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Modeling sporadic ALS in iPSC-derived motor neurons identifies a potential therapeutic agent

Nature Medicinevolume 24pages15791589 (2018) | Download Citation


Amyotrophic lateral sclerosis (ALS) is a heterogeneous motor neuron disease for which no effective treatment is available, despite decades of research into SOD1-mutant familial ALS (FALS). The majority of ALS patients have no familial history, making the modeling of sporadic ALS (SALS) essential to the development of ALS therapeutics. However, as mutations underlying ALS pathogenesis have not yet been identified, it remains difficult to establish useful models of SALS. Using induced pluripotent stem cell (iPSC) technology to generate stem and differentiated cells retaining the patients’ full genetic information, we have established a large number of in vitro cellular models of SALS. These models showed phenotypic differences in their pattern of neuronal degeneration, types of abnormal protein aggregates, cell death mechanisms, and onset and progression of these phenotypes in vitro among cases. We therefore developed a system for case clustering capable of subdividing these heterogeneous SALS models by their in vitro characteristics. We further evaluated multiple-phenotype rescue of these subclassified SALS models using agents selected from non-SOD1 FALS models, and identified ropinirole as a potential therapeutic candidate. Integration of the datasets acquired in this study permitted the visualization of molecular pathologies shared across a wide range of SALS models.

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  1. 1.

    Zinman, L. & Cudkowicz, M. Emerging targets and treatments in amyotrophic lateral sclerosis. Lancet Neurol. 10, 481–490 (2011).

  2. 2.

    Rosen, D. R. et al. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 362, 59–62 (1993).

  3. 3.

    Hadano, S. et al. A gene encoding a putative GTPase regulator is mutated in familial amyotrophic lateral sclerosis 2. Nat. Genet. 29, 166–173 (2001).

  4. 4.

    Arai, T. et al. TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Biochem. Biophys. Res. Commun. 351, 602–611 (2006).

  5. 5.

    Neumann, M. et al. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314, 130–133 (2006).

  6. 6.

    Kwiatkowski, T. J.Jr et al. Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis. Science 323, 1205–1208 (2009).

  7. 7.

    Vance, C. et al. Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science 323, 1208–1211 (2009).

  8. 8.

    Maruyama, H. et al. Mutations of optineurin in amyotrophic lateral sclerosis. Nature 465, 223–226 (2010).

  9. 9.

    Da Cruz, S. & Cleveland, D. W. Understanding the role of TDP-43 and FUS/TLS in ALS and beyond. Curr. Opin. Neurobiol. 21, 904–919 (2011).

  10. 10.

    Liu, H. N. et al. Lack of evidence of monomer/misfolded superoxide dismutase-1 in sporadic amyotrophic lateral sclerosis. Ann. Neurol. 66, 75–80 (2009).

  11. 11.

    Kabashi, E. et al. FUS and TARDBP but not SOD1 interact in genetic models of amyotrophic lateral sclerosis. PLoS Genet. 7, e1002214 (2011).

  12. 12.

    Da Cruz, S. et al. Misfolded SOD1 is not a primary component of sporadic ALS. Acta Neuropathol. 134, 97–111 (2017).

  13. 13.

    Philips, T. & Rothstein, J. D. Rodent models of amyotrophic lateral sclerosis. Curr. Protoc. Pharmacol. 69, 5.67.61–21 (2015).

  14. 14.

    Mizusawa, H. et al. Focal accumulation of phosphorylated neurofilaments within anterior horn cell in familial amyotrophic lateral sclerosis. Acta Neuropathol. 79, 37–43 (1989).

  15. 15.

    Okamoto, K., Hirai, S., Ishiguro, K., Kawarabayashi, T. & Takatama, M. Light and electron microscopic and immunohistochemical observations of the Onuf’s nucleus of amyotrophic lateral sclerosis. Acta Neuropathol. 81, 610–614 (1991).

  16. 16.

    Renton, A. E., Chio, A. & Traynor, B. J. State of play in amyotrophic lateral sclerosis genetics. Nat. Neurosci. 17, 17–23 (2014).

  17. 17.

    Iida, A. et al. Replication analysis of SNPs on 9p21.2 and 19p13.3 with amyotrophic lateral sclerosis in East Asians. Neurobiol. Aging 32, 757.e713–754 (2011).

  18. 18.

    Manolio, T. A. et al. Finding the missing heritability of complex diseases. Nature 461, 747–753 (2009).

  19. 19.

