Amyloid Aggregates,Presenilins, and Alzheimer's Disease

The cooperative action of three proteases is required to process the APP protein (695–770 amino acids) into small β-amyloid peptides (Aβ, 40–42 amino acids). Aβ aggregates are found in the senile plaques of patients with Alzheimer's disease and play a major role in the onset of this disorder. The functional analysis of several factors that contribute to the production and aggregation of Aβ has enhanced our knowledge of the mechanism of amyloid formation and increased the potential for effective therapeutic treatment.     

Copper(II) ions-immobilized virus-like hollow covalent organic frameworks for highly efficient capture and sensitive analysis of amyloid beta-peptide 1–42 by MALDI-MS

Amyloid beta-peptide 1–42 (Aβ1–42) is one of the biomarkers of Alzheimer's disease, and its selective capture and quantitative detection are important for diagnosis and treatment of Alzheimer's disease. Herein, copper(II) ions-immobilized virus-like hollow covalent organic frameworks (V-HCOFs@Cu²⁺) were synthesized by a facile approach. The as-prepared V-HCOFs@Cu²⁺ showed unique morphology, ultra-high specific surface (2552 m²/g), uniform mesoporous structure (3.2 nm), superior chemical stability and abundant binding sites. Based on these excellent properties, the V-HCOFs@Cu²⁺ could be adopted as an ideal enrichment probe for highly efficient capture of Aβ1–42, exhibiting high adsorption capacity (320 mg/g), and fast adsorption equilibration time (3 min). In addition, an attractive approach of the V-HCOFs@Cu²⁺-based matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was developed for the rapid screening and quantitative analysis of Aβ1–42 in human serum by using C-peptide as an internal standard, which exhibited low limit of detection (LOD, 0.2 fmol/µL), and satisfactory recovery. This work provides an alternative solution for enrichment of biomarkers and also offers the potential applications of COFs in clinical analysis     

Detection of amyloid-beta by Fmoc-KLVFF self-assembled fluorescent nanoparticles for Alzheimer's disease diagnosis

The abnormal aggregation of amyloid-beta(Aβ) has been widely believed to play an important role in the pathogenesis of Alz heimer's disease(AD),which is also recognized as one of the main biomarkers for AD diagnosis.The peptide sequence Lys-Leu-Val-Phe-Phe(KLVFF) is considered as the main driver of the fibrillation of Aβ,which also can be utilized to target Aβ and inhibit its aggregation.In this study,KLVFF and Fmoc-KLVFF fluorescent nanoparticles were self-assembled through zinc coordination and π-πstacking.The recognition of Aβ aggregates including oligomers and fibrils by fluorescent nanoparticles can be realized through aromatic,hydrophobic,and hydrogen-bond interactions.The fluorescent nanoprobes can distinguish Aβ aggregation formats and detect Aβ at the limit of 1 pg/mL(S/N=3).Hence,the detection of Aβ aggregates by fluorescent peptide nanoparticles has great potential for AD diagnosis and progression prediction.     

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.     

The α and β domains of human metallothionein-3 co-operatively protect against Aβ1-42-Cu2+ cytotoxicity

Cytotoxicity of A β with redox active metals in neuronal cells has been implicated in the progression of Alzheimer’s disease （AD）.Zn₇MT-3 protects cell against Aβ-Cu²⁺ toxicity.The roles of single domain proteins（α/β） andα-βdomain-domain interaction of Zn₇MT-3 in its anti-Aβ_1-42-Cu²⁺ toxicity activity were investigated herein.Aβ_1-42 and four mutants of human MT3 （α/βdomain,β（MT3）-α（MTl） and A31-34） were prepared and characterized.Aβ_1-42-Cu²⁺ induced hydroxyl radical and ROS production with/without Zn-MTs were measured by fluorescence spectroscopy and DCFH-DA in living cells,respectively.These results indicate that the two domains form a co-operative unit and each of them is indispensable in conducting its bioactivity.     

