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A screen for regulators of survival of motor neuron protein levels

Nature Chemical Biology volume 7, pages 544–552 (2011)Cite this article

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Abstract

The motor neuron disease spinal muscular atrophy (SMA) results from mutations that lead to low levels of the ubiquitously expressed protein survival of motor neuron (SMN). An ever-increasing collection of data suggests that therapeutics that elevate SMN may be effective in treating SMA. We executed an image-based screen of annotated chemical libraries and discovered several classes of compounds that were able to increase cellular SMN. Among the most important was the RTK–PI3K–AKT–GSK-3 signaling cascade. Chemical inhibitors of glycogen synthase kinase 3 (GSK-3) and short hairpin RNAs (shRNAs) directed against this target elevated SMN levels primarily by stabilizing the protein. It was particularly notable that GSK-3 chemical inhibitors were also effective in motor neurons, not only in elevating SMN levels, but also in blocking the death that was produced when SMN was acutely reduced by an SMN-specific shRNA. Thus, we have established a screen capable of detecting drug-like compounds that correct the main phenotypic change underlying SMA.

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Figure 1: Development of a high-content assay for SMN.
Figure 2: Identification of compounds that increase SMN in different intracellular compartments.
Figure 3: Compounds that elevate intracellular Na+ or Ca2+ increase SMN levels.
Figure 4: Growth factors increase SMN levels.
Figure 5: PDGF increases SMN through a signaling pathway involving PI3K activation and GSK-3 inhibition.
Figure 6: Inhibition of GSK-3 increases SMN.
Figure 7: GSK-3 inhibition increases SMN levels by decreasing the rate of SMN degradation.
Figure 8: GSK-3 inhibitors increase SMN levels in mouse ES cell–derived motor neurons and prolong survival after shRNA knockdown of SMN.

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References

  1. Lefebvre, S. et al. Identification and characterization of a spinal muscular atrophy-determining gene. Cell 80, 155–165 (1995).

    Article  CAS  PubMed  Google Scholar 

  2. Lefebvre, S. et al. Correlation between severity and SMN protein level in spinal muscular atrophy. Nat. Genet. 16, 265–269 (1997).

    Article  CAS  PubMed  Google Scholar 

  3. Lorson, C.L., Hahnen, E., Androphy, E.J. & Wirth, B. A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy. Proc. Natl. Acad. Sci. USA 96, 6307–6311 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Le, T.T. et al. SMNDelta7, the major product of the centromeric survival motor neuron (SMN2) gene, extends survival in mice with spinal muscular atrophy and associates with full-length SMN. Hum. Mol. Genet. 14, 845–857 (2005).

    Article  CAS  PubMed  Google Scholar 

  5. Patrizi, A.L. et al. SMN protein analysis in fibroblast, amniocyte and CVS cultures from spinal muscular atrophy patients and its relevance for diagnosis. Eur. J. Hum. Genet. 7, 301–309 (1999).

    Article  CAS  PubMed  Google Scholar 

  6. Nguyen thi, M. et al. A two-site ELISA can quantify upregulation of SMN protein by drugs for spinal muscular atrophy. Neurology 71, 1757–1763 (2008).

    Article  Google Scholar 

  7. Schrank, B. et al. Inactivation of the survival motor neuron gene, a candidate gene for human spinal muscular atrophy, leads to massive cell death in early mouse embryos. Proc. Natl. Acad. Sci. USA 94, 9920–9925 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Zhang, Z. et al. SMN deficiency causes tissue-specific perturbations in the repertoire of snRNAs and widespread defects in splicing. Cell 133, 585–600 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Liu, Q. & Dreyfuss, G. A novel nuclear structure containing the survival of motor neurons protein. EMBO J. 15, 3555–3565 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Pellizzoni, L., Kataoka, N., Charroux, B. & Dreyfuss, G. A novel function for SMN, the spinal muscular atrophy disease gene product, in pre-mRNA splicing. Cell 95, 615–624 (1998).

