An autocatalytic reaction as a model for the kinetics of the aggregation of beta-amyloid

Alzheimer's disease is the commonest form of senile dementia, affecting almost 20 million people worldwide. This neurodegenerative disorder is characterized by amyloid deposition in senile plaques, composed primarily of fibrils of an aggregated peptide, beta-amyloid. Fibrillation of beta-amyloid is a nucleation-dependent polymerization process, which is controlled by two kinetics parameters: the nucleation rate and the elongation or growth rate. As the kinetics of fibrillation is strongly dependent on the presence of trace amounts of fibrils, we suggest that the aggregation of beta-amyloid is a model of autocatalytic reaction. A mathematical analysis, permitting quantitative monitoring of the kinetics of fibrillogenesis of beta-amyloid, nucleation, and elongation constants, is presented. The model was checked by applying it to the aggregation of the fragment 1-40 of the beta-amyloid. Understanding of these rate constants may facilitate the study of the effect of substances used for controlling fibril creation and growth. The disaggregating effect of dodecyl trimethylammonium bromide, a cationic surfactant, was easily quantified by means of the model.     

Kinetics of inhibition of beta-amyloid aggregation by transthyretin

Deposition of beta-amyloid (Abeta) fibrils is an early event in the neurodegenerative processes associated with Alzheimer's disease. According to the "amyloid cascade" hypothesis, Abeta aggregation, and its subsequent deposition as fibrils, is the underlying cause of disease. Abeta is a proteolytic product of amyloid precursor protein (APP); several mutations in APP have been identified that are associated with early onset of disease. Transgenic mice overexpressing APP with the Swedish mutation develop numerous plaques but, surprisingly, lack the neurofibrillary tangles and neuronal loss characteristic of Alzheimer's disease, in apparent contradiction of the amyloid cascade hypothesis. However, recent studies suggest that coproduction of sAPPalpha, an alternative proteolytic product of APP, increases synthesis of transthyretin that, in turn, interacts directly with Abeta to inhibit its toxicity. Here we report results from biophysical analysis of Abeta aggregation kinetics in the presence of transthryetin. At substoichiometric ratios, transthyretin drastically decreased the rate of aggregation without affecting the fraction of Abeta in the aggregate pool. Detailed analysis of the data using a mathematical model demonstrated that the decrease in aggregation rate was due to both a decrease in the rate of elongation relative to the rate of initiation of filaments and a decrease in lateral association of filaments to fibrils. Tryptophan quenching data indicated that transthyretin binds weakly to Abeta, with an estimated apparent KS of 2300 M-1. Taken together, the data support a hypothesis wherein transthyretin preferentially binds to aggregated rather than monomeric Abeta and arrests further growth of the aggregates.     

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.     

Thermodynamic analysis of the aggregation propensity of oxidized Alzheimer's beta-amyloid variants

We have determined the critical concentrations of a set of 18 variants of Alzheimer's Abeta(1-40) peptide, each carrying a different residue at position 18. We find that the critical concentrations depend on the hydrophobicity and beta-sheet propensity of residue 18, and therefore on properties that we identified previously to affect also the kinetics by which these peptides aggregate. Since the critical concentrations can be related to the Gibbs free energy of aggregation (DeltaG), these data imply a link between the thermodynamics and the kinetics of aggregation in that sequences that form very stable aggregates are also those that form such aggregates very rapidly.     

Nucleation-dependent tau filament formation: the importance of dimerization and an estimation of elementary rate constants

Filamentous inclusions composed of the microtubule-associated protein tau are found in Alzheimer disease and other tauopathic neurodegenerative diseases, but the mechanisms underlying their formation from full-length protein monomer under physiological conditions are unclear. To address this issue, the fibrillization of recombinant full-length four-repeat human tau was examined in vitro as a function of time and submicromolar tau concentrations using electron microscopy assay methods and a small-molecule inducer of aggregation, thiazine red. Data were then fit to a simple homogeneous nucleation model with rate constant constraints established from filament dissociation rate, critical concentration, and mass-per-unit length measurements. The model was then tested by comparing the predicted time-dependent evolution of length distributions to experimental data. Results indicated that once assembly-competent conformations were attained, the rate-limiting step in the fibrillization pathway was tau dimer formation. Filament elongation then proceeded by addition of tau monomers to nascent filament ends. Filaments isolated at reaction plateau contained approximately 2 tau protomers/beta-strand spacing on the basis of mass-per-unit length measurements. The model suggests four key steps in the aggregation pathway that must be surmounted for tau filaments to form in disease.     

