For Alzheimerologists, the striking cryo-EM images of tau fibrils—with their back-to-back C-shaped rungs of tau—are emblazoned in the mind. In contrast, a new study proposes that not only do tau aggregates strike many poses in the AD brain, but that tau folds also set the pace of disease progression. Published January 5 in Science Translational Medicine, the study married antibodies with biophysical techniques to identify different conformations of tau in the hippocampi of people with AD. Led by Jiri Safar at Case Western Reserve University School of Medicine in Cleveland, the scientists found evidence of tau aggregates of various shapes and sizes. Among people with a fast-progressing form of AD, a distinct cadre of misfolded tau strains dominated. These replicated rapidly and comprised mostly the 4R tau isoform. The authors propose that tau’s distinct twists could sway the rate of AD progression, and influence the effectiveness of tau-targeted therapies.

  • Antibodies predict a diverse array of tau forms in people with AD.
  • Distinct contortions of tau detected in rapidly progressive AD.
  • Progression-associated conformers contained mostly the 4R isoform.

“What we found was an exceptional diversity,” Safar told Alzforum. “There is not a single entity, but a spectrum of disease-associated tau conformations.”

Michel Goedert and Sjors Scheres of the MRC Laboratory of Molecular Biology, Cambridge, England, U.K., wondered if the cores of the tau fibrils differed, or if variations in their outer—the so-called “fuzzy coat” that does not integrate into the fibril core—might explain the findings. Goedert and Scheres have used cryo-EM to resolve the distinct folds tau takes in different tauopathies, including AD, and to date the fibril cores from different patients with the same disease have largely been the same (Jun 2017 news; Oct 2021 news). 

Over the years, studies in cell culture and in transgenic mouse models have suggested that different conformations, or “strains” of tau, can faithfully propagate their unique shape via templated misfolding, much as prions do (May 2014 news; Nov 2017 news). Cryo-EM studies of tau plucked from postmortem brain samples have revealed a limited number of distinct tau folds that each track remarkably well with the type of neurodegenerative tauopathy they cause (Shi et al., 2021). 

In their study, first author Chae Kim and colleagues asked whether tau contorts into many different shapes among people with the same tauopathy, and even within the same person. If so, could such differences relate to how fast tauopathy blazes through the brain? They extracted tau from the hippocampi of 40 people who had died with AD, including 23 who had suffered from a rapidly progressive form of the disease. Initially referred to the U.S. National Prion Disease Pathology Surveillance Center because they were suspected to have prion disease, these patients were later found to have had AD, based on neuropathological examination. On average, people with this form of AD died within 14 months of onset, while those with more typical AD lived for nearly seven years. At death, there were no significant differences in tau tangle burden or its distribution between those with slow versus fast forms of the disease.

What about tau conformation? The researchers used indirect approaches to address this. One, called conformation-dependent immunoassay (CDI), labels tau with antibodies to epitopes that are buried within the folds of the protein under native conditions and become exposed when the protein is denatured. By measuring the ratio of antibody binding under denaturing versus native conditions, scientists can surmise how unfolded the protein is, and estimate how many different forms exist. In a related method, called conformational stability assay (CSA), extracted tau is exposed to increasing concentrations of the denaturing agent guanidine-HCl. Every conformer of tau unfolds at a slightly different concentration of the agent, revealing the array of potential structures present in a sample. The authors applied these methods to each fraction of tau separated by size on a sucrose gradient. Together, this generated estimates of the size and structural diversity of tau forms in each brain sample, though no information on the structure of individual conformers, as can be had from cryo-EM.

In a nutshell, the scientists found a diverse portfolio of folded taus within each brain sample. Across all samples, there were roughly equal proportions of 3R and 4R isoforms among tau monomers, while 4R predominated within insoluble tau aggregates regardless of size. The 4R isoform—which contains four repeats of the microtubule binding domain—made up 80 percent of tau incorporated into insoluble, protease-resistant aggregates. Notably, different forms of this insoluble, 4R-predominant tau existed in people with rapidly progressing versus typical AD. Together, the data suggested that distinct structures—formed mostly from protease-resistant 4R tau—are associated with rapid progression of AD.

Might these differ in seeding potency? In in-vitro seeding assays, and in biosensor cell lines that measure tau aggregate formation, tau extracted from people with rapidly progressing AD more potently triggered aggregation of normally folded tau. The source of this greater seeding potential primarily lay in fractions of insoluble 4R tau. The findings hint that the way tau misfolds could set the pace of AD progression, but do not answer the question of which structural attributes might accelerate tauopathy.

Safar said that future studies will need to zero in on specific forms of tau associated with rapid progression. He plans to more closely map structural specifics, including post-translational modifications, of tau strains using hydroxyl radical footprinting, a technique his group has used to probe characteristics of prion strains (Siddiqi et al., 2021). If disease-accelerating strains of tau can be detected in biofluids, they could serve as prognostic biomarkers, Safar said, adding that strain-specific methods are already being employed to detect different types of prion disease (see Foutz et al., 2016). 

Scheres and Goedert used the study to offer a reminder of how the definition of the word conformer is changing. While the broad definition of the term includes any proteins with the same sequence but different structure, a narrower definition has evolved in prion research. “In this context, it represents a misfolded conformation of an aggregated protein that provides the template for other copies of that protein to misfold and become part of the aggregate, leading to spreading of the misfolded protein,” they wrote. “Although the molecular mechanisms of templated seeding are not fully understood, it is assumed that the ordered cores of amyloids provide the conformations for templated seeding of new proteins. Therefore, it is most likely that the structures of the ordered cores of amyloids define conformers,” they wrote (full comment below).

