Inhibits Aβ Fibril Formation Research Articles

Synthesis of Some Benzothiazole Derivatives Based on 3-hydroxypyridine-4-one and Benzaldehydeand Evaluation of their β-amyloid Aggregation Inhibition using both Experimental Methods and Molecular Dynamic Simulation.

Some novel inhibitors based on the (benzo[d]thiazol-2-yl)-1-phenylmethanimine derivatives were designed to reduce the aggregation process in Alzheimer's disease. These structures seem to mimic stilbene-like scaffold, while the benzothiazole moiety "locks" the thioflavin T binding site. Other inhibitors were designed based on 2-((benzo[d]thiazol-2-ylimino)methyl)-5-(benzyloxy)-1-methylpyridin-4(H)-one derivatives. Benzo[d]thiazol-2-amine derivatives were prepared by the reaction of aniline derivatives with ammonium thiocyanate in the presence of bromine/acetic acid. Then, the reaction of amines with benzaldehyde derivatives and 5-(benzyloxy)-1-methyl-4-oxo-1,4-dihydropyridine-2-carbaldehyde gave the desired compounds. The plate reader-based fibrillation assay was done to evaluate the inhibition of Aβ aggregation. Also, molecular dynamicsimulation was carried out to clarify the interaction manner of the designed compounds with Aβ formation. The biological evaluation proved 5a and 7e as the best inhibitor of the Aβ aggregation. compound 5a in concentration of 50 µM inhibited Aβ fibril formation better than 7e. MD simulation elucidated that the Aβ aggregation inhibitors in different concentrations represented different binding conformations throughout the entire or in one area of Aβ. MD showed the ligands in lower concentrations accumulate in an area of Aβ aggregations and separate one fibril from the aggregated Aβ. On the contrary, in higher concentrations, the ligands tend to be located through the entire Aβ.

Metal Binding of Alzheimer's Amyloid-β and Its Effect on Peptide Self-Assembly.

ConspectusMetal ions have been identified as key factors modulating the aggregation of amyloid-β peptide (Aβ) implicated in Alzheimer's disease (AD). The presence of elevated levels of metal ions in the amyloid plaques in AD patients supports the notion that the dysfunction of metal homeostasis is connected to the development of AD pathology. Here, recent findings from high- and low-resolution biophysical techniques are put into perspective, providing detailed insights into the molecular structures and dynamics of metal-bound Aβ complexes and the effect of metal ions on the Aβ aggregation process. In particular, the development of theoretical kinetic models deducing different microscopic nucleation events from the macroscopic aggregation behavior has enabled deciphering of the effect of metal ions on specific nucleation processes. In addition to these macroscopic measurements of bulk aggregation to quantify microscopic rates, recent NMR studies have revealed details about the structures and dynamics of metal-Aβ complexes, thereby linking structural events to bulk aggregation. Interestingly, transition-metal ions, such as copper, zinc, and silver ions, form a compact complex with the N-terminal part of monomeric Aβ, respectively, where the metal-bound "folded" state is in dynamic equilibrium with an "unfolded" state. The rates and thermodynamic features of these exchange dynamics have been determined by using NMR relaxation dispersion experiments. Additionally, the application of specifically tailored paramagnetic NMR experiments on the Cu(II)-Aβ complex has been fruitful in obtaining structural constraints within the blind sphere of conventional NMR experiments. This enables the determination of molecular structures of the "folded" Cu(II)-coordinated N-terminal region of Aβ. Furthermore, the discussed transition-metal ions modulate Aβ self-assembly in a concentration-dependent manner, where low metal ion concentrations inhibit Aβ fibril formation, while at high metal ion concentrations other processes occur, resulting in amorphous aggregate formation. Remarkably, the metal-Aβ interaction predominately reduces one specific nucleation step, the fibril-end elongation, whereas primary and surface-catalyzed secondary nucleation mechanisms are less affected. Specific inhibition of fibril-end elongation theoretically predicts an enhanced generation of Aβ oligomers, which is an interesting contribution to understanding metal-Aβ-associated neurotoxic effects. Taken together, the metal binding process creates a metal-bound Aβ complex, which is seemingly inert to aggregation. This process hence efficiently reduces the aggregation-prone peptide pool, which on the macroscopic level is reflected as slower aggregation kinetics. Thus, the specific binding of metals to the Aβ monomer can be linked to the macroscopic inhibitory effect on Aβ bulk aggregation, providing a molecular understanding of the Aβ aggregation mechanism in the presence of metal ions, where the metal ion can be seen as a minimalist agent against Aβ self-assembly. These insights can help to target Aβ aggregation in vivo, where metal ions are key factors modulating the Aβ self-assembly and Aβ-associated neurotoxicity.

