Thioflavin-T Assay Research Articles

Canine SOD1 harboring E40K or T18S mutations promotes protein aggregation without reducing the global structural stability.

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease associated with aggregation of superoxide dismutase 1 (SOD1) protein. More than 160 mutations in human SOD1 have been identified in familial ALS and extensively characterized in previous studies. Here, we investigated the effects of T18S and E40K mutations on protein aggregation of canine SOD1. These two mutations are exclusively found in canine degenerative myelopathy (an ALS-like neurodegenerative disease in dogs), whose phenotype is unknown at the level of protein folding. Interestingly, the T18S and E40K mutations did not alter far-UV CD spectrum, enzymatic activity, or global structural stability of canine SOD1. However, thioflavin-T assay and transmission electron microscopy analysis revealed that these mutations promote formation of fibrous aggregates, in particular in the Cu2+/Zn2+-unbound state. These evidence suggested that the T18S and E40K mutations promote protein aggregation through a unique mechanism, possibly involving destabilization of the local structure, reduction of net negative charge, or production of disulfide-linked oligomers.

Secondary Metabolites from Plants Possessing Inhibitory Properties against Beta-Amyloid Aggregation as Revealed by Thioflavin-T Assay and Correlations with Investigations on Transgenic Mouse Models of Alzheimer's Disease.

Alzheimer’s disease is a neurodegenerative disorder for which there is a continuous search of drugs able to reduce or stop the cognitive decline. Beta-amyloid peptides are composed of 40 and 42 amino acids and are considered a major cause of neuronal toxicity. They are prone to aggregation, yielding oligomers and fibrils through the inter-molecular binding between the amino acid sequences (17–42) of multiple amyloid-beta molecules. Additionally, amyloid deposition causes cerebral amyloid angiopathy. The present study aims to identify, in the existing literature, natural plant derived products possessing inhibitory properties against aggregation. The studies searched proved the anti-aggregating effects by the thioflavin T assay and through behavioral, biochemical, and histological analysis carried out upon administration of natural chemical compounds to transgenic mouse models of Alzheimer’s disease. According to our present study results, fifteen secondary metabolites from plants were identified which presented both evidence coming from the thioflavin T assay and transgenic mouse models developing Alzheimer’s disease and six additional metabolites were mentioned due to their inhibitory effects against fibrillogenesis. Among them, epigallocatechin-3-gallate, luteolin, myricetin, and silibinin were proven to lower the aggregation to less than 40%.

Computational exploration and experimental validation to identify a dual inhibitor of cholinesterase and amyloid-beta for the treatment of Alzheimer's disease.

The cholinesterases are essential targets implicated in the pathogenesis of Alzheimer's disease (AD). In the present study, virtual screening and molecular docking are performed to identify the potential hits. Docking-post processing (DPP) and pose filtration protocols against AChE and BChE resulted in three hits (AW00308, HTS04089, and JFD03947). Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) and molecular dynamics simulation analysis affirmed the stability and binding pattern of the docked complex JFD03947, which was further synthesized and evaluated for in vitro cholinesterase inhibition (AChE, IC50 = 0.062 µM; BChE, IC50 = 1.482 µM) activity. The enzyme kinetics study of the JFD03947 against hAChE and hBChE suggested a mixed type of inhibition. The results of thioflavin T-assay also elicited anti-Aβ aggregation activity by JFD03947. Further, biological evaluation of identified compound JFD03947 also showed neuroprotective ability against the SH-SY5Y neuroblastoma cell lines.

Resveratrol-mediated cleavage of amyloid β1–42 peptide: potential relevance to Alzheimer's disease

