Natural Compounds against Neurodegenerative Diseases: Molecular Characterization of the Interaction of Catechins from Green Tea with Aβ1–42, PrP106–126, and Ataxin‐3 Oligomers

By combining NMR spectroscopy, transmission electron microscopy, and circular dichroism we have identified the structural determinants involved in the interaction of green tea catechins with Aβ1–42, PrP106–126, and ataxin‐3 oligomers. The data allow the elucidation of their mechanism of action, showing that the flavan‐3‐ol unit of catechins is essential for interaction. At the same time, the gallate moiety, when present, seems to increase the affinity for the target proteins. These results provide important information for the rational design of new compounds with anti‐amyloidogenic activity and/or molecular tools for the specific targeting of amyloid aggregates in vivo.     

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.     

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.     

Tetracycline prevents Aβ oligomer toxicity through an atypical supramolecular interaction

The antibiotic tetracycline was reported to possess an anti-amyloidogenic activity on a variety of amyloidogenic proteins both in in vitro and in vivo models. To unveil the mechanism of action of tetracycline on Aβ1-40 and Aβ1-42 at both molecular and supramolecular levels, we carried out a series of experiments using NMR spectroscopy, FTIR spectroscopy, dynamic laser light-scattering (DLS) and atomic force microscopy (AFM). Firstly we showed that the co-incubation of Aβ1-42 oligomers with tetracycline hinders the toxicity towards N2a cell lines in a dose-dependent manner. Therefore, the nature of the interaction between the drug and Aβ oligomers was investigated. To carry out NMR and FTIR studies we have prepared Aβ peptide solutions containing assemblies ranging from monomers to large oligomers. Saturation transfer difference (STD) NMR experiments have shown that tetracycline did not interact with monomers at variance with oligomers. Noteworthy, in this latter case we observed that this interaction was very peculiar since the transfer of magnetization from Aβ oligomers to tetracycline involved all drug protons. In addition, intermolecular cross-peaks between tetracycline and Aβ were not observed in NOESY spectra, indicating the absence of a specific binding site and suggesting the occurrence of a supramolecular interaction. DLS and AFM studies supported this hypothesis since the co-dissolution of Aβ peptides and tetracycline triggered the immediate formation of new aggregates that improved the solubility of Aβ peptides, preventing in this way the progression of the amyloid cascade. Moreover, competitive NMR binding experiments showed for the first time that tetracycline competes with thioflavin T (ThT) in the binding to Aβ peptides. Our data shed light on a novel mechanism of anti-amyloidogenic activity displayed by tetracycline, governed by hydrophobic and charge multiparticle interactions.     

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.     

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.     

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.     

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.     

Significant Structural Differences between Transient Amyloid‐β Oligomers and Less‐Toxic Fibrils in Regions Known To Harbor Familial Alzheimer′s Mutations

Small oligomers of the amyloid β (Aβ) peptide, rather than the monomers or the fibrils, are suspected to initiate Alzheimer′s disease (AD). However, their low concentration and transient nature under physiological conditions have made structural investigations difficult. A method for addressing such problems has been developed by combining rapid fluorescence techniques with slower two‐dimensional solid‐state NMR methods. The smallest Aβ₄₀ oligomers that demonstrate a potential sign of toxicity, namely, an enhanced affinity for cell membranes, were thus probed. The two hydrophobic regions (residues 10–21 and 30–40) have already attained the conformation that is observed in the fibrils. However, the turn region (residues 22–29) and the N‐terminal tail (residues 1–9) are strikingly different. Notably, ten of eleven known Aβ mutants that are linked to familial AD map to these two regions. Our results provide potential structural cues for AD therapeutics and also suggest a general method for determining transient protein structures.     

Charge regulation phenomenon predicted from the modeling of polypeptide electrophoretic mobilities as a relevant mechanism of amyloid‐beta peptide oligomerization

Electrophoretic mobilities of amyloid‐beta (1‐40) and (1‐42) peptides and their aggregates are modeled to study the amyloidogenic pathway associated with Alzheimer´s Disease. The near molecule pH generated by the intraparticle charge regulation phenomenon during the oligomerization of amyloid‐beta (1‐40) and (1‐42) peptides is evaluated and discussed as a relevant mechanism supporting the “amyloid cascade hypothesis” proposed in the literature. A theoretical framework associated with the oligomerization of amyloid‐beta peptides including simple scaling laws and the consideration of electrokinetic and hydrodynamic global properties of oligomers is presented. The central finding is the explanation of the near molecule pH change toward the pI when the oligomerization number increases. These results allow one to rationalize consecutive physical stages that validate the amyloid cascade hypothesis. Concluding remarks involving mainly the effects of pair and intraparticle charge regulation phenomena on the amyloidogenic pathway with some suggestions for future research are provided.     

