Cerebrospinal fluid amyloid beta peptide patterns in Alzheimer's disease patients and nondemented controls depend on sample pretreatment: indication of carrier-mediated epitope masking of amyloid beta peptides

A quantitative urea-based amyloid beta (Abeta)-sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Western immunoblot (Abeta-SDS-PAGE/immunoblot) reveals highly conserved and disease-specific Abeta peptide patterns (Abeta 1-37, 1-38, 1-39, 1-40, 1-42) in Alzheimer's disease (AD) patients and nondemented controls. For further standardization of this method, we analyzed cerebrospinal fluid (CSF) of eight probable AD patients and seven nondemented controls using different preanalytical procedures for Abeta-SDS-PAGE/immunoblot and Abeta1-42-enzyme linked immunosorbent assay (ELISA). Both diagnostic groups were discriminated significantly by absolute levels of Abeta1-42 and ratios of Abeta1-42/40, 1-42/38, 1-42/39. Preanalytical freezing of CSF led to a highly significant loss of all Abeta peptide species. This effect was most pronounced for Abeta1-42 and completely prevented by SDS-heat denaturation prior to freezing. Prolonged storage of SDS-heat denatured CSF led to a selective loss of Abeta1-42 and impaired the discrimination of diagnostic groups as measured by Abeta-SDS-PAGE/immunoblot. Neither freezing nor storage significantly affected absolute Abeta1-42 levels as determined by Abeta1-42-ELISA, but both impaired the discrimination of diagnostic groups. Hence, we suggest immediate analysis of samples for Abeta1-42-ELISA, analysis after a short freezing interval for Abeta-SDS-PAGE/immunoblot, and avoidance of prolonged storage intervals. Remarkably, Abeta-SDS-PAGE/immunoblot measured threefold higher levels of Abeta1-42 in CSF than Abeta1-42-ELISA. In summary, our results indicate carrier-mediated epitope masking of Abeta1-42.     

Examining the levels of ganglioside and cholesterol in cell membrane on attenuation the cytotoxicity of beta-amyloid peptide

The deposition of beta-amyloid (Abeta) on cell membranes is considered as one of the primary factors in having Alzheimer's disease (AD). Recent studies have suggested that certain components of plasma membrane, ganglioside and cholesterol could accelerate the accumulation of Abeta on the plasma membranes. However, the effect of cholesterol and ganglioside (GM1) on Abeta cytotoxicity is still a controversial issue. The aim of this study is to understand the roles of GM1 and cholesterol in AD by using PC12, a neuron-like cell. The effects of the sequence, conformation, and concentration of Abeta on cytotoxicity were also investigated. Monomeric Abeta could attack the plasma membrane resulting in cytotoxicity, however, fibrillar Abeta was found to be less toxic. Our results showed that Abeta (1-40) was more toxic than Abeta (25-35) and the cytotoxicity of Abeta was proportional to its concentration. Besides, the depletion of GM1 from plasma membrane, it would block the Abeta-induced cytotoxicity. Decreasing the cholesterol level by around 30% could attenuate the cytotoxicity of Abeta. These findings validate our idea that the cholesterol could stabilize the lateral pressure derived from the formation of GM1-Abeta complex on the membrane surface. Furthermore, both GM1 and cholesterol are essential in mechanism of Abeta accumulation and could modulate the cytotoxicity of monomeric Abeta.     

Molecular dynamics study of the beta amyloid peptide of Alzheimer's disease and its divalent copper complexes

The Abeta1-42 monomer structure was assessed with a 790 ns molecular dynamics (MD) simulation, and the results were compared with the NMR experiment on Abeta10-35 and Abeta1-40. Previous theoretical work in a model of the His13-His14 region of Abeta defined the possible Cu(II) binding geometries at this site (Raffa et al. J. Biol. Inorg. Chem. 2005, 10, 887-902). MD simulations totalling almost 2 micros were also carried out on Cu(II)/Abeta1-42 systems, using the ab initio structures as templates for the copper binding site. This work finds that the copper-free Abeta1-42 system may stabilize after approximately 350 ns into a collapsed coil conformation, and we find good agreement with some, but not all, of the structural features determined experimentally for the Abeta10-35 and Abeta1-40 peptides. The results of the Cu(II)/Abeta1-42 systems are compared to the Cu(II)-free Abeta1-42 simulation.     

