[Abstract unavailable

The epitope on tau protein recognized by the monoclonal antibody Alz50 was defined through internal deletion mutagenesis and quantified by affinity measurements. The epitope is discontinuous and requires both a previously identified N-terminal segment and the microtubule binding region for efficient binding of Alz50. The interaction between these regions is consistent with an intramolecular reaction mechanism, suggesting that Alz50 binding depends on the conformation of individual tau monomers. The results suggest that tau adopts a distinct conformation when polymerized into filaments and that this conformation is recognized selectively by Alz50.Alz50 is an IgM-class monoclonal antibody that stains the fibrillar pathology (dystrophic neurites, neurofibrillary tangles, and neuropil threads) commonly observed in postmortem histological analysis of Alzheimer's disease (AD) 1 brain (1, 2). Because of these properties, it has emerged as an important tool for gauging the temporal and spatial severity of Alzheimer's disease pathology (3, 4). The major components of the fibrillar pathology are straight and paired helical filaments (PHF) (5), which themselves are comprised largely of hyperphosphorylated forms of the microtubule-associated protein tau (6 -10). Previous studies have shown that Alz50 reacts with tau and that its epitope is located at the N terminus (11-13) in a region conserved in all known splice variants of human tau (14). Indeed, Alz50 has been shown to react with tau proteins isolated from normal brain (15), recombinant sources (12), and PHFs (8) by Western analysis. Nonetheless, the ability of Alz50 to label distinct populations of neurons in normal human brain (16), fetal brain (17), and early stage neurofibrillary degeneration (4, 18) suggests that Alz50 selectively recognizes a distinct subset of tau proteins.To place the many observations on Alz50 immunocytochemistry into a structural context, we reinvestigated its epitope selectivity in vitro. The results suggest that individual tau monomers adopt a specific conformation preceding or during filament formation that is selectively recognized by Alz50. EXPERIMENTAL PROCEDURESMaterials-All monoclonal antibodies were prepared from supernatants of hybridoma cells grown in serum-free medium. Supernatants containing Alz50 were pooled, precipitated with 45% ammonium sulfate, resuspended in TBS (50 mM Tris HCl, pH 7.5, 50 mM NaCl, and 1 mM EGTA), and dialyzed twice against 100 volumes of TBS. The dialysate was clarified by centrifugation and redialyzed against 5 mM sodium phosphate, pH 7.5, to precipitate the IgM. The resultant fine precipitate was resuspended in S300 buffer (50 mM Tris HCl, pH 7.5, 700 mM NaCl, and 1 mM EGTA), dispersed with 30 strokes of a glass-Teflon homogenizer, and loaded onto a 400-ml (2.6 ϫ 100-cm) S300HR gel filtration column equilibrated and run in S300 buffer. The IgM fraction emerging in the void volume was pooled, dialyzed against storage buffer (10 mM HEPES, pH 7.4, 50% glycerol, and 150 mM NaCl), and stored at Ϫ20°C until use...

Alzheimer's disease is defined in part by the intraneuronal accumulation of filaments comprised of the microtubule-associated protein tau. In vitro, fibrillization of recombinant tau can be induced by treatment with various agents, including phosphotransferases, polyanionic compounds, and fatty acids. Here we characterize the structural features required for the fatty acid class of tau fibrillization inducer using recombinant fulllength tau protein, arachidonic acid, and a series of straight chain anionic, cationic, and nonionic detergents. Induction of measurable tau fibrillization required an alkyl chain length of at least 12 carbons and a negative charge consisting of carboxylate, sulfonate, or sulfate moieties. All detergents and fatty acids were micellar at active concentrations, due to a profound, taudependent depression of their critical micelle concentrations. Anionic surfaces larger than detergent micelles, such as those supplied by phosphatidylserine vesicles, also induced tau fibrillization with resultant filaments originating from their surface. These data suggest that anionic surfaces presented as micelles or vesicles can serve to nucleate tau fibrillization, that this mechanism underlies the activity of fatty acid inducers, and that anionic membranes may serve this function in vivo.

Parkinson's disease is characterized by the aggregation of ␣-synuclein into filamentous forms within affected neurons of the basal ganglia. Fibrillization of purified recombinant ␣-synuclein is inefficient in vitro but can be enhanced by the addition of various agents including glycosaminoglycans and polycations. Here we report that fatty acids and structurally related anionic detergents greatly accelerate fibrillization of recombinant ␣-synuclein at low micromolar concentrations with lag times as short as 11 min and apparent first order growth rate constants as fast as 10.4 h ؊1 . All detergents and fatty acids were micellar at active concentrations because of an ␣-synuclein-dependent depression of their critical micelle concentrations. Other anionic surfaces, such as those supplied by anionic phospholipid vesicles, also induced ␣-synuclein fibrillization, with resultant filaments originating from their surface. These data suggest that anionic surfaces presented as micelles or vesicles can serve to nucleate ␣-synuclein fibrillization, that this mechanism underlies the inducer activity of anionic surfactants, and that anionic membranes may serve this function in vivo.

The mechanism through which arachidonic acid induces the polymerization of tau protein into filaments under reducing conditions was characterized through a combination of fluorescence spectroscopy and electron microscopy. Results show that polymerization follows a ligand-mediated mechanism, where binding of arachidonic acid is an obligate step preceding tau-tau interaction. Homopolymerization begins with rapid (on the order of seconds) nucleation, followed by a slower elongation phase (on the order of hours). Although essentially all synthetic filaments have straight morphology at early time points, they interact with thioflavin-S and monoclonal antibody Alz50 much like authentic paired helical filaments, suggesting that the conformation of tau protein is similar in the two filament forms. Over a period of days, synthetic straight filaments gradually adopt paired helical morphology. These results define a novel pathway of tau filament formation under reducing conditions, where oxidation may contribute to final paired helical morphology, but is not a necessary prerequisite for efficient nucleation or elongation of tau filaments.