Arginine Methylation Distinguishes ALS-FUS From FTLD-FUS
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Some researchers have come to think of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) as two manifestations of the same pathology, but Christian Haass and Dorothee Dormann dissected out differences between the two in presentations at the 8th International Conference on Frontotemporal Dementias, held 5-7 September in Manchester, U.K. The scientists from Ludwig-Maximilians-Universität in Munich, Germany, also described their study in the EMBO Journal online September 11. They found that in cases of ALS due to FUS mutations, and frontotemporal lobar degeneration (FTLD) linked to FUS proteinopathy, the cellular mechanisms of disease are somewhat distinct. Both diseases feature FUS aggregates and stress granules, yet abnormal—aka subnormal—arginine methylation of FUS marks only FTLD, not ALS, pathology. Arginine methylation and subsequent defects in nuclear import might become a new area of investigation in neurodegenerative diseases, the researchers said.
In healthy cells, FUS is a nuclear protein. Via a nuclear localization sequence (NLS) on its carboxyl-terminal end, it hooks up with the nuclear shuttle transportin 1 to enter the nucleus. In some cases of ALS, however, mutations to the NLS render the protein cytosolic (ALS-FUS). There is also a subset of FTLD in which FUS is stuck in the cytoplasm (FTLD-FUS), but since its NLS is normal, something else must explain its mislocalization (see Urwin et al., 2010). Since FUS is known to be subject to post-translational methylation, Dormann, in Haass’ lab, investigated whether those methyl groups might influence its cellular address.
She started by treating HeLa cells expressing FUS NLS mutants with AdOx, a broad-spectrum methylation inhibitor. Despite having the mutation, FUS now traveled its normal path to the nucleus. The finding suggests that methylation powerfully influences FUS retention in the cytosol.
Thinking methylation might interfere with the transportin-FUS interaction, Dormann incubated recombinant transportin with either methylated or unmethylated synthetic FUS peptides. That confirmed that the methyls inhibit transportin binding to FUS; the unmethylated FUS peptides bound transportin most tightly. Further work revealed a new, large transportin-binding domain on FUS that is sensitive to arginine methylation, expanding scientists’ view of how transportin binds its cargo.
To investigate what this might mean to human disease, the team created antibodies specific for methylated FUS. In control brain sections, methylated FUS appeared only in the nucleus. In ALS cases, methylated FUS was present not only in the nucleus, but also in the cytoplasmic aggregates. In FTLD, methylated FUS was again nuclear—but the FUS-positive inclusions in the cytosol were unmethylated.
The different methylated states of the aggregated FUS suggest two different pathways for inclusion formation in ALS and FTLD, Haass said in his presentation. The ALS mechanism is fairly straightforward. Dormann and Haass posit that FUS’ defective NLS prevents it from binding transportin and sequesters it in the cytoplasm, where it accumulates and aggregates.
The situation is more complex in FTLD, the scientists suggest. In addition to the loss of FUS methylation Dormann discovered, previous studies have shown that when FUS aggregates in FTLD, it brings with it TAF15 and EWS, related members of the FET family of proteins, which are also subject to arginine methylation (see ARF related news story on Neumann et al., 2011). This trio shares transportin as a nuclear importer, and transportin itself also appears in the cytoplasmic inclusions in FTLD-FUS (Neumann et al., 2012). In this form of FTLD, therefore, it appears that FUS, TAF15, and EWS all suffer from some generalized nuclear import defect. All three, Haass and Dormann propose, are hypomethylated. With no methyl groups to interfere, they bind extra tightly to transportin and somehow drag it into cytoplasmic inclusions. FTLD-FUS is marked by a more generalized defect in transportin-mediated nuclear import, the authors suggest.
The work goes a long way toward solving a puzzle about ALS and FTLD, commented Emanuele Buratti of the International Centre for Genetic Engineering and Biotechnology in Trieste, Italy, in an e-mail to Alzforum. “Although ALS-FUS and FTLD-FUS were shown to have overlapping clinical phenotype and neuropathology, there was no explanation for why individuals affected by the same protein pathology developed one form of the disease as opposed to the other,” wrote Buratti, who was not involved in the study (see full comment, below).
