Vaquer-Alicea J, Manon VA, Bommareddy V, Kunach P, Gupta A, Monistrol J, Perez VA, Tran HT, Saez-Calveras N, Du S, Batra S, Stoddard D, White CL 3rd, Joachimiak LA, Shahmoradian SH, Diamond MI. Functional classification of tauopathy strains reveals the role of protofilament core residues. Sci Adv. 2025 Jan 24;11(4):eadp5978. Epub 2025 Jan 22 PubMed.
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MRC Laboratory of Molecular Biology
MRC Laboratory of Molecular Biology
This paper by Vaquer-Alicea et al. describes the development of a new method to classify brain homogenates from different tauopathies, without the need for electron cryo-microscopy structure determination. Previously, cryo-EM structures of tau filaments from the brains of individuals with distinct tauopathies revealed that specific tau folds characterize different diseases, leading to a structure-based classification of disease (Shi et al., 2021) and thereby adding a molecular level of neuropathology (Scheres et al., 2023). However, because cryo-EM is expensive, and requires relatively large amounts of sample, cryo-EM structure determination of brain-derived filaments would be unwieldy as part of routine postmortem neuropathology. Although the different tau folds suggest that it should be possible to develop molecules that bind specifically to the different filaments in the future, such molecules are currently not available.
The method introduced by Vaquer-Alicea et al. provides an alternative route toward a generally applicable technique to classify tauopathy brain samples. The same group had previously introduced tau biosensor cells, HEK293T cells that overexpress truncated constructs of human mutant tau, coupled to fluorescent labels. When seeded with small amounts of tau filaments—from recombinant tau or from tauopathy brain homogenates—the seeded aggregation of tau in these cells can be monitored using fluorescence resonance energy transfer (FRET). In the current paper, the authors show that the seeded tau aggregates in the biosensor cells are indeed amyloid filaments, and they introduce a clever use of alanine scanning that allows for specific readouts when tau filaments with different structures are used as seeds.
For brain homogenates of 21 individuals with four different tauopathies, the authors convincingly show that seeded aggregation in their biosensor cells varies with the position of alanine mutations in the tau sequence, in a tau fold-dependent manner. Thereby, this method holds the exciting potential to be developed into a widely applicable test for the postmortem diagnosis of tauopathies.
Because the fluorescent protein labels hampered cryo-EM structure determination, the authors could not determine the structures of the seeded aggregates that form inside the biosensor cells. Previously, we showed that seeded aggregation of overexpressed human tau in SH-S5Y5 cells yielded filaments that resembled, but were not identical, to the filaments that were used as seeds (Tarutani et al., 2023). It is possible that the same is also true for the HEK293T biosensor cells. Therefore, care should be taken in the interpretation of the alanine scan results in terms of the structures of the input seeds. Future cryo-EM structure determination of the seeded aggregates, possibly without their fluorescent labels, would resolve these uncertainties, and would add valuable information to answer the question what determines the formation of specific tau folds in the different diseases.
References:
Shi Y, Zhang W, Yang Y, Murzin AG, Falcon B, Kotecha A, van Beers M, Tarutani A, Kametani F, Garringer HJ, Vidal R, Hallinan GI, Lashley T, Saito Y, Murayama S, Yoshida M, Tanaka H, Kakita A, Ikeuchi T, Robinson AC, Mann DM, Kovacs GG, Revesz T, Ghetti B, Hasegawa M, Goedert M, Scheres SH. Structure-based classification of tauopathies. Nature. 2021 Oct;598(7880):359-363. Epub 2021 Sep 29 PubMed.
Scheres SH, Ryskeldi-Falcon B, Goedert M. Molecular pathology of neurodegenerative diseases by cryo-EM of amyloids. Nature. 2023 Sep;621(7980):701-710. Epub 2023 Sep 27 PubMed.
Tarutani A, Lövestam S, Zhang X, Kotecha A, Robinson AC, Mann DM, Saito Y, Murayama S, Tomita T, Goedert M, Scheres SH, Hasegawa M. Cryo-EM structures of tau filaments from SH-SY5Y cells seeded with brain extracts from cases of Alzheimer's disease and corticobasal degeneration. FEBS Open Bio. 2023 Aug;13(8):1394-1404. Epub 2023 Jul 7 PubMed.
View all comments by Michel GoedertEmory University
The strain phenomenon—i.e., the concept that a single proteopathic agent can engender multiple manifestations of disease—has long been an enigmatic piece of the prion puzzle. In microbiology, “strain” classically refers to variant microbes within a given species; whereas microbial strain differences are genetically encoded, evidence has gradually accumulated that the essential feature of prion strains is variation of the 3D structure of the misfolded prion protein. In recent years, this concept has increasingly been applied to the tau and Aβ proteins, which have prion-like functionality and are central to Alzheimer's disease. However, the precise mechanisms driving strain-like variation have been uncertain.
With the emergence of powerful methods for visualizing the architecture of folded proteins (in particular cryo-electron microscopy), the molecular structural basis of proteopathic strains is coming into focus. Cryo-EM beautifully shows how the amino acids are arranged in amyloids, but it can only furnish limited information on how specific amino acids influence the protean nature of amyloidogenic proteins. Using an ingenious tau "biosensor" assay and alanine substitution scanning, Vaquer-Alicea and colleagues report that different tauopathies (including Alzheimer's) can be differentiated by the pattern of incorporation of systematically mutated (alanine-substituted) tau into intracellular tau aggregates.
In this paradigm, alanine acts as an inert residue that functionally neutralizes the location of the substituted amino acid, thereby reporting on the role of each amino acid in the interaction of tau monomers with pre-existing tau assemblies within cells. Thus, whereas cryo-EM yields information on the overall structure of proteopathic strains, the alanine substitution/biosensor method highlights the amino acids that most strongly govern strain-like properties.
This approach is a welcome addition to the technical armamentarium for defining the molecular basis of proteopathic strains, and it furnishes persuasive new support for the key role of prionic mechanisms in tauopathies. The findings underscore the importance of amino acids in the amyloid core for defining the strain-like variations of tau. They also affirm the influence of the cellular context—the host cells—on the prion-like propagation of tau strains.
Discerning the architecture of proteopathic strains is an important step toward understanding the clinical and pathological heterogeneity of Alzheimer's disease and probably many other degenerative disorders. In this regard, the alanine substitution/tau biosensor system might serve as a prototype for the development of new models for interrogating strain-like variations in diverse pathogenic proteins.
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