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Monomethylated and unmethylated FUS exhibit increased binding to Transportin and distinguish FTLD-FUS from ALS-FUS

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Abstract

Deposition of the nuclear DNA/RNA-binding protein Fused in sarcoma (FUS) in cytosolic inclusions is a common hallmark of some cases of frontotemporal lobar degeneration (FTLD-FUS) and amyotrophic lateral sclerosis (ALS-FUS). Whether both diseases also share common pathological mechanisms is currently unclear. Based on our previous finding that FUS deposits are hypomethylated in FTLD-FUS but not in ALS-FUS, we have now investigated whether genetic or pharmacological inactivation of Protein arginine methyltransferase 1 (PRMT1) activity results in unmethylated FUS or in alternatively methylated forms of FUS. To do so, we generated FUS-specific monoclonal antibodies that specifically recognize unmethylated arginine (UMA), monomethylated arginine (MMA) or asymmetrically dimethylated arginine (ADMA). Loss of PRMT1 indeed not only results in an increase of UMA FUS and a decrease of ADMA FUS, but also in a significant increase of MMA FUS. Compared to ADMA FUS, UMA and MMA FUS exhibit much higher binding affinities to Transportin-1, the nuclear import receptor of FUS, as measured by pull-down assays and isothermal titration calorimetry. Moreover, we show that MMA FUS occurs exclusively in FTLD-FUS, but not in ALS-FUS. Our findings therefore provide additional evidence that FTLD-FUS and ALS-FUS are caused by distinct disease mechanisms although both share FUS deposits as a common denominator.

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Acknowledgments

We thank Alice Suelzen for technical assistance. We thank H. Earl Ruley (Vanderbilt University School of Medicine; Nashville, TN, USA) for kind gift of mouse embryonic stem cells (mES) (PRMT1 knock-out and wild-type controls) and Elmar Wahle (Martin-Luther-Universität Halle-Wittenberg, Germany) for gift of reagents.

This work was supported by the European Research Council under the European Union’s Seventh Framework Program (FP7/2007–2013)/ERC Grant Agreement No. 321366-Amyloid (advanced grant to C.H.), the Deutsche Forschungsgemeinschaft (German Research Foundation) within the framework of the Munich Cluster for Systems Neurology (EXC 1010 SyNergy D.D., C.H.) and the Emmy Noether program DO 1804/1-1 (to D.D.), and the general legacy of Mrs. Ammer (to the Ludwig-Maximilians-University/the chair of C.H.) and the German Helmholtz Association (Grant VH-VI-510 to C.H. and M.N.; Grant W2/W3-036 to M.N.). M.S. was supported by a grant from the Fondo de Investigación Sanitaria (FI09/00732), Instituto Carlos III, Madrid, Spain. T.M. was supported by the Bavarian Ministry of Sciences, Research and the Arts (Bavarian Molecular Biosystems Research Network), the German Research Foundation (Emmy Noether program MA 5703/1-1), the Centre for Integrated Protein Science Munich (CIPSM), the President’s International Fellowship Initiative of CAS (No:2015VBB045), and the National Natural Science Foundation of China (No. 31450110423). We gratefully acknowledge the support of the NOMIS foundation (to M.D.R.), the Holcim Stiftung zur Föderung der wissenschaftlichen Fortbildung (to M.D.R.), and the Fondation Dufloteau (to M.D.R.).

Author information

Author notes

  1. Claudia Abou-Ajram & Dorothee Dormann

    Present address: BioMedical Center (BMC), Lehrstuhl Zellbiologie (Anatomie III), Großhaderner Strasse 9, 82152, Planegg-Martinsried, Germany

Authors and Affiliations

  1. Biomedical Center (BMC), Biochemistry, Ludwig-Maximilians-University Munich, Feodor-Lynen Strasse 17, 81377, Munich, Germany

    Marc Suárez-Calvet, Claudia Abou-Ajram, Eva Funk, Dorothee Dormann & Christian Haass

  2. Department of Neuropathology, University of Tübingen, 72076, Tübingen, Germany

    Manuela Neumann

  3. DZNE, German Center for Neurodegenerative Diseases, 72076, Tübingen, Germany

    Manuela Neumann

  4. Department of Psychiatry and Psychotherapy, Ludwig-Maximilians-University Munich, 80336, Munich, Germany

    Thomas Arzberger

  5. Center for Neuropathology and Prion Research, Ludwig-Maximilians-University Munich, 81377, Munich, Germany

    Thomas Arzberger

  6. German Center for Neurodegenerative Diseases (DZNE) Munich, Feodor-Lynen Strasse 17, 81377, Munich, Germany

    Marc Suárez-Calvet, Thomas Arzberger, Hannelore Hartmann, Dieter Edbauer & Christian Haass

  7. Munich Cluster for Systems Neurology (SyNergy), 81377, Munich, Germany

    Hannelore Hartmann, Dieter Edbauer, Dorothee Dormann & Christian Haass

  8. Universitat Autònoma de Barcelona, 08193, Bellaterra, Barcelona, Spain

    Marc Suárez-Calvet

  9. Institute of Molecular Immunology, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), 81377, Munich, Germany

    Elisabeth Kremmer

  10. Department of Chemistry, Center for Integrated Protein Science Munich (CIPSM), Technische Universität München, Lichtenbergstr.4, 85747, Garching, Germany