Whether the versions of tau that emerged in his study differ in their core structures or only their fuzzy coat is unclear. However, that tau strains from people with rapidly progressing AD had superior seeding capacity suggests that their structural differences could have a functional impact on propagation, Safar said. However, Scheres and Goedert point out that the tau constructs used in these seeding assays lacked the eight C-terminal amino acids that make up the core of the tau fibril, meaning they may not faithfully recapitulate seeding in the AD brain.—Jessica Shugart

 

Comments

  1. What is a conformer?

    In chemistry, conformers, or conformational isomers, are sets of molecules having the same bond connectivity sequences that can interconvert by rotation around one or more (sigma) bonds. However, in biology, and in particular the prion field, “conformer” has acquired a narrower meaning. In this context, the word represents a misfolded conformation of an aggregated protein that provides the template for other copies of that protein to misfold and become part of the aggregate, leading to spreading of the misfolded protein (Prusiner, 1998). Misfolding of the prion protein leads to the formation of amyloids, which are helical aggregates with a characteristic cross-β quaternary structure. Different conformers, or prion strains, can spread independently, and have been associated with distinct diseases.

    In recent years, it has become clear that other amyloids, such as assembled tau, Aβ, α-synuclein and TDP-43, can also spread through the brain in a prion-like manner. Solid-state NMR, and in particular cryo-EM, have recently obtained a wealth of structural data on amyloid filaments, including those extracted from diseased tissues (Tycko, 2000; Scheres et al., 2020). Amyloids have an ordered core, containing β-sheets that stretch along the direction of the helical axis. In addition, they have a so-called fuzzy coat, where amino acids beyond the N- and C-terminal borders of the ordered core are less structured, i.e., they can adopt a wide range of different conformations. Although the molecular mechanisms of templated seeding are not fully understood, it is assumed that the ordered cores of amyloids provide the conformations for templated seeding of new proteins. Therefore, it is most likely that the structures of the ordered cores of amyloids define conformers.

    Kim et al. argue that there are “distinct populations of tau conformers” in their sarkosyl-insoluble preparations of assembled tau from Alzheimer’s disease brains. (The paper's title mentions rapidly progressing sporadic AD, but similar data is presented for less rapidly progressing disease.) These claims are based on indirect measurements of conformation, involving conformation-dependent immunoassays and conformational stability assays. However, decades-old negative-stain EM reports, as well as more recent cryo-EM studies, of sarkosyl-insoluble fractions from AD brain, have failed to provide evidence for the presence of clouds of distinct conformations in tau filament cores. Only two types of AD filaments, with a common protofilament fold, have consistently been identified: paired helical and straight filaments (Crowther, 1991; Fitzpatrick et al., 2017). The same is true of cases of primary age-related tauopathy, with short intervals between memory impairment and death (Shi et al., 2021).

    We therefore wonder whether the observations that have led Kim et al. to conclude that there are distinct populations of tau conformers in their samples could be due to effects that originated in the fuzzy coat of the aggregates, rather than in distinct conformations of their ordered cores. We would therefore discourage use of the term “distinct conformers” to describe these entities. Although they would, strictly speaking, still be distinct conformers in the wider meaning of the word in chemistry, this is likely not so in the context of prion-like spreading.

    Possibly related to this, we note that the K18/K19 tau constructs that are used in the seeding experiments described by Kim et al. cannot faithfully replicate the AD seed structures, since they lack eight C-terminal residues from the ordered core of AD filaments.

    References:

    . Prions. Proc Natl Acad Sci U S A. 1998 Nov 10;95(23):13363-83. PubMed.

    . Solid-state NMR as a probe of amyloid fibril structure. Curr Opin Chem Biol. 2000 Oct;4(5):500-6. PubMed.

    . Cryo-EM structures of tau filaments. Curr Opin Struct Biol. 2020 Oct;64:17-25. Epub 2020 Jun 27 PubMed.

    . Straight and paired helical filaments in Alzheimer disease have a common structural unit. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2288-92. PubMed.

    . Cryo-EM structures of tau filaments from Alzheimer's disease. Nature. 2017 Jul 13;547(7662):185-190. Epub 2017 Jul 5 PubMed.

    . Cryo-EM structures of tau filaments from Alzheimer's disease with PET ligand APN-1607. Acta Neuropathol. 2021 May;141(5):697-708. Epub 2021 Mar 16 PubMed.

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References

News Citations

  1. Tau Filaments from the Alzheimer’s Brain Revealed at Atomic Resolution
  2. Flock of New Folds Fills in Tauopathy Family Tree
  3. Like Prions, Tau Strains Are True to Form
  4. Human Tau Strains Propagate Faithfully in Wild-Type Mice

Paper Citations

  1. . Structure-based classification of tauopathies. Nature. 2021 Oct;598(7880):359-363. Epub 2021 Sep 29 PubMed.
  2. . Structurally distinct external solvent-exposed domains drive replication of major human prions. PLoS Pathog. 2021 Jun;17(6):e1009642. Epub 2021 Jun 17 PubMed.
  3. . Diagnostic and Prognostic Value of Human Prion Detection in Cerebrospinal Fluid. Ann Neurol. 2016 Nov 28; PubMed.

Further Reading

No Available Further Reading

Primary Papers

  1. . Distinct populations of highly potent TAU seed conformers in rapidly progressing Alzheimer's disease. Sci Transl Med. 2022 Jan 5;14(626):eabg0253. PubMed.