阿尔茨海默氏症致病蛋白-Aβ_((12-28))肽段的NMR研究

Aβ肽的多聚化和纤维化在阿尔茨海默氏症(Alzheimer's diseas)的发生中起关键作用, 其中以Aβ_((1-42))的致病作用为最强,因此阻断其聚集成为阿尔茨海默氏症一种潜在的治疗方式. 作者在研究中发现某些化合物可以结合于Aβ_((12-28))肽段. 该文采用~1H-~1H COSY、 TOCSY、 ROESY和~(15)N-HSQC多种核磁分析方法, 对此肽段的~1H和~(15)N NMR谱信号进行了归属和详细分析, 为进一步研究其与小分子抑制剂的相互作用提供了基础.     

Cu2+ Coordination of Covalently Cross-linked β-Amyloid Dimers

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by deposition of extracellular amyloid plaques comprised from fibrillar aggregates of the amyloid-β peptide (Aβ). Cu²⁺ interactions with Aβ appear to be involved in both the production of reactive oxygen species and the misfolding of Aβ into oligomeric intermediates including covalently cross-linked dimers. We have previously investigated the Cu²⁺ coordination of Aβ monomers in detail, whilst others have shown that Aβ fibrils coordinate Cu²⁺ in a similar manner to Aβ monomers. However, the coordination of low-molecular-weight Aβ species, which are believed to be responsible for neuronal dysfunction in AD, has not been widely investigated. Here, we report the first study of Cu²⁺ coordination by synthetic Aβ dimers containing an artificial diaminopimelic acid (DAP) or a dityrosine cross-link at residue 10. Our preliminary findings show that dityrosine cross-linking imparts unique structural constraints, resulting in Cu²⁺ coordination distinct from Aβ monomers and fibrils, which may be relevant to the greater toxicity of low-molecular-weight Aβ oligomers in AD.     

Biaryl scaffold-focused virtual screening for anti-aggregatory and neuroprotective effects in Alzheimer’s disease

Background

Alzheimer’s disease (AD) is a primary cause of dementia in ageing population affecting more than 35 million people around the globe. It is a chronic neurodegenerative disease caused by defected folding and aggregation of amyloid beta (Aβ) protein. Aβ is formed by the cleavage of membrane embedded amyloid precursor protein (APP) by using enzyme ‘transmembrane aspartyl protease, β-secretase’. Inhibition of β-secretase is a viable strategy to prevent neurotoxicity in AD. Another strategy in the treatment of AD is inhibition of acetylcholinesterase. This inhibition reduces the degradation of acetylcholine and temporarily restores the cholinergic function of neurons and improves cognitive function. Monoamine oxidase and higher glutamate levels are also found to be linked with Aβ peptide related oxidative stress. Oxidative stress leads to reduced activity of glutamate synthase resulting in significantly higher level of glutamate in brain. The aim of this study is to perform in silico screening of a virtual library of biaryl scaffold containing compounds potentially used for the treatment of AD. Screening was done against the primary targets of AD therapeutics, acetylcholinesterase, β-secretase (BACE1), Monoamine oxidases (MAO) and N-Methyl-D-aspartate (NMDA) receptor. Compounds were screened for their inhibitory potential by employing molecular docking approach using AutoDock vina. Binding energy scores were embodied in the heatmap to display varies strengths of interactions of the ligands targeting AD.

Results

Several ligands showed notable interaction with at least two targets, but the strong interaction with all the targets is shown by very few ligands. The pharmacokinetics of the interacting ligands was also predicted. The interacting ligands have good drug-likeness and brain availability essential for drugs with intracranial targets.

Conclusion

These results suggest that biaryl scaffold may be pliable to drug development for neuroprotection in AD and that the synthesis of further analogues to optimize these properties should be considered.