“Although the mechanisms are different, the common feature, FUS intracellular mislocalization, supports the concept of ALS and FTLD being closely related, but not simply a single spectrum,” added Ian Mackenzie of Vancouver General Hospital in Canada, a study coauthor, in another e-mail.
The work is specific to ALS and FTLD with FUS pathology, Haass noted in an interview with Alzforum. Coauthor Manuela Neumann of the German Center for Neurodegenerative Diseases in Tübingen speculated that it might have broader significance as well. “Given that a lot of RNA binding proteins are arginine methylated, and [considering] the increasing number of RNA binding proteins involved in ALS and FTD, it is possible that dysregulation of arginine methylation might be involved also in non-FUSopathies,” she wrote in an e-mail to Alzforum (see full comment below).—Amber Dance
References
News Citations
Paper Citations
- Urwin H, Josephs KA, Rohrer JD, Mackenzie IR, Neumann M, Authier A, Seelaar H, van Swieten JC, Brown JM, Johannsen P, Nielsen JE, Holm IE, , Dickson DW, Rademakers R, Graff-Radford NR, Parisi JE, Petersen RC, Hatanpaa KJ, White CL, Weiner MF, Geser F, Van Deerlin VM, Trojanowski JQ, Miller BL, Seeley WW, van der Zee J, Kumar-Singh S, Engelborghs S, De Deyn PP, Van Broeckhoven C, Bigio EH, Deng HX, Halliday GM, Kril JJ, Munoz DG, Mann DM, Pickering-Brown SM, Doodeman V, Adamson G, Ghazi-Noori S, Fisher EM, Holton JL, Revesz T, Rossor MN, Collinge J, Mead S, Isaacs AM. FUS pathology defines the majority of tau- and TDP-43-negative frontotemporal lobar degeneration. Acta Neuropathol. 2010 Jul;120(1):33-41. PubMed.
- Neumann M, Bentmann E, Dormann D, Jawaid A, Dejesus-Hernandez M, Ansorge O, Roeber S, Kretzschmar HA, Munoz DG, Kusaka H, Yokota O, Ang LC, Bilbao J, Rademakers R, Haass C, Mackenzie IR. FET proteins TAF15 and EWS are selective markers that distinguish FTLD with FUS pathology from amyotrophic lateral sclerosis with FUS mutations. Brain. 2011 Sep;134(Pt 9):2595-609. PubMed.
- Neumann M, Valori CF, Ansorge O, Kretzschmar HA, Munoz DG, Kusaka H, Yokota O, Ishihara K, Ang LC, Bilbao JM, Mackenzie IR. Transportin 1 accumulates specifically with FET proteins but no other transportin cargos in FTLD-FUS and is absent in FUS inclusions in ALS with FUS mutations. Acta Neuropathol. 2012 Nov;124(5):705-16. PubMed.
Further Reading
Papers
- Mackenzie IR, Neumann M. FET proteins in frontotemporal dementia and amyotrophic lateral sclerosis. Brain Res. 2011 Dec 13; PubMed.
- Rademakers R, Neumann M, Mackenzie IR. Advances in understanding the molecular basis of frontotemporal dementia. Nat Rev Neurol. 2012 Jun 26;8(8):423-34. PubMed.
Primary Papers
- Dormann D, Madl T, Valori CF, Bentmann E, Tahirovic S, Abou-Ajram C, Kremmer E, Ansorge O, Mackenzie IR, Neumann M, Haass C. Arginine methylation next to the PY-NLS modulates Transportin binding and nuclear import of FUS. EMBO J. 2012 Sep 11; PubMed.
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Comments
International Centre for Genetic Engineering and Biotechnology
This is a key paper by the Haass lab, raising several important issues regarding our understanding of pathological mechanisms mediated by the FUS nuclear factor in ALS and FTLD (termed ALS-FUS and FTLD-FUS, respectively). Rather puzzlingly, in fact, was that although ALS-FUS and FTLD-FUS were shown to have overlapping clinical phenotype and neuropathology, there was no explanation for why individuals affected by the same protein pathology developed one form of the disease as opposed to the other. In this work, the convincing demonstration that the FUS protein is methylated in ALS-FUS-related cases but not in FTLD-FUS-related cases has gone a long way in helping to address this long-standing question. Indeed, together with recent findings that the FUS protein co-aggregates with two FET family proteins and the transportin receptor itself in FTLD-FUS cases, but not in ALS-FUS, the study by Dorothee Dormann, Christian Haass, and colleagues provides additional evidence that these two diseases may actually be initiated by distinct pathomechanisms. Most importantly, they implicate alterations in arginine methylation in pathogenesis, an observation that will have profound influence on future research in this field.
