Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A regenerative approach to the treatment of multiple sclerosis

Subjects

Abstract

Progressive phases of multiple sclerosis are associated with inhibited differentiation of the progenitor cell population that generates the mature oligodendrocytes required for remyelination and disease remission. To identify selective inducers of oligodendrocyte differentiation, we performed an image-based screen for myelin basic protein (MBP) expression using primary rat optic-nerve-derived progenitor cells. Here we show that among the most effective compounds identifed was benztropine, which significantly decreases clinical severity in the experimental autoimmune encephalomyelitis (EAE) model of relapsing-remitting multiple sclerosis when administered alone or in combination with approved immunosuppressive treatments for multiple sclerosis. Evidence from a cuprizone-induced model of demyelination, in vitro and in vivo T-cell assays and EAE adoptive transfer experiments indicated that the observed efficacy of this drug results directly from an enhancement of remyelination rather than immune suppression. Pharmacological studies indicate that benztropine functions by a mechanism that involves direct antagonism of M1 and/or M3 muscarinic receptors. These studies should facilitate the development of effective new therapies for the treatment of multiple sclerosis that complement established immunosuppressive approaches.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Benztropine induces oligodendrocyte precursor cell differentiation and in vitro myelination of co-cultured axons.
Figure 2: Benztropine decreases disease severity in the PLP-induced EAE model.
Figure 3: Benztropine-induced remyelination in the PLP-induced EAE model.
Figure 4: Benztropine treatment enhances remyelination in the cuprizone model.

Similar content being viewed by others

References

  1. Franklin, R. J. & Ffrench-Constant, C. Remyelination in the CNS: from biology to therapy. Nature Rev. Neurosci. 9, 839–855 (2008)

    CAS  Google Scholar 

  2. Franklin, R. J. Why does remyelination fail in multiple sclerosis? Nature Rev. Neurosci. 3, 705–714 (2002)

    CAS  Google Scholar 

  3. Nunes, M. C. et al. Identification and isolation of multipotential neural progenitor cells from the subcortical white matter of the adult human brain. Nature Med. 9, 439–447 (2003)

    CAS  PubMed  Google Scholar 

  4. Belachew, S. et al. Postnatal NG2 proteoglycan-expressing progenitor cells are intrinsically multipotent and generate functional neurons. J. Cell Biol. 161, 169–186 (2003)

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Huang, J. K. et al. Retinoid X receptor gamma signaling accelerates CNS remyelination. Nature Neurosci. 14, 45–53 (2011)

    CAS  PubMed  Google Scholar 

  6. Gensert, J. M. & Goldman, J. E. Endogenous progenitors remyelinate demyelinated axons in the adult CNS. Neuron 19, 197–203 (1997)

    CAS  PubMed  Google Scholar 

  7. Horner, P. J. et al. Proliferation and differentiation of progenitor cells throughout the intact adult rat spinal cord. J. Neurosci. 20, 2218–2228 (2000)

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Kremer, D., Aktas, O., Hartung, H. P. & Kury, P. The complex world of oligodendroglial differentiation inhibitors. Ann. Neurol. 69, 602–618 (2011)

    CAS  PubMed  Google Scholar 

  9. Patel, J. R. & Klein, R. S. Mediators of oligodendrocyte differentiation during remyelination. FEBS Lett. 585, 3730–3737 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Chang, A., Tourtellotte, W. W., Rudick, R. & Trapp, B. D. Premyelinating oligodendrocytes in chronic lesions of multiple sclerosis. N. Engl. J. Med. 346, 165–173 (2002)

    PubMed  Google Scholar 

  11. Chari, D. M., Huang, W. L. & Blakemore, W. F. Dysfunctional oligodendrocyte progenitor cell (OPC) populations may inhibit repopulation of OPC depleted tissue. J. Neurosci. Res. 73, 787–793 (2003)