     

Using a portable total reflection X-ray fluorescence system for a multielement analysis of Swiss mice brains with experimental Alzheimer's disease induced by β-amyloid oligomers

Alzheimer's disease (AD) is a progressive and irreversible disorder whose pathological features include β-amyloid (Aβ) plaques and neuronal and synaptic loss. Metals such as iron, copper, and zinc are increased in the brains of patients with AD. Those metals can interact with Aβ, resulting in the promotion of Aβ deposition and formation of plaque. However, no study analyzing the effects of single injection of Aβ soluble oligomers (AβOs) in the elements' homeostasis in mice was developed. Total reflection X-ray fluorescence (TXRF) is a multielement analytical technique that can be utilized to identify and quantify trace elements present in a sample at very low concentrations. In this study, in order to evaluate the concentration of metals in brain regions of Swiss mice, three groups of female mice and three of male mice were studied: control, AD10, and AD100. The AD groups received an AβOs intracerebroventricular injection so as to induce experimental AD. Afterwards, a craniotomy was performed, and six brain compartments were dissected and evaluated. TXRF measurements were performed using a portable TXRF system that uses an X-ray tube with a molybdenum anode and a detector Si-PIN. It is proved to determine the following elements' concentrations: phosphorus, sulfur, potassium, iron, copper, zinc, and rubidium. Results showed differences in the elemental concentration in some brain regions between AD groups. These alterations suggest that AβOs act quickly, even before the amyloid plaques' formation, explaining cognitive deficits independently of amyloid plaques. This study helped to understand that this modification on elemental concentration can be influenced by AβOs.     

Neuroprotective effects of edaravone-administration on 6-OHDA-treated dopaminergic neurons

Background

A recent human clinical trial of an Alzheimer's disease (AD) vaccine using amyloid beta (Aβ) 1–42 plus QS-21 adjuvant produced some positive results, but was halted due to meningoencephalitis in some participants. The development of a vaccine with mutant Aβ peptides that avoids the use of an adjuvant may result in an effective and safer human vaccine.

Results

All peptides tested showed high antibody responses, were long-lasting, and demonstrated good memory response. Epitope mapping indicated that peptide mutation did not lead to epitope switching. Mutant peptides induced different inflammation responses as evidenced by cytokine profiles. Ig isotyping indicated that adjuvant-free vaccination with peptides drove an adequate Th2 response. All anti-sera from vaccinated mice cross-reacted with human Aβ in APP/PS1 transgenic mouse brain tissue.

Conclusion

Our study demonstrated that an adjuvant-free vaccine with different Aβ peptides can be an effective and safe vaccination approach against AD. This study represents the first report of adjuvant-free vaccines utilizing Aβ peptides carrying diverse mutations in the T-cell epitope. These largely positive results provide encouragement for the future of the development of human vaccinations for AD.     

Isolation of mineralizing Nestin+ Nkx6.1+ vascular muscular cells from the adult human spinal cord

Background

Evidence suggests that rheumatoid arthritis (RA) may enhance or reduce the progression of Alzheimer's disease (AD). The present study was performed to directly explore the effects of collagen-induced rheumatoid arthritis (CIA) on amyloid plaque formation, microglial activation, and microvascular pathology in the cortex and hippocampus of the double transgenic APP/PS1 mouse model for AD. Wild-type or APP/PS1 mice that received type II collagen (CII) in complete Freund's adjuvant (CFA) at 2 months of age revealed characteristics of RA, such as joint swelling, synovitis, and cartilage and bone degradation 4 months later. Joint pathology was accompanied by sustained induction of IL-1β and TNF-α in plasma over 4 weeks after administration of CII in CFA.

Results

CIA reduced levels of soluble and insoluble amyloid beta (Aβ) peptides and amyloid plaque formation in the cortex and hippocampus of APP/PS1 mice, which correlated with increased blood brain barrier disruption, Iba-1-positive microglia, and CD45-positive microglia/macrophages. In contrast, CIA reduced vessel density and length with features of microvascular pathology, including vascular segments, thinner vessels, and atrophic string vessels.

Conclusions

The present findings suggest that RA may exert beneficial effects against Aβ burden and harmful effects on microvascular pathology in AD.     

