amyloid-beta-peptides and Protein-Aggregation--Pathological

Amyloids are a broad class of proteins and peptides that can misfold and assemble into long unbranched fibrils with a cross-β conformation. These misfolding and aggregation events are associated with the onset of a variety of human diseases, among them, Alzheimer's disease, Parkinson's disease, and Huntington disease. Our understanding of amyloids has been greatly supported by fluorescent molecular probes, such as thioflavin-T, which shows an increase in fluorescence emission upon binding to fibrillar aggregates. Since the first application of thioflavin-T in amyloid studies nearly 30 years ago, many probes have emerged exhibiting a variety of responses to amyloids, such as intensity changes, shifts in fluorescence maxima, and variations in lifetimes, among many others. These probes have shed light on a variety of topics including the kinetics of amyloid aggregation, the effectiveness of amyloid aggregation inhibitors, the elucidation of binding sites in amyloid structures, and the staining of amyloids aggregates in vitro, ex vivo, and in vivo. In this Review, we discuss the design, properties, and application of photoactive probes used to study amyloid aggregation, as well as the challenges faced by current probes and techniques, and the novel approaches that are emerging to address these challenges.

Topics: Amyloid beta-Peptides; Amyloidogenic Proteins; Animals; Benzothiazoles; Binding Sites; Fluorescent Dyes; Humans; Models, Molecular; Peptide Fragments; Protein Aggregates; Protein Aggregation, Pathological; Spectrometry, Fluorescence

The abnormal accumulation of amyloid-β (Aβ) peptide in the brain is one of the most important hallmarks of Alzheimer's disease. Aβ is an aggregation-prone and toxic polypeptide with 39-43 residues, derived from the amyloid precursor protein proteolysis process. According to the amyloid hypothesis, abnormal accumulation of Aβ in the brain is the primary influence driving Alzheimer's disease pathologies. Among all kinds of Aβ isoforms, Aβ40 and Aβ42 are believed to be the most important ones. Although these two kinds of Aβ differ only in two amino acid residues, recent studies show that they differ significantly in their metabolism, physiological functions, toxicities, and aggregation mechanism. In this review, we mainly summarize the similarities and differences between Aβ42 and Aβ40, recent studies on selective inhibitors as well as probes will also be mentioned.

Topics: Alzheimer Disease; Amino Acid Sequence; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Antibodies, Monoclonal; Brain Chemistry; Humans; Molecular Probes; Molecular Sequence Data; Peptide Fragments; Protein Aggregation, Pathological; Protein Structure, Secondary; Proteolysis; Styrenes; Thiazoles

Aberrant proteins or peptide aggregates form soluble oligomers or nanofibrils that can cause a wide range of amyloidosis diseases, including Alzheimer's disease (AD). The mechanisms of their cytotoxicity, however, remain controversial and poorly understood, greatly hindering the development of AD drugs. Here we report a comprehensive evaluation of the cytotoxicity of the aggregates by meta-analysis. The analysis indicates that the cytotoxicity of the aggregates converges in a narrower range in the mass concentrations than in the molar concentrations, suggesting that it is the weight of the aggregates rather than the number of the molecules that dictates the cytotoxicity. This new perspective implies that these aggregates are likely to have non-specific interactions with cells to cause cell death. The comparison of several existing theories regarding cellular volumes supports that the aggregates may result in crowding effect and increase the free energy, thus resulting in instability of the cells.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloidosis; Humans; Peptide Fragments; Protein Aggregates; Protein Aggregation, Pathological

Neurotoxicity of amyloid beta (Aβ) species generated in early stages of aggregation has been associated with development of Alzheimer's disease (AD). Consequently, the field of action of compounds that can identify and inhibit the formation of these species has enlarged considerably. This study investigates the effect and influence of the luminescent, water soluble metal complex cis-[Ru(phen)

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Cell Survival; Coordination Complexes; Microscopy, Electron, Transmission; PC12 Cells; Peptide Fragments; Protein Aggregation, Pathological; Rats; Ruthenium; Solubility; Water

Fibril formation of amyloid β (Aβ) peptides is one of the key molecular events connected to Alzheimer's disease. The pathway of formation and mechanism of action of Aβ aggregates in biological systems is still object of very active research. To this end, systematic modifications of the Phe

Topics: Amino Acids; Amyloid; Amyloid beta-Peptides; Humans; Hydrophobic and Hydrophilic Interactions; Mutation; Neurons; Peptide Fragments; Protein Aggregation, Pathological; Protein Conformation; Spectrum Analysis

Alzheimer's disease (AD) and cataract represent two common protein misfolding diseases closely associated with aging. Growing evidence suggests that these two diseases may be interrelated with each other through cross-sequence interactions between β-amyloid (Aβ) peptide and the short aggregating peptides derived from proteolytic breakdown of α-crystallin. αΑ(66-80) is one of several peptides produced by the proteolytic breakdown of α-crystallin in aged eye lens. Although it is evident that the Aβ(1-40) and αΑ(66-80) coexist in aged eye lenses and both the peptides are known to form macromolecular assemblies, their cross-sequence interaction and the seeding behavior are not known. In this study, the aggregation behavior of αΑ(66-80) has been examined in the presence of Aβ(1-40) on using thioflavin T (ThT) based aggregation kinetics. The presence of monomeric Aβ(1-40) augmented the aggregation kinetics of αΑ(66-80) and reduced the lag time of αΑ(66-80) aggregation. However, the addition of Aβ(1-40) or αΑ(66-80) fibrils (seeds) didn't result in any change in the rate of αΑ(66-80) aggregation. In this in vitro study, we could show that the presence Aβ(1-40) has substantial effect on the aggregation of αΑ(66-80), which suggests a possible interaction between AD and cataract pathologies.

Topics: Amyloid beta-Peptides; Cataract; Crystallins; Humans; Peptide Fragments; Protein Aggregation, Pathological

Amyloid β-peptide (Aβ) oligomerization is believed to contribute to the neuronal dysfunction in Alzheimer disease (AD). Despite decades of research, many details of Aβ oligomerization in neurons still need to be revealed. Förster resonance energy transfer (FRET) is a simple but effective way to study molecular interactions. Here, we used a confocal microscope with a sensitive Airyscan detector for FRET detection. By live cell FRET imaging, we detected Aβ42 oligomerization in primary neurons. The neurons were incubated with fluorescently labeled Aβ42 in the cell culture medium for 24 h. Aβ42 were internalized and oligomerized in the lysosomes/late endosomes in a concentration-dependent manner. Both the cellular uptake and intracellular oligomerization of Aβ42 were significantly higher than for Aβ40. These findings provide a better understanding of Aβ42 oligomerization in neurons.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Endosomes; Fluorescence Resonance Energy Transfer; Hippocampus; Humans; Lysosomes; Mice; Mice, Inbred C57BL; Neurons; PC12 Cells; Peptide Fragments; Protein Aggregation, Pathological; Rats

Recent investigations suggest that soluble oligomeric amyloid β (Aβ) species may be involved in early onset of Alzheimer's disease (AD). Using systematic proline replacement, solid-state NMR, and ESR, we identified a toxic turn at position 22 and 23 of Aβ42, the most potent neurotoxic Aβ species. Through radicalization, the toxic turn can induce formation of the C-terminal hydrophobic core to obtain putative Aβ42 dimers and trimers. Synthesized dimer and trimer models showed that the C-terminal hydrophobic core plays a critical role in the formation of high molecular weight oligomers with neurotoxicity. Accordingly, an anti-toxic turn antibody (24B3) that selectively recognizes a toxic dimer model of E22P-Aβ42 was developed. Sandwich enzyme-linked immunosorbent assay with 24B3 and 82E1 detected a significantly higher ratio of Aβ42 with a toxic turn to total Aβ42 in cerebrospinal fluid of AD patients compared with controls, suggesting that 24B3 could be useful for early onset of AD diagnosis.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Antibodies, Monoclonal; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Humans; Mice; Models, Molecular; Neurofibrillary Tangles; Peptide Fragments; Plaque, Amyloid; Proline; Protein Aggregates; Protein Aggregation, Pathological; Protein Structure, Tertiary

A hallmark of Alzheimer's disease (AD) is the formation of senile plaques comprised of the β-amyloid (Aβ) peptide. Aβ fibrillization is a complex nucleation-dependent process involving a variety of metastable intermediate aggregates and features the formation of inter- and intramolecular salt bridges involving lysine residues, K16 and K28. Cationic lysine residues also mediate protein-lipid interactions via association with anionic lipid headgroups. As several toxic mechanisms attributed to Aβ involve membrane interactions, the impact of acetylation on Aβ

Topics: Acetylation; Alzheimer Disease; Amyloid beta-Peptides; Animals; Lipid Bilayers; Lysine; Mice; Peptide Fragments; Protein Aggregation, Pathological

Self-interaction, chaperone binding and posttranslational modification of amyloid-beta (Aβ) is essential in the initiation and propagation of Aβ aggregation. Aggregation results in insoluble Aβ deposits characteristic of Alzheimer's disease (AD) brain lesions, i.e. senile plaques and cerebral amyloid angiopathy. Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes posttranslational modifications including the formation of covalent ε-(γ-glutamyl)lysine isopeptide bonds (molecular crosslinks), and colocalizes with Aβ deposits in AD. Two independent groups recently found that apart from the induction of Aβ oligomerization, the blood-derived transglutaminase member FXIIIa forms stable protein-protein complexes with Aβ independent of the transamidation reaction. Here, we investigated whether also tTG forms rigid protein complexes with Aβ in the absence of catalytic activation. We found that both Aβ

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Amyloid beta-Peptides; Brain; GTP-Binding Proteins; Humans; Peptide Fragments; Protein Aggregation, Pathological; Protein Binding; Protein Glutamine gamma Glutamyltransferase 2; Transglutaminases

The formation of amyloid fibrils from soluble peptide is a hallmark of many neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Characterization of the microscopic reaction processes that underlie these phenomena have yielded insights into the progression of such diseases and may inform rational approaches for the design of drugs to halt them. Experimental evidence suggests that most of these reaction processes are intrinsically catalytic in nature and may display enzymelike saturation effects under conditions typical of biological systems, yet a unified modeling framework accounting for these saturation effects is still lacking. In this paper, we therefore present a universal kinetic model for biofilament formation in which every fundamental process in the reaction network can be catalytic. The single closed-form expression derived is capable of describing with high accuracy a wide range of mechanisms of biofilament formation and providing the first integrated rate law of a system in which multiple reaction processes are saturated. Moreover, its unprecedented mathematical simplicity permits us to very clearly interpret the effects of increasing saturation on the overall kinetics. The effectiveness of the model is illustrated by fitting it to the data of in vitro Aβ40 aggregation. Remarkably, we find that primary nucleation becomes saturated, demonstrating that it must be heterogeneous, occurring at interfaces and not in solution.

