FRET-based Tau seeding assay does not represent prion-like templated assembly of Tau fibers

Abstract

Abbreviations: AD Alzheimer disease AFM atomic force microscopy CNS central nervous system FRET fluorescence resonance energy transfer GFP green fluorescent protein PHF paired helical filament Tau FL full-length Tau protein, largest isoform in human CNS (Uniprot P10636-F, htau40) Tau FLΔK full-length Tau protein with pro-aggregant mutation ΔK280 Tau RD Tau repeat domain (amyloidogenic domain) Tau RDΔK Tau repeat domain with pro-aggregant mutation ΔK280 Sf9 cell line from Spodoptera frugiperda STEM scanning transmission electron microscopy EM electron microscopy MPL mass per length Wt wildtype AbstractTau aggregation into amyloid fibers based on the cross-beta structure is a hallmark of several Tauopathies, including Alzheimer Disease (AD). Trans-cellular propagation of Tau with pathological conformation has been suggested as a key disease mechanism. This is thought to cause the spreading of Tau pathology in AD by templated conversion of naive Tau in recipient cells into a pathological state, followed by assembly of pathological Tau fibers, similar to the mechanism proposed for prion pathogenesis. In cell cultures, the process is usually monitored by a FRET assay where the recipient cell expresses the Tau repeat domain (Tau RD , with pro-aggregant mutation, e.g., ΔK280 or P301L, ~13.5 kDa) fused to GFP-based FRET pairs (YFP or CFP, ~28 kD). Since the diameter of the reporter GFP (~3 nm) is ~6.5 times larger than the β-strand distance (0.47nm), this points to a potential steric clash. Hence, we investigated the influence of GFP tagged (N-or C-terminally) Tau RD and Tau FL (full-length Tau) on their aggregation behavior in vitro. Using biophysical methods (light scattering, atomic force microscopy (AFM), and scanningtransmission electron microscopy (STEM)), we found that the assembly of Tau RDΔK -GFP was severely inhibited, even in the presence of nucleation enhancers (heparin and/or pre-formed PHFs from Tau RDΔK ). Some rare fiber-like particles had a very different subunit packing from proper PHFs, as judged by STEM. The mass per length (MPL) values of Tau RDΔK fibrils are equivalent to 4.45 molecules/nm, close to the expected value for a paired-helical fiber with 2 protofilaments and cross-β structure. By contrast, the elongated particles formed by Tau RDΔK -GFP have MPL values around ~2, less than half of the values expected for PHFs, indicating that the subunit packing is distinct. Thus, both kinetic and structural observations are incompatible with a model whereby external Tau can form a template for PHF assembly of Tau-GFP in recipient cells. As a consequence, the observed local increase of FRET in recipient cells must be caused by other signalling processes.

The AD tau core spontaneously self-assembles and recruits full-length tau to filaments

“…In addition, our findings provide clarity for a controversy surrounding the tau biosensor cell line. In particular, a recent study demonstrated that GFP interferes with the in vitro assembly of recombinant tau due to steric hindrance (Kaniyappan et al, 2020), leading the authors to speculate that the presence of fluorophore tags would similarly Cell Reports 34, 108843, March 16, 2021 9 Article ll perturb tau assembly in cell models, questioning the interpretation of changes in FRET signal in the biosensor cell line used in the current study. Moreover, the authors note the inefficient induction of FRET by purified tau assemblies in comparison with tissue lysates indicate that factors other than tau may be responsible for inducing FRET in biosensor cells, such as cytokines (Kaniyappan et al, 2020).…”

“…In particular, a recent study demonstrated that GFP interferes with the in vitro assembly of recombinant tau due to steric hindrance (Kaniyappan et al, 2020), leading the authors to speculate that the presence of fluorophore tags would similarly Cell Reports 34, 108843, March 16, 2021 9 Article ll perturb tau assembly in cell models, questioning the interpretation of changes in FRET signal in the biosensor cell line used in the current study. Moreover, the authors note the inefficient induction of FRET by purified tau assemblies in comparison with tissue lysates indicate that factors other than tau may be responsible for inducing FRET in biosensor cells, such as cytokines (Kaniyappan et al, 2020). However, our observation that purified AD tau core filaments and AD tau core-induced full-length tau filaments both promote significant increases in FRET positivity in the tau biosensor cell line clearly indicates the response is mediated by tau, with a higher FRET signal associated with more aggregation-prone forms of tau, consistent with the idea that FRET is an indication of seeding activity in this cell model.…”

“…However, our observation that purified AD tau core filaments and AD tau core-induced full-length tau filaments both promote significant increases in FRET positivity in the tau biosensor cell line clearly indicates the response is mediated by tau, with a higher FRET signal associated with more aggregation-prone forms of tau, consistent with the idea that FRET is an indication of seeding activity in this cell model. While the resulting structure of the aggregated forms of tau that are FRET positive is unlikely to recapitulate brain-derived tau filaments, as heparin-induced tau filaments also do not recapitulate the structure of brain-derived tau filaments (Fichou et al, 2018;Zhang et al, 2019), the effect of GFP on heparin-induced tau filament formation in vitro (Kaniyappan et al, 2020) may be physiologically irrelevant.…”

“…To do so, we cloned the CBD core (amino acids 274-380 corresponding to full-length 4R2N tau isoform) (Arakhamia et al, 2020;Zhang et al, 2020) and PiD core (amino acids 254-274; 306-378 corresponding to fulllength 4R2N isoform; PiD core is 3R, so residues 275-305 encoded by exon 10 are missing) (Falcon et al, 2018a) into a recombinant protein vector, and we optimized the purification protocol for each core protein. In addition, we also purified the K18 tau fragment (amino acids 244-368 of full-length 4R2N isoform; corresponds to the microtubule-binding domain of 4R tau) to determine how filament assembly compares with the CBD and PiD cores, as the K18 fragment is widely used to model tau aggregation (Barré and Eliezer, 2013;Holmes et al, 2014;Iba et al, 2013;Kaniyappan et al, 2020;Karikari et al, 2017Karikari et al, , 2019Sanders et al, 2014;Shammas et al, 2015;Weismiller et al, 2018;Zhang et al, 2019). We first monitored thioflavin signal with shaking at 37 C to assess the kinetics of aggregation of the different core proteins ( Figure 6A).…”

Quantitative Methods for the Detection of Tau Seeding Activity in Human Biofluids

“…Thus, some optimization needs to be done to improve the assay sensitivity and tailor it for a clinical use with reasonable volume of CSF. Such optimization may include ways to enhance the aggregation of FRET probes, including using tau constructs that may be more disease-relevant such as fragments covering the whole structure of amyloid core of tau aggregates ( Fitzpatrick et al, 2017 ), or adapting the linker between fluorescent reporters and the tau protein to avoid steric hindrance ( Kaniyappan et al, 2020a ). In addition, adapting the fluorescent protein pairs to increase the FRET efficiency may also increase the signal produced by reporter cell lines ( Bajar et al, 2016 ).…”