Solid‐Phase Synthesis and Characterization of N‐Terminally Elongated Aβ−3–x‐Peptides

In addition to the prototypic amyloid‐β (Aβ) peptides Aβ_1–40 and Aβ_1–42, several Aβ variants differing in their amino and carboxy termini have been described. Synthetic availability of an Aβ variant is often the key to study its role under physiological or pathological conditions. Herein, we report a protocol for the efficient solid‐phase peptide synthesis of the N‐terminally elongated Aβ‐peptides Aβ_?3–38, Aβ_?3–40, and Aβ_?3–42. Biophysical characterization by NMR spectroscopy, CD spectroscopy, an aggregation assay, and electron microscopy revealed that all three peptides were prone to aggregation into amyloid fibrils. Immunoprecipitation, followed by mass spectrometry, indicated that Aβ_?3–38 and Aβ_?3–40 are generated by transfected cells even in the presence of a tripartite β‐site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor. The elongated Aβ peptides starting at Val(?3) can be separated from N‐terminally‐truncated Aβ forms by high‐resolution isoelectric‐focusing techniques, despite virtually identical isoelectric points. The synthetic Aβ variants and the methods presented here are providing tools to advance our understanding of the potential roles of N‐terminally elongated Aβ variants in Alzheimer's disease.     

Spectroscopic Evidence for Polymorphic Aggregates Formed by Amyloid‐β Fragments

Understanding the structure of amyloid‐β (Aβ) aggregates is a key step towards elucidating the pathology of Alzheimer’s disease. In this work, three fragments of the Aβ_1–42 protein, Aβ_1–25 (DAEFRHDSGYEVHHQKLVFFAEDVG), Aβ_25–35 (GSNKGAIIGLM), and Aβ_33–42 (GLMVGGVVIA), were synthesized, and their aggregated structures were examined by linear infrared spectroscopy in the amide‐I (mainly the C?O stretching) region. The structures of the formed aggregates were found to be both sequence and pH dependent. The results suggest that instead of forming matured fibrils, as in the case of full‐length Aβ_1–42, both Aβ_1–25 and Aβ_33–42 form a mixture of threadlike β‐sheet fibril, soluble β‐sheet oligomer, and random coil structures. The β‐sheet conformations were found to be mainly antiparallel for the former and both parallel and antiparallel for the latter. However, the Aβ_25–35 fragment was found to form assembled fibrils containing predominantly parallel β‐sheets. The conformation and morphology of the aggregates were also confirmed by circular dichroism measurements and transmission electron microscopy. Factors influencing the structures of the aggregates formed by the Aβ fragments were discussed.     

Direct Nanospectroscopic Verification of the Amyloid Aggregation Pathway

The aggregation pathways of neurodegenerative peptides determine the disease etiology, and their better understanding can lead to strategies for early disease treatment. Previous research has allowed modelling of hypothetic aggregation pathways. However, their direct experimental observation has been elusive owing to methodological limitations. Herein, we demonstrate that nanoscale chemical mapping by tip‐enhanced Raman spectroscopy of single amyloid fibrils at various stages of aggregation captures the fibril formation process. We identify changes in TERS/Raman marker bands for Aβ_1‐42, including the amide III band (above 1255 cm^?1 for turns/random coil and below 1255 cm^?1 for β‐sheet conformation). The spatial distribution of β‐sheets in aggregates is determined, allowing verification of a particular fibrillogenesis pathway, starting from aggregation of monomers to meta‐stable oligomers, which then rearrange to ordered β‐sheets, already at the oligomeric or protofibrillar stage.     

Observation of metastable Aβ amyloid protofibrils by atomic force microscopy

Background: Brain amyloid plaque, a diagnostic feature of Alzheimer's disease (AD), contains an insoluble fibrillar core that is composed primarily of variants of the β-amyloid protein (Aβ). As Aβ amyloid fibrils may initiate neurodegeneration, the inhibition of fibril formation is a possible therapeutic strategy. Very little is known about the early steps of the process, however.Results: Atomic force microscopy was used to follow amyloid fibril formation in vitro by the Aβ variants Aβ1-40 and Aβ1-42. Both variants first form small ordered aggregates that grow slowly and then rapidly disappear, while prototypical amyloid fibrils of two discrete morphologies appear. Aβ1-42 aggregates much more rapidly than Aβ1-40, which is consistent with its connection to early-onset AD. We propose that the metastable intermediate species be called Aβ amyloid protofibrils.Conclusions: Aβ protofibrils are likely to be intermediates in the in vitro assembly of Aβ amyloid fibrils, but their in vivo role has yet to be determined. Numerous reports of a nonfibrillar form of Aβ aggregate in the brains of individuals who are predisposed to AD suggest the existence of a precursor form, possibly the protofibril. Thus, stabilization of Aβ protofibrils may be a useful therapeutic strategy.     