Topics: Amyloid; Amyloid beta-Peptides; Catalysis; Humans; Kinetics; Models, Chemical; Peptide Fragments; Protein Aggregation, Pathological

Herpes zoster is associated with an increased dementia and neovascular macular degeneration risk and a decline in glycemic control in diabetes mellitus. Because amyloid is present and pathogenic in these diseases, we quantified amyloid, Aβ40, Aβ42, and amylin in 14 zoster and 10 control plasmas. Compared with controls, zoster plasma had significantly elevated amyloid that correlated with Aβ42 and amylin levels and increased amyloid aggregation with addition of exogenous Aβ42 or amylin. These results suggest that zoster plasma contains factor(s) that promotes aggregation of amyloidogenic peptides, potentially contributing to the toxic amyloid burden and explaining accelerated disease progression following zoster.

Topics: Adult; Aged; Aged, 80 and over; Amyloid beta-Peptides; Case-Control Studies; Female; Gene Expression; Herpes Zoster; Herpesvirus 3, Human; Host-Pathogen Interactions; Humans; Islet Amyloid Polypeptide; Male; Middle Aged; Peptide Fragments; Protein Aggregates; Protein Aggregation, Pathological

High purity and sequence homogeneity of intrinsically disordered proteins are prerequisites for reproducible studies of the kinetics and equilibrium of their self-assembly reactions. Starting from the pure state enables quantitative studies of intrinsic and extrinsic factors in the process to understand its molecular determinants. Here we outline detailed protocols for recombinant expression and purification of ultra-pure amyloid β peptide (Aβ) in sequence homogeneous form, which allows for the setup of reproducible kinetic self-assembly experiments.

Topics: Amyloid beta-Peptides; Escherichia coli; Genetic Vectors; Humans; Intrinsically Disordered Proteins; Kinetics; Peptide Fragments; Protein Aggregates; Protein Aggregation, Pathological; Recombinant Proteins; Reproducibility of Results; Transformation, Bacterial

Cerebral amyloid angiopathy (CAA) and β-amyloid (Aβ) deposition in the brain parenchyma are hallmarks of Alzheimer's disease (AD). We previously reported that platelets contribute to Aβ aggregation in cerebral vessels by secreting the factor clusterin upon binding of Aβ40 to the fibrinogen receptor integrin α

Topics: Adult; Alzheimer Disease; Amyloid beta-Peptides; Animals; Blood Platelets; Cells, Cultured; Fibrinogen; Humans; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Peptide Fragments; Platelet Aggregation; Platelet Glycoprotein GPIIb-IIIa Complex; Platelet Membrane Glycoproteins; Protein Aggregation, Pathological; Protein Binding; Receptors, Collagen; Signal Transduction

Familial forms of Alzheimer's disease (AD) are caused by mutations in the presenilin genes or in the gene encoding for the amyloid precursor protein (APP). Proteolytic cleavage of APP generates the β-amyloid peptide (Aβ), which aggregates into amyloid plaques, one of the major hallmarks of AD. APP mutations within the Aβ sequence, so-called intra-Aβ mutations, cluster around position E693 of APP, which corresponds to position E22 in the Aβ sequence. One of these mutations is the Osaka mutation, E693Δ, which has unique aggregation properties with patients showing unusually low brain amyloid levels on amyloid PET scans. Despite intense research on the pathomechanisms of different intra-Aβ mutants, our knowledge is limited due to controversial findings in various studies. Here, we investigated in an ex vivo experimental system the neuro- and synaptotoxic properties of two intra-Aβ mutants with different intrinsic aggregation propensities, the Osaka mutation E22Δ and the Arctic mutation E22G, and compared them to wild-type (wt) Aβ. Experiments in hippocampal slice cultures from transgenic mice were complemented by treating wild-type slices with recombinantly produced Aβ40 or Aβ42 containing the respective intra-Aβ mutations. Our analyses revealed that wt Aβ and E22G Aβ, both recombinant and transgenic, caused a loss of dendritic spines along with an increase in tau phosphorylation and tau-dependent neurodegeneration. In all experiments, the 42-residue variants of wt and E22G Aβ showed stronger effects than the respective Aβ40 isoforms. In contrast, E22Δ Aβ neither reduced dendritic spine density nor resulted in increased tau phosphorylation or neuronal cell death in our ex vivo system. Our findings suggest that the previously reported major differences in the aggregation kinetics between E22G and E22Δ Aβ are likely reflected in different disease pathomechanisms.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Cell Death; Dendritic Spines; Hippocampus; Humans; In Vitro Techniques; Kinetics; Mice; Mice, Transgenic; Mutant Proteins; Mutation; Neurons; Peptide Fragments; Phosphorylation; Protein Aggregation, Pathological; Recombinant Proteins; Synapses; tau Proteins

The present study aimed to explore the effects of histone deacetylase 6 (HDAC6) on brain injury in rats induced by apolipoprotein E4 (APOE4) and amyloid β protein alloform 1‑40 (Aβ1‑40) copolymerization. The rats were randomly divided into four groups: Control group, sham group, APOE4 + Aβ1‑40 co‑injection group (model group) and HDAC6 inhibitor group (HDAC6 group). The brain injury model was established by co‑injection of APOE4 + Aβ1‑40. Morris water maze experiment was used to observe the spatial memory and learning the ability of rats. Histological changes of the hippocampus were observed by hematoxylin and eosin staining. The mRNA expression levels of choline acetyltransferase (ChAT) and HDAC6 were detected by reverse transcription‑quantitative PCR. Immunohistochemistry was used to detect the protein expression of HDAC6. Western blotting was used to detect the protein expression levels of HDAC6, microtubule‑associated protein tau and glycogen synthase kinase 3β (GSK3β). APOE4 and Aβ1‑40 co‑aggregation decreased the short‑term spatial memory and learning ability of rats, whereas inhibition of HDAC6 activity attenuated the injury. Inhibition of HDAC6 activity resulted in an attenuation of the APOE4 and Aβ1‑40 co‑aggregation‑induced increase in the number of dysplastic hippocampal cells. Further experiments demonstrated that APOE4 and Aβ1‑40 co‑aggregation decreased the expression levels of ChAT mRNA, and the phosphorylation levels of tau GSK3β protein in the hippocampus, whereas inhibition of HDAC6 activity resulted in increased expression of ChAT mRNA, tau protein and GSK3β phosphorylation. The inhibition of HDAC6 activity was also demonstrated to reduce brain injury induced by APOE4 and Aβ1‑40 co‑aggregation in model rats.

Topics: Amyloid beta-Peptides; Animals; Apolipoprotein E4; Brain Injuries; Choline O-Acetyltransferase; Gene Expression Regulation, Enzymologic; Hippocampus; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Male; Maze Learning; Peptide Fragments; Protein Aggregation, Pathological; Rats; Rats, Sprague-Dawley; Spatial Memory; tau Proteins

Aggregation states of amyloid beta peptides for amyloid beta A β 1 - 40 to A β 1 - 42 and A β p 3 - 42 are investigated through small angle neutron scattering (SANS). The knowledge of these small peptides and their aggregation state are of key importance for the comprehension of neurodegenerative diseases (e.g., Alzheimer's disease). The SANS technique allows to study the size and fractal nature of the monomers, oligomers and fibrils of the three different peptides. Results show that all the investigated peptides have monomers with a radius of gyration of the order of 10 Å, while the oligomers and fibrils display differences in size and aggregation ability, with A β p 3 - 42 showing larger oligomers. These properties are strictly related to the toxicity of the corresponding amyloid peptide and indeed to the development of the associated disease.

Topics: Amyloid; Amyloid beta-Peptides; Peptide Fragments; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding; Protein Conformation; Protein Multimerization; Spectrum Analysis

Amyloids are ubiquitous protein aggregates sharing common internal structural features; they are present in all organisms, from prokaryotes to eukaryotes, where they play physiological or pathological roles. Importantly, amyloids, which are generated by aggregation of a range of distinct proteins, could be a key factor in a number of major human disorders, the so-called conformational diseases. Because all amyloids exhibit similar cross-β motifs, one may envisage that molecules capable of blocking the formation of β-sheet structures could abolish aggregation of all amyloid proteins, albeit with different efficacies. Herein, two different β-sheet blockers were tested against a selection of amyloidogenic proteins, encompassing all the major types of amyloid-based disorders. Analysis of their blocking efficiency, using a simple but contrasted cell-based screening procedure, unequivocally confirms that they indeed behave as aggregation pan-inhibitors. The significant inhibitory effects observed for these compounds against all tested amyloidogenic proteins could spur a broader biological evaluation of other known and new amyloid aggregation inhibitors to further determine the potential use of this class of compounds for the universal treatment of conformational diseases.

