Significant Upregulation of Alzheimer's β‐Amyloid Levels in a Living System Induced by Extracellular Elastin Polypeptides

Alzheimer's disease (AD) is a neurodegenerative disorder and the primary cause of age‐related dementia. The etiology of AD is complex and has not been completely elucidated. Herein, we report that treatment with elastin‐like polypeptides (ELPs), a component of the brain extracellular matrix (ECM), significantly increased the levels of AD‐related amyloid‐β peptides (Aβ) both in vitro and in vivo. Regarding the molecular mechanism(s), the upregulation of Aβ levels was related to increased proteolytic processing of the amyloid precursor protein. Furthermore, nesting tests demonstrated that the ELP‐treated animals showed significant neurobehavioral deficits with cognitive impairment. These results suggest that the elastin is associated with AD‐related pathological and behavioral changes. This finding presents a new aspect for Alzheimer's amyloidosis event and provides a great promise in developing ELP‐based model systems to better understand the pathogenesis of AD.     

Detection of Aβ Monomers and Oligomers: Early Diagnosis of Alzheimer's Disease

Alzheimer's disease (AD), as the most common progressive neurodegenerative disorder, is pathologically characterized by deposition of extracellular plaque composed of amyloid‐β peptide (Aβ). Different assembled states of Aβ have been considered as both important biomarkers and drug targets for the diagnosis and therapy of AD. Recent studies demonstrate that small, diffusible Aβ oligomers formed by aggregation of Aβ monomers are the major toxic agents in AD. Therefore, the development of reliable assays for Aβ (both monomers and oligomers) will be important for the early differential diagnosis of dementia, predicting the progression of AD, as well as monitoring the effectiveness of novel anti‐Aβ drugs for AD. In this review, we summarize the recent progress made in the development of techniques for detection of Aβ monomers and oligomers. In particular, the principles governing the design of these sensors are classified and summarized. Moreover, the advantages and disadvantages of the assays are evaluated. This review also discusses the improvements and challenges for application of these assays in the early diagnosis of AD.     

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.     

Resistance of Cu(Aβ4–16) to Copper Capture by Metallothionein‐3 Supports a Function for the Aβ4–42 Peptide as a Synaptic CuII Scavenger

Aβ4‐42 is a major species of Aβ peptide in the brains of both healthy individuals and those affected by Alzheimer's disease. It has recently been demonstrated to bind Cu^II with an affinity approximately 3000 times higher than the commonly studied Aβ1‐42 and Aβ1‐40 peptides, which are implicated in the pathogenesis of Alzheimer's disease. Metallothionein‐3, a protein considered to orchestrate copper and zinc metabolism in the brain and provide antioxidant protection, was shown to extract Cu^II from Aβ1‐40 when acting in its native Zn₇MT‐3 form. This reaction is assumed to underlie the neuroprotective effect of Zn₇MT‐3 against Aβ toxicity. In this work, we used the truncated model peptides Aβ1‐16 and Aβ4‐16 to demonstrate that the high‐affinity Cu^II complex of Aβ4‐16 is resistant to Zn₇MT‐3 reactivity. This indicates that the analogous complex of the full‐length peptide Cu(Aβ4‐42) will not yield copper to MT‐3 in the brain, thus supporting the concept of a physiological role for Aβ4‐42 as a Cu^II scavenger in the synaptic cleft.     

Atomic‐Resolution Three‐Dimensional Structure of Amyloid β Fibrils Bearing the Osaka Mutation

Despite its central importance for understanding the molecular basis of Alzheimer's disease (AD), high‐resolution structural information on amyloid β‐peptide (Aβ) fibrils, which are intimately linked with AD, is scarce. We report an atomic‐resolution fibril structure of the Aβ1‐40 peptide with the Osaka mutation (E22Δ), associated with early‐onset AD. The structure, which differs substantially from all previously proposed models, is based on a large number of unambiguous intra‐ and intermolecular solid‐state NMR distance restraints.     

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.     

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.     

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.     

