Micropillar arrays as a high-throughput screening platform for therapeutics in multiple sclerosis

Abstract

Functional screening for compounds that promote remyelination represents a major hurdle in the development of rational therapeutics for multiple sclerosis. Screening for remyelination is problematic, as myelination requires the presence of axons. Standard methods do not resolve cell-autonomous effects and are not suited for high-throughput formats. Here we describe a binary indicant for myelination using micropillar arrays (BIMA). Engineered with conical dimensions, micropillars permit resolution of the extent and length of membrane wrapping from a single two-dimensional image. Confocal imaging acquired from the base to the tip of the pillars allows for detection of concentric wrapping observed as 'rings' of myelin. The platform is formatted in 96-well plates, amenable to semiautomated random acquisition and automated detection and quantification. Upon screening 1,000 bioactive molecules, we identified a cluster of antimuscarinic compounds that enhance oligodendrocyte differentiation and remyelination. Our findings demonstrate a new high-throughput screening platform for potential regenerative therapeutics in multiple sclerosis.

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Figure 1: Conception and fabrication of micropillar arrays for modeling myelination.
Figure 2: BIMA.
Figure 3: High-throughput screening of bioactive compounds for differentiation and membrane wrapping identifies a cluster of antimuscarinic compounds.
Figure 4: Validation of clemastine and benzatropine with purified oligodendroglia cultured alone or with purified DRG neurons.
Figure 5: Clemastine enhances the kinetics of remyelination and promotes remyelination in mice after gliotoxic injury with lysolecithin.

References

  1. 1

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

    CAS  Article  Google Scholar 

  2. 2

    Keirstead, H.S. & Blakemore, W. Identification of post-mitotic oligodendrocytes incapable of remyelination within the demyelinated adult spinal cord. J. Neuropathol. Exp. Neurol. 56, 1191–1201 (1997).

    CAS  Article  Google Scholar 

  3. 3

    Keirstead, H.S., Levine, J.M. & Blakemore, W.F. Response of the oligodendrocyte progenitor cell population (defined by NG2 labelling) to demyelination of the adult spinal cord. Glia 22, 161–170 (1998).

    CAS  Article  Google Scholar 

  4. 4

    Scolding, N. et al. Oligodendrocyte progenitors are present in the normal adult human CNS and in the lesions of multiple sclerosis. Brain 121, 2221–2228 (1998).

    Article  Google Scholar 

  5. 5

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

    CAS  Article  Google Scholar 

  6. 6

    Chang, A., Nishiyama, A., Peterson, J., Prineas, J. & Trapp, B.D. NG2-positive oligodendrocyte progenitor cells in adult human brain and multiple sclerosis lesions. J. Neurosci. 20, 6404–6412 (2000).

    CAS  Article  Google Scholar 

  7. 7

    Shi, J., Marinovich, A. & Barres, B.A. Purification and characterization of adult oligodendrocyte precursor cells from the rat optic nerve. J. Neurosci. 18, 4627–4636 (1998).

    CAS  Article  Google Scholar 

  8. 8

    Tang, D.G., Tokumoto, Y.M. & Raff, M.C. Long-term culture of purified postnatal oligodendrocyte precursor cells. Evidence for an intrinsic maturation program that plays out over months. J. Cell Biol. 148, 971–984 (2000).

    CAS  Article  Google Scholar 

  9. 9

    Zawadzka, M. et al. CNS-resident glial progenitor/stem cells produce Schwann cells as well as oligodendrocytes during repair of CNS demyelination. Cell Stem Cell 6, 578–590 (2010).

    CAS  Article  Google Scholar 

  10. 10

    Wolswijk, G. & Noble, M. Cooperation between PDGF and FGF converts slowly dividing O-2Aadult progenitor cells to rapidly dividing cells with characteristics of O-2Aperinatal progenitor cells. J. Cell Biol. 118, 889–900 (1992).

    CAS  Article  Google Scholar 

  11. 11

    Woodruff, R.H., Fruttiger, M., Richardson, W.D. & Franklin, R.J.M. Platelet-derived growth factor regulates oligodendrocyte progenitor numbers in adult CNS and their response following CNS demyelination. Mol. Cell. Neurosci. 25, 252–262 (2004).

    CAS  Article  Google Scholar 

  12. 12

    Colello, R.J. & Pott, U. Signals that initiate myelination in the developing mammalian nervous system. Mol. Neurobiol. 15, 83–100 (1997).

    CAS  Article  Google Scholar 

  13. 13

    Friede, R.L. Control of myelin formation by axon caliber (with a model of the control mechanism). J. Comp. Neurol. 144, 233–252 (1972).

