Tau R2 and R3 are essential regions for tau aggregation, seeding and propagation.

Tauopathies are characterised by intracellular deposits of fibrillar tau tangles. However, the interneuronal spread of pathological tau species precedes the development of major tau burdens. Two amyloid motifs, VQIINK in repeat 2 and VQIVYK in repeat 3, of tau repeat domain, assemble into β-sheet-rich fibrils on their own but alone do not form seed-competent fibrils. In contrast, the entire R3 region self-aggregates and forms seed-competent fibrils. Our study aimed to identify the minimal regions in the tau repeat domain that define seeding and its impact on intracellular tau phosphorylation and aggregation. Using peptides of individual repeats, we show that R2, like R3, forms seed-competent fibrils when assembled in the presence of heparin. However, R3, but not R2, forms seed-competent fibrils when assembled without heparin, even though both R2 and R3 have identical N-terminal hexapeptide and cysteine residue sequences. Moreover, cysteine to alanine substitution in R3 abrogates its self-aggregation and seeding potency. Tau RD P301S biosensor cells and Tau P301L (0N4R)-expressing HEK293 cells seeded with R2 and R3 fibrils show the induction of pathological phosphorylation of tau at Ser262/Ser396/Ser404 positions and oligomerisation of native tau. Protein fractions of biosensor cells seeded with R2 and R3 fibrils reseed endogenous tau aggregation when introduced into a fresh set of biosensor cells. Our findings suggest that R3 may be the minimal region for pathological seed generation under physiological conditions, whereas R2 might need polyanionic cofactors to generate pathogenic seeds. Lastly, R2 and R3 fibrils induce template-induced misfolding and pathological hyperphosphorylation of intracellular tau, making intracellular tau seed-competent.