The Src/c-Abl pathway is a potential therapeutic target in amyotrophic lateral sclerosis
A stepping stone to ALS drug discovery
ALS is a heterogeneous motor neuron disease for which there is no treatment and for which a common therapeutic target has yet to be identified. In a new study, Imamura et al. developed a drug screen using motor neurons generated from ALS patient induced pluripotent stem cells (iPSCs). They screened existing drugs and showed that inhibitors of Src/c-Abl kinases promoted autophagy and rescued ALS motor neurons from degeneration. One of the drugs was effective for promoting survival of motor neurons derived from ALS patients with different genetic mutations. The Src/c-Abl pathway may be a potential therapeutic target for developing new drugs to treat ALS.
Abstract
Amyotrophic lateral sclerosis (ALS), a fatal disease causing progressive loss of motor neurons, still has no effective treatment. We developed a phenotypic screen to repurpose existing drugs using ALS motor neuron survival as readout. Motor neurons were generated from induced pluripotent stem cells (iPSCs) derived from an ALS patient with a mutation in superoxide dismutase 1 (SOD1). Results of the screen showed that more than half of the hits targeted the Src/c-Abl signaling pathway. Src/c-Abl inhibitors increased survival of ALS iPSC-derived motor neurons in vitro. Knockdown of Src or c-Abl with small interfering RNAs (siRNAs) also rescued ALS motor neuron degeneration. One of the hits, bosutinib, boosted autophagy, reduced the amount of misfolded mutant SOD1 protein, and attenuated altered expression of mitochondrial genes. Bosutinib also increased survival in vitro of ALS iPSC-derived motor neurons from patients with sporadic ALS or other forms of familial ALS caused by mutations in TAR DNA binding protein (TDP-43) or repeat expansions in C9orf72. Furthermore, bosutinib treatment modestly extended survival of a mouse model of ALS with an SOD1 mutation, suggesting that Src/c-Abl may be a potentially useful target for developing new drugs to treat ALS.
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Supplementary Material
Summary
Materials and methods
Fig. S1. Generation of iPSCs from control and ALS patients.
Fig. S2. Characterization of motor neurons and genetic correction of mutant SOD1 iPSCs.
Fig. S3. Investigation of the effects of Src/c-Abl inhibitors.
Fig. S4. mRNA expression changes after bosutinib treatment by single-cell analysis.
Fig. S5. Decrease in misfolded proteins after bosutinib treatment.
Fig. S6. Analysis of postmortem ALS spinal cord tissue.
Table S1. List of iPSC clones.
Table S2. Sequence variations in exon regions for sporadic ALS.
Table S3. List of hit compounds.
Table S4. Genes highly expressed in mutant SOD1 ALS motor neurons identified by single-cell RNA-seq.
Table S5. Genes highly expressed in control motor neurons identified by single-cell RNA-seq.
Table S6. List of postmortem spinal cord tissue for immunohistochemistry.
Table S7. List of postmortem spinal cord tissue for ELISA.
Table S8. Primer list for editing of SOD1 gene.
Table S9. Primer list for quantitative PCR.
Resources
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Science Translational Medicine
Volume 9 | Issue 391
May 2017
May 2017
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Copyright © 2017, American Association for the Advancement of Science.
Submission history
Received: 15 April 2015
Accepted: 13 December 2016
Acknowledgments
We thank all of our co-workers and collaborators including T. Enami, R. Shibukawa, M. Funayama, M. Kawada, K. Goto, H. Houlden, E. Preza, and C. Okada for their technical support. We acknowledge P. Karagiannis for critical reading of the paper and N. Endo and R. Taniguchi for their administrative support. Funding: This work was funded in part by a grant from the iPS Cell Research Fund (S.Y.); the Program for Intractable Diseases Research utilizing Disease-specific iPS cells from the Japan Agency for Medical Research and Development (AMED) (H. Inoue); the Research Center Network for Realization of Regenerative Medicine from AMED (A.H., S.Y., and H. Inoue); Research Project for Practical Applications of Regenerative Medicine from AMED (A.O., T.E., and H. Inoue); Parkinson’s UK Senior Fellowship (F-0902) (T. Kunath); grant-in-aid for scientific research from the Japan Society for the Promotion of Science (15H04270; H. Ito, 15H05581; A.H.); and the Daiichi Sankyo Foundation of Life Science (H. Inoue). Author contributions: H. Inoue conceived the project; K.I. and H. Inoue designed the experiments; K.I., K.T., T.Y., T. Kondo, and S. Kitaoka performed cell culture, molecular experiments, and compound screen; A.W. performed single-cell analysis; K.I. and A. Tanaka performed animal experiments; S. Kaneko, T.A., and H. Ito performed human-sample analysis; N.O., M.H., and H.A. performed resequencing; K.I., A.W., T.Y., D.W., and H. Inoue analyzed the data; K.W., A.H., A.O., T. Kunath., S.W., T.E., T.F., H.N., K.H., H. Ichijo, J-.P.J., and S. Kaneko contributed reagents, materials, and analysis tools; Y.I., M.M., H.T., A. Tamaoka, H.F., K.M., K.O., and R.K. recruited patients; D.W., R.T., and S.Y. provided critical reading and scientific discussions; K.I. and H. Inoue wrote the paper. Competing interests: S.Y. is an unpaid scientific advisor to iPS Academia Japan. Kyoto University has filed patents related to this manuscript: PCT application PCT/JP2014/058142, entitled “Pluripotent stem cells for neuronal differentiation” with K.I. and H. Inoue as coinventors; and PCT application PCT/JP2016/050883, entitled “Agent for preventing and/or treating Amyotrophic lateral sclerosis” with K.I. and H. Inoue as coinventors. All other authors declare that they have no competing interests.
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