Aβ Fibrils Research Articles

Aggregation Dynamics of a 150 kDa Aβ42 Oligomer: Insights from Cryo Electron Microscopy and Multimodal Analysis

Protein misfolding is a widespread phenomenon that can result in the formation of protein aggregates, which are markers of various disease states, including Alzheimer's disease (AD). In AD, amyloid beta (Aβ) peptides are key players in the disease's progression, particularly the 40- and 42 residue variants, Aβ40 and Aβ42. These peptides aggregate to form amyloid plaques and contribute to neuronal toxicity. Recent research has shifted attention from solely Aβ fibrils to also include Aβ protofibrils and oligomers as potentially critical pathogenic agents. Particularly, oligomers demonstrate more significant toxicity compared to other Aβ specie. Hence, there is an increased interest in studying the correlation between toxicity and their structure and aggregation pathway. The present study investigates the aggregation of a 150kDa Aβ42 oligomer that does not lead to fibril formation. Using negative stain transmission electron microscopy (TEM), size exclusion chromatography (SEC), dynamic light scattering (DLS), and cryo-electron microscopy (cryo-EM), we demonstrate that 150kDa Aβ42 oligomers form higher-order string-like assemblies over time. These strings are unique from the classical Aβ fibrils. The significance of our work lies in elucidating molecular behavior of a novel non-fibrillar form of Aβ42 aggregate.

Brazilian kefir fraction mitigates the Alzheimer-like phenotype in Drosophila melanogaster with β-amyloid overexpression model

Alzheimer’s disease (AD) is a progressive neurodegenerative condition and the primary form of dementia among elderly people. The amyloidogenic hypothesis is the main theory that explains this phenomenon and describes the extracellular accumulation of amyloid beta (Aβ) peptides. Model organisms such as Drosophila melanogaster have been utilized to improve the understanding of this disease and its treatment. This study evaluated the effects of peptide and metabolic fractions of Brazilian kefir on a strain of D. melanogaster that expresses human Aβ peptide 1–42 in the eye. The parameters assessed included ommatidial organization, vacuole area, retinal thickness, and Aβ peptide quantification. The present study revealed that the fractions, particularly the peptidic fraction, significantly reduced the vacuole area and increased the retina thickness in treated flies, indicating an improvement in neurodegeneration phenotype. The peptidic fraction was also found to alter Aβ aggregation dynamics, inhibiting Aβ fibril formation, as revealed by dynamic light scattering. This study demonstrated that kefir fractions, particularly the peptidic fraction < 10 kDa, have the potential to regulate Aβ aggregation and alleviate neurodegeneration in a Drosophila melanogaster AD-like model. These findings suggest that kefir fractions could be viable for the bioprospection of novel drug prototypes for AD treatment, providing valuable insights into strategies targeting Aβ aggregation and neurodegeneration in AD.

Accelerated Alzheimer's Aβ-42 secondary nucleation chronologically visualized on fibril surfaces.

Protein fibril surfaces tend to generate toxic oligomers catalytically. To date, efforts to study the accelerated aggregation steps involved with Alzheimer's disease-linked amyloid-β (Aβ)-42 proteins on fibril surfaces have mainly relied on fluorophore-based analytics. Here, we visualize rare secondary nucleation events on the surface of Aβ-42 fibrils from embryonic to endpoint stages using liquid-based atomic force microscopy. Nanoscale imaging supported by atomic-scale molecular simulations tracked the adsorption and proliferation of oligomeric assemblies at nonperiodically spaced catalytic sites on the fibril surface. Upon confirming that fibril edges are preferential binding sites for oligomers during embryonic stages, the secondary fibrillar size changes were quantified during the growth stages. Notably, a small population of fibrils that displayed higher surface catalytic activity was identified as superspreaders. Profiling secondary fibrils during endpoint stages revealed a nearly threefold increase in their surface corrugation, a parameter we exploit to classify fibril subpopulations.

Single-Molecule Kinetic Observation of Antibody Interactions with Growing Amyloid β Fibrils.