Aggregation of amyloid β1–42 (Aβ1–42) peptide within the brain is considered one of the main causes of the neuropathological changes associated with Alzheimer's disease. Resveratrol is a well-known antioxidant but has also been reported to bind to Aβ1–42 peptide, thereby reducing aggregation. However, little is known of the precise mechanism by which resveratrol reduces Aβ1–42 peptide aggregation. Using the thioflavin-T assay, the ability of resveratrol to reduce the extent of Aβ1–42 peptide aggregation was investigated. The findings of the present study demonstrate that interaction of resveratrol with Aβ1–42 peptide resulted in the cleavage of Aβ1–42 peptide into smaller fragments, as detected by matrix assisted laser desorption ionization-time of flight mass spectrometry. Atomic force microscopy analyses revealed Aβ1–42 peptide, under control conditions, aggregated into oligomers, protofibrils, and fibrils, whereas there was a distinct lack of these structures when Aβ1–42 peptide was incubated with resveratrol. Following 10 days incubation of Aβ1–42 peptide with resveratrol, particles with a mean z-height of 1.940 nm (range 0.675–3.275 nm) were observed, which are characteristic of shorter peptide species. In cell-based studies, resveratrol significantly reduced the cytotoxicity of Aβ1–42 peptide toward SH-SY5Y human neuroblastoma cells, suggesting a protective effect of the polyphenol. We therefore propose a novel mechanism by which resveratrol disrupts Aβ1–42 aggregation by mediating fragmentation of Aβ1–42 into smaller peptides, which have no propensity to aggregate further.

Purification of Amyloid Fibers Formed by the Tetracycline Resistant Soil Bacterium, Microbacterium oryzae

Amyloid fibers are proteins formed by repeating protein monomers which stack to create a cross‐beta sheet quaternary structure. Though the unregulated production of amyloid fibers is often associated with diseases such as Alzheimer’s and Parkinson’s, amyloid fibers are also produced in a regulated manner by bacteria in biofilms. We isolated a number of tetracycline resistant soil bacteria from a pig farm in upstate New York and screened them for biofilm production with the crystal violet assay and amyloid formation using a Thioflavin T (ThT) assay. We focused on one species, Microbacterium oryzae isolate #29, which had the highest amyloid production. Five days of growth at ambient temperature in liquid R2A medium maximizes amyloid fiber recovery. An isolation procedure utilizing sonication and differential centrifugation to separate fibers from the cell (Jordal et al. 2009) was adapted for use with M. oryzae isolate #29. This procedure is being modified further to increase the percent recovery and improve purity. Two detergents, Triton X‐100 (TX‐100) and hexadecyltrimethylammonium bromide (CTAB), were assessed as a means of disrupting the cell membranes to separate fibers from soluble proteins during the procedure. The fluorescence of the detergents below and above their critical micelle concentrations in the presence of ThT was investigated to determine whether adding a detergent treatment step would interfere with the ThT assay, as is the case for the commonly used detergent SDS. Additionally, sonication time was assessed to see if more fibers could be released from the cell. Preliminary results suggest that while neither detergent interferes with the ThT assay only the TX‐100 appears to improve yield, and that a longer sonication treatment results in a greater percent recovery in the ultracentrifuge pellet. Fibers will be purified further using preparatory SDS‐PAGE and depolymerized using formic acid. The purified fibers can be imaged by atomic force microscopy and the amyloid protein monomers will be sequenced.Support or Funding InformationSt. Lawrence University Fellowship Program, Departments of Chemistry and Biology, St. Lawrence University, Canton, NY 13617

Small molecules breaking down islet amyloid polypeptide self‐assembly

Amyloid deposition is a recognised feature of the islets in the majority of type 2 diabetic patients. These deposits originate from islet amyloid polypeptide (IAPP). Normally, IAPP is secreted along with insulin from pancreatic β‐cells and has hormonal functions related to satiety. However, IAPP is one of the most potent amyloidogenic proteins. Deposition of IAPP is associated with β‐cell death and diabetes progression. Drugs to inhibit pancreatic amyloidosis are not available. We have prepared a small molecule screening library to inhibit pancreatic amyloidosis. Our central hypothesis is that IAPP fibril formation (fibrillization) and deposition can be modulated by IAPP‐interactive compounds. We aim to establish structure‐activity relationships by which our small molecules impede IAPP fibrillization. Our primary approach, thioflavin T (ThT) assay, represented a major tool for the determination of IAPP fibrillization and inhibition by small molecules. For the most potent molecules, the abrogation of cytotoxic intermediate species (i.e. oligomerization leading to fibril formation) was determined with photo‐induced cross‐linking of unmodified protein. To confirm the inhibitory effect morphologically, electron microscopy analysis was performed to detect IAPP fibrils directly. Disaggregation of preformed fibrils by different compounds was studied with ThT assays and electron microscopy. The 1st‐tier screening method (ThT assay) identified several lead compounds to target IAPP. The oligomerization of IAPP was abrogated by JF‐19‐029. JF‐19‐029 and NBMI‐19‐013 deteriorated preformed fibrils. Few molecules have been reported to break down mature fibrils. We hope to open a new therapeutic approach to reduce type 2 diabetes progression.

