Kaniyappan S, Tepper K, Biernat J, Chandupatla RR, Hübschmann S, Irsen S, Bicher S, Klatt C, Mandelkow EM, Mandelkow E. FRET-based tau seeding assay does not represent prion-like templated assembly of tau fibers. bioRxiv March 25, 2020. BioRxiv.

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  1. Understanding mechanisms of tau aggregation continues to be an important avenue of ongoing research. A challenge we all face in modeling tau aggregation as it occurs in AD and ADRD is that the milieu of the neuron is much more complicated than that occurring in vitro or even in cell lines. This differential complexity between neurons and in vitro milieus raises strong challenges for modeling the actual process that gives rise to tau pathology and toxic tau oligomers.

    The manuscript by Kaniyappan et al. comes from the Mandelkow laboratory and highlights important differences between tau aggregation occurring in the brain and tau aggregation occurring in FRET-based biosensor lines, such as those developed by the Diamond group. The Mandelkow team shows that the presence of fluorescent proteins in the chimeric recombinant tau constructs used in the FRET-based biosensor lines gives rise to tau fibrils that are structurally very different than native tau aggregates occurring in vitro or in the brain. This is perhaps not surprising, because the fluorescent proteins are larger than the small tau peptides (such as the K18 peptide) that drive tau aggregation in the FRET-based biosensor lines. Thus, the fluorescent proteins take up space and change the structure of the resulting filament. 

    The differences in filament structure pose important restrictions on how one can interpret results from such biosensor lines. The Mandelkow team correctly points out that one cannot use these biosensor lines as the basis of structural studies of tau filament formation.

    However, these biosensor lines have other uses that remain viable, even if the resulting tau filaments do not reflect those occurring in AD and ADRD. One important use is as a sensitive assay to detect the presence of aggregation-competent tau in brain tissues (e.g., from transgenic mice) or in experiments using other cultured cells. The FRET-based biosensors robustly form aggregates when seeded with aggregation-competent tau. In this scenario, the biosensor line is used to detect a particular biochemical species, and the nature of the resulting signal is less important than the sensitivity and specificity of detection.  Using an ELISA assay presents a good analogy. The signal from the ELISA assay reflects the presence of tau oligomers or aggregates, but the actual signal itself is structurally very different than the tau oligomer/aggregate.

    A second use of the biosensor lines is to detect conformational differences in tau aggregates. In this scenario, the structure of the resulting biosensor aggregate is less important than the pattern of accumulation of tau aggregates, which tends to selectively reflect the initiating tau aggregate conformations.

    View all comments by Benjamin Wolozin

  2. Having speed-read the tau controversy, I would conclude that the findings of the Mandelkow group are reasonable within the context of their experimental conditions. The problem is one of comparing apples and oranges, as pointed out by Diamond et al. The Diamond model of in vivo (cell culture) seeding is well validated in multiple labs.

    To settle the issue, it would be useful to run controlled comparisons of technical differences, such as the linker length for the fluorescent molecules, in the Mandelkow paradigm. It would also be useful if the Mandelkow group could perform cell culture experiments using the Diamond model.  I really like the degree of control you can get with in vitro paradigms such as the Mandelkows’. These are quite useful in finely dissecting molecular mechanisms, but the cellular environment can complicate things considerably.

    View all comments by Lary Walker

  3. There really never was a question that the conformational structure of the FRET-based bioreporter would be the same as tau aggregates in the brain–the former is short and has large fluorescent proteins attached, the latter is full-length and has innumerable post-translational modifications. Indeed, even recombinant tau, if aggregated with heparin, forms a very different structure than PHF.

    The key question is whether the FRET reporter assay allows one to examine biological processes that are of import to the disease. The work of Diamond and numerous other labs suggests that the answer is “yes” in many circumstances, although the Mandelkow data elegantly shows that the answer is “no” in terms of detailed conformational studies intended to model the seed that was introduced. 

    Thus, like most models, its utility depends heavily on understanding its strengths and weaknesses, and seeing how they impact the experimental question at hand.