    CAS  Article  Google Scholar 

  14. 14

    Voyvodic, J.T. Target size regulates calibre and myelination of sympathetic axons. Nature 342, 430–433 (1989).

    CAS  Article  Google Scholar 

  15. 15

    Michailov, G.V. et al. Axonal neuregulin-1 regulates myelin sheath thickness. Science 304, 700–703 (2004).

    CAS  Article  Google Scholar 

  16. 16

    Taveggia, C. et al. Neuregulin-1 type III determines the ensheathment fate of axons. Neuron 47, 681–694 (2005).

    CAS  Article  Google Scholar 

  17. 17

    Rosenberg, S.S., Kelland, E.E., Tokar, E., De la Torre, A.R. & Chan, J.R. The geometric and spatial constraints of the microenvironment induce oligodendrocyte differentiation. Proc. Natl. Acad. Sci. USA 105, 14662–14667 (2008).

    CAS  Article  Google Scholar 

  18. 18

    Lee, S. et al. A culture system to study oligodendrocyte myelination processes using engineered nanofibers. Nat. Methods 9, 917–922 (2012).

    CAS  Article  Google Scholar 

  19. 19

    Lee, S., Chong, S.Y.C., Tuck, S.J., Corey, J.M. & Chan, J.R. A rapid and reproducible assay for modeling myelination by oligodendrocytes using engineered nanofibers. Nat. Protoc. 8, 771–782 (2013).

    Article  Google Scholar 

  20. 20

    Fancy, S.P.J. et al. Axin2 as regulatory and therapeutic target in newborn brain injury and remyelination. Nat. Neurosci. 14, 1009–1016 (2011).

    CAS  Article  Google Scholar 

  21. 21

    Chan, J.R. et al. NGF controls axonal receptivity to myelination by Schwann cells or oligodendrocytes. Neuron 43, 183–191 (2004).

    CAS  Article  Google Scholar 

  22. 22

    Fancy, S.P.J. et al. Dysregulation of the Wnt pathway inhibits timely myelination and remyelination in the mammalian CNS. Genes Dev. 23, 1571–1585 (2009).

    CAS  Article  Google Scholar 

  23. 23

    Chong, S.Y.C. et al. Neurite outgrowth inhibitor Nogo-A establishes spatial segregation and extent of oligodendrocyte myelination. Proc. Natl. Acad. Sci. USA 109, 1299–1304 (2012).

    CAS  Article  Google Scholar 

  24. 24

    Etxeberria, A., Mangin, J.M., Aguirre, A. & Gallo, V. Adult-born SVZ progenitors receive transient synapses during remyelination in corpus callosum. Nat. Neurosci. 13, 287–289 (2010).

    CAS  Article  Google Scholar 

  25. 25

    Buckley, C.E., Goldsmith, P. & Franklin, R.J.M. Zebrafish myelination: a transparent model for remyelination? Dis. Model. Mech. 1, 221–228 (2008).

    CAS  Article  Google Scholar 

  26. 26

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

    CAS  Article  Google Scholar 

  27. 27

    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  Article  Google Scholar 

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Acknowledgements

We thank the Multiple Sclerosis Research Group at the University of California, San Francisco (UCSF) for support, advice and insightful discussions. This work was supported by the US National Multiple Sclerosis Society Harry Weaver Neuroscience Scholar Award (JF 2142-A2/T), UCSF CTSI Catalyst Award for Innovation, gifts from friends of the Multiple Sclerosis Research Group at UCSF and the Joint Research Fund for Overseas Chinese Young Scholars (NSCF, 31228011). The rabbit monoclonal antibody to PDGFRα was a gift from W.B. Stallcup (Sanford Burnham Medical Research Institute).

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F.M., S.P.J.F., Y.-A.A.S., J.N., C.Z., E.M., S.L. and J.R.C. performed experiments. F.M., S.P.J.F., B.P., L.X., R.J.M.F., S.L.H. and J.R.C. provided reagents. F.M., S.P.J.F., S.R.M., S.A.R., A.E., R.J.M.F., A.G., S.L.H. and J.R.C. provided intellectual contributions. F.M., S.P.J.F. and J.R.C. analyzed the data and wrote the paper.

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Correspondence to Jonah R Chan.

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The authors declare no competing financial interests.

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Mei, F., Fancy, S., Shen, YA. et al. Micropillar arrays as a high-throughput screening platform for therapeutics in multiple sclerosis. Nat Med 20, 954–960 (2014). https://doi.org/10.1038/nm.3618

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