Discovery of Chemicals to Either Clear or Indicate Amyloid Aggregates by Targeting Memory‐Impairing Anti‐Parallel Aβ Dimers

Amyloid‐β (Aβ) oligomers are implicated in Alzheimer disease (AD). However, their unstable nature and heterogeneous state disrupts elucidation of their explicit role in AD progression, impeding the development of tools targeting soluble Aβ oligomers. Herein parallel and anti‐parallel variants of Aβ(1–40) dimers were designed and synthesized, and their pathogenic properties in AD models characterized. Anti‐parallel dimers induced cognitive impairments with increased amyloidogenesis and cytotoxicity, and this dimer was then used in a screening platform. Through screening, two FDA‐approved drugs, Oxytetracycline and Sunitinib, were identified to dissociate Aβ oligomers and plaques to monomers in 5XFAD transgenic mice. In addition, fluorescent Astrophloxine was shown to detect aggregated Aβ in brain tissue and cerebrospinal fluid samples of AD mice. This screening platform provides a stable and homogeneous environment for observing Aβ interactions with dimer‐specific molecules.     

Free Superoxide is an Intermediate in the Production of H2O2 by Copper(I)‐Aβ Peptide and O2

Oxidative stress is considered as an important factor and an early event in the etiology of Alzheimer's disease (AD). Cu bound to the peptide amyloid‐β (Aβ) is found in AD brains, and Cu‐Aβ could contribute to this oxidative stress, as it is able to produce in vitro H₂O₂ and HO^. in the presence of oxygen and biological reducing agents such as ascorbate. The mechanism of Cu‐Aβ‐catalyzed H₂O₂ production is however not known, although it was proposed that H₂O₂ is directly formed from O₂ via a 2‐electron process. Here, we implement an electrochemical setup and use the specificity of superoxide dismutase‐1 (SOD1) to show, for the first time, that H₂O₂ production by Cu‐Aβ in the presence of ascorbate occurs mainly via a free O₂^.? intermediate. This finding radically changes the view on the catalytic mechanism of H₂O₂ production by Cu‐Aβ, and opens the possibility that Cu‐Aβ‐catalyzed O₂^.? contributes to oxidative stress in AD, and hence may be of interest.     

Organoplatinum‐Substituted Polyoxometalate Inhibits β‐amyloid Aggregation for Alzheimer's Therapy

Aggregated β‐amyloid (Aβ) is widely considered as a key factor in triggering progressive loss of neuronal function in Alzheimer's disease (AD), so targeting and inhibiting Aβ aggregation has been broadly recognized as an efficient therapeutic strategy for curing AD. Herein, we designed and prepared an organic platinum‐substituted polyoxometalate, (Me₄N)₃[PW₁₁O₄₀(SiC₃H₆NH₂)₂PtCl₂] (abbreviated as Pt^II‐PW₁₁) for inhibiting Aβ₄₂ aggregation. The mechanism of inhibition on Aβ₄₂ aggregation by Pt^II‐PW₁₁ was attributed to the multiple interactions of Pt^II‐PW₁₁ with Aβ₄₂ including coordination interaction of Pt²⁺ in Pt^II‐PW₁₁ with amino group in Aβ₄₂, electrostatic attraction, hydrogen bonding and van der Waals force. In cell‐based assay, Pt^II‐PW₁₁ displayed remarkable neuroprotective effect for Aβ₄₂ aggregation‐induced cytotoxicity, leading to increase of cell viability from 49 % to 67 % at a dosage of 8 μm . More importantly, the Pt^II‐PW₁₁ greatly reduced Aβ deposition and rescued memory loss in APP/PS1 transgenic AD model mice without noticeable cytotoxicity, demonstrating its potential as drugs for AD treatment.     

Gold‐Nanoparticle‐Based Multifunctional Amyloid‐β Inhibitor against Alzheimer’s Disease

Targeting amyloid‐β (Aβ)‐induced complex neurotoxicity has received considerable attention in the therapeutic and preventive treatment of Alzheimer’s disease (AD). The complex pathogenesis of AD suggests that it requires comprehensive treatment, and drugs with multiple functions against AD are more desirable. Herein, AuNPs@POMD‐pep (AuNPs: gold nanoparticles, POMD: polyoxometalate with Wells–Dawson structure, pep: peptide) were designed as a novel multifunctional Aβ inhibitor. AuNPs@POMD‐pep shows synergistic effects in inhibiting Aβ aggregation, dissociating Aβ fibrils and decreasing Aβ‐mediated peroxidase activity and Aβ‐induced cytotoxicity. By taking advantage of AuNPs as vehicles that can cross the blood–brain barrier (BBB), AuNPs@POMD‐pep can cross the BBB and thus overcome the drawbacks of small‐molecule anti‐AD drugs. Thus, this work provides new insights into the design and synthesis of inorganic nanoparticles as multifunctional therapeutic agents for treatment of AD.     

