Arginine Methylation Distinguishes ALS-FUS From FTLD-FUS

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).

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 transporting binding and nuclear import of FUS. EMBO J. 2012 Sep 11. Abstract

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