An online nano-LC-ESI-FTICR-MS method for comprehensive characterization of endogenous fragments from amyloid β and amyloid precursor protein in human and cat cerebrospinal fluid

Amyloid precursor protein (APP) is the precursor protein to amyloid β (Aβ), the main constituent of senile plaques in Alzheimer's disease (AD). Endogenous Aβ peptides reflect the APP processing, and greater knowledge of different APP degradation pathways is important to understand the mechanism underlying AD pathology. When one analyzes longer Aβ peptides by low-energy collision-induced dissociation tandem mass spectrometry (MS/MS), mainly long b-fragments are observed, limiting the possibility to determine variations such as amino acid variants or post-translational modifications (PTMs) within the N-terminal half of the peptide. However, by using electron capture dissociation (ECD), we obtained a more comprehensive sequence coverage for several APP/Aβ peptide species, thus enabling a deeper characterization of possible variants and PTMs. Abnormal APP/Aβ processing has also been described in the lysosomal storage disease Niemann-Pick type C and the major large animal used for studying this disease is cat. By ECD MS/MS, a substitution of Asp7 → Glu in cat Aβ was identified. Further, sialylated core 1 like O-glycans at Tyr10, recently discovered in human Aβ (a previously unknown glycosylation type), were identified also in cat cerebrospinal fluid (CSF). It is therefore likely that this unusual type of glycosylation is common for (at least) species belonging to the magnorder Boreoeutheria. We here describe a detailed characterization of endogenous APP/Aβ peptide species in CSF by using an online top-down MS-based method.     

The Aggregation Potential of the 1–15- and 1–16-Fragments of the Amyloid β Peptide and Their Influence on the Aggregation of Aβ40

The aggregation of amyloid β (Aβ) peptide is important in Alzheimer’s disease. Shorter Aβ fragments may reduce Aβ’s cytotoxicity and are used in diagnostics. The aggregation of Aβ16 is controversial; Liu et al. (J. Neurosci. Res. 75:162–171, 2004) and Liao et al. (FEBS Lett. 581:1161–1165, 2007) find that Aβ16 does not aggregate and reduces Aβ’s cytotoxicity, Du et al. (J. Alzheimer’s Dis. 27:401–413, 2011) reports that Aβ16 aggregates and that Aβ16 oligomers are toxic to cells. Here the aggregation potential of two shorter fragments, Aβ15 and Aβ16, and their influence on Aβ40 is measured by electron paramagnetic resonance (EPR) spectroscopy and the ThioT fluorescence assay (ThioT). Continuous-wave, 9 GHz EPR measurements and ThioT results reveal that neither Aβ15 nor Aβ16 aggregate by themselves and that they do not affect Aβ40 aggregation.     

Monitoring Alzheimer Amyloid Peptide Aggregation by EPR

Plaques containing the aggregated β-Amyloid (Aβ) peptide in the brain are the main indicators of Alzheimer’s disease. Fibrils, the building blocks of plaques, can also be produced in vitro and consist of a regular arrangement of the peptide. The initial steps of fibril formation are not well understood and could involve smaller aggregates (oligomers) of Aβ. Such oligomers have even been implicated as the toxic agents. Here, a method to study oligomers on the time scale of aggregation is suggested. We have labeled the 40 residue Aβ peptide variant containing an N-terminal cysteine (cys-Aβ) with the MTSL [1-oxyl-2,2,5,5-tetramethyl-Δ-pyrroline-3-methyl] methanethiosulfonate spin label (SL-Aβ). Fibril formation in solutions of pure SL-Aβ and of SL-Aβ mixed with Aβ was shown by Congo-red binding and electron microscopy. Continuous-wave 9 GHz electron paramagnetic resonance reveals three fractions of different spin-label mobility: one attributed to monomeric Aβ, one to a multimer (8–15 monomers), and the last one to larger aggregates or fibrils. The approach, in principle, allows detection of oligomers on the time scale of aggregation.     

Probing the role of metal cations on the aggregation behavior of amyloid β-peptide at a single molecule level by AFM

