Tau propagation/seeding assay based on TauRD-GFP FRET pairs does not represent templated assembly of PHFs

Senthilvelrajan Kaniyappan1, Katharina Tepper1, Jacek Biernat1, Ram Reddy Chandupatla1, Sabrina Hübschmann1, Stephan Irsen2, Sandra Bicher2, Christoph Klatt2, Eva-Maria Mandelkow1, Eckhard Mandelkow1

1 DZNE, Bonn
2 CAESAR, Bonn

Tau aggregation into amyloid-like fibers based on 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 [1]. 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 (TauRD, 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) TauRD and TauFL (full length Tau) on their aggregation behavior in vitro. Using light scattering (DLS), atomic force microscopy (AFM) and scanning-transmission electron microscopy (STEM), we found that assembly of TauRDΔK-GFP was severely inhibited, even in presence of nucleation enhancers (heparin). Some rare short fiber-like particles occurred but had a very different subunit packing, as judged by STEM. 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.