Studies on the cross‐interaction between hIAPP and Aβ25–35 and the aggregation process in binary mixture by electrospray ionization‐ion mobility‐mass spectrometry

A wealth of epidemiological evidence indicates a strong link between type 2 diabetes (T2D) and Alzheimer's disease (AD). The fiber deposition with cross‐β‐sheet structure formed by self‐aggregation and misfolding of amyloidogenic peptides is a common hallmark of both diseases. For the patients with T2D, the fibrils are mainly found in the islets of Langerhans that results from the accumulation of human islet amyloid polypeptide (hIAPP). The major component of aggregates located in the brain of AD patients is amyloid‐β (Aβ). Many biophysical and physiological properties are shared by hIAPP and Aβ, and both peptides show similar cytotoxic mechanisms. Therefore, it is meaningful to investigate the possible cross‐interactions of hIAPP and Aβ in both diseases. In this article, the segment 25–35 of Aβ was selected because Aβ_25–35 was a core region in the process of amyloid formation and showed similar aggregation tendency and toxicity with full‐length Aβ. The electrospray ionization‐ion mobility‐mass spectrometry analysis and thioflavin T fluorescence kinetic analysis combined with transmission electron microscopy were used to explore the effects of the coexistence of Aβ_25–35 and hIAPP on the self‐aggregation of both peptides and whether there was co‐assembly in fibrillation. The results indicated that the aggregation of hIAPP and Aβ_25–35 had two nucleation stages in the binary mixtures. hIAPP and Aβ_25–35 had a high binding affinity and a series of hetero‐oligomers formed in the mixtures of hIAPP and Aβ_25–35 in the early stage. The cross‐reaction between hIAPP monomers and Aβ_25–35 monomers as well as a little of oligomers during primary nucleation stage could accelerate the aggregation of Aβ_25–35. However, owing to the obvious difference in aggregation ability between hIAPP and Aβ_25–35, this cross‐interaction had no significant impact on the self‐assembly of hIAPP. Our study may offer a better understanding for exploring the molecular mechanism of the association between AD and T2D observed in clinical and epidemiological studies and developing therapeutic strategies against amyloid diseases.     

Amyloid formation from an α-helix peptide bundle is seeded by 3(10)-helix aggregates

Transformation of proteins and peptides to fibrillar aggregates rich in β sheets underlies many diseases, but mechanistic details of these structural transitions are poorly understood. To simulate aggregation, four equivalents of a water‐soluble, α‐helical (65 %) amphipathic peptide (AEQLLQEAEQLLQEL) were assembled in parallel on an oxazole‐containing macrocyclic scaffold. The resulting 4α‐helix bundle is monomeric and even more α helical (85 %), but it is also unstable at pH 4 and undergoes concentration‐dependent conversion to β‐sheet aggregates and amyloid fibrils. Fibrils twist and grow with time, remaining flexible like rope (>1 μm long, 5–50 nm wide) with multiple strings (2 nm), before ageing to matted fibers. At pH 7 the fibrils revert back to soluble monomeric 4α‐helix bundles. During α→β folding we were able to detect soluble 3₁₀ helices in solution by using 2D‐NMR, CD and FTIR spectroscopy. This intermediate satisfies the need for peptide elongation, from the compressed α helix to the fully extended β strand/sheet, and is driven here by 3₁₀‐helix aggregation triggered in this case by template‐promoted helical bundling and by hydrogen‐bonding glutamic acid side chains. A mechanism involving α?α₄?(3₁₀)₄?(3₁₀)_n?(β)_n?m(β)_n equilibria is plausible for this peptide and also for peptides lacking hydrogen‐bonding side chains, with unfavourable equilibria slowing the α→β conversion.     

