Aβ Aggregation Kinetics Research Articles

Secondary Modification of S100B Influences Anti Amyloid-β Aggregation Activity and Alzheimer's Disease Pathology.

Proteinaceous aggregates accumulate in neurodegenerative diseases such as Alzheimer's Disease (AD), inducing cellular defense mechanisms and altering the redox status. S100 pro-inflammatory cytokines, particularly S100B, are activated during AD, but recent findings reveal an unconventional molecular chaperone role for S100B in hindering Aβ aggregation and toxicity. This suggests a potential protective role for S100B at the onset of Aβ proteotoxicity, occurring in a complex biochemical environment prone to oxidative damage. Herein, we report an investigation in which extracellular oxidative conditions are mimicked to test if the susceptibility of S100B to oxidation influences its protective activities. Resorting to mild oxidation of S100B, we observed methionine oxidation as inferred from mass spectrometry, but no cysteine-mediated crosslinking. Structural analysis showed that the folding, structure, and stability of oxidized S100B were not affected, and nor was its quaternary structure. However, studies on Aβ aggregation kinetics indicated that oxidized S100B was more effective in preventing aggregation, potentially linked to the oxidation of Met residues within the S100:Aβ binding cleft that favors interactions. Using a cell culture model to analyze the S100B functions in a highly oxidative milieu, as in AD, we observed that Aβ toxicity is rescued by the co-administration of oxidized S100B to a greater extent than by S100B. Additionally, results suggest a disrupted positive feedback loop involving S100B which is caused by its oxidation, leading to the downstream regulation of IL-17 and IFN-α2 expression as mediated by S100B.

Patterning amyloid-β aggregation under the effect of acetylcholinesterase using a biological nanopore - an in vitro study

Aggregation of amyloid-β peptide (Aβ) is hypothesized to be the primary cause of Alzheimer's disease (AD) progression. Aβ aggregation has been widely studied using conventional sensing tools like emission fluorescence, electron microscopy, mass spectroscopy, and circular dichroism. However, none of these techniques can provide cost-efficient, highly sensitive quantification of Aβ aggregation kinetics at the molecular level. Among the influences on Aβ aggregation of interest to disease progression is the acceleration of Aβ aggregation by acetylcholinesterase (AChE), which is present in the brain and inflicts the fast progression of disease due to its direct interaction with Aβ. In this work, we demonstrate the ability of a biological nanopore to map and quantify AChE accelerated aggregation of Aβ monomers to mixed oligomers and small soluble aggregates with single-molecule precision. This method will allow future work on testing direct and indirect effects of therapeutic drugs on AChE accelerated Aβ aggregation as well as disease prognosis.

Effects of zinc and carnosine on aggregation kinetics of Amyloid-β40 peptide

The accumulation and amyloid formation of amyloid-β (Aβ) peptides is closely associated with the pathology of Alzheimer's disease. The physiological environment wherein Aβ aggregation happens is crowded with a large variety of metal ions including Zn2+. In this study, we investigated the role of Zn2+ in regulating the aggregation kinetics of Aβ40 peptide. Our results show that Zn2+ can shift a typical single sigmoidal aggregation kinetics of Aβ40 to a biphasic aggregation process. Zn2+ aids in initiating the rapid self-assembly of monomers to form oligomeric intermediates, which further grow into amyloid fibrils in the first aggregation phase. The presence of Zn2+ also retards the appearance of the second aggregation phase in a concentration dependent manner. In addition, our results show that a natural dipeptide, carnosine, can greatly alleviate the effect of Zn2+ on Aβ aggregation kinetics, most likely by coordinating with the metal ion to form chelates. These results suggest a potential in vivo protective effect of carnosine against the cytotoxicity of Aβ by suppressing Zn2+-induced rapid formation of Aβ oligomers.

Taylor Dispersion Analysis and Atomic Force Microscopy Provide a Quantitative Insight into the Aggregation Kinetics of Aβ (1-40)/Aβ (1-42) Amyloid Peptide Mixtures.

