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C9ORF72 poly(GA) aggregates sequester and impair HR23 and nucleocytoplasmic transport proteins

Abstract

Neuronal inclusions of poly(GA), a protein unconventionally translated from G4C2 repeat expansions in C9ORF72, are abundant in patients with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) caused by this mutation. To investigate poly(GA) toxicity, we generated mice that exhibit poly(GA) pathology, neurodegeneration and behavioral abnormalities reminiscent of FTD and ALS. These phenotypes occurred in the absence of TDP-43 pathology and required poly(GA) aggregation. HR23 proteins involved in proteasomal degradation and proteins involved in nucleocytoplasmic transport were sequestered by poly(GA) in these mice. HR23A and HR23B similarly colocalized to poly(GA) inclusions in C9ORF72 expansion carriers. Sequestration was accompanied by an accumulation of ubiquitinated proteins and decreased xeroderma pigmentosum C (XPC) levels in mice, indicative of HR23A and HR23B dysfunction. Restoring HR23B levels attenuated poly(GA) aggregation and rescued poly(GA)-induced toxicity in neuronal cultures. These data demonstrate that sequestration and impairment of nuclear HR23 and nucleocytoplasmic transport proteins is an outcome of, and a contributor to, poly(GA) pathology.

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Figure 1: Disrupting the conformation of poly(GA) proteins inhibits poly(GA) protein aggregation and toxicity.
Figure 2: Expression of poly(GA) proteins in mouse brains results in the formation of ubiquitin-positive poly(GA) inclusions.
Figure 3: Poly(GA) proteins sequester HR23 and nucleocytoplasmic transport proteins into inclusions.
Figure 4: Poly(GA) proteins cause ubiquitinated proteins to accumulate, decrease the stability of XPC proteins, and sequester XPC into inclusions.
Figure 5: Poly(GA) mice develop brain atrophy, neuronal loss and neurodegeneration.
Figure 6: Astrogliosis is observed in poly(GA) mouse brain.
Figure 7: Poly(GA) mice develop motor deficits, hyperactivity, anxiety and cognitive defects.
Figure 8: Exogenous HR23B attenuates poly(GA) aggregation and poly(GA)-induced neurotoxicity.

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Acknowledgements

We are grateful to all patients who agreed to donate post-mortem tissue. This work was supported by the US National Institutes of Health (NIH) National Institute on Aging (R01AG026251 (L.P.)); NIH National Institute of Neurological Disorders and Stroke (R21NS079807 (Y.-J.Z. and J.D.F.); R21NS089979 (T.F.G. and K.B.B.); F32NS087842 (J.J.); R01NS080882 (R.R.); R01NS085207 (J.D.R.); U54NS091046 (J.D.R.); R01NS063964 (L.P.); R01NS077402 (L.P.), R21NS084528 (L.P.); P01NS084974 (L.P., D.D., K.B.B. and R.R.); R01NS088689 (L.P.)); National Institute of Environmental Health Services (R01ES20395 (L.P.); Department of Defense (ALSRP AL130125 (L.P.)); Mayo Clinic Foundation (L.P.); Mayo Clinic Center for Individualized Medicine (L.P. and K.B.B.); Alzheimer's Association (NIRP-14-304425 (Y.-J.Z.); NIRP-12-259289 (J.D.F.)); Amyotrophic Lateral Sclerosis Association (Y.-J.Z., T.F.G., K.B.B., D.W.C. and L.P.); Robert Packard Center for ALS Research at Johns Hopkins (J.D.R. and L.P.), Target ALS (C.L.-T., J.D.R. and L.P.); Brain Science Institute (J.D.R.); the Ludwig Institute for Cancer Research (D.W.C. and C.L.T.), and the European Union's Seventh Framework Programme (FP7/2014-2019 grant 617198 (D.E.)). J.C.G. is the recipient of a National Science Foundation Graduate Research Fellowship Award, a Thomas Shortman Training Fund Graduate Scholarship and an Axol Science Scholarship.

