Beta-amyloid Fibrils Research Articles

High resolution structural characterization of Aβ42 amyloid fibrils by magic angle spinning NMR.

The presence of amyloid plaques composed of amyloid beta (Aβ) fibrils is a hallmark of Alzheimer’s disease (AD). The Aβ peptide is present as several length variants with two common alloforms consisting of 40 and 42 amino acids, denoted Aβ1–40 and Aβ1–42, respectively. While there have been numerous reports that structurally characterize fibrils of Aβ1–40, very little is known about the structure of amyloid fibrils of Aβ1–42, which are considered the more toxic alloform involved in AD. We have prepared isotopically 13C/15N labeled AβM01–42 fibrils in vitro from recombinant protein and examined their 13C–13C and 13C–15N magic angle spinning (MAS) NMR spectra. In contrast to several other studies of Aβ fibrils, we observe spectra with excellent resolution and a single set of chemical shifts, suggesting the presence of a single fibril morphology. We report the initial structural characterization of AβM01–42 fibrils utilizing 13C and 15N shift assignments of 38 of the 43 residues, including the backbone and side chains, obtained through a series of cross-polarization based 2D and 3D 13C–13C, 13C–15N MAS NMR experiments for rigid residues along with J-based 2D TOBSY experiments for dynamic residues. We find that the first ∼5 residues are dynamic and most efficiently detected in a J-based TOBSY spectrum. In contrast, residues 16–42 are easily observed in cross-polarization experiments and most likely form the amyloid core. Calculation of ψ and φ dihedral angles from the chemical shift assignments indicate that 4 β-strands are present in the fibril’s secondary structure.

A Novel Tetradentate Ruthenium(II) Complex Containing Tris(2- Pyridylmethyl)amine (tpa) As An Inhibitor Of Beta-amyloid Fibrillation.

We report herein the synthesis and application of a novel tetradentate ruthenium(II) complex 1 containing a tris(2-pyridylmethyl)amine (tpa) ligand as an inhibitor of β-amyloid fibrillogenesis. [Ru(tpa)(bt)]ClO(4) 1 (bt =2-acetylbenzo[b]thiophene-3-olate) showed significant inhibition of Aβ(1-40) peptide aggregation in vitro, which was confirmed by a Thioflavin T assay and transmission electron microscopy imaging.

Dual Inhibition And Monitoring Of Beta-amyloid Fibrillation By A Luminescent Iridium(III) Complex.

In this study, we report the synthesis and application of the novel cyclometallated luminescent Ir(III) complex 1 bearing the C^C^C ligand as a probe and inhibitor of Aβ fibrillation. We envisage that this kinetically-inert Ir(III) complex may be potentially developed as a dual-purpose probe and inhibitor of Aβ aggregation for Alzheimer's disease diagnosis and treatment.

Injection of insulin amyloid fibrils in the hippocampus of male Wistar rats: report on memory impairment and formation of amyloid plaques.

Amyloid fibrils result from a particular type of protein aggregation, and have been linked with various disorders, including neurodegenerative ones. In the case of Alzheimer's disease, amyloid beta (abeta) fibrils are detected in patients' brain, in the amyloid plaques. These fibrils can be produced in vitro, and their injection into animals' brains generates an animal model of Alzheimer's disease. Based on the structural similarity of amyloid fibrils that are formed from different proteins, we hypothesized that injecting insulin amyloid fibrils into rats' brains could result in amyloid plaque formation. Fourteen male Wistar rats were divided into control and experimental groups (n = 7). The experimental group was bilaterally injected with insulin amyloid in the hippocampus. Seven days after injection, a shuttle box test was performed and the experimental group's memory was found to be impaired. Histological investigation of these rats' brain showed the formation of amyloid plaques in the hippocampus. A limited test has provided preliminary evidence for the stability of these plaques up to 35 days. Further complementary studies are required to fully validate the proposed procedure, which is simple and relatively low cost, and could be suggested as an alternative to models generated with abeta fibrils.

Protein corona composition of gold nanoparticles/nanorods affects amyloid beta fibrillation process.

