Intronic hexanucleotide expansions in C9ORF72 are common in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, but it is unknown whether loss of function, toxicity by the expanded RNA or dipeptides from non-ATG-initiated translation are responsible for the pathophysiology. We determined the interactome of C9ORF72 in motor neurons and found that C9ORF72 was present in a complex with cofilin and other actin binding proteins. Phosphorylation of cofilin was enhanced in C9ORF72-depleted motor neurons, in patient-derived lymphoblastoid cells, induced pluripotent stem cell–derived motor neurons and post-mortem brain samples from ALS patients. C9ORF72 modulates the activity of the small GTPases Arf6 and Rac1, resulting in enhanced activity of LIM-kinases 1 and 2 (LIMK1/2). This results in reduced axonal actin dynamics in C9ORF72-depleted motor neurons. Dominant negative Arf6 rescues this defect, suggesting that C9ORF72 acts as a modulator of small GTPases in a pathway that regulates axonal actin dynamics.
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We thank R. Sendtner, H. Troll, E. Spirk, and N. Rachor for skillful technical assistance, J. Rieckmann, M. Moradi and P. Lüningschör for discussions, R. Rush from Flinders University, Adelaide, Australia for donating the p75NTR antibody and R. Blum from the Institute of Clinical Neurobiology, University of Wuerzburg for the GFP-actin construct and advice. This work was supported by the European Community′s Health Seventh Framework Programme (FP7) under grant agreement no. 259867 (M.S., P.J.S.), the Hermann-und-Lilly Schilling Stiftung im Stifterverband der Deutschen Industrie (M.S.), a grant by the Deutsche Gesellschaft für Muskelerkrankungen, IBC He 2/2 (A. Hermann, M.S.), The Bavarian Excellence Program ForIPS (M.S.), The DFG SPP 1738 (M.S.), the MeDDrive of the Medical Faculty of the Technische Universität Dresden (A. Hermann), BIOCREA GmbH to A. Hermann, the Helmholtz Virtual Institute program RNA Dysmetabolism in ALS, NOMIS Foundation (A. Hermann) and FTD (VI-510) (A. Hermann), and an unrestricted grant by a family of a deceased ALS patient (A. Hermann).
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Confirmation of C9ORF72 overexpression and knockdown in cultured mouse motor neurons.
A) Diagram of hu-C9ORF72 overexpression vector. (B) Diagram of C9ORF72 shRNA vector with co-expression of GFP under the CMV promoter. (C) Quantification of C9ORF72 RNA expression levels by qPCR in motor neurons infected with and without lentivirus with scrambled shRNA and C9ORF72 shRNA (ANOVA with Bonferroni posthoc test, mean ± s.e.m., F (2, 21)=3.98, p=>0.999, p=<0.001, p=<0.001, n=6 independent experiments). (D) Protein quantification of C9ORF72 protein levels in cultured motor neurons after overexpression or knockdown of C9ORF72. This figure shows one representative blot from n=6 independent experiments. (E) Quantification of the western blots, as shown in D. n=6 independent experiments. F (2, 21)=3.98, p=>0.999, p=<0.001, p=<0.001 ***, P < 0.001; ANOVA with Bonferroni posthoc test, mean ± s.e.m.
Supplementary Figure 2 Altered C9ORF72 expression does not affect motor neuron survival, and overexpression of human C9ORF72 can rescue the axon length defect after knockdown of endogenous mouse C9ORF72.
A) Survival of cultured mouse motor neurons transduced with lentiviral vectors for C9ORF72 overexpression or knockdown, as indicated. Cells were cultured with or without 5ng/ml BDNF, as indicated. Graph shows data from n=4 independent experiments, 100 cells per condition assayed. Kruskal-Wallis statistic= 31.7, p=0.0126, p=>0.999, p=0.0207, p=>0.999, p=0.0374, p=>0.999, p=0.0089. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ANOVA with Kruskal-Wallis test, mean ± s.e.m. (B) Representative image of control motor neurons or motor neurons after lentiviral C9ORF72 overexpression or knockdown at 7d in vitro in the presence of BDNF, stained for tubulin to visualize processes. Bars, 100μm. (C) Human C9ORF72-HA overexpression rescues defective axon elongation caused by knockdown of mouse C9ORF72 after 7d in vitro culture (ANOVA with Bonferroni posthoc test, The central line represents the median, the box limits the interquartile range, and the whiskers the minimum and maximum, F (4, 414) =21.93, p= < 0.001, p=<0.001, p=<0.001, p=>0.999, p=0.935, *** P<0.001, n=4 independent experiments, 100 cells per condition assayed). NS: not significant.