With the development of nanotechnology, understanding of intermolecular interactions on a single molecule level by atomic force spectroscopy (AFM) has played an important role in molecular biology and biomedical science. In recent years, some research suggested that the presence of metal cations is an important regulator in the processes of misfolding and aggregation of the amyloid β-protein (Aβ), which may be an important etiological factor of Alzheimer’s disease. However, the knowledge on the principle of interactions between Aβ and metal cations at the single molecule level is still poor understood. In this paper, the amyloid β-protein (Aβ) was fabricated on substrate of mixed thiol-modified gold nanoparticles using self-assembled monolayer method and the adhesion force in the longitudinal direction between metal cations and Aβ42 were investigated by AFM. The role of metal ions on Aβ aggregation is discussed from the perspective of single molecular force. The force results showed that the specific adhesion force F _i and the nonspecific force F ₀ between a single Aβ–Aβ pair in control experiment were calculated as 42 ± 3 and 80 pN, respectively. However, F _i between a single Aβ–Aβ pair in the presence of Cu²⁺, Zn²⁺, Ca²⁺ and Al³⁺ increased dramatically to 84 ± 6, 89 ± 3, 73 ± 5, 95 ± 5 pN successively, which indicated that unbinding between Aβ proteins is accelerated in the presence of metal cations. What is more, the imaging results showed that substoichiometric copper cations accelerate the formation of fibrils within 3 days. The combined atomic force spectroscopy and imaging analysis indicate that metal cations play a role in promoting the aggregating behavior of Aβ42.     

Different Conformation of Thiol Protease Inhibitor During Amyloid Formation: Inhibition by Curcumin and Quercetin

Cystatins are thiol proteinase inhibitors ubiquitously present in mammalian body and serve various important physiological functions. In the present study, we examined the effects of acid denaturation on newly identified thiol protease inhibitors from the lungs of Capra hircus (Goat) with a focus on protein conformational changes and amyloid fibril formation. Acid denaturation as studied by CD (Circular Dichroism) and fluorescence spectroscopy showed that purified inhibitor named GLC (Goat Lung Cystatin) populates three partly unfolded species, a native like state at pH?3.0, a partly unfolded intermediate at pH2.0, and unstructured unfolded state at pH?1.0, from each of which amyloid like fibrils grow as assessed by thioflavin T (ThT) spectroscopy. The result showed, native like structure formed at pH?3.0 is more responsive towards amyloid formation when compare to other conformation of proteins. Morphology of the protein species incubated for amyloid process was observed using transmission electron microscopy (TEM). Moreover, anti-fibrillogenic effects of curcumin and quercetin were analysed using ThT binding assay. Curcumin and quercetin produced a concentration dependent decline inThT fluorescence suggesting deaggregation of the fibrils. When added prior to amyloid fibril initiation 50 μM curcumin inhibited amyloid aggregation. However, more quercetin is needed to prevent the same extent of fibrillation. Implications for therapeutics in view of polyphenols as essential nutrients are suggested in lung diseases.     

Alzheimer's disease: biological aspects, therapeutic perspectives and diagnostic tools

Alzheimer's disease (AD) is the most common form of dementia among older people. Dementia is an irreversible brain disorder that seriously affects a person's ability to carry out daily activities. It is characterized by loss of cognitive functioning and behavioral abilities, to such an extent that it interferes with the daily life and activities of the affected patients. Although it is still unknown how the disease process begins, it seems that brain damage starts a decade or more before problems become evident. Scientific data seem to indicate that changes in the generation or the degradation of the amyloid-b peptide (Aβ) lead to the formation of aggregated structures that are the triggering molecular events in the pathogenic cascade of AD. This review summarizes some characteristic features of Aβ misfolding and aggregation and how cell damage and death mechanisms are induced by these supramolecular and toxic structures. Further, some interventions for the early diagnosis of AD are described and in the last part the potential therapeutic strategies adoptable to slow down, or better block, the progression of the pathology are reported.     

Protonation state and free energy calculation of HIV-1 protease–inhibitor complex based on electrostatic polarisation effect

The protonation states of catalytic Asp25/25′ residues remarkably affect the binding mechanism of the HIV-1 protease–inhibitor complex. Here we report a molecular dynamics simulation study, which includes electrostatic polarisation effect, to investigate the influence of Asp25/25′ protonation states upon the binding free energy of the HIV-1 protease and a C₂-symmetric inhibitor. Good agreements are obtained on inhibitor structure, hydrogen bond network, and binding free energy between our theoretical calculations and the experimental data. The calculations show that the Asp25 residue is deprotonated, and the Asp25′ residue is protonated. Our results reveal that the Asp25/25′ residues can have different protonation states when binding to different inhibitors although the protease and the inhibitors have the same symmetry. This study offers some insights into understanding the protonation state of HIV-1 protease–inhibitor complex, which could be helpful in designing new inhibitor molecules.     

