Observation of multi-step conformation switching in beta-amyloid peptide aggregation by fluorescence resonance energy transfer

We have observed the conformation switching of Abeta(11-25) in the course of amyloid aggregation by employing time-resolved fluorescence resonance energy transfer (FRET). The amyloid peptides undergo multi-step conformational changes during self-assembling such as random coil (monomers), collapsed coil (multimers), micellar structure, and extended beta-sheet in fibrils. We first identified the critical micelle concentration of Abeta(11-25) that occurs at ca. 3 microM for pH 5.0 and ca. 70 microM for pH 7.4. Our experimental results show clearly that the end-to-end distance of micellar Abeta(11-25) becomes much shorter than that of the collapsed coil or fibril structure.     

Demonstration of an anti-oxidative stress mechanism of quetiapine: implications for the treatment of Alzheimer's disease

We have shown that quetiapine, a new antipsychotic drug, protects cultured cells against oxidative stress-related cytotoxicities induced by amyloid beta (Abeta)25-35, and that quetiapine prevents memory impairment and decreases Abeta plaques in the brains of amyloid precursor protein (APP)/presenilin-1 (PS-1) double-mutant mice. The aim of this study was to understand why quetiapine has these protective effects. Because the cytotoxicity of both Abeta(25-35) and Abeta(1-40) requires fibril formation, our first experiments determined the effect of quetiapine on Abeta(25-35) aggregation. Quetiapine inhibited Abeta(25-35) aggregation in cell-free aqueous solutions and blocked the fibrillar aggregation of Abeta(25-35), as observed under an electron microscope. We then investigated why quetiapine inhibits Abeta(25-35) aggregation. During the aggregation of Abeta(25-35), a hydroxyl radical (OH*) was released, which in turn amplified Abeta(25-35) aggregation. Quetiapine blocked OH*-induced Abeta(25-35) aggregation and scavenged the OH* produced in the Fenton system and in the Abeta(25-35) solution, as analyzed using electron paramagnetic resonance spectroscopy. Furthermore, new compounds formed by quetiapine and OH* were observed in MS analysis. Finally, we applied Abeta(25-35) to PC12 cells to observe the effect of quetiapine on living cells. Abeta(25-35) increased levels of intracellular reactive oxygen species and calcium in PC12 cells and caused cell death, but these toxic effects were prevented by quetiapine. These results demonstrate an anti-oxidative stress mechanism of quetiapine, which contributes to its protective effects observed in our previous studies and explains the effectiveness of this drug for Alzheimer's disease patients with psychiatric and behavioral complications.     

Inhibition of beta-amyloid-induced neurotoxicity by imidazopyridoindoles derived from a synthetic combinatorial library

Alzheimer's disease is a progressive neurodegenerative disorder characterized by the deposit of amyloid fibrils in the brain that result from the self-aggregative polymerization of the beta-amyloid peptide (Abeta). Evidence of a direct correlation between the ability of Abeta to form stable aggregates in aqueous solution and its neurotoxicity has been reported. The cytotoxic effects of Abeta have been attributed to the aggregation properties of a domain corresponding to the peptide fragment Abeta25-35. In an effort to generate novel inhibitors of Abeta neurotoxicity and/or aggregation, a mixture-based synthetic combinatorial library composed of 23 375 imidazopyridoindoles was generated and screened for inhibition of Abeta25-35 neurotoxicity toward the rat pheochromocytoma PC-12 cell line. The effect of the identified lead compounds on Abeta25-35 aggregation was then evaluated by means of circular dichroism (CD) and thioflavin-T fluorescence spectroscopy. Their activity against Abeta1-42 neurotoxicity toward the PC-12 cell line was also determined. The most active imidazopyridoindoles inhibited both Abeta25-35 and Abeta1-42 neurotoxicity in the low- to mid-micromolar range. Furthermore, inhibition of the random coil to beta-sheet transition and self-aggregation of Abeta25-35 was observed by CD and fluorescence spectroscopy, supporting the relationship between inhibition of the Abeta aggregation process and neurotoxicity.     