Understanding the dynamic assembly process of amyloid β (Aβ) during fibril formation is essential for developing effective therapeutic strategies against Alzheimer's disease. Here, we employed high-speed atomic force microscopy to observe the growth of Aβ fibrils at the single-molecule level, focusing specifically on their interaction with anti-Aβ antibodies. Our findings show that fibril growth consists of intermittent periods of elongation and pausing, which are dictated by the alternating addition of Aβ monomers to protofilaments. We highlight the distinctive interaction of antibody 4396C, which specifically binds to the fibril ends in the paused state, suggesting a unique mechanism to hinder fibril elongation. Through real-time visualization of fibril growth and antibody interactions combined with molecular simulation, this study provides a refined understanding of Aβ assembly during fibril formation and suggests novel strategies for Alzheimer's therapy aimed at inhibiting the fibril elongation.

Bifunctional Copper Chelators Capable of Reducing Aβ Aggregation and Aβ-Induced Oxidative Stress.

Five bifunctional copper chelating agents, Alz-(1-5), designed to prevent beta-amyloid (Aβ) aggregation, were synthesized, and the leader compound (Alz-5) was chosen. Alz-5 acts as a bifunctional chelator that can interact with various Aβ aggregates and reduce their neurotoxicity. Reactive oxygen species measurements provided by the Pt-nanoelectrode technique in single Aβ42-affected human neuroblastoma SH-SY5Y cells revealed significant antioxidant activity of Alz-5. AFM data obtained on Aβ42 fibrils clearly indicate the antiaggregating property of Alz-5. To gain insights into the changes in the physiomechanical properties of Aβ42-affected cells, as well as in order to evaluate the antiaggregating ability of Alz-5, Young's modulus mapping on living SH-SY5Y cells affected consequently by Aβ42 and Alz-5 was conducted, and the ability of Alz-5 to decrease cell rigidity induced by Aβ42 was indisputably proven. Low cell toxicity and antioxidating properties, in conjunction with AFM and SICM-based biophysical provided on Aβ42-affected SH-SY5Y cells, support Alz-5 as a potential inhibitor of Aβ aggregation.

Dual-Carbon Dots Composite: A Multifunctional Photo-Propelled Nanomotor Against Alzheimer's β-Amyloid.

The abnormal accumulation of β-amyloid protein (Aβ) is considered as the main pathological hallmark of Alzheimer's disease (AD). The design of potent multifunctional theranostic agents targeting Aβ is one of the effective strategies for AD prevention and treatment. Nanomotors as intelligent, advanced, and multifunctional nanoplatforms can perform many complex tasks, but their application in AD theranostics is rare. Herein, sub-10nm multifunctional dual-carbon dots composites (ERCD) with photo-propelled nanomotor behavior are fabricated by conjugating near-infrared (NIR) carbon dots (RCD) of thermogenic and photodynamic capability with the previously reported epigallocatechin gallate-derived carbonized polymer dots (ECD). ERCD-1 (ECD:RCD = 1:2.5) showed potent inhibitory capability similar to ECD in the absence of NIR light, and exhibited photooxygenation activity and nanomotor behavior powered by "self-thermophoretic force" under NIR irradiation, significantly enhancing the inhibition, disaggregation, and photooxygenation capabilities. The nanomotor suppressed Aβ fibrillization and rapidly disaggregated mature Aβ fibrils at very low concentrations (0.5µg mL-1). Moreover, the NIR-activated ERCD-1 imaged Aβ plaques in vivo and prolonged nematode lifespan by 6 d at 2µg mL-1. As a proof-of-concept, this work opened a new avenue to the design of multifunctional sub-10nm nanomotors targeting Aβ for AD theranostics.

Safety, tolerability, pharmacokinetics and pharmacodynamics of a single intravenous dose of SHR-1707 in healthy adult subjects: two randomized, double-blind, single-ascending-dose, phase 1 studies