    Mattis, V. B. & Svendsen, C. N. Induced pluripotent stem cells: a new revolution for clinical neurology?. Lancet Neurol. 10, 383–394 (2011).

  20. 20.

    Okano, H. & Yamanaka, S. iPS cell technologies: significance and applications to CNS regeneration and disease. Mol. Brain 7, 22 (2014).

  21. 21.

    Kim, K. et al. Epigenetic memory in induced pluripotent stem cells. Nature 467, 285–290 (2010).

  22. 22.

    Okuno, H. et al. Changeability of the fully methylated status of the 15q11.2 region in induced pluripotent stem cells derived from a patient with Prader–Willi syndrome. Congenit. Anom. (Kyoto) 57, 96–103 (2017).

  23. 23.

    Ichiyanagi, N. et al. Establishment of in vitro FUS-associated familial amyotrophic lateral sclerosis model using human induced pluripotent stem cells. Stem Cell Reports 6, 496–510 (2016).

  24. 24.

    Fujimori, K. et al. Escape from pluripotency via inhibition of TGF-β/BMP and activation of wnt signaling accelerates differentiation and aging in hPSC progeny cells. Stem Cell Reports 9, 1675–1691 (2017).

  25. 25.

    Fujimori, K. et al. Modeling neurological diseases with induced pluripotent cells reprogrammed from immortalized lymphoblastoid cell lines. Mol. Brain 9, 88 (2016).

  26. 26.

    Matsumoto, T. et al. Functional neurons generated from T cell-derived induced pluripotent stem cells for neurological disease modeling. Stem Cell Reports 6, 422–435 (2016).

  27. 27.

    Guo, W. et al. An ALS-associated mutation affecting TDP-43 enhances protein aggregation, fibril formation and neurotoxicity. Nat. Struct. Mol. Biol. 18, 822–830 (2011).

  28. 28.

    Kuzel, M. D. Ropinirole: a dopamine agonist for the treatment of Parkinson’s disease. Am. J. Health Syst. Pharm. 56, 217–224 (1999).

  29. 29.

    Abramova, N. A., Cassarino, D. S., Khan, S. M., Painter, T. W. & Bennett, J. PJr.. Inhibition by R( + ) or S(−) pramipexole of caspase activation and cell death induced by methylpyridinium ion or beta amyloid peptide in SH-SY5Y neuroblastoma. J. Neurosci. Res. 67, 494–500 (2002).

  30. 30.

    Danzeisen, R. et al. Targeted antioxidative and neuroprotective properties of the dopamine agonist pramipexole and its nondopaminergic enantiomer SND919CL2x [(+)2-amino-4,5,6,7-tetrahydro-6-Lpropylamino-benzathiazole dihydrochloride]. J. Pharmacol. Exp. Ther. 316, 189–199 (2006).

  31. 31.

    Ferrari-Toninelli, G., Maccarinelli, G., Uberti, D., Buerger, E. & Memo, M. Mitochondria-targeted antioxidant effects of S(−) and R(+) pramipexole. BMC Pharmacol. 10, 2 (2010).

  32. 32.

    Gu, M. et al. Pramipexole protects against apoptotic cell death by non-dopaminergic mechanisms. J. Neurochem. 91, 1075–1081 (2004).

  33. 33.

    Sethy, V. H., Wu, H., Oostveen, J. A. & Hall, E. D. Neuroprotective effects of the dopamine agonists pramipexole and bromocriptine in 3-acetylpyridine-treated rats. Brain Res. 754, 181–186 (1997).

  34. 34.

    Cassarino, D. S., Fall, C. P., Smith, T. S. & Bennett, J. P.Jr.. Pramipexole reduces reactive oxygen species production in vivo and in vitro and inhibits the mitochondrial permeability transition produced by the parkinsonian neurotoxin methylpyridinium ion. J. Neurochem. 71, 295–301 (1998).

  35. 35.

    Wang, H. et al. R+ pramipexole as a mitochondrially focused neuroprotectant: initial early phase studies in ALS. Amyotroph. Lateral Scler. 9, 50––58 (2008).

  36. 36.

    Cheah, B. C. & Kiernan, M. C. Dexpramipexole, the R(+) enantiomer of pramipexole, for the potential treatment of amyotrophic lateral sclerosis. IDrugs 13, 911–920 (2010).

  37. 37.

    Corcia, P. & Gordon, P. H. Amyotrophic lateral sclerosis and the clinical potential of dexpramipexole. Ther. Clin. Risk Manag. 8, 359–366 (2012).

