Improved preparation of amyloid-beta peptides using DBU as Nalpha-Fmoc deprotection reagent.

Previous studies have shown the amyloid peptides, Abeta 1-40/42, to be exceptionally difficult to assemble by Fmoc-solid phase peptide synthesis due to the high hydrophobicity of the C-terminal segment and resulting on-resin aggregation. We found that the use of the stronger and more efficient base, DBU, at a concentration of 2% in DMF for Nalpha-Fmoc deprotection allowed substantially improved continuous flow solid phase assembly of the model peptide Abeta 29-40/42 fragments. This suggested that, at least for these sequences, incomplete deprotection was a greater problem than incomplete amino acid acylation. This base was then used during the synthesis of both Abeta 1-40 and Abeta 1-42, up to and including Ser8, from which point 20% piperidine in DMF was utilized so as to avoid potential aspartimide formation at Asp7. By this means, the deprotection efficiency through the difficult C-terminal portion of the sequence was much improved and resulted in increased availability of terminal amino groups for acylation. This simple strategy that obviates the need for special conditions significantly improved crude peptide quality and allowed considerable facilitation of subsequent purification.     

The Alzheimer's peptides Abeta40 and 42 adopt distinct conformations in water: a combined MD / NMR study

The role of peptides Abeta40 and Abeta42 in the early pathogenesis of Alzheimer's disease (AD) is frequently emphasized in the literature. It is known that Abeta42 is more prone to aggregation than Abeta40, even though they differ in only two (IA) amino acid residues at the C-terminal end. A direct comparison of the ensembles of conformations adopted by the monomers in solution has been limited by the inherent flexibility of the unfolded peptides. Here, we characterize the conformations of Abeta40 and Abeta42 in water by using a combination of molecular dynamics (MD) and measured scalar (3)J(HNHalpha) data from NMR experiments. We perform replica exchange MD (REMD) simulations and find that classical forcefields reproduce the NMR data quantitatively when the sampling is extended to the microseconds time-scale. Using the quantitative agreement of the NMR data as a validation of the model, we proceed to compare the conformational ensembles of the Abeta40 and Abeta42 peptide monomers. Our analysis confirms the existence of structured regions within the otherwise flexible Abeta peptides. We find that the C terminus of Abeta42 is more structured than that of Abeta40. The formation of a beta-hairpin in the sequence (31)IIGLMVGGVVIA involving short strands at residues 31-34 and 38-41 (in bold) reduces the C-terminal flexibility of the Abeta42 peptide and may be responsible for the higher propensity of this peptide to form amyloids.     

The aggregation kinetics of Alzheimer's beta-amyloid peptide is controlled by stochastic nucleation

We report here a recombinant expression system that allows production of large quantities of Alzheimer's Abeta(1-40) peptide. The material is competent to dissolve in water solutions with "random-coil properties," although its conformation and factual oligomerization state are determined by the physico-chemical solution conditions. When dissolved in 50 mM sodium phosphate buffer (pH 7.4) at 37 degrees C, the peptide is able to undergo a nucleated polymerization reaction. The aggregation profile is characteristically bipartite, consisting of lag and growth phase. From these curves we determined the lag time as well as the rate of aggregation. Both values were found to depend on peptide concentration and addition or formation of seeds. Moreover, they can vary considerably between apparently identical samples. These data imply that the nucleation event is under influence of a stochastic factor that can manifest itself in profound macroscopic differences in the aggregation kinetics of otherwise indistinguishable samples.     

Effect of different salt ions on the propensity of aggregation and on the structure of Alzheimer's abeta(1-40) amyloid fibrils

The formation of amyloid fibrils and other polypeptide aggregates depends strongly on the physico-chemical environment. One such factor affecting aggregation is the presence and concentration of salt ions. We have examined the effects of salt ions on the aggregation propensity of Alzheimer's Abeta(1-40) peptide and on the structure of the dissolved and of the fibrillar peptide. All salts examined promote aggregation strongly. The most pronounced effect is seen within the cationic series, i.e. for MgCl2. Evaluation of different possible explanations suggests that Abeta(1-40) aggregation depends on direct interaction between ions and Abeta(1-40) peptide, and correlates with ion-induced changes of the surface tension. Salts have profound effects on the fibril structure. In the presence of salts, fibrils are associated with smaller diameters, narrower crossover distances and lower amide I maxima. Since Abeta(1-40) aggregation responds to salts in a manner unlike that for other polypeptides, such as glucagon, beta2-microglobulin or alpha-synuclein; these data argue that there is no fully uniform way in which salts affect aggregation of different polypeptide chains. These observations are important for understanding and predicting aggregation on the basis of simple physico-chemical properties.     

