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GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport


The GGGGCC (G4C2) repeat expansion in a noncoding region of C9orf72 is the most common cause of sporadic and familial forms of amyotrophic lateral sclerosis and frontotemporal dementia1,2. The basis for pathogenesis is unknown. To elucidate the consequences of G4C2 repeat expansion in a tractable genetic system, we generated transgenic fly lines expressing 8, 28 or 58 G4C2-repeat-containing transcripts that do not have a translation start site (AUG) but contain an open-reading frame for green fluorescent protein to detect repeat-associated non-AUG (RAN) translation. We show that these transgenic animals display dosage-dependent, repeat-length-dependent degeneration in neuronal tissues and RAN translation of dipeptide repeat (DPR) proteins, as observed in patients with C9orf72-related disease. This model was used in a large-scale, unbiased genetic screen, ultimately leading to the identification of 18 genetic modifiers that encode components of the nuclear pore complex (NPC), as well as the machinery that coordinates the export of nuclear RNA and the import of nuclear proteins. Consistent with these results, we found morphological abnormalities in the architecture of the nuclear envelope in cells expressing expanded G4C2 repeats in vitro and in vivo. Moreover, we identified a substantial defect in RNA export resulting in retention of RNA in the nuclei of Drosophila cells expressing expanded G4C2 repeats and also in mammalian cells, including aged induced pluripotent stem-cell-derived neurons from patients with C9orf72-related disease. These studies show that a primary consequence of G4C2 repeat expansion is the compromise of nucleocytoplasmic transport through the nuclear pore, revealing a novel mechanism of neurodegeneration.

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Figure 1: G4C2 repeats induces length- and dosage-dependent degeneration in Drosophila.
Figure 2: Genetic screen identifies multiple modifiers of (G4C2)58 toxicity in the nucleocytoplasmic transport pathway.
Figure 3: Drosophila salivary gland cells expressing (G4C2)58 exhibit nuclear envelope abnormalities and accumulation of nuclear RNA.
Figure 4: Accumulation of nuclear RNA in human cells expressing G4C2 repeat expansion.

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We thank the Bloomington Drosophila Stock Center, the VDRC Stock Center, K. McKim and E. Baehrecke for fly lines, V. Budnik for Lamin C antibody, as well as the Cell and Tissue Imaging Center at St Jude Children’s Research Hospital and the University of Massachusetts Medical School Confocal Core for assistance. This work was supported by grants from Target ALS, The Packard Center for ALS Research at the Johns Hopkins University, and the ALS Association to F.-B.G., and J.P.T., and ALS Therapy Alliance, NIH (N079725) to F.-B.G., NIH (NS079725 and AG019724) to B.L.M., and the American-Lebanese-Syrian Associated Charities to J.P.T.

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Authors and Affiliations



F.-B.G. and J.P.T. conceived and supervised the project. B.D.F., Y.L., H.J.K., F.-B.G. and J.P.T. wrote the manuscript. B.D.F. and Y.L. performed the genetic screen and validation. B.D.F., Y.L., N.C.K., N.B. and K.-H.L. characterized Drosophila phenotypes and performed the assays characterizing RNA export in human cells. S.A. established human fibroblast cell lines, R.L.-G. generated some iPSC lines; S.A. and R.L.-G. performed cortical neuron differentiation. M.V. and B.D.F. conducted FISH experiments. L.P., B.L.M. and P.C.W. provided key reagents.

Corresponding authors

Correspondence to Fen-Biao Gao or J. Paul Taylor.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Expression of G4C2 repeats induces length-dependent phenotypes in Drosophila.

a, Expression of (G4C2)58 in Drosophila motor neurons using the OK371‐GAL4 driver leads to a significant reduction in active zones as immunostained by the anti‐Bruchpilot antibody NC82 and anti-HRP. Scale bar, 50 μm. b, Quantification of active zones (n = 6 individual larvae for control, 4 for 2× (G4C2)8, and 6 for 2× (G4C2)58). Values are mean ± s.e.m. ** P < 0.01, one-way ANOVA, Tukey’s post hoc test. c, Pan neuronal expression of (G4C2)58 repeats induces dosage-dependent decrease in larval size (left) and locomotor activity measured in 30 s (right) when (G4C2)58 is expressed in all neurons using the elav-GAL4 driver. d, Quantification of the distance travelled by third instar larvae reveals expressing two copies of (G4C2)58 results in a significant deficit in locomotor activity. Values are mean ± s.e.m. (n = 7 individual larvae for control, 4 for 1× (G4C2)58, and 5 for 2′ (G4C2)58). **P < 0.01, one-way ANOVA, Tukey’s post hoc test. e, Pan neuronal expression of (G4C2)58 repeats in Drosophila neurons using the elav-GAL4 driver leads to a significant reduction in the bouton number. Bouton number was quantified by examining the presynaptic (anti-HRP) and postsynaptic (anti-DLG1) markers (left). Scale bar, 50 μm. f, Quantification of bouton number (left) and muscle size (right) reveal that both are significantly reduced in Drosophila larvae expressing (G4C2)58 repeats. Values are mean ± s.e.m. (n = 6 individual larvae for control, 5 for 2× (G4C2)8, and 6 for 2× (G4C2)58), ** P < 0.01, one-way ANOVA, Tukey’s post hoc test. g, Expression of (G4C2)28 and (G4C2)58 but not (G4C2)8 in the muscle using the MHC-GAL4 driver leads to loss of wing control in adult flies (n = 30 individual Drosophila for control, 21 for (G4C2)8, 50 for (G4C2)28, and 24 for (G4C2)58). This phenotype was assessed by examining the permanent wing posture of live adult flies.