Topics: Amyloid; Amyloid beta-Peptides; Animals; Drug Discovery; Escherichia coli; Humans; Neuroprotective Agents; Peptide Fragments; Protein Aggregation, Pathological; Protein Structure, Secondary

Sporadic cerebral amyloid angiopathy (CAA) is characterized by cerebrovascular amyloid-β (Aβ) deposition, which leads to lobar hemorrhage and dementia. Biological molecules affecting the development of CAA have not been fully characterized. In this study, we performed proteome analysis of biopsied leptomeningeal and cortical vessels obtained from 6 CAA patients and 5 non-CAA patients who underwent surgery for large lobar hemorrhages. We found that 6 proteins, including Aβ, apolipoprotein E (apoE), clusterin (CLU), albumin, complement C4 and vitronectin were significantly upregulated in the vessels of CAA patients as compared to non-CAA patients. ApoE and CLU were found in all CAA patients. We next examined the effects of apoE and CLU on the early phase of Aβ aggregation, using a simple yet powerful in vitro model of CAA, which recapitulates the intramural periarterial drainage pathway model. We found that physiological concentrations of apoE and CLU delayed the initiation time of amyloid growth kinetics in a concentration-dependent manner. These data indicate that apoE and CLU may act as extracellular chaperones to inhibit Aβ amyloid deposition in CAA.

Topics: Aged; Aged, 80 and over; Amyloid beta-Peptides; Apolipoproteins E; Brain; Cerebral Amyloid Angiopathy; Clusterin; Female; Humans; Kaplan-Meier Estimate; Male; Middle Aged; Peptide Fragments; Protein Aggregation, Pathological; Proteome

Alzheimer's disease (AD) is pathologically characterized by the formation of extracellular senile plaques, predominately comprised of aggregated β-amyloid (Aβ), deposited in the brain. Aβ aggregation can result in a myriad of distinct aggregate species, from soluble oligomers to insoluble fibrils. Aβ strongly interacts with membranes, which can be linked to a variety of potential toxic mechanisms associated with AD. Oxidative damage accompanies the formation of Aβ aggregates, with a 10-50% proportion of Aβ aggregates being oxidized in vivo. Hydrogen peroxide (H

Topics: Amyloid beta-Peptides; Brain; Cell Membrane; Humans; Hydrogen Peroxide; Lipid Metabolism; Lipid Peroxidation; Models, Molecular; Oxidation-Reduction; Peptide Fragments; Protein Aggregates; Protein Aggregation, Pathological

The variation of amyloid β peptide (Aβ) concentration and Aβ aggregation are closely associated with the etiology of Alzheimer's diseases (AD). The interaction of Aβ with the monosialoganglioside-rich neuronal cell membrane has been suggested to influence Aβ aggregation. Therefore, studies on the mechanism of Aβ and sialic acids (SA) interaction would greatly contribute to better understanding the pathogenesis of AD. Herein, we report a novel approach for Aβ-SA interaction analysis and highly sensitive Aβ detection by mimicing the cell surface presentation of SA clusters through engineering of SA-modified peptide nanofiber (SANF). The SANF displayed well-ordered 1D nanostructure with high density of SA on surface. Using FAM-labeled Aβ fragments of Aβ

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Cattle; Glycopeptides; Humans; N-Acetylneuraminic Acid; Nanofibers; Peptide Fragments; Protein Aggregates; Protein Aggregation, Pathological; Spectrometry, Fluorescence

Complex amyloid aggregation of amyloid-β (1-40) (Aβ

Topics: Alzheimer Disease; Amyloid; Amyloid beta-Peptides; Humans; Peptide Fragments; Protein Aggregation, Pathological; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Folding; Protein Stability; Signal Transduction; Solubility

Zinc-induced aggregation of amyloid-β peptides (Aβ) is considered to contribute to the pathogenesis of Alzheimer's disease. While glycosaminoglycans (GAGs) that are commonly present in interneuronal space are known to enhance Aβ self-aggregation in vitro, the impact of GAGs on the formation of zinc-induced amorphous Aβ aggregates has not yet been thoroughly studied. Here, employing dynamic light scattering, bis-ANS fluorimetry, and sedimentation assays, we demonstrate that heparin serving as a representative GAG modulates the kinetics of zinc-induced Aβ42 aggregation in vitro by slowing the rate of aggregate formation and aggregate size growth. By using synthetic Aβ16 peptides to model the Aβ metal-binding domain (MBD), heparin was found to effectively interact with MBDs in complex with zinc ions. We suggest that heparin adsorbs to the surface of growing zinc-induced Aβ42 aggregates via electrostatic interactions, thus creating a steric hindrance that inhibits further inclusion of monomeric and/or oligomeric zinc-Aβ42 complexes. Furthermore, the adsorbed heparin can interfere with the zinc-bridging mechanism of Aβ42 aggregation, requiring the formation of two zinc-mediated interaction interfaces in the MBD. As revealed by computer simulations of the zinc-Aβ16 homodimer complexed with a heparin chain, heparin can interact with the MBD via polar contacts with residues Arg-5 and Tyr-10, resulting in a conformational rearrangement that hampers the formation of the second zinc-mediated interaction in the MBD interface. The findings of this study suggest that GAGs, which are common in the in vivo macromolecular environment, may have a substantial impact on the time course of zinc-induced Aβ aggregation.

Topics: Amyloid beta-Peptides; Heparin; Ions; Kinetics; Molecular Dynamics Simulation; Peptide Fragments; Protein Aggregates; Protein Aggregation, Pathological; Static Electricity; Zinc

We have developed a novel functional nucleic acid aptamer to amyloid-β peptide 1-40 (Aβ1-40) and investigated its potential to detect Aβ peptide fragments in neuropathologically confirmed Alzheimer brain hippocampus tissues samples. Our results demonstrate that the aptamer candidate RNV95 could detect tetrameric/pentameric low-molecular-weight Aβ aggregates in autopsy hippocampal tissue from two neuropathologically confirmed Alzheimer disease cases. Although these are preliminary observations, detailed investigations are under way. This is the first demonstration of aptamer-Aβ binding in Alzheimer brain tissues.

Topics: Alzheimer Disease; Amyloid; Amyloid beta-Peptides; Aptamers, Nucleotide; Hippocampus; Humans; Peptide Fragments; Plaque, Amyloid; Protein Aggregation, Pathological; Protein Binding; Protein Multimerization

The multicomponent nature of neuronal plaques in Alzheimer's disease signifies the possible recruitment of non-Aβ candidates during the amyloid growth of Aβ peptides. Here, we show that amyloid fibrils of Aβ

Topics: Amyloid; Amyloid beta-Peptides; Amyloidosis; Humans; In Vitro Techniques; Models, Molecular; Molecular Docking Simulation; Peptide Fragments; Plaque, Amyloid; Protein Aggregates; Protein Aggregation, Pathological; Protein Interaction Domains and Motifs

A catecholamine neurotransmitter, dopamine (DA), is suggested to be linked to the pathology of dementia; however, the involvement of DA and its structural analogues in the pathogenesis of Alzheimer's disease (AD), the most common form of dementia, composed of multiple pathogenic factors has not been clear. Herein, we report that DA and its rationally designed structural derivatives (1-6) based on DA's oxidative transformation are able to modulate multiple pathological elements found in AD [i.e., metal ions, metal-free amyloid-β (Aβ), metal-bound Aβ (metal-Aβ), and reactive oxygen species (ROS)], with demonstration of detailed molecular-level mechanisms. Our multidisciplinary studies validate that the protective effects of DA and its derivatives on Aβ aggregation and Aβ-mediated toxicity are induced by their oxidative transformation with concomitant ROS generation under aerobic conditions. In particular, DA and the derivatives (i.e., 3 and 4) show their noticeable anti-amyloidogenic ability toward metal-free Aβ and/or metal-Aβ, verified to occur via their oxidative transformation that facilitates Aβ oxidation. Moreover, in primary pan-microglial marker (CD11b)-positive cells, the major producers of inflammatory mediators in the brain, DA and its derivatives significantly diminish inflammation and oxidative stress triggered by lipopolysaccharides and Aβ through the reduced induction of inflammatory mediators as well as upregulated expression of heme oxygenase-1, the enzyme responsible for production of antioxidants. Collectively, we illuminate how DA and its derivatives could prevent multiple pathological features found in AD. The overall studies could advance our understanding regarding distinct roles of neurotransmitters in AD and identify key interactions for alleviation of AD pathology.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Copper; Dopamine; Humans; Inflammation; Metals; Microglia; Oxidative Stress; Peptide Fragments; Protein Aggregation, Pathological; Reactive Oxygen Species; Zinc

Increasing evidence shows that β-amyloid (Aβ) peptides, which are associated with Alzheimer disease (AD), are heavily glycated in patients, suggesting a role of this irreversible nonenzymatic post-translational modification in pathology. Previous reports have shown that glycation increases the toxicity of the Aβ peptides, although little is known about the mechanism. Here, we used the natural metabolic by-product methylglyoxal as a glycating agent and exploited various spectroscopic methods and atomic force microscopy to study how glycation affects the structures of the Aβ40 and Aβ42 peptides, the aggregation pathway, and the morphologies of the resulting aggregates. We found that glycation significantly slows down but does not prevent β-conversion to mature fibers. We propose that the previously reported higher toxicity of the glycated Aβ peptides could be explained by a longer persistence in an oligomeric form, usually believed to be the toxic species.

