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The C9orf72 repeat expansion disrupts nucleocytoplasmic transport


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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Figure 1: Genetic interaction between G4C2 repeats and nucleocytoplasmic transport machinery.
Figure 2: RanGAP binds to G4C2 repeats and is mislocalized along with NPC components.
Figure 3: C9orf72 HRE disrupts the nuclear/cytoplasmic Ran gradient.
Figure 4: C9orf72 HRE causes nucleocytoplasmic transport defects.
Figure 5: Pharmacological rescue of nucleocytoplasmic transport defects.


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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

Authors and Affiliations



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.

Corresponding authors

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

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Genetic interaction between G4C2 repeats and components of the nucleocytoplasmic transport machinery.

a, External eye morphology of 1-day-old (left column) and 15-day-old (left column) flies. Phalloidin staining of the retina of newly eclosed (middle column, magnified in right column) and 15-day-old (middle column, magnified in right column) flies is shown. Flies expressing 30 G4C2 repeats together with (from top row) RanGAPSD(GOF), RanGAP RNAi, RanGEF overexpression, RanGEF RNAi, importin-α overexpression, or exportin RNAi. Genotypes (from top row): (1) GMR-GAL4, UAS-(G4C2)30/RanGAPSD ; (2) GMR-GAL4, UAS-(G4C2)30/+; UAS-RanGAP RNAi/+; (3) GMR-GAL4, UAS-(G4C2)30/+; UAS-RanGEF/+; (4) GMR-GAL4, UAS-(G4C2)30/UAS-RanGEF RNAi; (5) GMR-GAL4, UAS-(G4C2)30/UAS-imp-α2; (6) GMR-GAL4, UAS-(G4C2)30/+; UAS-Exportin RNAi/+ (BL31353). b, c, Quantification of G4C2 mRNA levels by qRT–PCR. d, Flight assay. The top of the graduated cylinder is ‘0’, and thus decreased landing height represents better flight ability. Genotypes (from left lane): (1) and (2) UAS-(G4C2)30/+; elavGS-GAL4/+; (3) UAS-(G4C2)30/+; elavGS-GAL4/UAS-RanGAP. Number of flies (n) tested indicated in column. *P < 0.05; **P < 0.01.

Extended Data Figure 2 RanGAP does not rescue developmental defects caused by G4C2 repeats.

a, Staining of the active zone component Bruchpilot (Brp) was used to identify active zones in the type Ib NMJ of muscle 4 in abdominal segments 3 and 4. b, Quantification of active zone number. c, Electrophysiological recording of NMJ in muscle 6/7 of abdominal segments 3 and 4. dg, Evoked junctional potential (EJP) (d), miniature EJP (mEJP) amplitude (e), quantal content (f), and mEJP frequencies (g) are shown. Genotypes: (1) Ctrl, OK371-GAL4/+; (2) (G4C2)30, OK371-GAL4/+; UAS-(G4C2)30/+; (3) (G4C2)30 RanGAP OE, OK371-GAL4/+; UAS-(G4C2)30/UAS-RanGAP. *P < 0.05; **P < 0.01; ****P < 0.0001.

Extended Data Figure 3 Dot blot of GR and GP dipeptide proteins.

Dot blot of GR (a) and GP (b) compared with actin control. hs indicates heat-shock GAL4, and a heat shock was required to induce detectable polyGR as described7. A transgenic line UAS-(G4C2)36 previously shown to generate polyGR and polyGP DPRs under certain conditions was used as a positive control7.

Extended Data Figure 4 RanGAP/RanGAP1 binds to G4C2 repeats.

a, SDS–PAGE showing purified human RanGAP1. b, EMSA for RanGAP1 with (CUG)20, (C4G2)10, or (G4C2)10 RNA hairpins. c, EMSA for RanGAP1 with increasing length of repeats that were annealed in the presence of K+ to promote RNA G-quadruplex formation. d, Plot of the fraction bound from the EMSAs performed with RanGAP1 and RNA repeats shown in b and c. Similar RNA nucleotide lengths but different binding preferences indicate that RanGAP1 has a structure- and sequence-dependent RNA binding mode (top panel). All data were fit using a hyperbolic and linear regression, then the RanGAP1 binding model determine based on the r2 values for the best fit (n = 2). The length-dependent binding of RanGAP1 fits best to a hyperbolic regression, which demonstrates specific binding to the (G4C2) n G-quadruplex conformation, and the fraction bound increases with increasing nucleotide length (bottom panel). The fraction bound for the RNA hairpins fit best to a linear regression, which indicates nonspecific or less specific binding to RanGAP1. The k1/2 values for specific binding of RanGAP1 to the G-quadruplex RNA conformation are 162, 39 and 11 nM for (G4C2)4, (G4C2)6.5 and (G4C2)10, respectively. e, The RanGAP1–(G4C2)10 RNA G-quadruplex complex is resistant to nonspecific RNA competitors and antisense oligonucleotides (n = 1).

