Drug-based modulation of endogenous stem cells promotes functional remyelination in vivo

Abstract

Multiple sclerosis involves an aberrant autoimmune response and progressive failure of remyelination in the central nervous system. Prevention of neural degeneration and subsequent disability requires remyelination through the generation of new oligodendrocytes, but current treatments exclusively target the immune system. Oligodendrocyte progenitor cells are stem cells in the central nervous system and the principal source of myelinating oligodendrocytes. These cells are abundant in demyelinated regions of patients with multiple sclerosis, yet fail to differentiate, thereby representing a cellular target for pharmacological intervention. To discover therapeutic compounds for enhancing myelination from endogenous oligodendrocyte progenitor cells, we screened a library of bioactive small molecules on mouse pluripotent epiblast stem-cell-derived oligodendrocyte progenitor cells. Here we show seven drugs function at nanomolar doses selectively to enhance the generation of mature oligodendrocytes from progenitor cells in vitro. Two drugs, miconazole and clobetasol, are effective in promoting precocious myelination in organotypic cerebellar slice cultures, and in vivo in early postnatal mouse pups. Systemic delivery of each of the two drugs significantly increases the number of new oligodendrocytes and enhances remyelination in a lysolecithin-induced mouse model of focal demyelination. Administering each of the two drugs at the peak of disease in an experimental autoimmune encephalomyelitis mouse model of chronic progressive multiple sclerosis results in striking reversal of disease severity. Immune response assays show that miconazole functions directly as a remyelinating drug with no effect on the immune system, whereas clobetasol is a potent immunosuppressant as well as a remyelinating agent. Mechanistic studies show that miconazole and clobetasol function in oligodendrocyte progenitor cells through mitogen-activated protein kinase and glucocorticoid receptor signalling, respectively. Furthermore, both drugs enhance the generation of human oligodendrocytes from human oligodendrocyte progenitor cells in vitro. Collectively, our results provide a rationale for testing miconazole and clobetasol, or structurally modified derivatives, to enhance remyelination in patients.

Extended Data Fig. 1. Performance of the primary screen

a, Representative flow cytometry plots showing co-expression of NG2 and CD140a in both batches of EpiSC-derived OPCs used for this study. The batches of EpiSC-derived OPCs were sorted to purity (circled areas of plots) prior to use in this study. b, RNAseq expression heatmap showing down regulation of pluripotent stem cell transcripts and up regulation of OPC transcripts when EpiSCs were differentiated into OPCs. Fragments per kilobase exon per million reads (FPKM) for each transcript are shown as compared to in vivo isolated mouse OPCs. c, Quantification of DMSO (v/v) tolerance of EpiSC derived-OPCs in 96-well plates shown as mean ± SEM. For reference, 0.05% (v/v) DMSO was used as vehicle for all in vitro experiments in this study. n = 16 wells per group with >690 cells scored per well. d, Quantification of cell viability of thyroid hormone (positive control) and DMSO vehicle treatments per well across all 10 assay plates shown as mean ± SEM. n = 80 wells per group with >6,800 cells scored per well. e, Signal to background (S/B) mean values with standard deviation (s.d.) of controls from the entire screen. n = 80 wells per group. f, Raw data of MBP process length from the primary screen for thyroid hormone treatment and DMSO vehicle across all plates shown as mean ± SD. n = 8 wells per group with >6,800 cells scored per well. g, Raw data of MBP process intensity from the primary screen for thyroid hormone treatment and DMSO vehicle shown as mean ± SD. n = 8 wells per group with >6,800 cells scored per well.

