Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Poly(GR) impairs protein translation and stress granule dynamics in C9orf72-associated frontotemporal dementia and amyotrophic lateral sclerosis

Nature Medicine volume 24, pages 1136–1142 (2018)Cite this article

Subjects

Abstract

The major genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is a C9orf72 G4C2 repeat expansion1,2. Proposed mechanisms by which the expansion causes c9FTD/ALS include toxicity from repeat-containing RNA and from dipeptide repeat proteins translated from these transcripts. To investigate the contribution of poly(GR) dipeptide repeat proteins to c9FTD/ALS pathogenesis in a mammalian in vivo model, we generated mice that expressed GFP–(GR)100 in the brain. GFP–(GR)100 mice developed age-dependent neurodegeneration, brain atrophy, and motor and memory deficits through the accumulation of diffuse, cytoplasmic poly(GR). Poly(GR) co-localized with ribosomal subunits and the translation initiation factor eIF3η in GFP–(GR)100 mice and, of importance, in c9FTD/ALS patients. Combined with the differential expression of ribosome-associated genes in GFP–(GR)100 mice, these findings demonstrate poly(GR)-mediated ribosomal distress. Indeed, poly(GR) inhibited canonical and non-canonical protein translation in HEK293T cells, and also induced the formation of stress granules and delayed their disassembly. These data suggest that poly(GR) contributes to c9FTD/ALS by impairing protein translation and stress granule dynamics, consequently causing chronic cellular stress and preventing cells from mounting an effective stress response. Decreasing poly(GR) and/or interrupting interactions between poly(GR) and ribosomal and stress granule-associated proteins may thus represent potential therapeutic strategies to restore homeostasis.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: GFP–(GR)100 mice exhibited neurodegeneration and behavioral deficits.
Fig. 2: Poly(GR) proteins co-localized with ribosomal proteins in GFP–(GR)100 mice and c9FTD/ALS patients.
Fig. 3: Transcriptome analyses revealed altered ribosome pathways in GFP–(GR)100 mice.
Fig. 4: Expression of GFP–(GR)100 impaired canonical and non-canonical translation.

References

  1. DeJesus-Hernandez, M. et al. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72, 245–256 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Renton, A. E. et al. A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72, 257–268 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Gendron, T. F., Belzil, V. V., Zhang, Y. J. & Petrucelli, L. Mechanisms of toxicity in C9FTLD/ALS. Acta Neuropathol. 127, 359–376 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Zhang, K. et al. The C9orf72 repeat expansion disrupts nucleocytoplasmic transport. Nature 525, 56–61 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Donnelly, C. J. et al. RNA toxicity from the ALS/FTD C9ORF72 expansion is mitigated by antisense intervention. Neuron 80, 415–428 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Cooper-Knock, J. et al. Sequestration of multiple RNA recognition motif-containing proteins by C9orf72 repeat expansions. Brain 137, 2040–2051 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Sareen, D. et al. Targeting RNA foci in iPSC-derived motor neurons from ALS patients with a C9ORF72 repeat expansion. Sci. Transl. Med. 5, 208ra149 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Conlon, E. G. et al. The C9ORF72 GGGGCC expansion forms RNA G-quadruplex inclusions and sequesters hnRNP H to disrupt splicing in ALS brains. Elife 5(2016).

  9. Lee, Y. B. et al. Hexanucleotide repeats in ALS/FTD form length-dependent RNA foci, sequester RNA binding proteins, and are neurotoxic. Cell Rep. 5, 1178–1186 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Mori, K. et al. hnRNP A3 binds to GGGGCC repeats and is a constituent of p62-positive/TDP43-negative inclusions in the hippocampus of patients with C9orf72 mutations. Acta Neuropathol. 125, 413–423 (2013).

    Article  CAS  PubMed  Google Scholar 

  11. Ash, P. E. et al. Unconventional translation of C9ORF72 GGGGCC expansion generates insoluble polypeptides specific to c9FTD/ALS. Neuron 77, 639–646 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Gendron, T. F. et al. Antisense transcripts of the expanded C9ORF72 hexanucleotide repeat form nuclear RNA foci and undergo repeat-associated non-ATG translation in c9FTD/ALS. Acta Neuropathol. 126, 829–844 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Mori, K. et al. Bidirectional transcripts of the expanded C9orf72hexanucleotide repeat are translated into aggregating dipeptide repeat proteins. Acta Neuropathol. 126, 881–893 (2013).

    Article  CAS  PubMed  Google Scholar 

  14. Mori, K. et al. The C9orf72GGGGCC repeat is translated into aggregating dipeptide-repeat proteins in FTLD/ALS. Science 339, 1335–1338 (2013).