Aggregation of amyloid β peptides is known to be one of the main processes responsible for Alzheimer's disease. The resulting dementia is believed to be due in part to the formation of potentially toxic oligomers. However, the study of such intermediates and the understanding of how they form are very challenging because they are heterogeneous and transient in nature. Unfortunately, few techniques can quantify, in real time, the proportion and the size of the different soluble species during the aggregation process. In a previous work (Deleanu et al. Anal. Chem. 2021, 93, 6523-6533), we showed the potential of Taylor dispersion analysis (TDA) in amyloid speciation during the aggregation process of Aβ (1-40) and Aβ (1-42). The current work aims at exploring in detail the aggregation of amyloid Aβ (1-40):Aβ (1-42) peptide mixtures with different proportions of each peptide (1:0, 3:1, 1:1, 1:3, and 0:1) using TDA and atomic force microscopy (AFM). TDA allowed for monitoring the kinetics of the amyloid assembly and quantifying the transient intermediates. Complementarily, AFM allowed the formation of insoluble fibrils to be visualized. Together, the two techniques enabled us to study the influence of the peptide ratios on the kinetics and the formation of potentially toxic oligomeric species.

Tau/Aβ chimera peptides: A Thioflavin-T and MALDI-TOF study of Aβ amyloidosis in the presence of Cu(II) or Zn(II) ions and total lipid brain extract (TLBE) vesicles

Currently, Alzheimer's Disease (AD) is a complex neurodegenerative condition, with limited therapeutic options. Several factors, like Amyloid β (Aβ) aggregation, tau protein hyperphosphorylation, bio-metals dyshomeostasis and oxidative stress contribute to AD pathogenesis. These pathogenic processes might occur in the aqueous phase but also on neuronal membranes. Thus, investigating the connection between Aβ and biomembranes, becomes important for unveiling the molecular mechanism underlying Aβ amyloidosis as a critical event in AD pathology. In this work, the interaction of two peptides, made up with hybrid sequences from Tau protein 9−16 (EVMEDHAG) or 26−33 (QGGYTMHQ) N-terminal domain and Aβ16−20 (KLVFF) hydrophobic region, with full length Aβ40 or Aβ42 peptides is reported. The studied "chimera" peptides Ac-EVMEDHAGKLVFF-NH2 (τ9−16-KL) and Ac-QGGYTMHQKLVFF-NH2 (τ26−33-KL) are endowed with Aβ recognition and metal ion interaction capabilities provided by the tau or Aβ sequences, respectively. These peptides were characterized in previous study along with their metal dependent interaction and amyloidogenesis, either in the presence or absence of metal ion and artificial membranes made up with Total Lipid Brain Extract (TLBE) components, (Sciacca et al., 2020). In the present paper, the ability of the two peptides to inhibit Aβ aggregation is studied using composite experimental conditions including aqueous solution, the presence of metal ions (Cu or Zn), the presence of lipid vesicles mimicking neuronal membranes as well as the co-presence of metals and TLBE artificial membranes. We used Thioflavine-T (ThT) fluorescence or MALDI-TOF spectrometry analysis of Aβ limited proteolysis to respectively monitor the Aβ aggregation kinetic or validation of the Aβ interacting regions. We demonstrate that τ9−16-KL and τ26−33-KL peptides differently affect Aβ aggregation kinetics, with the tau sequence playing a crucial role. The results are discussed in terms of chimera's peptides hydrophobicity and electrostatic driven interactions at the aqueous/membrane interface.

Degradation of Alzheimer’s Amyloid-β by a Catalytically Inactive Insulin-Degrading Enzyme

