In vitro tau fibrillization: mapping protein regions

We have investigated the propensity to form fibrillar aggregates of a variety of fragments and variants of the tau protein under the influence of a tau fibrillization inducer: coenzyme Q(0). To better identify fibrillization hotspots, we compare the polymerization propensity of tau fragments containing the sequence of putative hotspots with that of tau variants with that same sequence deleted. We also investigate the effects of biologically occurring modifications such as phosphorylation and deamidation. We found that residues 305 to 335 are essential for in vitro tau fibrillization. Residues 306 to 311 facilitate in vitro assembly, but are not sufficient to mimic the in vivo fibrillization of tau. Furthermore, the propensity of the 306-311 sequence to form fibrils is highly decreased by chemical modifications of tyrosine 310 that are commonly found in vivo.     

Potent inhibition of tau fibrillization with a multivalent ligand

Small-molecule inhibitors of tau fibrillization are under investigation as tools for interrogating the tau aggregation pathway and as potential therapeutic agents for Alzheimer's disease. Established inhibitors include thiacarbocyanine dyes, which can inhibit recombinant tau fibrillization in the presence of anionic surfactant aggregation inducers. In an effort to increase inhibitory potency, a cyclic bis-thiacarbocyanine molecule containing two thiacarbocyanine moieties was synthesized and characterized with respect to tau fibrillization inhibitory activity by electron microscopy and ligand aggregation state by absorbance spectroscopy. Results showed that the inhibitory activity of the bis-thiacarbocyanine was qualitatively similar to a monomeric cyanine dye, but was more potent with 50% inhibition achieved at approximately 80nM concentration. At all concentrations tested in aqueous solution, the bis-thiacarbocyanine collapsed to form a closed clamshell structure. However, the presence of tau protein selectively stabilized the open conformation. These results suggest that the inhibitory activity of bis-thiacarbocyanine results from multivalency, and reveal a route to more potent tau aggregation inhibitors.     

Inhibition of tau fibrillization by oleocanthal via reaction with the amino groups of tau

Tau is a microtubule-associated protein that promotes microtubule assembly and stability. In Alzheimer's disease and related tauopathies, tau fibrillizes and aggregates into neurofibrillary tangles. Recently, oleocanthal isolated from extra virgin olive oil was found to display non-steroidal anti-inflammatory activity similar to ibuprofen. As our unpublished data indicates an inhibitory effect of oleocanthal on amyloid β peptide fibrillization, we reasoned that it might inhibit tau fibrillization as well. Herein, we demonstrate that oleocanthal abrogates fibrillization of tau by locking tau into the naturally unfolded state. Using PHF6 consisting of the amino acid residues VQIVYK, a hexapeptide within the third repeat of tau that is essential for fibrillization, we show that oleocanthal forms an adduct with the lysine via initial Schiff base formation. Structure and function studies demonstrate that the two aldehyde groups of oleocanthal are required for the inhibitory activity. These two aldehyde groups show certain specificity when titrated with free lysine and oleocanthal does not significantly affect the normal function of tau. These findings provide a potential scheme for the development of novel therapies for neurodegenerative tauopathies.     

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.     

Characterization of two VQIXXK motifs for tau fibrillization in vitro

Tau proteins are building blocks of the filaments that form neurofibrillary tangles of Alzheimer's disease (AD) and related neurodegenerative tauopathies. It was recently reported that two VQIXXK motifs in the microtubule (MT) binding region, named PHF6 and PHF6*, are responsible for tau fibrillization. However, the exact role each of these motifs plays in this process has not been analyzed in detail. Using a recombinant human tau fragment containing only the four MT-binding repeats (K18), we show that deletion of either PHF6 or PHF6* affected tau assembly but only PHF6 is essential for filament formation, suggesting a critical role of this motif. To determine the amino acid residues within PHF6 that are required for tau fibrillization, a series of deletion and mutation constructs targeting this motif were generated. Deletion of VQI in either PHF6 or PHF6* lessened but did not eliminate K18 fibrillization. However, removal of the single K311 residue from PHF6 completely abrogated the fibril formation of K18. K311D mutation of K18 inhibited tau filament formation, while K311A and K311R mutations had no effect. These data imply that charge change at position 311 is important in tau fibril formation. A similar requirement of nonnegative charge at this position for fibrillization was observed with the full-length human tau isoform (T40), and data from these studies indicate that the formation of fibrils by T40K311D and T40K311P mutants is repressed at the nucleation phase. These findings provide important insights into the mechanisms of tau fibrillization and suggest targets for AD drug discovery to ameliorate neurodegeneration mediated by filamentous tau pathologies.     

Electron microscopy as a quantitative method for investigating tau fibrillization

Fibrillization of tau protein is a hallmark lesion in Alzheimer's disease. To clarify the utility of electron microscopy as a quantitative assay for tau fibrillization in vitro, the interaction between synthetic tau filaments and carbon/formvar-coated grids was characterized in detail. Filament adsorption onto grids was hyperbolic when analyzed as a function of time or bulk protein concentration, with no evidence for competitive displacement or elution from other components in the reaction mixture. Filament length measurements were linear with filament concentration so long as the concentration of total tau protein in the sample was held constant, suggesting that measurement of filament lengths was accurate under these conditions. Furthermore, exponential filament length distributions were not significantly affected by adsorption time or filament concentration, suggesting that preferential binding among filaments of differing lengths was minimal. However, monomeric tau protein was found to be a strong competitor of filament adsorption, indicating that comparison of filament length measurements at different bulk tau concentrations should be interpreted with caution.     

A static laser light scattering assay for surfactant-induced tau fibrillization

Static light scattering is an important solution-based method for assaying spontaneous protein aggregation reactions. But the reliability of the measurements when conducted in the presence of fibrillization inducers has been questioned. Here the utility of static laser light scattering for quantitative assay of anionic micelle-induced protein fibrillization was characterized using tau protein, the major component of neurofibrillary lesions of Alzheimer's disease. Both inducer micellization and tau fibrillization made significant contributions to light scattering intensity. The intensity arising solely from micellization was quantified using proteins that promoted inducer micellization but could not fibrillize, such as mixed histones and assembly-incompetent mutant htau40(I277P/I308P). When corrected for micellization, reaction progress curves for wild-type tau fibrillization were sigmoidal and correlated well with measurements of total filament length made by transmission electron microscopy. The utility of the improved laser light scattering assay was demonstrated by quantifying the effect of inducer concentration on tau assembly kinetics using a three-parameter Gompertz growth function. Results showed that alkyl sulfate detergent accelerated tau nucleation as reflected by shorter lag times and modulated pre-nuclear equilibria to yield more filament mass at reaction equilibrium.     

C-terminal truncation modulates both nucleation and extension phases of tau fibrillization

Proteolytic post-translational modification has been proposed as an early stage event in the aggregation of τ protein and formation of neurofibrillary lesions in Alzheimer’s disease. Caspases and other proteases cleave τ in vivo at discrete locations including Asp⁴²¹ and Glu³⁹¹. Both cleavage products are prone to aggregation relative to wild-type, full-length τ protein. To determine the mechanism underlying this effect, the fibrillization of τ truncated after Asp⁴²¹ and Glu³⁹¹ residues was characterized in a full-length four-repeat τ background using quantitative electron microscopy methods under homogeneous nucleation conditions. Both C-terminal truncations decreased critical concentration relative to full-length τ, resulting in more filament mass at reaction plateau. Moreover, truncation directly augmented the efficiency of the nucleation reaction. The results suggest the mechanism through which C-terminal proteolysis can modulate τ filament accumulation depending on whether it precedes or follows nucleation.     

Potency of a tau fibrillization inhibitor is influenced by its aggregation state

Tau fibrillization is a potential therapeutic target for Alzheimer's and other neurodegenerative diseases. Several small-molecule inhibitors of tau aggregation have been developed for this purpose. One of them, 3,3'-bis(beta-hydroxyethyl)-9-ethyl-5,5'-dimethoxythiacarbocyanine iodide (N744), is a cationic thiacarbocyanine dye that inhibits recombinant tau filament formation when present at submicromolar concentrations. To prepare dosing regimens for testing N744 activity in biological models, its full concentration-effect relationship in the range 0.01-60muM was examined in vitro by electron microscopy and laser light scattering methods. Results revealed that N744 concentration dependence was biphasic, with fibrillization inhibitory activity appearing at submicromolar concentration, but with relief of inhibition and increases in fibrillization apparent above 10muM. Therefore, fibrillization was inhibited 50% only over a narrow concentration range, which was further reduced by filament stabilizing modifications such as tau pseudophosphorylation. N744 inhibitory activity also was paralleled by changes in its aggregation state, with dimer predominating at inhibitory concentrations and large dye aggregates appearing at high concentrations. Ligand dimerization was promoted by the presence of tau protein, which lowered the equilibrium dissociation constant for dimerization more than an order of magnitude relative to controls. The results suggest that ligand aggregation may play an important role in both inhibitory and disinhibitory phases of the concentration-effect curve, and may lead to complex dose-response relationships in model systems.     

TMAO promotes fibrillization and microtubule assembly activity in the C-terminal repeat region of tau

Alzheimer's disease most closely correlates with the appearance of the neurofibrillary tangles (NFTs), intracellular fibrous aggregates of the microtubule-associated protein, tau. Under native conditions, tau is an unstructured protein, and its physical characterization has revealed no clues about the three-dimensional structural determinants essential for aggregation or microtubule binding. We have found that the natural osmolyte trimethylamine N-oxide (TMAO) induces secondary structure in a C-terminal fragment of tau (tau(187)) and greatly promotes both self-aggregation and microtubule (MT) assembly activity. These processes could be distinguished, however, by a single-amino acid substitution (Tyr(310) --> Ala), which severely inhibited aggregation but had no effect on MT assembly activity. The inability of this mutant to aggregate could be completely reversed by TMAO. We propose a model in which TMAO induces partial order in tau(187), resulting in conformers that may correspond to on-pathway intermediates of either aggregation or tau-dependent MT assembly or both. These studies set the stage for future high-resolution structural characterization of these intermediates and the basis by which Tyr(310) may direct pathologic versus normal tau function.     

Pseudophosphorylation and glycation of tau protein enhance but do not trigger fibrillization in vitro

Alzheimer's disease is defined in part by the intraneuronal aggregation of tau protein into filamentous lesions. The pathway is accompanied by posttranslational modifications including phosphorylation and glycation, each of which has been shown to promote tau fibrillization in vitro when present at high stoichiometry. To clarify the site-specific impact of posttranslational modification on tau fibrillization, the ability of recombinant full-length four repeat tau protein (htau40) and 11 pseudophosphorylation mutants to fibrillize in the presence of anionic inducer was assayed in vitro using transmission electron microscopy and laser light scattering assays. Tau glycated with d-glucose was examined as well. Both glycated tau and pseudophosphorylation mutants S199E, T212E, S214E, double mutant T212E/S214E, and triple mutant S199E/S202E/T205E yielded increased filament mass at equilibrium relative to wild-type tau. Increases in filament mass correlated strongly with decreases in critical concentration, indicating that both pseudophosphorylation and glycation promoted fibrillization by shifting equilibrium toward the fibrillized state. Analysis of reaction time courses further revealed that increases in filament mass were not associated with reduced lag times, indicating that these posttranslational modifications did not promote filament nucleation. The results suggest that site-specific posttranslational modifications can stabilize filaments once they nucleate, and thereby support their accumulation at low intracellular tau concentrations.     

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.     

Mechanism of amylin fibrillization enhancement by heparin

We characterized the interaction of amylin with heparin fragments of defined length, which model the glycosaminoglycan chains associated with amyloid deposits found in type 2 diabetes. Binding of heparin fragments to the positively charged N-terminal half of monomeric amylin depends on the concentration of negatively charged saccharides but is independent of oligosaccharide length. By contrast, amylin fibrillogenesis has a sigmoidal dependence on heparin fragment length, with an enhancement observed for oligosaccharides longer than four monomers and a leveling off of effects beyond 12 monomers. The length dependence suggests that the negatively charged helical structure of heparin electrostatically complements the positively charged surface of the fibrillar amylin cross-β structure. Fluorescence resonance energy transfer and total internal reflection fluorescence microscopy experiments indicate that heparin associates with amylin fibrils, rather than enhancing fibrillogenesis catalytically. Short heparin fragments containing two- or eight-saccharide monomers protect against amylin cytotoxicity toward a MIN6 mouse cell model of pancreatic β-cells.     

Altering Peptide fibrillization by polymer conjugation

A strategy is presented that exploits the ability of synthetic polymers of different nature to disturb the strong self-assembly capabilities of amyloid based β-sheet forming peptides. Following a convergent approach, the peptides of interest were synthesized via solid-phase peptide synthesis (SPPS) and the polymers via reversible addition-fragmentation chain transfer (RAFT) polymerization, followed by a copper(I) catalyzed azide-alkyne cycloaddition (CuAAC) to generate the desired peptide-polymer conjugates. This study focuses on a modified version of the core sequence of the β-amyloid peptide (Aβ), Aβ(16-20) (KLVFF). The influence of attaching short poly(N-isopropylacrylamide) and poly(hydroxyethylacrylate) to the peptide sequences on the self-assembly properties of the hybrid materials were studied via infrared spectroscopy, TEM, circular dichroism and SAXS. The findings indicate that attaching these polymers disturbs the strong self-assembly properties of the biomolecules to a certain degree and permits to influence the aggregation of the peptides based on their β-sheets forming abilities. This study presents an innovative route toward targeted and controlled assembly of amyloid-like fibers to drive the formation of polymeric nanomaterials.     

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.     

Surface-modified magnetite nanoparticles affect lysozyme amyloid fibrillization

BackgroundThe surface of nanoparticles (NPs) is an important factor affecting the process of poly/peptides' amyloid aggregation. We have investigated the in vitro effect of trisodium citrate (TC), gum arabic (GA) and citric acid (CA) surface-modified magnetite nanoparticles (COAT-MNPs) on hen egg-white lysozyme (HEWL) amyloid fibrillization and mature HEWL fibrils.MethodsDynamic light scattering (DLS) was used to characterize the physico-chemical properties of studied COAT-MNPs and determine the adsorption potential of their surface towards HEWL. The anti-amyloid properties were studied using thioflavin T (ThT) and tryptophan (Trp) intrinsic fluorescence assays, and atomic force microscopy (AFM). The morphology of amyloid aggregates was analyzed using Gwyddion software. The cytotoxicity of COAT-MNPs was determined utilizing Trypan blue (TB) assay.ResultsAgents used for surface modification affect the COAT-MNPs physico-chemical properties and modulate their anti-amyloid potential. The results from ThT and intrinsic fluorescence showed that the inhibitory activities result from the more favorable interactions of COAT-MNPs with early pre-amyloid species, presumably reducing nuclei and oligomers formation necessary for amyloid fibrillization. COAT-MNPs also possess destroying potential, which is presumably caused by the interaction with hydrophobic residues of the fibrils, resulting in the interruption of an interface between β-sheets stabilizing the amyloid fibrils.ConclusionCOAT-MNPs were able to inhibit HEWL fibrillization and destroy mature fibrils with different efficacy depending on their properties, TC-MNPs being the most potent nanoparticles.General significanceThe study reports findings regarding the general impact of nanoparticles' surface modifications on the amyloid aggregation of proteins.     

In-situ atomic force microscopy study of beta-amyloid fibrillization

We report the use of atomic force microscopy to observe the initial stages of beta-amyloid fibrillization in situ. The growth of individual beta-amyloid protofibrils on a mica substrate was followed over several hours. The first in situ visualization of protofibril formation from single aggregate units of beta-amyloid is reported. The growth of these protofibrils through the subsequent addition of these aggregate units is also observed. Growth of the protofibrils is bi-directional and the outgrowth of protofibrils from a common amyloid/heterogeneous core is also observed. Elongation also occurred by the addition of protofibrils from solution. This data provides an exciting insight into the early stages of beta-amyloid fibrillization and can be used to enhance the understanding of the mechanism(s) by which beta-amyloid fibrillizes and may consequently enable inhibition of one or more stages of fibrillization as a potential therapeutic strategy.