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

April 2016
Acta Neuropathologica 131(4)

April 2016
131(4)

DOI:10.1007/s00401-016-1544-2

Authors:

Marc Suárez-Calvet

BarcelonaBeta Brain Research Center (BBRC)

Manuela Neumann

University of Tuebingen

Thomas Arzberger

Ludwig-Maximilians-University of Munich

Show all 19 authorsHide

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.

Characterization of monoclonal antibodies specific for UMA and MMA FUS. a Schematic figure of the FUS protein. FUS contains multiple functional domains: a QGSY-rich transcriptional activation domain (glutamine, glycine, serine and tyrosine rich domain), three RGG domains (arginine-glycine-glycine repeats); a RRM domain (RNA recognition motif), a ZnF domain (zinc finger domain), and the PY-NLS (proline-tyrosine nuclear localization signal). We generated antibodies against the differently methylated RGG3 domain (FUS473–503) with all nine arginines in three different forms of methylation modifications: UMA (red), MMA (blue) or ADMA (green). An asterisk indicates a methyl group. b Schematic representation of arginine methylation steps. All PRMTs (type I, II and III) are capable to add a methyl group and generate a monomethylated arginine (MMA). Subsequently, type I PRMTs deposit the asymmetrically dimethylated arginine (ADMA) signature, whereas type II PRMTs deposit the symmetrically dimethylated ariginine (SMDA) signature. Type III PRMT (PRMT7) can only generate MMA. c HeLa cells were transfected with siRNAs against FUS, EWS, TAF-15 or a non-targeting control and were subsequently treated with AdOx or left untreated. 48 h post-transfection cell lysates were analysed by immunoblotting with the antibodies raised against UMA FUS (14G1), MMA FUS (15E11) and ADMA (9G6). All of them show a signal at the predicted molecular weight of FUS (arrow) that disappears upon knockdown of FUS. Whereas ADMA FUS antibody (9G6) shows immunoreactivity to untreated cells, UMA FUS (14G1) and MMA FUS (15E11) antibodies only reacted with lysates derived from AdOx-treated cells. These antibodies do not show cross-reactivity against EWS and TAF-15. Tubulin was used as a loading control and FUS, EWS and TAF-15 antibodies were used to examine knockdown efficiency. Asterisks: unspecific bands

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MMA FUS antibody labels inclusions in FTLD-FUS but not in ALS-FUS. Double-label immunofluorescence with MMA FUS antibody 15E11 (green), a pan-FUS antibody (red) and nuclear counterstaining with Hoechst (blue). The majority of FUS-positive cytoplasmic and intranuclear inclusions in aFTLD-U (dentate gyrus), NIFID (dentate gyrus) and BIBD (frontal cortex) are co-labelled by the MMA FUS-specific antibody, in striking contrast to the FUS-positive inclusions in ALS-FUS cases with different mutations, which are not labelled with the MMA FUS antibody. Scale bar 50 µm

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MMA FUS immunohistochemistry in FTLD-FUS brains. Immunohistochemical detection of MMA FUS in the superior frontal gyrus of a FTLD-FUS (NIFID) case using the 15E11 antibody. a In the FTLD-FUS case neuronal cytoplasmic inclusions are mainly found in the outer cortical layers (layers II and III); (insert represents zoomed stippled rectangle). b, c 15E11 positive inclusions are not detectable in the frontal cortex of a control or a FTLD-TDP case. d–g As seen for UMA FUS (Fig. 9) cytoplasmic inclusions can be dot-like (black arrow), ring-like (violet arrow), crescent (red arrow) or triangular (green arrow). There is a non-specific staining of axonal tracks in the FTLD-FUS (h), the control (i) and a FTLD-TDP case (j), which is most obvious in the subcortical white matter (asterisks in h, i and j). This background staining most likely reflects cross-reactivity with additional MMA proteins (besides MMA FUS) as shown by this antibody also in immunoblots (Fig. 1c; Fig. 2a). Scale bars in a–c, represent 200 µm, scale bars in d–g 50 µm, scale bars in h–j 500 µm