Inhibitory activities of phenylpropanoids from Lycopus lucidus on amyloid aggregation related to Alzheimer's disease and type 2 diabetes.

The number of patients with Alzheimer's disease (AD) and type 2 diabetes (T2D) is increasing rapidly, and thus more research has been focused on the relationship between these two age-related chronic diseases. According to the amyloid hypothesis, prevention of the aggregation of amyloid β (Aβ) and human islet amyloid polypeptide (hIAPP) is a promising strategy for AD and T2D. In this study, thioflavin-T assay and transmission electron microscopy were performed to evaluate the inhibitory effect of three phenylpropanoids isolated from Lycopus lucidus-schizotenuin A and lycopic acids A and B-on both Aβ and hIAPP fibrillization. All tested compounds exhibited similarly strong inhibitory activity toward amyloid aggregation. These results suggested that catechol moieties play important roles in the inhibition of amyloid plaque formation.

Surface Functionalization of Gold Nanorods Improves Nanostructure Assemblies on Amyloid Fibril Scaffolds

A hybrid scaffold containing gold nanorods and lysozyme amyloid fibrils has been fabricated, and the effect of surface modification on improving nanostructure assembly on the biological template has been investigated. The nanohybrid system was characterized by monitoring surface plasmon resonance bands, dynamic light scattering spectroscopy, Thioflavin-T assay, and transmission electron microscopy. Surface of gold nanorods (GNRs) was modified with polystyrene sulfonate (PSS), and possible difference in assembly of the pristine and modified nanostructures was compared upon interaction with amyloid fibrils. Analysis of transmission electron microscopy showed that changing the surface charge of GNRs with biocompatible polymer improved electrostatic interactions between the nanostructures and amyloid fibril templates. Analysis of cell viability assays also showed that surface functionalization of GNRs remarkably improved biocompatibility of the nanoscaffold. Results of this study encourage utilization of modification strategies to fabricate a new generation of nanoscaffolds with fruitful applications in regenerative medicine.

Formation and physicochemical properties of amyloid fibrils from soy protein

Amyloid-like fibrils from food proteins possess unique functional properties for food and many other uses. This study reports the effect of hydrolytic heating (pH 2.0, 85 °C, 0–24 h) and incubation times (0–7 days) on the formation and physicochemical properties of amyloid fibrils based on soy protein isolates (SPI). The SPI hydrolysates and fibrils were characterized through AFM, Thioflavin T (ThT) fluorescence, SDS-PAGE, FTIR, solubility, particle size, and DSC. Stable amyloid-like protein fibrils were formed with 8–10 h of hydrolytic heating at 85 °C followed by 3 days of incubation at room temperature, as observed under AFM and confirmed with ThT assay. The fibrils contained significantly higher amounts of regular secondary structures than SPI. Incubation of the hydrolysates led to a slight increase of average particle sizes. Protein solubility near the isoelectric point (approximately pH 4.8) increased with longer hydrolytic heating (0–24 h). The hydrolysates and fibrils exhibited better gelling properties than the SPI. The DSC results revealed that hydrolysates from longer hydrolytic heating times (12 and 24 h) possessed stronger aggregation potential during heat treatment. This study provides useful information to manipulate the formation of protein fibrils and will benefit future research to explore their potential applications.

Real-Time BODIPY-Binding Assay To Screen Inhibitors of the Early Oligomerization Process of Aβ1-42 Peptide.

Misfolding and aggregation of amyloid β1–42 peptide (Aβ1–42) play a central role in the pathogenesis of Alzheimer's disease (AD). Targeting the highly cytotoxic oligomeric species formed during the early stages of the aggregation process represents a promising therapeutic strategy to reduce the toxicity associated with Aβ1–42. Currently, the thioflavin T (ThT) assay is the only established spectrofluorometric method to screen aggregation inhibitors. The success of the ThT assay is that it can detect Aβ1–42 aggregates with high β‐sheet content, such as protofibrils or fibrils, which appear in the late aggregation steps. Unfortunately, by using the ThT assay, the detection of inhibitors of early soluble oligomers that present a low β‐sheet character is challenging. Herein, a new, facile, and robust boron‐dipyrromethene (BODIPY) real‐time assay suitable for 96‐well plate format, which allows screening of compounds as selective inhibitors of the formation of Aβ1–42 oligomers, is reported. These inhibitors decrease the cellular toxicity of Aβ1–42, although they fail in the ThT assay. The findings have been confirmed and validated by structural analysis and cell viability assays under comparable experimental conditions. It is demonstrated that the BODIPY assay is a convenient method to screen and discover new candidate compounds that slow down or stop the pathological early oligomerization process and are active in the cellular assay. Therefore, it is a suitable complementary screening method of the current ThT assay.

