Serial amplification of tau filaments using Alzheimer’s brain homogenates and C322A or C322S recombinant tau

The assembly of tau into amyloid filaments is a defining characteristic of Alzheimer’s disease (AD) and other tauopathies. Cryo-electron microscopy (cryo-EM) showed that specific tau folds characterise different diseases, and that in vitro models often yield filaments with folds that do not replicate those that form in disease. Here, we investigated the aggregation of full-length recombinant 0N3R tau, using wild-type, or mutations C322A or C322S and a real-time quaking-induced conversion (RT-QuIC) assay with brain homogenate seeding. The assembly of C322A 0N3R tau resulted in filaments with a structure resembling the AD fold in paired helical filaments (PHFs), but with a more open C-shaped core attributed to the C322A mutation. C322S 0N3R tau formed structurally more distinct filaments with respect to PHFs, with an ordered carboxy-terminal region. Both mutant filaments retained the ability to seed a second round of aggregation. Meanwhile, wild-type 0N3R tau exhibited poor reproducibility and formed predominantly unfolded aggregates. Our findings emphasise the need for optimised assembly conditions to obtain disease-relevant filament folds. Refining these methodologies could enhance our understanding of the molecular origins of tauopathies and facilitate the development of targeted therapeutic strategies for these conditions.