Extended Data Fig. 2. Drug hit ranking and validation

a, Chart ranking the 22 primary drug hits (Single Dose Rank) into 4 tiers based on calculation of half maximal effective concentration (EC₅₀) to induce PLP1+ oligodendrocytes from OPCs and concentration at which 50% of the cells were lost (50% Tox) calculated from a seven-point dose treatment. n = 4 wells per dose per drug using independently sourced drug and separate OPC batch from the primary screen. Tiers ranged from the most potent and least toxic effectors to the least potent and most toxic: Tier A (green), Tier B (grey), Tier C (orange) and Tier D (red). 1536-well format external validation of 14 of 16 tested hits is also shown. Drugs were further ranked into groups of High (green), Medium (grey), and Low (orange) based on their ability to increase MBP+ axonal ensheathment in mouse cerebellar brain slices relative to vehicle (DMSO) treated controls as measured by HCA. NT = not tested. b, External validation whole 1536-well images of MBP+ (green) oligodendrocytes generated from OPCs after 72 hours of treatment. GE InCell HCA is shown with processes traced in yellow and nuclei in blue.

Extended Data Fig. 3. Primary screen structure-activity analysis

Chemoinformatic identification of two substructures consistently enriched in high performing drugs in the OPC assay. Numerical activity rank in the primary screen is indicated with the top 22 shown in green, 23–50 shown in grey, and >51 in red. a, (1,3) diazoles, mono-substituted at the 1-position showed consistent activity on OPCs. b, c, (1,3) diazoles, poly-substituted at 2 or more of the R groups (b), or (1,2,4) triazoles, mono-substituted at the 1-position (c) showed no activity on OPCs. d, The sterane base structure showed enrichment in the top performing hits.

Extended Data Fig. 4. Histological assessment of remyelination in the LPC-induced model of demyelination

a, Representative electron micrographs showing remyelinated axons within lesions of miconazole treated mice at eight dpl. Scale bar, 2 µm. b, Toluidine blue stained histological sections showing the extent of remyelination in the lesions of treated animals at twelve dpl. Normal uninjured myelin appears to the left of the black dashed line demarcating the definitive lesion edge. Scale bar, 20 µm.

Extended Data Fig. 5. Miconazole and clobetasol enhance myelination in vivo

a and b, Representative immunohistochemical images of the lateral corpus callosum (CC) of postnatal day day mouse pups that had been injected i.p. daily for four days previously starting on postnatal day two with vehicle, clobetasol (2 mg/kg), or miconazole (10 mg/kg). CC1 (red) marks newly generated oligodendrocytes (a) and MBP (green) shows the extent of developmental myelination (b). Clobetasol and miconazole treatment each induce a marked increase in the number of CC1 positive oligodendrocytes in the lateral corpus callosum (a) and a significant increase in the length of the corpus callosum covered with aligned MBP+ fibers (b). Scale bar, 200 µm. Two-tailed t-test *p≤0.05 and **p≤0.01. Striatum is marked as Str. All graphs are presented as mean ± SEM.

Extended Data Fig. 6. RNAseq time course of drug treated OPCs

a, Volcano plots of all genes from OPCs treated with clobetasol or miconazole relative to vehicle control, with differentially expressed genes highlighted (red). Significance (measured as – log₁₀[q-value]) is plotted in relationship to expression change (log₂[treatment/vehicle]). Time course was after two, six and twelve hours of drug treatment. b, Venn diagrams depicting the overlap of genes differentially expressed at any time point and increased in treatments vs vehicle (left), as well as those decreased in treatments vs vehicle (right). c, Significant canonical pathways perturbed by each drug treatment according to Ingenuity Pathway Analysis.

Extended Data Fig. 7. Global phosphoproteomic analysis of miconazole-treated OPCs

a and b, OPCs treated with miconazole for one (a) or five (b) hours followed by global phosphoproteomic analysis. Proteins highlighted in green were observed to have a two-fold or greater increase in phosphorylation while those highlighted in red were observed to have a two-fold or greater decrease in phosphorylation as compared to time point matched vehicle treated controls. Proteins highlighted in grey were detected in the analysis but were not changed compared to vehicle control. See Supplementary Table 3 for the full phosphoproteomic dataset. c, Quantification of the percentage of MBP+ oligodendrocytes differentiated from mouse OPCs after 72 hours of treatment with DMSO, miconazole (1 µM), or voriconazole (7 doses 0.01–6.7 µM). n = 6 wells per condition with >6,000 cells scored per well. Graph presented as mean ± SEM. The chemical structure of voriconazole is shown.

