Differential aggregation and seeding of tau microtubule-binding region fragments in vitro.

The microtubule-binding repeat region (MTBR) of tau is central to pathological aggregation and propagation in Alzheimer's disease (AD). Cryo-electron microscopy has defined residues 306-378, encompassing R3 and R4, as the ordered fibril core of AD tau; however, whether this structural core also represents the most potent seeding unit remains unclear. We generated recombinant tau fragments beginning at R1, R2, or R3 and compared their amyloid-forming and cellular seeding activities. Thioflavin T assays showed that R3-R4 and R3-378, corresponding to the AD fibril core, exhibited strong amyloid-forming activity. In contrast, FRET-based cellular assays showed maximal seeding with R2-starting fragments. Extension of R2-containing fragments into R1 markedly reduced seeding activity, indicating that R2 accessibility and sequence context, rather than the mere presence of R2, influence seed competence. These findings demonstrate a dissociation between sequence determinants of mature fibril formation and cellular tau seeding and identify exposure of R2 as a potential feature of propagation-competent tau.