Binding of nonsteroidal anti‐inflammatory drugs to Aβ fibril

Nonsteroidal anti‐inflammatory drugs are considered as potential therapeutic agents against Alzheimer's disease. Using replica exchange molecular dynamics and atomistic implicit solvent model, we studied the mechanisms of binding of naproxen and ibuprofen to the Aβ fibril derived from solid‐state NMR measurements. The binding temperature of naproxen is found to be almost 40 K higher than of ibuprofen implicating higher binding affinity of naproxen. The key factor, which enhances naproxen binding, is strong interactions between ligands bound to the surface of the fibril. The naphthalene ring in naproxen appears to provide a dominant contribution to ligand‐ligand interactions. In contrast, ligand‐fibril interactions cannot explain differences in the binding affinities of naproxen and ibuprofen. The concave fibril edge with the groove is identified as the primary binding location for both ligands. We show that confinement of the ligands to the groove facilitates ligand‐ligand interactions that lowers the energy of the ligands bound to the concave edge compared with those bound to the convex edge. Our simulations appear to provide microscopic rationale for the differing binding affinities of naproxen and ibuprofen observed experimentally. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Comparison of neurotoxicity of different aggregated forms of Aβ40, Aβ42 and Aβ43 in cell cultures

The abnormal deposition of amyloid‐β (Aβ) peptides in the brain is the main neuropathological hallmark of Alzheimer's disease (AD). Amyloid deposits are formed by a heterogeneous mixture of Aβ peptides, among which the most studied are Aβ40 and Aβ42. Aβ40 is abundantly produced in the human brain, but the level of Aβ42 is remarkably increased in the brain of AD patients. Aside from Aβ40 and Aβ42, recent data have raised the possibility that Aβ43 peptides may be instrumental in AD pathogenesis. Besides its length, whether the Aβ aggregated form accounts for the neurotoxicity is also particularly controversial. Aβ fibrils are generally considered as key pathogenic substances in AD pathogenesis. Nevertheless, recent data implicated soluble Aβ oligomers as the main cause of synaptic dysfunction and memory loss in AD. To further address this uncertainty, we analyzed the neurotoxicity of different Aβ species and Aβ forms at the cellular level. The results showed that Aβ42 could form oligomers significantly faster than Aβ40 and Aβ43 and Aβ42 oligomers showed the greatest level of neurotoxicity. Regardless of the length of Aβ peptides, Aβ oligomers induced significantly higher cytotoxicity compared with the other two Aβ forms. Surprisingly, the neurotoxicity of fibrils in PC12 cells was only marginally but not significantly stronger than monomers, contrary to previous reports. Altogether, our findings demonstrate the high pathogenicity of Aβ42 among the three Aβ species and support the idea that Aβ42 oligomers contribute to the pathological events leading to neurodegeneration in AD. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Aβ seeding potency peaks in the early stages of cerebral β‐amyloidosis

Little is known about the extent to which pathogenic factors drive the development of Alzheimer's disease (AD) at different stages of the long preclinical and clinical phases. Given that the aggregation of the β‐amyloid peptide (Aβ) is an important factor in AD pathogenesis, we asked whether Aβ seeds from brain extracts of mice at different stages of amyloid deposition differ in their biological activity. Specifically, we assessed the effect of age on Aβ seeding activity in two mouse models of cerebral Aβ amyloidosis (APPPS1 and APP23) with different ages of onset and rates of progression of Aβ deposition. Brain extracts from these mice were serially diluted and inoculated into host mice. Strikingly, the seeding activity (seeding dose SD₅₀) in extracts from donor mice of both models reached a plateau relatively early in the amyloidogenic process. When normalized to total brain Aβ, the resulting specific seeding activity sharply peaked at the initial phase of Aβ deposition, which in turn is characterized by a temporary several‐fold increase in the Aβ42/Aβ40 ratio. At all stages, the specific seeding activity of the APPPS1 extract was higher compared to that of APP23 brain extract, consistent with a more important contribution of Aβ42 than Aβ40 to seed activity. Our findings indicate that the Aβ seeding potency is greatest early in the pathogenic cascade and diminishes as Aβ increasingly accumulates in brain. The present results provide experimental support for directing anti‐Aβ therapeutics to the earliest stage of the pathogenic cascade, preferably before the onset of amyloid deposition.     