Extended Data Figure 5 RanGAP/RanGAP1 is mislocalized in C9-ALS S2 and iPS cells.

a, RanGAP mislocalization with (G4C2)30 expression is not caused by apoptosis. S2 cells transfected with RanGAP–HA (first column) or RanGAP–HA and (G4C2)30 (second column) were co-stained with HA (red), cleaved Dcp-1 (green) and TO-PRO3 (blue). As a control, S2 cells treated with DMSO (third column) or actinomycin (right column) are co-stained with cleaved Dcp-1 (green) and TO-PRO3 (blue). b, S2 cells transfected with G4C2 were co-stained with a Ran antibody (red) and TO-PRO3 (blue). c, Abnormal aggregated RanGAP1 is variably observed in C9-ALS iPSC neurons and is largely absent from control iPSC neurons. Arrows indicate abnormal RanGAP1 staining. d, Single microscopic plane of aggregated RanGAP1 co-localized with Nup205 at the nuclear membrane (Lamin B) in C9-ALS iPSC neurons. Single immuno-label view in right panels for Nup205 and RanGap1, with xy and xz projections. e, Cytoplasmic RanGAP1 aggregates can co-localize with ubiquitin in C9-ALS iPSC neurons.

Extended Data Figure 6 Electrophysiological and immunocytochemical characterization of iPSC neurons and astroglia.

a, IR-DIC images of iPSC neurons from control (left panel) and C9orf72 (right panel) patient cells (a′). Representative action potentials in response to somatic current injections (70 pA) in iPSC neurons (b′d′). The majority of cells from both groups displayed either single, adaptive or repetitive responses, as demonstrated previously49. These action potentials were blocked by TTX treatment. Normal (e′) and C9orf72 (f′) patient cells displayed mEPSCs that were sensitive to NBQX treatment, suggesting functional synaptic input. Resting membrane potential, membrane capacitance, and membrane resistance were comparable in both groups (g′i′). b, Quantification of iPSC neuron markers showing glutamatergic and Islet-1+ iPSC neurons. c, iPSCs differentiated into neurons include phenotypic markers such as Islet-1, HB9, ChAT (choline acetyl transferase, motor neuron); Tuj1, MAP2, SMI32 (cytoskeletal), VGLUT1 (vesicular glutamate transporter 1), NMDAR1 (NMDA receptor), and synaptic markers SYT1 (synaptotagmin) and SYP (synaptophysin). d, Astroglia markers include ALDH1 (universal astroglial marker) and GFAP (reactive astroglia).

Extended Data Figure 7 Additional human RanGAP1 and Nup107 pathology in C9-ALS brain.

a, b, C9orf72 motor cortex (b) reveals aberrant nuclear localization of RanGAP1, compared to a non C9 control tissue (a), including various nuclear aggregate pathologies seen at higher power in C9orf72 ALS motor cortex (d) as compared to control (c). e, Aberrant RanGAP1 nuclear aggregates were not readily observed in C9-ALS cerebellar cortex molecular layer (ML), Purkinje cells (PK) or granule cell (GL) layer when compared to non C9-ALS control cerebellum. Number in the upper right of each panel identifies autopsy specimen (Supplementary Table 2). f, Nup107 was also aggregated at the nuclear membrane in C9-ALS motor cortex cells when compared to non C9 control tissues.