It is known that insulin-degrading-enzyme (IDE) plays a crucial role in the clearance of Alzheimer’s amyloid-β (Aβ). The cysteine-free IDE mutant (cf-E111Q-IDE) is catalytically inactive against insulin, but its effect on Aβ degradation is unknown that would help in the allosteric modulation of the enzyme activity. Herein, the degradation of Aβ(1–40) by cf-E111Q-IDE via a non-chaperone mechanism is demonstrated by NMR and LC-MS, and the aggregation of fragmented peptides is characterized using fluorescence and electron microscopy. cf-E111Q-IDE presented a reduced effect on the aggregation kinetics of Aβ(1–40) when compared with the wild-type IDE. Whereas LC-MS and diffusion ordered NMR spectroscopy revealed the generation of Aβ fragments by both wild-type and cf-E111Q-IDE. The aggregation propensities and the difference in the morphological phenotype of the full-length Aβ(1–40) and its fragments are explained using multi-microseconds molecular dynamics simulations. Notably, our results reveal that zinc binding to Aβ(1–40) inactivates cf-E111Q-IDE’s catalytic function, whereas zinc removal restores its function as evidenced from high-speed AFM, electron microscopy, chromatography, and NMR results. These findings emphasize the catalytic role of cf-E111Q-IDE on Aβ degradation and urge the development of zinc chelators as an alternative therapeutic strategy that switches on/off IDE’s function.

Cobalt(III) Schiff base complexes stabilize non-fibrillar amyloid-β aggregates with reduced toxicity

The aggregation of amyloid-β (Aβ) is believed to be foundational to the pathogenesis of Alzheimer's disease (AD). In vitro aggregation kinetics have been shown to correlate with rates of disease progression in both AD patients and animal models, thus proving to be a useful metric for testing Aβ-targeted therapeutics. Here we present evidence of cobalt(III) Schiff base complex ([Co(acetylacetonate)(NH3)2]Cl; Co(III)-sb) modulation of Aβ aggregation kinetics by a variety of complementary techniques. These include Thioflavin T (ThT) fluorescence, circular dichroism (CD) spectroscopy, transmission electron microscopy (TEM), and atomic force microscopy (AFM). Our data was fitted to kinetic rate laws using a mathematical model developed by Knowles et al. in order to extract mechanistic information about the effect of Co(III)-sb on aggregation kinetics. Our analysis revealed that Co(III)-sb alters Aβ aggregation by decreasing the polymerization rate and increasing the nucleation rate, suggesting that Co(III)-sb causes Aβ to rapidly stabilize oligomeric species with reduced elongation into mature fibrils. This result was corroborated by TEM and AFM of Aβ aggregates in vitro. We also demonstrate that Aβ aggregate mixtures produced in the presence of Co(III)-sb exhibit decreased cytotoxicity compared to untreated samples.

Amyloid-β Interactions with Lipid Rafts in Biomimetic Systems: A Review of Laboratory Methods.

Biomimetic lipid bilayer systems are a useful tool for modeling specific properties of cellular membranes in order to answer key questions about their structure and functions. This approach has prompted scientists from all over the world to create more and more sophisticated model systems in order to decipher the complex lateral and transverse organization of cellular plasma membranes. Among a variety of existing biomembrane domains, lipid rafts are defined as small, dynamic, and ordered assemblies of lipids and proteins, enriched in cholesterol and sphingolipids. Lipid rafts appear to be involved in the development of Alzheimer's disease (AD) by affecting the aggregation of the amyloid-β (Aβ) peptide at neuronal membranes thereby forming toxic oligomeric species. In this review, we summarize the laboratory methods which allow to study the interaction of Aβ with lipid rafts. We describe step by step protocols to form giant (GUVs) and large unilamellar vesicles (LUVs) containing raft-mimicking domains surrounded by membrane nonraft regions. Using fluorescence microscopy GUV imaging protocols, one can design experiments to visualize micron-scale raft-like domains, to determine the micron-scale demixing temperature of a given lipid mixture, construct phase diagram, and photogenerate domains in order to assess the dynamics of raft formation and raft size distribution. LUV fluorescence spectroscopy protocols with proper data analysis can be used to measure molecular packing of raft/nonraft regions of the membrane, to report on nanoscale raft formation and determine nanoscale demixing temperature. Because handling of the Aβ requires dedicated laboratory experience, we present illustrated protocols for Aβ-stock aliquoting, Aβ aqueous solubilization, oligomer preparation, determination of the Aβ concentration before and after filtration. Thioflavin binding, dynamic light scattering, and transmission electron microscopy protocols are described as complementary methods to detect Aβ aggregation kinetics, aggregate sizes, and morphologies of observed aggregates.