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Authors and Affiliations

Authors

Contributions

L.P. and Y.-J.Z. contributed to the conception and design. Y.-J.Z. performed immunoblots, quantitative reverse-transcription PCR (qRT-PCR), co-immunoprecipitation and behavioral tests; T.F.G. completed anti-GA antibody generation and characterization, and performed poly(GA) assays with L.D.; J.C.G. and J.D.R. performed immunofluorescence staining for RanGAP1 and Pom121. H.S. performed ICV injection and behavioral tests; Y.-F.X. performed silver staining, immunofluorescence staining and primary neuronal cultures; Y.-F.X. and Z.S.W. quantified the Purkinje cells in cerebellum. M.E.M. and A.M.L. quantified neuronal loss and gliosis burden; M.S. and G.B. contributed to ELISA; W.-L.L. performed immunoEM; J.G. and A.G. performed immunofluorescence staining and immunoblotting; J.N.S. prepared primary neurons; K.J.-W. made plasmids; J.T. and M.Y. harvested mice and prepared brain lysates; E.A.P. produced AAV1; J.C. aided with ICV injections; M.C.-C. performed immunohistochemistry staining; A.K. and J.D.F. contributed to behavioral tests; J.D.B. and C.A.D. contributed to the purification of recombinant protein and the transmission electron microscopy study; J.J., C.L.-T., D.E. and D.W.C. characterized and provided anti-poly(GA) antibodies. R.R., K.B.B. and D.W.D. contributed to the tissue collection. C.D.L. analyzed data. L.P., Y.-J.Z., T.F.G. and R.B.K. analyzed data and wrote the manuscript.

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Correspondence to Leonard Petrucelli.

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Integrated supplementary information

Supplementary Figure 1 Neuropathology of poly(GA) inclusions in the brains of GFP-(GA)50 mice.

Immunohistochemical analysis in the indicated brain regions of GFP-(GA)50 mice with (a) anti-GFP antibody or (b) anti-GA antibody. Scale bars, 20 μm. (c) Double immunofluorescence staining in the cortex of GFP-(GA)50 mice for the indicated proteins. The arrows point to MAP2-positive neurons and the arrowhead to an astrocyte. Scale bars, 10 μm. (d) Immunohistochemical analysis in the cortex and hippocampus of GFP, GFP-(GA)50 and GFP-(GA)50-mut mice with an anti-ubiquitin antibody. Scale bars, 20 μm.

Supplementary Figure 2 HR23, RanGAP1 and Pom121 form inclusions in the brains of GFP-(GA)50 and (G4C2)66 mice.

(a) Immunohistochemical analysis of HR23A and HR23B proteins in the hippocampus of 6 month-old GFP, GFP-(GA)50 and GFP-(GA)50-mut mice. Scale bars, 20 μm. (b) Double immunofluorescence staining for GFP-(GA)50 and HR23A or HR23B in the hippocampus of 6 month-old GFP-(GA)50 mice. Scale bars, 5 μm. (c) Immunofluorescence staining for HR23B in the cortex of 6 month-old (G4C2)66 mice. Scale bar, 10 μm. (d) Representative images of immunohistochemical analysis of HR23A and HR23B in the hippocampus of c9FTD/ALS subjects (n = 7) or healthy controls (n = 4). Scale bars, 20 μm. (e) Double immunofluorescence staining for RanGAP1 and either HR23A or HR23B in the cortex of 6 month-old GFP-(GA)50 mice. Scale bars, 10 μm. (f) Immunofluorescence staining for RanGAP1 or Pom121 in the cortex of 6 month-old (G4C2)66 mice. Scale bars, 10 μm.

Supplementary Figure 3 HR23 proteins interact with, and are sequestered by, poly(GA) proteins.

(a) Cytoplasmic and nuclear fractions were prepared from HEK293T cells exogenously expressing GFP, GFP-(GA)50 or GFP-(GA)50-mut, followed by immunoblots analysis using the indicated antibodies. Tubulin and Lamin A/C were used as cytoplasmic and nuclear markers, respectively. (b) Protein complexes were immunoprecipitated from the indicated input lysates (top left) from HEK293T cells exogenously expressing GFP, GFP-(GA)50 or GFP-(GA)50-mut with antibodies to GFP, HR23A or HR23B, followed by immunoblot analysis using the indicated antibodies. (c) Protein complexes were immunoprecipitated from the indicated input lysates (left, top and bottom) from HEK293T cells exogenously expressing GFP, GFP-(GA)50, GFP-(GR)50, or GFP-(GP)47 with an antibody to HR23B, followed by immunoblot analysis using antibodies to GFP and poly(GR). (d) Double immunofluorescence staining for HR23B and poly(GA), poly(GP) or poly(GR) in brains of 6 month-old (G4C2)66 mice. Scale bar, 5 μm. (e) Double immunofluorescence staining for HR23B and poly(GA), poly(GP) or poly(GR) in the hippocampus of c9FTD/ALS patients. Scale bar, 5 μm. For a-c, full-length immunoblots are presented in Supplementary Figure 10.

Supplementary Figure 4 XPC is sequestered into poly(GA) inclusions in the hippocampus of GFP-(GA)50 mice.