    CAS  PubMed  Google Scholar 

  12. Wolswijk, G. Chronic stage multiple sclerosis lesions contain a relatively quiescent population of oligodendrocyte precursor cells. J. Neurosci. 18, 601–609 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Kuhlmann, T. et al. Differentiation block of oligodendroglial progenitor cells as a cause for remyelination failure in chronic multiple sclerosis. Brain 131, 1749–1758 (2008)

    CAS  PubMed  Google Scholar 

  14. Hart, I. K., Richardson, W. D., Bolsover, S. R. & Raff, M. C. PDGF and intracellular signaling in the timing of oligodendrocyte differentiation. J. Cell Biol. 109, 3411–3417 (1989)

    CAS  PubMed  Google Scholar 

  15. Billon, N., Tokumoto, Y., Forrest, D. & Raff, M. Role of thyroid hormone receptors in timing oligodendrocyte differentiation. Dev. Biol. 235, 110–120 (2001)

    CAS  PubMed  Google Scholar 

  16. Tokumoto, Y. M., Tang, D. G. & Raff, M. C. Two molecularly distinct intracellular pathways to oligodendrocyte differentiation: role of a p53 family protein. EMBO J. 20, 5261–5268 (2001)

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Fernandez, M. et al. Thyroid hormone administration enhances remyelination in chronic demyelinating inflammatory disease. Proc. Natl Acad. Sci. USA 101, 16363–16368 (2004)

    ADS  CAS  PubMed  Google Scholar 

  18. Calzà, L., Fernandez, M. & Giardino, L. Cellular approaches to central nervous system remyelination stimulation: thyroid hormone to promote myelin repair via endogenous stem and precursor cells. J. Mol. Endocrinol. 44, 13–23 (2010)

    PubMed  Google Scholar 

  19. Barres, B. A., Lazar, M. A. & Raff, M. C. A novel role for thyroid hormone, glucocorticoids and retinoic acid in timing oligodendrocyte development. Development 120, 1097–1108 (1994)

    CAS  PubMed  Google Scholar 

  20. Buckley, C. E. et al. Drug reprofiling using zebrafish identifies novel compounds with potential pro-myelination effects. Neuropharmacology 59, 149–159 (2010)

    CAS  PubMed  Google Scholar 

  21. Ibanez, C. et al. Steroids and the reversal of age-associated changes in myelination and remyelination. Prog. Neurobiol. 71, 49–56 (2003)

    CAS  PubMed  Google Scholar 

  22. Baer, A. S. et al. Myelin-mediated inhibition of oligodendrocyte precursor differentiation can be overcome by pharmacological modulation of Fyn-RhoA and protein kinase C signalling. Brain 132, 465–481 (2009)

    PubMed  PubMed Central  Google Scholar 

  23. Joubert, L. et al. Chemical inducers and transcriptional markers of oligodendrocyte differentiation. J. Neurosci. Res. 88, 2546–2557 (2010)

    CAS  PubMed  Google Scholar 

  24. Gard, A. L. & Pfeiffer, S. E. Two proliferative stages of the oligodendrocyte lineage (A2B5+O4and O4+GalC) under different mitogenic control. Neuron 5, 615–625 (1990)

    CAS  PubMed  Google Scholar 

  25. Pfeiffer, S. E., Warrington, A. E. & Bansal, R. The oligodendrocyte and its many cellular processes. Trends Cell Biol. 3, 191–197 (1993)

    CAS  PubMed  Google Scholar 

  26. Gaspard, N. et al. Generation of cortical neurons from mouse embryonic stem cells. Nature Protocols 4, 1454–1463 (2009)

    CAS  PubMed  Google Scholar 

  27. Barres, B. A. et al. Cell death and control of cell survival in the oligodendrocyte lineage. Cell 70, 31–46 (1992)

    CAS  PubMed  Google Scholar 

  28. Eshleman, A. J., Henningsen, R. A., Neve, K. A. & Janowsky, A. Release of dopamine via the human transporter. Mol. Pharmacol. 45, 312–316 (1994)

