Does TDP-43 Oligomerize and Coax Aβ to Do the Same?

TDP-43 self-assembles into oligomers that are able to recruit Aβ to form oligomers of its own, claims a paper in the September 12 Nature Communications. Using a new TDP-43 oligomer antibody, the authors report binding in postmortem brain samples from people who had frontotemporal dementia. Senior author Yun-Ru (Ruby) Chen of Academia Sinica in Taipei, Taiwan, suggests that these multimers may be toxic, as has been proposed for other oligomers implicated in neurodegeneration. The new antibody, or one like it, might help doctors diagnose or even one day treat TDP-43 proteinopathy, Chen speculated.

“Many people knew that these oligomers existed … this is the first comprehensive study,” noted Rakez Kayed of the University of Texas Medical Branch in Galveston (Johnson et al., 2009; Guo et al., 2011; Choksi et al., 2013).

The finding that TDP-43 causes Aβ to oligomerize is new, added Kayez, who was not involved in the current work. If confirmed, it would complement recent work by Kayez and collaborators showing that Aβ can template TDP-43 aggregation (Guerrero-Muñoz et al., 2014). Kayez hopes that Chen’s new findings will inspire researchers to examine TDP-43 oligomers, TDP-43 mutations, and their potential toxicity more closely in animal models.

TDP-43 forms round oligomers, as seen by atomic force microscopy (scale bar=500 nanometers). In the upper left, a close-up of a ring-shaped oligomer (scale bar=50 nanometers). [Image courtesy of Fang et al., Nature Communications.]

TDP-43 Makes Round Oligomers
Chen and first author Yu-Sheng Fang have been working on TDP-43 biochemistry since researchers identified it as a major component in protein inclusions in FTD and ALS in 2006 (see Oct 2006 news story). They started by purifying recombinant human TDP-43 from Escherichia coli. While they expected their recombinant protein to produce a protein of the eponymous 43 kilodaltons, it immediately glommed together into structures more than 10 times that, according to size-exclusion chromatography. Because these aggregates were soluble, the researchers knew they were distinct from the filamentous, insoluble inclusion bodies that characterize FTD brain and ALS spinal cord.

Chen and Fang surmised they were looking at oligomers, and used the oligomer-specific antibody A11 to confirm their suspicions. A11 recognizes some conformation common to amyloid-forming oligomers regardless of the primary structure of the peptide, be it Aβ, α-synuclein, or other amyloidogenic protein (see Apr 2003 news story). Scientists still do not understand which epitope A11 recognizes, and some researchers get better results with it than others, noted David Teplow of the David Geffen School of Medicine at the University of California, Los Angeles, who was not involved in the work.

To investigate the oligomeric nature of TDP-43 more directly, Fang and colleagues used electron and atomic force microscopy to map them in three dimensions. The oligomers formed spherical and ring-shaped structures (see image above). They resembled previously characterized pathogenic Aβ and α-synuclein oligomers (Lashuel et al., 2002) but were larger, more than 50 nanometers across compared to two or three nanometers for typical Aβ oligomers. The TDP-43 structures also differed from Aβ in that they never adopted conformations that bound the dye thioflavin T, which binds to amyloid fibrils in beta sheet formation.

Next, Fang asked if oligomerization might alter TDP-43’s normal function as a DNA-binding protein. She compared the oligomers to a truncated TDP-43 that contained only the nucleic acid binding sites. While the shortened TDP-43 was able to attach itself to one of its target DNA sequences—transactivation response element from HIV—the oligomers bound the DNA more weakly. “That tells us the oligomer has a loss of the TDP-43 physiological function,” perhaps due to abnormal conformation or masking of the DNA-binding domain, Chen concluded.

The scientists also report evidence for a gain of toxic function. When Fang treated human neuroblastoma BE(2)-C cultures or primary mouse cortical neurons with the oligomers, about 20 percent of the cells died. The authors then injected oligomers into the hippocampus of live mice. Two weeks later, they examined the brains and observed a loss of cortical neurons where they injected the TDP-43.

Fang and Chen wondered if the TDP-43 oligomers they saw in vitro exist in the brains of people with FTD. To find out, they used the oligomers to create an antibody they called TDP-O. They claim that unlike A11, their antibody is specific for TDP-43 oligomers because it does not bind Aβ oligomers. They did not test TDP-O for reactivity against other amyloidogenic proteins, however, leading Teplow to suggest that it might still exhibit off-target binding. The authors bathed brain sections from FTD patients with TDP-O. Most TDP-43 antibodies detect fibrils, but not this one. Instead, it labeled oligomers that showed up as round structures under the electron microscope. When Fang and colleagues immunoprecipitated these structures with TDP-O, the structures also bound another TDP-43 antibody directed against the protein’s amino terminus, confirming they did indeed contain TDP-43.

TDP-43 Oligomers Attract Aβ
TDP-43 joins the ranks of many neurodegeneration-linked proteins that oligomerize, including Aβ, α-synuclein, and the prion protein PrP. These proteins cross-seed each other’s oligomerization (see Sep 2008 news story; Jul 2013 news story). Might TDP-43 do the same? Fang investigated by incubating TDP-43 oligomers with soluble Aβ. By itself, Aβ assembles into long fibrils. However, just a smidgen of TDP-43 inhibited that process, leading the authors to speculate that the Aβ got stuck in the oligomer stage. To check, they photo-crosslinked the Aβ and ran it on a protein gel. Without TDP-43, they saw Aβ monomers, dimers, trimers, and tetramers. After they added TDP-43, they observed Aβ pentamers and even larger species of uncertain makeup. In addition, when they looked at Aβ in the electron microscope, they saw it formed fibrils when alone, but only small orbs in the presence of TDP-43.

Chen cannot be sure what conformation these oligomers take, and whether they contain only Aβ or a combination of Aβ and TDP-43. Chen and Kayed agreed that this kind of cross-seeding may explain why TDP-43 inclusions are often present in AD brains (Amador-Ortiz et al., 2007; Uryu et al., 2008; Hu et al., 2008; Kadokura et al., 2009).

However, Teplow urged caution in interpreting these experiments. “I see no evidence of cross-seeding,” he said, raising several caveats. The authors used Aβ40, not Aβ42, which is more relevant to Alzheimer’s disease, and they didn’t use Aβ seeds as a positive control to ensure that they had the right conditions for seeding to occur. He also noted the transition from Aβ monomers to larger species did not rise proportionately with increasing TDP-43, suggesting the DNA binding protein may be superfluous. He agreed with the authors that TDP-43 seems to inhibit Aβ fibrillization, but was not convinced that it converted Aβ to oligomers.

Chen interprets her results to mean that TDP-43 oligomers are somehow involved in TDP-43 proteinopathies, and perhaps Alzheimer’s disease as well. She plans to investigate whether TDP-43-seeded Aβ species are toxic, and how the proposed cross-seeding occurs. For example, she would like to use the TDP-O antibody to look for TDP-43 oligomers in blood and cerebrospinal fluid from people with AD, ALS, and FTD and test if their levels correlate with phenotype, such as severity of symptoms.


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