(a) Immunohistochemical analysis of XPC in the hippocampus of GFP, GFP-(GA)50 and GFP-(GA)50-mut mice. Scale bar, 20 μm. (b) Double immunofluorescence staining of XPC and poly(GA) proteins in the hippocampus of GFP-(GA)50 mice. Scale bar, 5 μm.

Supplementary Figure 5 Analysis of brain morphology, body weight and motor cortex neurons in GFP-(GA)50 mice.

(a) Gross morphological analysis with hematoxylin and eosin staining of brains from 6 month-old GFP, GFP-(GA)50 and GFP-(GA)50-mut mice. Scale bar, 5 mm. (b) The mean body weight of 6 month-old male and female GFP, GFP-(GA)50 and GFP-(GA)50-mut mice, using 6–8 male mice or 4 female mice per group. Data are presented as mean ± s.e.m. Male mice: P < 0.0001, as analyzed by one-way ANOVA; ****P < 0.0001 and ***P = 0.0002, Tukey’s post-hoc analysis. Female mice: P = 0.0724, one-way ANOVA. n.s., not significant. (c) Immunohistochemical analysis of NeuN in layer V of the motor cortex of GFP, GFP-(GA)50 and GFP-(GA)50-mut mice. Scale bar, 30 μm.

Supplementary Figure 6 Poly(GA) inclusions in 4- to 6-week-old GFP-(GA)50 mice.

Immunohistochemical analysis of cortex and hippocampus of GFP, GFP-(GA)50 and GFP-(GA)50-mut mice with (a) anti-GFP antibody or (b) anti-ubiquitin antibody. Scale bars, 20 μm.

Supplementary Figure 7 No signs of neurodegeneration were observed in 4- to 6-week-old GFP-(GA)50 mice.

(a) Graph showing the mean brain weight of mice expressing GFP, GFP-(GA)50 or GFP-(GA)50-mut (n = 5–7 per group). (b) The mean body weight of male and female GFP, GFP-(GA)50 and GFP-(GA)50-mut mice using 2–5 male mice or 1–3 female mice. (c) Representative images of NeuN-labeled cells in the motor cortex and hippocampus of GFP, GFP-(GA)50 or GFP-(GA)50-mut mice. Scale bars, 200 μm. (d) Quantification of the number of NeuN-positive cells in the cortex (left) or motor cortex (right) of GFP, GFP-(GA)50 or GFP-(GA)50-mut mice (n = 5–7 per group). (e) Quantification of the number of Purkinje cells in the cerebellum of GFP, GFP-(GA)50 or GFP-(GA)50-mut mice (n = 5–7 per group). (f) Representative images of GFAP staining to identify reactive astrocytes in the motor cortex and hippocampus of GFP, GFP-(GA)50 or GFP-(GA)50-mut mice. Scale bars, 100 μm. Data are presented as mean ± s.e.m., and analyzed by one-way ANOVA; P = 0.0988 (a), P = 0.7026 (b), P = 0.0563 (d, Cortex), P = 0.1609 (d, Motor cortex) and P = 0.9042 (e). n.s., not significant.

Supplementary Figure 8 Exogenous HR23B does not decrease poly(GR) levels nor attenuate poly(GR)-induced neurotoxicity.

(a) Immunoblot and (b) densitometric analysis of immunoblots for the indicated proteins to determine their levels of expression in primary neurons transduced to express GFP-(GR)50 or GFP in the presence or absence of exogenous Myc-tagged HR23B. Data are presented as mean ± s.e.m. from 3 separate experiments. In b, left: P = 0.0005, one-way ANOVA; P = 0.6252 (GFP-(GR)50+Vector vs. GFP-(GR)50+HR23B-Myc), Tukey’s post-hoc analysis. Right: P < 0.0001, one-way ANOVA; ****P < 0.0001 and P = 0.7798 (GFP-(GR)50+Vector vs. GFP-(GR)50+HR23B-Myc), Tukey’s post-hoc analysis. n.s., not significant. For a, full-length immunoblots are presented in Supplementary Figure 10.

Supplementary Figure 9 Full-length immunoblots for main figures.

The region delineated by the box on each blot is the image shown in the corresponding figure.

Supplementary Figure 10 Full-length immunoblots for supplementary figures.

The region delineated by the box on each blot is the image shown in the corresponding figure.

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Supplementary Figures 1–10 and Supplementary Tables 1–3 (PDF 1548 kb)

Supplementary Methods Checklist (PDF 514 kb)

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Zhang, YJ., Gendron, T., Grima, J. et al. C9ORF72 poly(GA) aggregates sequester and impair HR23 and nucleocytoplasmic transport proteins. Nat Neurosci 19, 668–677 (2016). https://doi.org/10.1038/nn.4272

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