    CAS  PubMed  Google Scholar 

  29. McKearney, J. W. Stimulant actions of histamine H1 antagonists on operant behavior in the squirrel monkey. Psychopharmacol. 77, 156–158 (1982)

    CAS  Google Scholar 

  30. Agoston, G. E. et al. Novel N-substituted 3α-[bis(4'-fluorophenyl)methoxy]tropane analogues: selective ligands for the dopamine transporter. J. Med. Chem. 40, 4329–4339 (1997)

    CAS  PubMed  Google Scholar 

  31. De Angelis, F., Bernardo, A., Magnaghi, V., Minghetti, L. & Tata, A. M. Muscarinic receptor subtypes as potential targets to modulate oligodendrocyte progenitor survival, proliferation and differentiation. Dev. Neurobiol. 72, 713–728 (2012)

    CAS  PubMed  Google Scholar 

  32. Stidworthy, M. F. et al. Notch1 and Jagged1 are expressed after CNS demyelination, but are not a major rate-determining factor during remyelination. Brain 127, 1928–1941 (2004)

    PubMed  Google Scholar 

  33. Taveggia, C., Feltri, M. L. & Wrabetz, L. Signals to promote myelin formation and repair. Nature Rev. Neurol. 6, 276–287 (2010)

    Google Scholar 

  34. Ragheb, F. et al. Pharmacological and functional characterization of muscarinic receptor subtypes in developing oligodendrocytes. J. Neurochem. 77, 1396–1406 (2001)

    CAS  PubMed  Google Scholar 

  35. Felder, C. C. Muscarinic acetylcholine receptors: signal transduction through multiple effectors. FASEB J. 9, 619–625 (1995)

    CAS  PubMed  Google Scholar 

  36. Owens, T. & Sriram, S. The immunology of multiple sclerosis and its animal model, experimental allergic encephalomyelitis. Neurol. Clin. 13, 51–73 (1995)

    CAS  PubMed  Google Scholar 

  37. Lawson, B. R. et al. Inhibition of transmethylation down-regulates CD4 T cell activation and curtails development of autoimmunity in a model system. J. Immunol. 178, 5366–5374 (2007)

    CAS  PubMed  Google Scholar 

  38. Mix, E., Meyer-Rienecker, H. & Zettl, U. K. Animal models of multiple sclerosis for the development and validation of novel therapies—potential and limitations. J. Neurol. 255 (Suppl 6). 7–14 (2008)

    CAS  PubMed  Google Scholar 

  39. Steinman, L. & Zamvil, S. S. Virtues and pitfalls of EAE for the development of therapies for multiple sclerosis. Trends Immunol. 26, 565–571 (2005)

    CAS  PubMed  Google Scholar 

  40. Aharoni, R. et al. Distinct pathological patterns in relapsing-remitting and chronic models of experimental autoimmune enchephalomyelitis and the neuroprotective effect of glatiramer acetate. J. Autoimmun. 37, 228–241 (2011)

    CAS  PubMed  Google Scholar 

  41. Liu, L. et al. Myelin repair is accelerated by inactivating CXCR2 on nonhematopoietic cells. J. Neurosci. 30, 9074–9083 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Mi, S. et al. LINGO-1 antagonist promotes spinal cord remyelination and axonal integrity in MOG-induced experimental autoimmune encephalomyelitis. Nature Med. 13, 1228–1233 (2007)

    CAS  PubMed  Google Scholar 

  43. Kawashima, K. & Fujii, T. Basic and clinical aspects of non-neuronal acetylcholine: overview of non-neuronal cholinergic systems and their biological significance. J. Pharmacol. Sci. 106, 167–173 (2008)

    CAS  PubMed  Google Scholar 

  44. Stern, J. N. et al. Promoting tolerance to proteolipid protein-induced experimental autoimmune encephalomyelitis through targeting dendritic cells. Proc. Natl Acad. Sci. USA 107, 17280–17285 (2010)