Structural Interaction of Apolipoprotein A-I Mimetic Peptide with Amyloid-β Generates Toxic Hetero-oligomers

Apolipoproteins are involved in pathological conditions of Alzheimer's disease (AD), and it has been reported that truncated apolipoprotein fragments and β-amyloid (Aβ) peptides coexist as neurotoxic heteromers within the plaques. Therefore, it is important to investigate these complexes at the molecular level to better understand their properties and roles in the pathology of AD. Here, we present a mechanistic insight into such heteromerization using a structurally homologue apolipoprotein fragment of apoA-I (4F) complexed with Aβ(M1-42) and characterize their toxicity. The 4F peptide slows down the aggregation kinetics of Aβ(M1-42) by constraining its structural plasticity. NMR and CD experiments identified 4F-Aβ(M1-42) heteromers comprised of unstructured Aβ(M1-42) and helical 4F. A uniform two-fold reduction in 15N/1H NMR signal intensities of Aβ(M1-42) with no observable chemical shift perturbation indicated the formation of a large complex, which was further confirmed by diffusion NMR experiments. Microsecond-scale atomistic molecular dynamics simulations showed that 4F interaction with Aβ(M1-42) is electrostatically driven and induces unfolding of Aβ(M1-42). Neurotoxicity profiling of Aβ(M1-42) complexed with 4F confirms a significant reduction in cell viability and neurite growth. Thus, the molecular architecture of heteromerization between 4F and Aβ(M1-42) discovered in this study provides evidence toward our understanding of the role of apolipoproteins or their truncated fragments in exacerbating AD pathology.

Effects of Cu(II) on the aggregation of amyloid-β.

Aberrant aggregation of the Aβ protein is a hallmark of Alzheimer's disease (AD), but no complete characterization of the molecular level pathogenesis has been achieved. A promising hypothesis is that dysfunction of metal ion homeostasis, and consequently, the undesired interaction of metal ions with Aβ, may be central to the development of AD. Qualitatively, most data indicate that Cu(II) induces rapid self-assembly of both Aβ40 and Aβ42 during the initial phase of the aggregation, while at longer time scales fibrillation may occur, depending on the experimental conditions. For Aβ40 and Cu(II):Aβ ≤ 1, most data imply that low concentration of Aβ40 favors nucleation and rapid fibril elongation, while high concentration of Aβ40 favors formation of amorphous aggregates. However, there are conflicting reports on this issue. For Aβ42 and Cu(II):Aβ ≤ 1, there is consensus that the lag time is extended upon addition of Cu(II). For Cu(II):Aβ > 1, the lag time is increased upon interaction with Cu(II), and in most cases fibrillation is not observed, presumably because Cu(II) occupies a second more solvent-exposed binding site, which is more prone to form metal ion-bridged species and cause rapid formation of non-fibrillar aggregates. The interesting N-terminally truncated Aβ11-40 with high affinity for Cu(II), exhibits delay of fibrillation upon addition of 0.4eq. Cu(II). In our view, there are still problems achieving reproducible results in this field, and we provide a shortlist of some of the pitfalls. Finally, we propose a consensus model for the effects of Cu(II) on the aggregation kinetics of Aβ.

Effects of the Aphanizomenon flos-aquae Extract (Klamin®) on a Neurodegeneration Cellular Model

Cyanobacteria have been recognized as a source of bioactive molecules to be employed in nutraceuticals, pharmaceuticals, and functional foods. An extract of Aphanizomenon flos-aquae (AFA), commercialized as Klamin®, was subjected to chemical analysis to determine its compounds. The AFA extract Klamin® resulted to be nontoxic, also at high doses, when administered onto LAN5 neuronal cells. Its scavenging properties against ROS generation were evaluated by using DCFH-DA assay, and its mitochondrial protective role was determined by JC-1 and MitoSOX assays. Klamin® exerts a protective role against beta amyloid- (Aβ-) induced toxicity and against oxidative stress. Anti-inflammatory properties were demonstrated by NFβB nuclear localization and activation of IL-6 and IL-1β inflammatory cytokines through ELISA. Finally, by using thioflavin T (ThT) and fluorimetric measures, we found that Klamin® interferes with Aβ aggregation kinetics, supporting the formation of smaller and nontoxic structures compared to toxic Aβ aggregates alone. Altogether, these data indicate that the AFA extract may play a protective role against mechanisms leading to neurodegeneration.