para‐Sulfonatocalix[n]arenes Inhibit Amyloid β‐Peptide Fibrillation and Reduce Amyloid Cytotoxicity

Amyloid β‐peptide (Aβ) fibrillation is a major hallmark of Alzheimer's disease (AD). Inhibition of Aβ fibrillation is thus considered to be an effective strategy for AD prevention and treatment. Here we show that para ‐sulfonatocalix[n ]arenes (SC[n ]A, n =4, 6, 8), a class of amphiphilic calixarene derivatives, can bind to Aβ₄₂ through nonspecific and multipoint hydrophobic interactions. Their binding leads to a pronounced delay in β‐sheet adoption and formation of multiple secondary structures of the peptide, accompanied by changes at the level of the fibrillary architecture. Furthermore, the ζ‐potential value of Aβ₄₂ incubated with SC[6/8]A decreased, which correlated with the reduction of amyloid cytotoxicity. Overall, the SC[n ]A effectively inhibits Aβ₄₂ fibrillation and reduces amyloid cytotoxicity, and SC[8]A showed the best performance among the three macrocycles, possibly owing to its having the strongest interactions with Aβ₄₂.     

Harnessing Intramolecular Rotation To Enhance Two‐photon Imaging of Aβ Plaques through Minimizing Background Fluorescence

The aggregation of amyloid beta (Aβ) proteins in senile plaques is a critical event during the development of Alzheimer's disease, and the postmortem detection of Aβ‐rich proteinaceous deposits through fluorescent staining remains one of the most robust diagnostic tools. In animal models, fluorescence imaging can be employed to follow the progression of the disease, and among the different imaging methods, two‐photon microscopy (TPM) has emerged as one of the most powerful. To date, several near‐infrared‐emissive two‐photon dyes with a high affinity for Aβ fibrils have been developed, but there has often been a tradeoff between excellent two‐photon cross‐sections and large fluorescence signal‐to‐background ratios. In the current work, we introduced a twisted intramolecular charge state (TICT)‐based de‐excitation pathway, which results in a remarkable fluorescence increase of around 167‐fold in the presence of Aβ fibrils, while maintaining an excellent two‐photon cross section, thereby enabling high‐contrast ex vivo and in vivo TPM imaging. Overall, the results suggest that adopting TICT de‐excitation in two‐photon fluorophores may represent a general method to overcome the tradeoff between probe brightness and signal‐to‐background ratio.     

An online nano‐LC‐ESI‐FTICR‐MS method for comprehensive characterization of endogenous fragments from amyloid β and amyloid precursor protein in human and cat cerebrospinal fluid

Amyloid precursor protein (APP) is the precursor protein to amyloid β (Aβ), the main constituent of senile plaques in Alzheimer's disease (AD). Endogenous Aβ peptides reflect the APP processing, and greater knowledge of different APP degradation pathways is important to understand the mechanism underlying AD pathology. When one analyzes longer Aβ peptides by low‐energy collision‐induced dissociation tandem mass spectrometry (MS/MS), mainly long b‐fragments are observed, limiting the possibility to determine variations such as amino acid variants or post‐translational modifications (PTMs) within the N‐terminal half of the peptide. However, by using electron capture dissociation (ECD), we obtained a more comprehensive sequence coverage for several APP/Aβ peptide species, thus enabling a deeper characterization of possible variants and PTMs. Abnormal APP/Aβ processing has also been described in the lysosomal storage disease Niemann–Pick type C and the major large animal used for studying this disease is cat. By ECD MS/MS, a substitution of Asp7 → Glu in cat Aβ was identified. Further, sialylated core 1 like O‐glycans at Tyr10, recently discovered in human Aβ (a previously unknown glycosylation type), were identified also in cat cerebrospinal fluid (CSF). It is therefore likely that this unusual type of glycosylation is common for (at least) species belonging to the magnorder Boreoeutheria. We here describe a detailed characterization of endogenous APP/Aβ peptide species in CSF by using an online top‐down MS‐based method. Copyright © 2012 John Wiley & Sons, Ltd.     