Genetic Reduction of Insulin Signaling Mitigates Amyloid-β Deposition by Promoting Expression of Extracellular Matrix Proteins in the Brain.

The insulin/IGF-1 signaling (IIS) regulates a wide range of biological processes, including aging and lifespan, and has also been implicated in the pathogenesis of Alzheimer's disease (AD). We and others have reported that reduced signaling by genetic ablation of the molecules involved in IIS (e.g., insulin receptor substrate 2 [IRS-2]) markedly mitigates amyloid plaque formation in the brains of mouse models of AD, although the molecular underpinnings of the amelioration remain unsolved. Here, we revealed, by a transcriptomic analysis of the male murine cerebral cortices, that the expression of genes encoding extracellular matrix (ECM) was significantly upregulated by the loss of IRS-2. Insulin signaling activity negatively regulated the phosphorylation of Smad2 and Smad3 in the brain, and suppressed TGF-β/Smad-dependent expression of a subset of ECM genes in brain-derived cells. The ECM proteins inhibited Aβ fibril formation in vitro, and IRS-2 deficiency suppressed the aggregation process of Aβ in the brains of male APP transgenic mice as revealed by injection of aggregation seeds in vivo Our results propose a novel mechanism in AD pathophysiology whereby IIS modifies Aβ aggregation and amyloid pathology by altering the expression of ECM genes in the brain.SIGNIFICANCE STATEMENT The insulin/IGF-1 signaling (IIS) has been recognized as a regulator of aging, a leading risk factor for the onset of Alzheimer's disease (AD). In AD mouse models, genetic deletion of key IIS molecules markedly reduces the amyloid plaque formation in the brain, although the molecular underpinnings of this amelioration remain elusive. We found that the deficiency of insulin receptor substrate 2 leads to an increase in the expression of various extracellular matrices (ECMs) in the brain, potentially through TGF-β/Smad signaling. Furthermore, some of those ECMs exhibited the potential to inhibit amyloid plaque accumulation by disrupting the formation of Aβ fibrils. This study presents a novel mechanism by which IIS regulates Aβ accumulation, which may involve altered brain ECM expression.

Protective effects of Radix Stellariae extract against Alzheimer's disease via autophagy activation in Caenorhabditis elegans and cellular models

Enhancing the clearance of proteins associated with Alzheimer's disease (AD) emerges as a promising approach for AD therapeutics. This study explores the potential of Radix Stellariae, a traditional Chinese medicine, in treating AD. Utilizing transgenic C. elegans models of AD, we demonstrated that a 75% ethanol extract of Radix Stellariae (RSE) (at 50 µg/mL) effectively diminishes Aβ and Tau protein expression, and alleviates their induced impairments including paralysis, behavioral dysfunction, neurotoxicity, and ROS accumulation. Additionally, RSE enhances the stress resistance of C. elegans. Further investigations revealed that RSE promotes autophagy, a critical cellular process for protein degradation, in these models. We found that inhibiting autophagy-related genes negated the neuroprotective effects of RSE, suggesting a central role for autophagy in the actions of RSE. In PC-12 cells, we observed that RSE not only inhibited Aβ fibril formation but also promoted the degradation of AD-related proteins and reduced their cytotoxicity. Mechanistically, RSE was found to induce autophagy via modulating PI3K/AKT/mTOR and AMPK/mTOR signaling pathways. Importantly, inhibiting autophagy counteracted the beneficial effects of RSE on the clearance of AD-associated proteins. Moreover, we identified Dichotomine B, a β-carboline alkaloid, as a key active constituent of RSE in mitigating AD pathology in C. elegans at concentrations ranging from 50 to 1000 µM. Collectively, our study presents novel discoveries that RSE alleviates AD pathology and toxicity primarily by inducing autophagy, both in vivo and in vitro. These findings open up new avenues for exploring the therapeutic potential of RSE and its active component, Dichotomine B, in treating neurodegenerative diseases like AD.