Early phase of amyloid beta42-induced cytotoxicity in neuronal cells is associated with vacuole formation and enhancement of exocytosis

Amyloid beta (Abeta) neurotoxicity is believed to play a critical role in the pathogenesis of Alzheimer's disease (AD) mainly because of its deposition in AD brain and its neuronal toxicity. However, there have been discrepancies in Abeta-induced cytotoxicity studies, depending on the assay methods. Comparative analysis of Abeta42-induced in vitro cytotoxicity might be useful to elucidate the etiological role of Abeta in the pathogenesis of AD. In this study, MTT, CCK-8, calcein-AM/EthD-1 assays as well as thorough microscopic examinations were comparatively performed after Abeta42 treatment in a neuronal precursor cells (NT2) and a somatic cells (EcR293). Extensive formation of vacuoles was observed at the very early stage of Abeta42 treatment in both cells. Early observation of Abeta42 toxicity as seen in vacuole formation was also shown in MTT assay, but not in CCK-8 and calcein-AM/EthD-1 assays. In addition, Abeta42 treatment dramatically accelerated MTT formazan exocytosis, implying its effect on the extensive formation of cytoplasmic vacuoles. Abeta42 seems to cause indirect inhibition on the intracellular MTT reduction as well as vacuole formation and exocytosis enhancement. Following the acute cellular dysfunction induced by Abeta42, the prolonged treatment of micromolar concentration of Abeta42 resulted in slight inhibition on redox and esterase activity. The early Abeta42-induced vacuolated morphology and later chronic cytotoxic effect in neuronal cell might be linked to the chronic neurodegeneration caused by the accumulation of Abeta42 in AD patients' brain.     

Effect of lysine-28 side-chain acetylation on the nanomechanical behavior of alzheimer amyloid beta25-35 fibrils

Amyloid fibrils are self-associating filamentous structures formed from the 39- to 42-residue-long amyloid beta peptide (Abeta peptide). The deposition of Abeta fibrils is one of the most important factors in the pathogenesis of Alzheimer's disease. Abeta25-35 is a fibril-forming peptide that is thought to represent the biologically active, toxic form of the full-length Abeta peptide. We have recently shown that beta sheets can be mechanically unzipped from the fibril surface with constant forces in a reversible transition, and the unzipping forces differ in fibrils composed of different peptides. In the present work, we explored the effect of epsilon-amino acetylation of the Lys28 residue on the magnitude of the unzipping force of Abeta25-35 fibrils. Although the gross structure of the Lys28-acetylated (Abeta25-35_K28Ac) and wild-type Abeta25-35 (Abeta25-35wt) fibrils were similar, as revealed by atomic force microscopy, the fundamental unzipping forces were significantly lower for Abeta25-35_K28Ac (20 +/- 4 pN SD) than for Abeta25-35wt (42 +/- 9 pN SD). Simulations based on a simple two-state model suggest that the decreased unzipping forces, caused most likely by steric constraints, are likely due to a destabilized zippered state of the fibril.     

Amyloid beta-protein: monomer structure and early aggregation states of Abeta42 and its Pro19 alloform

