Following in the footsteps of tau and α-synuclein, TDP-43, too, appears to twist into distinct amyloid arrangements in different neurodegenerative conditions. According to a paper published in Nature on August 2, filaments of TDP-43 in brain samples from people with frontotemporal lobar degeneration (FTLD) type A share a protofilament core that folds into a chevron-like shape. This new amyloid fold markedly differs from the double spiral contortion previously found in people with a form of FTLD accompanied by amyotrophic lateral sclerosis. The authors, led by Benjamin Ryskeldi-Falcon of the MRC Laboratory of Molecular Biology in Cambridge, U.K., also zeroed in on a post-translational tweak—citrullination of an arginine residue—that may trigger its formation.
- In three people with FTLD-A, the core of TDP-43 filaments folds into a chevron pattern.
- This fold is distinct from the one TDP-43 takes in people with FTD/ALS.
- Modification of a core arginine residue may facilitate the chevron shape.
To Ryskeldi-Falcon’s mind, the protofilament folds taken by TDP-43 in different forms of FTLD support an emerging theme in neurodegenerative proteinopathies, i.e., that distinct filament structures define different diseases.
Along with filaments of TDP-43, the researchers also detected TMEM106b fibrils in the same brain samples. This corroborates three previous studies that identified TMEM106b fibrils in brain samples from people with different forms of FTLD and other neurodegenerative diseases. One group had claimed that the abundant TDP-43 aggregates in these samples were not fibrillar (Apr 2022 news on Jiang et al., 2022). Now that filaments of both proteins have been clearly identified in the same brain samples, it seems that the discrepancy likely comes down to different biochemical extraction procedures, Ryskeldi-Falcon told Alzforum.
To Christian Haass of the German Center for Neurodegenerative Diseases in Munich, the unambiguous presence of TDP-43 filaments in different pathological forms of FTLD further supports a pathological nature of these aggregates, and unveils new opportunities for therapeutic targeting (comment below).
FTLD comprises a spectrum of disorders marked by extreme heterogeneity at both the neuropathological and clinical level. About half of cases carry TDP-43 inclusions, which, depending on their morphology and distribution within the brain, are classified into four types, dubbed A-D (Neumann et al., 2021). Previously, Ryskeldi-Falcon and colleagues had resolved the structure of TDP-43 filaments in people with both FTLD/ALS who had TDP-43 type B pathology, in which moderate numbers of neuronal cytoplasmic inclusions of TDP-43 gather in both the superficial and deep layers of the cortex, in the absence of dystrophic neurites. Whether plucked from the frontal or motor cortices, this brand of TDP-43 filament contained a core that twisted into a double-spiral fold (Dec 2021 news). Would the RNA-binding protein twist into the same configuration in people with different neuropathological and clinical manifestations of this proteinopathy?
To find out, first author Diana Arseni and colleagues turned the cryo-electron microscope on filaments extracted from brain samples rife with the most common form of TDP-43 pathology, i.e., type A. Abundant neuronal cytoplasmic inclusions of the protein, which are concentrated in the superficial layers of the cortex and accompanied by short, thick dystrophic neurites, characterize this disease. Type A TDP-43 pathology mostly strikes people with FTD who do not have ALS. Brain samples were provided by Masato Hasegawa of Tokyo Metropolitan University and Bernardino Ghetti of Indiana University in Indianapolis. Three samples were used for cryo-EM, including one whose clinical presentation was behavioral variant of FTD, and two others who suffered from non-fluent variant of primary progressive aphasia. The latter two carried a progranulin mutation.
Arseni spotted TDP-43 filaments lurking in all three samples. The filaments twisted to the right along their axes. Their cores consisted of single molecules of TDP-43 stacked on top of each other. Other fibril protofilaments often consist of dimers, as in the case of tau and Aβ filaments (Jul 2017 news; Jan 2022 news).
With cryo-EM, the scientists resolved the shape of each TDP-43 molecule in this ordered core, revealing that a stretch of residues from R272 to Q360 of the low-complexity domain of TDP-43 bends and folds over itself four times to form five “layers” (image below).
Behold the Chevron Fold. In filaments from three people with type A FTLD (top), a portion of TDP-43’s low-complexity domain folds over itself to create five layers (top). A model of the protein (bottom) shows that layers 3, 4, and 5 each form a V. Together, all three form a chevron, the authors suggest. [Courtesy of Arnesi et al., Nature, 2023.]
This five-layer structure centers around a hydrophobic stretch of residues that form a kinked β-strand within the fourth layer. This central β-strand forms steric zippers with amino acids in the neighboring third and fifth layers of the fold. Each of these three zipped-up sections of the protein form a V. Together they create a shape akin to a chevron, i.e., three Vs in a row. Layers 1 and 2 at the core's N-terminus comprise shorter β-strands that do not partake in the chevron.
Residues that lie N- and C-terminal to the ordered core form an unstructured fuzzy coat around the filaments, which was not resolved by cryo-EM. In addition to unresolved segments of this coat, non-proteinaceous densities mingled between the first, second, and third layers of the protofilament core, suggesting the presence of possible co-factors.