Peptide backbone-copper ring structure: A molecular insight into copper-induced amyloid toxicity

Copper ions can promote amyloid diseases that are associated with amyloid peptides, such as type 2 diabetes (T2D), Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). However, the underlying molecular mechanism remains obscure. Here we present that Cu²⁺ is able to specifically bind to the backbone of T2D-related human islet amyloid polypeptide (hIAPP) by forming a ring structure, which causes the reduction of Cu²⁺ to Cu⁺ to produce reactive oxygen species (ROS) and the modulation of hIAPP aggregation. Nuclear magnetic resonance spectroscopy showed that Cu²⁺ bound to the backbone of a turn region, His18—Ser21, which is critical for hIAPP aggregation. Ab initio calculations and x-ray absorption fine structure analyses revealed that Cu²⁺ simultaneously bound with both the amide nitrogen and carbonyl oxygen on the peptide backbone, resulting in a ring structure, and causing the reduction of Cu²⁺ to Cu⁺ to form a hIAPP-Cu⁺ complex. 2',7'-dichlorodihydrofluorescin diacetate fluorescence measurements further indicated that this complex led to enhanced ROS levels in rat insulinoma cells. Additionally, thioflavin T fluorescence and atomic force microscopy measurements denoted that the backbone-Cu ring structure largely modulated hIAPP aggregation, including the inhibition of hIAPP fibrillation and the promotion of peptide oligomerization. These findings shed new light on the molecular mechanism of Cu²⁺-induced amyloid toxicity involving both the enhancement of ROS and the modulation of hIAPP aggregation.     

A rational approach to selective pharmacophore designing: an innovative strategy for specific recognition of Gsk3β

We propose a novel cheminformatics approach that combines structure and ligand-based design to identify target-specific pharmacophores with well-defined exclusion ability. Our strategy includes the prediction of selective interactions, developing structure, and knowledge-based selective pharmacophore models, followed by database screening and molecular docking. This unique strategy was employed in addressing the off-target toxicity of Gsk3β and CDKs. The connections of Gsk3β in eukaryotic cell apoptosis and the extensive potency of Gsk3β inhibitors to block cell death have made it a potential drug-discovery target for many grievous human disorders. Gsk3β is phylogenetically very closely related to the CDKs, such as CDK1 and CDK2, which are suggested to be the off-target proteins of Gsk3β inhibitors. Here, we have employed novel computational approaches in designing the ligand candidates that are potentially inhibitory against Gsk3β, with well-defined the exclusion ability to CDKs. A structure-ligand -based selective pharmacophore was modeled. This model was used to retrieve molecules from the zinc database. The hits retrieved were further screened by molecular docking and protein-ligand interaction fingerprints. Based on these results, four molecules were predicted as selective Gsk3β antagonists. It is anticipated that this unique approach can be extended to investigate any protein-ligand specificity.     

Tunable inhibition of β-amyloid peptides by fast green molecules

The aggregation of β-amyloid (Aβ) protein into toxic intermediates and mature fibrils is considered to be one of the main causes of Alzheimer's disease (AD). Small molecules as one of blockers are expected to be the potential drug treatment for the disease. However, the nucleation process in molecular assembly is less informative in the literatures. In this work, the formation of Aβ (16-22) peptides was investigated with the presence of small molecule of fast green (FG) at the initial aggregation stage. The results exhibited the tunable inhibitory ability of FG molecules on Aβ (16-22) peptides. Atomic force microscopy (AFM) demonstrated that the inhibitory effect would be dependent on the dose of FG molecules, which could delay the lag time (nucleation) and form single layer conjugates. Spectral measurements further showed that the β-sheet secondary structure of Aβ (16-22) reduced dramatically after the presence of FG molecules. Instead, non-β-sheet nanosheets were formed when the FG/Aβ (16-22) ratio reached 1:1. In addition, the cytotoxicity of aggregates reduced greatly with the presence of FG molecules compared with the Aβ (16-22) fibrils. Overall, this study provided a method for suppressing the toxic amyloid aggregates by FG molecules efficiently, and also showed a strategy for fabrication of two-dimensional materials by small molecules.     