    View all comments by Bradley Hyman

  4. This is an interesting study as it provides valuable information on the nature of the tau aggregates that underlie the fluorescent signal one detects in this seeding assay. In addition, it provides a very useful characterization of how fluorophores linked to different tau constructs influence aggregation. The results of this study suggest that a deeper characterization of the different tau seeding assays might be worthwhile. Perhaps assays that lead to accumulation of tau fibrils are more sensitive biosensors (e.g., generate more signal)? It might also be necessary for certain studies to use seeding-based biosensor assays that generate fibrils that more closely resemble those of tauopathy patients, for example to study how tau seeding impacts the cell or induces tau propagation to other cells.

    It should also be noted that the main conclusion of this article might only apply to biosensor assays with the repeat domain of tau linked to the fluorophore. The data in the manuscript show that fibrils do form when the fluorophore is linked to the N-terminal or C-terminal of full-length tau. It remains to be determined if aggregates composed of full-length tau linked to fluorophores—induced by human brain-derived seeds—more closely resemble the fibrils found in the brains of tauopathy patients.

    In addition to the previously posted comments to this article, it is worth noting that other versions of this assay use labelled antibodies to stain the tau aggregates after seeding to obtain a FRET signal. The observed characteristics of this assay are similar to the original tau biosensor assay (Courade et al, 2018). 

    References:

    Courade JP, Angers R, Mairet-Coello G, Pacico N, Tyson K, Lightwood D, Munro R, McMillan D, Griffin R, Baker T, Starkie D, Nan R, Westwood M, Mushikiwabo ML, Jung S, Odede G, Sweeney B, Popplewell A, Burgess G, Downey P, Citron M. Epitope determines efficacy of therapeutic anti-Tau antibodies in a functional assay with human Alzheimer Tau. Acta Neuropathol. 2018 Nov;136(5):729-745. Epub 2018 Sep 20 PubMed.

    View all comments by Thomas Vogels

  5. In our paper, now in press at Mol. Neurodegeneration, we argue that the repeat domain of Tau, when coupled to GFP, cannot form amyloid-like Tau filaments because of steric hindrance. GFP is too big to fit onto the tight cross-β structure of an amyloid fiber; analogous observations have been made with other amyloid proteins. This implies that the local foci, observed in a Diamond-type sensor cell expressing FRET pairs of TauRD-XFP, are not caused by amyloid Tau fibers. This contradicts the hypothesis that the propagation of Tau pathology is based on a prion-like propagation of Tau assembly. However, it would be compatible with a local accumulation of TauRD-XFP in a non-amyloid state.

    Several colleagues have made the counterargument that the sensor cell reaction is a reliable indicator of some pathological property in the Tau preparations used to trigger the FRET reaction (e.g., extracts from AD brains or transgenic mouse models). We agree (and say so in the paper), but this only means that the sensor cell is a useful diagnostic tool.

    By analogy, in ALS research arsenite is used to induce local foci of XFP-labeled FUS in the cytoplasm, but this does not mean that arsenite templates the amyloid assembly of FUS; rather, it works indirectly by triggering stress granules containing FUS-XFP.

    The implication of our argument is that not Tau itself, but some other Tau-related agent may be the trigger for the FRET reaction. In support of this, the same type of FRET reaction can be triggered by non-Tau agents, e.g. pro-inflammatory cytokines; therefore the trigger is not unique. 

    In essence, we argue that one should strictly distinguish between the spreading of Tau pathology, which is generally accepted by Braak staging, and the spreading of Tau protein by templated assembly, which is a matter of debate, and generally adopt a less Tau-centric view on the origins of Tau pathology. After all, only 25 percent of AD diversity can be ascribed to Tau, as shown by Dujardin et al., 2020.

    References:

    Dujardin S, Commins C, Lathuiliere A, Beerepoot P, Fernandes AR, Kamath TV, De Los Santos MB, Klickstein N, Corjuc DL, Corjuc BT, Dooley PM, Viode A, Oakley DH, Moore BD, Mullin K, Jean-Gilles D, Clark R, Atchison K, Moore R, Chibnik LB, Tanzi RE, Frosch MP, Serrano-Pozo A, Elwood F, Steen JA, Kennedy ME, Hyman BT. Tau molecular diversity contributes to clinical heterogeneity in Alzheimer's disease. Nat Med. 2020 Aug;26(8):1256-1263. Epub 2020 Jun 22 PubMed. Correction.

    View all comments by Eckhard Mandelkow

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  1. Widely Used Tau Seeding Assay Challenged