All the TDP-43 filaments observed from the three samples contained a chevron fold at their core. However, in about 5 percent of fibrils, the chevron fold took a slightly different configuration at its N-terminus and at the turn between the third and fourth layers. Interestingly, the scientists spotted this “minority chevron” within fibrils containing the predominant fold, suggesting the two were compatible variations, rather than entirely distinct folds. The chevron fold was markedly distinct from the fold Ryskeldi-Falcon’s group had previously described in people with FTLD type B pathology, although it did involve a similar segment of the protein (image below).
“This important study now provides the direct proof for different TDP-43 conformations as the probable molecular basis for the phenotypic variability among TDP-43 proteinopathies,” wrote Manuela Neumann of the German Center for Neurodegenerative Diseases in Tübingen (comment below). Neumann discovered the TDP-43 aggregates that led to the identification of the different neuropathological subtypes of FTLD (Neumann et al., 2006).
Variations on a Fold. The chevron structure (left) assumed by TDP-43 in filaments in FTLD type A differs from the spiral (right) found in FTLD type B filaments. [Courtesy of Arseni et al., Nature, 2023.]
What makes TDP-43 fold into this distinct structure? This question remains unanswered. A clue might come from R293, an arginine residue sandwiched snugly between layers 1 and 2 of the protofilament fold. With its positive charge only partially compensated for by surrounding peptide groups, this arginine should be “very unhappy” in this position, Ryskeldi-Falcon said. The only explanation for its placement? A post-translational modification that neutralizes its charge, he said. In the case of arginine, citrullination, aka deamination, would fit the bill. Indeed, mass spectrometry confirmed that R293 was citrullinated in TDP-43 from brain samples of people with FTLD type A pathology. Ryskeldi-Falcon proposed that without this modification, the chevron fold could not have formed.
Mass spec also detected some R293 residues donning a methyl group, a modification that would be expected to fit within the slightly altered chevron fold taken by a minority of protofilaments. Ryskeldi-Falcon proposed that these post-translational modifications likely govern how the fold forms, as well as its variations.
Colin Masters and Victor Streltsov of the University of Melbourne agreed. They went a step further. “This suggests a broader concept where specific disease-related, post-translational modification patterns determine the amyloid filament conformation of distinct proteins in neurodegenerative diseases,” they wrote (comment below).
Beyond this single arginine residue in TDP-43, emerging evidence suggests that citrullination could play a broader role in the function and aggregation of proteins involved in neurodegenerative disease. For example, recent studies have reported an uptick in the activity of protein arginine deaminases—the enzymes that perform citrullination—in the brains of people with different neurodegenerative conditions (Feb 2018 news; Ishigami et al., 2005; Mondal et al., 2021). Other emerging studies cast citrullination as a potential cause of harmful autoimmune reactions that underlie diseases such as multiple sclerosis (Martín Monreal et al., 2023).
Maj-Linda Selenica of the University of Kentucky in Lexington studies the role of citrullination on TDP-43 function and aggregation. She told Alzforum that her group has found 11 of the 20 TDP-43 arginine residues can become citrullinated, and the modification appears to strongly influence the protein's liquid-liquid phase separation, pushing it to condense into small droplets. Selenica’s group has generated antibodies specific for these different modified forms. Preliminary findings suggest that TDP-43 citrullination rises in people with limbic predominant TDP-43 encephalopathy (LATE), a proteinopathy that causes cognitive decline and often co-occurs with AD. Selenica was excited to see that citrullination appears to be involved in the aggregation of TDP-43 in FTD, and believes targeting the pathway could hold therapeutic promise.
In addition to TDP-43 fibrils, Arseni also detected TMEM106b fibrils lurking in all three of the brain samples. These did not co-localize with the TDP-43 filaments. Their structure was consistent with the shape previously spotted among people with FTD/ALS type B pathology, as well as among people with other neurodegenerative diseases and even those without a proteinopathy (Apr 2022 news).
Thus far, TMEM106b filament structures do not appear specific to disease type, in contrast to fibrils of TDP-43 and other proteins, including tau, and α-synuclein. “The available evidence is consistent with the age-dependent accumulation of TMEM106B filaments in the human brain,” the authors concluded. —Jessica Shugart
- Surprise! TMEM106b Fibrils Found in Neurodegenerative Diseases
- Double Spiral Sets TDP-43 Apart from Other Amyloids
- Tau Filaments from the Alzheimer’s Brain Revealed at Atomic Resolution
- Cryo-EM Unveils Distinct Aβ42 Fibril Structures for Sporadic, Familial AD
- Citrullination, Anyone? New Gene Implicated in ALS
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No Available Further Reading
- Arseni D, Chen R, Murzin AG, Peak-Chew SY, Garringer HJ, Newell KL, Kametani F, Robinson AC, Vidal R, Ghetti B, Hasegawa M, Ryskeldi-Falcon B. TDP-43 forms amyloid filaments with a distinct fold in type A FTLD-TDP. Nature. 2023 Aug;620(7975):898-903. Epub 2023 Aug 2 PubMed.