  38. 38.

    Cudkowicz, M. E. et al. Dexpramipexole versus placebo for patients with amyotrophic lateral sclerosis (EMPOWER): a randomised, double-blind, phase 3 trial. Lancet Neurol. 12, 1059–1067 (2013).

  39. 39.

    Iida, A. et al. A functional variant in ZNF512B is associated with susceptibility to amyotrophic lateral sclerosis in Japanese. Hum. Mol. Genet. 20, 3684–3692 (2011).

  40. 40.

    Nakamura, R. et al. Neck weakness is a potent prognostic factor in sporadic amyotrophic lateral sclerosis patients. J. Neurol. Neurosurg. Psychiatry 84, 1365–1371 (2013).

  41. 41.

    Watanabe, H. et al. A rapid functional decline type of amyotrophic lateral sclerosis is linked to low expression of TTN. J. Neurol. Neurosurg. Psychiatry 87, 851–858 (2016).

  42. 42.

    Yang, W. S. & Stockwell, B. R. Ferroptosis: death by lipid peroxidation. Trends Cell Biol. 26, 165–176 (2016).

  43. 43.

    Radak, Z., Zhao, Z., Goto, S. & Koltai, E. Age-associated neurodegeneration and oxidative damage to lipids, proteins and DNA. Mol. Aspects Med. 32, 305–315 (2011).

  44. 44.

    Reed, T. T. Lipid peroxidation and neurodegenerative disease. Free Radic. Biol. Med. 51, 1302–1319 (2011).

  45. 45.

    Alves, C. J. et al. Gene expression profiling for human iPS-derived motor neurons from sporadic ALS patients reveals a strong association between mitochondrial functions and neurodegeneration. Front Cell Neurosci. 9, 289 (2015).

  46. 46.

    Burkhardt, M. F. et al. A cellular model for sporadic ALS using patient-derived induced pluripotent stem cells. Mol. Cell Neurosci. 56, 355–364 (2013).

  47. 47.

    Imamura, K. et al. The Src/c-Abl pathway is a potential therapeutic target in amyotrophic lateral sclerosis. Sci. Transl. Med. 9, (2017).

  48. 48.

    Ravits, J. et al. Deciphering amyotrophic lateral sclerosis: what phenotype, neuropathology and genetics are telling us about pathogenesis. Amyotroph. Lateral Scler. Frontotemporal Degener. 14 (Suppl 1), 5–18 (2013).

  49. 49.

    Mackenzie, I. R. et al. Pathological TDP-43 distinguishes sporadic amyotrophic lateral sclerosis from amyotrophic lateral sclerosis with SOD1 mutations. Ann. Neurol. 61, 427–434 (2007).

  50. 50.

    Borasio, G. D. et al. Dopaminergic deficit in amyotrophic lateral sclerosis assessed with [I-123] IPT single photon emission computed tomography. J Neurol Neurosurg. Psychiatry 65, 263–265 (1998).

  51. 51.

    Cooper, R. L. & Neckameyer, W. S. Dopaminergic modulation of motor neuron activity and neuromuscular function in Drosophila melanogaster. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 122, 199–210 (1999).

  52. 52.

    Yuan, N. & Lee, D. Suppression of excitatory cholinergic synaptic transmission by Drosophila dopamine D1-like receptors. Eur. J. Neurosci. 26, 2417–2427 (2007).

  53. 53.

    Reimer, M. M. et al. Dopamine from the brain promotes spinal motor neuron generation during development and adult regeneration. Dev. Cell 25, 478–491 (2013).

  54. 54.

    Simpson, E. P., Henry, Y. K., Henkel, J. S., Smith, R. G. & Appel, S. H. Increased lipid peroxidation in sera of ALS patients: a potential biomarker of disease burden. Neurology 62, 1758–1765 (2004).

  55. 55.

    Chen, L., Hambright, W. S., Na, R. & Ran, Q. Ablation of the ferroptosis inhibitor glutathione peroxidase 4 in neurons results in rapid motor neuron degeneration and paralysis. J. Biol. Chem. 290, 28097–28106 (2015).

  56. 56.

    Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

  57. 57.

    Imaizumi, Y. et al. Mitochondrial dysfunction associated with increased oxidative stress and alpha-synuclein accumulation in PARK2 iPSC-derived neurons and postmortem brain tissue. Mol. Brain 5, 35 (2012).

  58. 58.