Topics: Alzheimer Disease; Amino Acid Sequence; Amyloid; Amyloid beta-Peptides; Glycosylation; Humans; Peptide Fragments; Protein Aggregation, Pathological; Protein Conformation; Protein Processing, Post-Translational; Sequence Homology

Small oligomers formed during the aggregation of certain peptides and proteins are highly cytotoxic in numerous neurodegenerative disorders. Because of their transient nature and conformational heterogeneity, however, the structural and biological features of these oligomers are still poorly understood. Here, we describe a method of generating stable oligomers formed by the Alzheimer's Aβ

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Caenorhabditis elegans; Cell Line, Tumor; Cell Survival; Humans; In Vitro Techniques; Movement; Neuroblastoma; Neurons; Peptide Fragments; Polymers; Protein Aggregates; Protein Aggregation, Pathological; Protein Conformation, beta-Strand; Zinc

Aggregation of amyloid-β (Aβ) protein stimulated by Cu

Topics: Amyloid beta-Peptides; Carnosine; Cell Line, Tumor; Chelating Agents; Copper; Humans; Oligopeptides; Peptide Fragments; Protein Aggregation, Pathological; Reactive Oxygen Species

Protein aggregation is a hallmark of several neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. It has been shown that lysine residues play a key role in the formation of these aggregates. Thus, the ability to disrupt aggregate formation by covalently modifying lysine residues could lead to the discovery of therapeutically relevant antiamyloidogenesis compounds. Herein, we demonstrate that an ortho-iminoquinone (IQ) can be utilized to inhibit amyloid aggregation. Using alpha-synuclein and Aβ

Topics: alpha-Synuclein; Amyloid beta-Peptides; Animals; Catechin; Cell Survival; Cells, Cultured; Chickens; Dopaminergic Neurons; HEK293 Cells; Humans; Lysine; Methionine; Mice; Micrococcus luteus; Microtubule-Associated Proteins; Muramidase; Neuroprotective Agents; Oxidation-Reduction; Peptide Fragments; Protein Aggregation, Pathological; Quinones; Tyrosine 3-Monooxygenase

There are three specific regions in the Amyloid beta (Aβ) peptide sequence where variations cause enhanced toxicity in Alzheimer's disease: the N-terminus, the central salt bridge, and the C-terminus. Here, we investigate if there is a close conformational connection between these three regions, which may suggest a concerted mechanism of toxicity. We measure the effects of Zn

Topics: Amyloid; Amyloid beta-Peptides; Animals; Cations, Divalent; Cell Survival; Cells, Cultured; Cerebral Cortex; Curcumin; Microscopy, Electron, Transmission; Neurons; Neuroprotective Agents; Nuclear Magnetic Resonance, Biomolecular; Peptide Fragments; Protein Aggregation, Pathological; Protein Conformation; Rats; Spectrometry, Fluorescence; Zinc

The self-assembling of the amyloid β (Aβ) peptide into neurotoxic aggregates is considered a central event in the pathogenesis of Alzheimer's disease (AD). Based on the "amyloid hypothesis", many efforts have been devoted to designing molecules able to halt disease progression by inhibiting Aβ self-assembly. Here, we combine biophysical (ThT assays, TEM and AFM imaging), biochemical (WB and ESI-MS), and computational (all-atom molecular dynamics) techniques to investigate the capacity of four optically pure components of the natural product silymarin (silybin A, silybin B, 2,3-dehydrosilybin A, 2,3-dehydrosilybin B) to inhibit Aβ aggregation. Despite TEM analysis demonstrated that all the four investigated flavonoids prevent the formation of mature fibrils, ThT assays, WB and AFM investigations showed that only silybin B was able to halt the growth of small-sized protofibrils thus promoting the formation of large, amorphous aggregates. Molecular dynamics (MD) simulations indicated that silybin B interacts mainly with the C-terminal hydrophobic segment

Topics: Amyloid beta-Peptides; Animals; Animals, Genetically Modified; Blotting, Western; Caenorhabditis elegans; Dose-Response Relationship, Drug; Microscopy, Atomic Force; Microscopy, Electron, Transmission; Molecular Dynamics Simulation; Molecular Structure; Neuroprotective Agents; Peptide Fragments; Protein Aggregation, Pathological; Protein Conformation; Silybin; Silymarin

The interaction of the intrinsically disordered polypeptide islet amyloid polypeptide (IAPP), which is associated with type 2 diabetes (T2D), with the Alzheimer's disease amyloid-β (Aβ) peptide modulates their self-assembly into amyloid fibrils and may link the pathogeneses of these two cell-degenerative diseases. However, the molecular determinants of this interaction remain elusive. Using a systematic alanine scan approach, fluorescence spectroscopy, and other biophysical methods, including heterocomplex pulldown assays, far-UV CD spectroscopy, the thioflavin T binding assay, transmission EM, and molecular dynamics simulations, here we identified single aromatic/hydrophobic residues within the amyloid core IAPP region as hot spots or key residues of its cross-interaction with Aβ40(42) peptide. Importantly, we also find that none of these residues in isolation plays a key role in IAPP self-assembly, whereas simultaneous substitution of four aromatic/hydrophobic residues with Ala dramatically impairs both IAPP self-assembly and hetero-assembly with Aβ40(42). Furthermore, our experiments yielded several novel IAPP analogs, whose sequences are highly similar to that of IAPP but have distinct amyloid self- or cross-interaction potentials. The identified similarities and major differences controlling IAPP cross-peptide interaction with Aβ40(42)

Topics: Amino Acid Substitution; Amino Acids; Amino Acids, Aromatic; Amyloid beta-Peptides; Circular Dichroism; Humans; Hydrophobic and Hydrophilic Interactions; Islet Amyloid Polypeptide; Kinetics; Methylation; Microscopy, Electron, Transmission; Models, Molecular; Molecular Dynamics Simulation; Peptide Fragments; Protein Aggregation, Pathological; Protein Stability; Protein Structure, Secondary; Solid-Phase Synthesis Techniques; Solubility; Spectrometry, Fluorescence

Alzheimer's disease (AD) is the most prevalent disorder of senile dementia mainly characterized by amyloid-beta peptide (Aβ) deposits in the brain. Cannabinoids are relevant to AD as they exert several beneficial effects in many models of this disease. Still, whether the endocannabinoid system is either up- or down-regulated in AD has not yet been fully elucidated. Thus, the aim of the present paper was to analyze endocannabinoid 2-arachidonoylglycerol (2-AG) metabolism in cerebral cortex synaptosomes incubated with Aβ oligomers or fibrils. These Aβ conformations were obtained by "aging" the 1-40 fragment of the peptide under different agitation and time conditions. A diminished availability of 2-AG resulting from a significant decrease in diacylglycerol lipase (DAGL) activity was observed in the presence of large Aβ

Topics: Amyloid beta-Peptides; Animals; Arachidonic Acids; Cerebral Cortex; Endocannabinoids; Glycerides; Humans; Lipid Peroxidation; Lipoprotein Lipase; Microscopy, Electron, Transmission; Mitochondria; Peptide Fragments; Protein Aggregation, Pathological; Rats, Wistar; Synaptosomes

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

Topics: Algorithms; Amino Acid Substitution; Amyloid beta-Peptides; Cathepsin B; Cystatin C; Fluorescence Resonance Energy Transfer; Humans; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Nerve Tissue Proteins; Osmolar Concentration; Peptide Fragments; Protein Aggregation, Pathological; Protein Interaction Domains and Motifs; Proteolysis; Recombinant Proteins; Reproducibility of Results; Solubility

Using a predictive coarse-grained protein force field, we compute and compare the free energy landscapes and relative stabilities of amyloid-β protein (1-42) and amyloid-β protein (1-40) in their monomeric and oligomeric forms up to the octamer. At the same concentration, the aggregation free energy profile of Aβ42 is more downhill, with a computed solubility that is about 10 times smaller than that of Aβ40. At a concentration of 40 μM, the clear free energy barrier between the pre-fibrillar tetramer form and the fibrillar pentamer in the Aβ40 aggregation landscape disappears for Aβ42, suggesting that the Aβ42 tetramer has a more diverse structural range. To further compare the landscapes, we develop a cluster analysis based on the structural similarity between configurations and use it to construct an oligomerization map that captures the paths of easy interconversion between different but structurally similar states of oligomers for both species. A taxonomy of the oligomer species based on β-sheet stacking topologies is proposed. The comparison of the two oligomerization maps highlights several key differences in the landscapes that can be attributed to the two additional C-terminal residues that Aβ40 lacks. In general, the two terminal residues strongly stabilize the oligomeric structures for Aβ42 relative to Aβ40, and greatly facilitate the conversion from pre-fibrillar trimers to fibrillar tetramers.

Topics: Amyloid beta-Peptides; Humans; Models, Molecular; Peptide Fragments; Protein Aggregation, Pathological; Thermodynamics

A series of 7-substituted coumarin derivatives were designed and synthesised to display ChE and MAO-B inhibitory activity. The compounds consisted out of a coumarin structure (MAO-B inhibitor) and benzyl-, piperidine-, N-benzylpiperidine- or p-bromo-N-benzylpiperizine moiety, resembling the N-benzylpiperidine function of donepezil (ChE inhibitor), connected via an alkyl ether linkage at the 7 position. The biological assay results indicated that all the compounds (1-25) displayed selective inhibition to hMAO-B over hMAO-A, with the benzyloxy series (1-8, 10-13) showing nano-molar hMAO-B inhibition (IC

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Benzyl Compounds; Binding Sites; Cholinesterase Inhibitors; Coumarins; Dose-Response Relationship, Drug; Humans; Monoamine Oxidase; Monoamine Oxidase Inhibitors; Peptide Fragments; Piperidines; Protein Aggregation, Pathological; Protein Binding; Structure-Activity Relationship

Low pH has a strong stabilising effect on the fibrillar assembly of amyloid β, which is associated with Alzheimer's disease. The stabilising effect is already pronounced at pH 6.0, suggesting that protonation of histidines might mediate this effect. Through the systematic substitution of the three native histidines in Aβ for alanines, we have evaluated their role in fibril stability. Using surface plasmon resonance, we show that at neutral pH the fibrillar forms of all His-Ala variants are destabilised by a factor of 4-12 compared to wild-type Aβ. However, none of the His-Ala Aβ variants impair the stabilising effect of the fibril at low pH.

