Abstract

An intronic GGGGCC repeat expansion in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the pathogenic mechanism of this repeat remains unclear. Using human induced motor neurons (iMNs), we found that repeat-expanded C9ORF72 was haploinsufficient in ALS. We found that C9ORF72 interacted with endosomes and was required for normal vesicle trafficking and lysosomal biogenesis in motor neurons. Repeat expansion reduced C9ORF72 expression, triggering neurodegeneration through two mechanisms: accumulation of glutamate receptors, leading to excitotoxicity, and impaired clearance of neurotoxic dipeptide repeat proteins derived from the repeat expansion. Thus, cooperativity between gain- and loss-of-function mechanisms led to neurodegeneration. Restoring C9ORF72 levels or augmenting its function with constitutively active RAB5 or chemical modulators of RAB5 effectors rescued patient neuron survival and ameliorated neurodegenerative processes in both gain- and loss-of-function C9ORF72 mouse models. Thus, modulating vesicle trafficking was able to rescue neurodegeneration caused by the C9ORF72 repeat expansion. Coupled with rare mutations in ALS2, FIG4, CHMP2B, OPTN and SQSTM1, our results reveal mechanistic convergence on vesicle trafficking in ALS and FTD.

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Acknowledgements

We thank the NINDS Biorepository at Coriell Institute for providing the following cell lines for this study: ND12133, ND03231, ND01751, ND11976, ND03719, ND00184, ND5280, ND06769, ND10689, ND12099, ND14954, ND08957, ND12100 and ND014587. We thank H. Chui and C. Miller (University of Southern California Alzheimer's Disease Research Center) and N. Shneider (Columbia University Medical Center) for control and C9ORF72 patient tissue. We thank the Choi Family Therapeutic Screening Facility for chemical screening support and the Translational Imaging Center at USC for imaging support. We thank M. Koppers, Y, Adolfs, C. van der Meer and M. Broekhoven for help with mouse breeding and kainate injection experiments. We thank S. Waguri (Fukushima Medical University) for providing the M6PR-GFP construct. We thank C, Buser for assistance with electron microscopy. We also thank S. Alworth (DRVision Technologies), K. Hebestreit and R. Bhatnagar (Verge Genomics), B. Baloh (Cedars Sinai Medical Center), J. O'Rourke (Cedars Sinai Medical Center), C. Donnelly, C. Tong, A. McMahon and Q. Liu-Michael for reagents, technical support and discussions. E.Y.S. is a Walter V. and Idun Berry Postdoctoral Fellow. K.A.S. was supported in part by a Muscular Dystrophy Association Development Grant. L.M. was supported by NIH grant T32DC009975-04. This work was supported by NIH grants AG039452, AG023084 and NS034467 to B.V.Z. R.J.P. was supported by grants from ALS Foundation Netherlands (TOTALS), Epilepsiefonds (12-08, 15-05), and VICI grant Netherlands Organization for Scientific Research (NWO). This work was also supported by NIH grants R00NS077435 and R01NS097850, US Department of Defense grant W81XWH-15-1-0187, and grants from the Donald E. and Delia B. Baxter Foundation, the Tau Consortium, the Frick Foundation for ALS Research, the Muscular Dystrophy Association, the New York Stem Cell Foundation, the USC Keck School of Medicine Regenerative Medicine Initiative, the USC Broad Innovation Award, and the Southern California Clinical and Translational Science Institute to J.K.I. J.K.I. is a New York Stem Cell Foundation-Robertson Investigator.

Author information

Author notes

    • Yingxiao Shi
    •  & Shaoyu Lin

    These authors contributed equally to this work.

Affiliations

  1. Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.

    • Yingxiao Shi
    • , Shaoyu Lin
    • , Kim A Staats
    • , Yichen Li
    • , Wen-Hsuan Chang
    • , Shu-Ting Hung
    • , Eric Hendricks
    • , Gabriel R Linares
    • , Louise Menendez
    • , Tohru Sugawara
    • , Phillip Woolwine
    • , Mickey Huang
    • , Michael J Cowan
    • , Brandon Ge
    • , Nicole Koutsodendris
    • , Kaitlin P Sandor
    • , Jacob Komberg
    • , Valerie Hennes
    • , Carina Seah
    •  & Justin K Ichida
  2. Eli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research at USC, Los Angeles, California, USA.

