GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport

Abstract

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 dementia. 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.

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

a, G₄C₂ 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. 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 seconds (right) when G₄C₂-58 is expressed in all neurons using the elav-GAL4 driver. d, Quantification of the distance traveled 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=3 trials, more than 4 larvae per group in each trial. ** 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 buoton number (left) and muscle size (right) reveal both are significantly reduced in Drosophila larvae expressing G₄C₂-58 repeats. Values are mean ± s.e.m. n ≥ 5, ** 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 > 20 for all genotypes). This phenotype was assessed by examining the permanent wing posture of live adult flies.

Extended Data Figure 2. RAN translation is observed in the 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 bars: 25 μm. g, Expression of G₄C₂-58 in pan neuronal cells by elav driver results in the nuclear and cytoplasmic inclusions. Scale bar: 25 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 nontoxic 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 Ran (T24N) 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 significantly 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 vs. middle). Crm1 knockdown in the absence of G₄C₂-58 repeats does not produce a rough eye phenotype (left). 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.

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 knock down of identified genes. b, Phenotypes demonstrating enhancement of the G₄C₂-58 rough eye phenotype by RNAi knock down of identified genes. c, Knock down 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-transefected cells (absence of GFP signal) do not show an increase in nuclear RNA in either G₄C₂-8 or G₄C₂-58 transfected cell. Scale bars: 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 iPSCs 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. Bars: 50 μm. b, qRT-PCR analysis ofexpression levels of pluripotent stem cell markers SOX2 and Nanog in these iPSCs lines showing no statistical differences between these lines and human embryonic stem cell line H9. c, After differentiation into cortical neurons about 90% of cells in these cultures are MAP2-positive neurons. d, Quantification of average percentage of MAP2-positive neurons and there is no difference between control and C9ORF72 cultures. e, Quantification of average percentage of VGLUT-positive excitatory neurons among all neurons and there is no difference between control and C9ORF72 cultures. Scale bars in all panels: 50 mm.

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 analyzed. b, After in vitro spontaneous differentiation of control and C9 carrier iPSC lines, cells were staining for α-fetoprotein (AFP, endoderm), desmin (mesoderm), βIII-tubulin (ectoderm), and Hoechst (nuclei). All lines showed differentiation towards derivates of three germ layers. Scale bars: 20 mm.

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

a and b, Total cellular RNA was measured by FISH in fibroblasts derived from either control (a) subjects or patients (b) with G₄C₂ repeat expansion. Scale bars: 25 μm. c, Quantification shows no statistical difference in the observed nuclear to cytoplasmic RNA ratio in patient vs. control fibroblasts. n=16 for all lines.

Extended Data Figure 10. qRT-PCR Analysis

a–f. 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., *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., *p < 0.05 by Student’s t-test.

Figure 1. G₄C₂ repeats induces length- and dosage-dependent degeneration in Drosophila

a, Constructs expressing 8, 28, or 58 copies of G₄C₂ repeats. b, G₄C₂-58 causes a rough eye phenotype. c, Two copies of G₄C₂-58 expressed in motor neurons results in a decrease in larval size (left panel) and locomotor activity (right panel). d, Quantification of the distance traveled by larvae expressing repeats in motor neurons. Values are mean ± s.e.m., n = 3 trials, ** p < 0.01, by One Way ANOVA, Tukey’s Post Hoc test. e, Expression of two copies of G₄C₂-58 in motor neurons reduces bouton number. Scale bar: 25 μm. f, Quantification of bouton number and muscle size in larvae expressing G₄C₂ repeats. Values are mean ± s.e.m., n = 6, ** p < 0.01, by One Way ANOVA, Tukey’s Post Hoc test.

Figure 2. Genetic screen identifies multiple modifiers of G₄C₂-58 toxicity in the nucleocytoplasmic transport pathway

a, G₄C₂-58 expression driven by GMR-GAL4 causes a rough eye phenotype (top right) that was enhanced by either Df(2R)1725/+ or Df(2R)1735/+(middle). b, Overlapping genomic region of deficiency lines Df(2R)1725 or Df(2R)1735. c, RNAi knockdown (Nup50^20824/GD and Nup50^100564/KK) and a genetic allele (Nup50^KG09557) identify Nup50 as an enhancer. d, The deficiency Df(3R)Antp17 (green) but not others (grey) suppressed the G₄C₂-58 eye phenotype. e and f, Identification of Ref as a suppressor. g, Table summarizing modifier genes and their known functions. h, Suppressors (green) and enhancers (red) of G₄C₂-58 toxicity in the nucleocytoplasmic trafficking pathway.

Figure 3. Drosophila salivary gland cells expressing G₄C₂-58 exhibit nuclear envelope abnormalities and accumulation of nuclear RNA

a, The effect of G₄C₂-58 expression on Nup107 localization. Scale bar: 50 μm. b, G₄C₂-58 expression causes abnormal nuclear envelope morphology. Scale bar: 50 μm. c, Quantification of the frayed nuclear envelope phenotype in cells expressing either G₄C₂-8 (n = 251) or G₄C₂-58 (n = 127). Values are mean ± s.e.m., ** p < 0.001 by Student’s t test. d, Accumulation of total nuclear RNA relative to cytoplasmic RNA (red). Knockdown of Ref1 led to a partial rescue of nuclear RNA accumulation. Scale bar: 25 μm. e and f, Quantification of relative intensity of total nuclear RNA vs. cytoplasmic RNA. Values are mean ± s.e.m., n = 30 cells, * p < 0.01, ** p < 0.001 by One Way ANOVA, Tukey’s Post Hoc test.

Figure 4. Accumulation of nuclear RNA in human cells expressing G₄C₂ repeat expansion

a, Reduced accumulation of newly synthesized RNA in the cytoplasm of cells transfected with G₄C₂-58 (white arrows) (imaged at 60 min). Yellow arrows: untransfected cells. b, Quantification of cytoplasmic RNA intensity following metabolic labeling of newly synthesized RNA (0 min: n > 24, 30 min: n > 7, 60 min: n > 25). ** p < 0.01, *** p < 0.001 by One Way ANOVA, Tukey’s Post Hoc test. c, Total RNA intensity following labeling of newly synthesized RNA at time point 0 min. d, PolyA⁺ RNA was measured by FISH in transiently transfected HeLa cells. Scale bar: 50 μm. e, Quantification of cells containing polyA⁺ RNA puncta, n = 30 cells per condition. f, Total cellular RNA in 2-month old iPSCs-derived cortical neurons. Scale bar: 25 μm. g, Quantification of the cytoplasmic RNA ratio in patient cortical neurons vs. controls. Values are mean ± s.e.m., n > 15 cells per line, ** p < 0.01 by Student’s t test.