Extended Data Figure 2 RAN translation is observed in Drosophila expressing G4C2 repeats.

a, Western blot revealing translation of RAN poly-dipeptides in flies expressing (G4C2)58 in the eye. RAN poly-dipeptides were not found in flies expressing (G4C2)8 or control flies. There was minimal expression of GFP-positive product observed in flies expressing (G4C2)28. GFP-expressing flies (lane 1) were used as a positive control for the anti-GFP antibody. b, Western blot showing production of RAN product when (G4C2)28 and (G4C2)58 but not (G4C2)8 repeats are expressed in the muscle. RAN products were visualized with anti-GFP antibody (left) and anti-poly(GP) antibody (right). c, The RAN product poly-GP–GFP from flies expressing (G4C2)58 in the muscle form large visible inclusions as visualized under light sheet fluorescent microscopy (left) and by confocal microscopy (right). Scale bar, 50 μm. d, Expression of (G4C2)58 in the salivary gland cells results in the formation of large nuclear inclusions and smaller cytoplasmic inclusions. Scale bar, 50 μm. e, f, Expression of (G4C2)58 in the ventral ganglion by OK371 driver results in the formation of nuclear and cytoplasmic inclusions, whereas GFP shows diffused nuclear and cytoplasmic localization. Lamin staining shows nuclear membrane, and CD8–RFP shows plasma membrane. Scale bar, 10 μm. g, Expression of (G4C2)58 in pan neuronal cells by elav driver results in the nuclear and cytoplasmic inclusions. Scale bar, 10 μm.

Extended Data Figure 3 Ectopic expression of poly(GR) but not poly(GA) or poly(GP) peptides are toxic in Drosophila.

a, Transgenic Drosophila were generated that express ATG-driven poly(GA), poly(GR) and poly(GP) peptides with an N-terminal GFP tag (top). Expression of GFP–(GA)50 and GFP–(GP)47 were non-toxic when expressed in the eye with GMR-GAL4 whereas GFP–(GR)50 expression resulted in >95% lethality with surviving adults having severely degenerated eyes (bottom). b, Western blot showing the expression of (G4C2)58, GFP–(GA)50, GFP–(GR)50 and GFP–(GP)47 as visualized in muscle by anti-GFP antibody. c, Western blot showing the expression of poly(GP) in muscle of flies expressing (G4C2)58 and GFP–(GP)47 but not GFP–(GA)50, GFP–(GR)50 and control flies as visualized by anti-GP antibody. d, Dot blot analysis of RAN peptides in muscle revealing expression of poly(GA) only in GFP–(GA)50 flies, expression of poly(GR) in (G4C2)58 and GFP–(GR)50 flies. As expected, anti-sense DPR poly(PR) was not found in any of the lysates. The background protein signal was used as a loading control.

Extended Data Figure 4 Nuclear import and export is altered by (G4C2)58 expression.

a, A threonine to asparagine substitution at residue 24 in the Ran protein abolishes the affinity for GTP and reduces its affinity for GDP. Hence, the RanT24N is always in either a nucleotide-free state or in its inactive, GDP-bound state, and acts as dominant negative. RANT24N expression driven by GMR-GAL4 causes a mild eye phenotype when expressed in the absence of (G4C2)58 (upper row, right panel). The (G4C2)58 rough eye phenotype is strongly enhanced by dominant-negative RanT24N expression (middle row, left panel). The (G4C2)58 eye phenotype is strongly enhanced by knockdown of Nup153 by two independent RNAi lines (middle row, two right panels). The (G4C2)58 eye phenotype is also mildly enhanced by knockdown of transportin (Trn) (bottom row). b, Knockdown of Crm1 in flies expressing (G4C2)58 induces a mild enhancement of the (G4C2)58 eye phenotype (left versus middle). Crm1 knockdown in the absence of (G4C2)58 repeats does not produce a rough eye phenotype (right). c, Expression of two copies of (G4C2)58 in the Drosophila motor neurons leads to reduced viability (50%). Chemical inhibition of Crm1 with Leptomycin B (500 nM) enhances (G4C2)58 toxicity resulting in reduced viability (23%). Leptomycin B does not impede viability (100%) in Drosophila expressing GFP. n is displayed on the graph and represents the individual pupae from two separate experiments.