Topics: Amino Acid Substitution; Amyloid; Amyloid beta-Peptides; Histidine; Humans; Hydrogen-Ion Concentration; Kinetics; Microscopy, Electron, Transmission; Mutation; Peptide Fragments; Protein Aggregation, Pathological; Protein Stability; Recombinant Proteins; Surface Plasmon Resonance

The self-assembly of two derivatives of KLVFF, a fragment Aβ(16-20) of the amyloid beta (Aβ) peptide, is investigated and recovery of viability of neuroblastoma cells exposed to Aβ (1-42) is observed at sub-stoichiometric peptide concentrations. Fluorescence assays show that NH

Topics: Amino Acid Sequence; Amyloid beta-Peptides; Amyloidosis; Animals; Cell Survival; Molecular Structure; Neurons; Peptide Fragments; Protein Aggregates; Protein Aggregation, Pathological; Protein Structure, Secondary; Rats; Spectrometry, Mass, Electrospray Ionization; Spectroscopy, Fourier Transform Infrared; X-Ray Diffraction

Brain accumulation of the amyloid-β protein (Aβ) and synapse loss are neuropathological hallmarks of Alzheimer disease (AD). Aβ oligomers (AβOs) are synaptotoxins that build up in the brains of patients and are thought to contribute to memory impairment in AD. Thus, identification of novel synaptic components that are targeted by AβOs may contribute to the elucidation of disease-relevant mechanisms. Trans-synaptic interactions between neurexins (Nrxs) and neuroligins (NLs) are essential for synapse structure, stability, and function, and reduced NL levels have been associated recently with AD. Here we investigated whether the interaction of AβOs with Nrxs or NLs mediates synapse damage and cognitive impairment in AD models. We found that AβOs interact with different isoforms of Nrx and NL, including Nrx2α and NL1. Anti-Nrx2α and anti-NL1 antibodies reduced AβO binding to hippocampal neurons and prevented AβO-induced neuronal oxidative stress and synapse loss. Anti-Nrx2α and anti-NL1 antibodies further blocked memory impairment induced by AβOs in mice. The results indicate that Nrx2α and NL1 are targets of AβOs and that prevention of this interaction reduces the deleterious impact of AβOs on synapses and cognition. Identification of Nrx2α and NL1 as synaptic components that interact with AβOs may pave the way for development of novel approaches aimed at halting synapse failure and cognitive loss in AD.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Brain; Cell Adhesion Molecules, Neuronal; Cells, Cultured; Disease Models, Animal; Humans; Male; Mice; Nerve Tissue Proteins; Peptide Fragments; Protein Aggregation, Pathological; Rats; Rats, Wistar; Synapses

The intracellular and extracellular accumulation of disordered proteins and aggregated proteins occurs in many protein conformational diseases, such as aging-related neurodegeneration and alcoholic liver diseases. However, the conventional methods to study protein structural changes are limited for the rapid detection and monitoring of protein aggregation because of long incubation times (i.e., usually several days), complicated sample pretreatment steps, and expensive instrumentation. Here, we describe an ultrafast colorimetric method for the real-time monitoring of protein structure evolution and the determination of predominant structures via nanoparticle-assisted protein aggregation. During the aggregation process, nanoparticles act as nucleation cores, which form networks depending on the structures of the protein aggregates, and accelerate the kinetics of the protein aggregation. Simultaneously, these nanoparticles exhibit colorimetric responses according to their embedded shapes (e.g., fibrillar and amorphous) on the protein aggregates. We observed distinct spectral shifts and concomitant colorimetric responses of concentration- and type-dependent protein aggregation with the naked eye within a few minutes (<2 min) under acidic conditions. Moreover, the morphological transitions from small aggregates to larger aggregates of nanoparticle-assisted protein aggregates were visualized with dark-field microscope imaging, which show a similar trend with that of protein aggregates formed without the aid of nanoparticles. Finally we show that our proposed method can be utilized to screen the protein aggregation propensity under a variety of conditions such as different pH levels, high temperature, and chemicals. These findings suggest that the proposed method is an easy way to study the molecular biophysics of protein aggregation and to rapidly screen anti-aggregation drugs for protein conformational diseases.

Topics: Amyloid beta-Peptides; Gold; Humans; Hydrogen-Ion Concentration; Metal Nanoparticles; Peptide Fragments; Protein Aggregation, Pathological; Protein Conformation

Oligomeric assemblies are postulated to be proximate neurotoxic species in human diseases associated with aberrant protein aggregation. Their heterogeneous and transient nature makes their structural characterization difficult. Size distributions of oligomers of several amyloidogenic proteins, including amyloid β-protein (Aβ) relevant to Alzheimer's disease (AD), have been previously characterized in vitro by photo-induced cross-linking of unmodified proteins (PICUP) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Due to non-physiological conditions associated with the PICUP chemistry, Aβ oligomers cross-linked by PICUP may not be representative of in vivo conditions. Here, we examine an alternative Copper and Hydrogen peroxide Induced Cross-linking of Unmodified Proteins (CHICUP), which utilizes naturally occurring divalent copper ions and hydrogen peroxide and does not require photo activation. Our results demonstrate that CHICUP and PICUP applied to the two predominant Aβ alloforms, Aβ40 and Aβ42, result in similar oligomer size distributions. Thioflavin T fluorescence data and atomic force microscopy images demonstrate that both CHICUP and PICUP stabilize Aβ oligomers and attenuate fibril formation. Relative to noncross-linked peptides, CHICUP-treated Aβ40 and Aβ42 cause prolonged disruption to biomimetic lipid vesicles. CHICUP-stabilized Aβ oligomers link the amyloid cascade, metal, and oxidative stress hypotheses of AD into a more comprehensive understanding of the molecular basis of AD pathology. Because copper and hydrogen peroxide are elevated in the AD brain, CHICUP-stabilized Aβ oligomers are biologically relevant and should be further explored as a new therapeutic target.

Topics: Alzheimer Disease; Amyloid; Amyloid beta-Peptides; Copper; Humans; Hydrogen Peroxide; Microscopy, Atomic Force; Peptide Fragments; Protein Aggregation, Pathological; Protein Folding

Amyloid β (Aβ) peptide aggregation is the main molecular mechanism underlying the development of Alzheimer's disease, the most widespread form of senile dementia worldwide. Increasing evidence suggests that the key factor leading to impaired neuronal function is accumulation of water-soluble Aβ oligomers rather than formation of the senile plaques created by the deposition of large fibrillary aggregates of Aβ. However, several questions remain about the preliminary steps and the progression of Aβ oligomerization.. We show that the initial stages of the aggregation of fluorescently labeled Aβ can be determined with a high degree of precision and at physiological (i.e., nanomolar) concentrations by using either steady-state fluorimetry or time-correlated single-photon counting.. We study the dependence of the oligomerization extent and rate on the Aβ concentration. We determine the chemical binding affinity of fluorescently labeled Aβ for liposomes that have been recently shown to be pharmacologically active in vivo, reducing the Aβ burden within the brain. We also probe their capacity to hinder the Aβ oligomerization process in vitro.. We introduced a fluorescence assay allowing investigation of the earliest steps of Aβ oligomerization, the peptide involved in Alzheimer's disease. The assay proved to be sensitive even at Aβ concentrations as low as those physiologically observed in the cerebrospinal fluid.. This work represents an extensive and quantitative study on the initial events of Aβ oligomerization at physiological concentration. It may enhance our comprehension of the molecular mechanisms leading to Alzheimer's disease, thus paving the way to novel therapeutic strategies.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Humans; Liposomes; Peptide Fragments; Protein Aggregation, Pathological; Spectrometry, Fluorescence

Brain deposition of Aβ in the form of amyloid plaques is a pathological hallmark of Alzheimer's disease. There are two major species of Aβ in the brain: Aβ42 and Aβ40. Although Aβ40 is several-fold more abundant than Aβ42 in soluble form, Aβ42 is the major component of amyloid plaques. Structural knowledge of Aβ42 fibrils is important both for understanding the process of Aβ aggregation and for designing fibril-targeting drugs. Here we report site-specific structural information of Aβ42 fibrils at 22 residue positions based on electron paramagnetic resonance data. In combination with structure prediction program Rosetta, we modeled Aβ42 fibril structure at atomic resolution. Our Aβ42 fibril model consists of four parallel in-register β-sheets: βN (residues ∼7-13), β1 (residues ∼17-20), β2 (residues ∼32-36), and βC (residues 39-41). The region of β1-loop-β2 in Aβ42 fibrils adopts similar structure as that in Aβ40 fibrils. This is consistent with our cross seeding data that Aβ42 fibril seeds shortened the lag phase of Aβ40 fibrillization. On the other hand, Aβ42 fibrils contain a C-terminal β-arc-β motif with a special turn, termed "arc", at residues 37-38, which is absent in Aβ40 fibrils. Our results can explain both the higher aggregation propensity of Aβ42 and the importance of Aβ42 to Aβ40 ratio in the pathogenesis of Alzheimer's disease.