Pseudophosphorylation of tau protein directly modulates its aggregation kinetics

Hyperphosphorylation of tau protein is associated with neurofibrillary lesion formation in Alzheimer's disease and other tauopathic neurodegenerative diseases. It fosters lesion formation by increasing the concentration of free tau available for aggregation and by directly modulating the tau aggregation reaction. To clarify how negative charge incorporation into tau directly affects aggregation behavior, the fibrillization of pseudophosphorylation mutant T212E prepared in a full-length four-repeat tau background was examined in vitro as a function of time and submicromolar tau concentrations using electron microscopy assay methods. Kinetic constants for nucleation and extension phases of aggregation were then estimated by direct measurement and mathematical simulation. Kinetic analysis revealed that pseudophosphorylation increased tau aggregation rate by increasing the rate of filament nucleation. In addition, it increased aggregation propensity by stabilizing mature filaments against disaggregation. The data suggest that incorporation of negative charge into the T212 site can directly promote tau filament formation at multiple steps in the aggregation pathway.     

Protofibril formation of amyloid beta-protein at low pH via a non-cooperative elongation mechanism

Deposition of the amyloid beta-protein (Abeta) in senile or diffuse plaques is a distinctive feature of Alzheimer's disease. The role of Abeta aggregates in the etiology of the disease is still controversial. The formation of linear aggregates, known as amyloid fibrils, has been proposed as the onset and the cause of pathological deposition. Yet, recent findings suggest that a more crucial role is played by prefibrillar oligomeric assemblies of Abeta that are highly toxic in the extracellular environment. In the present work, the mechanism of protofibril formation is studied at pH 3.1, starting from a solution of oligomeric precursors. By combining static light scattering and photon correlation spectroscopy, the growth of the mass and the size of aggregates are determined at different temperatures. Analysis and scaling of kinetic data reveal that under the studied conditions protofibrils are formed via a single non-cooperative elongation mechanism, not prompted by nucleation. This process is well described as a linear colloidal aggregation due to diffusion and coalescence of growing aggregates. The rate of elongation follows an Arrhenius law with an activation enthalpy of 15 kcal mol(-1). Such a value points to a conformational change of peptides or oligomers being involved in binding to protofibrils or in general to a local reorganization of each aggregate. These results contribute to establishing a clearer relation at the molecular level between the fibrillation mechanism and fibrillar precursors. The observation of a non-cooperative aggregation pathway supports the hypothesis that amyloid formation may represent an escape route from a dangerous condition, induced by the presence of toxic oligomeric species.     

Urea modulation of beta-amyloid fibril growth: experimental studies and kinetic models

Aggregation of beta-amyloid (Abeta) into fibrillar deposits is widely believed to initiate a cascade of adverse biological responses associated with Alzheimer's disease. Although it was once assumed that the mature fibril was the toxic form of Abeta, recent evidence supports the hypothesis that Abeta oligomers, intermediates in the fibrillogenic pathway, are the dominant toxic species. In this work we used urea to reduce the driving force for Abeta aggregation, in an effort to isolate stable intermediate species. The effect of urea on secondary structure, size distribution, aggregation kinetics, and aggregate morphology was examined. With increasing urea concentration, beta-sheet content and the fraction of aggregated peptide decreased, the average size of aggregates was reduced, and the morphology of aggregates changed from linear to a globular/linear mixture and then to globular. The data were analyzed using a previously published model of Abeta aggregation kinetics. The model and data were consistent with the hypothesis that the globular aggregates were intermediates in the amyloidogenesis pathway rather than alternatively aggregated species. Increasing the urea concentration from 0.4 M to 2 M decreased the rate of filament initiation the most; between 2 M and 4 M urea the largest change was in partitioning between the nonamyloid and amyloid pathways, and between 4 M and 6 M urea, the most significant change was a reduction in the rate of filament elongation.     