    Article  CAS  PubMed  Google Scholar 

  11. König, H., Matter, N., Bader, R., Thiele, W. & Muller, F. Splicing segregation: the minor spliceosome acts outside the nucleus and controls cell proliferation. Cell 131, 718–729 (2007).

    Article  PubMed  Google Scholar 

  12. Pessa, H.K. et al. Minor spliceosome components are predominantly localized in the nucleus. Proc. Natl. Acad. Sci. USA 105, 8655–8660 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Steitz, J.A. et al. Where in the cell is the minor spliceosome? Proc. Natl. Acad. Sci. USA 105, 8485–8486 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Rossoll, W. et al. Smn, the spinal muscular atrophy-determining gene product, modulates axon growth and localization of beta-actin mRNA in growth cones of motoneurons. J. Cell Biol. 163, 801–812 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Zhang, H. et al. Multiprotein complexes of the survival of motor neuron protein SMN with Gemins traffic to neuronal processes and growth cones of motor neurons. J. Neurosci. 26, 8622–8632 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. McWhorter, M.L., Monani, U.R., Burghes, A.H. & Beattie, C.E. Knockdown of the survival motor neuron (Smn) protein in zebrafish causes defects in motor axon outgrowth and pathfinding. J. Cell Biol. 162, 919–931 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Jablonka, S., Beck, M., Lechner, B.D., Mayer, C. & Sendtner, M. Defective Ca2+ channel clustering in axon terminals disturbs excitability in motoneurons in spinal muscular atrophy. J. Cell Biol. 179, 139–149 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Burghes, A.H. & Beattie, C.E. Spinal muscular atrophy: why do low levels of survival motor neuron protein make motor neurons sick? Nat. Rev. Neurosci. 10, 597–609 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Avila, A.M. et al. Trichostatin A increases SMN expression and survival in a mouse model of spinal muscular atrophy. J. Clin. Invest. 117, 659–671 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Jarecki, J. et al. Diverse small-molecule modulators of SMN expression found by high-throughput compound screening: early leads towards a therapeutic for spinal muscular atrophy. Hum. Mol. Genet. 14, 2003–2018 (2005).

    Article  CAS  PubMed  Google Scholar 

  21. Andreassi, C. et al. Aclarubicin treatment restores SMN levels to cells derived from type I spinal muscular atrophy patients. Hum. Mol. Genet. 10, 2841–2849 (2001).

    Article  CAS  PubMed  Google Scholar 

  22. Wan, L., Ottinger, E., Cho, S. & Dreyfuss, G. Inactivation of the SMN complex by oxidative stress. Mol. Cell 31, 244–254 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Mattis, V.B. et al. Novel aminoglycosides increase SMN levels in spinal muscular atrophy fibroblasts. Hum. Genet. 120, 589–601 (2006).

    Article  CAS  PubMed  Google Scholar 

  24. Chang, H.C. et al. Modeling spinal muscular atrophy in Drosophila. PLoS ONE 3, e3209 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Burnett, B.G. et al. Regulation of SMN protein stability. Mol. Cell. Biol. 29, 1107–1115 (2009).

    Article  CAS  PubMed  Google Scholar 

  26. Chen, S. et al. A small molecule that directs differentiation of human ESCs into the pancreatic lineage. Nat. Chem. Biol. 5, 258–265 (2009).

    Article  CAS  PubMed  Google Scholar 

  27. Ichida, J.K. et al. A small-molecule inhibitor of TGF-β signaling replaces sox2 in reprogramming by inducing nanog. Cell Stem Cell 5, 491–503 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Chang, H.C., Hung, W.C., Chuang, Y.J. & Jong, Y.J. Degradation of survival motor neuron (SMN) protein is mediated via the ubiquitin/proteasome pathway. Neurochem. Int. 45, 1107–1112 (2004).

    Article  CAS  PubMed  Google Scholar 

  29. Pullen, M.A., Brooks, D.P. & Edwards, R.M. Characterization of the neutralizing activity of digoxin-specific Fab toward ouabain-like steroids. J. Pharmacol. Exp. Ther. 310, 319–325 (2004).