    Christoph Göbl, Moritz Resch, Benjamin Bourgeois & Tobias Madl

  11. Institute of Structural Biology, Helmholtz Zentrum München, 85764, Neuherberg, Germany

    Christoph Göbl, Moritz Resch, Benjamin Bourgeois & Tobias Madl

  12. Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, 8010, Graz, Austria

    Tobias Madl

  13. Omics Center Graz, BioTechMed, 8010, Graz, Austria

    Tobias Madl

  14. Department of Chemistry and Biochemistry, University of Bern, 3012, Bern, Switzerland

    Stefan Reber, Daniel Jutzi & Marc-David Ruepp

  15. Graduate School for Cellular and Biomedical Sciences, University of Bern, 3012, Bern, Switzerland

    Stefan Reber

  16. Department of Pathology, Vancouver General Hospital, University of British Columbia, Vancouver, Canada

    Ian R. A. Mackenzie

  17. Department of Neuropathology, John Radcliffe Hospital, Oxford, UK

    Olaf Ansorge

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  1. Marc Suárez-Calvet
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401_2016_1544_MOESM1_ESM.eps

Supplementary material 1 (EPS 17351 kb) Supplementary Fig. 1 To verify specificity in mES cells, the UMA FUS (14G1) (a) and MMA FUS (15E11) (b) antibodies were pre-incubated with increasing concentrations of the three peptides used for immunization: UMA FUS473-503, MMA FUS473-503 and ADMA FUS473-503. Subsequently, immunoblots on mES cell lysates using the UMA FUS antibody or MMA FUS antibody not pre-preincubated with any peptide (first lane) or pre-incubated with increasing concentrations of each of the peptides (second to forth lane) were performed. The specific band recognized by the UMA and MMA FUS antibodies selectively disappeared when it was pre-incubated with the UMA FUS473-503 or MMA FUS473-503 peptide, respectively. Asterisks indicate unspecific bands

401_2016_1544_MOESM2_ESM.eps

Supplementary material 2 (EPS 1894 kb) Supplementary Fig. 2 Isothermal titration calorimetry (ITC) titrations of Transportin-1 with differentially arginine methylated synthetic FUS. A FUS peptide solution was titrated into a Transportin-1 solution and the heat release upon binding of the peptide to Transportin-1 was detected. From the heat release, the characteristic thermodynamic parameters of interactions in solution including binding affinity, enthalpy changes (ΔH), and entropy changes (ΔS) can be determined. Experimental calorimetric data of the binding of FUS473-503 (synthetic) to Transportin-1 is shown. The arginine methylation status is indicated. The experiment was carried out at 25 °C. Dilution heats measured by titrating FUS into the corresponding buffer were in the range of the heat effects observed at the end of the titration (data not shown) and were subtracted for the analysis

401_2016_1544_MOESM3_ESM.tif

Supplementary material 3 (TIFF 17265 kb) Supplementary Fig. 3 (a) siRNAs against FUS, EWS, TAF-15 or a non-targeting control were transfected into HeLa cells, and cells were either left untreated or treated with AdOx. Lysates were analysed 48 h post-transfection by immunoblotting with the antibody raised against UMA FUS (2A3). This antibody only reacts with lysates derived from AdOx-treated cells and the signal disappears upon knockdown of FUS. Tubulin was used as a loading control and FUS, EWS and TAF-15 antibodies were used to examine knockdown efficiency. Immunohistochemical detection of UMA FUS in the superior frontal gyrus of a FTLD-FUS (NIFID) case using the 2A3 antibody. (b) In the FTLD-FUS case there are abundant neuronal cytoplasmic inclusions, which are not detectable in the frontal cortex of a control case (c). (d-f) Higher magnification reveals the diversity of NCI morphology as seen for 14G1 antibody (Fig. 9). Scale bars in b, c = 100 µm, scale bars in d-f = 50 µm

401_2016_1544_MOESM4_ESM.tif

Supplementary material 4 (TIFF 17346 kb) Supplementary Fig. 4 (a) siRNAs against FUS, EWS, TAF-15 or a non-targeting control were transfected into HeLa cells, and cells were either left untreated or treated with AdOx. Lysates were analysed 48 h post-transfection by immunoblotting with the antibody raised against MMA FUS (18E11). This antibody only reacts with lysates derived from AdOx-treated cells and the signal disappears upon knockdown of FUS. Tubulin was used as a loading control and FUS, EWS and TAF-15 antibodies were used to examine knockdown efficiency. Asterisks: unspecific bands. Immunohistochemical detection of MMA FUS in the superior frontal gyrus of a FTLD-FUS (NIFID) case using the 18E11 antibody. (b) In the FTLD-FUS case there are abundant neuronal cytoplasmic inclusions that are not detectable in the frontal cortex of a control case (c). (d-f) Higher magnification reveals the diversity of NCI morphology as seen for 15E11 antibody (Fig. 11). Scale bars in b, c = 100 µm, scale bars in d-f = 50 µm

401_2016_1544_MOESM5_ESM.tif

Supplementary material 5 (TIFF 25017 kb) Supplementary Fig. 5 Double immunofluorescence with the UMA FUS antibody 14G1 (a) or MMA FUS antibody 15E11 (b) (green), a TRN-specific antibody (red) and nuclear counterstaining with DAPI (blue) in a FTLD-FUS (NIFID) case. UMA FUS or MMA FUS-positive inclusions consistently showed co-labeling for TRN. Scale bar: 50 µm

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Suárez-Calvet, M., Neumann, M., Arzberger, T. et al. Monomethylated and unmethylated FUS exhibit increased binding to Transportin and distinguish FTLD-FUS from ALS-FUS . Acta Neuropathol 131, 587–604 (2016). https://doi.org/10.1007/s00401-016-1544-2

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