Mutagenic analysis of the nucleation propensity of oxidized Alzheimer's beta-amyloid peptide

The formation of polypeptide aggregates represents a nucleated polymerization reaction in which an initial nucleation event (lag phase) is followed by the extension of newly formed nuclei into larger aggregates, including fibrils (growth phase). The efficiencies of these reactions relate to the lag time (lag phase) and to the rate of aggregation (growth phase), which can be determined from experimental aggregation curves. Here we present a mutagenic analysis in which we replace valine 18 of the Alzheimer's Abeta (1-40) peptide with 17 different amino acids and determine its effect on the lag time, and therefore, on the propensity of nucleation. Comparison with various physico-chemical properties shows that nucleation is affected in a predictable manner depending on the beta-sheet propensity and hydrophobicity of residue 18. In addition, we observe a direct proportionality between the lag time and the rate of aggregation. These data imply that the two reactions, nucleation and polymerization, are governed by very similar physicochemical principles and that they involve the formation of the same types of noncovalent interactions.     

Is aggregation of beta-amyloid peptides a mis-functioning of a current interaction process?

In a previous study, Hughes et al. [Proc. Natl. Acad. Sci. USA 93 (1996) 2065-2070] demonstrated that the amyloid peptide is able to interact with itself in a two-hybrid system and that interaction is specific. They further supported that the method could be used to define the sequences that might be important in nucleation-dependent aggregation. The sequence of the amyloid peptide can be split into four clusters, two hydrophilic (1-16 and 22-28) and two hydrophobic (17-21 and 29-42). We designed by molecular modeling and tested by the two-hybrid approach, series of mutations spread all over the sequence and changing the distribution of hydrophobicity and/or the spatial hindrance. In the two-hybrid assay, interaction of native Abeta is reproduced. Screening of mutations demonstrates that the C-domain (residues 29-40 (42)), the median domain (residues 17-22) and the N-domain (1-16) are all crucial for interaction. This demonstrates that almost all fragments of the amyloid peptide but a loop (residues 23-28) and the C-term amino acid are important for the native interaction. We support that the folded three-dimensional (3D) structure is the Abeta-Abeta interacting species, that the whole sequence is involved in that 3D fold which has a low secondary structure propensity and a high susceptibility to mutations and thus should have a low stability. The native fold of Abeta could be stabilized in Abeta-Abeta complexes which could in other circumstances facilitate the nucleation event of aggregation that leads to the formation of stable senile plaques.     

Role of aggregation conditions in structure, stability, and toxicity of intermediates in the Abeta fibril formation pathway

beta-amyloid peptide (Abeta) is one of the main protein components of senile plaques associated with Alzheimer's disease (AD). Abeta readily aggregates to forms fibrils and other aggregated species that have been shown to be toxic in a number of studies. In particular, soluble oligomeric forms are closely related to neurotoxicity. However, the relationship between neurotoxicity and the size of Abeta aggregates or oligomers is still under investigation. In this article, we show that different Abeta incubation conditions in vitro can affect the rate of Abeta fibril formation, the conformation and stability of intermediates in the aggregation pathway, and toxicity of aggregated species formed. When gently agitated, Abeta aggregates faster than Abeta prepared under quiescent conditions, forming fibrils. The morphology of fibrils formed at the end of aggregation with or without agitation, as observed in electron micrographs, is somewhat different. Interestingly, intermediates or oligomers formed during Abeta aggregation differ greatly under agitated and quiescent conditions. Unfolding studies in guanidine hydrochloride indicate that fibrils formed under quiescent conditions are more stable to unfolding in detergent than aggregation associated oligomers or Abeta fibrils formed with agitation. In addition, Abeta fibrils formed under quiescent conditions were less toxic to differentiated SH-SY5Y cells than the Abeta aggregation associated oligomers or fibrils formed with agitation. These results highlight differences between Abeta aggregation intermediates formed under different conditions and provide insight into the structure and stability of toxic Abeta oligomers.     