A mechanistic model to predict effects of cathepsin B and cystatin C on β-amyloid aggregation and degradation

β-Amyloid (Aβ) aggregation is thought to initiate a cascade of neurodegenerative events in Alzheimer's disease (AD). Much effort is underway to develop strategies to reduce Aβ concentration or inhibit aggregation. Cathepsin B (CatB) proteolytically degrades Aβ into non-aggregating fragments but is potently inhibited by cystatin C (CysC). It has been suggested that decreasing CysC would facilitate Aβ clearance by relieving CatB inhibition. However, CysC binds Aβ and inhibits Aβ aggregation, suggesting that an intervention that increases CysC would prevent Aβ aggregation. Both approaches have been tested in animal models, yielding contradictory results, possibly because of the opposing influences of CysC on Aβ degradation versus aggregation. Here, we sought to develop a model that quantitatively predicts the effects of CysC and CatB on Aβ aggregation. Aβ aggregation kinetics in the absence of CatB or CysC was measured. The rate constant for Aβ degradation by CatB and the equilibrium constant for binding of CysC to Aβ were determined. We derived a mathematical model that combines material balances and kinetic rate equations. The model accurately predicted Aβ aggregation kinetics at various CatB and CysC concentrations. We derived approximate expressions for the half-times of degradation and aggregation and show that their ratio can be used to estimate, at any given Aβ, CatB, or CysC concentration, whether Aβ aggregation or degradation will result. Our results may be useful for designing experiments and interpreting results from investigations of manipulation of CysC concentration as an AD therapy.

Lipid membranes catalyse the fibril formation of the amyloid-β (1–42) peptide through lipid-fibril interactions that reinforce secondary pathways

Alzheimer's disease is associated with the aggregation of amyloid-β (Aβ) peptides into oligomers and fibrils. We have explored how model lipid membranes modulate the rate and mechanisms of Aβ(1–42) self-assembly, in order to shed light on how this pathological reaction may occur in the lipid-rich environments that the peptide encounters in the brain. Using a combination of in vitro biophysical experiments and theoretical approaches, we show that zwitterionic DOPC lipid vesicles accelerate the Aβ(1–42) fibril growth rate by interacting specifically with the growing fibrils. We probe this interaction with help of a purpose-developed Förster resonance energy transfer assay that monitors the proximity between a fibril-specific dye and fluorescent lipids in the lipid vesicle membrane. To further rationalise these findings we use mathematical models to fit the aggregation kinetics of Aβ(1–42) and find that lipid vesicles alter specific mechanistic steps in the aggregation reaction; they augment monomer-dependent secondary nucleation at the surface of existing fibrils and facilitate monomer-independent catalytic processes consistent with fibril fragmentation. We further show that DOPC vesicles have no effect on primary nucleation. This finding is consistent with experiments showing that Aβ(1–42) monomers do not directly bind to the lipid bilayer. Taken together, our results show that plain lipid membranes with charge and composition that is representative of outer cell membranes can significantly augment autocatalytic steps in the self-assembly of Aβ(1–42) into fibrils. This new insight suggests that strategies to reduce fibril-lipid interactions in the brain may have therapeutic value.

Familial Alzheimer’s Disease Mutations within the Amyloid Precursor Protein Alter the Aggregation and Conformation of the Amyloid-β Peptide