Recognition sequence design for peptidyl modulators of beta-amyloid aggregation and toxicity

beta-Amyloid (Abeta), the primary protein component of Alzheimer's plaques, is neurotoxic when aggregated into fibrils. We have devised a modular strategy for generating compounds that inhibit Abeta toxicity, based on linking a recognition element for Abeta to a disrupting element designed to interfere with Abeta aggregation. One such compound, with the 15-25 sequence of Abeta as the recognition element and a lysine hexamer as the disrupting element, altered Abeta aggregation kinetics and protected cells from Abeta toxicity [Ghanta et al. (1996) J. Biol. Chem. 271, 29525]. To optimize the recognition element, peptides of 4-8 residues composed of overlapping sequences within the 15-25 domain were synthesized, along with hybrid compounds containing those recognition sequences coupled to a lysine hexamer. None of the recognition peptides altered Abeta aggregation kinetics and only two, KLVFF and KLVF, had any protective effect against Abeta toxicity. The hybrid peptide KLVFF-KKKKKK dramatically altered Abeta aggregation kinetics and aggregate morphology and provided significantly improved protection against Abeta toxicity compared to the recognition peptide alone. In contrast, FAEDVG-KKKKKK possessed only modest inhibitory activity and had no marked effect on Abeta aggregation. The scrambled sequence VLFKF was nearly as effective a recognition domain as KLVFF, suggesting the hydrophobic characteristics of the recognition sequence are critical. None of the cytoprotective peptides prevented Abeta aggregation; rather, they increased aggregate size and altered aggregate morphology. These results suggest that coupling recognition with disrupting elements is an effective generalizable strategy for the creation of Abeta inhibitors. Significantly, prevention of Abeta aggregation may not be required for prevention of toxicity.     

Oligomerization of amyloid Abeta16-22 peptides using hydrogen bonds and hydrophobicity forces

The 16-22 amino-acid fragment of the beta-amyloid peptide associated with the Alzheimer's disease, Abeta, is capable of forming amyloid fibrils. Here we study the aggregation mechanism of Abeta16-22 peptides by unbiased thermodynamic simulations at the atomic level for systems of one, three, and six Abeta16-22 peptides. We find that the isolated Abeta16-22 peptide is mainly a random coil in the sense that both the alpha-helix and beta-strand contents are low, whereas the three- and six-chain systems form aggregated structures with a high beta-sheet content. Furthermore, in agreement with experiments on Abeta16-22 fibrils, we find that large parallel beta-sheets are unlikely to form. For the six-chain system, the aggregated structures can have many different shapes, but certain particularly stable shapes can be identified.     

Membrane interactions and the effect of metal ions of the amyloidogenic fragment Abeta(25-35) in comparison to Abeta(1-42)

Abeta(1-42) peptide, found as aggregated species in Alzheimer's disease brain, is linked to the onset of Alzheimer's disease. Many reports have linked metals to inducing Abeta aggregation and amyloid plaque formation. Abeta(25-35), a fragment from the C-terminal end of Abeta(1-42), lacks the metal coordinating sites found in the full-length peptide and is neurotoxic to cortical cortex cell cultures. We report solid-state NMR studies of Abeta(25-35) in model lipid membrane systems of anionic phospholipids and cholesterol, and compare structural changes to those of Abeta(1-42). When added after vesicle formation, Abeta(25-35) was found to interact with the lipid headgroups and slightly perturb the lipid acyl-chain region; when Abeta(25-35) was included during vesicle formation, it inserted deeper into the bilayer. While Abeta(25-35) retained the same beta-sheet structure irrespective of the mode of addition, the longer Abeta(1-42) appeared to have an increase in beta-sheet structure at the C-terminus when added to phospholipid liposomes after vesicle formation. Since the Abeta(25-35) fragment is also neurotoxic, the full-length peptide may have more than one pathway for toxicity.     

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.     

Abeta(31-35) peptide induce apoptosis in PC 12 cells: contrast with Abeta(25-35) peptide and examination of underlying mechanisms

The toxic behaviour of the two shorter sequences of the native Abeta amyloid peptide required for cytotoxicity i.e., Abeta(31-35) and Abeta(25-35) peptides, was studied. We have shown that Abeta(31-35) peptide induces neurotoxicity in undifferentiated PC 12 cell via an apoptotic cell death pathway, including caspase activation and DNA fragmentation. Abeta(25-35) peptide, like the shorter amyloid peptide has the ability to induce neurotoxicity, as evaluated by the MTS reduction assay and by adherent cell count, but the Abeta(25-35) peptide-induced neurotoxicity is not associated with any biochemical features of apoptosis. The differences observed between the neurotoxic properties of Abeta(31-35) and Abeta(25-35) peptides might result on their different ability to be internalised within the neuronal cells. Furthermore, this study reveals that the redox state of methionine residue, C-terminal in Abeta(31-35) and Abeta(25-35) peptides affect in a different way the toxic behaviour of these two short amyloid fragments. Taken together our results suggest that Abeta(31-35) peptide induces cell death by apoptosis, unlike the Abeta(25-35) peptide and that role played by methionine-35 in Abeta induced neurotoxicity might be related to the Abeta aggregation state.     