Study of the interaction of unaggregated and aggregated amyloid β protein (10–21) with outward potassium channel

Metal ion-induced aggregation of Aβ into insoluble plaques is a central factor in Alzheimer’s disease. Zn²⁺ is the only physiologically available transition metal ion responsible for aggregating A β at pH 7.4. To make it clear that the neurotoxicity of Zn²⁺-induced aggregation of Aβ on neurons is the key to understand Aβ mechanism of action further. In this paper, we choose Aβ (10–21) as the model fragment to research hippocampal CA1 pyramidal neurons. For the first time, we adopt the combination of spectral analysis with patch-clamp technique for the preliminary study of the mutual relations of Zn²⁺, Aβ and ion channel from the cell level. The following expounds upon the effects and mode of action of two forms (unaggregated and aggregated) of Aβ (10–21) on hippocampus outward potassium channel three processes (activation, inactivation and reactivation). It also shows the molecular mechanics of AD from the channel level. These results are significant for the further study of Aβ nosogenesis and the development of new types of target drugs for the treatment of AD.     

Target-driven supramolecular self-assembly for selective amyloid-β photooxygenation against Alzheimer's disease

Photo-oxygenation of β-amyloid (Aβ) has been considered an efficient way to inhibit Aβ aggregation in Alzheimer''s disease (AD). However, current photosensitizers cannot simultaneously achieve enhanced blood–brain barrier (BBB) permeability and selective photooxygenation of Aβ, leading to poor therapeutic efficacy, severe off-target toxicity, and substandard bioavailability. Herein, an Aβ target-driven supramolecular self-assembly (PKNPs) with enhanced BBB penetrability and switchable photoactivity is designed and demonstrated to be effective in preventing Aβ aggregation in vivo. PKNPs are prepared by the self-assembly of the Aβ-targeting peptide KLVFF and an FDA-approved porphyrin derivative (5-(4-carboxyphenyl)-10,15,20-triphenylporphyrin). Due to the photothermal effect of PKNPs, the BBB permeability of PKNPs under irradiation is 8.5-fold higher than that of porphyrin alone. Moreover, upon selective interaction with Aβ, PKNPs undergo morphological change from the spherical to the amorphous form, resulting in a smart transformation from photothermal activity to photodynamic activity. Consequently, the disassembled PKNPs can selectively oxygenate Aβ without affecting off-target proteins (insulin, bovine serum albumin, and human serum albumin). The well-designed PKNPs exhibit not only improved BBB permeability but also highly selective Aβ photooxygenation. Furthermore, in vivo experiments demonstrate that PKNPs can alleviate Aβ-induced neurotoxicity and prolong the life span of the commonly used AD transgenic Caenorhabditis elegans CL2006. Our work may open a new path for using supramolecular self-assemblies as switchable phototheranostics for the selective and effective prevention of Aβ aggregation and related neurotoxicity in AD.

Photo-oxygenation of β-amyloid (Aβ) has been considered an efficient way to inhibit Aβ aggregation in Alzheimer''s disease (AD). We present the first example of Aβ-responsive photodynamic therapy to treatment of AD by using PKNPs self-assemblies.     

Contradictory effect of gold nanoparticle-decorated molybdenum sulfide nanocomposites on amyloid-β-40 aggregation

The effect of gold nanoparticle-decorated molybdenum sulfide (AuNP-MoS₂) nanocomposites on amyloid-β-40 (Aβ₄₀) aggregation was investigated. The interesting discovery was that the effect of AuNP-MoS₂ nanocomposites on Aβ₄₀ aggregation was contradictory. Low concentration of AuNP-MoS₂ nanocomposites could enhance the nucleus formation of Aβ₄₀ peptides and accelerate Aβ₄₀ fibrils aggregation. However, although high concentration of AuNP-MoS₂ nanocomposites could enhance the nucleus formation of Aβ₄₀ peptides, it eventually inhibited Aβ₄₀ aggregation process. It might be attributed to the interaction between AuNP-MoS₂ nanocomposites and Aβ₄₀ peptides. For low concentration of AuNP-MoS₂ nanocomposites, it was acted as nuclei, resulting in the acceleration of the nucleation process. However, the structural flexibility of Aβ₄₀ peptides was limited as the concentration of AuNP-MoS₂ nanocomposites was increased, resulting in the inhibition of Aβ₄₀ aggregation. These findings suggested that AuNP-MoS₂ nanocomposites might have a great potential to design new multifunctional material for future treatment of amyloid-related diseases.     