    ADS  CAS  PubMed  Google Scholar 

  45. Steelman, A. J., Thompson, J. P. & Li, J. Demyelination and remyelination in anatomically distinct regions of the corpus callosum following cuprizone intoxication. Neurosci. Res. 72, 32–42 (2012)

    CAS  PubMed  Google Scholar 

  46. Matsushima, G. K. & Morell, P. The neurotoxicant, cuprizone, as a model to study demyelination and remyelination in the central nervous system. Brain Pathol. 11, 107–116 (2001)

    CAS  PubMed  Google Scholar 

  47. Kappos, L. et al. Oral fingolimod (FTY720) for relapsing multiple sclerosis. N. Engl. J. Med. 355, 1124–1140 (2006)

    CAS  PubMed  Google Scholar 

  48. Durelli, L. et al. Every-other-day interferon beta-1b versus once-weekly interferon beta-1a for multiple sclerosis: results of a 2-year prospective randomised multicentre study (INCOMIN). Lancet 359, 1453–1460 (2002)

    CAS  PubMed  Google Scholar 

  49. Noronha, A., Toscas, A. & Jensen, M. A. Interferon β decreases T cell activation and interferon γ production in multiple sclerosis. J. Neuroimmunol. 46, 145–153 (1993)

    CAS  PubMed  Google Scholar 

  50. Brinkmann, V. et al. The immune modulator FTY720 targets sphingosine 1-phosphate receptors. J. Biol. Chem. 277, 21453–21457 (2002)

    CAS  PubMed  Google Scholar 

  51. Kondo, T. & Raff, M. The Id4 HLH protein and the timing of oligodendrocyte differentiation. EMBO J. 19, 1998–2007 (2000)

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Wang, S., Sdrulla, A., Johnson, J. E., Yokota, Y. & Barres, B. A. A role for the helix-loop-helix protein Id2 in the control of oligodendrocyte development. Neuron 29, 603–614 (2001)

    CAS  PubMed  Google Scholar 

  53. Pang, Y. et al. Neuron-oligodendrocyte myelination co-culture derived from embryonic rat spinal cord and cerebral cortex. Brain Behav. 2, 53–67 (2012)

    PubMed  PubMed Central  Google Scholar 

  54. Izrael, M. et al. Human oligodendrocytes derived from embryonic stem cells: Effect of noggin on phenotypic differentiation in vitro and on myelination in vivo. Mol. Cell. Neurosci. 34, 310–323 (2007)

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the Skaggs Institute for Chemical Biology and the California Institute for Regenerative Medicine grant TR3-05617 (to P.G.S), the California Institute for Regenerative Medicine (TG2-01165) and National Science Foundation pre-doctoral fellowships (to V.A.D and C.A.L, respectively). We are grateful to T. Hasnat, M. Chadwell, W. Kiosses and M. Wood for technical support. This is manuscript number 21786 of The Scripps Research Institute.

Author information

Authors and Affiliations

Authors

Contributions

L.L.L., P.G.S., C.A.L. and V.A.D. initiated the project and developed strategy. V.A.D., V.T., C.C.G., B.K., H.J.K., K.P., J.G.S. and I.A. performed the experiments. L.L.L., P.G.S. and V.A.D.wrote the manuscript. B.R.L., C.A.L., A.N.T., F.H.G. and T.K. contributed essential ideas and comments.