(A) Western blot analysis of HEK293 and NSC-34 cells overexpressing huC9ORF72-HA. (B) Western blot analysis of immunoprecipitates of huC9ORF72-HA protein. Transduced huC9ORF72 was precipitated with antibodies against HA, resulting blots were stained with antibodies against huC9ORF72. (C) Confirmative blot of samples used for LC-MS analysis. Samples were pulled down with HA antibody and the blots were probed with C9ORF72 antibody. 1% of immunoprecipitation sample and 10% of input was used for the analysis.
(A) HA pulldown of C9ORF72-HA protein from motor neurons cultured for 7d in vitro and immunoblotting for C9ORF72 and cofilin. (B) Immunoprecipitation of endogenous C9ORF72 from cultured motor neurons. The resulting immunoblot exposed to cofilin antibodies confirms the interaction of C9ORF72 with cofilin. (C) Motor neurons after 7d in vitro stained for C9ORF72-HA, cofilin and phalloidin. Bars 100μm. (D) Colocalization of C9ORF72-HA with cofilin in axonal growth cones of motor neurons grown for 5d in vitro. Bars 10μm.
Supplementary Figure 5 Construction and efficacy of viral constructs for C9ORF72 knockdown and coexpression of GFP-actin.
(A) Representative scheme of the C9ORF72 shRNA vector with co-expression of GFP-actin under the CMV promoter. (B) Motor neurons transduced with C9ORF72 shRNA or scrambled shRNA lentiviruses were detected at 7d in vitro by co expressed GFP-actin. GFP-actin co-localizes with actin and tubulin in axonal processes. Bars 100μm. (C) Western blot analysis of cultured motor neurons at 7d in vitro transduced with shRNA for scrambled and C9ORF72 with coexpression of GFP and GFP-actin. The blot also shows the effect of knockdown on C9ORF72 protein levels when extracts from cells transduced with shRNA viruses are compared with cells carrying control scrambled constructs.
(A) C9ORF72 iPSC colonies express characteristic pluripotency markers as shown by an immunofluorescence staining of Oct4, TRA-1-60 and TRA-1-81. (B) C9ORF72 iPSCs (#1=patient1; #2=patient2) are successfully silenced upon reprogramming. FIB, fibroblasts (C) C9ORF72 cells successfully differentiate into cells from all there germ layers as shown by the expression of the germ layer markers Catenin (Endoderm), alpha-SMA/FN (Mesoderm; FN= fibronectin) and TUJ1 (Ectoderm). Scale bars 50µm.
(A) iPSCs differentiated into motor neuron include phenotypic markers such as Islet-1, HB9, Tuj1, MAP2 both in control and C9-ALS derived neurons. Bars 100μm.
Supplementary Figures 1–9 (PDF 1717 kb)
Protein table: List of identified C9ORF72 interactors (XLSX 330 kb)
GO term analysis of identified C9ORF72 interactors (XLSX 11 kb)
Live cell imaging of the axonal growth cone in a human control iPS cell derived motoneuron transduced with GFP-actin sh scrambled (AVI 675 kb)
Live cell imaging of the axonal growth cone in a human C9-ALS iPS cell derived motoneuron transduced with GFP-actin sh scrambled (AVI 766 kb)
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Sivadasan, R., Hornburg, D., Drepper, C. et al. C9ORF72 interaction with cofilin modulates actin dynamics in motor neurons. Nat Neurosci 19, 1610–1618 (2016). https://doi.org/10.1038/nn.4407