Circular dichroism and aggregation studies of amyloid beta (11-8) fragment and its variants

Aggregation of Abeta peptides is a seminal event in Alzheimer's disease. Detailed understanding of Abeta assembly would facilitate the targeting and design of fibrillogenesis inhibitors. Here comparative conformational and aggregation studies using CD spectroscopy and thioflavine T fluorescence assay are presented. As a model peptide, the 11-28 fragment of Abeta was used. This model peptide is known to contain the core region responsible for Abeta aggregation. The structural and aggregational behaviour of the peptide was compared with the properties of its variants corresponding to natural, clinically relevant mutants at positions 21-23 (A21G, E22K, E22G, E22Q and D23N). In HFIP (hexafluoro-2-propanol), a strong alpha-helix inducer, the CD spectra revealed an unexpectedly high amount of beta-sheet conformation. The aggregation process of Abeta(11-28) variants provoked by water addition to HFIP was found to be consistent with a model of an alpha-helix-containing intermediate. The aggregation propensity of all Abeta(11-28) variants was also compared and discussed.     

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.     

Modeling and simulation of fed-batch protein refolding process

The simplified kinetic model that assumes competition between first-order folding and third-order aggregation was used to model the fed-batch refolding of denatured-reduced lysozyme. It was found that the model was able to describe the process at limited concentration ranges, i.e., 1-2 and 5-7 mg mL(-)(1), respectively, at extensive guanidinium chloride (GdmCl) concentrations and controlled concentrations of oxidizing and reducing agents. The folding or aggregation rate constant was different at the two protein concentration ranges and strongly dependent on the denaturant concentration. As a result, both rate constants at the two concentration ranges were expressed as functions of GdmCl concentration. The rate constants determined by fed-batch experiments could be employed for the prediction of the fed-batch process but were not able to be extended to a batch refolding by direct dilution. Computer simulations show that the denaturant concentration and fed-batch flow rate are important factors influencing the refolding yield. Prolonged fed-batch time is beneficial to keep the transient intermediate concentration at a low level and to increase the yield of correctly folded protein. This is of importance when the denaturant concentration in refolding buffer solution is low. Thus, at a low denaturant concentration, fed-batch time should be sufficiently long, whereas at an appropriately high GdmCl concentration, a short fed-batch time or a high feed rate of the denatured protein is effective to give a high refolding yield.     

Characterization of early stage intermediates in the nucleation phase of Aβ aggregation

Alzheimer's disease (AD) is a common form of dementia, which is characterized by the presence of extracellular amyloid plaques comprising the amyloid β peptide (Aβ). Although the mechanism underlying AD pathogenesis remains elusive, accumulating evidence suggests that the process of amyloid fibril formation is a surface-mediated event, which plays an important role in AD onset and progression. In this study, the mechanism of Aβ aggregation on hydrophobic surfaces was investigated with dual polarization interferometry (DPI), which provides real-time information on early stages of the aggregation process. Aggregation was monitored on a hydrophobic C18 surface and a polar silicon oxynitride surface. The DPI results showed a characteristic Aβ aggregation pattern involving a decrease in the density of Aβ at the surface followed by an increase in the thickness on the hydrophobic C18 chip. Most importantly, the DPI measurements provided unique information on the early stages of Aβ aggregation, which is characterized by the presence of initially slow nucleus formation process followed by exponential fibril elongation. The dimensions of the putative nucleus corresponded to a thickness of ～5 nm for both Aβ40 and Aβ42, which may represent about 10-15 molecules. The results thus support the nucleation-dependent polymerization model as indicated by the presence of a nucleation phase followed by an exponential growth phase. These results are the first reported measurements of the real-time changes in Aβ molecular structure during the early stages of amyloid formation at the nanometer level.     