Sodium Oligomannate Electrostatically Binds to Aβ and Blocks Its Aggregation.

GV-971 (sodium oligomannate) is a China Food and Drug Administration (CFDA)-approved drug for treating Alzheimer's disease, and it could inhibit Aβ fibril formation in vitro and in mouse studies. To elucidate the mechanisms for understanding how GV-971 modulates Aβ's aggregation, we conducted a systematic biochemical and biophysical study of Aβ40/Aβ42:GV-971 systems. The integrating analysis of previously published data and our results suggests that the multisite electrostatic interactions between GV-971's carboxylic groups and Aβ40/Aβ42's three histidine residues might play a dominant role in driving the binding of GV-971 to Aβ. The fuzzy-type electrostatic interactions between GV-971 and Aβ are expected to protect Aβ from aggregation potentially through breaking the histidine-mediated inter-Aβ electrostatic interactions. Meanwhile, since GV-971's binding exhibited a slight downregulation effect on the flexibility of Aβ's histidine-colonized fragment, which potentially favors Aβ aggregation, we conclude that the dynamics alteration plays a minor role in GV-971's modulation on Aβ aggregation.

α-Enolase reduces cerebrovascular Aβ deposits by protecting Aβ amyloid formation.

Cerebral amyloid angiopathy (CAA) is characterized by cerebrovascular amyloid β (Aβ) deposits and causes dementia and cerebral hemorrhage. Although α-enolase (ENO1) was shown to possess multifunctional roles, its exact functions in CAA pathogenesis have not been determined. In this study, we focused on ENO1, a well-known glycolytic enzyme, which was previously identified via a proteomic approach as an upregulated protein in brain samples from patients with Alzheimer's disease (AD). We utilized the thioflavin T fluorescence assay and transmission electron microscopy to monitor the effects of ENO1 on amyloid formation by Aβ peptides. We also cultured murine primary cerebrovascular smooth muscle cells to determine the effects of ENO1 on Aβ cytotoxicity. To investigate the effects of ENO1 in vivo, we infused ENO1 or a vehicle control into the brains of APP23 mice, atransgenicmodelofAD/CAA, using a continuous infusion system, followed by a cognitive test and pathological and biochemical analyses. We found that novel functions of ENO1 included interacting with Aβ and inhibiting its fibril formation, disrupting Aβ fibrils, and weakening the cytotoxic effects of these fibrils via proteolytic degradation of Aβ peptide. We also demonstrated that infusion of ENO1 into APP23 mouse brains reduced cerebrovascular Aβ deposits and improved cognitive impairment. In addition, we found that enzymatically inactivated ENO1 failed to inhibit Aβ fibril formation and fibril disruption. The proteolytic activity of ENO1 may thus underlie the enzyme's cytoprotective effect and clearance of Aβ from the brain, and ENO1 may be a therapeutic target in CAA.

A Cationic Gallium Phthalocyanine Inhibits Amyloid β Peptide Fibril Formation.