Protein fibrillation process (e.g., from amyloid beta (Aβ) and α-synuclein) is the main cause of several catastrophic neurodegenerative diseases such as Alzheimer's and Parkinson diseases. During the past few decades, nanoparticles (NPs) were recognized as one of the most promising tools for inhibiting the progress of the disease by controlling the fibrillation kinetic process; for instance, gold NPs have a strong capability to inhibit Aβ fibrillations. It is now well understood that a layer of biomolecules would cover the surface of NPs (so called "protein corona") upon the interaction of NPs with protein sources. Due to the fact that the biological species (e.g., cells and amyloidal proteins) "see" the protein corona coated NPs rather than the pristine coated particles, one should monitor the fibrillation process of amyloidal proteins in the presence of corona coated NPs (and not pristine coated ones). Therefore, the previously obtained data on NPs effects on the fibrillation process should be modified to achieve a more reliable and predictable in vivo results. Herein, we probed the effects of various gold NPs (with different sizes and shapes) on the fibrillation process of Aβ in the presence and absence of protein sources (i.e., serum and plasma). We found that the protein corona formed a shell at the surface of gold NPs, regardless of their size and shape, reducing the access of Aβ to the gold inhibitory surface and, therefore, affecting the rate of Aβ fibril formation. More specifically, the anti-fibrillation potencies of various corona coated gold NPs were strongly dependent on the protein source and their concentrations (10% serum/plasma (simulation of an in vitro milieu) and 100% serum/plasma (simulation of an in vivo milieu)).

Effects of Crowding Agents and Volume Exclusion on Amyloid Beta Fibrillation

Several common neurodegenerative disorders can be attributed to pathological protein aggregation, including Alzheimer Disease, the most common form of dementia. It is known that macromolecular crowding promotes protein associations, including the formation of complexes and aggregates, both through theoretical works and in vitro. Therefore a possible contributing factor in the onset of Alzheimer Disease and other amyloidoses could be an increase in crowding content inside cells. In this work, we carried out computational simulations demonstrating the promotion of a generalized aggregation reaction model for amyloid beta fibrillation by increasing crowding agent concentration within biologically relevant values. In our in silico experiment, we implemented simulations with emulating the biophysico chemical properties of the intracellular environment, including nonspecific affinity of reacting species to crowding agents, varying size and shape of crowding agents, glymphatic removal and new synthesis of reactants, and others, demonstrating conditions in which aggregation can be more highly dependent on crowding agent concentration. Our work suggests loss of cellular crowding or volume control as a precursor to pathological aggregation.

Neuroprotection by association of palmitoylethanolamide with luteolin in experimental Alzheimer's disease models: the control of neuroinflammation.

Alzheimer's disease (AD) is the most common neurodegenerative disorder. Its neuropathological hallmarks include deposition of beta amyloid (Aβ) fibrils in senile plaques. Numerous biochemical events, leading to Aβ neurotoxicity in AD, have been proposed and it seems that neuroinflammation plays a prominent role among these. Thus, since inflammatory processes and oxidative stress are considered to play an important role in neuroinflammatory disorders and in AD pathology, in the present work we decided to test a new composite, which is a formulation constituted of an anti-inflammatory compound such as palmitoylethanolamide (PEA) and the well recognized antioxidant flavonoid luteolin (Lut), subjected to an ultra-micronization process, here designated co-ultraPEALut. We investigated the effect of co-ultraPEALut in both an in vitro and ex vivo organotypic model of AD. For the in vitro model, we used human neuronal cells, obtained by differentiating SH-SY5Y neuroblastoma cells into sustainable neuronal morphology. These well differentiated cells express features specific to mature neurons, such as synaptic structures and functional axonal vesicle transport, making this new concept for in vitro differentiation valuable for many neuroscientific research areas, including AD. Differentiated SH-SY5Y cells were pre-treated with co-ultraPEALut (reference concentrations: 27, 2.7 and 0.27 µM PEA) for 2 h. AD features were induced by Aβ₁₋₄₂ stimulation (1 µM). Twenty-four hours later cell vitality was evaluated by the colorimetric MTT assay, whereas the neuroinflammation underling AD was observed by Western blot analysis for IκBα degradation and nuclear factor-κB traslocation, as well as glial fibrillary acidic protein expression. For the organotypic model of AD, hippocampal slice cultures were prepared from mice at postnatal day 6 and after 21 days of culturing the slices were pre-treated with co-ultraPEALut (reference concentrations: 27, 2.7 and 0.27 µM PEA) for 2 h and then incubated with Aβ₁₋₄₂ (1 µg/ml) for 24 h. Pre-treatment with co-ultraPEALut significantly reduced inducible nitric oxide synthase and glial fibrillary acidic protein expression, restored neuronal nitric oxide synthase and brainderived neurotrophic factor and reduced the apoptosis. Taken together our results clearly showed that co-ultraPEALut is able to blunt Aβ-induced astrocyte activation and to exert a marked protective effect on glial cells. These findings suggest that the association of co-ultraPEALut may provide an effective strategy for AD.