Rational design of amyloid β peptide–binding proteins: Pseudo‐Aβ β‐sheet surface presented in green fluorescent protein binds tightly and preferentially to structured Aβ

Some neurodegenerative diseases such as Alzheimer disease (AD) and Parkinson disease are caused by protein misfolding. In AD, amyloid β‐peptide (Aβ) is thought to be a toxic agent by self‐assembling into a variety of aggregates involving soluble oligomeric intermediates and amyloid fibrils. Here, we have designed several green fluorescent protein (GFP) variants that contain pseudo‐Aβ β‐sheet surfaces and evaluated their abilities to bind to Aβ and inhibit Aβ oligomerization. Two GFP variants P13H and AP93Q bound tightly to Aβ, K_d = 260 nM and K_d = 420 nM, respectively. Moreover, P13H and AP93Q were capable of efficiently suppressing the generation of toxic Aβ oligomers as shown by a cell viability assay. By combining the P13H and AP93Q mutations, a super variant SFAB4 comprising four strands of Aβ‐derived sequences was designed and bound more tightly to Aβ (K_d = 100 nM) than those having only two pseudo‐Aβ strands. The SFAB4 protein preferentially recognized the soluble oligomeric intermediates of Aβ more than both unstructured monomer and mature amyloid fibrils. Thus, the design strategy for embedding pseudo‐Aβ β‐sheet structures onto a protein surface arranged in the β‐barrel structure is useful to construct molecules capable of binding tightly to Aβ and inhibiting its aggregation. This strategy may provide implication for the diagnostic and therapeutic development in the treatment of AD. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Brains of rhesus monkeys display Aβ deposits and glial pathology while lacking Aβ dimers and other Alzheimer's pathologies

Cerebral amyloid beta (Aβ) deposits are the main early pathology of Alzheimer's disease (AD). However, abundant Aβ deposits also occur spontaneously in the brains of many healthy people who are free of AD with advancing aging. A crucial unanswered question in AD prevention is why AD does not develop in some elderly people, despite the presence of Aβ deposits. The answer may lie in the composition of Aβ oligomer isoforms in the Aβ deposits of healthy brains, which are different from AD brains. However, which Aβ oligomer triggers the transformation from aging to AD pathogenesis is still under debate. Some researchers insist that the Aβ 12‐mer causes AD pathology, while others suggest that the Aβ dimer is the crucial molecule in AD pathology. Aged rhesus monkeys spontaneously develop Aβ deposits in the brain with striking similarities to those of aged humans. Thus, rhesus monkeys are an ideal natural model to study the composition of Aβ oligomer isoforms and their downstream effects on AD pathology. In this study, we found that Aβ deposits in aged monkey brains included 3‐mer, 5‐mer, 9‐mer, 10‐mer, and 12‐mer oligomers, but not 2‐mer oligomers. The Aβ deposits, which were devoid of Aβ dimers, induced glial pathology (microgliosis, abnormal microglia morphology, and astrocytosis), but not the subsequent downstream pathologies of AD, including Tau pathology, neurodegeneration, and synapse loss. Our results indicate that the Aβ dimer plays an important role in AD pathogenesis. Thus, targeting the Aβ dimer is a promising strategy for preventing AD.     

Inhibition of Aβ42 aggregation using peptides selected from combinatorial libraries

Increasing evidence suggests that the aggregation of the small peptide Aβ42 plays an important role in the development of Alzheimer's disease. Inhibiting the initial aggregation of Aβ42 may be an effective treatment for preventing, or slowing, the onset of the disease. Using an in vivo screen based on the enzyme EGFP, we have searched through two combinatorially diverse peptide libraries to identify peptides capable of inhibiting Aβ42 aggregation. From this initial screen, three candidate peptides were selected and characterized. ThT studies indicated that the selected peptides were capable of inhibiting amyloid aggregation. Additional ThT studies showed that one of the selected peptides was capable of disaggregating preformed Aβ42 fibers. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Identification of key stabilizing interactions of amyloid-β oligomers based on fragment molecular orbital calculations on macrocyclic β-hairpin peptides