Identifying Phlorofucofuroeckol-A as a Dual Inhibitor of Amyloid-β25-35 Self-Aggregation and Insulin Glycation: Elucidation of the Molecular Mechanism of Action.

Both amyloid-β (Aβ) and insulin are amyloidogenic peptides, and they play a critical role in Alzheimer’s disease (AD) and type-2 diabetes (T2D). Misfolded or aggregated Aβ and glycated insulin are commonly found in AD and T2D patients, respectively, and exhibit neurotoxicity and oxidative stress. The present study examined the anti-Aβ25-35 aggregation and anti-insulin glycation activities of five phlorotannins isolated from Ecklonia stolonifera. Thioflavin-T assay results suggest that eckol, dioxinodehydroeckol, dieckol, and phlorofucofuroeckol-A (PFFA) significantly inhibit Aβ25-35 self-assembly. Molecular docking and dynamic simulation analyses confirmed that these phlorotannins have a strong potential to interact with Aβ25-35 peptides and interrupt their self-assembly and conformational transformation, thereby inhibiting Aβ25-35 aggregation. In addition, PFFA dose-dependently inhibited d-ribose and d-glucose induced non-enzymatic insulin glycation. To understand the molecular mechanism for insulin glycation and its inhibition, we predicted the binding site of PFFA in insulin via computational analysis. Interestingly, PFFA strongly interacted with the Phe1 in insulin chain-B, and this interaction could block d-glucose access to the glycation site of insulin. Taken together, our novel findings suggest that phlorofucofuroeckol-A could be a new scaffold for AD treatment by inhibiting the formation of β-sheet rich structures in Aβ25-35 and advanced glycation end-products (AGEs) in insulin.

α-Synuclein chaperone suppresses nucleation and amyloidogenesis of prion protein

Protein misfolding diseases are a group of devastating disorders characterized by structural conversion of a soluble protein into an amyloid-like aggregate. Typically, the structural conversion occurs by misfolding of a single disease-associated protein, such as α-synuclein (αS) in Parkinson’s disease, amyloid-β in Alzheimer’s disease, and prion protein (PrP) in transmissible spongiform encephalopathies (TSEs). However, accumulating evidence has implicated that cross-interactions between heterologous amyloidogenic proteins dramatically impact on amyloidogenesis and disease pathology. Here we show αS in a monomeric state can suppress amyloidogenesis of PrP in vitro. Thioflavin-T assays and transmission electron miscopy revealed that monomeric αS inhibits the nucleation step of amyloidogenesis without inhibiting the growing step. Surface plasmon resonance and co-sedimentation assays neither detected interaction between αS and monomeric PrP nor fibrillar PrP. These results suggested that αS suppress amyloidogenesis of PrP by binding to a transiently accumulated intermediate, such as a partially unfolded state. Moreover, we found that oligomeric αS, which was recently suggested to interact with PrP, also did not interact with PrP. Taken together, our study revealed a chaperon-like activity of αS against PrP amyloidogenesis, suggesting a possible involvement of αS in the pathology of TSEs.

The triphenylmethane dye brilliant blue G is only moderately effective at inhibiting amyloid formation by human amylin or at disaggregating amylin amyloid fibrils, but interferes with amyloid assays; Implications for inhibitor design.

