⁺ cells in the optic nerve in control (A) is lower compared with TF-treated tadpoles (B). (C) Remyelination was assayed by counting the number of GFP⁺ cells per optic nerve on day 3 of the repair period. Treatment of tadpoles with TF at concentrations ranging between 1 µM and 100 µM improved remyelination up to 2.25-fold compared with spontaneous recovery (control) set as 1. Data shown are mean ± SEM, n = 5–8 tadpoles per group. *p < 0.05; **p < 0.01 (1-way ANOVA, Dunn post hoc test. Scale bar in A and B = 20 µm. MBP-GFP-NTR = myelin basic protein–GFP-nitroreductase; TF = teriflunomide.

Demyelination was induced by lysophosphatidylcholine on day 0, and differentiation was assessed on day 11. (A and B) Triple immunolabeling for Olig2, MBP, and CC1 in the spinal cord lesion of control (NaCl)-treated mice (A) and TF-treated mice (B). The lesion limits are indicated by white dashed lines. (C and D) Immunolabeling for Olig2 in the core of the lesion of control (NaCl)-treated mice (C) and TF-treated mice (D). (E) No difference in the number of Olig2⁺cells/mm² is observed between the groups. (F and G) Immunolabeling for CC1 in the core of the lesion of control (NaCl)-treated mice (F) and TF-treated mice (G). (H) Significantly, more CC1⁺ cells are present in the lesion of TF-treated mice compared with control. (I and J) Merge showing the colabeling for Olig2 and CC1. White arrowheads indicate cells coexpressing Olig2 and CC1. (K) The percentage of Olig2⁺ cells coexpressing CC1 is significantly increased in the lesion of TF treated mice compared with control. Data shown are mean ± SEM, n = 5 mice per group. *p < 0.05; ***p < 0.001 (Student 2-tailed unpaired t test). Scale bar = 10 µm. MBP = myelin basic protein; TF = teriflunomide.