    Article  CAS  PubMed  Google Scholar 

  30. Dodson, A.W., Taylor, T.J., Knipe, D.M. & Coen, D.M. Inhibitors of the sodium potassium ATPase that impair herpes simplex virus replication identified via a chemical screening approach. Virology 366, 340–348 (2007).

    Article  CAS  PubMed  Google Scholar 

  31. Burt, J.M. & Langer, G.A. Ca++ distribution after Na+ pump inhibition in cultured neonatal rat myocardial cells. Circ. Res. 51, 543–550 (1982).

    Article  CAS  PubMed  Google Scholar 

  32. Wasserstrom, J.A. & Aistrup, G.L. Digitalis: new actions for an old drug. Am. J. Physiol. Heart Circ. Physiol. 289, H1781–H1793 (2005).

    Article  CAS  PubMed  Google Scholar 

  33. Cohen, P. & Goedert, M. GSK3 inhibitors: development and therapeutic potential. Nat. Rev. Drug Discov. 3, 479–487 (2004).

    Article  CAS  PubMed  Google Scholar 

  34. Fang, X. et al. Phosphorylation and inactivation of glycogen synthase kinase 3 by protein kinase A. Proc. Natl. Acad. Sci. USA 97, 11960–11965 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Kunick, C. et al. Structure-aided optimization of kinase inhibitors derived from alsterpaullone. ChemBioChem 6, 541–549 (2005).

    Article  CAS  PubMed  Google Scholar 

  36. Cheng, H., Woodgett, J., Maamari, M. & Force, T. Targeting GSK-3 family members in the heart: a very sharp double-edged sword. J. Mol. Cell Cardiol. published online, doi:10.1016/j.yjmcc.2010.11.020 (13 December 2010).

  37. Xu, C., Kim, N.G. & Gumbiner, B.M. Regulation of protein stability by GSK3 mediated phosphorylation. Cell Cycle 8, 4032–4039 (2009).

    Article  CAS  PubMed  Google Scholar 

  38. Pearson, R.B. & Kemp, B.E. Protein kinase phosphorylation site sequences and consensus specificity motifs: tabulations. Methods Enzymol. 200, 62–81 (1991).

    Article  CAS  PubMed  Google Scholar 

  39. Badorff, C., Seeger, F.H., Zeiher, A.M. & Dimmeler, S. Glycogen synthase kinase 3beta inhibits myocardin-dependent transcription and hypertrophy induction through site-specific phosphorylation. Circ. Res. 97, 645–654 (2005).

    Article  CAS  PubMed  Google Scholar 

  40. Cho, S. & Dreyfuss, G. A degron created by SMN2 exon 7 skipping is a principal contributor to spinal muscular atrophy severity. Genes Dev. 24, 438–442 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Wichterle, H., Lieberam, I., Porter, J.A. & Jessell, T.M. Directed differentiation of embryonic stem cells into motor neurons. Cell 110, 385–397 (2002).

    Article  CAS  PubMed  Google Scholar 

  42. Shafey, D., Cote, P.D. & Kothary, R. Hypomorphic Smn knockdown C2C12 myoblasts reveal intrinsic defects in myoblast fusion and myotube morphology. Exp. Cell Res. 311, 49–61 (2005).

    Article  CAS  PubMed  Google Scholar 

  43. Shafey, D., MacKenzie, A.E. & Kothary, R. Neurodevelopmental abnormalities in neurosphere-derived neural stem cells from SMN-depleted mice. J. Neurosci. Res. 86, 2839–2847 (2008).

    Article  CAS  PubMed  Google Scholar 

  44. Pearson, G. et al. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr. Rev. 22, 153–183 (2001).

    CAS  PubMed  Google Scholar 

  45. Farooq, F., Balabanian, S., Liu, X., Holcik, M. & MacKenzie, A. p38 mitogen-activated protein kinase stabilizes SMN mRNA through RNA binding protein HuR. Hum. Mol. Genet. 18, 4035–4045 (2009).