Design of peptidyl compounds that affect beta-amyloid aggregation: importance of surface tension and context

Self-association of beta-amyloid (Abeta) peptide into cross-beta-sheet fibrils induces cellular toxicity in vitro and is linked with progression of Alzheimer's disease. Previously, we demonstrated that hybrid peptides, containing a recognition domain that binds to Abeta and a disrupting domain consisting of a chain of charged amino acids, inhibited Abeta-associated toxicity in vitro and increased the rate of Abeta aggregation. In this work we examine the design parameter space of the disrupting domain. Using KLVFFKKKKKK as a base case, we tested hybrid compounds with a branched rather than linear lysine oligomer, with l-lysine replaced by d-lysine, and with lysine replaced by diaminopropionic acid. We synthesized a compound with a novel anionic disrupting domain that contained cysteine thiols oxidized to sulfates, as well as other compounds in which alkyl or ether chains were appended to KLVFF. In all cases, the hybrid compound's ability to increase solvent surface tension was the strongest predictor of its effect on Abeta aggregation kinetics. Finally, we investigated the effects of arginine on Abeta aggregation. Arginine is a well-known chaotrope but increases surface tension of water. Arginine modestly decreased Abeta aggregation. In contrast, RRRRRR slightly, and KLVFFRRRRRR greatly, increased Abeta aggregation. Thus, the influence of arginine on Abeta aggregation depends strongly on the context in which it is presented. The effect of arginine, RRRRRR, and KLVFFRRRRRR on Abeta aggregation was examined in detail using laser light scattering, circular dichroism spectroscopy, Fourier transform infrared spectroscopy, thioflavin T fluorescence, and transmission electron microscopy.     

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.     

N-Methylated peptide inhibitors of beta-amyloid aggregation and toxicity. Optimization of the inhibitor structure

The key pathogenic event in the onset of Alzheimer's disease (AD) is believed to be the aggregation of the beta-amyloid (Abeta) peptide into toxic oligomers. Molecules that interfere with this process may therefore act as therapeutic agents for the treatment of AD. N-Methylated peptides (meptides) are a general class of peptide aggregation inhibitors that act by binding to one face of the aggregating peptide but are unable to hydrogen bond on the other face, because of the N-methyl group replacing a backbone NH group. Here, we optimize the structure of meptide inhibitors of Abeta aggregation, starting with the KLVFF sequence that is known to bind to Abeta. We varied the meptide length, N-methylation sites, acetylation, and amidation of the N and C termini, side-chain identity, and chirality, via five compound libraries. Inhibitor activity was tested by thioflavin T binding, affinity chromatography, electron microscopy, and an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide toxicity assay. We found that inhibitors should have all d chirality, have a free N terminus but an amidated C terminus, and have large, branched hydrophobic side chains at positions 1-4, while the side chain at position 5 was less important. A single N-methyl group was necessary and sufficient. The most active compound, d-[(chGly)-(Tyr)-(chGly)-(chGly)-(mLeu)]-NH(2), was more active than all previously reported peptide inhibitors. Its related non-N-methylated analogues were insoluble and toxic.     

Electrophysiologic properties of channels induced by Abeta25-35 in planar lipid bilayers

Abeta25-35, a fragment of the neurotoxic amyloid beta protein Abeta1-42 found in the brain of Alzheimer patients, possesses amyloidogenic, neurotoxins and channel forming abilities similar to that of Abeta1-42. We have previously reported that Abeta25-35 formed voltage-dependent, relatively nonselective, ion-permeable channels in planar lipid bilayers. Here, we show that Abeta25-35 formed channels in both solvent-containing and solvent-free bilayers. We also report that for Abeta25-35, channel forming activity was dependent on ionic strength, membrane lipid composition, and peptide concentration, but not on pH. Lower ionic strength and negatively charged lipids increased channel formation activity, while cholesterol decreased activity. The nonlinear function relating [Abeta25-35] and membrane activity suggests that aggregation of at least three monomers is required for channel formation.     