Solution structures in aqueous SDS micelles of two amyloid beta peptides of A beta(1-28) mutated at the alpha-secretase cleavage site (K16E, K16F)

NMRsolution structures are reported for two mutants (K16E, K16F) of the soluble amyloid beta peptide Abeta(1-28). The structural effects of these mutations of a positively charged residue to anionic and hydrophobic residues at the alpha-secretase cleavage site (Lys16-Leu17) were examined in the membrane-simulating solvent aqueous SDS micelles. Overall the three-dimensional structures were similar to that for the native Abeta(1-28) sequence in that they contained an unstructured N-terminus and a helical C-terminus. These structural elements are similar to those seen in the corresponding regions of full-length Abeta peptides Abeta(1-40) and Abeta(1-42), showing that the shorter peptides are valid model systems. The K16E mutation, which might be expected to stabilize the macrodipole of the helix, slightly increased the helix length (residues 13-24) relative to the K16F mutation, which shortened the helix to between residues 16 and 24. The observed sequence-dependent control over conformation in this region provides an insight into possible conformational switching roles of mutations in the amyloid precursor protein from which Abeta peptides are derived. In addition, if conformational transitions from helix to random coil to sheet precede aggregation of Abeta peptides in vivo, as they do in vitro, the conformation-inducing effects of mutations at Lys16 may also influence aggregation and fibril formation.     

Unique physicochemical profile of beta-amyloid peptide variant Abeta1-40E22G protofibrils: conceivable neuropathogen in arctic mutant carriers

A new early-onset form of Alzheimer's disease (AD) was described recently where a point mutation was discovered in codon 693 of the beta-amyloid (Abeta) precursor protein gene, the Arctic mutation. The mutation translates into a single amino acid substitution, glutamic acid-->glycine, in position 22 of the Abeta peptide. The mutation carriers have lower plasma levels of Abeta than normal, while in vitro studies show that Abeta1-40E22G protofibril formation is significantly enhanced. We have explored the nature of the Abeta1-40E22G peptide in more detail, in particular the protofibrils. Using size-exclusion chromatography (SEC) and circular dichroism spectroscopy (CD) kinetic and secondary structural characteristics were compared with other Abeta1-40 peptides and the Abeta12-28 fragment, all having single amino acid substitutions in position 22. We have found that Abeta1-40E22G protofibrils are a group of comparatively stabile beta-sheet-containing oligomers with a heterogeneous size distribution, ranging from >100 kDa to >3000 kDa. Small Abeta1-40E22G protofibrils are generated about 400 times faster than large ones. Salt promotes their formation, which significantly exceeds all the other peptides studied here, including the Dutch mutation Abeta1-40E22Q. Position 22 substitutions had significant effects on aggregation kinetics of Abeta1-40 and in Abeta12-28, although the qualitative aspects of the effects differed between the native peptide and the fragment, as no protofibrils were formed by the fragments. The rank order of protofibril formation of Abeta1-40 and its variants was the same as the rank order of the length of the nucleation/lag phase of the Abeta12-28 fragments, E22V>E22A?E22G>E22Q?E22, and correlated with the degree of hydrophobicity of the position 22 substituent. The molecular mass of peptide monomers and protofibrils were estimated better in SEC studies using linear rather than globular calibration standards. The characteristics of the Abeta1-40E22G suggest an important role for the peptide in the neuropathogenesis in the Arctic form of AD.     

Interactions of Alzheimer amyloid-beta peptides with glycosaminoglycans effects on fibril nucleation and growth.

Proteoglycans and their constituent glycosaminoglycans are associated with all amyloid deposits and may be involved in the amyloidogenic pathway. In Alzheimer's disease, plaques are composed of the amyloid-beta peptide and are associated with at least four different proteoglycans. Using CD spectroscopy, fluorescence spectroscopy and electron microscopy, we examined glycosaminoglycan interaction with the amyloid-beta peptides 1-40 (Abeta40) and 1-42 (Abeta42) to determine the effects on peptide conformation and fibril formation. Monomeric amyloid-beta peptides in trifluoroethanol, when diluted in aqueous buffer, undergo a slow random to amyloidogenic beta sheet transition. In the presence of heparin, heparan sulfate, keratan sulfate or chondroitin sulfates, this transition was accelerated with Abeta42 rapidly adopting a beta-sheet conformation. This was accompanied by the appearance of well-defined amyloid fibrils indicating an enhanced nucleation of Abeta42. Incubation of preformed Abeta42 fibrils with glycosaminoglycans resulted in extensive lateral aggregation and precipitation of the fibrils. The glycosaminoglycans differed in their relative activities with the chondroitin sulfates producing the most pronounced effects. The less amyloidogenic Abeta40 isoform did not show an immediate structural transition that was dependent upon the shielding effect by the phosphate counter ion. Removal or substitution of phosphate resulted in similar glycosaminoglycan-induced conformational and aggregation changes. These findings clearly demonstrate that glycosaminoglycans act at the earliest stage of fibril formation, namely amyloid-beta nucleation, and are not simply involved in the lateral aggregation of preformed fibrils or nonspecific adhesion to plaques. The identification of a structure-activity relationship between amyloid-beta and the different glycosaminoglycans, as well as the condition dependence for glycosaminoglycan binding, are important for the successful development and evaluation of glycosaminoglycan-specific therapeutic interventions.     