The amyloid beta-protein (Abeta) is a seminal neuropathic agent in Alzheimer's disease (AD). Recent evidence points to soluble Abeta oligomers as the probable neurotoxic species. Among the naturally occurring Abeta peptides, the 42-residue form Abeta42 is linked particularly strongly with AD, even though it is produced at approximately 10% of the levels of the more abundant 40-residue form Abeta40. Here, we apply mass spectrometry and ion mobility to the study of Abeta42 and its Pro19 alloform. The Phe19 --> Pro19 substitution blocks fibril formation by [Pro19]Abeta42. Evidence indicates that solution-like structures of Abeta monomers are electrosprayed and characterized. Unfiltered solutions of Abeta42 produce only monomers and large oligomers, whereas [Pro19]Abeta42 solutions produce abundant monomers, dimers, trimers, and tetramers but no large oligomers. When passed through a 10,000 amu filter and immediately sampled, Abeta42 solutions produce monomers, dimers, tetramers, hexamers, and an aggregate of two hexamers that may be the first step in protofibril formation. These results are consistent with recently published photochemical cross-linking data and lend support to recent aggregation mechanisms proposed by Bitan, Teplow, and co-workers [J. Biol. Chem. 2003, 278, 34882-34889].     

Conformational restriction via cyclization in beta-amyloid peptide Abeta(1-28) leads to an inhibitor of Abeta(1-28) amyloidogenesis and cytotoxicity

The aggregation process of beta-amyloid peptide Abeta into amyloid is strongly associated with the pathology of Alzheimer's disease (AD). Aggregation may involve a transition of an alpha helix in Abeta(1-28) into beta sheets and interactions between residues 18-20 of the "Abeta amyloid core." We applied an i, i+4 cyclic conformational constraint to the Abeta amyloid core and devised side chain-to-side chain lactam-bridged cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28). In contrast to Abeta(1-28) and [Lys(17), Asp(21)]Abeta(1-28), cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28) was not able to form beta sheets and cytotoxic amyloid aggregates. Cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28) was able to interact with Abeta(1-28) and to inhibit amyloid formation and cytotoxicity. Cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28) also interacted with Abeta(1-40) and interfered with its amyloidogenesis. Cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28) or similarly constrained Abeta sequences may find therapeutic and diagnostic applications in AD.     

On-line high-performance liquid chromatography/mass spectrometric investigation of amyloid-beta peptide variants found in Alzheimer's disease

Abeta peptides are the major components of amyloid deposits in Alzheimer's disease. The presence of N-terminally truncated Abeta variants in amyloid may be a critical factor in Alzheimer's disease pathogenesis. These Abeta variants are less soluble and more amyloidogenic than full-length Abeta, making their separation, purification and identification difficult. High-performance liquid chromatography (HPLC) at elevated temperatures, coupled to electrospray ionization (ES) mass spectrometry (MS), enables rapid separation and identification of N-terminally truncated Abeta variants. This methodology provides a potential tool for exploring the importance of these Abeta variants in both the pathogenesis and diagnosis of Alzheimer's disease.     

Why Is the C-terminus of Abeta(1-42) more unfolded than that of Abeta(1-40)? Clues from hydrophobic interaction

Abeta(1-40) and Abeta(1-42) are the main forms of amyloid beta (Abeta) peptides in the brain of Alzheimer's patients; however, the latter possesses much stronger aggregation and deposition propensity than the former, which is partially attributed to the more unfolded C-terminus of Abeta(1-42) than that of Abeta(1-40). To explore the physical basis underlying the different dynamic behaviors of both Abeta peptides, parallel molecular dynamics (MD) simulations on Abeta(1-40) and Abeta(1-42) were performed to investigate their thermal unfolding processes. It is revealed that the addition of residues 41 and 42 in Abeta(1-42) disrupts the C-terminal hydrophobic core, which triggers the unraveling of the C-terminal helix of Abeta(1-42). This conclusion is supported by the MD simulation on the I41A mutant of Abeta(1-42), in which the C-terminal helix possesses relatively higher conformational stability than that of wild type Abeta(1-42) owing to the change in hydrophobic interaction patterns.     