BackgroundSHR-1707 is a novel humanized anti-Aβ IgG1 monoclonal antibody that binds to Aβ fibrils and monomers to block the formation of Aβ plaques or to promote the microglial phagocytosis of Aβ. Preclinical studies showed that SHR-1707 reduced brain Aβ deposition in 5xFAD transgenic mice. Herein, we conducted two phase 1 studies to evaluate the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of a single intravenous dose of SHR-1707 in healthy adult subjects.MethodsTwo randomized, double-blind, single-ascending-dose, phase 1 studies were conducted in China (Study CHN) and Australia (Study AUS). Study CHN consisted of 2 parts. In Part 1, eligible healthy young adults (18–45 years) were sequentially randomized 8:2 to receive SHR-1707 (five cohorts: 2, 6, 20, 40, and 60 mg/kg) or placebo in each cohort; in Part 2, elderly subjects (55–80 years) were randomized 8:4 to receive SHR-1707 (20 mg/kg) or placebo. A similar design was used in Study AUS, but with only healthy young adults enrolled across three dosing cohorts (2, 20, and 60 mg/kg).ResultsSixty-two (part 1/2, n = 50/12; age range, 18–42/55–63 years) and 30 subjects (age range, 18–42 years) received SHR-1707 or placebo in Study CHN and Study AUS, respectively. In Study CHN, all treatment-related adverse events (TRAEs) were mild, with the most common being transient laboratory abnormalities. In Study AUS, TRAEs were mostly mild (1 moderate event each with SHR-1707/placebo); the most common TRAEs with SHR-1707 were dysgeusia and fatigue (8.3% each). In both studies, the exposure of SHR-1707 increased in a slightly greater than dose-proportional manner over the dose range of 2–60 mg/kg in young adults; there was a dose-dependent increase in plasma Aβ42 concentration following SHR-1707 administration compared with the placebo group. The safety and PK and PD profiles of SHR-1707 in the elderly subjects were consistent with the younger counterpart at the same dose level. No ethnic difference in safety, PK and PD of SHR-1707 was observed.ConclusionsA single intravenous dose of SHR-1707 at 2–60 mg/kg was safe and well tolerated in healthy young adult and elderly subjects. The PK and PD profiles are supportive for further clinical development.Trial registrationNCT04973189 (retrospectively registered on Jul.21, 2021) and NCT04745104 (registered on Feb.6, 2021) on clinicaltrials.gov.

Deciphering the Morphological Difference of Amyloid-β Fibrils in Familial and Sporadic Alzheimer's Diseases.

The aggregation of amyloid-β (Aβ) into amyloid fibrils is the major pathological hallmark of Alzheimer's disease (AD). Aβ fibrils can adopt a variety of morphologies, the relative populations of which are recently found to be associated with different AD subtypes such as familial and sporadic AD (fAD and sAD, respectively). The two AD subtypes differ in their ages of onset, AD-related genetic predispositions, and dominant Aβ fibril morphologies. We postulate that these disease subtype-dependent fibril morphology differences can be attributed to the intrinsic fibril properties and interacting molecules in the environment. Using atomistic discrete molecular dynamics simulations, we demonstrated that the fAD-dominant morphology exhibited a lower free-energy barrier for fibril growth but also a lower stability compared with the sAD-dominant fibril morphology, resulting in the time-dependent population change consistent with experimental observations. Additionally, we studied the effect of the Bri2 BRICHOS domain, an endogenous protein that has been reported to inhibit Aβ aggregation by preferential binding to fibrils, as one of the possible environmental factors. The Bri2 BRICHOS domain showed stronger binding to the fAD-dominant fibril than the sAD-dominant fibril in silico, suggesting a more effective suppression of fAD-dominant fibril formation. This result explains the high population of the sAD-dominant fibril morphology in sporadic cases with normal Bri2 functions. Genetic predisposition in fAD, on the other hand, might impair or overwhelm Bri2 functions, leading to a high population of fAD-associated fibril morphology. Together, our computational findings provide a theoretical framework for elucidating the AD subtypes entailed by distinct dominant amyloid fibril morphologies.

Comment on "Extracellular Vesicles Slow Down Aβ(1-42) Aggregation by Interfering with the Amyloid Fibril Elongation Step".

Halipi et al. explored the impact of extracellular vesicles (EVs) on amyloid-β (Aβ) aggregation. They concluded that EVs reduce Aβ aggregation, as seen by shorter and thicker fibrils. While we agree with the complex role of EVs in Alzheimer's disease, we are sceptical of the claim that EVs slow down Aβ aggregation, noting missing key references. Previous literature rather suggests that EVs (derived from neuronal cell lines) accelerate the process of Aβ fibrillation and plaque formation. Halipi et al.'s findings may be skewed due to the lack of essential neuronally expressed Aβ-binding partners, like the prion protein (PrPC) in their EV samples. The commentary, in the light of included original experiments and cited literature, suggests that membrane proteins like PrPC are crucial to fully understand the role of EVs in Aβ aggregation, and Halipi et al.'s conclusions should be reexamined in light of these factors.