Amyloid β (Aβ) peptide deposition is considered as the main cause of Alzheimer's disease (AD). Previously, we have shown that a Zn containing neutral phthalocyanine (Zn-Pc) inhibits Aβ fibril formation. The objective of this study is to investigate the effects of a cationic gallium containing Pc (GaCl-Pc) on Aβ fibril formation process. Aβ fibril formation was induced by incubating synthetic Aβ peptides in a fibril forming buffer, and the amount of fibril was evaluated by ThT fluorescence assay. GaCl-Pc dosedependently inhibited both Aβ1-40 and Aβ1-42 fibril formation. It mainly inhibited the elongation phase of Aβ1-42 fibril formation kinetics, but not the lag phase. Western blotting results showed that it did not inhibit its oligomerization process, rather increased it. Additionally, GaCl-Pc destabilized preformed Aβ1- 42 fibrils dose-dependently in vitro condition, and decreased Aβ levels in the brain slice culture of APP transgenic AD model mice (J20 strain). Near-infrared scanning results showed that GaCl-Pc had the ability to bind to Aβ1-42. MTT assay demonstrated that GaCl-Pc did not have toxicity towards a neuronal cell line (A1) in culture rather, showed protective effects on Aβ-induced toxicity. Moreover, it dosedependently decreased Aβ-induced reactive oxygen species levels in A1 culture. Thus, our result demonstrated that GaCl-Pc decreased Aβ aggregation and destabilized the preformed fibrils. Since cationic molecules show a better ability to cross the blood-brain barrier, cationic GaCl-Pc could be important for the therapy of AD.

Quercetin-loaded selenium nanoparticles inhibit amyloid-β aggregation and exhibit antioxidant activity

Abnormal aggregation of amyloid-β (Aβ) particles and oxidative stress are pathological hallmarks of Alzheimer’s disease (AD). Quercetin (Que), a flavonoid antioxidant, has been widely used to inhibit Aβ fibril formation and can potentially attenuate oxidative stress. However, poor aqueous solubility, extensive first-pass metabolism, and low permeability of the blood-brain barrier (BBB) limit its clinical application. Herein, we introduce a simple strategy to develop nanocomposites (NC) for drug delivery. This method involves combining Que and Na2SeO3 to obtain selenium nanoparticles. These nanoparticles are then modified using a combination of acacia and polysorbate 80 (P80-Que@Se NC). This recently developed nanocomposite has the potential to enhance drug delivery across the blood-brain barrier (BBB) and polysorbate 80 act as a pharmaceutical excipient that increases the aqueous solubility of Que. In vitro results demonstrated that P80-Que@Se had high aqueous solubility compared to individual Que, and could effectively inhibit Aβ fibrillation. In vitro Cell Counting Kit (CCK)-8 analysis indicated that P80-Que@Se nanocomposites could protect PC12 cells from H2O2-induced cell death. Additionally, P80-Que@Se had high 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and exhibited excellent antioxidant activity. This study suggested that P80-Que@Se NCs may aid in the treatment of AD and have broad implications for future NC-based studies and applications. In brief, we believe that this reliable drug delivery method will serve as a useful template for researchers designing therapies to be delivered across the BBB.

The fruit of Crataegus pinnatifida ameliorates memory deficits in β-amyloid protein-induced Alzheimer's disease mouse model

Ethnopharmacological relevanceThe fruit of Crataegus pinnatifida is a traditional medicine widely used as digestive drug in East Asia. Although Chinese herbal medicine used it for mental health, scientific evidence does not exist, yet. Aims of studyThe aim of this study is to show that the ethanol extract of the fruit of Crataegus pinnatifida (CPE) has neuroprotective effect on Alzheimer’ disease model mice. Materials and methodsIntracerebroventricular injection of Aβ was used to induce Alzheimer's disease-like pathology. Passive avoidance and Y-maze tasks were used to examine the effect of CPE on memory impairments by Aβ. Immunohistochemistry was used to examine the effect of CPE on glial activation. ThT assay was used to observe the effect of CPE on Aβ aggregation. MTT and LDH release assays were utilized to examine effects of CPE on Aβ-induced cytotoxicity. ResultsCPE prevented memory deficit in Aβ-induced memory impairment model. Moreover, CPE prevented glial activation in the hippocampus of Aβ-injected model. In in vitro test, CPE inhibited Aβ fibril formation in a concentration-dependent manner. CPE also caused disaggregation of Aβ fibrils. Along with this, CPE blocked neuronal cell death induced by Aβ. ConclusionsCollectively, these experimental findings demonstrated that CPE could be a candidate for development of AD therapy.

Novel Therapeutic Potentials of Taxifolin for Amyloid-β-associated Neurodegenerative Diseases and Other Diseases: Recent Advances and Future Perspectives.