    • Yingxiao Shi
    • , Shaoyu Lin
    • , Kim A Staats
    • , Yichen Li
    • , Wen-Hsuan Chang
    • , Shu-Ting Hung
    • , Eric Hendricks
    • , Gabriel R Linares
    • , Louise Menendez
    • , Tohru Sugawara
    • , Phillip Woolwine
    • , Mickey Huang
    • , Michael J Cowan
    • , Brandon Ge
    • , Nicole Koutsodendris
    • , Kaitlin P Sandor
    • , Jacob Komberg
    • , Valerie Hennes
    • , Carina Seah
    •  & Justin K Ichida
  3. Zilkha Neurogenetic Institute, Keck School of Medicine of the University of Southern California, Los Angeles, California, USA.

    • Yingxiao Shi
    • , Shaoyu Lin
    • , Kim A Staats
    • , Yichen Li
    • , Wen-Hsuan Chang
    • , Shu-Ting Hung
    • , Eric Hendricks
    • , Gabriel R Linares
    • , Yaoming Wang
    • , Kassandra Kisler
    • , Brent Wilkinson
    • , Louise Menendez
    • , Tohru Sugawara
    • , Phillip Woolwine
    • , Mickey Huang
    • , Michael J Cowan
    • , Brandon Ge
    • , Nicole Koutsodendris
    • , Kaitlin P Sandor
    • , Jacob Komberg
    • , Valerie Hennes
    • , Carina Seah
    • , Amy R Nelson
    • , Marcelo Coba
    • , Berislav V Zlokovic
    •  & Justin K Ichida
  4. Department of Physiology and Biophysics, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.

    • Yaoming Wang
    • , Kassandra Kisler
    • , Amy R Nelson
    •  & Berislav V Zlokovic
  5. Department of Pathology, Stanford University School of Medicine, Stanford, California, USA.

    • Esther Y Son
  6. Jefferson Weinberg ALS Center, Vickie and Jack Farber Institute for Neuroscience, Department of Neuroscience, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.

    • Xinmei Wen
    •  & Davide Trotti
  7. Department of Translational Neuroscience, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, the Netherlands.

    • Vamshidhar R Vangoor
    • , Ketharini Senthilkumar
    •  & R Jeroen Pasterkamp
  8. DRVision Technologies, LLC, Bellvue, Washington, USA.

    • Tze-Yuan Cheng
    •  & Shih-Jong J Lee
  9. Icagen Corporation, Oro Valley, Arizona, USA.

    • Paul R August
  10. Verge Genomics, San Francisco, California, USA.

    • Jason A Chen
    • , Nicholas Wisniewski
    • , Victor Hanson-Smith
    • , T Grant Belgard
    •  & Alice Zhang
  11. Department of Psychiatry and Behavioral Sciences, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.

    • Marcelo Coba
  12. National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA.

    • Chris Grunseich
    •  & Michael E Ward
  13. Department of Neurology, University Medical Center Utrecht, Utrecht, the Netherlands.

    • Leonard H van den Berg

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Contributions

Y.S., S.L., Y.L. and J.K.I. conceived the project. Y.S., S.L., E.Y.S., Y.L., L.M., K.A.S., V.R.V., K.S., S.J.J.L., P.R.A., M.C., R.J.P., D.T., B.V.Z. and J.K.I. designed the experiments. Y.S., S.L., W.-H.C., E.Y.S., Y.L., S.-T.H., E.H., G.R.L., T.S., M.H., C.S., A.R.N., T.-Y.C., Y.W., K.K., B.W., L.M., M.J.C., B.G., K.P.S., J. K., N.K., X.W., V.H., A.R.N., K.A.S., V.R.V., K.S., R.J.P. and J.K.I. performed experiments and interpreted data. K.K. performed all electrophysiological studies and P.W., J.A.C., N.H.-S., N.W., T.G.B., A.Z. and K.A.S. performed RNA-Seq analysis. Y.S., S.L., E.Y.S., K.A.S. and J.K.I. prepared the manuscript. C.G. and M.W. developed the method of inducing iMNs using the Dox-NIL construct. All of the authors discussed the results and commented on the manuscript.