Conformational solution studies of the SDS micelle-bound 11-28 fragment of two Alzheimer's beta-amyloid variants (E22K and A21G) using CD, NMR, and MD techniques

The beta-amyloid (Abeta) is the major peptide constituent of neuritic plaques in Alzheimer's disease (AD) and its aggregation is believed to play a central role in the pathogenesis of the disease. Naturally occurring mutations resulting in changes in the Abeta sequence (pos. 21-23) are associated with familial AD-like diseases with extensive cerebrovascular pathology. It was proved that the mutations alter the aggregation ability of Abeta and its neurotoxicity. Among five mutations at positions 21-23 there are two mutations with distinct clinical characteristics and potentially distinct pathogenic mechanism-the Italian (E22K) and the Flemish (A21G) mutations. In our studies we have examined the structures of the 11-28 fragment of the Italian and Flemish Abeta variants. The fragment was chosen because it has been shown to be the most important for amyloid fibril formation. The detailed structure of both variants Abeta(11-28) was determined using CD, 2D NMR, and molecular dynamics techniques under water-SDS micelle conditions. The NMR analysis revealed two distinct sets of proton resonances for the peptides. The studies of both peptides pointed out the existence of well-defined alpha-helical conformation in the Italian mutant, whereas the Flemish was found to be unstructured with the possibility of a bent structure in the central part of the peptide.     

Mutagenesis of the central hydrophobic cluster in Abeta42 Alzheimer's peptide. Side-chain properties correlate with aggregation propensities

Protein misfolding and deposition underlie an increasing number of debilitating human disorders. Alzheimer's disease is pathologically characterized by the presence of numerous insoluble amyloid plaques in the brain, composed primarily of the 42 amino acid human beta-amyloid peptide (Abeta42). Disease-linked mutations in Abeta42 occur in or near a central hydrophobic cluster comprising residues 17-21. We exploited the ability of green fluorescent protein to act as a reporter of the aggregation of upstream fused Abeta42 variants to characterize the effects of a large set of single-point mutations at the central position of this hydrophobic sequence as well as substitutions linked to early onset of the disease located in or close to this region. The aggregational properties of the different protein variants clearly correlated with changes in the intrinsic physicochemical properties of the side chains at the point of mutation. Reduction in hydrophobicity and beta-sheet propensity resulted in an increase of in vivo fluorescence indicating disruption of aggregation, as confirmed by the in vitro analysis of synthetic Abeta42 variants. The results confirm the key role played by the central hydrophobic stretch on Abeta42 deposition and support the hypothesis that sequence tunes the aggregation propensities of polypeptides.     

Aggregation of liposomes induced by the toxic peptides Alzheimer's Abetas, human amylin and prion (106-126): facilitation by membrane-bound GM1 ganglioside

To compare both the peptide molecular self-aggregation and the interaction with membrane lipids of the Alzheimer's amyloid beta (Abeta)40, Abeta42 peptides, and the cytotoxic peptides human amylin and prion (106-126) peptides, we applied a liposome aggregation technology. The kinetics of the changes in the optical density (DeltaOD) of liposome suspensions generated by the aggregation of liposomes induced by these peptides, allowed us to comparatively analyze their phospholipid affinity and self-aggregation. The kinetic curves showed an initial nonlinear region where d(DeltaOD)/dt followed first order kinetics corresponding to the binding of the peptides to the membrane of the liposome, a linear region where d(DeltaOD)/dt was constant, corresponding to the interaction between two membrane-bound peptide molecules, and a final slower increasing nonlinear region that corresponds to nucleation or seeding of aggregation. The analysis of the aggregation curves demonstrated that amylin and prion peptides also showed affinity for the acidic phospholipid phosphatidylserine (PS), as it has previously been shown for the Alzheimer's Abeta40, Abeta42 peptides. Abeta42 showed the highest, and amylin the lowest, affinity for the liposome membrane. When bound to the membrane of the liposomes, all the peptides preserved the self-aggregation characteristics observed in solution. Aging the Abeta40 and Abeta42 peptide solutions that permit molecular self-aggregation reduced their capacity to induce liposome aggregation. The self-aggregation of membrane-bound prion molecules was several orders of magnitude higher than that observed for the other toxic peptides. Incorporation of the ganglioside GM1 into the membrane of liposomes enhanced the peptide-induced liposome aggregation. Kinetic analysis revealed that this enhancement was due to facilitation of the formation of bridges between membrane-bound peptide molecules, demonstrating that the peptide-membrane interaction and the peptide amyloidogenesis are independent functions performed at separate molecular regions.     

