Aggregated and highly phosphorylated tau protein is a pathological hallmark of Alzheimer's disease (AD) and other tauopathies. We identified motifs of alternating polar and apolar amino acids within the microtubule-binding repeats of tau which were interrupted by small breaking stretches. Minimal mutation of these breaking sequences yielded a unique instantly aggregating tau mutant containing longer stretches of polar/ apolar amino acids without losing its microtubule-binding capacity. These modifications produced rapid aggregation and cytotoxicity with accompanying occurrence of pathologic tau phosphoepitopes (AT8, AT180, AT270, AT100, Ser 422 , and PHF-1) and conformational epitopes (MC-1 and Alz50) in cells. Similar to pathological tau in the pretangle state, toxicity appeared to occur early without the requirement for extensive fibril formation. Thus, our mutant protein provides a novel platform for the investigation of the molecular mechanisms for toxicity and cellular behavior of pathologically aggregated tau proteins and the identification of its interaction partners.Aggregates of hyperphosphorylated tau protein are a pathological hallmark of many neurodegenerative diseases, which include Alzheimer disease (AD), 3 frontotemporal dementia with Parkinson syndrome linked to chromosome 17 (FTDP-17), and others (1, 2). These aggregates can become entangled in the form of neurofibrillary tangles that are held responsible for neuronal cell death via yet unknown intracellular mechanisms.The tau protein exists as a naturally unfolded protein that is associated with microtubules. However, within pathological aggregates like neurofibrillary tangles, a -strand conformation can be identified (3, 4). The physiological function of tau involves the binding and stabilization of the microtubule cytoskeleton. It exists as several splicing isoforms in the central nervous system. The expression levels of the different isoforms vary during the development and maturation of the central nervous system. This probably serves to modulate neuronal microtubule stability and function.The tau isoforms contain three or four C-terminal microtubule-binding repeats (MTBRs) and one or two acidic residues at the N terminus. The latter domains are not directly involved in the binding of microtubules, whereas the MTBRs of tau bind to the acidic C terminus of the tubulin monomers. The naturally unfolded nature of tau is probably explained by the fact that an extended conformation allows its interaction with several tubulin dimers (5).In the course of several diseases of the nervous system, tau molecules aggregate in the form of pathological multimers. Isolated tau protein exhibits a tendency to aggregate in the form of fibrils when incubated in the presence of polyanionic compounds or fatty acid-like molecules (3). It has been shown that the third MTBR contains a sequence that aggregates in isolated form (6), and accordingly, the MTBRs have been shown to be located in the core of fibrils by tryptophan fluorescence scanning spectroscopy (4). Ho...