Analyzing the electronic states and inter-/intra-molecular interactions of amyloid oligomers expand our understanding of the molecular basis of Alzheimer's disease and other amyloid diseases. In the current study, several high-resolution crystal structures of oligomeric assemblies of Aβ-derived peptides have been studied by the ab initio fragment molecular orbital (FMO) method. The FMO method provides comprehensive details of the molecular interactions between the residues of the amyloid oligomers at the quantum mechanical level. Based on the calculations, two sequential aromatic residues (F19 and F20) and negatively charged E22 on the central region of Aβ have been identified as key residues in oligomer stabilization and potential interesting pharmacophores for preventing oligomer formation.     

Studies on the in Vitro Assembly of Aβ 1–40: Implications for the Search for Aβ Fibril Formation Inhibitors

The progressive deposition of the amyloid β peptide (Aβ) in fibrillar form is a key feature in the development of the pathology in Alzheimer's disease (AD). We have characterized the time course of Aβ fibril formation using a variety of assays and under different experimental conditions. We describe in detail the morphological development of the Aβ polymerization process from pseudo-spherical structures and protofibrils to mature thioflavin-T-positive/Congo red-positive amyloid fibrils. Moreover, we structurally characterize the various polymorphic fibrillar assemblies using transmission electron microscopy and determine their mass using scanning transmission electron microscopy. These results provide the framework for future investigations into how target compounds may interfere with the polymerization process. Such substances might have a therapeutic potential in AD.     

A synthetic peptide corresponding to a region of the human pericentriolar material 1 (PCM‐1) protein binds β‐amyloid (Aβ1‐42) oligomers

We have recently reported that a ~19‐kDa polypeptide, rPK‐4, is a protein kinase Cs inhibitor that is 89% homologous to the 1171–1323 amino acid region of the 228‐kDa human pericentriolar material‐1 (PCM‐1) protein (Chakravarthy et al. 2012). We have now discovered that rPK‐4 binds oligomeric amyloid‐β peptide (Aβ)_1‐42 with high affinity. Most importantly, a PCM‐1‐selective antibody co‐precipitated Aβ and amyloid β precursor protein (AβPP) from cerebral cortices and hippocampi from AD (Alzheimer's disease) transgenic mice that produce human AβPP and Aβ_1‐42, suggesting that PCM‐1 may interact with amyloid precursor protein/Aβ in vivo. We have identified rPK‐4′s Aβ‐binding domain using a set of overlapping synthetic peptides. We have found with ELISA, dot‐blot, and polyacrylamide gel electrophoresis techniques that a ~ 5 kDa synthetic peptide, amyloid binding peptide (ABP)‐p4‐5 binds Aβ_1‐42 at nM levels. Most importantly, ABP‐p4‐5, like rPK‐4, appears to preferentially bind Aβ_1‐42 oligomers, believed to be the toxic AD‐drivers. As expected from these observations, ABP‐p4‐5 prevented Aβ_1‐42 from killing human SH‐SY5Y neuroblastoma cells via apoptosis. These findings indicate that ABP‐p4‐5 is a possible candidate therapeutic for AD.     

Wild‐type,Flemish, and Dutch amyloid‐β exhibit different cytotoxicities depending on Aβ40 to Aβ42 interaction time and concentration ratio

Addition of amyloid β (Aβ) peptide Aβ40 to Aβ42 can delay Aβ42 aggregation, but consequent cytotoxicity has been reported to be enhanced or diminished. In the present study, we found that cytotoxicity was enhanced when human neuroblastoma SH‐SY5Y cells were incubated in a mixture of wt Aβ42 and Aβ40wt at a ratio of 1 : 10–20 (0.1 : 1–2 μM) for 24–36 h, whereas the enhancement was detected in cells incubated for longer times (48–60 h) with the less amyloidogenic Flemish Aβ40 variant or in cells incubated for as short as 12 h with the more amyloidogenic Dutch variant. Reductions in cytotoxicity by Aβ40 were most prominently observed in the Flemish and wt Aβ40/Aβ42 mixture at ratio 1 : 20 incubated for a short time (~12 h). The most cytotoxic Aβ40/Aβ42 mixtures were enriched in Aβ protofibril‐like structures, implying a strong correlation between cytotoxicity and this structure, the formation of which was dependent on amyloidogenic properties and incubation time. The consequences of the interactions were probably because of the different amyloidogenic properties of the Aβ40 variants, rather than to those of Aβ42, because aggregation rates of Aβ40 variants were highly dependent on sequence, whereas those of Aβ42 variants were not. These studies highlight a potential role for Aβ40 in cytotoxicity and provide novel mechanistic insights into the pathogenesis of each familial Alzheimer's disease‐associated Aβ40 variant. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