Corresponding authors

Correspondence to Brian R. Lawson, Peter G. Schultz or Luke L. Lairson.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 High-throughput screen to identify inducers of OPC differentiation.

a, Rat primary OPCs in basal differentiation media treated with DMSO (<0.1%) or thyroid hormone (T3; 1 μM) for 6 days in culture, fixed and stained using antibodies for myelin basic protein (MBP), 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNP) and oligodendrocyte marker O4. A2B5+ OPCs differentiated into immature oligodendrocytes that express CNP and O4, but not MBP, upon reduction of PDGF-AA. T3 added as a positive control induced differentiation to mature cells that express MBP. Scale bars, 100 μm. b, Schematic representation of the high-throughput screening platform used to identify inducers of OPC differentiation. c, Inducers of OPC differentiation identified as hits from a screen of known biologically active compounds. Scale bars, 100 μm; inset, 40 μm.

Extended Data Figure 2 Benztropine induces dose-dependent OPC differentiation in vitro to mature oligodendrocytes.

a, Dose response assay used to confirm primary screening activity of benztropine and determine potency (EC50). OPCs were treated with benztropine and immunostained using antibodies for MBP (n = 3, mean and s.d.). b, Images showing dose-dependent induction of OPC differentiation after treatment with benztropine. (Scale bars, 100 μm; inset, 40 μm). OPCs in basal differentiation media treated with DMSO (<0.1%), T3 (1 μM) or benztropine (1.5 μM) for 6 days and analysed for MBP and MOG expression by western blot (c) and by qRT–PCR (d) (n = 3, mean and s.d.). e, OPCs were plated in differentiation medium and treated with DMSO (<0.1%), benztropine (1.5 μM) or T3 (1 μM) for 6 days. Cells were fixed and immunostained for myelin basic protein (MBP), myelin oligodendroglial glycoprotein (MOG), CNP, oligodendrocyte marker O1, oligodendrocyte marker O4, glial marker SOX10, proteolipid peptide (PLP), OLIG1 and OLIG2. Representative images showing expression of mature oligodendrocyte markers in benztropine- and T3-treated cells, but not DMSO-treated cells. Scale bars, 100 μm; inset, 40 μm. f, Expression of cell cycle genes by qRT–PCR. (n = 3, mean and s.d., *P < 0.05, t-test). g, OPCs plated in basal differentiation medium and treated with benztropine (1.5 μM) on various days (0, 1, 2, 3, 4 and 5), fixed on day 6 and immunostained for MBP (n = 3, mean and s.d.). h, OPCs plated in basal differentiation medium, treated with benztropine (1.5 μM) on the same day, fixed on various days (3, 4, 5 and 6) following compound treatment and immunostained for MBP (n = 3, mean and s.d.).

Extended Data Figure 3 Benztropine induces OPC differentiation and in vitro myelination through M1/M3 muscarinic receptor antagonism and has no effect on histamine or nicotinic signalling.

a, Mouse OPCs co-cultured with mouse cortex-derived cells in the presence of DMSO or benztropine and immunostained for MBP (red) and nuclei with Hoechst 33342 (blue). Scale bars, 100 μm. b, Quantification of MBP staining of mouse OPCs treated with DMSO or benztropine. c, Analysis of myelination in OPCs with neurons co-culture. Arrowheads point to regions of myelination. Scale bars, 20 μm. d, Quantification of fraction of myelinating oligodendrocytes in OPCs with neurons co-cultures (n = 10, mean and s.e.m., **P < 0.01, ANOVA with Bonferroni correction). OPCs co-treated with benztropine (1.5 μM) and carbachol (2.3 μM) for 6 days and stained for MBP (green) (Scale bars, 100 μm; inset, 40 μm). e, Antagonism of benztropine-induced OPC differentiation by muscarinic agonist carbachol. f, Quantification of MBP staining of OPCs co-treated with benztropine (1.5 μM) and muscarinic receptor agonist carbachol for 6 days under basal differentiation conditions (n = 3, mean and s.d.). gk, OPCs plated co-treated with benztropine (0.8 μM) and either nicotine (g), histamine (h), histamine receptor agonist histamine trifluoromethyl toluidide (HTMT) (i), dopamine receptor agonist quinpirole (j) or dopamine receptor antagonist haloperidol (k) (n = 3, mean and s.d., ns = not significant). l, Various nicotinic receptor antagonists have no effect on OPC differentiation. m, Benztropine blocks carbachol- and muscarine-induced activation of Notch signalling measured by western blot for intracellular domain of Notch1. (a.u., arbitrary unit, n = 3, mean and s.d., *P < 0.05, t-test). n, Naive whole rat brain and rat primary OPCs treated with DMSO (<0.1%) or T3 (1 μM) for 6 days tested for expression of muscarinic receptors and choline acetyl transferase (ChAT) by PCR using gene-specific primers. o, Quantification of M1, M2, M3, M4 and ChAT expression by qRT-PCR. (n = 3, mean and s.d., expression fold change normalized to OPCs). p, OPCs treated with benztropine (25 μM) and pelleted for western blot analysis of total protein. q, Carbachol induced a dose-dependent increase in intracellular Ca2+ levels, whereas benztropine and atropine (a muscarinic antagonist) dose-dependently blocked carbachol (50 μM) induced calcium influx through antagonism of M1/M3 muscarinic receptors. r, Benztropine (13 μM) had no effect on the levels of cAMP. Forskolin is a positive control for increasing intracellular cAMP (n = 3, mean and s.d., *P < 0.05, t-test).