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Loss of PRMT1 elicits a switch in the methylation pattern of FUS from ADMA to MMA and UMA. a Whole cell lysates of PRMT1 knockout (PRMT1 −/−) and wild-type (PRMT1 +/+) mES cells (duplicates from two independent experiments) were prepared and immunoblotted with antibodies specific for ADMA FUS (9G6), MMA FUS (15E11) and UMA FUS (14G1). A decrease in ADMA FUS paralleled by an increase in UMA and MMA FUS is observed in PRMT1 −/− mES cells. Membranes were blotted with anti-Tubulin and anti-total FUS antibodies to visualize equal loading and with a PRMT1-specific antibody to confirm PRMT1 knockout. Asterisks indicate unspecific bands. b, c Whole cell lysates were subjected to immunoprecipitation (IP) with an anti-FUS antibody and an isotype control antibody. Precipitated proteins were analysed by immunoblotting (IB) with a general anti-MMA antibody (D5A12) that recognizes monomethylated RGG motifs. This demonstrates an increase in MMA FUS in PRMT1 −/− cells (top panel). The input (whole cell lysates) is shown in the left panel. Equal amounts of FUS protein were immunoprecipitated from PRMT1 +/+ and PRMT1 −/− cells (bottom panel). The same immunoprecipitated material was also immunoblotted with another anti-MMA antibody (Me-R4-100) raised against monomethylated arginine. This confirmed an increase in MMA FUS upon PRMT1 knockout (PRMT1 −/−). d Immunoprecipitation (IP) of monomethylated proteins followed by immunoblotting (IB) for FUS confirmed an increase of MMA FUS in PRMT1 −/− cells

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Neuronal FUS switches its methylation pattern from ADMA to MMA and UMA upon treatment with AdOx. Rat cortical neurons (DIV 7) were treated with AdOx (10 µM) for 48 h or left untreated and examined by immunoblotting with antibodies specific for UMA FUS (14G1), MMA FUS (15E11) and ADMA FUS (9G6). In line with the results observed in HeLa and mES cells, UMA FUS (14G1) and MMA FUS (15E11) were selectively increased upon treatment with AdOx, whereas ADMA FUS was decreased. Tubulin was used as a loading control. Duplicates from two independent experiments are shown. Asterisks: unspecific bands

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1 3

Acta Neuropathol (2016) 131:587–604

DOI 10.1007/s00401-016-1544-2

ORIGINAL PAPER

Monomethylated and unmethylated FUS exhibit increased

binding to Transportin and distinguish FTLD‑FUS

from ALS‑FUS

Marc Suárez‑Calvet1,7,9 · Manuela Neumann3,4 · Thomas Arzberger5,6,7 ·

Claudia Abou‑Ajram1,2 · Eva Funk1 · Hannelore Hartmann7,8 · Dieter Edbauer7,8 ·

Elisabeth Kremmer10 · Christoph Göbl11,12 · Moritz Resch11,12 ·

Benjamin Bourgeois11,12 · Tobias Madl11,12,13,14 · Stefan Reber15,16 ·

Daniel Jutzi15 · Marc‑David Ruepp15 · Ian R. A. Mackenzie17 · Olaf Ansorge18 ·

Dorothee Dormann1,2,8 · Christian Haass1,7,8

Received: 31 July 2015 / Revised: 29 January 2016 / Accepted: 29 January 2016 / Published online: 19 February 2016

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 Pro-

tein 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 argi-

nine (UMA), monomethylated arginine (MMA) or asym-

metrically dimethylated arginine (ADMA). Loss of PRMT1

indeed not only results in an increase of UMA FUS and a

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 sclero-

sis (ALS-FUS). Whether both diseases also share common

pathological mechanisms is currently unclear. Based on our

Electronic supplementary material The online version of this

article (doi:10.1007/s00401-016-1544-2) contains supplementary

material, which is available to authorized users.

