17 June 2011. TDP-43, linked to both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, could be joining the ranks of amyloid proteins—at least from an in vitro perspective. The protein is thought to form non-amyloid aggregates in people and animals, but a synthetic piece of TDP-43 makes amyloid in a test tube, report a team of researchers from the Chinese Academy of Science in Beijing and the Northwestern University Feinberg School of Medicine in Chicago, Illinois. Small, amyloid-like oligomers might push TDP-43 to become toxic, the authors suggest in a paper in the June 12 Nature Structural & Molecular Biology online. Their results also lend support to the concept that TDP-43 could function like a prion, converting native protein to a malformed version in a pathology that spreads from one cell to the next. Also, in an extension of previous results, scientists from the University of Montréal report in the June Archives of Neurology further evidence linking an excess of polyglutamine repeats in ataxin 2 to ALS. Ataxin 2 binds TDP-43 via bridging RNAs.
Looks Like a Prion, Acts Like a Prion….
The TDP-43 work was led by joint first authors Weirui Guo, Yanbo Chen, and Xiaohong Zhou under the guidance of co-senior authors Qi Xu, of the Peking Union Medical College in Beijing, and Jane Wu, of both the Chinese Academy of Science and Northwestern University. The researchers carried out a detailed biochemical analysis of TDP-43 from a variety of sources: autopsy samples from people who had frontotemporal lobar dementia or were cognitively normal; human embryonic kidney cell lines stably transfected with a wild-type or disease-linked A315T TDP-43 construct; and Escherichia coli expressing recombinant wild-type or A315T protein.
They fractionated protein complexes based on weight and found that TDP-43 and its fragments showed up in complexes as large as 667 kDa and as small as 14 kDa, suggesting it forms oligomers of different sizes. They also noted that TDP-43-A315T, and to a lesser extent wild-type protein, often appeared as a phosphorylation-dependent 75 kDa species. The authors concluded it represents a hyperphosphorylated version of the protein, although the exact structure remains to be determined. The A315T mutant, the researchers suggest, causes disease because of its increased propensity to become phosphorylated. In addition, TDP-43 fragments, particularly the A315T ones, resisted degradation by detergent and proteases.
Prions are degradation-resistant, oligomerizing proteins. Other researchers have suggested that TDP-43 contains a prion-like sequence (see ARF related news story on Sun et al., 2011 and Ju et al., 2011; ARF related news story on Fuentealba et al., 2010; Udan and Baloh, 2011; Cushman et al., 2010). In their own sequence analysis, the Beijing-Chicago team found that the TDP-43 carboxyl terminus, known to be involved in toxicity, has prion-like segments. They used molecular dynamics simulation to model how different carboxyl-terminal fragments might behave, and discovered that the 46-mer Q286-Q331—particularly the A315T version—formed β sheets, like amyloids. Another research group reported last year that TDP-43 makes amyloid-like structures (Chen et al., 2010).
To test for amyloid formation, the authors synthesized the 46-mers and agitated them at 37 degrees Celsius in solution with the amyloid marker thioflavin T, which bound to the peptides. Using electron and atomic force microscopy, they determined that, when incubated for several days, both the wild-type and A315T TDP-43 peptides came together in fibrils. They treated primary mouse neural cultures with each peptide and found that the wild-type was toxic, A315T even more so.
The researchers suggest one possible extrapolation of their results is that amyloid oligomers of TDP-43 induce misfolding of other TDP-43 molecules, ultimately destroying neurons. The A315T substitution, and perhaps other known mutations in the carboxyl-terminal domain, could enhance this propensity to oligomerize.
However, there is little evidence that this scenario plays out in vivo. In fact, the amyloids Wu and colleagues observed are unlike those described in people who died of ALS or FTLD. The current study reports on non-ubiquitinated amyloid aggregates, while ubiquitinated non-amyloid TDP-43 aggregates are the hallmark of TDP-43 proteinopathies (see ARF related news story on Neumann et al., 2006). Perhaps pathologists using thioflavin T are missing the signal, Wu suggested: “Either the epitope is somehow not exposed, or not formed.” Indeed, some amyloids do not bind to thioflavin T or Congo red, but those are rare, said James Shorter of the University of Pennsylvania.
Shorter and his UPenn colleague Aaron Gitler were not involved in the current work. In an e-mail to ARF, Gitler noted that TDP-43 is an intracellular protein, so it is not clear how the extracellular toxicity of TDP-43 fragments relates to disease. Wu and colleagues are not ready to propose a precise mechanism for the toxicity of these amyloid fibrils, she said.
One thing is clear, Shorter said: The Q286-Q331 region is key to TDP-43’s toxic activities. “I do not doubt that that region is very important in the misfolding and aggregation,” he said.
Mounting Evidence for Ataxin 2 in ALS
Other research groups are converging on the importance of TDP-43’s carboxyl end (see also ARF related news story on Zhang et al., 2009) and its prion potential. Similarly, multiple teams have now published evidence that repeats in ataxin 2 contribute to ALS, as first reported by Gitler and colleagues (see ARF related news story on Elden et al., 2010). The Archives of Neurology study, led by first author Hussein Daoud and senior author Guy Rouleau, is the latest in a string of studies to follow up on that study (Ross et al., 2011; Van Damme et al., 2011; Yu et al., 2011; Lee et al., 2011; Fischbeck and Pulst, 2011; see Gitler comment, below). The Montréal team, in a study of more than 1,000 people with ALS and healthy controls, found that having 29 or more CAG repeats was associated with an ALS diagnosis.—Amber Dance.
Guo W, Chen Y, Zhou X, Kar A, Ray P, Chen X, Rao EJ, Yang M, Ye H, Zhu L, Jianghong Liu, Xu M, Yang Y, Wang C, Zhang D, Bibio EH, Mesulam M, Shen Y, Xu Q, Fushimi K, Wu JY. An ALS-associated mutation affecting TDP-43 enhances protein aggregation, fibril formation and neurotoxicity. Nat Struct Mol Biol. 2011 Jun 12. Abstract
Daoud H, Belzil V, Martins S, Sabbagh M, Provencher P, Lacomblez L, Meininger V, Camu W, Dupré N, Dion PA, Rouleau GA. Association of long ATXN2 CAG repeat sizes with increased risk of amyotrophic lateral sclerosis. Arch Neurol. 2011 Jun;68(8):739-42. Abstract