Destabilisation of Alzheimer's amyloid-β protofibrils by Baicalein: mechanistic insights from all-atom molecular dynamics simulations.

Alzheimer's disease (AD) is the most common form of dementia and the fifth leading cause of death globally. Aggregation and deposition of neurotoxic Aβ fibrils in the neural tissues of the brain is a key hallmark in AD pathogenesis. Destabilisation studies of the amyloid-peptide by various natural molecules are highly relevant due to their neuroprotective and therapeutic potential for AD. We performed molecular dynamics (MD) simulation to investigate the destabilisation mechanism of amyloidogenic protofilament intermediate by Baicalein (BCL), a naturally occurring flavonoid. We found that the BCL molecule formed strong hydrophobic contacts with non-polar residues, specifically F19, A21, V24, and I32 of Chain A and B of the pentameric protofibril. Upon binding, it competed with the native hydrophobic contacts of the Aβ protein. BCL loosened the tight packing of the hydrophobic core by disrupting the hydrogen bonds and the prominent D23-K28 inter-chain salt bridges of the protofibril. The decrease in the structural stability of Aβ protofibrils was confirmed by the increased RMSD, radius of gyration, solvent accessible surface area (SASA), and reduced β-sheet content. PCA indicated that the presence of the BCL molecule intensified protofibril motions, particularly affecting residues in Chain A and B regions. Our findings propose that BCL would be a potent destabiliser of Aβ protofilament, and may be considered as a therapeutic agent in treating AD.

Discovering Effective Chiral Dipeptides against Aβ(1-42) Aggregation by the Computational Screening Strategy.

The β-sheet-breaker (BSB) peptides inhibiting amyloidogenic aggregation have been extensively studied. However, the inhibition efficacy of ultrashort chiral dipeptides remains inadequately understood. In this study, we proposed a computational screening strategy to identify chiral dipeptides as BSB with optimal antiaggregation performance against Aβ(1-42) aggregation. We constructed a complete dipeptide library encompassing all possible chiral sequence arrangements and then filtered the library by cascaded molecular docking-molecular dynamics (MD) simulation. Our screening strategy discovered dipeptide DWDP (superscript for chirality) that displayed strong interactions with Aβ fibrils and inhibitory effects on Aβ aggregation, validated by subsequent experiments. Mechanistic investigation by both MD and replica-exchange molecular dynamics (REMD) simulations revealed that DWDP interacts with Aβ by hydrophobic contacts and hydrogen bonds and thus inhibits Aβ intermolecular contacts and salt bridge formation, therefore inhibiting Aβ aggregation and disrupting Aβ aggregates. Totally, our strategy presents a viable approach to discover potential dipeptides with effective antiaggregation ability as potential therapeutic agents for Alzheimer's disease.

Extraction of In-Cell β-Amyloid Fibrillar Aggregates for Studying Molecular-Level Structural Propagations Using Solid-State NMR Spectroscopy.

Molecular-level structural polymorphisms of β-amyloid (Aβ) fibrils have recently been recognized as pathologically significant. High-resolution solid-state nuclear magnetic resonance (ssNMR) spectroscopy has been utilized to study these structural polymorphisms, particularly in ex-vivo fibrils seeded from amyloid extracts of post-mortem brain tissues of Alzheimer's disease (AD) patients. One unaddressed question in current ex-vivo seeding protocol is whether fibrillation from exogenous monomeric Aβ peptides, added to the extracted seeds, can be quantitatively suppressed. Addressing this issue is critical because uncontrolled fibrillation could introduce biased molecular structural polymorphisms in the resulting fibrils. Here, we present a workflow to optimize the key parameters of ex-vivo seeding protocols, focusing on the quantification of amyloid extraction and the selection of exogenous monomeric Aβ concentrations to minimize nonseeded fibrillation. We validate this workflow using three structurally different 40-residue Aβ (Aβ40) fibrillar seeds, demonstrating their ability to propagate their structural features to exogenous wild-type Aβ40.

