Where’s the FUS —Evidence for Sporadic ALS Role Creates Stir

Although most cases of amyotrophic lateral sclerosis have no evidence of inheritance, scientists are finding valuable clues to the disease among familial cases. The latest evidence transitioning from the genetic to the mechanistic suggests that the ALS gene FUS also participates in the sporadic form of the disease. In a paper posted online April 14 in the Annals of Neurology, researchers from the Northwestern University Feinberg School of Medicine in Chicago, Illinois, reported the presence of aggregates of FUS, or fused in sarcoma, in autopsy tissue samples from patients with ALS and frontotemporal lobar dementia, a related condition.

The findings are getting mixed reviews. This is a unification of familial with sporadic ALS in a most direct fashion, said study senior author Teepu Siddique. Han-Xiang Deng was the first author. Other scientists question the antibody Deng used, and the validity of the results. This is intriguing, but it needs a lot of additional, more rigorous investigation to understand where there is true significance, said Ian Mackenzie of the University of British Columbia in Vancouver, who was not among the authors.

Familial ALS makes up only 10 percent of all cases; FUS mutations account for some 4 or 5 percent of the familial portion (see ARF related news story on Vance et al., 2009 and Kwiatkowski et al., 2009). Mutations in TDP-43 account for a further 3 or 4 percent of familial cases, and the enzyme superoxide dismutase 1 (SOD1) holds the lion’s share, causing one-fifth of familial ALS. Both FUS and TDP-43 are involved in RNA processing and trafficking. FUS is primarily nuclear, but is also present in the cytoplasm. FUS and TDP-43 mutations can affect the spinal cord, causing ALS; the brain, causing frontotemporal lobar dementia; or a combination of the two. With this study, Siddique hopes to solidify mechanistic links between genetic ALS and the vast majority of cases that are sporadic. This is a common theme in disease research; similarly, the discovery of Alzheimer-linked mutations in APP and presenilin informed research on sporadic AD.

Deng and colleagues collected tissue from 100 cases in all. Tissues included spinal cord samples from 52 people with sporadic ALS; 16 with familial ALS; 10 who had both ALS and dementia, such as parkinsonism; and six healthy controls. In addition, they examined frontal lobe or hippocampal sections from 11 people with FTLD and five more healthy controls.

Initially, the researchers tested nine different FUS antibodies on their ALS spinal cord sections. They found that one, a rabbit polyclonal antibody from Proteintech Group, Inc., in Chicago, gave them the strongest signal, and continued their analyses with this antibody. Control cases without ALS also showed no FUS-positive inclusions in the spinal cord. Deng and colleagues saw FUS staining in the skein-like inclusions typical of ALS in all familial cases, with the exception of four associated with SOD1 mutations. ALS may not be one disease, Siddique said; these data place SOD1-based ALS in its own category.

Deng and colleagues observed FUS-positive inclusions in two familial ALS cases of known genetic cause, one with a FUS mutation and one with a TDP-43 mutation. Moreover, they saw FUS in the inclusions of all sporadic ALS samples. Based on confocal immunofluorescence microscopy, FUS and TDP-43 colocalized in virtually all skein-like and dense inclusions, Siddique wrote in an e-mail to ARF. This discovery is rather surprising, because the authors of a previous study found that TDP-43 did not appear with FUS in inclusions, and suggested the two proteins fit into different pathological pathways (Vance et al., 2009).

In brain samples from people who had FTLD, nine cases showed inclusions positive for both TDP-43 and FUS. However, two further cases of a rare kind of FTLD with FUS pathology showed no positive staining for TDP-43 in the inclusions, matching previous results (see ARF related news story; Seelaar et al., 2010). Control samples had no FUS pathology.

Siddique suggested that FUS in ALS parallels tau and amyloid-β in Alzheimer disease or α-synuclein in Parkinson’s. In each of these neurodegenerative conditions, rare genetic mutations cause disease, but those same proteins, when wild-type, participate in pathology in all cases.

This is an important observation, wrote Manuela Neumann of the University Hospital of Zrich in Switzerland of the paper, in an e-mail to ARF. However, there are some open questions and discrepancies with previous studies, which in my opinion need to be addressed in more detailbefore definite conclusions can be drawn.

Mackenzie suggested three possible interpretations of Deng’s work. First, the results could be correct. It is certainly plausible, Neumann wrote, that only certain FUS epitopes are available in TDP-43 inclusions, and other antibody studies missed them. In that case, scientists must rethink their understanding of TDP-43 and FUS, and figure out how previous studies went wrong.

Second, Mackenzie suggested, FUS might be present in TDP-43 inclusions, but only as an innocent bystander swept up in the aggregates. As an example of this bystander effect, he noted that the prion-containing plaques of Creutzfeld-Jakob disease often collect a little amyloid-β, but that amyloid is not necessarily part of the pathology. It could simply have been in the wrong place at the wrong time, as a growing aggregate attached to everything nearby—and the same might happen to FUS when TDP-43 starts aggregating.

Third, the FUS staining in Deng’s paper could be a technical artifact, if the antibody is not labeling FUS at all. Neumann also raised this possibility: This antibody might cross-react with any other protein component in TDP-43 inclusions or detect, for example, specific conformational domains similar between FUS and TDP-43, a fact not completely unlikely, given the striking structural and functional similarities of both, she wrote. In other words, Proteintech’s FUS antibody might actually be a rather poor TDP-43 antibody. FUS and TDP-43 do not share sequence homology, but because of their similar functions, they might still have similar epitopes.

Siddique noted that the group used two other FUS antibodies that stained tissue samples similarly to Proteintech’s, although the signal was weaker. He also pointed to the two cases of FUS-positive, TDP-43-negative FTLD as evidence that the FUS signal was not dependent on the presence of TDP-43. On a Western blot, a standard TDP-43 antibody and Proteintech’s FUS antibody do not recognize bands of the same size, but that does not entirely rule out cross-reactivity, Neumann wrote. The Proteintech antibody might still pick up TDP-43, or an associated protein, in immunohistochemistry samples.

Siddique cites his group’s unusual sample processing methods as an explanation for the divergent results. Most researchers boil or microwave tissue in a process called antigen retrieval, which opens up protein epitopes to make them more accessible to antibodies. The Siddique lab prefers an alternate approach with a device called a decloaking chamber, rather like a pressure cooker, which heats samples to 125 degrees Celsius for 20 minutes. This technique works better for some antigens, Siddique said, and if other researchers use boiling or microwaving, they might not see results that match his.

If validated, Deng’s results suggest that scientists must look for a common pathway uniting FUS, TDP-43, and sporadic ALS. These kinds of studies, connecting rare family genetics to a widespread disease, are important investigations, Siddique said. Other recent genetic studies have linked optineurin (see ARF related news story on Maruyama et al., 2010) and D-amino acid oxidase (see ARF related news story on Mitchell et al., 2010) to ALS, and those proteins may have significance beyond the families where they were easy to spot. The low-hanging fruit of familial disease can provide you with insights into the wider sporadic disorder, Siddique said.

Deng HX, Zhai H, Bigio EH, Yan J, Fecto F, Ajroud K, Mishra M, Ajroud-Driss S, Heller S, Sufit R, Siddique N, Mugaini E, Siddique T. FUS-immunoreactive inclusions are a common feature in sporadic and non-SOD1 familial amyotrophic lateral sclerosis. Ann Neurol. 2010 Apr 14.

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