    Article  CAS  PubMed  Google Scholar 

  46. Yeh, H.J. et al. Developmental expression of the platelet-derived growth factor alpha-receptor gene in mammalian central nervous system. Proc. Natl. Acad. Sci. USA 90, 1952–1956 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Rak, K. et al. Valproic acid blocks excitability in SMA type I mouse motor neurons. Neurobiol. Dis. 36, 477–487 (2009).

    Article  CAS  PubMed  Google Scholar 

  48. Bain, J. et al. The selectivity of protein kinase inhibitors: a further update. Biochem. J. 408, 297–315 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Lahusen, T., De Siervi, A., Kunick, C. & Senderowicz, A.M. Alsterpaullone, a novel cyclin-dependent kinase inhibitor, induces apoptosis by activation of caspase-9 due to perturbation in mitochondrial membrane potential. Mol. Carcinog. 36, 183–194 (2003).

    Article  CAS  PubMed  Google Scholar 

  50. Takadera, T., Ohtsuka, M. & Aoki, H. Chelation of extracellular calcium-induced cell death was prevented by glycogen synthase kinase-3 inhibitors in PC12 cells. Cell. Mol. Neurobiol. 30, 193–198 (2009).

    Article  PubMed  Google Scholar 

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Acknowledgements

The authors would like to thank K. Kotkow, Y. M. Yang and K. Chen for helpful comments, K. Krumholz for technical help and J. LaLonde for editorial assistance. Additionally, we would like to thank R. Sivasankaran and C. Song at Novartis for sharing the conditions of the splicing assay design. The work was supported by the Spinal Muscular Atrophy Foundation, by the Harvard Stem Cell Institute and by a P01 grant (P01NS066888-01A1) from the US National Institute of Neurological Disorders and Stroke (NINDS). Mouse Hb9∷CFP was a gift from K. Eggan, Harvard University.

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Author notes

  1. Amy D Sinor-Anderson, Wen-Ning Zhao & Patrick W Heiser

    Present address: Present addresses: Thermo Fisher Scientific, Pittsburgh, Pennsylvania, USA (A.D.S.-A.), Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts, USA (W.-N.Z.) and Ferring Pharmaceuticals, Parsippany, New Jersey, USA (P.W.H.).,

Authors and Affiliations

  1. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.,

    Nina R Makhortova, Monica Hayhurst, Antonio Cerqueira, Amy D Sinor-Anderson, Wen-Ning Zhao, Patrick W Heiser & Lee L Rubin

  2. Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, USA

    Nina R Makhortova, Monica Hayhurst, Antonio Cerqueira, Anthony C Arvanites, Lance S Davidow, Kelvin Lam, Hien D Ngo & Lee L Rubin

  3. Children's Hospital Boston, F.M. Kirby Neurobiology Center, Boston, Massachusetts, USA

    Zachary O Waldon & Judith A Steen

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Contributions

N.R.M., M.H., A.C., A.D.S.-A., W.-N.Z. and P.W.H. designed, carried out the experiments and analyzed results. H.D.N. carried out experiments. A.C.A., L.S.D. and K.L. analyzed screening results. Z.O.W. and J.A.S. performed MS and analyzed results. L.L.R. designed experiments and analyzed results. N.R.M., M.H. and L.L.R. wrote the manuscript.

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Correspondence to Lee L Rubin.

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Competing interests

L.L.R. is a founder of iPierian and a member of its Scientific Advisory Board.

Supplementary information

Supplementary Text and Figures

Supplementary Methods and Supplementary Results (PDF 2084 kb)

Supplementary Data 1

Chemical structures of primary screen hit compounds (XLSX 847 kb)

Supplementary Data 2

Chemical structures of additional compounds used in this study (XLSX 65 kb)

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Makhortova, N., Hayhurst, M., Cerqueira, A. et al. A screen for regulators of survival of motor neuron protein levels. Nat Chem Biol 7, 544–552 (2011). https://doi.org/10.1038/nchembio.595

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