Molecular mechanisms of amyloid formation in living systems

Fibrillar protein aggregation is a hallmark of a variety of human diseases. Examples include the deposition of amyloid-β and tau in Alzheimer''s disease, and that of α-synuclein in Parkinson''s disease. The molecular mechanisms by which soluble proteins form amyloid fibrils have been extensively studied in the test tube. These investigations have revealed the microscopic steps underlying amyloid formation, and the role of factors such as chaperones that modulate these processes. This perspective explores the question to what extent the mechanisms of amyloid formation elucidated in vitro apply to human disease. The answer is not yet clear, and may differ depending on the protein and the associated disease. Nevertheless, there are striking qualitative similarities between the aggregation behaviour of proteins in vitro and the development of the related diseases. Limited quantitative data obtained in model organisms such as Caenorhabditis elegans support the notion that aggregation mechanisms in vivo can be interpreted using the same biophysical principles established in vitro. These results may however be biased by the high overexpression levels typically used in animal models of protein aggregation diseases. Molecular chaperones have been found to suppress protein aggregation in animal models, but their mechanisms of action have not yet been quantitatively analysed. Several mechanisms are proposed by which the decline of protein quality control with organismal age, but also the intrinsic nature of the aggregation process may contribute to the kinetics of protein aggregation observed in human disease.

The molecular mechanisms of amyloid formation have been studied extensively in test tube reactions. This perspective article addresses the question to what extent these mechanisms apply to the complex situation in living cells and organisms.     

Helical γ-Peptide Foldamers as Dual Inhibitors of Amyloid-β Peptide and Islet Amyloid Polypeptide Oligomerization and Fibrillization

Type 2 diabetes (T2D) and Alzheimer's disease (AD) belong to the 10 deadliest diseases and are sorely lacking in effective treatments. Both pathologies are part of the degenerative disorders named amyloidoses, which involve the misfolding and the aggregation of amyloid peptides, hIAPP for T2D and Aβ_1-42 for AD. While hIAPP and Aβ_1-42 inhibitors have been essentially designed to target β-sheet-rich structures composing the toxic amyloid oligomers and fibrils of these peptides, the strategy aiming at trapping the non-toxic monomers in their helical native conformation has been rarely explored. We report herein the first example of helical foldamers as dual inhibitors of hIAPP and Aβ_1-42 aggregation and able to preserve the monomeric species of both amyloid peptides. A foldamer composed of 4-amino(methyl)-1,3-thiazole-5-carboxylic acid (ATC) units, adopting a 9-helix structure reminiscent of 3₁₀ helix, was remarkable as demonstrated by biophysical assays combining thioflavin-T fluorescence, transmission electronic microscopy, capillary electrophoresis and mass spectrometry.     

Molecular dynamic studies of amyloid-beta interactions with curcumin and Cu<Superscript>2+</Superscript> ions

Amyloid-beta (Aβ) peptide readily forms aggregates that are associated with Alzheimer’s disease. Transition metals play a key role in this process. Recently, it has been shown that curcumin (CUA), a polyphenolic phytochemical, inhibits the aggregation of Aβ peptide. However, interactions of Aβ peptide with metal ions or CUA are not entirely clear. In this work, molecular dynamics (MD) simulations were carried out to clear the nature of interactions between the 42-residue Aβ peptide (Aβ-42) and Cu²⁺ ions and CUA. Altogether nine different models were investigated, and more than 2 µs of the simulation data were analyzed. The models represent the possible modes of arrangement between Aβ-42 and Cu²⁺ ions and CUA, respectively, and were used to shed light on the Aβ-42 conformational behavior in the presence of Cu²⁺ ions and CUA molecules. Obtained data clearly showed that the presence of a CUA molecule or a higher concentration of copper ions significantly affect the conformational behavior of Aβ-42. Calculations showed that the change of the His13 protonation state (Aβ(H13δ)-Cu²⁺, Aβ(H13δ)-Cu²⁺ -CUA models) leads to higher occurrence of the Asp23-Lys28 salt bridge. Analyzes of trajectories revealed that C-terminal β-sheet structures occurred significantly less frequently, and CUA promoted the stabilization of the α-helical structure. Further, calculations of the Aβ-42 complex with CUA and Cu²⁺ ions showed that CUA can chelate the Cu²⁺ ion and directly interact with Aβ, which may explain why CUA acts as an inhibitor of Aβ aggregation.     

