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The Src/c-Abl pathway is a potential therapeutic target in amyotrophic lateral sclerosis

Science Translational Medicine
24 May 2017
Vol 9, Issue 391

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.


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


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.
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Published In

Science Translational Medicine
Volume 9 | Issue 391
May 2017

Submission history

Received: 15 April 2015
Accepted: 13 December 2016


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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.



Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan.
Department of Clinical Neuroscience, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima 770-8503, Japan.
Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan.
Kayoko Tsukita
Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan.
Knut Woltjen
Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan.
Hakubi Center for Advanced Research, Kyoto University, Kyoto 606-8501, Japan.
Takuya Yamamoto
Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan.
Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto 606-8507, Japan.
Akitsu Hotta
Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan.
Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto 606-8507, Japan.
Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, Saitama 332-0012, Japan.
Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan.
Shiho Kitaoka
Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan.
Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan.
Akito Tanaka
Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan.
Dai Watanabe
Department of Biological Sciences, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
Division of Neurology, Department of Internal Medicine, Jichi Medical University, Tochigi 329-0498, Japan.
Hiroshi Takuma
Department of Neurology, Faculty of Medicine, University of Tsukuba, Tsukuba 305-8575, Japan.
Akira Tamaoka
Department of Neurology, Faculty of Medicine, University of Tsukuba, Tsukuba 305-8575, Japan.
MRC Centre for Regenerative Medicine, School of Biological Sciences, University of Edinburgh, 5 Little France Drive, Edinburgh EH16 4UU, UK.
Selina Wray
Department of Molecular Neuroscience, University College London Institute of Neurology, Queen Square, London WC1N 3BG, UK.
Department of Neurology, Kochi Medical School, Kochi University, Kochi 783-8505, Japan.
Takumi Era
Department of Cell Modulation, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 860-0811, Japan.
Kouki Makioka
Department of Neurology, Gunma University Graduate School of Medicine, Maebashi 371-8511, Japan.
Koichi Okamoto
Geriatrics Research Institute and Hospital, Maebashi 371-0847, Japan.
Takao Fujisawa
Cell Signaling, Graduate School of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
Department of Medical Sciences, University of Miyazaki, Miyazaki 889-1601, Japan.
Cell Signaling, Graduate School of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
Hidenori Ichijo
Cell Signaling, Graduate School of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
Jean-Pierre Julien
Department of Psychiatry and Neurosciences, Research Centre of Mental Health Institute of Quebec (IUSMQ), Laval University, Québec, Canada.
Nanako Obata
Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan.
Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan.
Haruhiko Akiyama
Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan.
Satoshi Kaneko
Department of Neurology, Kansai Medical University, Hirakata 573-1191, Japan.
Takashi Ayaki
Department of Neurology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan.
Department of Neurology, Wakayama Medical University, Kimiidera, Wakayama 641-8509, Japan.
Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto 606-8507, Japan.
Department of Neurology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan.
Shinya Yamanaka
Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan.
Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA.
Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan.


Corresponding author. Email: [email protected]

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