Extended Data Figure 4 Benztropine dose-dependently reduces clinical severity and induces remyelination in the PLP-induced EAE model.

a, Clinical severity scores of EAE mice treated with various doses of benztropine in the prophylactic mode (n = 8, mean and s.e.m.). b, EAE mice treated with benztropine (10 mg per kg) or vehicle in the therapeutic mode and spinal cord sections from mice representative of the average group scores during the relapse phase of EAE stained with Luxol fast blue and H&E, or Luxol fast blue only. Arrows point to regions of lymphocyte infiltration (LFB + H&E) or demyelination (LFB). Scale bars represent 100 μm. EAE mice treated with benztropine (10 mg per kg) or vehicle in prophylactic mode. c, Spinal cord sections from mice representative of the average group scores on day 8, 11 and 14 immunostained with antibodies specific to CD45 and GSTπ. d, Mean clinical scores of mice at the time of spinal cord isolation and quantification of the infiltrated areas (CD45+) and number of GSTπ+ cells (n = 8, mean and s.e.m., **P < 0.01, t-test). Scale bars, 100 μm. e, EAE mice treated with benztropine (10 mg per kg) or vehicle in prophylactic mode and spinal cord sections from mice representative of the average group scores on day 11 and 14, immunostained with antibody specific to MBP. Arrows point to regions of lymphocyte infiltration. Scale bars, 100 μm. f, Electron microscopy images showing myelin around axons in normal mice, vehicle-treated mice and mice in remission. Scale bars as indicated. g, Analysis of electron microscopy images indicating distribution of axonal diameters measured for 4 groups. h, Analysis of electron microscopy images indicating distribution of g-ratios of axons for 4 groups. i, Scatterplot of g-ratios in relation to spinal cord axonal diameters (n = 1,000, ***P < 0.001, one-way ANOVA, exponential trend line). j, Quantification of the number of axons associated with oligodendrocytes (n = 25, mean and s.e.m., **P < 0.01, t-test). Oligodendrocytes were identified visually by their cytoplasmic processes wrapping around axons.

Extended Data Figure 5 Benztropine has no effect on in vitro and in vivo immunological responses in EAE mice.