Study of the interaction of Zn^2＋ with Aβ（10-21） by fluorescamine assay

Zinc may play a role as a co-factor in the pathogenesis of Alzheimer's disease(AD)through influencing the conformation and neurotoxicity of amyloidβ-protein(Aβ).Using the fluorescamine assay,we show for the first time that Zn~(2 )induced Aβ(10-21) aggregate in a concentration-dependent manner.These results indicate that Aβ(10-21)can be used as an in vitro model in Zn~(2 )- induced Aβaggregation and that the region 10-21 to be the minimal fragment of zinc-binding domain of full length Aβ(1-42).     

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.     

Metal–Organic Frameworks Harness Cu Chelating and Photooxidation Against Amyloid β Aggregation in Vivo

Recently, photooxygenation of amyloid β (Aβ) has emerged as an effective way to inhibit Aβ aggregation in Alzheimer's disease (AD) treatment. However, their further application has been highly obstructed by self-aggregation, no metal chelating ability, and poor protein-enrichment capacity. Herein, porphyrinic metal–organic frameworks (PMOFs) are utilized as a superior Cu^II chelating and photooxidation agent for inhibiting Aβ aggregation. We selected only four classical kinds of POMFs (Zr–MOF, Al–MOF, Ni–MOF, Hf–MOF) for further investigation in our study, which are stable in physiological conditions and exhibit excellent biocompatibility. Among them, Hf–MOF was the most efficient Aβ photooxidant. A possible explanation about the difference in capacity of ¹O₂ generation of these four PMOFs has been provided according to the experimental results and DFT calculations. Furthermore, Hf–MOFs are modified with Aβ-targeting peptide, LPFFD. This can not only enhance Hf–MOFs targeting cellular Aβ to decrease Aβ-induced cytotoxicity, but also improve Aβ photooxidation in the complicated living environment. More intriguingly, in vivo studies indicate that the well-designed LPFFD modified Hf–MOFs can decrease Aβ-induced neurotoxicity and extend the longevity of the commonly used transgenic AD model Caenorhabditis elegans CL2006. Our work may open a new avenue for using MOFs as neurotoxic-metal-chelating and photo-therapeutic agents for AD treatment.     

Direct electrochemical oxidation of amyloid-β peptides via tyrosine,histidine, and methionine residues

The direct electrochemical oxidation of synthetic peptides analogous to the amyloid-beta (Aβ) implicated in the pathogenesis of Alzheimer's disease has been demonstrated on carbon screen-printed electrodes in the phosphate buffer (pH 7.2) by using square wave voltammetry. The full-length peptide Aβ42 was found to produce two peaks at potentials of 0.6, 1.05, and a wave at about 1–1.5 V (vs. Ag/AgCl) assigned, respectively, to its Tyr, His, and Met residues. The correspondence between the oxidation signals and the appropriate residues was established based on the analysis of voltammograms obtained for the free Tyr, His, and Met amino acids; for the Aβ16 peptide (representing the Aβ metal-binding domain and lacking the Met residue); for Aβ16 mutants differing in the number of His residues; and for the rat Aβ16 lacking the Tyr residue. The oxidation signals were found to be proportional to the amino acid and peptide concentrations. The observed difference in the electrochemical behavior of Aβ peptides widens the application of direct electrochemistry to peptide differentiation and point mutation studies as well as to investigation of Aβ aggregation and complexing with metal ions.     

KLVFF peptide functionalized nanoparticles capture Aβ42 by co-assembly for decreasing cytotoxicity

The bis(pyrene)-Lys-Leu-Val-Phe-Phe-Gly-poly ethylene glycol (BP-KLVFFG-PEG) based nanoparticles capture Aβ₄₂ by recognition and co-assembly, the length of PEG chain in which leads to different morphologies of coassemblies and capture efficiency. The co-assembly strategy shows a decrease of cytotoxicity, potentially for Alzheimer's disease treatment.     