It can be easily foreseen, in fact, that this work will act as an important springboard for research in an area rather underappreciated until this moment: the post-translational modifications of these risk proteins. In this respect, it should be kept in mind that post-translational modifications are also common for non-FUS versions of these diseases, such as the aberrant phosphorylation and ubiquitination of TDP-43 in ALS and FTLD. Presently, very little is known regarding the effects these modifications have on the biological (nuclear import, export, aggregation, toxicity, etc.) or functional properties (pre-mRNA splicing, mRNA transport, and other RNA processing events) of all these disease proteins. That gap in our knowledge should be addressed as soon as possible, since, as the authors show, it may hold the key for crucial advancements in our understanding of these diseases.
Of course, like all seminal findings of this kind, these observations also spark a number of questions about which we can only speculate at the moment. These range from the very specific, such as whether different methylation patterns may be connected not just with disease but also with other characteristics such as age of onset, to the very general, such as identifying the different molecular pathomechanisms that must be activated in either case. I therefore expect that future research will be very busy in all these directions.
University of Tübingen and DZNE AG Neumann
The paper clearly demonstrates that arginine methylation of FUS is an important modulator for nuclear import of FUS and its homologues, TAF15 and EWS, as demonstrated in vitro and in cell culture.
In my opinion, the most exciting finding is the difference we observed in the methylation status of FUS in the inclusions in the spectrum of FUSopathies using the novel methylation-specific antibodies. While FUS was methylated in cases with FUS mutations (that usually present with ALS), it seems to be un- or hypomethylated in FTLD-FUS.
Together with some other recent findings from my group, working together with Ian Mackenzie, on differences between FUSopathies with FUS mutations and those without (Neumann et al., 2011; Neumann et al., 2012), these data fit into the hypothesis we published in a recent review (Rademakers et al., 2012) of distinct pathomechanisms underlying inclusion body formation in FUSopathies by implying alterations of post-translational modifications of FET proteins as the most plausible scenario.
There are still a lot of open questions, since arginine methylation, in general, is not very well understood. Which of the methyltransferases is the main enzyme for arginine methylations of FET proteins in the brain? Are individual sites for arginine methylation more important than others? How reversible is the process?
Given the fact that a lot of RNA binding proteins are arginine methylated, and [considering] the increasing number of RNA binding proteins involved in ALS and FTD, it is possible that dysregulation of arginine methylation might be involved also in non-FUSopathies. However, currently there are no data demonstrating arginine methylation of TDP-43, the other most common FTD/ALS protein.
References:
Neumann M, Bentmann E, Dormann D, Jawaid A, Dejesus-Hernandez M, Ansorge O, Roeber S, Kretzschmar HA, Munoz DG, Kusaka H, Yokota O, Ang LC, Bilbao J, Rademakers R, Haass C, Mackenzie IR. FET proteins TAF15 and EWS are selective markers that distinguish FTLD with FUS pathology from amyotrophic lateral sclerosis with FUS mutations. Brain. 2011 Sep;134(Pt 9):2595-609. PubMed.
Neumann M, Valori CF, Ansorge O, Kretzschmar HA, Munoz DG, Kusaka H, Yokota O, Ishihara K, Ang LC, Bilbao JM, Mackenzie IR. Transportin 1 accumulates specifically with FET proteins but no other transportin cargos in FTLD-FUS and is absent in FUS inclusions in ALS with FUS mutations. Acta Neuropathol. 2012 Nov;124(5):705-16. PubMed.
Rademakers R, Neumann M, Mackenzie IR. Advances in understanding the molecular basis of frontotemporal dementia. Nat Rev Neurol. 2012 Jun 26;8(8):423-34. PubMed.
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