Most cases of Alzheimer's disease (AD) are sporadic, but a small percentage of AD cases, called familial AD (FAD), are associated with mutations in presenilin 1, presenilin 2, or the amyloid precursor protein. Amyloid precursor protein mutations falling within the amyloid-β (Aβ) sequence lead to a wide range of disease phenotypes. There is increasing evidence that distinct amyloid structures distinguished by amyloid conformation-dependent monoclonal antibodies have similarly distinct roles in pathology. It is possible that this phenotypic diversity of FAD associated with mutations within the Aβ sequence is due to differences in the conformations adopted by mutant Aβ peptides, but the effects of FAD mutations on aggregation kinetics and conformational and morphological changes of the Aβ peptide are poorly defined. To gain more insight into this possibility, we therefore investigated the effects of 11 FAD mutations on the aggregation kinetics of Aβ, as well as its ability to form distinct conformations recognized by a panel of amyloid conformation-specific monoclonal antibodies. We found that most FAD mutations increased the rate of aggregation of Aβ. The FAD mutations also led to the adoption of alternative amyloid conformations distinguished by monoclonal antibodies and resulted in the formation of distinct aggregate morphologies as determined by transmission electron microscopy. In addition, several of the mutant peptides displayed a large reduction in thioflavin T fluorescence, despite forming abundant fibrils indicating that thioflavin T is a probe of conformational polymorphisms rather than a reliable indicator of fibrillization. Taken together, these results indicate that FAD mutations falling within the Aβ sequence lead to dramatic changes in aggregation kinetics and influence the ability of Aβ to form immunologically and morphologically distinct amyloid structures.

Pyroglutamate-modified Aβ(3-42) affects aggregation kinetics of Aβ(1-42) by accelerating primary and secondary pathways.

The aggregation into amyloid fibrils of amyloid-β (Aβ) peptides is a hallmark of Alzheimer's disease. A variety of Aβ peptides have been discovered in vivo, with pyroglutamate-modified Aβ (pEAβ) forming a significant proportion. pEAβ is mainly localized in the core of plaques, suggesting a possible role in inducing and facilitating Aβ oligomerization and accumulation. Despite this potential importance, the aggregation mechanism of pEAβ and its influence on the aggregation kinetics of other Aβ variants have not yet been elucidated. Here we show that pEAβ(3-42) forms fibrils much faster than Aβ(1-42) and the critical concentration above which aggregation was observed was drastically decreased by one order of magnitude compared to Aβ(1-42). We elucidated the co-aggregation mechanism of Aβ(1-42) with pEAβ(3-42). At concentrations at which both species do not aggregate as homofibrils, mixtures of pEAβ(3-42) and Aβ(1-42) aggregate, suggesting the formation of mixed nuclei. We show that the presence of pEAβ(3-42) monomers increases the rate of primary nucleation of Aβ(1-42) and that fibrils of pEAβ(3-42) serve as highly efficient templates for elongation and catalytic surfaces for secondary nucleation of Aβ(1-42). On the other hand, the addition of Aβ(1-42) monomers drastically decelerates the primary and secondary nucleation of pEAβ(3-42) while not altering the pEAβ(3-42) elongation rate. In addition, even moderate concentrations of fibrillar Aβ(1-42) prevent pEAβ(3-42) aggregation, likely due to non-reactive binding of pEAβ(3-42) monomers to the surfaces of Aβ(1-42) fibrils. Thus, pEAβ(3-42) accelerates aggregation of Aβ(1-42) by affecting all individual reaction steps of the aggregation process while Aβ(1-42) dramatically slows down the primary and secondary nucleation of pEAβ(3-42).

Cerebrospinal fluid-induced retardation of amyloid β aggregation correlates with Alzheimer's disease and the APOE ε4 allele

Misfolding and aggregation of amyloid β (Aβ) are key features of Alzheimer's disease (AD) pathogenesis, but the molecular events controlling this process are not known in detail. In vivo, Aβ aggregation and plaque formation occur in the interstitial fluid of the brain extracellular matrix. This fluid communicates freely with cerebrospinal fluid (CSF). Here, we examined the effect of human CSF on Aβ aggregation kinetics in relation to AD diagnosis and carrier status of the apolipoprotein E (APOE) ε4 allele, the main genetic risk factor for sporadic AD. The aggregation of Aβ was inhibited in the presence of CSF and, surprisingly, the effect was more pronounced in APOE ε4 carriers. However, by fractionation of CSF using size exclusion chromatography, it became evident that it was not the ApoE protein itself that conveyed the inhibition, since the retarding species eluted at lower volume, corresponding to a much higher molecular weight, than ApoE monomers. Cholesterol quantification and immunoblotting identified high-density lipoprotein particles in the retarding fractions, indicating that such particles may be responsible for the inhibition. These results add information to the yet unresolved puzzle on how the risk factor of APOE ε4 functions in AD pathogenesis.