Amyloid–β peptides time-dependent structural modifications: AFM and voltammetric characterization

The human amyloid beta (Aβ) peptides, Aβ_1-40 and Aβ_1-42, structural modifications, from soluble monomers to fully formed fibrils through intermediate structures, were investigated, and the results were compared with those obtained for the inverse Aβ_40-1 and Aβ_42-1, mutant Aβ_1-40Phe¹⁰ and Aβ_1-40Nle³⁵, and rat Aβ_1-40Rat peptide sequences. The aggregation was followed at a slow rate, in chloride free media and room temperature, and revealed to be a sequence-structure process, dependent on the physicochemical properties of each Aβ peptide isoforms, and occurring at different rates and by different pathways. The fibrilization process was investigated by atomic force microscopy (AFM), via changes in the adsorption morphology from: (i) initially random coiled structures of ∼0.6 nm height, corresponding to the Aβ peptide monomers in random coil or in α-helix conformations, to (ii) aggregates and protofibrils of 1.5–6.0 nm height and (iii) two types of fibrils, corresponding to the Aβ peptide in a β-sheet configuration. The reactivity of the carbon electrode surface was considered. The hydrophobic surface induced rapid changes of the Aβ peptide conformations, and differences between the adsorbed fibrils, formed at the carbon surface (beaded, thin, <2.0 nm height) or in solution (long, smooth, thick, >2.0 nm height), were detected. Differential pulse voltammetry showed that, according to their primary structure, the Aβ peptides undergo oxidation in one or two steps, the first step corresponding to the tyrosine amino acids oxidation, and the second one to the histidine and methionine amino acids oxidation. The fibrilization process was electrochemically detected via the decrease of the Aβ peptide oxidation peak currents that occurred in a time dependent manner.     

Inhibiting and Reversing Amyloid‐β Peptide (1–40) Fibril Formation with Gramicidin S and Engineered Analogues

In Alzheimer’s disease, amyloid‐β (Aβ) peptides aggregate into extracellular fibrillar deposits. Although these deposits may not be the prime cause of the neurodegeneration that characterizes this disease, inhibition or dissolution of amyloid fibril formation by Aβ peptides is likely to affect its development. ThT fluorescence measurements and AFM images showed that the natural antibiotic gramicidin S significantly inhibited Aβ amyloid formation in vitro and could dissolve amyloids that had formed in the absence of the antibiotic. In silico docking suggested that gramicidin S, a cyclic decapeptide that adopts a β‐sheet conformation, binds to the Aβ peptide hairpin‐stacked fibril through β‐sheet interactions. This may explain why gramicidin S reduces fibril formation. Analogues of gramicidin S were also tested. An analogue with a potency that was four‐times higher than that of the natural product was identified.     

The Aggregation Pattern of Aβ1–40 is Altered by the Presence of N-Truncated Aβ4–40 and/or CuII in a Similar Way through Ionic Interactions

Alzheimer's disease (AD) is one of the most common of the multifactorial diseases and is characterized by a range of abnormal molecular processes, such as the accumulation of extracellular plaques containing the amyloid-β (Aβ) peptides and dyshomeostasis of copper in the brain. In this study, we have investigated the effect of Cu^II on the aggregation of Aβ_1–40 and Aβ_4–40, representing the two most prevalent families of Aβ peptides, that is, the full length and N-truncated peptides. Both families are similarly abundant in healthy and AD brains. For either of the studied peptides, substoichiometric Cu^II concentrations accelerated aggregation, whereas superstoichiometric Cu^II inhibited fibril formation, likely by stabilizing the oligomers. The addition of either Aβ_4–40 or substoichiometric Cu^II affected the aggregation profile of Aβ_1–40, by yielding shorter and thicker fibrils; amorphous aggregates were formed in the presence of a molar excess of Cu^II. The similarity of these two effects can be attributed to the increase in the positive charge on the Aβ N terminus, caused both by Cu^II complexation and N truncation at position 4. Our findings provide a better understanding of the biological Aβ aggregation process as these two Aβ species and Cu^II coexist and interact under physiological conditions.     