Amyloid-β (Aβ) has been closely implicated in the pathogenesis of cerebral amyloid angiopathy (CAA) and Alzheimer’s disease (AD), the major causes of dementia. Thus, Aβ could be a target for the treatment of these diseases, for which, currently, there are no established effective treatments. Taxifolin is a bioactive catechol-type flavonoid present in various plants, such as herbs, and it exhibits pleiotropic effects including anti-oxidant and anti-glycation activities. Recently, we have demonstrated that taxifolin inhibits Aβ fibril formation in vitro and have further shown that it improves cerebral blood flow, facilitating Aβ clearance in the brain and suppressing cognitive decline in a mouse model of CAA. These findings suggest the novel therapeutic potentials of taxifolin for CAA. Furthermore, recent extensive studies have reported several novel aspects of taxifolin supporting its potential as a therapeutic drug for AD and metabolic diseases with a high risk for dementia as well as for CAA. In this review, we have summarized the recent advances in taxifolin research based on in vitro, in vivo, and in silico approaches. Furthermore, we have discussed future research directions on the potential of taxifolin for use in novel therapeutic strategies for CAA, AD, and metabolic diseases with an increased risk for dementia.

Remarkable Brain Penetration of Cyclopentadienyl M(CO)3+ (M = 99mTc, Re) Derivatives of Benzothiazole and Benzimidazole Paves the Way for Their Application as Diagnostic, with Single-Photon-Emission Computed Tomography (SPECT), and Therapeutic Agents for Alzheimer's Disease.

The synthesis and evaluation of three novel 99mTc complexes (99mTc-1-3) and their corresponding Re complexes (Re-1-3), in which the phenyl ring of 2-phenylbenzothiazole or 2-phenylbenzimidazole is replaced by the cyclopentadienyl tricarbonyl [Cp99mTc(CO)3] core, are reported. Both 99mTc and Re complexes were prepared from the corresponding ferrocenyl derivatives, and the Re complexes were fully characterized by elemental analysis, spectroscopic methods, and X-ray crystallography. The complexes exhibit effective in vitro binding to β-amyloid (Aβ) plaques and fibrils, inhibit Aβ fibril formation, and significantly reduce Aβ-induced cytotoxicity and reactive oxygen species production in neuronal cell cultures. The brain uptake of the 99mTc complexes ranges between 7.94 and 3.99% ID/g at 2 min p.i., being the highest recorded for potential 99mTc Aβ plaque imaging probes in mice. Powered by their high brain uptake, the complexes represent strong theranostic candidates against Alzheimer's disease combining single-photon-emission computed tomography diagnostic (99mTc complexes) and antiamyloid therapeutic (Re complexes) potential.

Genesis of Neuroprotective Peptoid from Aβ30–34 Inhibits Aβ Aggregation and AChE Activity

Aβ peptide and hyper-phosphorylated microtubule associated protein (Tau) aggregation causes severe damage to both the neuron membrane and key signal processing microfilament (microtubule) in Alzheimer's disease (AD) brains. To date, the key challenge is to develop nontoxic, proteolytically stable amyloid inhibitors, which can simultaneously target multiple pathways involved in AD. Various attempts have been made in this direction; however, clinical outcomes of those attempts have been reported to be poor. Thus, we choose development of peptoid (N-substituted glycine oligomers)-based leads as potential AD therapeutics, which are easy to synthesize, found to be proteolytically stable, and exhibit excellent bioavailability. In this paper, we have designed and synthesized a new short peptoid for amyloid inhibition from 30-34 hydrophobic pocket of amyloid beta (Aβ) peptide. The peptoid selectively binds with 17-21 hydrophobic region of Aβ and inhibits Aβ fibril formation. Various in vitro assays suggested that our AI peptoid binds with tubulin/microtubule and promotes its polymerization and stability. This peptoid also inhibits AChE-induced Aβ fibril formation and provides significant neuroprotection against toxicity generated by nerve growth factor (NGF) deprived neurons derived from rat adrenal pheochromocytoma (PC12) cell line. Moreover, this peptoid shows serum stability and is noncytotoxic to primary rat cortical neurons.

