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

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.

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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.

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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.

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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.

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Correspondence to Leonard Petrucelli.

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Supplementary Information

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

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RAN-seq analysis

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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

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