A Hexameric Peptide Barrel as Building Block of Amyloid‐β Protofibrils

Oligomeric and protofibrillar aggregates formed by the amyloid‐β peptide (Aβ) are believed to be involved in the pathology of Alzheimer’s disease. Central to Alzheimer pathology is also the fact that the longer Aβ₄₂ peptide is more prone to aggregation than the more prevalent Aβ₄₀. Detailed structural studies of Aβ oligomers and protofibrils have been impeded by aggregate heterogeneity and instability. We previously engineered a variant of Aβ that forms stable protofibrils and here we use solid‐state NMR spectroscopy and molecular modeling to derive a structural model of these. NMR data are consistent with packing of residues 16 to 42 of Aβ protomers into hexameric barrel‐like oligomers within the protofibril. The core of the oligomers consists of all residues of the central and C‐terminal hydrophobic regions of Aβ, and hairpin loops extend from the core. The model accounts for why Aβ₄₂ forms oligomers and protofibrils more easily than Aβ₄₀.     

Natural Compounds against Alzheimer’s Disease: Molecular Recognition of Aβ1–42 Peptide by Salvia sclareoides Extract and its Major Component,Rosmarinic Acid,as Investigated by NMR

Amyloid peptides, Aβ1–40 and Aβ1–42, represent major molecular targets to develop potential drugs and diagnostic tools for Alzheimer’s Disease (AD). In fact, oligomeric and fibrillar aggregates generated by these peptides are amongst the principal components of amyloid plaques found post mortem in patients suffering from AD. Rosmarinic acid has been demonstrated to be effective in preventing the aggregation of amyloid peptides in vitro and to delay the progression of the disease in animal models. Nevertheless, no information is available about its molecular mechanism of action. Herein, we report the NMR characterization of the interaction of Salvia sclareoides extract and that of its major component, rosmarinic acid, with Aβ1–42 peptide, whose oligomers have been described as the most toxic Aβ species in vivo. Our data shed light on the structural determinants of rosmarinic acid–Aβ1–42 oligomers interaction, thus allowing the elucidation of its mechanism of action. They also provide important information for the rational design of new compounds with higher affinity for Aβ peptides to generate new anti‐amyloidogenic molecules and/or molecular tools for the specific targeting of amyloid aggregates in vivo. In addition, we identified methyl caffeate, another natural compound present in different plants and human diet, as a good ligand of Aβ1–42 oligomers, which also shows anti‐amyloidogenic activity. Finally, we demonstrated the possibility to exploit STD‐NMR and trNOESY experiments to screen extracts from natural sources for the presence of Aβ peptide ligands.     

The Molecular Structure of Alzheimer β‐Amyloid Fibrils Formed in the Presence of Phospholipid Vesicles

β‐amyloid (Aβ) fibrils are the major species involved in Alzheimer’s disease (AD). An atomic‐resolution molecular structure of Aβ40 fibrils formed in the presence of lipid vesicles was obtained by using magic angle spinning (MAS) solid‐state NMR spectroscopy. The fibril structures formed in the presence of the lipid vesicles are remarkably different from those formed in solution. These results provide insights into the molecular mechanism of Aβ aggregation in the presence of lipid vesicles.     

Application of an Electrochemical Method to Evaluation of Amyloid‐β Aggregation Inhibitors: Testing the RGKLVFFGR‐NH2 Peptide Antiaggregant

The aggregation of amyloid‐β peptide (Aβ) is believed to play a crucial role in the Alzheimer's disease (AD) pathogenesis and is considered as a therapeutic target for treating AD. The Aβ electrooxidation via a Tyr‐10 residue, sensitive to a depletion of a pool of Aβ monomers and oligomers in the course of Aβ aggregation, may be employed for testing natural and synthetic organic compounds (including short peptides) potentially able to inhibit the pathological Aβ aggregation (antiaggregants). In the present work, using the known peptide antiaggregant RGKLVFFGR‐NH₂ (OR2) and its scrambled variant KGLRVGFRF‐NH₂ as a control, we demonstrate that the electrochemical method based on electrooxidation of an Aβ42 Tyr‐10 residue, when combined with methods allowing for the evaluation of the Aβ42 aggregate structure and size, can provide essential information regarding the antiaggregant impact on Aβ42 aggregation. Electrochemical measurements were performed using square wave voltammetry on carbon screen printed electrodes whereas the Aβ42 aggregate structure and size were analyzed by means of the conventional thioflavin T (ThT) based fluorescence assay and dynamic light scattering. While inhibiting Aβ42 fibrillation as manifested by the unchanged level of ThT fluorescence, the OR2 peptide antiaggregant had no effect on the decrease of Aβ42 electrooxidation current in the course of Aβ42 aggregation. These observations suggest that OR2 does not stop the aggregation but redirects it into a pathway where amorphous rather than fibrillar aggregates are formed. Hence, the direct electrochemistry appears to offer a simple and cost‐effective approach for probing potential peptide antiaggregants, which is complementary to methods based on detecting Aβ aggregates.     