Binding of fullerenes to amyloid beta fibrils: size matters.

Binding affinity of fullerenes C20, C36, C60, C70 and C84 for amyloid beta fibrils is studied by docking and all-atom molecular dynamics simulations with the Amber force field and water model TIP3P. Using the molecular mechanic-Poisson Boltzmann surface area method one can demonstrate that the binding free energy linearly decreases with the number of carbon atoms of fullerene, i.e. the larger is the fullerene size, the higher is the binding affinity. Overall, fullerenes bind to Aβ9-40 fibrils stronger than to Aβ17-42. The number of water molecules trapped in the interior of 12Aβ9-40 fibrils was found to be lower than inside pentamer 5Aβ17-42. C60 destroys Aβ17-42 fibril structure to a greater extent compared to other fullerenes. Our study revealed that the van der Waals interaction dominates over the electrostatic interaction and non-polar residues of amyloid beta peptides play the significant role in interaction with fullerenes providing novel insight into the development of drug candidates against Alzheimer's disease.

Unambiguous Determination of the Absolute Configuration of Dimeric Stilbene Glucosides from the Rhizomes of Gnetum africanum.

Dimeric stilbene glucosides 1-3 [two diastereomers of (-)-gnemonoside A (1a and 1b), (-)-gnemonoside C (2), and (-)-gnemonoside D (3)] as well as a mixture of the two enantiomers of gnetin C (4) were isolated from the rhizomes of Gnetum africanum. The two enantiomers of gnetin C, (+)-4 and (-)-4, were obtained from the aglycones of 1a and 1b, respectively. The configurations of these stilbenoids were investigated by NMR and vibrational circular dichroism (VCD) experiments. The absolute configurations of (-)-1a, (-)-2, (-)-3, and (-)-4 were established as 7aS,8aS by VCD spectroscopy in combination with density functional theory calculations. The antiamyloidogenic activity of the isolated stilbenes was also evaluated versus beta-amyloid fibrils. The four glucosides of gnetin C (1a, 1b, 2, and 3) were found to be the most active compounds, with inhibition percentages of 56, 56, 58, and 54 at 10 μM, respectively.

Reelin delays amyloid-beta fibril formation and rescues cognitive deficits in a model of Alzheimer’s disease

Reelin is an extracellular matrix protein that is crucial for neural development and adult brain plasticity. While the Reelin signalling cascade has been reported to be associated with Alzheimer's disease (AD), the role of Reelin in this pathology is not understood. Here we use an in vitro approach to show that Reelin interacts with amyloid-β (Aβ42) soluble species, delays Aβ42 fibril formation and is recruited into amyloid fibrils. Furthermore, Reelin protects against both the neuronal death and dendritic spine loss induced by Aβ42 oligomers. In mice carrying the APP(Swe/Ind) mutation (J20 mice), Reelin overexpression delays amyloid plaque formation and rescues the recognition memory deficits. Our results indicate that by interacting with Aβ42 soluble species, delaying Aβ plaque formation, protecting against neuronal death and dendritic spine loss and preventing AD cognitive deficits, the Reelin pathway deserves consideration as a therapeutic target for the treatment of AD pathogenesis.

Is there a sodium effect in fibrillar amyloid-β oligomers?

Hall mark in Alzheimer’s Disease (AD) is the aggregation of amyloid-β (Aβ) peptide into oligomers and fibrils. Nowadays, the soluble oligomers are believed to be the most neurotoxic species, probably the causative agents in AD. Amyloid-beta fibrils on the other hand may serve as reservoirs for small toxic oligomers. While it is well known from experiment, that the aggregation process is modulated by salt concentration in solution, the molecular details of the underlying interactions are not. Salts occur ubiquitously in physiological environments and are known to have profound effects on the solubility of proteins (Hofmeister series). Monovalent alkali metal ions exhibit a more subtle effect on Aβ aggregation in experiment than doubly charged species [1,2]. In this contribution we investigate the presence of the so-called ‘sodium-effect’ in fibrillar Aβ oligomers. This effect was originally described for dendrimer micelles formation modulating the self-organization of amphiphilic carboxylates: Na+ forms bridging complexes with carboxylate groups, in contrast to K+ [3]. Molecular dynamics simulations for a systematic series of single and double layer fibrillar Aβ oligomers in aqueous 150mM salt solution provide insights about the stabilizing interactions between the cations and charged Aβ key residues (e.g. Glu22). Comparison with a previous computational study at low ion concentration [4] show similarities and differences on a structural level. Interestingly, ions access the Aβ water channel region via entry paths suggested previously [4].