Synthesis and Characterization of Acylated Polycaprolactone (PCL) Nanospheres and Investigation of Their Influence on Aggregation of Amyloid Proteins

Acylated polycaprolactone (PCL) nanospheres were fabricated and employed to interact with amyloid-β-(25–35) peptides (Aβ_25–35), "peptide 11 of the 40 peptide full amyloid-β". The nanospheres were characterized by scanning electron microscopy (SEM), attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, and dynamiclightscattering (DLS), all of which confirmed that the acylated polycaprolactone (Ac-PCL) nanospheres were successfully fabricated. The effect of the nanospheres on the aggregation of Aβ_25–35 peptides was investigated by thioflavin T fluorescence measurements. The result showed that, without nanospheres, the Aβ_25–35 peptides aggregated gradually from monomers and oligomers to long fibrils with increasing incubation time. In comparison, the nanospheres were effective in interfering with fibrillogenesis and aggregation of amyloid-β. We suggest this study may contribute to the development of new therapeutic strategies against amyloid-related disorders.     

Entrapment of Aβ(1-40) peptide in unstructured aggregates

Recognizing the complexity of the fibrillogenesis process provides a solid ground for the development of therapeutic strategies aimed at preventing or inhibiting protein-protein aggregation. Under this perspective, it is meaningful to identify the possible aggregation pathways and their relative products. We found that Aβ-peptide dissolved in a pH 7.4 solution at small peptide concentration and low ionic strength forms globular aggregates without typical amyloid β-conformation. ThT binding kinetics was used to monitor aggregate formation. Circular dichroism spectroscopy, AFM imaging, static and dynamic light scattering were used for structural and morphological characterization of the aggregates. They appear stable or at least metastable with respect to fiber growth, therefore appearing as an incidental product in the pathway of fibrillogenesis.     

Study on the Amyloid A $\beta$42 with Accelerated Molecular Dynamics Simulations *

One major cause of Alzheimer's disease (AD) is evidently due to the aggregation and deposition of amyloid $\beta$ peptides (A$\beta$) in the brain tissue of the patient. Preventing misfolding and self-aggregation of A$\beta$ protein can reduce the formation of highly toxic polymer, which is important for the treatment of AD. Among them, the $\alpha$-helix consisting of 42 residues (A$\beta$42) is the main component of senile plaques in AD. In this paper, 500 ns accelerated molecular dynamics are performed at different temperatures (300 K, 350 K, 400 K, 450 K) to study of the effect of temperature-induced conformation changes of A$\beta$42 protein during the unfolding process respectively.     

Structure‐Making Effects of Metal Nanoparticles in Amyloid Peptide Fibrillation

There is growing concern that nanoparticles (NPs) may accelerate amyloid protein aggregation and thus cause amyloid‐related diseases. Here, the potential of silver and gold NPs is explored (diameter 20 nm) on the aggregation of the amyloid peptide sequences NNFGAIL from human islet amyloid polypeptide and the yeast prion protein sequence GNNQQNY, which are both the sequences of the full systems, which are able to aggregate into characteristic amyloid cross‐beta sheet fibrillar structures. Here, it is shown that silver and gold NPs in physiological aqueous solution at ambient temperatures accelerate the aggregation kinetics of both peptides significantly (in vitro). Scanning electron microscopy and X‐ray diffraction provide solid evidence for a “structure‐making” effect of the NPs. In particular, we are able to image the initial peptide corona and measure its structural reorganization in time‐resolved kinetic experiments. After a conversion time Δt, the coated NPs appear to act as templates or seeds for rapid fibrillation. Interestingly, cross‐fibrillation experiments with different peptide‐coated NPs (pcNPs) reveal that they can efficiently induce aggregation of similar peptides once the pcNPs are structurally converted. It is discussed that these structurally converted pcNPs may display similar kinetic features as toxic and aggregation inducing oligomers/protofibrils in normal amyloid aggregation, without being transient and very low‐concentration species. Finally, we suggest and discuss a simple mechanistic picture with the biomolecule corona of NPs being central to the function of the coated NPs in amyloid fibrillation.