Understanding Osaka mutation polymorphic Aβ fibril response to static and oscillating electric fields: insights from computational modeling

Alzheimer’s disease (AD) is a prevalent neurodegenerative disorder, impacting millions of individuals worldwide. Among its defining characteristics is the accumulation of senile plaques within the brain’s gray matter, formed through the self-assembly of misfolded proteins contributing to the progressive symptoms of AD. This study investigates a polymorphic Aβ fibril under static and oscillating electric fields using molecular dynamics simulation. Specifically, we utilized a polymorphic fibrillar complex composed of two intertwined pentamer-strands of the Aβ1–40 peptide with the Osaka mutation (E22Δ), known for its toxicity and stable structure. Our findings demonstrate that a 0.3 and 0.4 V/nm electric field combined with a 0.20 GHz frequency effectively disrupts the polymorphic conformation of Aβ fibrils. Furthermore, we elucidate the molecular mechanisms underlying this disruption, providing insights into the potential therapeutic use of oscillating electric fields for AD. This research offers valuable insights into novel therapeutic approaches for combating AD pathology.

Amyloid Fibril Formation on Neuronal Cells in the Coexistence of Aβ40 and Aβ42.

The abnormal aggregation and subsequent deposition of amyloid β-protein (Aβ) in the brain are considered central to the pathogenesis of Alzheimer's disease. The two major species of Aβ are Aβ40 and Aβ42, present at an approximate ratio of 9: 1. Accumulating evidence suggests that neuronal membranes are an important platform of amyloidogenesis by Aβ. However, information on the aggregational behaviors of coexistent Aβ40 and Aβ42 on membranes is lacking. In this study, the aggregation and resultant cytotoxicity of coexistent Aβ40 and Aβ42 at a physiologically relevant ratio were investigated by fluorescence techniques. We found that the degree of coexistence of both Aβs in aggregates increased as the assembly proceeded, and reached a maximum in fibrils. However, the cytotoxicity of the mixed fibrils was weaker than that of Aβ42 fibrils, indicating that Aβ40 attenuates the toxicity of Aβ42 by forming mixed fibrils. In contrast, the degree of coexistence was significantly lower in aqueous phase aggregation, highlighting different aggregation mechanisms between in membranes and in the aqueous phase.

Curved Nanointerface Controls the Chiral Effect on Peptide Fibrillation.

Nanostructures with varying functionalities have been engineered to modulate the fibrillation of amyloid-β (Aβ) peptides. Nevertheless, the chirality effect at the curved nanointerfaces is seldom dissected. In this study, we systematically explored the curvature-modulated chiral effect on the regulation of Aβ1-42 fibrillation by using l/d-penicillamine-gold nanoparticles (l/d-PGNPs). According to the microscopic and spectroscopic analyses, Aβ1-42 fibrillation can be effectively suppressed by more curved (0.2 nm-1, 1/r) d-nanointerface (d-PGNPs5) with notable chiral selectivity, even at a low inhibitor/peptide (I/P) molar ratio (1:100). A greatly alleviated cytotoxic effect of Aβ1-42 peptides after the inhibition process is also revealed. The highly curved nanointerface drives the formation of multiple hydrogen bonds and promotes electrostatic interactions with Aβ1-42. Importantly, the curved d-nanointerface controls well the spatial arrangement of Pen motifs, making it incompatible with the fibrillation direction of Aβ1-42 and thus gaining enhanced efficiency on amyloid fibrillar modulation. This study provides valuable insights into the interactions between chirality and peptide-nanointerface effects, which are crucial for the development of inhibitors in anti-β-amyloidosis.

Osmundacetone ameliorates Alzheimer's-like pathologies by inhibiting β-amyloid fibrillation, oxidative damage and neuroinflammation in APP/PS1 transgenic mice