Solid-state NMR sequential assignment of Osaka-mutant amyloid-beta (Aβ1−40 E22Δ) fibrils

Alzheimer's disease (AD) is the most common form of dementia. Aggregation of amyloid β (Aβ), a peptide of 39-43 residues length, into insoluble fibrils is considered to initiate the disease. Determination of the molecular structure of Aβ fibrils is technically challenging and is a significant goal in AD research that may lead to design of effective therapeutical inhibitors of Aβ aggregation. Here, we present chemical-shift assignments for fibrils formed by highly pure recombinant Aβ1-40 with the Osaka E22Δ mutation that is found in familial AD. We show that that all regions of the peptide are rigid, including the N-terminal part often believed to be flexible in Aβ wt.

A central role for dityrosine crosslinking of Amyloid-β in Alzheimer's disease.

BackgroundAlzheimer’s disease (AD) is characterized by the deposition of insoluble amyloid plaques in the neuropil composed of highly stable, self-assembled Amyloid-beta (Aβ) fibrils. Copper has been implicated to play a role in Alzheimer’s disease. Dimers of Aβ have been isolated from AD brain and have been shown to be neurotoxic.ResultsWe have investigated the formation of dityrosine cross-links in Aβ42 formed by covalent ortho-ortho coupling of two tyrosine residues under conditions of oxidative stress with elevated copper and shown that dityrosine can be formed in vitro in Aβ oligomers and fibrils and that these links further stabilize the fibrils. Dityrosine crosslinking was present in internalized Aβ in cell cultures treated with oligomeric Aβ42 using a specific antibody for dityrosine by immunogold labeling transmission electron microscopy. Results also revealed the prevalence of dityrosine crosslinks in amyloid plaques in brain tissue and in cerebrospinal fluid from AD patients.ConclusionsAβ dimers may be stabilized by dityrosine crosslinking. These results indicate that dityrosine cross-links may play an important role in the pathogenesis of Alzheimer’s disease and can be generated by reactive oxygen species catalyzed by Cu2+ ions. The observation of increased Aβ and dityrosine in CSF from AD patients suggests that this could be used as a potential biomarker of oxidative stress in AD.

Discovery of Dihydrochalcone as Potential Lead for Alzheimer’s Disease: In Silico and In Vitro Study

By the virtual screening method we have screened out Dihydrochalcone as a top-lead for the Alzheimer’s disease using the database of about 32364 natural compounds. The binding affinity of this ligand to amyloid beta (A) fibril has been thoroughly studied by computer simulation and experiment. Using the Thioflavin T (ThT) assay we have obtained the inhibition constant IC50 M. This result is in good agreement with the estimation of the binding free energy obtained by the molecular mechanic-Poisson Boltzmann surface area method and all-atom simulation with the force field CHARMM 27 and water model TIP3P. Cell viability assays indicated that Dihydrochalcone could effectively reduce the cytotoxicity induced by A. Thus, both in silico and in vitro studies show that Dihydrochalcone is a potential drug for the Alzheimers disease.

In silico and in vitro studies to elucidate the role of Cu2+ and galanthamine as the limiting step in the amyloid beta (1–42) fibrillation process

The formation of fibrils and oligomers of amyloid beta (Aβ) with 42 amino acid residues (Aβ 1-42 ) is the most important pathophysiological event associated with Alzheimer's disease (AD). The formation of Aβ fibrils and oligomers requires a conformational change from an α-helix to a β-sheet conformation, which is encouraged by the formation of a salt bridge between Asp 23 or Glu 22 and Lys 28. Recently, Cu(2+) and various drugs used for AD treatment, such as galanthamine (Reminyl(®) ), have been reported to inhibit the formation of Aβ fibrils. However, the mechanism of this inhibition remains unclear. Therefore, the aim of this work was to explore how Cu(2+) and galanthamine prevent the formation of Aβ1-42 fibrils using molecular dynamics (MD) simulations (20 ns) and in vitro studies using fluorescence and circular dichroism (CD) spectroscopies. The MD simulations revealed that Aβ1-42 acquires a characteristic U-shape before the α-helix to β-sheet conformational change. The formation of a salt bridge between Asp 23 and Lys 28 was also observed beginning at 5 ns. However, the MD simulations of Aβ 1-42 in the presence of Cu(2+) or galanthamine demonstrated that both ligands prevent the formation of the salt bridge by either binding to Glu 22 and Asp 23 (Cu(2+) ) or to Lys 28 (galanthamine), which prevents Aβ 1-42 from adopting the U-characteristic conformation that allows the amino acids to transition to a β-sheet conformation. The docking results revealed that the conformation obtained by the MD simulation of a monomer from the 1Z0Q structure can form similar interactions to those obtained from the 2BGE structure in the oligomers. The in vitro studies demonstrated that Aβ remains in an unfolded conformation when Cu(2+) and galanthamine are used. Then, ligands that bind Asp 23 or Glu 22 and Lys 28 could therefore be used to prevent β turn formation and, consequently, the formation of Aβ fibrils.