    Article  CAS  PubMed  Google Scholar 

  15. Zu, T. et al. RAN proteins and RNA foci from antisense transcripts in C9ORF72 ALS and frontotemporal dementia. Proc. Natl Acad. Sci. USA 110, E4968–E4977 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Saberi, S. et al. Sense-encoded poly-GR dipeptide repeat proteins correlate to neurodegeneration and uniquely co-localize with TDP-43 in dendrites of repeat-expanded C9orf72 amyotrophic lateral sclerosis. Acta Neuropathol. 135, 459–474 (2018).

    Article  CAS  PubMed  Google Scholar 

  17. Jovicic, A. et al. Modifiers of C9orf72 dipeptide repeat toxicity connect nucleocytoplasmic transport defects to FTD/ALS. Nat. Neurosci. 18, 1226–1229 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kanekura, K. et al. Poly-dipeptides encoded by the C9ORF72 repeats block global protein translation. Hum. Mol. Genet. 25, 1803–1813 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Kwon, I. et al. Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells. Science 345, 1139–1145 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Lee, K. H. et al. C9orf72 dipeptide repeats impair the assembly, dynamics, and function of membrane-less organelles. Cell 167, 774–788.e17 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Tao, Z. et al. Nucleolar stress and impaired stress granule formation contribute to C9orf72 RAN translation-induced cytotoxicity. Hum. Mol. Genet. 24, 2426–2441 (2015).

    Article  CAS  PubMed  Google Scholar 

  22. Yamakawa, M. et al. Characterization of the dipeptide repeat protein in the molecular pathogenesis of c9FTD/ALS. Hum. Mol. Genet. 15, 1630–1645 (2014).

    Google Scholar 

  23. Lopez-Gonzalez, R. et al. Poly(GR) in C9ORF72-related ALS/FTD compromises mitochondrial function and increases oxidative stress and DNA damage in iPSC-derived motor neurons. Neuron 92, 383–391 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Freibaum, B. D. et al. GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport. Nature 525, 129–133 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Boeynaems, S. et al. Drosophila screen connects nuclear transport genes to DPR pathology in c9ALS/FTD. Sci. Rep. 6, 20877 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Shi, K. Y. et al. Toxic PRn poly-dipeptides encoded by the C9orf72 repeat expansion block nuclear import and export. Proc. Natl Acad. Sci. USA 114, E1111–E1117 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Chew, J. et al. Neurodegeneration. C9ORF72 repeat expansions in mice cause TDP-43 pathology, neuronal loss, and behavioral deficits. Science 348, 1151–1154 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Yang, D. et al. FTD/ALS-associated poly(GR) protein impairs the Notch pathway and is recruited by poly(GA) into cytoplasmic inclusions. Acta Neuropathol. 130, 525–535 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Schmidt, E. K., Clavarino, G., Ceppi, M. & Pierre, P. SUnSET, a nonradioactive method to monitor protein synthesis. Nat. Methods 6, 275–277 (2009).

    Article  CAS  PubMed  Google Scholar 

  30. Landry, D. M., Hertz, M. I. & Thompson, S. R. RPS25 is essential for translation initiation by the Dicistroviridae and hepatitis C viral IRESs. Genes Dev. 23, 2753–2764 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Hertz, M. I., Landry, D. M., Willis, A. E., Luo, G. & Thompson, S. R. Ribosomal protein S25 dependency reveals a common mechanism for diverse internal ribosome entry sites and ribosome shunting. Mol. Cell. Biol. 33, 1016–1026 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Zhang, Y. J. et al. C9ORF72 poly(GA) aggregates sequester and impair HR23 and nucleocytoplasmic transport proteins. Nat. Neurosci. 19, 668–677 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Mizielinska, S. et al. C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins. Science 345, 1192–1194 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Lin, Y. et al. Toxic PR poly-dipeptides encoded by the C9orf72 repeat expansion target LC domain polymers. Cell 167, 789–802.e12 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Cheng, W. et al. C9ORF72 GGGGCC repeat-associated non-AUG translation is upregulated by stress through eIF2alpha phosphorylation. Nat. Commun. 9, 51 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. Green, K. M. et al. RAN translation at C9orf72-associated repeat expansions is selectively enhanced by the integrated stress response. Nat. Commun. 8, 2005 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Zhang, Y. J. et al. Aggregation-prone c9FTD/ALS poly(GA) RAN-translated proteins cause neurotoxicity by inducing ER stress. Acta Neuropathol. 128, 505–524 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Prudencio, M. et al. Distinct brain transcriptome profiles in C9orf72-associated and sporadic ALS. Nat. Neurosci. 18, 1175–1182 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Kalari, K. R. et al. MAP-RSeq: Mayo Analysis Pipeline for RNA sequencing. BMC Bioinformatics 15, 224 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Hansen, K. D., Irizarry, R. A. & Wu, Z. Removing technical variability in RNA-seq data using conditional quantile normalization. Biostatistics 13, 204–216 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  42. Langfelder, P. & Horvath, S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics 9, 559 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Gendron, T. F. et al. Cerebellar c9RAN proteins associate with clinical and neuropathological characteristics of C9ORF72 repeat expansion carriers. Acta Neuropathol. 130, 559–573 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Kramer, N. J. et al. Spt4 selectively regulates the expression of C9orf72 sense and antisense mutant transcripts. Science 353, 708–712 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are grateful to all patients who agreed to donate post-mortem tissue. This work was supported by the National Institutes of Health/National Institute of Neurological Disorders and Stroke (R35NS097273 (L.P.); P01NS084974 (L.P., D.W.D., R.R. and B.O.); P01NS099114 (T.F.G. and L.P.); R01NS088689 (L.P.); R35NS097263(10) (A.D.G.)); the Mayo Clinic Foundation (L.P.); the Amyotrophic Lateral Sclerosis Association (T.F.G., L.P., Y.-J.Z. and M.P.), the Robert Packard Center for ALS Research at Johns Hopkins (A.D.G. and L.P.) and the Target ALS Foundation (T.F.G., A.D.G., L.P. and Y.-J.Z.). We would like to thank J. N. Stankowski, E. A. Perkerson, L. Rousseau and V. Phillips for technical support. This manuscript is dedicated to Dr Antimo D’Aniello.