Molecular Structure of Amyloid Fibrils Formed by Residues 127 to 147 of the Human Prion Protein

Amyloid fibrils are filamentous and insoluble forms of peptides or proteins. Proline has long been considered to be incompatible with the cross‐β structural motif of amyloid fibrils. On the basis of solid‐state NMR spectroscopy data, we present a structural model of an in‐register parallel β sheet for the amyloid fibrils formed from a human prion protein fragment, huPrP_127–47. We have developed a simple solid‐state NMR spectroscopy technique to identify solvent‐protected backbone amide protons in a H/D exchange experiment without disaggregating the amyloid fibrils, from which we find that proline residue P₁₃₇ does not disrupt the β‐sheet structure from G₁₂₇ to G₁₄₂. We suggest that the resultant kink at P₁₃₇ generates a twist between adjacent peptide strands to maintain hydrogen bonding in the β‐sheet regions flanking the P₁₃₇ residue. Although proline can be well integrated into the cross‐β structure of amyloid fibrils, the kink formed at the position of the proline residue will considerably weaken the hydrogen bonding between the neighboring strands, especially when the mutation site is near the central region of a β sheet.     

Understanding Amyloid‐β Oligomerization at the Molecular Level: The Role of the Fibril Surface

The aggregation of the amyloid β‐peptide into fibrils is a complex process that involves mechanisms such as primary and secondary nucleation, fibril elongation and fibril fragmentation. Some of these processes generate neurotoxic Aβ oligomers, which are involved in the development of Alzheimer's disease. Recent experimental studies have emphasized the role of the fibril as a catalytic surface for the production of highly toxic oligomers during secondary nucleation. By using molecular dynamics simulations, we show that it is the hydrophobic fibril region that causes the structural changes required for catalyzing the formation of β‐sheet‐rich Aβ1‐42 oligomers on the fibril surface. These results reveal, for the first time, the molecular basis of the secondary nucleation pathway.     

Combined Experimental and Simulation Studies Suggest a Revised Mode of Action of the Anti‐Alzheimer Disease Drug NQ‐Trp

Inhibition of the aggregation of the monomeric peptide β‐amyloid (Aβ) into oligomers is a widely studied therapeutic approach in Alzheimer’s disease (AD). Many small molecules have been reported to work in this way, including 1,4‐naphthoquinon‐2‐yl‐L ‐tryptophan (NQ‐Trp). NQ‐Trp has been reported to inhibit aggregation, to rescue cells from Aβ toxicity, and showed complete phenotypic recovery in an in vivo AD model. In this work we investigated its molecular mechanism by using a combined approach of experimental and theoretical studies, and obtained converging results. NQ‐Trp is a relatively weak inhibitor and the fluorescence data obtained by employing the fluorophore widely used to monitor aggregation into fibrils can be misinterpreted due to the inner filter effect. Simulations and NMR experiments showed that NQ‐Trp has no specific “binding site“‐type interaction with mono‐ and dimeric Aβ, which could explain its low inhibitory efficiency. This suggests that the reported anti‐AD activity of NQ‐Trp‐type molecules in in vivo models has to involve another mechanism. This study has revealed the potential pitfalls in the development of aggregation inhibitors for amyloidogenic peptides, which are of general interest for all the molecules studied in the context of inhibiting the formation of toxic aggregates.     