N‐terminal truncated pyroglutamyl β amyloid peptide Aβpy3‐42 shows a faster aggregation kinetics than the full‐length Aβ1‐42

We tested directly the differences in the aggregation kinetics of three important β amyloid peptides, the full‐length Aβ1‐42, and the two N‐terminal truncated and pyroglutamil modified Aβpy3‐42 and Aβpy11‐42 found in different relative concentrations in the brains in normal aging and in Alzheimer disease. By following the circular dichroism signal and the ThT fluorescence of the solution in phosphate buffer, we found substantially faster aggregation kinetics for Aβpy3‐42. This behavior is due to the particular sequence of this peptide, which is also responsible for the specific oligomeric aggregation states, found by TEM, during the fibrillization process, which are very different from those of Aβ1‐42, more prone to fibril formation. In addition, Aβpy3‐42 is found here to have an inhibitory effect on Aβ1‐42 fibrillogenesis, coherently with its known greater infective power. This is an indication of the important role of this peptide in the aggregation process of β‐peptides in Alzheimer disease. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 861–873, 2009. This article was originally published online as an accepted preprint. The “Published Online“ date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Stability of Aβ‐fibril fragments in the presence of fatty acids

We consider the effect of lauric acid on the stability of various fibril‐like assemblies of Aβ peptides. For this purpose, we have performed molecular dynamics simulations of these assemblies either in complex with lauric acid or without presence of the ligand. While we do not observe a stabilizing effect on Aβ₄₀‐fibrils, we find that addition of lauric acid strengthens the stability of fibrils built from the triple‐stranded S‐shaped Aβ₄₂‐peptides considered to be more toxic. Or results may help to understand how the specifics of the brain‐environment modulate amyloid formation and propagation.     

Basic conformers in β‐peptides

The conformation of oligomers of β‐amino acids of the general type Ac‐[β‐Xaa]_n‐NHMe (β‐Xaa = β‐Ala, β‐Aib, and β‐Abu; n = 1–4) was systematically examined at different levels of ab initio molecular orbital theory (HF/6‐31G*, HF/3‐21G). The solvent influence was considered employing two quantum‐mechanical self‐consistent reaction field models. The results show a wide variety of possibilities for the formation of characteristic elements of secondary structure in β‐peptides. Most of them can be derived from the monomer units of blocked β‐peptides with n = 1. The stability and geometries of the β‐peptide structures are considerably influenced by the side‐chain positions, by the configurations at the C^α‐ and C^β‐atoms of the β‐amino acid constituents, and especially by environmental effects. Structure peculiarities of β‐peptides, in particular those of various helix alternatives, are discussed in relation to typical elements of secondary structure in α‐peptides. © 1999 John Wiley & Sons, Inc. Biopoly 50: 167–184, 1999     

Effect of structural parameters of peptides on dimer formation and highly oxidized side products in the oxidation of thiols of linear analogues of human β‐defensin 3 by DMSO