A molecular switch in amyloid assembly: Met35 and amyloid beta-protein oligomerization

Aberrant protein oligomerization is an important pathogenetic process in vivo. In Alzheimer's disease (AD), the amyloid beta-protein (Abeta) forms neurotoxic oligomers. The predominant in vivo Abeta alloforms, Abeta40 and Abeta42, have distinct oligomerization pathways. Abeta42 monomers oligomerize into pentamer/hexamer units (paranuclei) which self-associate to form larger oligomers. Abeta40 does not form these paranuclei, a fact which may explain the particularly strong linkage of Abeta42 with AD. Here, we sought to determine the structural elements controlling paranucleus formation as a first step toward the development of strategies for treating AD. Because oxidation of Met(35) is associated with altered Abeta assembly, we examined the role of Met(35) in controlling Abeta oligomerization. Oxidation of Met(35) in Abeta42 blocked paranucleus formation and produced oligomers indistinguishable in size and morphology from those produced by Abeta40. Systematic structural alterations of the C(gamma)(35)-substituent group revealed that its electronic nature, rather than its size (van der Waals volume), was the factor controlling oligomerization pathway choice. Preventing assembly of toxic Abeta42 paranuclei through selective oxidation of Met(35) thus represents a potential therapeutic approach for AD.     

Formation and stabilization model of the 42-mer Abeta radical: implications for the long-lasting oxidative stress in Alzheimer's disease

Amyloid fibrils mainly consist of 40-mer and 42-mer peptides (Abeta40, Abeta42). Abeta42 is believed to play a crucial role in the pathogenesis of Alzheimer's disease because its aggregative ability and neurotoxicity are considerably greater than those of Abeta40. The neurotoxicity of Abeta peptides involving the generation of free radicals is closely related to the S-oxidized radical cation of Met-35. However, the cation's origin and mechanism of stabilization remain unclear. Recently, structural models of fibrillar Abeta42 and Abeta40 based on systematic proline replacement have been proposed by our group [Morimoto, A.; et al. J. Biol. Chem. 2004, 279, 52781] and Wetzel's group [Williams, A. D.; et al. J. Mol. Biol. 2004, 335, 833], respectively. A major difference between these models is that our model of Abeta42 has a C-terminal beta-sheet region. Our biophysical study on Abeta42 using electron spin resonance (ESR) suggests that the S-oxidized radical cation of Met-35 could be generated by the reduction of the tyrosyl radical at Tyr-10 through a turn structure at positions 22 and 23, and stabilized by a C-terminal carboxylate anion through an intramolecular beta-sheet at positions 35-37 and 40-42 to form a C-terminal core that would lead to aggregation. A time-course analysis of the generation of radicals using ESR suggests that stabilization of the radicals by aggregation might be a main reason for the long-lasting oxidative stress of Abeta42. In contrast, the S-oxidized radical cation of Abeta40 is too short-lived to induce potent neurotoxicity because no such stabilization of radicals occurs in Abeta40.     

Electrophoretic separation of amyloid beta peptides in plasma

In this prospective study, for the first time we have separated and quantified amyloid beta (Abeta) peptides in the plasma of patients with Alzheimer's disease (AD, n = 8) and age- and environment-matched healthy controls (n = 9) with urea-based Abeta-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/immunoblot. In addition to the Abeta peptides 1-37/38/39/40/42, which we recently identified as regular constituents of human cerebrospinal fluid (CSF), we have observed a novel electrophoretic band migrating slightly cathodically to Abeta1-42. Since a standard peptide with the amino acid sequence Abeta2-40 migrates in the same position, we hypothesize that this plasma-specific band may correspond to Abeta2-40. The concentration of Abeta peptides in the plasma has been approximately 100-fold lower compared to the CSF. Interestingly, the concentration of the two shortest peptides and the longest one of these considered here (i.e., Abeta1-37/38/42) have increased significantly when the samples have been frozen at -80 degrees C before immunoprecipitation, while the 'middle-length' peptides (i.e., Abeta1-39/40) have not been affected by this procedure. We have not observed significant differences of the Abeta peptides concentrations between AD and control subjects. Our method can be used to investigate the significance of plasma Abeta peptides in neurodegenerative disorders, and to monitor the efficiency of drugs with beta/gamma-secretase inhibitory potency.     