Backgroundβ-Amyloid (Aβ) fibrillation is critical for Aβ deposition and cytotoxicity during the progression of Alzheimer's disease (AD). Consequently, anti-Aβ monoclonal antibody drugs targeting Aβ oligomers and aggregation are considered potential therapeutic strategies for AD treatment. Similar to the working mechanisms of anti-Aβ monoclonal antibody drugs, our study identified osmundacetone (OAC), a small-molecule compound isolated from the traditional Chinese medicine Rhizoma Osmundae, as exerting anti-AD effects by targeting Aβ. PurposeThis study sought to determine whether OAC influences the Aβ burden in APP/PS1 mice and to identify potential regulatory mechanisms. MethodsFive-month-old APP/PS1 mice were injected intraperitoneally with OAC at a dose of 1 mg/kg for 12 weeks. The cognitive functions of the mice were assessed via the Morris water maze test and the open field test. Osmundacetone was analyzed via molecular docking, an isothermal dose‒response fingerprint-cellular context thermal shift assay, a thioflavine T fluorescence assay, and an atomic force microscopy assay to analyze the effects of OAC on Aβ fibrillation. Immunofluorescence, immunoblotting, and immunohistochemistry were used to assess Aβ clearance, AD pathology, oxidative stress, and inflammatory responses. ResultsThe innovative biochemical and physical data illustrated that the ability of OAC to inhibit Aβ fibrillation was accomplished by binding directly to Aβ, which differed from the majority of previously reported natural polyphenols that modulate the Aβ content and structure in an indirect manner. The inhibition of Aβ fibrosis by OAC subsequently promoted Aβ lysosomal degradation, resulting in a decreased Aβ burden in APP/PS1 mice. Furthermore, OAC treatment inhibited oxidative damage by upregulating glutathione peroxidase expression and attenuated the production of inflammatory factors by downregulating nuclear factor-kB phosphorylation in APP/PS1 mice. ConclusionThese findings demonstrate, for the first time, that OAC could reduce the brain Aβ burden in APP/PS1 mice by inhibiting Aβ fibrillation through direct binding to Aβ and improve cognitive dysfunction by attenuating oxidative damage and neuroinflammation. These findings indicate that OAC may be a promising candidate for the treatment of AD.

A Novel Tetrahydroacridine Derivative with Potent Acetylcholinesterase Inhibitory Properties and Dissociative Capability against Aβ42 Fibrils Confirmed by In Vitro Studies.

Alzheimer's disease (AD) is one of the most common causes of dementia, accounting for more than 60% of all cases. It is a neurodegenerative disease in which symptoms such as a decline in memory, thinking, learning, and organizing skills develop gradually over many years and eventually become more severe. To date, there is no effective treatment for the cause of Alzheimer's disease, and the existing pharmacological options primarily help manage symptoms. Treatment is mainly based on acetylcholinesterase (AChE) inhibitors such as donepezil, rivastigmine, and galantamine, which exhibit numerous adverse cardiovascular and gastrointestinal effects due to excessive stimulation of peripheral cholinergic activity involving muscarinic receptors. Therefore, in addition to the obvious drugs that act on the cause of the disease, new drugs based on AChE inhibition that show the fewest side effects are needed. One potential drug could be a new compound under study, tetrahydroacridine derivative (CHDA), which showed significant potential to inhibit the AChE enzyme in previous in vitro studies. The present study shows that while having very potent AChE inhibitory properties, CHDA is a compound with low toxicity to nerve cell culture and living organisms. In addition, it exhibits dissociative activity against amyloid β fibrils, which is extremely important for applications in Alzheimer's disease therapy.

A Small Molecule Impedes the Aβ1-42 Tetramer Neurotoxicity by Preserving Membrane Integrity: Microsecond Multiscale Simulations.

Amyloid-β (Aβ1-42) peptides aggregated into plaques deposited in the brain are the main hallmark of Alzheimer's disease (AD), a social and economic burden worldwide. In this context, insoluble Aβ1-42 fibrils are the main components of plaques. The recent trials that used approved AD drugs show that they can remove the fibrils from AD patients' brains, but they did not halt the course of the disease. Mounting evidence envisages that the soluble Aβ1-42 oligomers' interactions with the neuronal membrane trigger higher cell death than Aβ1-42 fibril interactions. Developing a compound that can alleviate the oligomer's toxicity is one of the most demanding tasks for curing the disease. We performed two molecular dynamics (MD) simulations in an explicit solvent model. In the first case, 55-μs of multiscale all-atom (AA)/coarse-grained (CG) MD simulations were carried out to decipher the impact of a previously described small anti-Aβ molecule, termed M30 (2-octahydroisoquinolin-2(1H)-ylethanamine), on an Aβ1-42 tetramer structure in close contact with a DMPC bilayer. In the second case, 15-μs AA/CG MD simulations were performed to rationalize the dynamics between Aβ1-42 and Aβ1-42-M30 tetramer complexes embedded in DMPC. On the membrane bilayer, we found that the Aβ1-42 tetramer penetrates the bilayer surface due to unrestricted conformational flexibility and many contacts with the membrane phosphate groups. In contrast, no Aβ1-42-M30 tetramer penetration was observed during the entire course of the simulation. In the case of the membrane-embedded Aβ1-42 tetramer, the integrity of the bottom bilayer leaflet was severely affected by the interactions between the negatively charged phosphate groups and the positively charged residues of the Aβ1-42 tetramer, resulting in a deep tetramer penetration into the bilayer hydrophobic region. These contacts were not observed in the case of the membrane-embedded Aβ1-42-M30 tetramer. It was noted that M30 molecules bind to Aβ1-42 tetramer through hydrogen bonds, resulting in a conformational stable Aβ1-42-M30 complex. The associated complex has reduced conformational changes and an enhanced rigidity that prevents the tetramer dissociation by interfering with the tetramer-membrane contacts. Our findings suggest that the M30 molecules could bind to Aβ1-42 tetramer resulting in a rigid structure, and that such complexes do not significantly perturb the membrane bilayer organization. These observations support the in vitro and in vivo experimental evidence that the M30 molecules prevent synaptotocity, improving AD-affected mice memory.