AçaÍ (Euterpe oleraceae Mart) berry extract protects neuronal cells against beta-amyloid toxicity and inhibits beta-amyloid fibril formation in vitro

AçaÍ (Euterpe oleraceae Mart) berry extract protects neuronal cells against beta-amyloid toxicity and inhibits beta-amyloid fibril formation in vitro

The Protein Corona Mediates the Impact of Nanomaterials and Slows Amyloid Beta Fibrillation

Put your coat on: It is well recognized that the surfaces of nanomaterials in biological media are covered by various biomolecules (e.g., proteins). A) The protein corona creates a shell over different nanomaterials, regardless of their physicochemical properties (e.g., composition and shape), resulting in reduced levels of amyloid beta fibril formation. B) Pristine nanomaterials might have acceleratory effects on the fibrillation of amyloid beta.

Anti-inflammatory and anti-amyloidogenic effects of a small molecule, 2,4-bis(p-hydroxyphenyl)-2-butenal in Tg2576 Alzheimer’s disease mice model

BackgroundAlzheimer’s disease (AD) is pathologically characterized by excessive accumulation of amyloid-beta (Aβ) fibrils within the brain and activation of astrocytes and microglial cells. In this study, we examined anti-inflammatory and anti-amyloidogenic effects of 2,4-bis(p-hydroxyphenyl)-2-butenal (HPB242), an anti-inflammatory compound produced by the tyrosine-fructose Maillard reaction.Methods12-month-old Tg2576 mice were treated with HPB242 (5 mg/kg) for 1 month and then cognitive function was assessed by the Morris water maze test and passive avoidance test. In addition, western blot analysis, Gel electromobility shift assay, immunostaining, immunofluorescence staining, ELISA and enzyme activity assays were used to examine the degree of Aβ deposition in the brains of Tg2576 mice. The Morris water maze task was analyzed using two-way ANOVA with repeated measures. Otherwise were analyzed by one-way ANOVA followed by Dunnett’s post hoc test.ResultsTreatment of HPB242 (5 mg/kg for 1 month) significantly attenuated cognitive impairments in Tg2576 transgenic mice. HPB242 also prevented amyloidogenesis in Tg2576 transgenic mice brains. This can be evidenced by Aβ accumulation, BACE1, APP and C99 expression and β-secretase activity. In addition, HPB242 suppresses the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) as well as activation of astrocytes and microglial cells. Furthermore, activation of nuclear factor-kappaB (NF-κB) and signal transducer and activator of transcription 1/3 (STAT1/3) in the brain was potently inhibited by HPB242.ConclusionsThus, these results suggest that HPB242 might be useful to intervene in development or progression of neurodegeneration in AD through its anti-inflammatory and anti-amyloidogenic effects.

N-Acetyl-l-cysteine capped quantum dots offer neuronal cell protection by inhibiting beta (1-40) amyloid fibrillation.

This is the first work that revealed the neuro-protective effect of functionalized quantum dots against the cytotoxicity induced by beta-amyloid peptides. This study gives insight into the future treatment of Alzheimer's disease. It opens many avenues for the development of the next generation nanotechnology for biomedical and therapeutic applications.

Physiological Temperature Has a Crucial Role in Amyloid Beta in the Absence and Presence of Hydrophobic and Hydrophilic Nanoparticles

Amyloid beta fibrillation can lead to major disorder of neurons processes and is associated with several neuronal diseases (e.g., Alzheimer's disease). We report here an importance of slight temperature changes, in the physiological range (35-42 °C), on the amyloid fibrillation process in the presence and absence of hydrophilic (silica) and hydrophobic (polystyrene) nanoparticles (NPs). The results highlight the fact that slight increases in temperature can induce inhibitory and acceleratory effects of hydrophobic and hydrophilic NPs on the fibrillation process, respectively. Using further in vivo considerations, the outcomes of this study can be used for considerable modifications on the current diagnosis and treatment approaches in amyloid-involved diseases.