Topics: Alzheimer Disease; Amyloid; Amyloid beta-Peptides; Electron Spin Resonance Spectroscopy; Humans; Microscopy, Electron, Transmission; Models, Molecular; Mutation; Peptide Fragments; Protein Aggregates; Protein Aggregation, Pathological; Protein Structure, Tertiary

Small proteins like amyloid beta (Aβ) monomers are related to neurodegenerative disorders by aggregation to insoluble fibrils. Small angle neutron scattering (SANS) is a nondestructive method to observe the aggregation process in solution. We show that SANS is able to resolve monomers of small molecular weight like Aβ for aggregation studies. We examine Aβ monomers after prolonged storing in d-hexafluoroisopropanol (dHFIP) by using SANS and dynamic light scattering (DLS). We determined the radius of gyration from SANS as 1.0±0.1 nm for Aβ1-40 and 1.6±0.1 nm for Aβ1-42 in agreement with 3D NMR structures in similar solvents suggesting a solvent surface layer with 5% increased density. After initial dissolution in dHFIP Aβ aggregates sediment with a major component of pure monomers showing a hydrodynamic radius of 1.8±0.3 nm for Aβ1-40 and 3.2±0.4 nm for Aβ1-42 including a surface layer of dHFIP solvent molecules.

Topics: Amyloid beta-Peptides; Humans; Hydrodynamics; Hydrogen Bonding; Models, Chemical; Models, Molecular; Neutron Diffraction; Peptide Fragments; Propanols; Protein Aggregation, Pathological; Protein Conformation; Scattering, Small Angle; Solvents

Cellular membrane disruption induced by β-amyloid (Aβ) peptides has been considered one of the major pathological mechanisms for Alzheimer disease. Mechanistic studies of the membrane disruption process at a high-resolution level, on the other hand, are hindered by the co-existence of multiple possible pathways, even in simplified model systems such as the phospholipid liposome. Therefore, separation of these pathways is crucial to achieve an in-depth understanding of the Aβ-induced membrane disruption process. This study, which utilized a combination of multiple biophysical techniques, shows that the peptide-to-lipid (P:L) molar ratio is an important factor that regulates the selection of dominant membrane disruption pathways in the presence of 40-residue Aβ peptides in liposomes. Three distinct pathways (fibrillation with membrane content leakage, vesicle fusion, and lipid uptake through a temporarily stable ionic channel) become dominant in model liposome systems under specific conditions. These individual systems are characterized by both the initial states of Aβ peptides and the P:L molar ratio. Our results demonstrated the possibility to generate simplified Aβ-membrane model systems with a homogeneous membrane disruption pathway, which will benefit high-resolution mechanistic studies in the future. Fundamentally, the possibility of pathway selection controlled by P:L suggests that the driving forces for Aβ aggregation and Aβ-membrane interactions may be similar at the molecular level.

Topics: Alzheimer Disease; Amyloid; Amyloid beta-Peptides; Cell Membrane; Circular Dichroism; Humans; Ion Channels; Lipid Bilayers; Liposomes; Magnetic Resonance Spectroscopy; Membrane Lipids; Microscopy, Confocal; Peptide Fragments; Phospholipids; Protein Aggregation, Pathological; Protein Binding; Spectrometry, Fluorescence

Amyloid-beta (Aβ) peptide oligomerization plays a central role in the pathogenesis of Alzheimer's disease (AD), and Aβ oligomers are collectively considered an appealing therapeutic target for the treatment of AD. However, the molecular mechanisms leading to the pathologic accumulation of oligomers are unclear, and the exact structural composition of oligomers is being debated. Using targeted and quantitative mass spectrometry, we reveal site-specific Aβ autocleavage during the early phase of aggregation, producing a typical Aβ fragment signature and that truncated Aβ peptides can form stable oligomeric complexes with full-length Aβ peptide. We show that the use of novel anti-Aβ antibodies raised against these truncated Aβ isoforms allows for monitoring and targeting the accumulation of truncated Aβ fragments. Antibody-enabled screening of transgenic models of AD as well as human postmortem brain tissue and cerebrospinal fluid revealed that aggregation-associated Aβ cleavage is a highly relevant clinical feature of AD.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Autoantibodies; Brain; Chromatography, Gel; Chromatography, Liquid; Electrophoresis, Polyacrylamide Gel; Humans; Immunoblotting; Mass Spectrometry; Mice, Transgenic; Peptide Fragments; Protein Aggregation, Pathological; Protein Isoforms; Protein Multimerization; Protein Structure, Secondary

Several studies have addressed the utility of cerebrospinal (CSF) α-synuclein levels as a potential biomarker of α-synuclein aggregation disorders. However, its relevance in the differential diagnostic context of neurodegenerative and movement disorders is still a contentious subject. Here, we report total CSF α-synuclein levels in a cohort of clinically diagnosed α-synuclein-related disorders encompassing Parkinson's disease, Parkinson's disease dementia, dementia with Lewy bodies and multiple system atrophy in comparison to essential tremor and neurological control cases. α-synuclein levels in α-synuclein-related disorders were significantly lower than in controls (p < 0.001). However, in the differential diagnostic context, only Parkinson's disease cases presented significant lower α-synuclein levels compared to essential tremor and neurological controls. In cases with clinically diagnosed α-synuclein pathology, CSF α-synuclein levels showed a moderate positive correlation with CSF tau and p-tau, but not with Aβ42 levels. Due to elevated CSF tau levels in dementia with Lewy bodies samples, tau/α-synuclein ratio showed a good clinical accuracy in discriminating controls from dementia with Lewy bodies cases (AUC = 0.8776) compared to single α-synuclein (AUC = 0.7192) and tau (AUC = 0.7739) levels. In conclusion, α-synuclein alone lacks of clinical value as a biomarker of α-synuclein-related disorders, but in combination with total tau, it may improve the diagnosis of dementia with Lewy bodies.

Topics: Aged; Amyloid beta-Peptides; Biomarkers; Enzyme-Linked Immunosorbent Assay; Female; Humans; Lewy Body Disease; Male; Middle Aged; Peptide Fragments; Protein Aggregation, Pathological; ROC Curve; Statistics, Nonparametric; tau Proteins

A predictive coarse-grained protein force field [associative memory, water-mediated, structure, and energy model for molecular dynamics (AWSEM)-MD] is used to study the energy landscapes and relative stabilities of amyloid-β protein (1-40) in the monomer and all of its oligomeric forms up to an octamer. We find that an isolated monomer is mainly disordered with a short α-helix formed at the central hydrophobic core region (L17-D23). A less stable hairpin structure, however, becomes increasingly more stable in oligomers, where hydrogen bonds can form between neighboring monomers. We explore the structure and stability of both prefibrillar oligomers that consist of mainly antiparallel β-sheets and fibrillar oligomers with only parallel β-sheets. Prefibrillar oligomers are polymorphic but typically take on a cylindrin-like shape composed of mostly antiparallel β-strands. At the concentration of the simulation, the aggregation free energy landscape is nearly downhill. We use umbrella sampling along a structural progress coordinate for interconversion between prefibrillar and fibrillar forms to identify a conversion pathway between these forms. The fibrillar oligomer only becomes favored over its prefibrillar counterpart in the pentamer where an interconversion bottleneck appears. The structural characterization of the pathway along with statistical mechanical perturbation theory allow us to evaluate the effects of concentration on the free energy landscape of aggregation as well as the effects of the Dutch and Arctic mutations associated with early onset of Alzheimer's disease.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Humans; Molecular Dynamics Simulation; Mutation; Peptide Fragments; Protein Aggregates; Protein Aggregation, Pathological; Protein Conformation; Protein Domains; Protein Folding; Protein Multimerization; Thermodynamics

Mutations within the β-amyloid peptide (Aβ) sequence that cause early onset familial Alzheimer's disease (FAD) have been shown to promote Aβ aggregation. How these FAD-related mutants increase the aggregative ability of Aβ is not fully understood. Here, we characterized the effect of the Arctic variant (E22G) on the conformational stability of Aβ using various forms of spectroscopy and kinetic analyses, including nuclear magnetic resonance (NMR), circular dichroism (CD) spectroscopy, Fourier-transform infrared (FT-IR) spectroscopy and transmission electron microscopy (TEM). The E22G mutation in the Arctic variant reduced the α-helical propensity and conformational stability of Aβ on residues 15-25. This mutation also caused an increase in both α-helix-to-β-strand conversion and fibril nucleation rates. Our results suggest that the α-helical propensity of residues 15-25 may play a determinant role in the aggregative ability of Aβ. This may provide a structural basis for understanding the molecular mechanism of Aβ aggregation.

Topics: Amino Acid Sequence; Amyloid beta-Peptides; Humans; Kinetics; Mutation, Missense; Peptide Fragments; Protein Aggregation, Pathological; Protein Structure, Secondary; Sodium Dodecyl Sulfate; Surface-Active Agents

Dysfunctional interactions of amyloid-β (Aβ) with Zn and Cu ions are proved to be related to the etiology of Alzheimer's disease (AD). Disruption of these metal-Aβ interactions using metal chelators holds considerable promise as a therapeutic strategy to combat this incurable disease. Herein, we report that two cyclam derivatives (L1 and L2) are capable of modulating Zn(2+)/Cu(2+)-mediated Aβ40 aggregation, reactive oxygen species (ROS) production, and neurotoxicity. These chelators were found to inhibit the metal-induced Aβ40 aggregation, dissociate metal-Aβ40 aggregates and restore the metal-induced β-sheet structure of Aβ40 to its random coil conformation, as observed by BCA protein assay, thioflavin T fluorescence and circular dichroism spectroscopy. Moreover, preliminary investigation of SH-SY5Y cells indicates that L1 and L2 can diminish the neurotoxicity of metal-Aβ40 species, control metal-Aβ40-triggered ROS production and protect cells against apoptosis. These observations warrant the further investigations of L1 and L2 as potential anti-AD agents.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Apoptosis; Cell Line; Chelating Agents; Copper; Heterocyclic Compounds; Humans; Metals; Neurons; Peptide Fragments; Polyamines; Protein Aggregates; Protein Aggregation, Pathological; Protein Conformation; Reactive Oxygen Species; Zinc