Surface-induced aggregation of beta amyloid peptide by co-substituted alkanethiol monolayers supported on gold

The primary pathological characteristic of Alzheimer's disease is the presence in the brain of self-assembled beta amyloid (Abeta) protein fibrils, consisting of 35-43 amino acid residues. The toxicity of the aggregated protein structures has previously been proposed to be related to the interaction of Abeta fibrils with neuronal membranes (phospholipid bilayers). Here, surfaces consisting of self-assembled alkanethiol monolayers with different end groups--supported on Au--are used to test the effect of surface chemistry on the structure and morphology of aggregates formed from an active fragment (Abeta10-35) of the Abeta peptide. The influence of monolayer nature (end group) on the aggregation of Abeta10-35 was examined using reflection-absorption infrared spectroscopy (RAIRS) and scanning force microscopy (SFM). Evaluation of the SFM and RAIRS data reveals the presence of Abeta10-35 protein on the various monolayer surfaces, with the surface protein possessing predominantly beta-sheet and random-coil conformations. Time-dependent studies of the extent of Abeta10-35 aggregation and deposition on the various surfaces and the effect of the monolayers on seeding of Abeta10-35 aggregates in solution are also discussed.     

Inhibition of amyloid beta protein fibrillation by polymeric nanoparticles

Copolymeric NiPAM:BAM nanoparticles of varying hydrophobicity were found to retard fibrillation of the Alzheimer's disease-associated amyloid beta protein (Abeta). We found that these nanoparticles affect mainly the nucleation step of Abeta fibrillation. The elongation step is largely unaffected by the particles, and once the Abeta is nucleated, the fibrillation process occurs with the same rate as in the absence of nanoparticles. The extension of the lag phase for fibrillation of Abeta is strongly dependent on both the amount and surface character of the nanoparticles. Surface plasmon resonance studies show that Abeta binds to the nanoparticles and provide rate and equilibrium constants for the interaction. Numerical analysis of the kinetic data for fibrillation suggests that binding of monomeric Abeta and prefibrillar oligomers to the nanoparticles prevents fibrillation. Moreover, we find that fibrillation of Abeta initiated in the absence of nanoparticles can be reversed by addition of nanoparticles up to a particular time point before mature fibrils appear.     

Ex situ atomic force microscopy analysis of beta-amyloid self-assembly and deposition on a synthetic template

The beta-amyloid (Abeta) deposition, which is the conversion of soluble Abeta peptides to insoluble plaques on a surface, is an essential pathological process in Alzheimer's disease (AD). The identification and characterization of possible environmental factors that may influence amyloid deposition in vivo are important to unveil the underlying etiology of AD. According to the amyloid cascade hypothesis, diffuse plaques are initial and visual deposits in the early event of AD, leading to amyloid plaques. To study amyloid deposition and growth in vitro, we prepared a synthetic template by immobilizing Abeta seeds on an N-hydroxysuccinimide ester-activated solid surface. According to our analysis with an ex situ atomic force microscope, the formation of amyloid plaque-like aggregates was mediated by the interaction between Abeta in a solution and on a synthetic template, suggesting that Abeta oligomers function well as seeds for amyloid deposition. It was observed that insoluble amyloid aggregates formed on the template surface serve as a sink of soluble Abeta in a solution as well as mediate the formation of intermediates in the pathway of amyloid fibrillization in a solution. Relative seeding efficiencies of fresh monomers, oligomers, and fully grown fibrils were analyzed by measuring the deposited plaque volume and its height distribution through atomic force microscopy. The result revealed that oligomeric forms of Abeta act more efficiently as seeds than monomers or fibrils do. Fluorescence spectroscopy with thioflavin T confirmed that amyloid aggregate formation proceeds in a concentration-dependent manner. Analysis with Fourier transform infrared spectroscopy indicated a progressive transition of soluble Abeta42 monomer to amyloid fibrils having antiparallel beta-sheet structure on the template. Furthermore, studies on the interaction between Abeta40 and 42, two major variants of Abeta derived from the amyloid precursor protein, showed that amyloid aggregate formation on the surface was accelerated further by the homogeneous association of soluble Abeta42 onto Abeta42 seeds than by other combinations. A slightly acidic condition was found to be unfavorable for amyloid formation. This study gives insight into understanding the effects of environmental factors on amyloid formation via the use of a synthetic template system.     