GM1 Ganglioside Inhibits β‐Amyloid Oligomerization Induced by Sphingomyelin

β‐Amyloid (Aβ) oligomers are neurotoxic and implicated in Alzheimer's disease. Neuronal plasma membranes may mediate formation of Aβ oligomers in vivo. Membrane components sphingomyelin and GM₁ have been shown to promote aggregation of Aβ; however, these studies were performed under extreme, non‐physiological conditions. We demonstrate that physiological levels of GM₁, organized in nanodomains do not seed oligomerization of Aβ₄₀ monomers. We show that sphingomyelin triggers oligomerization of Aβ₄₀ and that GM₁ is counteractive thus preventing oligomerization. We propose a molecular explanation that is supported by all‐atom molecular dynamics simulations. The preventive role of GM₁ in the oligomerization of Aβ₄₀ suggests that decreasing levels of GM₁ in the brain, for example, due to aging, could reduce protection against Aβ oligomerization and contribute to the onset of Alzheimer's disease.     

Label‐free Electrochemical Sensor for Ex‐vivo Monitoring of Alzheimer's Disease Biomarker

The beta‐amyloid (Aβ) peptide was used as an important biomarker for Alzheimer's disease (AD) diagnosis. The development of an accurate, selective, rapid, and highly sensitive technique for detecting of Aβ level is an important issue in biology, and medicine to assess human health risks. Here, gold nanoparticles (Au NPs) with different size were electrochemically deposited onto the indium tin oxide (ITO) substrate in the presence of different molecular weights of surfactants. The modified substrates were used as a high sensitive electrochemical sensor of in‐vitro as well as ex‐vivo monitoring of Aβ based on cyclic voltammetry and square wave voltammetry techniques. Our findings revealed that the modification of ITO electrode with Au NPs could enhance its sensor performance with high sensitivity for low concentration levels of Aβ over a wide linear range with a detection limit of about 20.7 ng/g, which is less than the concentration of insoluble Aβ40 (105.4±40.2 μg/g) in brain of AD induced. In addition, Au NPs/ITO modified electrodes have demonstrated ability to monitor Aβ in the brain extracted samples without any potential interference with other components. Raman spectroscopy has been used to confirm the presence of Aβ in the AD‐induced samples. Thus, it is applicable for analyzing ex‐vivo samples.     

Modification of a Small β‐Barrel Protein,To Give Pseudo‐Amyloid Structures,Inhibits Amyloid β‐Peptide Aggregation

Aggregation of amyloid β‐peptide (Aβ) is closely related to the pathogenesis of Alzheimer’s disease (AD). Although much effort has been devoted to the construction of molecules that inhibit the aggregation of Aβ1‐42, high doses are needed for the inhibition of Aβ aggregation in many cases. Previously, we reported that designed green fluorescent protein (GFP) analogues that gives pseudo‐Aβ β‐sheet structures can work as an aggregation inhibitor against Aβ. To further test this design strategy, we constructed protein analogues that mimic Aβ β‐sheet structures of amyloids by using insulin‐like growth factor 2 receptor domain 11 (IGF2R‐d11) as a scaffold. A designed protein, named IG11KK, which has a parallel configuration of Aβ‐like β sheets, can bind more preferentially to oligomeric Aβ1‐42 than the monomer. Moreover, IG11KK suppressed the aggregation of Aβ1‐42 efficiently, even though lower concentrations of IG11KK than Aβ were used. The aggregation kinetics of Aβ in the presence of the designed proteins revealed that IG11KK can work as an inhibitor not only for the early to middle stages, but also in the latter stage of Aβ aggregation owing to its favorable binding to oligomeric structures of Aβ. The design strategy using β‐barrel proteins such as IGF2R‐d11 and GFP is useful in generating excellent inhibitors of protein misfolding and amyloid formation.     

Introduction of a Fluorescent Probe to Amyloid‐β to Reveal Kinetic Insights into Its Interactions with Copper(II)

The kinetics of the interactions between amyloid‐β (Aβ) and metal ions are crucial to understanding the physiological and pathological roles of Aβ in the normal brain and in Alzheimer’s disease. Using the quenching of a fluorescent probe by Cu²⁺, the mechanism of Aβ/Cu²⁺ interactions in physiologically relevant conditions has been elucidated. Cu²⁺ binds to Aβ at a near diffusion‐limited rate, initially forming component I. The switching between component I and II occurs on the second timescale, with a significant energy barrier. Component I is much more reactive towards Cu²⁺ ligands and likely responsible for initial Aβ dimer formation. Clioquinol (CQ) is shown to sequester Cu²⁺ more effectively than other tested ligands. These findings have implications for the potential roles of Aβ in regulating neurotransmission, and for the screening of small molecules targeting Aβ–metal interactions.