Aggregation of Aβ(17–36) in the Presence of Naturally Occurring Phenolic Inhibitors Using Coarse-Grained Simulations

Although some naturally occurring polyphenols have been found to inhibit amyloid β (Aβ) fibril formation and reduce neuron cell toxicity in vitro, their exact inhibitory mechanism is unknown. In this work, discontinuous molecular dynamics combined with the PRIME20 force field and a newly built inhibitor model are performed to examine the effect of vanillin, resveratrol, curcumin, and epigallocatechin-3-gallate (EGCG) on the aggregation of Aβ(17–36) peptides. Four sets of peptide/inhibitor simulations are performed in which inhibitors (1) bind to Aβ(17–36) monomer (2) interfere with Aβ(17–36) oligomerization (3) disrupt a pre-formed Aβ(17–36) protofilament, and (4) prevent the growth of Aβ(17–36) protofilament. The single-ring compound, vanillin, slightly slows down but cannot inhibit the formation of a U-shaped Aβ(17–36) protofilament. The multiple-ring compounds, EGCG, resveratrol, and curcumin, redirect Aβ(17–36) from a fibrillar aggregate to an unstructured oligomer. The three aromatic groups of the EGCG molecule are in a stereo (nonplanar) configuration, helping it contact the N-terminal, middle, and C-terminal regions of the peptide. Resveratrol and curcumin bind only to the hydrophobic residues near peptide termini. The rank order of inhibitory effectiveness of Aβ(17–36) aggregation is as follows: EGCG > resveratrol > curcumin > vanillin, consistent with experimental findings on inhibiting full-length Aβ fibrillation. Furthermore, we learn that the inhibition effect of EGCG is specific to the peptide sequence, while those of resveratrol and curcumin are non-specific in that they stem from strong interference with hydrophobic side-chain association, regardless of the residues' location and peptide sequence. Our studies provide molecular-level insights into how polyphenols inhibit Aβ fibril formation, knowledge that could be useful for designing amyloid inhibitors.

Dual inhibition of BACE1 and Aβ aggregation by β-ecdysone: Application of a phytoecdysteroid scaffold in Alzheimer’s disease therapeutics

Current medications for the complex neurological disorder, Alzheimer’s disease (AD), can neither stop disease progression nor revert back disease pathogenesis. The present study demonstrates the applicability of a phytoecdysteroid, β-ecdysone, as a multi-potent agent in AD therapeutics. β-ecdysone strongly binds to the active site cavity of BACE1 with calculated dissociation constant of 1.75±0.1μM. Steady-state and time-resolved fluorescence spectroscopy reveal that binding of β-ecdysone induces conformational transition of the protein from open to closed form thereby blocking substrate binding. Even 500nM of the compound completely blocks the enzyme activity. Furthermore, β-ecdysone strongly inhibits Aβ aggregation, evident from ANS and ThT binding assay. Co-incubation of equimolar peptide and β-ecdysone completely inhibits Aβ fibril formation which is further complemented by the AFM study. Low systemic toxicity of β-ecdysone further extends the applicability of the compound as functional food and dietary supplement for disease management.

Curcumin Attenuates Amyloid-β Aggregate Toxicity and Modulates Amyloid-β Aggregation Pathway.

The abnormal misfolding and aggregation of amyloid-β (Aβ) peptides into β-sheet enriched insoluble deposits initiates a cascade of events leading to pathological processes and culminating in cognitive decline in Alzheimer's disease (AD). In particular, soluble oligomeric/prefibrillar Aβ have been shown to be potent neurotoxins. The naturally occurring polyphenol curcumin has been shown to exert a neuroprotective effect against age-related neurodegenerative diseases such as AD. However, its protective mechanism remains unclear. In this study, we investigated the effects of curcumin on the aggregation of Aβ40 as well as Aβ40 aggregate induced neurotoxicity. Our results show that the curcumin does not inhibit Aβ fibril formation, but rather enriches the population of "off-pathway" soluble oligomers and prefibrillar aggregates that were nontoxic. Curcumin also exerted a nonspecific neuroprotective effect, reducing toxicities induced by a range of Aβ conformers, including monomeric, oligomeric, prefibrillar, and fibrillar Aβ. The neuroprotective effect is possibly membrane-mediated, as curcumin reduced the extent of cell membrane permeabilization induced by Aβ aggregates. Taken together, our study shows that curcumin exerts its neuroprotective effect against Aβ induced toxicity through at least two concerted pathways, modifying the Aβ aggregation pathway toward the formation of nontoxic aggregates and ameliorating Aβ-induced toxicity possibly through a nonspecific pathway.