Modeling amyloid beta-peptide insertion into lipid bilayers

Inspired by recent suggestions that the Alzheimer's amyloid beta peptide (Abeta) can insert into cell membranes and form harmful ion channels, we model insertion of the 40- and 42-residue forms of the peptide into cell membranes using a Monte Carlo code which is specific at the amino acid level. We examine insertion of the regular Abeta peptide as well as mutants causing familial Alzheimer's disease, and find that all but one of the mutants change the insertion behavior by causing the peptide to spend more simulation steps in only one leaflet of the bilayer. We also find that Abeta42, because of the extra hydrophobic residues relative to Abeta40, is more likely to adopt this conformation than Abeta40 in both wild-type and mutant forms. We argue qualitatively why these effects happen. Here, we present our results and develop the hypothesis that this partial insertion increases the probability of harmful channel formation. This hypothesis can partly explain why these mutations are neurotoxic simply due to peptide insertion behavior. We further apply this model to various artificial Abeta mutants which have been examined experimentally, and offer testable experimental predictions contrasting the roles of aggregation and insertion with regard to toxicity of Abeta mutants. These can be used through further experiments to test our hypothesis.     

Absolute correlation between lag time and growth rate in the spontaneous formation of several amyloid-like aggregates and fibrils

The formation of polypeptide aggregates, including amyloid fibrils and prions, is a biochemical process of considerable interest in the context of its association with ageing and neurodegeneration. Aggregation occurs typically with a lag phase and a growth phase that reflect an underlying nucleation-polymerisation mechanism. While the propensity of nucleation can be estimated from the lag time t(l), the efficiency of growth is represented by the growth rate k(g). Here, I have analysed the absolute k(g) and t(l) values from a total of 298 samples prepared from insulin, glucagon and different sequence variants of the Alzheimer's Abeta(1-40) peptide. Although these samples differ in the conditions of aggregation, systematic comparison reveals an overall similarity in the plot of k(g)versus t(l). The plot fits readily with the simple equation k(g)=alpha/t(l) and by using a proportionality factor alpha of 4.5. In contrast to the individual values of k(g) and t(l) that depend substantially on sequential and environmental parameters, alpha seems much less affected by such factors. These data suggest mechanistic similarities in the nucleation behaviour of different amyloid-like fibrils and aggregates.     

A Cdk5 inhibitory peptide reduces tau hyperphosphorylation and apoptosis in neurons

The extracellular aggregation of amyloid beta (Abeta) peptides and the intracellular hyperphosphorylation of tau at specific epitopes are pathological hallmarks of neurodegenerative diseases such as Alzheimer's disease (AD). Cdk5 phosphorylates tau at AD-specific phospho-epitopes when it associates with p25. p25 is a truncated activator, which is produced from the physiological Cdk5 activator p35 upon exposure to Abeta peptides. We show that neuronal infections with Cdk5 inhibitory peptide (CIP) selectively inhibit p25/Cdk5 activity and suppress the aberrant tau phosphorylation in cortical neurons. Furthermore, Abeta(1-42)-induced apoptosis of these cortical neurons was also reduced by coinfection with CIP. Of particular importance is our finding that CIP did not inhibit endogenous or transfected p35/Cdk5 activity, nor did it inhibit the other cyclin-dependent kinases such as Cdc2, Cdk2, Cdk4 and Cdk6. These results, therefore, provide a strategy to address, and possibly ameliorate, the pathology of neurodegenerative diseases that may be a consequence of aberrant p25 activation of Cdk5, without affecting 'normal' Cdk5 activity.     

Alzheimer's Abeta peptides containing an isostructural backbone mutation afford distinct aggregate morphologies but analogous cytotoxicity. Evidence for a common low-abundance toxic structure(s)?

