The hexanucleotide repeat expansion (HRE) GGGGCC (G4C2) in C9orf72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent studies support an HRE RNA gain-of-function mechanism of neurotoxicity, and we previously identified protein interactors for the G4C2 RNA including RanGAP1. A candidate-based genetic screen in Drosophila expressing 30 G4C2 repeats identified RanGAP (Drosophila orthologue of human RanGAP1), a key regulator of nucleocytoplasmic transport, as a potent suppressor of neurodegeneration. Enhancing nuclear import or suppressing nuclear export of proteins also suppresses neurodegeneration. RanGAP physically interacts with HRE RNA and is mislocalized in HRE-expressing flies, neurons from C9orf72 ALS patient-derived induced pluripotent stem cells (iPSC-derived neurons), and in C9orf72 ALS patient brain tissue. Nuclear import is impaired as a result of HRE expression in the fly model and in C9orf72 iPSC-derived neurons, and these deficits are rescued by small molecules and antisense oligonucleotides targeting the HRE G-quadruplexes. Nucleocytoplasmic transport defects may be a fundamental pathway for ALS and FTD that is amenable to pharmacotherapeutic intervention.

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We thank S. Michaelis for discussions; K. Russell, M. Elrick and J. Ravits for human tissue and/or human histological studies. We also thank the Hetzer Laboratory for the CMV–NLS–tdTomato–NES construct; B. Ganetzky and C. Staber for the Drosophila RanGAP antibody; P. Jin for the UAS-(G4C2)30 fly stock; C. Svendsen for some control iPS cell lines; L. Petrucelli for the GP antibody; F. Hirth for the TBPH antibody. We thank the TRiP at Harvard Medical School (NIH/NIGMS R01-GM084947) for providing transgenic RNAi fly stocks used in this study. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. This work was supported by grants from NIH (R01 NS085207 and NS091046 to J.D.R. and R.S., R01 NS082563 to T.E.L., R01 NS074324 and NS089616 to J.W.), Brain Science Institute, Robert Packard Center for ALS Research at Johns Hopkins, Muscular Dystrophy Association (J.D.R.), Alzheimer’s Drug Discovery Foundation (J.D.R. and R.S.), Judith and Jean Pape Adams Charitable Foundation (J.W. and R.S.), Alzheimer’s Disease Research Center – Johns Hopkins (R.S.), Maryland TEDCO (C.J.D. and J.W.), Target ALS Springboard Fellowship (C.J.D.), William and Ella Owens Foundation (R.S.), and ALS Association (T.E.L., R.S. and J.D.R.). K.Z. is a Milton Safenowitz fellow in the ALS Association. A.R.H. is a fellow on an NIH training grant (CA009110) and a recipient of an NIH K99 award (NS091486). J.C.G. and S.J.M. are recipients of a National Science Foundation Graduate Research Fellowship Award and J.C.G. is a recipient of the Thomas Shortman Training Fund Graduate Scholarship.

Author information

Author notes

    • Ke Zhang
    •  & Christopher J. Donnelly

    These authors contributed equally to this work.

    • Thomas E. Lloyd
    •  & Jeffrey D. Rothstein

    These authors jointly supervised this work.


  1. Department of Neurology, School of Medicine, Johns Hopkins University, Maryland 21205, USA

    • Ke Zhang
    • , James B. Machamer
    • , Kathleen M. Cunningham
    • , Saksham Gupta
    • , Lyle W. Ostrow
    •  & Thomas E. Lloyd
  2. Brain Science Institute, School of Medicine, Johns Hopkins University, Maryland 21205, USA

    • Christopher J. Donnelly
    • , Jonathan C. Grima
    • , Elizabeth L. Daley
    • , Sean J. Miller
    • , Svetlana Vidensky
    • , Michael A. Thomas
    • , Rita Sattler
    •  & Jeffrey D. Rothstein
  3. Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Maryland 21205, USA

    • Aaron R. Haeusler
    • , Peter Steinwald
    • , Michael J. Matunis
    •  & Jiou Wang
  4. Department of Neuroscience, School of Medicine, Johns Hopkins University, Maryland 21205, USA

    • Jonathan C. Grima
    • , Ingie Hong
    • , Shu-Ling Chiu
    • , Richard L. Huganir
    • , Thomas E. Lloyd
    •  & Jeffrey D. Rothstein


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K.Z., C.J.D., R.S., T.E.L. and J.D.R. conceived the project. K.Z., C.J.D., A.R.H., J.W., R.S., T.E.L. and J.D.R. designed the experiments. K.Z. performed most studies related to Drosophila, with assistance from J.B.M., K.M.C. and S.G. C.J.D. performed studies employing iPSC neuronal cultures and human tissue with help from S.J.M., L.W.O. and J.C.G. A.R.H. performed the EMSA analysis. J.B.M. performed the fly NMJ and electrophysiologic analyses; I.H., S.-L.C. and R.L.H. performed and/or interpreted the human iPS electrophysiological analyses. J.C.G., E.L.D., S.V., M.A.T. and P.S. provided technical support. A.R.H., K.Z. and C.J.D. developed the figures. K.Z., C.J.D., A.R.H., M.J.M., J.W., R.S., T.E.L. and J.D.R. interpreted data and prepared the manuscript. K.Z. and C.J.D. contributed equally to this work. T.E.L. and J.D.R. contributed equally to this work. All authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Thomas E. Lloyd or Jeffrey D. Rothstein.

Extended data

Supplementary information

Excel files

  1. 1.

    Supplementary Table 1

    This table contains the genetic modifier screen of G4C2-mediated neurodegeneration.

  2. 2.

    Supplementary Table 4

    This table shows the number of C9ORF72 and control iPS quantified for each experiment.

PDF files

  1. 1.

    Supplementary Tables 2-3

    This file contains Supplementary Table 2, a list of C9ORF72 ALS and non-neurological control patient motor cortex and cerebellum employed in these studies and Supplementary Table 3, C9ORF72 and control iPS lines used in the study.

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