Attenuation of the Aggregation and Neurotoxicity of Amyloid‐β Peptides by Catalytic Photooxygenation

Alzheimer’s disease (AD), a progressive severe neurodegenerative disorder, is currently incurable, despite intensive efforts worldwide. Herein, we demonstrate that catalytic oxygenation of amyloid‐β peptides (Aβ) might be an effective approach to treat AD. Aβ1–42 was oxygenated under physiologically‐relevant conditions (pH 7.4, 37 °C) using a riboflavin catalyst and visible light irradiation, with modifications at the Tyr¹⁰, His¹³, His¹⁴, and Met³⁵ residues. The oxygenated Aβ1–42 exhibited considerably lower aggregation potency and neurotoxicity compared with native Aβ. Photooxygenation of Aβ can be performed even in the presence of cells, by using a selective flavin catalyst attached to an Aβ‐binding peptide; the Aβ cytotoxicity was attenuated in this case as well. Furthermore, oxygenated Aβ1–42 inhibited the aggregation and cytotoxicity of native Aβ.     

Structural Insight into IAPP‐Derived Amyloid Inhibitors and Their Mechanism of Action

Designed peptides derived from the islet amyloid polypeptide (IAPP) cross‐amyloid interaction surface with Aβ (termed interaction surface mimics or ISMs) have been shown to be highly potent inhibitors of Aβ amyloid self‐assembly. However, the molecular mechanism of their function is not well understood. Using solution‐state and solid‐state NMR spectroscopy in combination with ensemble‐averaged dynamics simulations and other biophysical methods including TEM, fluorescence spectroscopy and microscopy, and DLS, we characterize ISM structural preferences and interactions. We find that the ISM peptide R3‐GI is highly dynamic, can adopt a β‐like structure, and oligomerizes into colloid‐like assemblies in a process that is reminiscent of liquid–liquid phase separation (LLPS). Our results suggest that such assemblies yield multivalent surfaces for interactions with Aβ40. Sequestration of substrates into these colloid‐like structures provides a mechanistic basis for ISM function and the design of novel potent anti‐amyloid molecules.     

Flavonoids and Their Glycosides as Anti‐amyloidogenic Compounds: Aβ1–42 Interaction Studies to Gain New Insights into Their Potential for Alzheimer's Disease Prevention and Therapy

Combining NMR spectroscopy, transmission electron microscopy, biochemical and in vitro toxicity assays, we characterized the effect of flavonoid glycosylation, a chemical modification found very frequently in nature, on their ability to recognize and bind Aβ1–42 oligomers, preventing their aggregation and their neurotoxicity. Our data allow the elucidation of their structure–activity relationships, showing that glycosylation has a modest impact on flavonoid affinity for Aβ oligomers but, at the same time, increases both solubility and chemical stability, thus promoting their beneficial properties against Alzheimer's disease (AD). As flavonoids and their glycosides are widely available in natural foods, our results provide important information for the evaluation of the role of a flavonoid‐rich diet for the prevention of AD. In addition, the structural data collected can be exploited for the rational design of more potent Aβ oligomer inhibitors, useful for the development of new therapies against AD.     

Characterizing amyloid‐beta protein misfolding from molecular dynamics simulations with explicit water