    Egawa, N. et al. Drug screening for ALS using patient-specific induced pluripotent stem cells. Sci. Transl. Med. 4, 145ra104 (2012).

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The authors would like to thank W. Akamatsu (Juntendo University), D. Sipp, S. Morimoto, and Y. Nishimoto (Keio University) for providing invaluable comments on the project and all the members of the H.O. laboratory for their encouragement and kind support. We would also like to thank H. Inoue, S. Yamanaka, and M. Nakagawa (Kyoto University) for donating hiPSC clones (A21412, A21428, A3411, A3416, and 201B7) and M. Ishikawa (Keio University) for constructing and providing the existing drug library. This work was supported by funding from the Research Project for Practical Applications of Regenerative Medicine from the Japan Agency for Medical Research and Development (AMED) (grant nos. 15bk0104027h0003, 16bk0104016h0004, and 17bk0104016h0005 to H.O.), the Research Center Network for Realization Research Centers/Projects of Regenerative Medicine (the Program for Intractable Disease Research Utilizing Disease-specific iPS Cells and the Acceleration Program for Intractable Diseases Research Utilizing Disease-specific iPS Cells) from AMED (grant nos. 15bm0609003h0004, 16bm0609003h0005, 17bm0609003h0006 and 17bm0804003h0001 to H.O.), the research grants for the sporadic ALS patients registry study (JaCALS) from AMED (grant no. 17lk0201057h0002 to G.S.), a research grant on “Development of therapy for sporadic ALS from omics analyses using a large-scale clinical database, genomic DNA and immortalized cell repository of Japanese ALS patients from AMED” (grant no. 18ek0109284s0302 to G.S. and H.O.), an IBC grant from the Japan ALS Association (to H.O.), the Translational Research Network Program from AMED (to H.O.), the Practical Research Project for Rare/Intractable Diseases from AMED (grant nos. 18ek0109284s0302 to G.S. and H.O. and 18ek0109329h0001 to H.O.), the grant-in-aid project Scientific Research on Innovation Area (Brain Protein Aging and Dementia Control) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to G.S.and H.O.), Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (grant no. JP16J06437 to K.F.), the Keio University Grant-in-Aid for the Encouragement of Young Medical Scientists (to K.F.), and the Keio University Doctorate Student Grant-in-Aid Program (to K.F.).

Author information


  1. Department of Physiology, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan

    • Koki Fujimori
    • , Mitsuru Ishikawa
    •  & Hideyuki Okano
  2. The Institute of Medical Sciences, Tokai University, Isehara, Kanagawa, Japan

    • Asako Otomo
    •  & Shinji Hadano
  3. Department of Molecular Life Sciences, Tokai University School of Medicine, Isehara, Kanagawa, Japan

    • Asako Otomo
    •  & Shinji Hadano
  4. Micro/Nano Technology Center, Tokai University, Hiratsuka, Kanagawa, Japan

    • Asako Otomo
  5. Department of Neurology, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan

    • Naoki Atsuta
    • , Ryoichi Nakamura
    •  & Gen Sobue
  6. Department of Neurology, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan

    • Tetsuya Akiyama
    •  & Masashi Aoki
  7. Research Center for Brain and Nervous Diseases, Tokai University Graduate School of Medicine, Isehara, Kanagawa, Japan

    • Shinji Hadano
  8. Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan

    • Hideyuki Saya
  9. Research Division of Dementia and Neurodegenerative Disease, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan

    • Gen Sobue


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K.F. and H.O. designed the study. K.F., A.O., and S.H. established motor neuron differentiation protocols. K.F. generated iPSCs from 32 SALS patients, analyzed the in vitro pathology of FALS and SALS models, designed the phenotype-based clustering system, performed drug screening, conducted in vitro pharmacology, performed transcriptome analysis, and analyzed the data. K.F. and H.S. designed the drug screening system. M.I. established iPSCs from ALS patients carrying SOD1 mutations. N.A., R.N., T.A., M.A., and G.S. provided ALS patient samples and analyzed the data from clinical observations. H.S. provided the existing drug library for screening. Project management was conducted by S.H., M.A., H.S., G.S., and H.O. The manuscript was prepared by K.F. and H.O. All authors contributed to the final editing and approval of the manuscript.

Competing interests

H.O. is a paid Scientific Advisory Board Member at SanBio Co., Ltd. and K Pharma Inc.

Corresponding author

Correspondence to Hideyuki Okano.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–16 and Supplementary Tables 1–4

  2. Reporting Summary

  3. Supplementary Dataset 1

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