A screen for regulators of survival of motor neuron protein levels

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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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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 Hb9CFP was a gift from K. Eggan, Harvard University.

Author information

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.

Correspondence to Lee L Rubin.

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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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