Structure-Activity Relationship Studies of Isomeric 2,4-Diaminoquinazolines on β-Amyloid Aggregation Kinetics.

A library of isomeric 2,4-diaminoquinazoline (DAQ) derivatives were synthesized and evaluated for antiaggregation potential toward Aβ40/42. Structure-activity relationship data identified compound 3k (N (4)-(4-bromobenzyl)quinazoline-2,4-diamine) with a 4-bromobenzyl substituent as the most potent inhibitor (Aβ40 IC50 = 80 nM) and was almost 18-fold more potent compared to the reference agent curcumin (Aβ40 IC50 = 1.5 μM). The corresponding N (2)-isomer 4k (N (2)-(4-bromobenzyl)quinazoline-2,4-diamine) was also able to prevent Aβ aggregation (Aβ40 IC50 = 1.7 μM). However, compound 4k exhibited superior inhibition of Aβ42 aggregation (Aβ42 IC50 = 1.7 μM) compared to compound 3k (Aβ42 IC50 = 14.8 μM) and was ∼1.8-fold more potent compared to curcumin (Aβ42 IC50 = 3.1 μM). These results were supported by Aβ aggregation kinetics investigations and transmission electron microscopy studies, which demonstrate the suitability of DAQ ring system to develop antiamyloid agents as pharmacological tools to study Aβ aggregation.

Real-Time Monitoring of Alzheimer's-Related Amyloid Aggregation via Probe Enhancement-Fluorescence Correlation Spectroscopy.

This work describes the use of fluorescence correlation spectroscopy (FCS) and a novel amyloid-binding fluorescent probe, ARCAM 1, to monitor the aggregation of the Alzheimer's disease-associated amyloid β-peptide (Aβ). ARCAM 1 exhibits a large increase in fluorescence emission upon binding to Aβ assemblies, making it an excellent candidate for probe enhancement FCS (PE-FCS). ARCAM 1 binding does not change Aβ aggregation kinetics. It also exhibits greater dynamic range as a probe in reporting aggregate size by FCS in Aβ, when compared to thioflavin T (ThT) or an Aβ peptide modified with a fluorophore. Using fluorescent burst analysis (via PE-FCS) to follow aggregation of Aβ, we detected soluble aggregates at significantly earlier time points compared to typical bulk fluorescence measurements. Autocorrelation analysis revealed the size of these early Aβ assemblies. These results indicate that PE-FCS/ARCAM 1 based assays can detect and provide size characterization of small Aβ aggregation intermediates during the assembly process, which could enable monitoring and study of such aggregates that transiently accumulate in biofluids of patients with Alzheimer's and other neurodegenerative diseases.

Charge Dependent Retardation of Amyloid β Aggregation by Hydrophilic Proteins

The aggregation of amyloid β peptides (Aβ) into amyloid fibrils is implicated in the pathology of Alzheimer's disease. In light of the increasing number of proteins reported to retard Aβ fibril formation, we investigated the influence of small hydrophilic model proteins of different charge on Aβ aggregation kinetics and their interaction with Aβ. We followed the amyloid fibril formation of Aβ40 and Aβ42 using thioflavin T fluorescence in the presence of six charge variants of calbindin D9k and single-chain monellin. The formation of fibrils was verified with transmission electron microscopy. We observe retardation of the aggregation process from proteins with net charge +8, +2, -2, and -4, whereas no effect is observed for proteins with net charge of -6 and -8. The single-chain monellin mutant with the highest net charge, scMN+8, has the largest retarding effect on the amyloid fibril formation process, which is noticeably delayed at as low as a 0.01:1 scMN+8 to Aβ40 molar ratio. scMN+8 is also the mutant with the fastest association to Aβ40 as detected by surface plasmon resonance, although all retarding variants of calbindin D9k and single-chain monellin bind to Aβ40.

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.