Rapid Assembly of Alzheimer-like Paired Helical Filaments from Microtubule-Associated Protein Tau Monitored by Fluorescence in Solution

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Abstract
Alzheimer's disease is characterized by the progressive deposition of two types of fibers in the affected brains, the amyloid fibers (consisting of the Aβ peptide, generating the amyloid plaques) and paired helical filaments (PHFs, made up of tau protein, forming the neurofibrillary tangles). While the principles of amyloid aggregation are known in some detail, the investigation of PHF assembly has been hampered by the low efficiency of tau aggregation, the requirement of high protein concentrations, and the lack of suitable detection methods. Here we report a quantitative assay system that permits monitoring of the assembly of PHFs in real time by the fluorescence of dyes such as thioflavine 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 efficiently, but other tau isoforms or constructs form bona fide PHFs as well. The rate of assembly is greatly enhanced by polyanions such as RNA, heparin, and notably polyglutamate which resembles the acidic tail of tubulin. The assembly is optimal at pH ∼6 and low ionic strengths (<50 mM) and increases steeply with temperatures above 30 °C, indicating that it is an entropy-driven process.