Low cetyltrimethylammonium bromide concentrations induce reversible amorphous aggregation of tobacco mosaic virus and its coat protein at room temperature

Ordered and amorphous protein aggregation causes numerous diseases. Tobacco mosaic virus coat protein for many decades serves as the classical model of ordered protein aggregation ("polymerization"). It was also found to be highly prone to heat-induced amorphous aggregation and the rate of this aggregation could be easily manipulated by changes in solution ionic strength and temperature. Here, we report that rapid amorphous aggregation of this protein can be induced at 25 degrees C in phosphate buffer by low micromolar (start at about 15 microM) concentrations of cationic surfactant cetyltrimethylammonium bromide. At equilibrium four surfactant molecules bound to the protein subunit. As judged by circular dichroism and fluorescence spectroscopy data, the coat protein molecules retained their native structure upon the cetyltrimethylammonium bromide induced aggregation. No aggregation was observed at the higher surfactant concentrations (above 300 microM). Micromolar concentrations of anionic surfactant sodium dodecylsulfate rapidly reversed the cetyltrimethylammonium bromide induced aggregation of the coat protein due to formation of mixed surfactant-surfactant micelles. Cetyltrimethylammonium bromide (100-300 microM) also induced the reversible intact tobacco mosaic virus virion aggregation. The possible liability to the cetyltrimethylammonium bromide induced amorphous aggregation of other ordered aggregate-producing proteins has been discussed.     

Molecular interactions of dendrimers with amyloid peptides: pH dependence

The formation of amyloid plaques is a key pathological event in neurodegenerative disorders, such as prion and Alzheimer's diseases. Dendrimers are considered promising therapeutic agents in these disorders. In the present work, we have studied the effect of polypropyleneimine dendrimers on the formation of amyloid fibrils as a function of pH in order to gain further insight in the aggregation mechanism and its inhibition. Amyloid fibrils from prion peptide PrP 185-208 and Alzheimer's peptide Abeta 1-28 were produced in vitro, and their formation was monitored using the dye thioflavin T (ThT). The results showed that the level of protonation of His, Glu, and Asp residues is important for the final effect, especially at low dendrimer concentration when their inhibiting capacity depends on the pH. At the highest concentrations, dendrimers were very effective against fibril formations for both prion and Alzheimer's peptides.     

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.     

Amyloid-beta protofibrils differ from amyloid-beta aggregates induced in dilute hexafluoroisopropanol in stability and morphology

The brains of Alzheimer's disease (AD) patients contain large numbers of amyloid plaques that are rich in fibrils composed of 40- and 42-residue amyloid-beta (Abeta) peptides. Several lines of evidence indicate that fibrillar Abeta and especially soluble Abeta aggregates are important in the etiology of AD. Recent reports also stress that amyloid aggregates are polymorphic and that a single polypeptide can fold into multiple amyloid conformations. Here we demonstrate that Abeta-(1-40) can form soluble aggregates with predominant beta-structures that differ in stability and morphology. One class of aggregates involved soluble Abeta protofibrils, prepared by vigorous overnight agitation of monomeric Abeta-(1-40) at low ionic strength. Dilution of these aggregation reactions induced disaggregation to monomers as measured by size exclusion chromatography. Protofibril concentrations monitored by thioflavin T fluorescence decreased in at least two kinetic phases, with initial disaggregation (rate constant approximately 1 h(-1)) followed by a much slower secondary phase. Incubation of the reactions without agitation resulted in less disaggregation at slower rates, indicating that the protofibrils became progressively more stable over time. In fact, protofibrils isolated by size exclusion chromatography were completely stable and gave no disaggregation. A second class of soluble Abeta aggregates was generated rapidly (<10 min) in buffered 2% hexafluoroisopropanol (HFIP). These aggregates showed increased thioflavin T fluorescence and were rich in beta-structure by circular dichroism. Electron microscopy and atomic force microscopy revealed initial globular clusters that progressed over several days to soluble fibrous aggregates. When diluted out of HFIP, these aggregates initially were very unstable and disaggregated completely within 2 min. However, their stability increased as they progressed to fibers. Relative to Abeta protofibrils, the HFIP-induced aggregates seeded elongation by Abeta monomer deposition very poorly. The techniques used to distinguish these two classes of soluble Abeta aggregates may be useful in characterizing Abeta aggregates formed in vivo.     

Aggregation of deoxyhemoglobin subunits.