a, Benztropine and various muscarinic antagonists have no effect on in vitro T-cell proliferation measured using carboxyfluorescein succinimidyl ester (CFSE) labelling, whereas mycophenolate and FTY720 suppress T-cell proliferation as determined by the percentage of CD4+ T-cell-gated populations positive for the given marker. b, c, Various muscarinic antagonists have no effect on T-cell activation as measured by CD4+CD25+, CD4+CD69+, CD8+CD25+ and CD8+CD69+ cell populations. FTY720 and mycophenolate serve as positive controls for suppression of T-cell activation. d, Representative flow cytometry scatter plots show similar numbers of CD4+, CD8+, and CD44Hi cells in spleens isolated from vehicle- and benztropine-treated mice. e, f, Total splenocytes isolated from benztropine (10 mg per kg) or vehicle treated (14 days in the prophylactic mode) naive SJL/J (e) or EAE (f) mice analysed for various populations of immune cells and cytokine secretion. Benztropine treatment had no effect on the numbers of total splenocytes, CD4+ T cells, CD8+ T cells, CD4+CD44Hi T cells and CD8+CD44Hi T cells. Benztropine treatment showed a minor, but significant decrease in the number of B cells (n = 5, mean and s.e.m., *P < 0.05, t-test). Benztropine had no effect on cytokine production from CD4+ T cells expressing IL-2, IL-10, TNF-α or IFN-γ. (n = 5, mean and s.e.m.). g, Benztropine showed no effect on keyhole limpet hemocyanin protein conjugated to 2,4,6-trinitrophenyl hapten (TNP-KLH)-induced T-cell-dependent B-cell response. Mice were injected with TNP-KLH in adjuvant and treated with vehicle or benztropine (10 mg per kg) daily. Serum was isolated at various time points and anti-TNP-IgG levels were measured by ELISA. (3 replicate ELISAs, n = 5 mice per group, mean and s.e.m.).

Extended Data Figure 6 Benztropine does not affect derivation and in vitro polarization of macrophages from bone marrow derived monocytes.

a, Flow cytometry analysis of bone marrow derived monocytes treated in vitro with either DMSO (<0.1% v/v) or benztropine (5 µM) for 24 h followed by 24 h treatment with LPS (100 ng ml−1) plus IFNγ (20 ng ml−1) for the expression of M1 markers: CD86, MHC-II and CD80, or 24 h treatment with IL-4 plus IL-13 (20 ng ml−1 each) for the expression of M2 marker CD206. b, M1/M2 polarized macrophages re-stimulated using either LPS (100 ng ml−1) plus IFNγ (20 ng ml−1) (M1) or IL-4 plus IL-13 (20 ng ml−1 each) (M2) for 16 h in the presence of either benztropine (5 μM) or DMSO and analysed for the expression of M1 (CD80) or M2 (CD206) markers by flow cytometry. c, d, Treatment with LPS (100 ng ml−1) plus IFNγ (20 ng ml−1) induced the expression of the prototypical M1 cytokine TNF-α as detected by intracellular flow cytometry (c) and ELISA (d) with no significant differences between DMSO or benztropine (5 µM) treated cells (data representative of 2 replicate experiments).

Extended Data Figure 7 Benztropine does not affect in vivo polarization of macrophages in the spleen or spinal cord.

EAE mice were treated with benztropine (10 mg per kg) or vehicle for 14 days in the prophylactic mode. a, Mean clinical EAE scores for mice treated with vehicle or benztropine (n = 6, mean and s.e.m. for spleens and spinal cords, n = 12 for isolated spinal leukocytes analysis). be, Spleens and spinal leukocytes were isolated from the mice as described in Methods. Total RNA was isolated, reverse transcribed and gene expression was measured by qRT–PCR. Expression for each marker was normalized to the average gene expression of the vehicle group. No significant differences were observed in the expression of markers of macrophage polarization in the spleen (b), whole spinal cords (c) and leukocytes (d, e) isolated from spinal cords (n = 6 mice per group for spleens and spinal cords, n = 12 mice per group (n = 6 for qRT–PCR) for spinal leukocytes analysis. Error bars represent s.e.m.).