* Dorothee Dormann

dorothee.dormann@med.uni-muenchen.de

* Christian Haass

christian.haass@mail03.med.uni-muenchen.de

1 Biomedical Center (BMC), Biochemistry, Ludwig-

Maximilians-University Munich, Feodor-Lynen Strasse 17

81377 Munich, Germany

2 Present Address: BioMedical Center (BMC), Lehrstuhl

Zellbiologie (Anatomie III), Großhaderner Strasse 9,

82152 Planegg-Martinsried, Germany

3 Department of Neuropathology, University of Tübingen,

72076 Tübingen, Germany

4 DZNE, German Center for Neurodegenerative Diseases,

72076 Tübingen, Germany

5 Department of Psychiatry and Psychotherapy, Ludwig-

Maximilians-University Munich, 80336 Munich, Germany

6 Center for Neuropathology and Prion Research, Ludwig-

Maximilians-University Munich, 81377 Munich, Germany

7 German Center for Neurodegenerative Diseases (DZNE)

Munich, Feodor-Lynen Strasse 17, 81377 Munich, Germany

8 Munich Cluster for Systems Neurology (SyNergy),

81377 Munich, Germany

9 Universitat Autònoma de Barcelona, 08193 Bellaterra,

Barcelona, Spain

10 Institute of Molecular Immunology, Helmholtz Zentrum

München, German Research Center for Environmental

Health (GmbH), 81377 Munich, Germany

11 Department of Chemistry, Center for Integrated Protein

Science Munich (CIPSM), Technische Universität München,

Lichtenbergstr.4, 85747 Garching, Germany

12 Institute of Structural Biology, Helmholtz Zentrum München,

85764 Neuherberg, Germany

13 Institute of Molecular Biology and Biochemistry, Center

of Molecular Medicine, Medical University of Graz,

8010 Graz, Austria

14 Omics Center Graz, BioTechMed, 8010 Graz, Austria

15 Department of Chemistry and Biochemistry, University

of Bern, 3012 Bern, Switzerland

16 Graduate School for Cellular and Biomedical Sciences,

University of Bern, 3012 Bern, Switzerland

17 Department of Pathology, Vancouver General Hospital,

University of British Columbia, Vancouver, Canada

18 Department of Neuropathology, John Radcliffe Hospital,

Oxford, UK

Content courtesy of Springer Nature, terms of use apply. Rights reserved.

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NEUROPATH APPL NEURO

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Abnormal cytoplasmic accumulation of Fused in Sarcoma (FUS) in neurons defines subtypes of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). FUS is a member of the FET protein family that includes Ewing's sarcoma (EWS) and TATA-binding protein-associated factor 2N (TAF15). FET proteins are predominantly localized to the nucleus, where they bind RNA and DNA to modulate transcription, mRNA splicing, and DNA repair. In ALS cases with FUS inclusions (ALS-FUS), mutations in the FUS gene cause disease, whereas FTLD cases with FUS inclusions (FTLD-FUS) do not harbor FUS mutations. Notably, in FTLD-FUS, all FET proteins accumulate with their nuclear import receptor Transportin 1 (TRN1), in contrast ALS-FUS inclusions are exclusively positive for FUS. In the present study, we show that induction of DNA damage replicates several pathologic hallmarks of FTLD-FUS in immortalized human cells and primary human neurons and astrocytes. Treatment with the antibiotic calicheamicin γ1, which causes DNA double-strand breaks, leads to the cytoplasmic accumulation of FUS, TAF15, EWS, and TRN1. Moreover, cytoplasmic translocation of FUS is mediated by phosphorylation of its N terminus by the DNA-dependent protein kinase. Finally, we observed elevated levels of phospho-H2AX in FTLD-FUS brains, indicating that DNA damage occurs in patients. Together, our data reveal a novel regulatory mechanism for FUS localization in cells and suggest that DNA damage may contribute to the accumulation of FET proteins observed in human FTLD-FUS cases, but not in ALS-FUS.

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Protein methylation is a common posttranslational modification that mostly occurs on arginine and lysine residues. Arginine methylation has been reported to regulate RNA processing, gene transcription, DNA damage repair, protein translocation, and signal transduction. Lysine methylation is best known to regulate histone function and is involved in epigenetic regulation of gene transcription. To better study protein methylation, we have developed highly specific antibodies against monomethyl arginine, asymmetric dimethyl arginine, and monomethyl, dimethyl, and trimethyl lysine motifs respectively. These antibodies were used to perform immunoaffinity purification (IAP) of methyl peptides followed by LC-MS/MS analysis to identify and quantify arginine and lysine methylation sites in several model studies. Overall, we identified over 1000 arginine methylation sites in human cell lines and mouse tissues, and approximately 160 lysine methylation sites in human cell line HCT116. The methylation sites that were identified in this study exceed those found in the literature to date. Detailed analysis of arginine-methylated proteins observed in mouse brain compared to those found in mouse embryo shows tissue-specific distribution of arginine methylation, and extends the type of proteins that are known to be arginine methylated to many new protein types. Many arginine-methylated proteins that we identified from the brain including receptors, ion channels, transporters, and vesicle proteins, are involved in synaptic transmission, while the most abundant methylated proteins identified from mouse embryo are transcriptional regulators and RNA processing proteins.

