Anionic contribution for fibrous maturation of protofibrillar assemblies of the human tau repeat domain in a fluoroalcohol solution

Tau protein forms fibrous aggregates in the brain of patients with Alzheimer's disease. This type of aggregation in vitro is promoted efficiently by polyanions and anionic micelles. Here, we report another cosolvent system that induces the fibrous aggregation of human tau four-repeat domain (tau4RD). The protein aggregation was primarily achieved by a nonanionic agent 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), while the ionic condition was modified by inorganic salts. The aggregation analysis by three spectroscopic methods revealed a two-phase kinetics of the aggregation of tau4RD in the presence of HFIP at approximately 4-6%. Large increases in the light-scattering, the thioflavin-binding, and the secondary structure content of tau4RD have progressed within a few minutes at 37 degrees C, which was followed by another slower aggregation phase. Electron microscopic analysis demonstrated that the amorphous granules are formed in the faster step, which acquired a fibrous shape in the slower step in the solution containing NaCl. In the absence of the salt, however, the fibrous maturation was inhibited. Examination of various salt species in place of NaCl demonstrated that binding of anions to the precursor aggregates was essential for the fibrous maturation. On the basis of the results, we proposed an aggregation scheme of tau in which the formation of a thioflavin-binding intermediate occurred ahead of its fibrous maturation. The anionic environment was suggested to play a crucial role in the fibrous maturation and, therefore, could be an in vivo determinant of the morphology of the aggregates of tau.