Pathways of tau fibrillization.

New methods for analyzing tau fibrillization have yielded insights into the biochemical transitions involved in the process. Here we review the parallels between the sequential progression of tau fibrillization observed macroscopically in Alzheimer's disease (AD) lesions and the pathway of tau aggregation observed in vitro with purified tau preparations. In addition, pharmacological agents for further dissection of fibrillization mechanism and lesion formation are discussed.

Cyanine dye N744 inhibits tau fibrillization by blocking filament extension: implications for the treatment of tauopathic neurodegenerative diseases.

Tau fibrillization is a potential therapeutic target for Alzheimer's and other neurodegenerative diseases. Small molecules capable of both inhibiting aggregation and promoting filament disaggregation have been discovered, but knowledge of their mechanism of action and potential for testing in biological models is fragmentary. To clarify these issues, the interaction between a small-molecule inhibitor of tau fibrillization, 3,3'-bis(beta-hydroxyethyl)-9-ethyl-5,5'-dimethoxythiacarbocyanine iodide (N744), and full-length four-repeat tau protein was characterized in vitro using transmission electron microscopy and fluorescence spectroscopy. Analysis of reaction time courses performed in the presence of anionic fibrillization inducers revealed that increasing concentrations of N744 decreased the total filament length without modulating lag time, indicating that filament extension but not nucleation was affected by inhibitor under the conditions that were investigated. Critical concentration measurements confirmed that N744 shifted equilibria at filament ends away from the fibrillized state, resulting in endwise filament disaggregation when it was added to synthetic filaments. Both increasing bulk tau concentrations and filament stabilizing modifications such as pseudophosphorylation and glycation antagonized N744 activity. The results illustrate the importance of mechanism for the design and interpretation of pharmacological studies in biological models of tau aggregation.

Triggers of full-length tau aggregation: a role for partially folded intermediates.

Alzheimer's disease is characterized in part by the accumulation of full-length tau proteins into intracellular filamentous inclusions. To clarify the events that trigger lesion formation, the aggregation of recombinant full-length four-repeat tau (htau40) was examined in vitro under near-physiological conditions using transmission electron microscopy and spectroscopy methods. In the absence of exogenous inducers, tau protein behaved as an assembly-incompetent monomer with little tertiary structure. The addition of anionic inducers led to fibrillization with nucleation-dependent kinetics. On the basis of circular dichroism spectroscopy and reactivity with thioflavin S and 8-anilino-1-naphthalenesulfonic acid fluorescent probes, the inducer stabilized a monomeric species with the folding characteristics of a premolten globule state. Planar aromatic dyes capable of binding the intermediate state with high affinity were also capable of triggering fibrillization in the absence of other inducers. Dye-mediated aggregation was characterized by concentration-dependent decreases in lag time, indicating increased nucleation rates, and submicromolar critical concentrations, indicating a final equilibrium that favored the filamentous state. The data suggest that the rate-limiting barrier for filament formation from full-length tau is conformational and that the aggregation reaction is triggered by environmental conditions that stabilize assembly-competent conformations.

Evidence for an intermediate in tau filament formation.

Alzheimer's disease is defined in part by the intraneuronal accumulation of filaments comprised of the microtubule-associated protein tau. In vitro, fibrillization of full-length, unphosphorylated recombinant tau can be induced under near-physiological conditions by treatment with various agents, including anionic surfactants. Here we examine the pathway through which anionic surfactants promote tau fibrillization using a combination of electron microscopy and fluorescence spectroscopy. Protein and surfactant first interacted in solution to form micelles, which then provided negatively charged surfaces that accumulated tau aggregates. Surface aggregation of tau protein was followed by the time-dependent appearance of a thioflavin S reactive intermediate that accumulated over a period of hours. The intermediate was unstable in the absence of anionic surfaces, suggesting it was not filamentous. Fibrillization proceeded after intermediate formation with classic nucleation-dependent kinetics, consisting of lag phase followed by the exponential increase in filament lengths, followed by an equilibrium phase reached in approximately 24 h. The pathway did not require protein insertion into the micelle hydrophobic core or conformational change arising from mixed micelle formation, because anionic microspheres constructed from impermeable polystyrene were capable of qualitatively reproducing all aspects of the fibrillization reaction. It is proposed that the progression from amorphous aggregation through intermediate formation and fibrillization may underlie the activity of other inducers such as hyperphosphorylation and may be operative in vivo.

Anionic micelles and vesicles induce tau fibrillization in vitro.

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 full-length 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.

Rapid anionic micelle-mediated alpha-synuclein fibrillization in vitro.

Parkinson's disease is characterized by the aggregation of alpha-synuclein into filamentous forms within affected neurons of the basal ganglia. Fibrillization of purified recombinant alpha-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 alpha-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 alpha-synuclein-dependent depression of their critical micelle concentrations. Other anionic surfaces, such as those supplied by anionic phospholipid vesicles, also induced alpha-synuclein fibrillization, with resultant filaments originating from their surface. These data suggest that anionic surfaces presented as micelles or vesicles can serve to nucleate alpha-synuclein fibrillization, that this mechanism underlies the inducer activity of anionic surfactants, and that anionic membranes may serve this function in vivo.