Micellization of surfactin and its effect on the aggregate conformation of amyloid beta(1-40)

The aggregation of amyloid beta-peptide (Abeta(1-40)) into fibrils is a key pathological process associated with Alzheimer's disease. This work has investigated the micellization process of biosurfactant surfactin and its effect on the aggregation behavior of Abeta(1-40). The results show that surfactin has strong self-assembly ability to form micelles and the micelles tend to form larger aggregates. Surfactin adopts a beta-turn conformation at low micelle concentration but a beta-sheet conformation at high micelle concentration. The effect of surfactin on the Abeta(1-40) aggregation behavior exhibits a strong concentration-dependent fashion. Below the critical micelle concentration of surfactin, the electrostatic binding of surfactin monomers on Abeta(1-40) causes Abeta(1-40) molecules to unfold. Assisted by the hydrophobic interaction among surfactin monomers on the Abeta(1-40) chain, the conformation of Abeta(1-40) transfers to the beta-sheet structure, which promotes the formation of fibrils. At low surfactin micelle concentration, besides the electrostatic force and hydrophobic interaction, hydrogen bonds formed between surfactin micelles and adjacent Abeta(1-40) peptide chains may promote the ordered organization of these Abeta(1-40) peptide chains, thus leading to the formation of beta-sheets and fibrils to a great extent. At high surfactin micelle concentration, the separating of Abeta(1-40) chains by the excessive surfactin micelles and the aggregation of the complexes of Abeta(1-40) with surfactin micelles inhibit the formation of beta-sheets and fibrils.     

Thermodynamic studies on the interaction of antibodies with beta-amyloid peptide

Antibodies against beta-amyloid peptides (Abetas) are considered an important therapeutic opportunity in Alzheimer's disease. Despite the vast interest in Abeta no thermodynamic data on the interaction of antibodies with Abeta are available as yet. In the present study we use isothermal titration calorimetry (ITC) and surface plasmon resonance to provide a quantitative thermodynamic analysis of the interaction between soluble monomeric Abeta(1-40) and mouse monoclonal antibodies (mAb). Using four different antibodies directed against the N-terminal, middle, and C-terminal Abeta epitopes, we measured the thermodynamic parameters for the binding to Abeta. Each antibody species was found to have two independent and equal binding sites for Abeta with binding constants in the range of 10(7) to 10(8) M(-1). The binding reaction was essentially enthalpy driven with a reaction enthalpy of DeltaH(0)(Abeta) approximately -19 to -8 kcal/mol, indicating the formation of tight complexes. The loss in conformational freedom was supported by negative values for the reaction entropy DeltaS(0)(Abeta). We also measured the heat capacity change of the 1mAb:2Abeta reaction. DeltaC(0)(p, abeta) was large and negative but could not be explained exclusively by the hydrophobic effect. The free energy of binding was found to be linearly correlated with the size of the epitope.     

The N-domain of angiotensin-converting enzyme specifically hydrolyzes the Arg-5-His-6 bond of Alzheimer's Abeta-(1-16) peptide and its isoAsp-7 analogue with different efficiency as evidenced by quantitative matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Chronic imbalance between production and degradation of the human amyloid-beta peptide (Abeta) is assumed to play an important role in pathogenesis of Alzheimer's disease (AD). Post-translational modifications of Abeta could influence its interactions with specifically cleaving proteases and, therefore, perturb the Abeta homeostasis. The angiotensin-converting enzyme (ACE) was previously shown to degrade non-modified Abeta in vitro and in cells. In the presented work, we investigated the effect of isomerization of Asp-7, a common non-enzymatic age-related modification found in AD-associated Abeta species, on hydrolysis of Abeta by ACE. Two synthetic peptides corresponding to the Abeta region 1-16 with either Asp or isoAsp residues in position 7 were examined as monomeric soluble substrates for the N- as well as for the C-domain of ACE. The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) coupled with the (18)O-labeled internal standard approach has allowed us to show that (i) the N-domain of ACE (N-ACE), but not the C-domain, selectively cleaves the Arg-5-His-6 bond in both peptides, and that (ii) N-ACE hydrolyzes the isoAsp-7 analogue more efficiently than the non-modified one. Our results demonstrate a new endopeptidase activity of N-ACE as well as high preference of the domain to recognize and hydrolyze the isomerized Abeta species that were earlier suggested to promote AD pathogenesis. The results suggest the need for further analysis of biological effects of isomerized Abeta and its interaction with ACE in AD pathogenesis.     