(R)‐α‐trifluoromethylalanine containing short peptide in the inhibition of amyloid peptide fibrillation

The extracellular deposition of insoluble amyloid fibrils resulting from the aggregation of the amyloid-β (Aβ) is a pathological feature of neuronal loss in Alzheimer's disease (AD). Numerous small molecules have been reported to interfere with the process of Aβ aggregation. Compounds containing aromatic structures, hydrophobic amino acids and/or the α-aminoisobutyric acid (Aib) as β-sheet breaker elements have been reported to be effective inhibitors of Aβ aggregation. We synthesized two peptides, one containing the Aib amino acid and the other including its trifluoromethylated analog (R)-α-Trifluoromethylalanine ((R)-Tfm-Alanine) and we evaluated the impact of these peptides on Aβ amyloid formation. The compounds were tested by standard methods such as thioflavin-T fluorescence spectroscopy and transmission electron microscopy but also by circular dichroism, liquid state nuclear magnetic resonance (NMR) and NMR saturation transfer difference (STD) experiments to further characterize the effect of the two molecules on Aβ structure and on the kinetics of depletion of monomeric, soluble Aβ. Our results demonstrate that the peptide containing Aib reduces the quantity of aggregates containing β-sheet structure but slightly inhibits Aβ fibril formation, while the molecule including the trifluoromethyl (Tfm) group slows down the kinetics of Aβ fibril formation, delays the random coil to β-sheet structure transition and induces a change in the oligomerization pathway. These results suggest that the hydrophobic Tfm group has a better affinity with Aβ than the methyl groups of the Aib and that this Tfm group is effective and important in preventing the Aβ aggregation.

Few-layer bismuth selenides exfoliated by hemin inhibit amyloid-β1–42 fibril formation

Inhibiting amyloid-β (Aβ) fibril formation is the primary therapeutic strategy for Alzheimer’s disease. Several small molecules and nanomaterials have been used to inhibit Aβ fibril formation. However, insufficient inhibition efficiency or poor metabolization limits their further applications. Here, we used hemin to exfoliate few-layer Bi2Se3 in aqueous solution. Then we separated few-layer Bi2Se3 with different sizes and thicknesses by fractional centrifugation, and used them to attempt to inhibit Aβ1-42 aggregation. The results show that smaller and thinner few-layer Bi2Se3 had the highest inhibition efficiency. We further investigated the interaction between few-layer Bi2Se3 and Aβ1-42 monomers. The results indicate that the inhibition effect may be due to the high adsorption capacity of few-layer Bi2Se3 for Aβ1−42 monomers. Few-layer Bi2Se3 also decreased Aβ-mediated peroxidase-like activity and cytotoxicity according to in vitro neurotoxicity studies under physiological conditions. Therefore, our work shows the potential for applications of few-layer Bi2Se3 in the biomedical field.

Single chain variable fragment against aβ expressed in baculovirus inhibits abeta fibril elongation and promotes its disaggregation.