Extended Data Figure 8 C9orf72 HRE disrupts the cytoplasmic/nuclear Ran gradient.

a, Representative images of disrupted N/C Ran gradient in C9-ALS ChAT+ iPSC neurons. b, c, Representative images and quantification of control (top row) or C9-ALS iPSC neurons (bottom row) expressing Ran-GFP that are co-stained with Ran and MAP2. Both Ran antibody and Ran–GFP indicate a reduced N/C Ran ratio. d, Overexpression of RanGAP1–GFP rescues the N/C Ran ratio in C9-ALS iPSC neurons. e, Control iPSC neurons treated with tunicamycin show enhanced level of activated Caspase 3 in the soma but no change in N/C Ran localization compared to controls with vehicle treatment. f, RanGAP1 is not aggregated in control and C9-ALS iPSC astroglia. g, Representative image of N/C Ran in C9-ALS astrocytes when identified using the pan astroglial ALDH1 marker. h, N/C Ran is not altered in C9-ALS astroglia when comparing astrocytes of a similar size. i, Mean intensity fluorescence (MIF) of nuclear Ran does not differ in control or C9-ALS astroglia. j, Representative image of C9-ALS iPSC neuron with G4C2 RNA foci in approximately 40% of MAP2+ neurons at 50–70 DIV. Number of C9-ALS iPSC neurons with RNA foci is reduced with C9orf72 RNA targeting antisense oligonucleotides compared to scrambled/non-targeting antisense oligonucleotides to <10% of iPSC neurons. k, Antisense oligonucleotides that reduce G4C2 RNA foci also enhance N/C Ran and N/C TDP-43 ratios. *P < 0.05; **P < 0.01; ****P < 0.0001.

Extended Data Figure 9 C9orf72 HRE causes nucleocytoplasmic transport defects.

a, Quantification of the nuclear GFP intensity in Fig. 4a. b, Immunoblot of the GFP levels in Fig. 4a. c, Quantification of the TBPH N/C ratio in Fig. 4a. d, Wild-type control and (G4C2)30-expressing motor neurons expressing NLS–NES–GFP (left two columns) or NLS–ΔNES–GFP (right two columns) co-stained with a GFP antibody (green) and TO-PRO3 (blue) (top row). The GFP signal is shown separately in the bottom row. Genotypes (from left): (1) OK371-GAL4/UAS-NLS-NES-GFP (II); (2) OK371-GAL4/UAS-NLS-NES-GFP; UAS-(G4C2)30/+; (3) OK371-GAL4/+; UAS-NLS-NES(P12)-GFP/+; (4) OK371-GAL4/+; UAS-NLS-NES(P12)-GFP/UAS-(G4C2)30 .

Extended Data Figure 10 Model of C9orf72 mutation induced nucleocytoplasmic transport disruption.

a, In normal cases, RanGAP1 is tethered onto the NPC via RanBP2, where it activates RanGTP hydrolysis to produce RanGDP. RanGDP dissociates from and activates the Importin-αβ complex to import NLS–NES-containing protein cargos such as TDP-43. b, In the nucleus, RanGEF converts RanGDP to RanGTP that is required for the dissociation of the NLS–Importin-αβ complex and the export of NES protein cargoes. c, In C9-ALS, G4C2 HRE binds and sequesters RanGAP1, leading to an increase in cytoplasmic RanGTP. High cytoplasmic RanGTP prevents the formation of the NLS–Importin-αβ complex, thereby disrupting the N/C Ran gradient and impairing nuclear import of NLS-containing proteins. d, Dipeptide repeat proteins translated from the G4C2 RNA can be toxic when expressed at high levels but it is unclear whether they contribute to nucleocytoplasmic trafficking deficits in Drosophila since they are not detected at the time of degeneration. The C9orf72 HRE sense strand appears to be contributing to nucleocytoplasmic trafficking deficits in human iPSC neurons and fly model systems, as small molecules and antisense oligonucleotides targeting the sense RNA substantially suppress the nuclear import phenotypes and neurodegeneration as a result of the G4C2 repeat RNA expression. Overall, the data are most consistent with an RNA-mediated mechanism with evidence that includes: (1) RanGAP1 was identified as 1 of 19 sequence-specific interactors of G4C2 RNA; (2) RanGAP is a strong genetic modifier of G4C2 RNA-mediated degeneration in Drosophila under conditions in which polyGR and polyGP are not detected; (3) RanGAP directly and potently interacts with HRE RNA; and (4) G4C2 RNA foci can co-localize with RanGAP1.

Supplementary information

Supplementary Table 1

This table contains the genetic modifier screen of G4C2-mediated neurodegeneration. (XLSX 36 kb)

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. (PDF 148 kb)

Supplementary Table 4

This table shows the number of C9ORF72 and control iPS quantified for each experiment. (XLSX 40 kb)

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

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