Laminin isoforms differentially regulate adhesion, spreading, proliferation, and ERK activation of beta1 integrin-null cells

The presence of many laminin receptors of the beta1 integrin family on most cells makes it difficult to define the biological functions of other major laminin receptors such as integrin alpha6beta4 and dystroglycan. We therefore tested the binding of a beta1 integrin-null cell line GD25 to four different laminin variants. The cells were shown to produce dystroglycan, which based on affinity chromatography bound to laminin-1, -2/4, and -10/11, but not to laminin-5. The cells also expressed the integrin alpha6Abeta4A variant. GD25 beta1 integrin-null cells are known to bind poorly to laminin-1, but we demonstrate here that these cells bind avidly to laminin-2/4, -5, and -10/11. The initial binding at 20 min to each of these laminins could be inhibited by an integrin alpha6 antibody, but not by a dystroglycan antibody. Hence, integrin alpha6Abeta4A of GD25 cells was identified as a major receptor for initial GD25 cell adhesion to three out of four tested laminin isoforms. Remarkably, cell adhesion to laminin-5 failed to promote cell spreading, proliferation, and extracellular signal-regulated kinase (ERK) activation, whereas all these responses occurred in response to adhesion to laminin-2/4 or -10/11. The data establish GD25 cells as useful tools to define the role integrin alpha6Abeta4A and suggest that laminin isoforms have distinctly different capacities to promote cell adhesion and signaling via integrin alpha6Abeta4A.     

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.     

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.     

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.     

Single chain Fv antibodies against the 25-35 Abeta fragment inhibit aggregation and toxicity of Abeta42

Alzheimer's disease (AD) is characterized by the deposition of amyloid-beta (Abeta) protein in the brain. Immunization studies have demonstrated that anti-Abeta antibodies reduce Abeta deposition and improve clinical symptoms seen in AD. However, conventional antibody-based therapies risk an inflammatory response that can result in meningoencephalitis and cerebral hemorrhage. Here we report on the development of human-based single chain variable domain antibody fragments (scFvs) directed against the Abeta 25-35 region as potential therapeutics for AD that do not risk an inflammatory response. The 25-35 region of Abeta represents a promising therapeutic target since it promotes aggregation and is highly toxic. Two scFvs with differing affinities for Abeta were studied, and both inhibited aggregation of Abeta42 as determined by thioflavin T binding assay and atomic force microscopy analysis and blocked Abeta-induced toxicity toward human neuroblastoma SH-SY5Y cells as determined by MTT and LDH release assays. These results provide additional evidence that scFvs against Abeta provide an attractive alternative to more conventional antibody-based therapeutics for controlling aggregation and toxicity of Abeta.     

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.     

Solid state NMR reveals a pH-dependent antiparallel beta-sheet registry in fibrils formed by a beta-amyloid peptide

We report solid state nuclear magnetic resonance (NMR) measurements that probe the supramolecular organization of beta-sheets in the cross-beta motif of amyloid fibrils formed by residues 11-25 of the beta-amyloid peptide associated with Alzheimer's disease (Abeta(11-25)). Fibrils were prepared at pH 7.4 and pH 2.4. The solid state NMR data indicate that the central hydrophobic segment of Abeta(11-25) (sequence LVFFA) adopts a beta-strand conformation and participates in antiparallel beta-sheets at both pH values, but that the registry of intermolecular hydrogen bonds is pH-dependent. Moreover, both registries determined for Abeta(11-25) fibrils are different from the hydrogen bond registry in the antiparallel beta-sheets of Abeta(16-22) fibrils at pH 7.4 determined in earlier solid state NMR studies. In all three cases, the hydrogen bond registry is highly ordered, with no detectable "registry-shift" defects. These results suggest that the supramolecular organization of beta-sheets in amyloid fibrils is determined by a sensitive balance of multiple side-chain-side-chain interactions. Recent structural models for Abeta(11-25) fibrils based on X-ray fiber diffraction data are inconsistent with the solid state NMR data at both pH values.     

Structure of the 21-30 fragment of amyloid beta-protein

Folding and self-assembly of the 42-residue amyloid beta-protein (Abeta) are linked to Alzheimer's disease (AD). The 21-30 region of Abeta, Abeta(21-30), is resistant to proteolysis and is believed to nucleate the folding of full-length Abeta. The conformational space accessible to the Abeta(21-30) peptide is investigated by using replica exchange molecular dynamics simulations in explicit solvent. Conformations belonging to the global free energy minimum (the "native" state) from simulation are in good agreement with reported NMR structures. These conformations possess a bend motif spanning the central residues V24-K28. This bend is stabilized by a network of hydrogen bonds involving the side chain of residue D23 and the amide hydrogens of adjacent residues G25, S26, N27, and K28, as well as by a salt bridge formed between side chains of K28 and E22. The non-native states of this peptide are compact and retain a native-like bend topology. The persistence of structure in the denatured state may account for the resistance of this peptide to protease degradation and aggregation, even at elevated temperatures.