Extracellular deposition of amyloid‐beta (Aβ) protein, a fragment of membrane glycoprotein called β‐amyloid precursor transmembrane protein (βAPP), is the major characteristic for the Alzheimer's disease (AD). However, the structural and mechanistic information of forming Aβ protein aggregates in a lag phase in cell exterior has been still limited. Here, we have performed multiple all‐atom molecular dynamics simulations for physiological 42‐residue amyloid‐beta protein (Aβ42) in explicit water to characterize most plausible aggregation‐prone structure (APS) for the monomer and the very early conformational transitions for Aβ42 protein misfolding process in a lag phase. Monitoring the early sequential conformational transitions of Aβ42 misfolding in water, the APS for Aβ42 monomer is characterized by the observed correlation between the nonlocal backbone H‐bond formation and the hydrophobic side‐chain exposure. Characteristics on the nature of the APS of Aβ42 allow us to provide new insight into the higher aggregation propensity of Aβ42 over Aβ40, which is in agreement with the experiments. On the basis of the structural features of APS, we propose a plausible aggregation mechanism from APS of Aβ42 to form fibril. The structural and mechanistic observations based on these simulations agree with the recent NMR experiments and provide the driving force and structural origin for the Aβ42 aggregation process to cause AD. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2011     

Real‐time Amyloid Aggregation Monitoring with a Photonic Crystal‐based Approach

We propose the application of a new label‐free optical technique based on photonic nanostructures to real‐time monitor the amyloid‐beta 1‐42 (Aβ(1‐42)) fibrillization, including the early stages of the aggregation process, which are related to the onset of the Alzheimer’s Disease (AD). The aggregation of Aβ peptides into amyloid fibrils has commonly been associated with neuronal death, which culminates in the clinical features of the incurable degenerative AD. Recent studies revealed that cell toxicity is determined by the formation of soluble oligomeric forms of Aβ peptides in the early stages of aggregation. At this phase, classical amyloid detection techniques lack in sensitivity. Upon a chemical passivation of the sensing surface by means of polyethylene glycol, the proposed approach allows an accurate, real‐time monitoring of the refractive index variation of the solution, wherein Aβ(1‐42) peptides are aggregating. This measurement is directly related to the aggregation state of the peptide throughout oligomerization and subsequent fibrillization. Our findings open new perspectives in the understanding of the dynamics of amyloid formation, and validate this approach as a new and powerful method to screen aggregation at early stages.     

Aggregation of Beta‐Amyloid Peptides Proximal to Zwitterionic Lipid Bilayers

One of the hallmarks of Alzheimers disease is the deposition of amyloid plaques, which consist of β‐amyloid (Aβ) peptides in fibrillar states. Nonfibrillar Aβ aggregates have been considered as an important intermediate in the pathway of fibrillization, but little is known about the formation mechanism. The on‐pathway β‐sheet intermediates of Aβ₄₀ peptides can be trapped by incubating the peptides in liposomes formed by zwitterionic lipids. The aggregates of Aβ₄₀ peptides have been prepared at a peptide concentration of less than 10 μm . Solid‐state NMR spectroscopy data show that the backbone conformation of the aggregates is almost identical to that of the fibrils formed in free solution. In contrast to anionic lipids, zwitterionic lipids, which are typical of neuronal soma, did not induce any significant conformational difference in Aβ₄₀ fibrils. This liposome–Aβ system may serve as a useful model to study the fibril formation mechanism.     

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.     

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.     

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.     

Bifunctional compounds for controlling metal-mediated aggregation of the aβ42 peptide

Abnormal interactions of Cu and Zn ions with the amyloid β (Aβ) peptide are proposed to play an important role in the pathogenesis of Alzheimer's disease (AD). Disruption of these metal-peptide interactions using chemical agents holds considerable promise as a therapeutic strategy to combat this incurable disease. Reported herein are two bifunctional compounds (BFCs) L1 and L2 that contain both amyloid-binding and metal-chelating molecular motifs. Both L1 and L2 exhibit high stability constants for Cu(2+) and Zn(2+) and thus are good chelators for these metal ions. In addition, L1 and L2 show strong affinity toward Aβ species. Both compounds are efficient inhibitors of the metal-mediated aggregation of the Aβ(42) peptide and promote disaggregation of amyloid fibrils, as observed by ThT fluorescence, native gel electrophoresis/Western blotting, and transmission electron microscopy (TEM). Interestingly, the formation of soluble Aβ(42) oligomers in the presence of metal ions and BFCs leads to an increased cellular toxicity. These results suggest that for the Aβ(42) peptide-in contrast to the Aβ(40) peptide-the previously employed strategy of inhibiting Aβ aggregation and promoting amyloid fibril dissagregation may not be optimal for the development of potential AD therapeutics, due to formation of neurotoxic soluble Aβ(42) oligomers.