Amyloid beta (Abeta) peptide amyloidogenesis, involving the formation of numerous distinct quaternary structures, appears to cause Alzheimer's disease. However, the precise identification of the toxic structure(s) and the neurotoxicity mechanism(s) remains elusive. Mutating the Abeta 1-40 Phe19-Phe20 backbone amide bond to an isostructural E-olefin bond enables formation of spherical aggregates to the exclusion of detectable amyloid fibrils. Herein, the fibrillization and toxicity of amide-to-ester mutants of Abeta 1-40 at the 19-20 position and surrounding backbone amide bonds are compared to the fibrillization and toxicity of the 19-20 E-olefin Abeta analogue and wild type Abeta. Whereas isostructural amide-to-E-olefin mutations eliminate both the H-bond donor and acceptor capabilities, isostructural amide-to-ester mutations eliminate the donor while retaining the ester carbonyl as a weakened acceptor. None of the amide-to-ester Abeta 1-40 mutants prevent fibrillization; in fact several exhibit hastened amyloidogenesis. The 18-19 amide-to-ester substitution is the only backbone mutation within the hydrophobic core region of the fibril (residues 17-21) that significantly slows fibrillization. Despite forming different morphologies, the 19-20 E-olefin mutant, the 18-19 amide-to-ester mutant, and WT Abeta 1-40 fibrils all exhibit similar toxicities when applied to PC12 cells at 18 h into the aggregation reactions, as assessed by MTT metabolic activity measurements. This result suggests that a common but low abundance aggregate morphology, that is accessible to these Abeta analogues, mediates toxicity, or that several different aggregate morphologies are similarly toxic.     

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.     

Characterization of copper interactions with alzheimer amyloid beta peptides: identification of an attomolar-affinity copper binding site on amyloid beta1-42

Cu and Zn have been shown to accumulate in the brains of Alzheimer's disease patients. We have previously reported that Cu(2+) and Zn(2+) bind amyloid beta (Abeta), explaining their enrichment in plaque pathology. Here we detail the stoichiometries and binding affinities of multiple cooperative Cu(2+)-binding sites on synthetic Abeta1-40 and Abeta1-42. We have developed a ligand displacement technique (competitive metal capture analysis) that uses metal-chelator complexes to evaluate metal ion binding to Abeta, a notoriously self-aggregating peptide. This analysis indicated that there is a very-high-affinity Cu(2+)-binding site on Abeta1-42 (log K(app) = 17.2) that mediates peptide precipitation and that the tendency of this peptide to self-aggregate in aqueous solutions is due to the presence of trace Cu(2+) contamination (customarily approximately 0.1 microM). In contrast, Abeta1-40 has much lower affinity for Cu(2+) at this site (estimated log K(app) = 10.3), explaining why this peptide is less self-aggregating. The greater Cu(2+)-binding affinity of Abeta1-42 compared with Abeta1-40 is associated with significantly diminished negative cooperativity. The role of trace metal contamination in inducing Abeta precipitation was confirmed by the demonstration that Abeta peptide (10 microM) remained soluble for 5 days only in the presence of high-affinity Cu(2+)-selective chelators.     

Lipids revert inert Abeta amyloid fibrils to neurotoxic protofibrils that affect learning in mice

Although soluble oligomeric and protofibrillar assemblies of Abeta-amyloid peptide cause synaptotoxicity and potentially contribute to Alzheimer's disease (AD), the role of mature Abeta-fibrils in the amyloid plaques remains controversial. A widely held view in the field suggests that the fibrillization reaction proceeds 'forward' in a near-irreversible manner from the monomeric Abeta peptide through toxic protofibrillar intermediates, which subsequently mature into biologically inert amyloid fibrils that are found in plaques. Here, we show that natural lipids destabilize and rapidly resolubilize mature Abeta amyloid fibers. Interestingly, the equilibrium is not reversed toward monomeric Abeta but rather toward soluble amyloid protofibrils. We characterized these 'backward' Abeta protofibrils generated from mature Abeta fibers and compared them with previously identified 'forward' Abeta protofibrils obtained from the aggregation of fresh Abeta monomers. We find that backward protofibrils are biochemically and biophysically very similar to forward protofibrils: they consist of a wide range of molecular masses, are toxic to primary neurons and cause memory impairment and tau phosphorylation in mouse. In addition, they diffuse rapidly through the brain into areas relevant to AD. Our findings imply that amyloid plaques are potentially major sources of soluble toxic Abeta-aggregates that could readily be activated by exposure to biological lipids.