Extended Data Figure 5 Phenotypes of additional suppressors and enhancers of (G4C2)58.

a, Phenotypes demonstrating suppression of the (G4C2)58 rough eye phenotype by RNAi knockdown of identified genes. b, Phenotypes demonstrating enhancement of the (G4C2)58 rough eye phenotype by RNAi knockdown of identified genes. c, Knockdown of identified modifier genes shows little or no phenotype in the absence of G4C2 repeat expression.

Extended Data Figure 6 Impairment of nucleocytoplasmic shuttling in Drosophila and cultured human cell lines.

a, (G4C2)58 expression driven by Fkh-GAL4 causes an abnormal nuclear envelope as shown by Lamin C staining (bottom) in comparison to (G4C2)8 (top). Scale bar, 10 μm. b, Transfection of 293T cells with (G4C2)58 (bottom) but not (G4C2)8 (top) leads to an increase in nuclear RNA puncta as visualized with a total RNA-FISH probe. Non-transfected cells (absence of GFP signal) do not show an increase in nuclear RNA in either (G4C2)8 or (G4C2)58 transfected cells. Scale bar, 25 μm. c, Enlarged images showing slowed accumulation of newly synthesized RNA in the cytoplasm of HeLa cells expressing (G4C2)58. Scale bar, 25 μm.

Extended Data Figure 7 Characterization of newly generated integration-free iPSC lines.

a, iPSC lines from a control subject (line 11) and a G4C2 repeat expansion carrier (line 3 and line 8) express pluripotent markers SSEA-4, Nanog and Oct-4. Scale bar, 50 μm. b, qRT–PCR analysis of expression levels of pluripotent stem-cell markers SOX2 and Nanog in these iPSC lines showing no statistical differences between these lines and human embryonic stem-cell line H9. Relative mRNA levels are quantified from 3 independent experiments. c, After differentiation into cortical neurons about 90% of cells in these cultures are MAP2-positive neurons. Scale bar, 50 μm. d, Quantification of average percentage of MAP2-positive neurons and there is no difference between control and C9orf72 cultures. Average percentages were quantified from 3 independent experiments. e, Quantification of average percentage of VGLUT-positive excitatory neurons among all neurons; there is no difference between control and C9orf72 cultures. n = 3 independent experiments.

Extended Data Figure 8 Karyotyping analysis and pluripotency of newly generated iPSC lines.

a, G-band staining showing a normal karyotype for all the lines analysed. b, After in vitro spontaneous differentiation of control and C9 carrier iPSC lines, cells were stained for α-fetoprotein (endoderm), desmin (mesoderm), βIII-tubulin (ectoderm) and Hoechst (nuclei). All lines showed differentiation towards derivates of three germ layers. Scale bars, 20 μm.

Extended Data Figure 9 Accumulation of nuclear RNA is not seen in fibroblasts derived from patients with G4C2 repeat expansion.

a, b, Total cellular RNA was measured by FISH in fibroblasts derived from 4 control (a) subjects or 5 patients (b) with G4C2 repeat expansion. Scale bar, 25 μm. c, Quantification shows no statistical difference in the observed nuclear to cytoplasmic RNA ratio in patient versus control fibroblasts. n = 16 individual cells analysed for each line.

Extended Data Figure 10 qRT–PCR analysis.

af, qRT–PCR analysis demonstrating knockdown of selected modifiers in Drosophila eyes. mRNA levels of selected modifier (a, d), GAL4 (b, e) and (G4C2)58 (c, f) assayed by qRT–PCR in progeny resulting from wild type (w1118), classical mutant allele or UAS RNAi lines of selected modifiers mated with either GMR-GAL4 or GMR-GAL4/Cyo;UAS-G4C2-58-GFP/TM6 to induce knockdown of the selected gene. RNA was obtained from whole Drosophila head lysates. Gene expression levels are mean ± s.d. from n = 3 independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA, Tukey’s post hoc test. g, h, qRT–PCR analysis demonstrating knockdown of Ref1 and Nup50 in salivary gland. mRNA levels of selected modifier (left), GAL4 (middle) and (G4C2)58 (right) assayed by qRT–PCR in progeny resulting from either P(PZ)Ref102267 (g) or Nup5020824/GD (h) mated with Fkh-GAL4,UAS-G4C2-58-GFP/TM6. RNA was obtained from salivary gland lysates. Gene expression levels are mean ± s.d., n = 3 independent experiments. *P < 0.05 by Student’s t-test.

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Freibaum, B., Lu, Y., Lopez-Gonzalez, R. et al. GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport. Nature 525, 129–133 (2015).

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