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

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

Tacrines as Therapeutic Agents for Alzheimer's Disease. IV. The Tacripyrines and Related Annulated Tacrines

Notwithstanding the clinical use of tacrine was hampered by severe hepatotoxicity, tacrine still remains a reference scaffold in the search for new efficient drugs for Alzheimer's disease therapy. In this account we summarize the efforts toward the development and characterization of non‐hepatotoxic tacripyrines and related tacrine analogues resulting from the substitution of the benzene ring by a 1,4‐dihydropyridine, a 1,2,3,4‐tetrahydropyrimidine or a pyridone nucleus. These efforts have successfully led to the identification of a number of promising hits endowed with interesting multifunctional profiles. These include the 4′‐metoxytacripyrine (S)‐ ITH122 , able to target cholinesterases (ChEs), beta‐amyloid (Aβ) and Ca²⁺ channels, the racemic 3′‐methoxytacripyrimidine EB65F2 , the first fully balanced micromolar inhibitor of ChEs and Ca²⁺ channels, and tacripyrine (?)‐SCR1693 a GSK‐3β (enzyme involved in tau phosphorylation) inhibitor able to also lower Aβ production in N2a cells.     

Quantitative Aspects of Electrochemical Detection of Amyloid‐β Aggregation

The tyrosine based electrochemical analysis of synthetic amyloid‐β (Aβ) peptide – an analog of natural peptide implicated in Alzheimer's disease pathogenesis – was applied for a quantitative estimation of peptide aggregation in vitro. The analysis was carried out by square wave voltammetry (SWV) on carbon screen printed electrodes (SPE). The electrooxidation peak current (I_p) for Aβ42 peptide in different aggregation states was directly compared with the size and structure of Aβ42 aggregates occurring in the analyzed sample. Dynamic light scattering (DLS) and thioflavin T (ThT) based fluorescence assay were employed to estimate the size and structure of Aβ42 aggregates. The I_p was found to decrease in a linear fashion when the average diameter of aggregates and the relative ThT fluorescence in Aβ42 solutions exceeded 35 nm and 3, respectively, while being nearly constant below these values. It was suggested that the electrooxidation current is mostly generated by peptide monomers and that a depletion of the monomer pool due to inclusion of Aβ42 molecules in aggregates is responsible for the decrease of electrooxidation current. The direct electrochemistry is emerging as a method complementary to methods based on aggregates’ detection and commonly employed for monitoring Aβ aggregation. The work further enlarges the basis for application of the cost‐effective and rapid electrochemical techniques, such as SWV on carbon SPE, to in vitro studies of Aβ aggregation.     

Structural Dynamics of Amyloid β Peptide Binding to Acetylcholine Receptor and Virtual Screening for Effective Inhibitors

The interaction between Amyloid β (Aβ) peptide and acetylcholine receptor is the key for our understanding of how Aβ fragments block the ion channels within the synapses and thus induce Alzheimer's disease. Here, molecular docking and molecular dynamics (MD) simulations were performed for the structural dynamics of the docking complex consisting of Aβ and α7-nAChR (α7 nicotinic acetylcholine receptor), and the inter-molecular interactions between ligand and receptor were revealed. The results show that A\begin{document}$ \beta_{25-35} $\end{document} is bound to α7-nAChR through hydrogen bonds and complementary shape, and the A\begin{document}$ \beta_{25-35} $\end{document} fragments would easily assemble in the ion channel of \begin{document}$ \alpha $\end{document}7-nAChR, then block the ion transfer process and induce neuronal apoptosis. The simulated amide-I band of A\begin{document}$ \beta_{25-35} $\end{document} in the complex is located at 1650.5 cm\begin{document}$ ^{-1} $\end{document}, indicating the backbone of A\begin{document}$ \beta_{25-35} $\end{document} tends to present random coil conformation, which is consistent with the result obtained from cluster analysis. Currently existing drugs were used as templates for virtual screening, eight new drugs were designed and semi-flexible docking was performed for their performance. The results show that, the interactions between new drugs and \begin{document}$ \alpha $\end{document}7-nAChR are strong enough to inhibit the aggregation of A\begin{document}$ \beta_{25-35} $\end{document} fragments in the ion channel, and also be of great potential in the treatment of Alzheimer's disease.