Beta amyloid (Aβ) deposition is a pathological milestone of Alzheimer's disease (AD). This is facilitated by an isoform of Apolipoprotein E4 (ApoE4), which is a dominant risk factor for AD. However, current in vitro Aβ aggregation assays were performed in extreme conditions not linked to physiological conditions, to understand the mechanism of Aβ induced neurotoxicity. Here, we present a simple method for the ApoE4-mediated Aβ aggregation at physiological conditions using single gold nanoparticle based on localized surface plasmon resonance (LSPR). It can be directly observed by dark-field microscope or even by the naked eye. Following LSPR principles, we used ApoE4 inducing Aβ42 self-assemblies on gold nanoparticles (AuNPs) surface via their surface charge interaction. Using physiologically mimic cerebrospinal fluid, we determined a detection limit of 1.5 pM for Aβ42 corresponding to the ~2.9 nm LSPR-peak shift under ApoE4. Interestingly, the result also shows that ApoE4 induces the aggregation of Aβ42 more specifically and rapidly than that of Aβ40. This is the first biomimetic platform for real-time detection of Aβ aggregation, mimicking biological conditions, which can be used to investigate AD directly.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Apolipoprotein E4; Gold; Humans; Limit of Detection; Metal Nanoparticles; Peptide Fragments; Protein Aggregates; Protein Aggregation, Pathological; Surface Plasmon Resonance

The multifactorial pathogenesis of Alzheimer's disease (AD) implicates that multi-target-directed ligands (MTDLs) intervention may represent a promising therapy for AD. Amyloid-β (Aβ) aggregation and oxidative stress, two prominent neuropathological hallmarks in patients, play crucial roles in the neurotoxic cascade of this disease. In the present study, a series of novel (-)-meptazinol-melatonin hybrids were designed, synthesized and biologically characterized as potential MTDLs against AD. Among them, hybrids 7-7c displayed higher dual inhibitory potency toward cholinesterases (ChEs) and better oxygen radical absorbance capacity (ORAC) than the parental drugs. Furthermore, compound 7c could effectively inhibit Aβ self-aggregation, showed favorable safety and the blood-brain barrier (BBB) permeability. Therefore, 7c may serve as a valuable candidate that is worthy of further investigations in the treatment of AD.

Topics: Acetylcholinesterase; Amyloid beta-Peptides; Antioxidants; Cell Line, Tumor; Cell Membrane Permeability; Cell Survival; Cholinesterase Inhibitors; Drug Design; Humans; Melatonin; Meptazinol; Neurons; Neuroprotective Agents; Oxidative Stress; Peptide Fragments; Protein Aggregates; Protein Aggregation, Pathological; Reactive Oxygen Species

Aβ(1-40) peptide supramolecular assembly and fibril formation processes are widely recognized to have direct implications in the progression of Alzheimer's disease. The molecular basis of this biological process is still unknown and there is a strong need of developing effective strategies to control the occurring events. To this purpose the exploitation of small molecules interacting with Aβ aggregation represents one of the possible routes. Moreover, the use specific labeling has represented so far one of the most common and effective methods to investigate such a process. This possibility in turn rests on the reliability of the probe/labels involved. Here we present evidences of the effect of Thioflavin T (ThT), a worldwide used fluorescent dye to monitor amyloid growth, on the Aβ(1-40) conformation, stability and aggregation. By combining experimental information and Molecular Dynamics simulation results, we show that the presence of ThT in solution affects peptide conformation inducing peculiar supramolecular association. In particular ThT interactions with specific Aβ(1-40) residues promote a rigid partially-folded conformation which shifts the balance between different species in solution toward a more aggregation-prone ensemble of peptides, leading to aggregation. Our findings suggest ways for developing strategies to reverse and block aggregation or to stimulate supramolecular assembly and consequently reduce the presence of transient oligomers. This investigation underlines the need of developing label-free techniques for unbiased quantitative studies of Aβ(1-40) aggregation processes.

Topics: Alzheimer Disease; Amino Acid Sequence; Amyloid beta-Peptides; Benzothiazoles; Fluorescence Recovery After Photobleaching; Fluorescent Dyes; Humans; Molecular Dynamics Simulation; Molecular Sequence Data; Peptide Fragments; Protein Aggregates; Protein Aggregation, Pathological; Protein Conformation; Protein Multimerization; Protein Stability; Protein Structure, Secondary; Thiazoles

Recent studies show that the accumulation of redox-active Cu mediates the aggregation of amyloid β-peptide (Aβ) and conspicuous oxidative damage to the brain in Alzheimer's disease (AD). However, the key roles for Tyr 10 in Aβ-Cu(II) complex and its potential biological relevance to AD etiology under oxidative stress, were not stressed enough. Interestingly, our results indicated that Aβ40 (not Aβ16)-Cu(II) complex showed obviously enhanced peroxidase activity than free Cu(II). Although Tyr 10 was not the residue binding Cu(II), the mutation of Tyr 10 residue in Aβ40 decreased the peroxidase activity of Aβ40-Cu(II) complex, and the mutation of Tyr 10 could inhibit Aβ40 aggregation. Under oxidative and nitrative stress conditions, the Aβ-Cu(II) complex caused oxidation and nitration of the Aβ Tyr 10 residue through peroxidase-like reactions, where the formation of Cu(I) and hydroxyl radical (OH) was proposed as a chemical mechanism. We also showed that, when Aβ40 aggregates were bound to Cu(II), they retained peroxidase-like activity. Therefore, Tyr 10 residue is pivotal in Aβ-Cu(II) complex and shows important relevance to oxidative stress, implicating the novel significance of Tyr 10 residue as well as Aβ-Cu(II) complex in the pathology of AD.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Copper; Humans; Peptide Fragments; Peroxidase; Protein Aggregation, Pathological; Tyrosine

It has been suggested that the aggregation and cytotoxicity of amyloid-β (Aβ) peptide with transition-metal ions in neuronal cells is involved in the development and progression of Alzheimer's disease (AD). As the most abundant protein in blood plasma and in cerebrospinal fluid, human serum albumin (HSA) can bind Aβ in vivo and subsequently inhibit Aβ fibril growth. However, the roles of albumin in Cu-induced Aβ aggregation and toxicity, and its potential biological relevance to AD therapy, were not stressed enough. Here, we showed that HSA was capable of binding Cu (I) with much higher affinity than Aβ, competitively inhibiting the interaction of Aβ and Cu ions. In the presence of biological reducing agent ascorbate, HSA inhibited Cu (II)/Cu (I)-mediated Aβ40 aggregation, reactive oxygen species production, and neurotoxicity. However, in the absence of Cu (II)/Cu (I), HSA could not effectively inhibit Aβ40 aggregation and neurotoxicity at 24 h (or less) incubation time, but decreased Aβ40 aggregation at much longer incubation (120 h). Our data suggested that through competitively decreasing Cu-Aβ interaction, HSA could effectively inhibit Cu (II)/Cu (I)-induced Aβ40 aggregation and neurotoxicity, and play important roles in regulating redox balance as well as metal homeostasis in AD prevention and therapy.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Ascorbic Acid; Cell Line, Tumor; Cell Survival; Copper; Drug Interactions; Homeostasis; Humans; Neurons; Oxidation-Reduction; Peptide Fragments; Protein Aggregation, Pathological; Protein Binding; Reactive Oxygen Species; Serum Albumin

Although the N-terminal region of Amyloid β (Aβ) peptides plays dual roles as metal-coordinating sites and conformational modulator, few studies have been performed to explore the effects of mutations at this region on the overall conformational ensemble of Aβ and the binding propensity of metal ions. In this work, we focus on how three familial Alzheimer's disease mutations (D7H, D7N, and H6R) alter the structural characteristics and thermodynamic stabilities of Aβ42 using molecular dynamics simulations. We observe that each mutation displays increased β-sheet structures in both N and C termini. In particular, both the N terminus and central hydrophobic region of D7H can form stable β-hairpin structures with its C terminus. The conserved turn structure at Val²⁴-Lys²⁸ in all peptides and Zn²⁺-bound Aβ42 is confirmed as the common structural motif to nucleate folding of Aβ. Each mutant can significantly increase the solvation free energy and thus enhance the aggregation of Aβ monomers. The correlation dynamics between Aβ(1-16) and Aβ(17-42) fragments are elucidated by linking the domain motions with the corresponding structured conformations. We characterize the different populations of correlated domain motions for each mutant from a more macroscopic perspective, and unexpectedly find that Zn²⁺-bound Aβ42 ensemble shares the same populations as Aβ42, indicating that the binding of Zn²⁺ to Aβ follows the conformational selection mechanism, and thus is independent of domain motions, even though the structures of Aβ have been modified at a residue level.

Topics: Alzheimer Disease; Amino Acid Substitution; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Binding Sites; Energy Transfer; Humans; Hydrophobic and Hydrophilic Interactions; Kinetics; Models, Molecular; Molecular Dynamics Simulation; Mutation; Peptide Fragments; Protein Aggregation, Pathological; Protein Conformation; Protein Folding; Protein Stability; Solubility; Surface Properties; Zinc

The self-assembly of Aβ peptides into a range of conformationally heterogeneous amyloid states represents a fundamental event in Alzheimer's disease. Within these structures oligomeric intermediates are considered to be particularly pathogenic. To test this hypothesis we have used a conformational targeting approach where particular conformational states, such as oligomers or fibrils, are recognized in vivo by state-specific antibody fragments.. We show that oligomer targeting with the KW1 antibody fragment, but not fibril targeting with the B10 antibody fragment, affects toxicity in Aβ-expressing Drosophila melanogaster. The effect of KW1 is observed to occur selectively with flies expressing Aβ(1-40) and not with those expressing Aβ(1-42) or the arctic variant of Aβ(1-42) This finding is consistent with the binding preference of KW1 for Aβ(1-40) oligomers that has been established in vitro. Strikingly, and in contrast to the previously demonstrated in vitro ability of this antibody fragment to block oligomeric toxicity in long-term potentiation measurements, KW1 promotes toxicity in the flies rather than preventing it. This result shows the crucial importance of the environment in determining the influence of antibody binding on the nature and consequences of the protein misfolding and aggregation.. While our data support to the pathological relevance of oligomers, they highlight the issues to be addressed when developing inhibitory strategies that aim to neutralize these states by means of antagonistic binding agents.