Author information

Authors and Affiliations

  1. Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA

    Yong-Jie Zhang, Tania F. Gendron, Mark T. W. Ebbert, Aliesha D. O’Raw, Mei Yue, Karen Jansen-West, Mercedes Prudencio, Jeannie Chew, Casey N. Cook, Lillian M. Daughrity, Jimei Tong, Yuping Song, Sarah R. Pickles, Monica Castanedes-Casey, Aishe Kurti, Rosa Rademakers, Dennis W. Dickson, John D. Fryer & Leonard Petrucelli

  2. Neurobiology of Disease Graduate Program, Mayo Graduate School, Mayo Clinic College of Medicine, Rochester, MN, USA

    Yong-Jie Zhang, Tania F. Gendron, Mercedes Prudencio, Jeannie Chew, Casey N. Cook, Rosa Rademakers, Dennis W. Dickson, John D. Fryer & Leonard Petrucelli

  3. Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA

    Xu Zhang & Wenqian Hu

  4. Department of Neurology, Mayo Clinic, Jacksonville, FL, USA

    Bjorn Oskarsson

  5. Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA

    Aaron D. Gitler

Authors
  1. Yong-Jie Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tania F. Gendron
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mark T. W. Ebbert
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Aliesha D. O’Raw
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mei Yue
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Karen Jansen-West
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xu Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Mercedes Prudencio
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jeannie Chew
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Casey N. Cook
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Lillian M. Daughrity
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Jimei Tong
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Yuping Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Sarah R. Pickles
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Monica Castanedes-Casey
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Aishe Kurti
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Rosa Rademakers
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Bjorn Oskarsson
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Dennis W. Dickson
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Wenqian Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Aaron D. Gitler
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. John D. Fryer
    View author publications

    You can also search for this author in PubMed Google Scholar

  23. Leonard Petrucelli
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

L.P. and Y.-J.Z. contributed to the conception and design. Y.-J.Z. performed cell culture and treatments, preparation of lysates, western blot, RT-PCR, qPCR, immunofluorescence staining, SUnSET assay and FISH; T.F.G. and L.M.D. generated and/or performed poly(GP) and poly(GR) immunoassays; M.T.W.E. and J.D.F. analysed RNA-Seq data; A.D.O. performed intracerebroventricular injection, behavioural tests, and immunofluorescence staining and quantification of neuropathology and TIA-1-positive stress granules; M.Y. performed the quantification of neuropathology and RNA foci, and in vivo SUnSET assay; M.P. and Y.S. ran the RNA 6000 Nano kit to verify RNA integrity; W.H. and X.Z. contributed to the ribosome study; K.J.-W. made plasmids and AAV1 virus; J.C. and M.C.-C. performed immunohistochemistry staining; C.N.C. and S.R.P. performed the SUnSET assay. J.T. collected mouse tissues; A.K. and J.D.F. contributed to behavioural tests; R.R., B.O. and D.W.D. contributed to the tissue collection; A.D.G. assisted with data analysis; L.P., Y.-J.Z. T.F.G. and M.T.W.E. analyzed data and wrote the manuscript.

Corresponding author

Correspondence to Leonard Petrucelli.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Supplementary Figures and Tables

Supplementary Discussion, Supplementary Figures 1–12 and Supplementary Tables 1 and 2

Reporting Summary

Supplementary Dataset

RAN-seq analysis

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, YJ., Gendron, T.F., Ebbert, M.T.W. et al. Poly(GR) impairs protein translation and stress granule dynamics in C9orf72-associated frontotemporal dementia and amyotrophic lateral sclerosis. Nat Med 24, 1136–1142 (2018). https://doi.org/10.1038/s41591-018-0071-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41591-018-0071-1

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing