Methylene blue modulates huntingtin aggregation intermediates and is protective in Huntington's disease models.

Huntington's disease (HD) is a devastating neurodegenerative disorder with no disease-modifying treatments available. The disease is caused by expansion of a CAG trinucleotide repeat and manifests with progressive motor abnormalities, psychiatric symptoms, and cognitive decline. Expression of an expanded polyglutamine repeat within the Huntingtin (Htt) protein impacts numerous cellular processes, including protein folding and clearance. A hallmark of the disease is the progressive formation of inclusions that represent the culmination of a complex aggregation process. Methylene blue (MB), has been shown to modulate aggregation of amyloidogenic disease proteins. We investigated whether MB could impact mutant Htt-mediated aggregation and neurotoxicity. MB inhibited recombinant protein aggregation in vitro, even when added to preformed oligomers and fibrils. MB also decreased oligomer number and size and decreased accumulation of insoluble mutant Htt in cells. In functional assays, MB increased survival of primary cortical neurons transduced with mutant Htt, reduced neurodegeneration and aggregation in a Drosophila melanogaster model of HD, and reduced disease phenotypes in R6/2 HD modeled mice. Furthermore, MB treatment also promoted an increase in levels of BDNF RNA and protein in vivo. Thus, MB, which is well tolerated and used in humans, has therapeutic potential for HD.

Fibril specific, conformation dependent antibodies recognize a generic epitope common to amyloid fibrils and fibrillar oligomers that is absent in prefibrillar oligomers.

BACKGROUND: Amyloid-related degenerative diseases are associated with the accumulation of misfolded proteins as amyloid fibrils in tissue. In Alzheimer disease (AD), amyloid accumulates in several distinct types of insoluble plaque deposits, intracellular Abeta and as soluble oligomers and the relationships between these deposits and their pathological significance remains unclear. Conformation dependent antibodies have been reported that specifically recognize distinct assembly states of amyloids, including prefibrillar oligomers and fibrils. RESULTS: We immunized rabbits with a morphologically homogeneous population of Abeta42 fibrils. The resulting immune serum (OC) specifically recognizes fibrils, but not random coil monomer or prefibrillar oligomers, indicating fibrils display a distinct conformation dependent epitope that is absent in prefibrillar oligomers. The fibril epitope is also displayed by fibrils of other types of amyloids, indicating that the epitope is a generic feature of the polypeptide backbone. The fibril specific antibody also recognizes 100,000 x G soluble fibrillar oligomers ranging in size from dimer to greater than 250 kDa on western blots. The fibrillar oligomers recognized by OC are immunologically distinct from prefibrillar oligomers recognized by A11, even though their sizes overlap broadly, indicating that size is not a reliable indicator of oligomer conformation. The immune response to prefibrillar oligomers and fibrils is not sequence specific and antisera of the same specificity are produced in response to immunization with islet amyloid polypeptide prefibrillar oligomer mimics and fibrils. The fibril specific antibodies stain all types of amyloid deposits in human AD brain. Diffuse amyloid deposits stain intensely with anti-fibril antibody although they are thioflavin S negative, suggesting that they are indeed fibrillar in conformation. OC also stains islet amyloid deposits in transgenic mouse models of type II diabetes, demonstrating its generic specificity for amyloid fibrils. CONCLUSION: Since the fibril specific antibodies are conformation dependent, sequence-independent, and recognize epitopes that are distinct from those present in prefibrillar oligomers, they may have broad utility for detecting and characterizing the accumulation of amyloid fibrils and fibrillar type oligomers in degenerative diseases.

Methylene blue inhibits amyloid Abeta oligomerization by promoting fibrillization.

Amyloid plaques are hallmark neuropathological lesions in Alzheimer's disease, which consist of abnormally aggregated Abeta protein. Multiple Abeta aggregated species have been identified, and neurotoxicity appears to be correlated with the amount of nonfibrillar oligomers. Therefore, selective inhibition of Abeta oligomer formation has emerged as an attractive means of therapeutic intervention. To investigate whether small molecules can modulate aggregation to achieve selective inhibition of neurotoxic amyloid oligomers, Abeta aggregation was assayed in vitro in the presence of methylene blue, using immunoreactivity with the prefibrillar oligomer-specific antibody A11, transmission electron microscopy, and turbidity assays. Methylene blue inhibited oligomerization when used at substoichiometric concentrations relative to that of the Abeta monomer. Inhibition of Abeta oligomerization was achieved concomitant with promotion of fibrillization, suggesting that oligomer and fibril formation are distinct and competing pathways. Methylene blue-mediated promotion of fiber formation occurred via a dose-dependent decrease in the lag time and an increase in the fibrillization rate, consistent with promotion of both filament nucleation and elongation. Addition of methylene blue to preformed oligomers resulted in oligomer loss and promotion of fibrillization. The data show that Abeta oligomer formation is inhibited by promoting fibril formation, which suggests that the relative pathological significance of oligomers and fibrils may be tested in vivo using methylene blue. If Abeta oligomers represent the primary pathogenic species, then inhibition of this highly toxic species via promotion of formation of less toxic aggregates may be therapeutically useful.

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.

Anti-A beta 1-11 antibody binds to different beta-amyloid species, inhibits fibril formation, and disaggregates preformed fibrils but not the most toxic oligomers.

Different strategies proposed as therapy for Alzheimer disease (AD) have aimed to reduce the level of toxic forms of A beta peptide in the brain. Here, we directly analyze the therapeutic utility of the polyclonal anti-A beta(1-11) antibody induced in 3xTg-AD mice vaccinated with the second generation prototype epitope vaccine. Substoichiometric concentrations of purified anti-A beta(1-11) antibody prevented aggregation of A beta(42) and induced disaggregation of preformed A beta(42) fibrils down to nonfilamentous and nontoxic species. Anti-A beta(1-11) antibody delayed A beta(42) oligomer formation but ultimately appeared to stabilize nonfibrillar conformations, including oligomer-like assemblies. The reduced oligomer-mediated cytotoxicity observed upon preincubation of A beta oligomers with the anti-A beta(1-11) antibody in the absence of oligomer disaggregation suggests a possible oligomer rearrangement in the presence of the antibody. These in vitro observations suggest that preventive vaccination may protect from AD or may delay the onset of the disease, whereas therapeutic vaccination cannot disrupt the toxic oligomers and may only minimally alleviate preexisting AD pathology.

Small molecule inhibitors of aggregation indicate that amyloid beta oligomerization and fibrillization pathways are independent and distinct.

Alzheimer disease is characterized by the abnormal aggregation of amyloid beta peptide into extracellular fibrillar deposits known as amyloid plaques. Soluble oligomers have been observed at early time points preceding fibril formation, and these oligomers have been implicated as the primary pathological species rather than the mature fibrils. A significant issue that remains to be resolved is whether amyloid oligomers are an obligate intermediate on the pathway to fibril formation or represent an alternate assembly pathway that may or may not lead to fiber formation. To determine whether amyloid beta oligomers are obligate intermediates in the fibrillization pathway, we characterized the mechanism of action of amyloid beta aggregation inhibitors in terms of oligomer and fibril formation. Based on their effects, the small molecules segregated into three distinct classes: compounds that inhibit oligomerization but not fibrillization, compounds that inhibit fibrillization but not oligomerization, and compounds that inhibit both. Several compounds selectively inhibited oligomerization at substoichiometric concentrations relative to amyloid beta monomer, with some active in the low nanomolar range. These results indicate that oligomers are not an obligate intermediate in the fibril formation pathway. In addition, these data suggest that small molecule inhibitors are useful for clarifying the mechanisms underlying protein aggregation and may represent potential therapeutic agents that target fundamental disease mechanisms.

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.

Site-specific pseudophosphorylation modulates the rate of tau filament dissociation.

Hyperphosphorylation of tau is of fundamental importance for neurofibrillary lesion development in Alzheimer's disease, but the mechanisms through which it acts are not clear. Experiments with pseudophosphorylation mutants of full-length tau protein indicate that incorporation of negative charge into specific sites can modulate the aggregation reaction, and that this occurs by altering the critical concentration of assembly. Here, the kinetic origin of this effect was determined using quantitative electron microscopy methods and pseudophosphorylation mutant T212E in a full-length four-repeat tau background. On the basis of disaggregation rates, decreases in critical concentration resulted primarily from decreases in the dissociation rate constant. The results suggest a mechanism through which site-specific posttranslational modifications can modulate filament accumulation at low free intracellular tau concentrations.

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.

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.

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.

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.

Ligand-dependent inhibition and reversal of tau filament formation.

Alzheimer's disease is defined in part by the intraneuronal accumulation of filaments comprised of the microtubule associated protein tau. Because animal model studies suggest that a toxic gain of function accompanies tau aggregation in neurons, selective pharmacological inhibitors of the process may have utility in slowing neurodegeneration. Here, the properties of a candidate small molecule inhibitor of tau fibrillization, 3-(2-hydroxyethyl)-2-[2-[[3-(2-hydroxyethyl)-5-methoxy-2-benzothiazolylidene]methyl]-1-butenyl]-5-methoxybenzothiazolium (N744), were characterized in vitro using transmission electron microscopy. N744 inhibited arachidonic acid-induced aggregation of full-length, four-repeat tau protein at substoichiometric concentrations relative to total tau and with an IC(50) of approximately 300 nM. Inhibition was accompanied by a dose-dependent decrease in the number concentration of filaments, suggesting that N744 interfered with tau filament nucleation. Stoichiometric concentrations of N744 also promoted tau disaggregation when added to mature synthetic filaments. Disaggregation followed first-order kinetics and was accompanied by a steady decrease in filament number, suggesting that N744 promoted endwise loss of tau molecules with limited filament breakage. N744 at substoichiometric concentrations did not inhibit Abeta and alpha-synuclein aggregation, indicating it was tau selective under these conditions. Because of its activity in vitro, N744 may offer a pharmacological approach to the role of tau fibrillization in neurodegeneration.

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.

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.