Competing interests

J.K.I. and P.A. are co-founders of Acurastem, Inc. P.A. is an employee of Icagen Corporation. J.K.I. and P.A. declare that they are bound by confidentiality agreements that prevent them from disclosing details of their financial interests in this work. S.-J.L. is a founder of DRVision Technologies and T.-Y.C. is an employee of DRVision Technologies. A.Z. and J.A.C. are co-founders of Verge Genomics and A.Z., V.H.-S., N.W. and T.G.B. are employees of Verge Genomics.

Corresponding author

Correspondence to Justin K Ichida.

Supplementary information

PDF files

  1. 1.

    Supplementary Figures & Tables

    Supplementary Figures 1–17 & Supplementary Tables 1–6

  2. 2.

    Life Sciences Reporting Summary

Excel files

  1. 1.

    Supplementary Table 7

    RNA sequencing data of Hb9::RFP+ iMNs from CTRL2, C9- ALS1, CTRL2 C9ORF72+/-, and CTRL2 C9ORF72-/- iPSCs.

Videos

  1. 1.

    Channel rhodopsin neuromuscular junction assay: C9-ALS patient iMNs (C9-ALS2). Green light is activated and can be observed by an increase in overall brightness at the following times: 7-13 sec, 18-23 sec, 29-35 sec, 40-45 sec, 51-56 sec, 1:02-1:07. Light-induced myotube contraction can be observed during those intervals.

  2. 2.

    Channel rhodopsin neuromuscular junction assay: control iMNs (CTRL2). Green light is activated and can be observed by an increase in overall brightness at the following times: 7- 12 sec, 18-23 sec, 29-34 sec, 40-45 sec, 52-57 sec, 1:03-1:08. Light-induced myotube contraction can be observed during those intervals.

  3. 3.

    Time-lapse video of C9-ALS (C9-ALS3) iMN degeneration. Fluorescent neurons are Hb9::RFP+ iMNs. Frames were captured at 24-hour intervals.

  4. 4.

    Time-lapse video of control (CTRL2) iMN degeneration. Fluorescent neurons are Hb9::RFP+ iMNs. Frames were captured at 24-hour intervals.

  5. 5.

    Time-lapse video of M6PR-GFP+ vesicle trafficking in a control iMN. Frames were captured at a rate of 8 frames/sec over a 60 sec interval. The green line outlines the cell soma.

  6. 6.

    Time-lapse video of M6PR-GFP+ vesicle trafficking in a C9ORF72+/- (CTRL2, C9+/-) iMN. Frames were captured at a rate of 8 frames/sec over a 60 sec interval. The white line outlines the cell soma.

  7. 7.

    Time-lapse video of M6PR-GFP+ vesicle trafficking in a C9- ALS iMN. Frames were captured at a rate of 8 frames/sec over a 60 sec interval. The white line outlines the cell soma.

  8. 8.

    Time-lapse video of M6PR-GFP+ vesicle trafficking in a C9- ALS iMN expressing C9ORF72 isoform A. Frames were captured at a rate of 8 frames/sec over a 60 sec interval. The white line outlines the cell soma.

  9. 9.

    Time-lapse video of M6PR-GFP+ vesicle trafficking in a C9- ALS iMN expressing C9ORF72 isoform B. Frames were captured at a rate of 8 frames/sec over a 60 sec interval. The white line outlines the cell soma.

  10. 10.

    Time-lapse video of Gcamp6 fluorescence in control iMNs treated with glutamate. Frames were captured over a 24 second interval and the video was increased to 2x speed to facilitate viewing.

  11. 11.

    Time-lapse video of Gcamp6 fluorescence in C9-ALS iMNs treated with glutamate. Frames were captured over a 24 second interval and the video was increased to 2x speed to facilitate viewing.

  12. 12.

    Time-lapse video of Gcamp6 fluorescence in C9ORF72+/- iMNs treated with glutamate. Frames were captured over a 24 second interval and the video was increased to 2x speed to facilitate viewing.

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https://doi.org/10.1038/nm.4490