Stoichiometric inhibition of amyloid beta-protein aggregation with peptides containing alternating alpha,alpha-disubstituted amino acids

We have prepared two peptides based on the hydrophobic core (Lys-Leu-Val-Phe-Phe) of amyloid beta-protein (Abeta) that contain alpha,alpha-disubstituted amino acids at alternating positions, but differ in the positioning of the oligolysine chain (AMY-1, C-terminus; AMY-2, N-terminus). We have studied the effects of AMY-1 and AMY-2 on the aggregation of Abeta and find that, at stoichiometric concentrations, both peptides completely stop Abeta fibril growth. Equimolar mixtures of AMY-1 and Abeta form only globular aggregates as imaged by scanning force microscopy and transmission electron microscopy. These samples show no signs of protofibrillar or fibrillar material even after prolonged periods of time (4.5 months). Also, 10 mol % of AMY-1 prevents Abeta self-assembly for long periods of time; aged samples (4.5 months) show only a few protofibrillar or fibrillar aggregates. Circular dichroism spectroscopy of equimolar mixtures of AMY-1 and Abeta show that the secondary structure of the mixture changes over time and progresses to a predominantly beta-sheet structure, which is consistent with the design of these inhibitors preferring a sheet-like conformation. Changing the position of the charged tail on the peptide, AMY-2 interacts with Abeta differently in that equimolar mixtures form large ( approximately 1 mum) globular aggregates which do not progress to fibrils, but precipitate out of solution. The differences in the aggregation mediated by the two peptides is discussed in terms of a model where the inhibitors act as cosurfactants that interfere with the native ability of Abeta to self-assemble by disrupting hydrophobic interactions either at the C-terminus or N-terminus of Abeta.     

Design of peptides that form amyloid-like fibrils capturing amyloid beta1-42 peptides

Amyloid beta-peptide (Abeta) plays a critical role in Alzheimer's disease (AD). The monomeric state of Abeta can self-assemble into oligomers, protofibrils, and amyloid fibrils. Since the fibrils and soluble oligomers are believed to be responsible for AD, the construction of molecules capable of capturing these species could prove valuable as a means of detecting these potentially toxic species and of providing information pertinent for designing drugs effective against AD. To this aim, we have designed short peptides with various hydrophobicities based on the sequence of Abeta14-23, which is a critical region for amyloid fibril formation. The binding of the designed peptides to Abeta and the amplification of the formation of peptide amyloid-like fibrils coassembled with Abeta are elucidated. A fluorescence assay utilizing thioflavin T, known to bind specifically to amyloid fibrils, revealed that two designed peptides (LF and VF, with the leucine and valine residues, respectively, in the hydrophobic core region) could form amyloid-like fibrils effectively by using mature Abeta1-42 fibrils as nuclei. Peptide LF also coassembled with soluble Abeta oligomers into peptide fibrils. Various analyses, including immunostaining with gold nanoparticles, enzyme-linked immunosorbent assays, and size-exclusion chromatography, confirmed that the LF and VF peptides formed amyloid-like fibrils by capturing and incorporating Abeta1-42 aggregates into their peptide fibrils. In this system, small amounts of mature Abeta1-42 fibrils or soluble oligomers could be transformed into peptide fibrils and detected by amplifying the amyloid-like fibrils with the designed peptides.