Investigation of biomolecular dynamics by sensitivity-enhanced 1H–2H CPMAS NMR using matrix-free dynamic nuclear polarization

Molecular dynamics of functional groups contain valuable information about structural properties and functional activities in biomolecules. NMR spectroscopy is a sensitive tool for the investigation of molecular dynamics over a wide range of timescales and thus may deepen the understanding of the biomolecules of interest. Here, we present an approach to use DNP-enhanced 2H NMR to study dynamics of selectively deuterated methyl groups in insoluble proteins such as amyloid beta (Aβ) fibrils. We adopted and optimized the matrix-free DNP approach by varying the amount of added polarizing agent as well as the rehydration level of model proteins. We show that the DNP enhancement obtained in 1H–2H cross-polarization (CP) MAS spectra may increase the sensitivity for selectively deuterated Aβ fibril samples by more than one order of magnitude, accelerating the collection of spin-lattice relaxation data in the DNP-accessible temperature range between 100 and 150 K by up to 400-fold. However, below the coalescence temperature, which describes the transition from the fast to the slow exchange regime, the experimentally obtained relaxation time constants suffer from a paramagnetic relaxation enhancement effect due to the presence of the polarizing agent. This seems to be a general effect for biomolecules as it is also confirmed for two other protein model systems. Our demonstration opens the possibility to extend the scope of 2H NMR for dynamics measurements to effective concentrations and/or temperatures below what is currently accessible; however, the observed interplay between paramagnetic relaxation and molecular dynamics also emphasizes the necessity for a better understanding of relaxation effects in DNP-enhanced NMR.

Ratiometric Imaging Detection of Amyloid-β Fibrils by a Dual-Emissive Tris-Heteroleptic Ruthenium Complex.

Two dual fluorescent/phosphorescent tris-heteroleptic mononuclear Ru(ΙΙ) complexes (2 and 3) were designed and applied in amyloid-β (Aβ) sensing. These complexes have a general formula of [Ru(phen)(dppz)(L)](PF6)2, where L is (2-pyrazinyl)(2-pyridyl)(methyl)amine (H-L) with different substituents (-OMe for 2, -H for 3), phen is 1,10-phenanthroline, and dppz is dipyridophenazine, respectively. Compared with the previously reported ratiometric probe 1 with a di(pyrid-2-yl)(methyl)amine ligand, complex 2 can be employed for not only ratiometric emissive detection of Aβ aggregation but also ratiometric imaging detection of Aβ fibrils. In ratiometric emissive detection, as the incubation time of the Aβ sample (Aβ40 and Aβ42) was prolonged, a new phosphorescence emission band appeared with gradual enhancement of the emission intensity, while the fluorescence emission was basically unchanged, which could be treated as an intrinsic internal reference signal. In comparison, a larger ratiometric photoluminescence enhancement (I640/I440) was observed for Aβ40 aggregation with respect to Aβ42. In ratiometric imaging detection, the imaging signals obtained from the phosphorescence emission are much brighter than the fluorescence emission in both Aβ40 and Aβ42 fibrils. As indicated by molecular docking results, stronger interactions were found between complex 2 with Aβ40 fibrils, which included π/π, π/C-H, and π/H interactions between bidentate ligands dppz and phen with amino acid residues. Moreover, computational calculations were carried out to assist the interpretation of these experimental findings.