The purpose of this study was to examine the effects of structural parameters of peptides on their oxidation by DMSO, including location of cysteine, effect of adjunct group participation, molecular hydrophobicity, steric hindrance or the accessibility of thiol group and peptide conformation, on oxidation rates, dimer formation and associated side products. We designed and synthesized two series of linear cysteine‐containing analogues of human β‐defensin 3 (the C1‐peptides with cysteine at the N‐terminus residue 1, the C29‐peptides with cysteine located at residue 29 in the centre of peptide), which were used for preparation of disulphide‐linked homodimers. HPLC–ESI–MS was used to monitor the oxidation process and to characterize the molecular weights of dimers and side products of high oxidation. The formations of dimers and side products were dependent on the position of cysteines. Hydrophobicity generally rendered the thiol groups less accessible and hence exposed them to slow oxidation to form dimers (or even fail to form dimers during the timescale of observation). Molecular dynamics simulations showed that the exposure of cysteines (and sulphurs) of the C1‐peptides was much larger than for the C29‐peptides. The larger hydrophobic side chains tended to enable clustering of the side chains that sequester cysteine, particularly in the C29‐peptides, which provided a molecular explanation for the observed trends in oxidation rates. Together with molecular modelling, we propose a reaction mechanism to elucidate the oxidation results of these peptides. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

FTIR spectroscopic studies on aggregation process of the β‐amyloid 11–28 fragment and its variants

Aggregation of Aβ peptides is a seminal event in Alzheimer's disease. Detailed understanding of the Aβ assembly process would facilitate the targeting and design of fibrillogenesis inhibitors. Here, conformational studies using FTIR spectroscopy are presented. As a model peptide, the 11–28 fragment of Aβ was used. This model peptide is known to contain the core region responsible for Aβ aggregation. The structural behavior of the peptide during aggregation provoked by the addition of water to Aβ(11–28) solution in hexafluoroisopropanol was compared with the properties of its variants corresponding to natural, clinically relevant mutants at positions 21–23 (A21G, E22K, E22G, E22Q and D23N). The results showed that the aggregation of the peptides proceeds via a helical intermediate, and it is possible that the formation of α‐helical structures is preceded by creation of 3₁₀‐helix/3₁₀‐turn structures. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Neuroprotective effects of donepezil against Aβ42‐induced neuronal toxicity are mediated through not only enhancing PP2A activity but also regulating GSK‐3β and nAChRs activity

The main purpose of this study was to evaluate whether donepezil, acetylcholinesterase inhibitor, shown to play a protective role through inhibiting glycogen synthesis kinase‐3β (GSK‐3β) activity, could also exert neuroprotective effects by stimulating protein phosphatase 2A (PP2A) activity in the amyloid‐beta (Aβ)42‐induced neuronal toxicity model of Alzheimer's disease. In Aβ42‐induced toxic conditions, each PP2A and GSK‐3β activity measured at different times showed time‐dependent reverse pattern toward the direction of accelerating neuronal deaths with the passage of time. In addition, donepezil pre‐treatment showed dose‐dependent stepwise increase of neuronal viability and stimulation of PP2A activity. However, such effects on them were significantly reduced through the depletion of PP2A activity with either okadaic acid or PP2Ac siRNA. In spite of blocked PP2A activity in this Aβ42 insult, however, donepezil pretreatment showed additional significant recovering effect on neuronal viability when compared to the value without donepezil. Moreover, donepezil partially recovered its dephosphorylating effect on hyperphosphorylated tau induced by Aβ42. This observation led us to assume that additional mechanisms of donepezil, including its inhibitory effect on GSK‐3β activity and/or the activation role of nicotinic acetylcholine receptors (nAChRs), might be involved. Taken together, our results suggest that the neuroprotective effects of donepezil against Aβ42‐induced neurotoxicity are mediated through activation of PP2A, but its additional mechanisms including regulation of GSK‐3β and nAChRs activity would partially contribute to its effects.

     

Stability of secondary structural elements in a solvent-free environment: the α helix

The stability of the α helix as an element of secondary structure is examined in the absence of solvation, in the gas phase. Mass-analyzed ion kinetic energy (MIKE) spectrometry was applied to measure intercharge repulsion and intercharge distance in multiply protonated melittin, a polypeptide known to possess a stable helical structure in a number of different environments. The experimental results, interpreted in combination with molecular mechanics calculations, suggest that triply charged melittin retains its secondary structure in the gas phase. The stability if the α-helical conformation of the polypeptide in the absence of solvent molecules reflects the fact that a network of intrinsic helical hydrogen bonds is energetically more favorable than unfolded conformations. © 1997 Wiley-Liss, Inc.