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

Abstract

Tau 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 of nucleated polymerization proposed for prion pathogenesis. In cell cultures, the process is often monitored by a FRET assay where the recipient cell expresses the Tau repeat domain (Tau RD) with a pro-aggregant mutation, fused to GFP-based FRET pairs. Since the size of the reporter GFP (barrel of~3 nm × 4 nm) is~7 times larger than the β-strand distance (0.47 nm), this points to a potential steric clash. Hence, we investigated the influence of the GFP tag on Tau FL or Tau RD aggregation. Using biophysical methods (light scattering, atomic force microscopy (AFM), and scanning-transmission electron microscopy (STEM)), we found that the assembly of Tau RD-GFP was severely inhibited and incompatible with that of Alzheimer filaments. These observations argue against the hypothesis that the propagation of Tau pathology in AD is caused by the prion-like templated aggregation of Tau protein, transmitted via cell-to-cell spreading of Tau. Thus, even though the observed local increase of FRET in recipient cells may be a valid hallmark of a pathological reaction, our data argue that it is caused by a process distinct from assembly of Tau RD filaments.

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 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 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.…”

“…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 full-length 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, 2017 , 2019 ; Sanders 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 ).…”

“…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.…”

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

“…The addition of liposomes (lipofectamine) to facilitate the transduction of seeds into cells greatly increases the sensitivity of the biosensor assay but bypasses tau uptake mechanisms and therefore does not reflect seeding as it happens in the brain. A recent report suggests that the fusion of tau RD to fluorescent proteins may induce steric hindrance that avoids the elongation of tau aggregates into paired helical filaments ( Kaniyappan et al, 2020b ). The authors propose that the increase of FRET signal after exposure to seed-competent tau may result from cellular processes different from aggregation.…”