Extended Data Fig. 8. Miconazole and clobetasol enhance human OPC differentiation

a, Representative phase contrast image of a hESC colony cultured on matrigel. b, Representative phase contrast image of hESC-derived OPCs. c, hESC-derived OPCs stain positive for Sox10. d and e, Representative images of hESC-derived OPCs (d) and hiPSC-derived OPCs (e) treated with vehicle (DMSO), miconazole (1 µM), or clobetasol (5 µM) for 21 days stained for MBP (red). f and g, High content analysis of hESC-derived (f) and hiPSC-derived (g) OPCs differentiated in the presence of drugs or vehicle over 21 days. n = 3–5 wells per condition with >120 cells scored per well. Graphs presented as mean ± SEM. Scale bars, 100µm.

Extended Data Fig. 9. Effects of miconazole and clobetasol on immune cell survival and function

a–d, Quantification of cell proliferation (a, c) and differentiation (b, d) of naïve CD4⁺ T cells from unprimed SJL/J mice after activation with plate-bound anti-CD3 under Th1 (a, b) or Th17 (c, d) cell driving conditions. e–j, Ex vivo recall assays quantifying cell proliferation (ΔCPM) (e, h) along with interferon gamma (IFN-γ) (f, i) and IL-17 (g, j) cytokine production from lymphocytes of mice primed with PLP_139–151 (e–g) or MOG_35–55 (h–j). Cultures were treated with vehicle (DMSO), benztropine, clobetasol, or miconazole (10⁻⁹-10⁻⁵ M) and analyzed after four days. Four independent replicates are shown for each assay.

Extended Data Fig. 10. Histological improvements in MOG_35–55 EAE spinal cords after treatment with miconazole or clobetasol

a, b Representative images of luxol fast blue (LFB) staining (a) demonstrated a clear decrease in areas of white matter disruption in the spinal cords of drug treated animals which coincides with increased MBP staining (b). c, IBA1 staining showed a small reduction of immune cell infiltration into the lesion areas, especially in clobetasol treated animals, but not an abrogation, d and e, Representative toluidine blue stained images (d) and electron micrographs (e) revealed a reduction in the areas of demyelination in drug treated animals. Lesioned areas are outlined with black dotted lines. Insets in toluidine blue staining show higher magnification of myelination in the corresponding spinal cords. Scale bar, 100 µm (a–d) and 2 µm (e).

Figure 1. A pluripotent stem cell-based phenotypic screening platform to identify modulators of OPC differentiation and maturation

a, Representative images of vehicle and drug hit treated mouse EpiSC-derived OPCs from the primary screen. Nuclear (DAPI, blue) and MBP (red) staining along with high content analysis (HCA) to identify oligodendrocyte nuclei (green) and MBP+ processes (yellow). Scale bar, 100µm. b, Scatter plot of primary screen results displayed as normalized values of MBP process length and intensity for all 727 drugs with the 22 hits marked in red. Baseline (vehicle) was set at zero and thyroid hormone (positive control) was set at 100. c, Montaged images of whole postnatal day seven mouse cerebellar slices treated with drug or vehicle for five days and stained for MBP (green). Insets show a representative example of the HCA script used to identify and quantify MBP+ aligned fibers (light blue). Scale bar, 1 mm for whole slices and 100 µm for insets. d, Relative quantitation of HCA and western blot (WB) data from cerebellar slices treated for five days. For HCA screen, n = 1 with 6–12 slices averaged per group (also see Extended Data Fig. 2a). For western blot, n = 3 independent replicates of 12 slices per group. Values are mean for HCA and mean ± SEM for WB. e, Representative WB of MBP isoforms and β-Actin (loading control) of cerebellar slices treated for five days. Full blots are available in Supplementary Figure 1. f, Chemical structures of clobetasol and miconazole. Source Data is provided for Figure 1b, d.