Extended Data Figure 8 Benztropine does not affect clinical severity in an adoptive transfer model of EAE.

a, b, Incidence of adoptive transfer of EAE (a) and mean clinical EAE scores (b) in mice injected with splenocytes isolated from benztropine- or vehicle-treated donor groups. T cells obtained from either benztropine- (BT, 10 mg per kg) or vehicle-treated donor EAE mice and further expanded in the presence or absence of benztropine (5 μM) were able to adoptively transfer EAE to naive recipient mice. Benztropine-treated recipient mice showed little to no clinical symptoms of EAE compared to vehicle-treated recipient mice, whether injected with benztropine- or vehicle-treated donor splenocytes (n = 6 mice, mean and s.e.m., *P < 0.05, t-test). c, Schematic for the adoptive transfer EAE model. d, Table showing various groups and treatments. e, ELISA for anti-PLP IgG shows equivalent PLP response in donor mice treated with either vehicle or benztropine (n = 30, mean and s.e.m).

Extended Data Figure 9 Quantification of myelin staining in the cuprizone model.

a, Luxol fast blue (LFB) and H&E staining was performed on sections from the corpus callosum region of brains isolated from mice treated either with benztropine (10 mg per kg) or vehicle after 7 weeks of exposure to cuprizone. b, Images were converted to a 256 shade grey scale. c, The 256 shades of grey were divided into 5 bins of 50 shades each (1–50, 51–100, 105–150, 151–200 and 201–256). Number of objects in the corpus callosum region in each bin were counted using Image-Pro plus. d, Representative images of Image-Pro rendering of the quantification of objects in each bin. e, Quantification of Luxol fast blue staining on week 2 shows an increase in the darker pixels (1–50 and 51–100) with benztropine treatment along with corresponding reduction in the number of lighter pixels (151–200). Six images per mouse were analysed and four mice per group were used at each time point (mean and s.d., *P < 0.05, t-test). Scale bars, 200 μm.

Extended Data Figure 10 Effect of the addition of benztropine to interferon-β and FTY720 treatments.

a, b, EAE severity scores for mice treated with various doses of FTY720 (a) or interferon-β (b). c, Mice treated therapeutically with FTY720 (1 mg per kg) in combination with a sub-optimal dose of benztropine (BT, 2.5 mg per kg) show significantly decreased clinical severity compared to FTY720 (1 mg per kg) or benztropine (2.5 mg per kg) alone. d, EAE mice treated with interferon-β (IFN 10,000 U per mouse) in combination with benztropine (2.5 mg per kg) show significantly decreased clinical severity compared to interferon-β (IFN; 10,000 U per mouse) or benztropine (2.5 mg per kg) alone. e, EAE mice treated with a tenfold lower dose of FTY720 (0.1 mg per kg) in combination with benztropine (2.5 mg per kg). f, EAE mice treated with a tenfold lower dose of FTY720 (0.1 mg per kg) in combination with benztropine (2.5 mg per kg) show clinical severity comparable to optimal dose of FTY720 (1 mg per kg) (n = 8 mice per group, mean and s.e.m., *P < 0.05; t-test). g, i, Spinal cord sections from EAE mice treated with the indicated drug(s) for 14 days in the prophylactic mode and immunostained for CD45 (immune cells) and GSTπ (oligodendrocytes) showing infiltration (g) and oligodendrocytes (i). h, j, Quantification of the number of CD45+ (h) and GSTπ+ (j) cells showing a decrease in infiltrating cells with FTY720 treatment and an increase in oligodendrocytes numbers with benztropine treatment and synergy between benztropine (2.5 mg per kg) and FTY720 (0.1 mg per kg) (n = 5, mean and s.e.m., ns, not significant). Scale bars, 100 μm. k, Mean clinical EAE scores for mice at the time of spinal cord isolation (n = 8, mean and s.e.m).

Supplementary information

Supplementary Tables

This file contains Supplementary Tables 1-5. (PDF 321 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Deshmukh, V., Tardif, V., Lyssiotis, C. et al. A regenerative approach to the treatment of multiple sclerosis. Nature 502, 327–332 (2013). https://doi.org/10.1038/nature12647

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature12647

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research