The formation of deoxyhemoglobin was examined by measuring the heme spectral change that accompanies the aggregation of isolated alpha and beta chains. At low hemeconcentrations (less than 10(-5) M), tetramer formation can be described by two consecutive, second order reactions representing the aggregation of monomers followed by the association of alphabeta dimers. At neutral pH, the rates of monomer and dimer aggregation are roughly the same, approximately 5 X 10(5) M(-1) X(-1) at 20 degrees. Raising or lowering the pH results in a uniform decrease of both aggregation rates due presumably to repulsion of positively charged subunits at acid pH and repulsion of negatively charged subunits at alkaline pH. Addition of p-hydroxymercuribenzoate to alpha chains lowers the rate of monomer aggregation whereas addition of mercurials to the beta subunits appears to lower both the rate of monomer and the rate of dimer aggregation. At high heme concentrations (greater than 10(-5) M) or in the presence of organic phosphates, the rate of chain aggregation becomes limited, in part, by the slow dissociation of beta chain tetramers. In the case of inositol hexaphosphate, the rate of hemoglobin formation exhibits a bell-shaped dependence on phosphate concentration. When intermediate concentrations of inositol hexaphosphate (approximately 10(-4 M) are preincubated with beta subunits, a slow first order time course is observed and exhibits a half-time of about 8 min. As more inositol hexaphosphate is added, the chain aggregation reaction begins to occur more rapidly. Eventually at about 10(-2) M inositol hexaphospate, the time course becomes almost identical to that observed in the absence of phosphates. The increase in the velocity of the chain aggregation reaction at high phosphate concentrations suggests strongly that inositol hexaphosphate binds to beta monomers and, if added in sufficiently large amounts, promotes beta4 dissociation. A quantitative analysis of these results showed that the affinity of beta monomers for inositol hexaphosphate is the same as that of alphabeta dimers. Only when tetramers are formed, either alpha2beta2 or beta4, is a marked increase in affinity for inositol hexaphosphate observed.     

Effects of cytochalasin, phalloidin, and pH on the elongation of actin filaments

We used electron microscopy to measure the effects of cytochalasins, phalloidin, and pH on the rates of elongation at the barbed and pointed ends of actin filaments. In the case of the cytochalasins, we compared the effects on ATP- and ADP-actin monomers. Micromolar concentrations of either cytochalasin B (CB) or cytochalasin D (CD) inhibit elongation at both ends of the filament, about 95% at the barbed end and 50% at the pointed end, so that the two ends contribute about equally to the rate of growth. Half-maximal inhibition of elongation at the barbed end is at 0.1 microM CB and 0.02 microM CD for ATP-actin and at 0.1 microM CD for ADP-actin. At the pointed end, CD inhibits elongation by ATP-actin and ADP-actin about equally. At high (2 microM) concentrations, the cytochalasins reduce the association and dissociation rate constants in parallel for both ADP- and ATP-actin, so their effects on the critical concentrations are minimal. These observations confirm and extend those of Bonder and Mooseker [Bonder, E. M., & Mooseker, M. S. (1986) J. Cell Biol. 102, 282-288]. The dependence of the elongation rate on the concentration of both cytochalasin and actin can be explained quantitatively by a mechanism that includes the effects of cytochalasin binding to actin monomers [Godette, D. W., & Frieden, C. (1986) J. Biol. Chem. 261, 5974-5980] and a partial cap of the barbed end of the filament by the complex of ADP-actin and cytochalasin.(ABSTRACT TRUNCATED AT 250 WORDS)     

CLAC binds to aggregated Abeta and Abeta fragments, and attenuates fibril elongation

Deposition of amyloid beta-peptide (Abeta) into amyloid plaques is one of the invariant neuropathological features of Alzheimer's disease. Proteins that codeposit with Abeta are potentially important for the pathogenesis, and a recently discovered plaque-associated protein is the collagenous Alzheimer amyloid plaque component (CLAC). In this study, we investigated the molecular interactions between Abeta aggregates and CLAC using surface plasmon resonance spectroscopy and a solid-phase binding immunoassay. We found that CLAC binds to Abeta with high affinity, that the central region of Abeta is necessary and sufficient for CLAC interaction, and that the aggregation state of Abeta as well as the presence of negatively charged residues is important. We also show that this binding results in a reduced rate of fibril elongation. Taken together, we suggest that CLAC becomes involved at an intermediate stage in the pathogenesis by binding to Abeta fibrils, including fibrils formed from peptides with truncated N- or C-termini, and thereby slows their growth.