Topics: Amino Acid Sequence; Amyloid beta-Peptides; Animals; Animals, Genetically Modified; Antibodies; Cell Line, Tumor; Disease Models, Animal; Drosophila melanogaster; Drosophila Proteins; Eye; Hippocampus; Humans; Long-Term Potentiation; Mice; Mice, Inbred C57BL; Neuroblastoma; Neurotoxicity Syndromes; Peptide Fragments; Protein Aggregation, Pathological; Protein Binding; Protein Conformation

Polyamines promote the formation of the Aβ peptide amyloid fibers that are a hallmark of Alzheimer's disease. Here we show that polyamines interact with nonaggregated Aβ peptides, thereby reducing the peptide's hydrophobic surface. We characterized the associated conformational change through NMR titrations and molecular dynamics simulations. We found that even low concentrations of spermine, sperimidine, and putrescine fully protected SH-SY5Y (a neuronal cell model) against the most toxic conformational species of Aβ, even at an Aβ oligomer concentration that would otherwise kill half of the cells or even more. These observations lead us to conclude that polyamines interfere with the more toxic prefibrillar conformations and might protect cells by promoting the structural transition of Aβ toward its less toxic fibrillar state that we reported previously. Since polyamines are present in brain fluid at the concentrations where we observed all these effects, their activity needs to be taken into account in understanding the molecular processes related to the development of Alzheimer's disease.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Cell Line, Tumor; Cell Survival; Humans; Peptide Fragments; Polyamines; Protein Aggregation, Pathological; Protein Structure, Secondary

Numerous proteins have the ability to assemble into fibrillar aggregates which are of great interest, because they feature in scores of human diseases and many technological products. In the present work, we analyze the kinetics of protein fibrillation when the process is governed solely by elongation of initially appeared fibrils in the protein solution. We derive exact expressions for the time dependences of the fibrillation degree, the concentration of monomeric protein in the solution, and the average fibril size. Furthermore, we present formulas for the initial fibrillation rate and the half-fibrillation time in terms of experimentally controllable quantities. The results obtained provide a mechanistic insight into the kinetics of protein fibrillation mediated by fibril elongation. We confront theory with experiment and find that it allows a good description of available experimental data for fibrillation of the Alzheimer's disease-associated protein Aβ(1-40) and the yeast prion protein Sup35.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Humans; Peptide Fragments; Peptide Termination Factors; Protein Aggregates; Protein Aggregation, Pathological; Protein Multimerization; Saccharomyces cerevisiae Proteins

Insight into how amyloid β (Aβ) aggregation occurs in vivo is vital for understanding the molecular pathways that underlie Alzheimer's disease and requires new techniques that provide detailed kinetic and mechanistic information. Using noninvasive fluorescence lifetime recordings, we imaged the formation of Aβ(1-40) and Aβ(1-42) aggregates in live cells. For both peptides, the cellular uptake via endocytosis is rapid and spontaneous. They are then retained in lysosomes, where their accumulation leads to aggregation. The kinetics of Aβ(1-42) aggregation are considerably faster than those of Aβ(1-40) and, unlike those of the latter peptide, show no detectable lag phase. We used superresolution fluorescence imaging to examine the resulting aggregates and could observe compact amyloid structures, likely because of spatial confinement within cellular compartments. Taken together, these findings provide clues as to how Aβ aggregation may occur within neurons.

Topics: Amyloid beta-Peptides; Cell Survival; Humans; Kinetics; Peptide Fragments; Protein Aggregates; Protein Aggregation, Pathological; Tumor Cells, Cultured

Integration of two fluorescence imaging methods enables tracking of the formation of fibrillar Aβ peptide amyloid aggregates in neurons, as discussed by Esbjörner and colleagues in this issue of Chemistry & Biology. This approach has the potential to fundamentally improve our understanding of the onset and therapeutic intervention of neurodegenerative diseases.

Topics: Amyloid beta-Peptides; Humans; Peptide Fragments; Protein Aggregates; Protein Aggregation, Pathological

Alzheimer's disease (AD) is a progressive and age-related neurodegenerative disorder affecting brain cells and is the most common form of "dementia", because of the cognitive detriment which takes place. Neuronal disruption represents its major feature, due to the cytosolic accumulation of amyloid β-peptide (Aβ) which leads to senile plaques formation and intracellular neurofibrillary tangles. Many studies have focused on the design and therapeutic use of new molecules able to inhibit Aβ aggregation. In this context, we evaluated the ability of two recently synthesized series of N-alkyl carbazole derivatives to increase the Aβ soluble forms, through molecular docking simulations and in vitro experiments. Our data evidenced that two carbazole derivatives, the most active, adopt distinct binding modes involving key residues for Aβ fibrillization. They exhibit a good interfering activity on Aβ aggregation in mouse (N2a) cells, stably expressing wild-type human amyloid precursor protein (APP) 695. These preliminary results are promising and we are confident that the N-alkyl carbazole derivatives may encourage next future studies needed for enlarging the knowledge about the AD disease approach.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Carbazoles; Cell Line; Culture Media, Conditioned; Drug Evaluation, Preclinical; Humans; Mice; Molecular Docking Simulation; Peptide Fragments; Protein Aggregates; Protein Aggregation, Pathological; Protein Stability; Solubility

Protein/peptide oligomerization, cross-β strand fibrillation, and amyloid deposition play a critical role in many diseases, but despite extensive biophysical characterization, the structural and dynamic details of oligomerization and fibrillation of amyloidic peptides/proteins remain to be fully clarified. Here, we simultaneously monitored the atomic, molecular, and mesoscopic states of aggregating Alzheimer's amyloid β (Aβ) peptides over time, using a slow aggregation protocol and a fast aggregation protocol, and determined the cytotoxicity of the intermediate states. We show that in the early stage of fast fibrillation (the lag phase) the Aβ peptides coalesced into apparently unstructured globules (15-200 nm in diameter), which slowly grew larger. Then a sharp transition occurred, characterized by the first appearance of single fibrillar structures of approximately ≥100 nm. These fibrils emerged from the globules. Simultaneously, an increase was observed for the cross-β strand conformation that is characteristic of the fibrils that constitute mature amyloid. The number and size of single fibrils rapidly increased. Eventually, the fibrils coalesced into mature amyloid. Samples from the early lag phase of slow fibrillation conditions were especially toxic to cells, and this toxicity sharply decreased when fibrils formed and matured into amyloid. Our results suggest that the formation of fibrils may protect cells by reducing the toxic structures that appear in the early lag phase of fibrillation.

Topics: Alzheimer Disease; Amyloid; Amyloid beta-Peptides; Cell Line, Tumor; Cell Survival; Humans; Kinetics; Microscopy, Atomic Force; Nanostructures; Peptide Fragments; Protein Aggregation, Pathological

Alzheimer's disease (AD) is characterized by the abnormal aggregation of amyloid-β peptide (Aβ) in extracellular deposits known as senile plaques. The tyrosine residue (Tyr-10) is believed to be important in Aβ-induced neurotoxicity due to the formation of tyrosyl radicals. To reduce the likelihood of cross-linking, here we designed an Aβ-40 analogue (Aβ-40 Y10F) in which the tyrosine residue was substituted by a structurally similar residue, phenylalanine. The aggregation rate was determined by the Thioflavin T (ThT) assay, in which Aβ-40 Y10F populated an ensemble of folded conformations much quicker and stronger than the wild type Aβ. Biophysical tests subsequently confirmed the results of the ThT assay, suggesting the measured increase of β-aggregation may arise predominantly from enhancement of hydrophobicity upon substitution and thus the propensity of intrinsic β-sheet formation. Nevertheless, Aβ-40 Y10F exhibited remarkably decreased neurotoxicity compared to Aβ-40 which could be partly due to the reduced generation of hydrogen peroxide. These findings may lead to further understanding of the structural perturbation of Aβ to its fibrillation.

Topics: Alzheimer Disease; Amino Acid Substitution; Amyloid beta-Peptides; Animals; Cells, Cultured; Humans; Hydrogen Peroxide; Neurons; Peptide Fragments; Phenylalanine; Protein Aggregation, Pathological; Protein Folding; Protein Structure, Secondary; Rats, Sprague-Dawley; Tyrosine

The aggregation of β-amyloid peptide (Aβ) into fibrils plays an important role in the pathogenesis of Alzheimer's disease (AD). Metal ions including copper and zinc are closely connected to the precipitation and toxicity of Aβ. In this study, a surface plasmon resonance (SPR) biosensor was constructed to investigate the interactions between Aβ and metal ions. Aβ peptide was immobilized on the SPR chip surface through a preformed alkanethiol self-assembled monolayer (SAM). Our observations indicate that the immobilized Aβ undergoes a conformational change upon exposure to the metal ions. A difference in metal binding affinity between Aβ(1-28) and Aβ(1-42) was also detected. The results suggest that SPR is an effective method to characterize the interactions between Aβ and metal ions.

Topics: Amyloid beta-Peptides; Copper; Humans; Peptide Fragments; Protein Aggregation, Pathological; Protein Binding; Surface Plasmon Resonance