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C9ORF72 interaction with cofilin modulates actin dynamics in motor neurons

Abstract

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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Figure 1: C9ORF72 expression modulates axon growth in cultured motor neurons.
Figure 2: C9ORF72 interacts with cofilin.
Figure 3: Enhanced phospho-cofilin and reduced C9ORF72 in patient-derived cells and tissues.
Figure 4: C9ORF72 regulates actin dynamics.
Figure 5: Altered G/F actin ratio in motor neurons after overexpression and knockdown of C9ORF72.
Figure 6: C9ORF72 interaction with Arf6.
Figure 7: C9ORF72 depletion leads to activation of Arf6.
Figure 8: Reducing activity of Arf6 rescues Rac1 activation.

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Acknowledgements

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).

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R.S., M.S., C.D., D.H., F.M. and M.M. designed the experiments. R.S. developed lentiviruses, performed all experiments to characterize the function of C9ORF72 in cultured motor neurons. C.D. and A. Hansel helped with the initial generation of viral vectors for C9ORF72-HA overexpression and knockdown. D.H. did the LC-MS experiments and D.H., M.F. and M.M. were responsible for the analysis of the LC-MS results. N.F. helped with live cell imaging and analysis. S.J. collected and contributed the lymphoblastoid cell lines. A. Hermann, J.S. and X.L. contributed iPSCs and performed experiments with iPSC-derived motor neurons shown in Supplementary Figure 6. P.J.S. and P.G.I. collected and provided post-mortem tissues from ALS patients. R.S. and M.S. wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Michael Sendtner.

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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.

Supplementary Figure 3 Interactome of C9ORF72 in NSC34 cells.

(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.

Supplementary Figure 4 Interaction of C9ORF72 and cofilin in motor neurons.

(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.

Supplementary Figure 6 Characterization of the C9ORF72 patient-specific iPSC cell lines.

(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.

Supplementary Figure 7 Immunocytochemical characterization of iPSC neurons.

(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 Figure 8 Full-length pictures of the blots presented in the main figures.

Supplementary Figure 9 Full-length pictures of the blots presented in the main and supplementary figures.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9 (PDF 1717 kb)

Supplementary Methods Checklist (PDF 471 kb)

Supplementary Table 1

Protein table: List of identified C9ORF72 interactors (XLSX 330 kb)

Supplementary Table 2

GO term analysis of identified C9ORF72 interactors (XLSX 11 kb)

Live cell imaging of the axonal growth cone in a motoneuron transduced with GFP-actin sh scrambled. (AVI 1815 kb)

Live cell imaging of the axonal growth cone in a motoneuron transduced with GFP-actin sh C9ORF72 (AVI 727 kb)

41593_2016_BFnn4407_MOESM59_ESM.avi

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)

41593_2016_BFnn4407_MOESM60_ESM.avi

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

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