RAPID ASSEMBLY OF ALZHEIMER-LIKE PAIRED HELICAL FILAMENTS FROM MICROTUBULE-ASSOCIATED PROTEIN-TAU MONITORED BY FLUORESCENCE IN SOLUTION

Alzheimer's disease is characterized by the progressive deposition of two types of fibers in the affected brains, the amyloid fibers (consis ting of the A beta peptide, generating the amyloid plaques) and paired helical filaments (PHFs, made up of tan protein, forming the neurofib rillary tangles). While the principles of amyloid aggregation are know n in some detail, the investigation of PHF assembly has been hampered by the low efficiency of tau aggregation, the requirement of high prot ein concentrations, and the lack of suitable detection methods. Here w e report a quantitative assay system that permits monitoring of the as sembly of PHFs in real time by the fluorescence of dyes such as thiofl avine S or T. Using this assay, we evaluated parameters that influence the efficiency of filament formation. Disulfide-linked dimers of tau constructs representing the repeat domain assemble into PHFs most effi ciently, but other tau isoforms or constructs form bona fide PHFs as w ell. The rate of assembly is greatly enhanced by polyanions such as RN A, heparin, and notably polyglutamate which resembles the acidic tail of tubulin. The assembly is optimal at pH similar to 6 and low ionic s trengths (<50 mM) and increases steeply with temperatures above 30 deg rees C, indicating that it is an entropy-driven process.