Figure 2. Miconazole and clobetasol each enhance remyelination in the LPC lesion mouse model

a, Representative immunohistochemical images of treated mice showing newly generated oligodendrocytes (CC1, red) and MBP (green) within the lesion (approximated by white dashed outline) at eight and twelve dpl. Scale bar, 200 µm. b, Quantitation of CC1+ oligodendrocytes per lesion area at eight dpl. Values are mean ± SEM. n = 3 mice per group. Two-tailed t-test *p<0.05. c, Representative electron micrographs showing remyelinated axons within lesions of drug treated mice at twelve dpl. Scale bar, 2 µm. d, Scatter plot of g-ratios of lesion axons at twelve dpl. n = 100 calculated from two mice per group. Percentage of lesion axons myelinated is indicated in the legend. Source Data is provided for Figure 2b, d.

Figure 3. Cellular and molecular effects of miconazole and clobetasol on mouse OPCs

a, Percent MBP+ oligodendrocytes generated from OPCs at 72 hours with treatments initiated at time points indicated. n = 6 wells per condition with >6,000 cells scored per well. b, Percent MBP+ oligodendrocytes generated from OPCs treated simulataneously and analyzed at time points indicated. n = 8 wells per condition with >1,700 cells scored per well. c, Percent GFAP+ astrocytes generated from OPCs at 72 hours of treatment. n = 4 wells per condition >2,900 cells scored per well. d, Heatmap depicting biochemical inhibition of muscarinic receptors M1-M5 displayed as percent inhibition with minimum (green) and maximum (red). e, Western blot of total GR and its phosphorylation at Ser220 (p-GR) in OPCs treated for one hour. f, Percent MBP+ oligodendrocytes generated from OPCs 72 hours after treatment. n = 6 wells per condition with >1,400 cells scored per well. g, Western blot of total ERK1/2 and their phosphorylation at Thr202/Tyr204 or Thr185/Tyr187 (p-ERK1/2) in cells (OPCs or mouse embryonic fibroblasts) treated for one hour. FGF served as a positive control for p-ERK1/2 induction. h, Western blot of total ERK1/2 and p-ERK1/2 in OPCs treated for one hour in the presence of the indicated pathway inhibitors. All graphs depict mean ± SEM. Full western blots are available in Supplementary Figure 2. Source Data is provided for Figure 3a–d, f.

Figure 4. Therapeutic efficacy of miconazole and clobetasol in mouse models of multiple sclerosis

a, Scoring of disease severity in relapsing remitting PLP_139–151-induced EAE mice treated beginning on day 13 (black arrow) and ending on day 29. n = 10 mice per group. Graph depicts mean daily disease score ± SEM. b, Flow cytometric-based quantification of spleen cell numbers at day 29 from the PLP_139–151 EAE cohort in (a). Values are mean ± SEM. n = 4–5 mice per group. c, Scoring of disease severity in chronic progressive MOG_35–55-induced EAE mice treated daily for ten days beginning at the peak of disease on day 15 (black arrow). n =12–16 mice per group. Graph depicts mean daily disease score ± SEM. d, Mean improvement of disease score (Δ) per animal (peak score minus ending score) of MOG_35–55 EAE cohort in (c). Also shown are external validation results in MOG_35–55 EAE from an independent contract laboratory. n = 12 mice per group. For all EAE experiments drugs were dosed daily by intraperitoneal (i.p.) injection: clobetasol (2 mg/kg), miconazole (10 mg/kg), benztropine (10 mg/kg), or FTY720 (1 mg/kg). All EAE disease scoring was 0=no abnormality; 1=limp tail; 2=limp tail and hind limb weakness; 3=hind limb paralysis; 4=hind limb paralysis and forelimb weakness; and 5=moribund. Two-tailed t-test *p-value <0.05 and **p-value <0.01 for drug treated groups compared to their respective vehicle treated group. Source Data is provided for Figure 4a–d.