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Motifs rich in arginines and glycines were recognized several decades ago to play functional roles and were termed glycine-arginine-rich (GAR) domains and/or RGG boxes. We review here the evolving functions of the RGG box along with several sequence variations that we collectively term the RGG/RG motif. Greater than 1,000 human proteins harbor the RGG/RG motif, and these proteins influence numerous physiological processes such as transcription, pre-mRNA splicing, DNA damage signaling, mRNA translation, and the regulation of apoptosis. In particular, we discuss the role of the RGG/RG motif in mediating nucleic acid and protein interactions, a function that is often regulated by arginine methylation and partner-binding proteins. The physiological relevance of the RGG/RG motif is highlighted by its association with several diseases including neurological and neuromuscular diseases and cancer. Herein, we discuss the evidence for the emerging diverse functionality of this important motif.

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Mar 2013
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Mutations in the gene encoding Fused in Sarcoma (FUS) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. FUS is a predominantly nuclear DNA and RNA binding protein that is involved in RNA processing. Large FUS immuno-reactive inclusions fill the perikaryon of surviving motor neurons of ALS patients carrying mutations at post-mortem. This sequestration of FUS is predicted to disrupt RNA processing and initiate neurodegeneration. Here we demonstrate that C-terminal ALS mutations disrupt the nuclear localising signal (NLS) of FUS resulting in cytoplasmic accumulation in transfected cells and patient fibroblasts. FUS mislocalisation is rescued by the addition of the wild-type FUS NLS to mutant proteins. We also show that oxidative stress recruits mutant FUS to cytoplasmic stress granules where it is able to bind and sequester wild-type FUS. While FUS interacts with itself directly by protein-protein interaction, the recruitment of FUS to stress granules and interaction with PABP is RNA dependent. These findings support a two-hit hypothesis whereby cytoplasmic mislocalisation of FUS protein, followed by cellular stress contributes to the formation of cytoplasmic aggregates that may sequester FUS, disrupt RNA processing and initiate motor neuron degeneration.

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Article

Full-text available

Feb 2013

Arginine methylation is a common posttranslational modification that is found on both histone and non-histone proteins. Three types of arginine methylation exist in mammalian cells: monomethylarginine (MMA), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). PRMT1 is the primary methyltransferase that deposits the ADMA mark, and it accounts for over 90% of this type of methylation. Here, we show that with the loss of PRMT1 activity, there are major increases in global MMA and SDMA levels, as detected by type-specific antibodies. Amino acid analysis confirms that MMA and SDMA levels accumulate when ADMA levels are reduced. These findings reveal the dynamic interplay between different arginine methylation types in the cells, and that the pre-existence of the dominant ADMA mark can block the occurrence of SDMA and MMA marks on the same substrate. This study provides clear evidence of competition for different arginine methylation types on the same substrates.

Nomenclature and nosology for neuropathologic subtypes of frontotemporal lobar degeneration: an update

Article

Jan 2010

Mutations in protein N-arginine methyltransferases are not the cause of FTLD-FUS

Article

Apr 2013

The nuclear protein fused in sarcoma (FUS) is found in cytoplasmic inclusions in a subset of patients with the neurodegenerative disorder frontotemporal lobar degeneration (FTLD-FUS). FUS contains a methylated arginine-glycine-glycine domain that is required for transport into the nucleus. Recent findings have shown that this domain is hypomethylated in patients with FTLD-FUS. To determine whether the cause of hypomethylation is the result of mutations in protein N-arginine methyltransferases (PRMTs), we selected 3 candidate genes (PRMT1, PRMT3, and PRMT8) and performed complete sequencing analysis and real-time polymerase chain reaction mRNA expression analysis in 20 FTLD-FUS cases. No mutations or statistically significant changes in expression were observed in our patient samples, suggesting that defects in PRMTs are not the cause of FTLD-FUS.

Fused in Sarcoma (FUS): An Oncogene Goes Awry in Neurodegeneration.

Article

Apr 2013
MOL CELL NEUROSCI

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

Abstract and Figures

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