The study investigates the self-assembly of hyperphosphorylated tau (AD-P-τ) into paired helical filaments (PHF) and straight filaments (SF) in vitro. AD-P-τ, which is abnormally hyperphosphorylated in Alzheimer's disease (AD) brains, self-aggregates into PHF-like structures under reducing conditions at pH 6.9 at 35°C. Dephosphorylation but not deglycosylation inhibits this self-assembly. Hyperphosphorylation also induces the self-assembly of all six isoforms of human tau into PHF and SF, and the microtubule binding domains/repeats region can self-assemble into PHF. The findings suggest that hyperphosphorylation neutralizes the inhibitory basic charges of flanking regions, promoting the self-assembly of tau into tangles of PHF and SF. This process is crucial in the neurofibrillary degeneration seen in AD and other tauopathies.The study investigates the self-assembly of hyperphosphorylated tau (AD-P-τ) into paired helical filaments (PHF) and straight filaments (SF) in vitro. AD-P-τ, which is abnormally hyperphosphorylated in Alzheimer's disease (AD) brains, self-aggregates into PHF-like structures under reducing conditions at pH 6.9 at 35°C. Dephosphorylation but not deglycosylation inhibits this self-assembly. Hyperphosphorylation also induces the self-assembly of all six isoforms of human tau into PHF and SF, and the microtubule binding domains/repeats region can self-assemble into PHF. The findings suggest that hyperphosphorylation neutralizes the inhibitory basic charges of flanking regions, promoting the self-assembly of tau into tangles of PHF and SF. This process is crucial in the neurofibrillary degeneration seen in AD and other tauopathies.