Develop a rapid, cell-based biosensor assay that classifies tauopathies based on how tau alanine point mutants incorporate into seeded aggregates.

Cryogenic electron microscopy (cryo-EM) has enabled a structure-based classification of tauopathies, but it is slow, resource-intensive, requires substantial tissue, and is not available for routine neuropathological use. We sought a practical alternative that infers fold-specific features from tauopathy material with fewer resources and material.

We engineered six alanine substitutions within the tau repeat domain and expressed each variant in tau biosensor cells. Brain-derived seeds from a library of 50 well-characterized tauopathy cases were used to induce aggregation in the biosensors. For each case, we quantified the incorporation of each mutant into seeded aggregates and used these data to distinguish strains associated with distinct tauopathies. We named this system the Tau Multiplex.

The Tau Multiplex assay correctly predicted the primary neuropathologic diagnosis for all tested cases and revealed heterogeneity often missed by standard approaches. Overall, 20% of samples showed the presence of co-dominant folds or atypical signatures that did not map to known entities. Notably, 43% of Pick’s disease cases exhibited an additional 4R pattern alongside the expected 3R profile, and 21% of Progressive Supranuclear Palsy cases displayed a distinct 4R signature relative to the canonical pattern. These findings indicate that multiple tau strains can coexist within a single patient and their relative abundance varies across diseases.