Alzheimer’s disease (AD) is the most common form of age-related dementia, and the most urgent problem is that it is currently incurable. Amyloid-β (Aβ) peptide is believed to play a major role in the pathogenesis of AD. We previously reported that an Aβ N-terminal amino acid targeting monoclonal antibody (MAb), A8, inhibits Aβ fibril formation and has potential as an immunotherapy for AD based on a mouse model. To further study the underlying mechanisms, we tested our hypothesis that the single chain fragment variable (scFv) without the Fc fragment is capable of regulating either Aβ aggregation or disaggregation in vitro. Here, a model of cell-free Aβ “on-pathway” aggregation was established and identified using PCR, Western blot, ELISA, transmission electron microscopy (TEM) and thioflavin T (ThT) binding analyses. His-tagged A8 scFvs was cloned and solubly expressed in baculovirus. Our data demonstrated that the Ni-NTA agarose affinity-purified A8 scFv inhibited the forward reaction of “on-pathway” aggregation and Aβ fibril maturation. The effect of A8 scFv on Aβ fibrillogenesis was markedly more significant when administered at the start of the Aβ folding reaction. Furthermore, the results also showed that pre-formed Aβ fibrils could be disaggregated via incubation with purified A8 scFv, which suggested that A8 scFv is involved in the reverse reaction of Aβ aggregation. Therefore, A8 scFv was capable of both inhibiting fibrillogenesis and disaggregating matured fibrils. Our present study provides valuable insight into the regulators of ultrastructural dynamics of cell-free “on-pathway” Aβ aggregation and will assist in the development of therapeutic strategies for AD.

A carboxylated Zn-phthalocyanine inhibits fibril formation of Alzheimer's amyloid β peptide.

Amyloid β (Aβ), a 39-42 amino acid peptide derived from amyloid precursor protein, is deposited as fibrils in Alzheimer's disease brains, and is considered to play a major role in the pathogenesis of the disease. We have investigated the effects of a water-soluble Zn-phthalocyanine, ZnPc(COONa)₈, a macrocyclic compound with near-infrared optical properties, on Aβ fibril formation in vitro. A thioflavin T fluorescence assay showed that ZnPc(COONa)₈ significantly inhibited Aβ fibril formation, increasing the lag time and dose-dependently decreasing the plateau level of fibril formation. Moreover, it destabilized pre-formed Aβ fibrils, resulting in an increase in low-molecular-weight species. After fibril formation in the presence of ZnPc(COONa)₈, immunoprecipitation of Aβ₁₋₄₂ using Aβ-specific antibody followed by near-infrared scanning demonstrated binding of ZnPc(COONa)₈ to Aβ₁₋₄₂. A study using the hydrophobic fluorescent probe 8-anilino-1-naphthalenesulfonic acid showed that ZnPc(COONa)8 decreased the hydrophobicity during Aβ₁₋₄₂ fibril formation. CD spectroscopy showed an increase in the α helix structure and a decrease in the β sheet structure of Aβ₁₋₄₀ in fibril-forming buffer containing ZnPc(COONa)₈. SDS/PAGE and a dot-blot immunoassay showed that ZnPc(COONa)₈ delayed the disappearance of low-molecular-weight species and the appearance of higher-molecular-weight oligomeric species of Aβ₁₋₄₂. A cell viability assay showed that ZnPc(COONa)₈ was not toxic to a neuronal cell line (A1), but instead protected A1 cells against Aβ₁₋₄₂-induced toxicity. Overall, our results indicate that ZnPc(COONa)₈ binds to Aβ and decreases the hydrophobicity, and this change is unfavorable for Aβ oligomerization and fibril formation.

The N-terminal region of myelin basic protein reduces fibrillar amyloid-β deposition in Tg-5xFAD mice

Alzheimer's disease is a progressive neurodegenerative disorder that is characterized by extensive deposition of fibrillar amyloid-β (Aβ) in the brain. Previously, myelin basic protein (MBP) was identified to be a potent inhibitor to Aβ fibril formation, and this inhibitory activity was localized to the N-terminal residues 1–64, a fragment designated MBP1. Here, we show that the modest neuronal expression of a fusion protein of the biologically active MBP1 fragment and the enhanced green fluorescent protein (MBP1-EGFP) significantly improved the performance of spatial learning memory in Tg-5xFAD mice, a model of pathologic Aβ accumulation in brain. The levels of insoluble Aβ and fibrillar amyloid were significantly reduced in bigenic Tg-5xFAD/Tg-MBP1-EGFP mice. Quantitative stereological analysis revealed that the reduction in amyloid was because of a reduction in the size of fibrillar plaques rather than a decrease in plaque numbers. The current findings support previous studies showing that MBP1 inhibits Aβ fibril formation in vitro and demonstrate the ability of MBP1 to reduce Aβ pathology and improve behavioral performance.