A Method for Evaluating the Level of Soluble β‐Amyloid(1–40/1–42) in Alzheimer’s Disease Based on the Binding of Gelsolin to β‐Amyloid Peptides

In the present work, a new electrochemical strategy for the sensitive and specific detection of soluble β‐amyloid Aβ_{(1–40/1–42)} peptides in a rat model of Alzheimer’s disease (AD) is described. In contrast to previous antibody‐based methods, β‐amyloid_{(1–40/1–42)} was quantified based on its binding to gelsolin, a secretory protein present in the cerebrospinal fluid (CSF) and plasma. The level of soluble β‐amyloid peptides in the CSF and various brain regions were found with this method to be lower in rats with AD than in normal rats.     

Tip‐Enhanced Raman Spectroscopy to Distinguish Toxic Oligomers from Aβ1–42 Fibrils at the Nanometer Scale

For the first time, natural Aβ_1–42 fibrils (WT) implicated in Alzheimer's disease, as well as two synthetic mutants forming less toxic amyloid fibrils (L34T) and highly toxic oligomers (oG37C), are chemically characterized at the scale of a single structure using tip‐enhanced Raman spectroscopy (TERS). While the proportion of TERS features associated with amino acid residues is similar for the three peptides, a careful examination of amide I and amide III bands allows us to clearly distinguish WT and L34T fibers organized in parallel β‐sheets from the small and more toxic oG37C oligomers organized in anti‐parallel β‐sheets.     

Intramolecular Charge Interactions as a Tool to Control the Coiled‐Coil‐to‐Amyloid Transformation

Under the influence of a changed environment, amyloid‐forming proteins partially unfold and assemble into insoluble β‐sheet rich fibrils. Molecular‐level characterization of these assembly processes has been proven to be very challenging, and for this reason several simplified model systems have been developed over recent years. Herein, we present a series of three de novo designed model peptides that adopt different conformations and aggregate morphologies depending on concentration, pH value, and ionic strength. The design strictly follows the characteristic heptad repeat of the α‐helical coiled‐coil structural motif. In all peptides, three valine residues, known to prefer the β‐sheet conformation, have been incorporated at the solvent‐exposed b, c, and f positions to make the system prone to amyloid formation. Additionally, pH‐controllable intramolecular electrostatic repulsions between equally charged lysine (peptide A) or glutamate (peptide B) residues were introduced along one side of the helical cylinder. The conformational behavior was monitored by circular dichroism spectroscopic analysis and thioflavin T fluorescence, and the resulting aggregates were further characterized by transmission electron microscopy. Whereas uninterrupted α‐helical aggregates are found at neutral pH, Coulomb repulsions between lysine residues in peptide A destabilize the helical conformation at acidic pH values and trigger an assembly into amyloid‐like fibrils. Peptide B features a glutamate‐based switch functionality and exhibits opposite pH‐dependent folding behavior. In this case, α‐helical aggregates are found under acidic conditions, whereas amyloids are formed at neutral pH. To further validate the pH switch concept, peptide C was designed by including serine residues, thus resulting in an equal distribution of charged residues. Surprisingly, amyloid formation is observed at all pH values investigated for peptide C. The results of further investigations into the effect of different salts, however, strongly support the crucial role of intramolecular charge repulsions in the model system presented herein.     

Structural and Material Properties of Amyloid Aβ40/42 Fibrils

In this study, structural and mechanical properties of a series of models of Aβ₄₂ (one‐ and two‐fold) and Aβ₄₀ (two‐ and three‐fold) fibrils have been computed by using all‐atom molecular dynamics simulations. Based on calculations of the twist angle (θ) and periodicity (v=360d/θ), oligomers formed by 20, 11, and 13 monomers were found to be the smallest realistic models of three‐fold Aβ₄₀, one‐fold Aβ₄₂, and two‐fold Aβ₄₂ fibrils, respectively. Our results predict that the Aβ₄₀ fibrils initially exist in two staggered conformations [STAG(+2) and STAG(+1)] and then undergo a [STAG(+2)→STAG(+1)] transformation in a size‐dependent manner. The length of the loop region consisting of the residues 23–29 shrinks with the elongation of both Aβ₄₀ and Aβ₄₂ fibrils. A comparison of the computed potential energy suggests that a two‐fold Aβ₄₀ aggregate is more stable than its three‐fold counterpart, and that Aβ₄₂ oligomers can exist only in one‐fold conformation for aggregates of more than 11 monomers in length. The computed Young′s modulus and yield strengths of 50 GPa and 0.95 GPa, respectively, show that these aggregates possess excellent material properties.