The development of inhibitors of islet amyloid formation is important as pancreatic amyloid deposition contributes to type-2 diabetes and islet transplant failure. The Alzheimer’s Aβ peptide and human amylin (h-amylin), the polypeptide responsible for amyloid formation in type-2 diabetes, share common physio-chemical features and some inhibitors of Aβ also inhibit amyloid formation by h-amylin and vice versa. Thus, a popular and potentially useful strategy to find lead compounds for anti-amylin amyloid agents is to examine compounds that have effects on Aβ amyloid formation. The triphenylmethane dye, brilliant blue G (BBG, Sodium;3-[[4-[(E)-[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]-2-methylcyclohexa-2,5-dien-1-ylidene]methyl]-N-ethyl-3-methylanilino]methyl]benzenesulfonate) has been shown to modulate Aβ amyloid formation and inhibit Aβ induced toxicity. However, the effects of BBG on h-amylin have not been examined, although other triphenylmethane derivatives inhibit h-amylin amyloid formation. The compound has only a modest impact on h-amylin amyloid formation unless it is added in significant excess. BBG also remodels preformed h-amylin amyloid fibrils if added in excess, however BBG has no significant effect on h-amylin induced toxicity towards cultured β-cells or cultured CHO-T cells except at high concentrations. BBG is shown to interfere with standard thioflavin-T assays of h-amylin amyloid formation and disaggregation, highlighting the difficulty of interpreting such experiments in the absence of other measurements. BBG also interferes with ANS based assays of h-amylin amyloid formation. The work highlights the differences between inhibition of Aβ and h-amylin amyloid formation, illustrates the limitation of using Aβ inhibitors as leads for h-amylin amyloid inhibitors, and reinforces the difficulties in interpreting dye binding assays of amyloid formation.

Free and bound Thioflavin T molecules with ultrafast relaxation: implications for assessment of protein binding and aggregation

Fluorescent dye Thioflavin T (ThT) is a widely used probe for the detection of amyloid fibrils, which are protein aggregates involved in the pathogenesis of neurodegenerative disorders. Upon the formation of a complex with amyloids, the fluorescence quantum yield of ThT increases 1000-fold due to a dramatic reduction of the nonradiative decay rate. This is accompanied by a remarkable change of ThT fluorescence lifetime τ from ~1 to ~1000 ps, thus making it possible to assess ThT binding to different systems using τ as an indicator. However, when measuring ThT interaction with proteins, one can observe that the binding affinity determined from the ThT fluorescence intensity’s dependence on protein concentration may be orders of magnitude lower than that determined using τ. Here we show that this discrepancy at least partly originates from a limited temporal resolution when determining the fluorescence lifetime of ThT in the ThT-protein system using the time-correlated single photon counting technique (TCSPC), which is usually characterized by a ~100 ps instrument response function. This results in the situation when a small fraction (~1%) of ThT molecules with a relatively slow decay (τ ~ 1000 ps) completely disguises the impact of ThT molecules with an ultrafast decay (τ ~ 1 ps) to the overall measured fluorescence decay curve. Moreover, using the femtosecond-resolved fluorescence up-conversion technique, we demonstrate that not only free ThT molecules but also a subpopulation of protein-bound ThT molecules exhibits fluorescence decay on a 1 ps timescale. The obtained results are of critical importance for a reliable interpretation of protein binding and aggregation experiments when using a ThT assay with a fluorescence lifetime determined by the TCSPC as an indicator.

Chemical characterization, antioxidant properties, cholinesterase inhibitory and anti-amyloidogenic activities of sulfated polysaccharides from some seaweeds

Sulfated polysaccharides were extracted from Ecklonia maxima (PKPM), Gelidium pristoides (PMNP) and Ulva rigida (PURL) and characterized using scanning electron microscope with energy dispersing x-ray (SEM-EDX), Fourier transform infrared spectroscopy (FTIR) and Gas Chromatography-coupled with mass spectrometry (GC-MS). The radical scavenging activity, metal chelating effects, enzyme inhibitory potentials and anti-amyloidogenic activities of the sulfated polysaccharide samples were also evaluated. The elemental analysis and FTIR results confirmed the presence of sulfate groups in the polysaccharides. Furthermore, GC-MS analysis revealed the presence of glucose, galactose, fucose, arabinose and xylose as the main monosaccharides present in the samples. PKPM, PMNP and PURL exhibited radical scavenging activities against DPPH, ABTS and OH radicals and were able to chelate Fe2+. The sulfated polysaccharides also showed inhibitory effects on acetylcholinesterase, butyrylcholinesterase and β-secretase activities. PKPM showed higher cholinesterase and β-secretase inhibitory activities than PURL and PMNP. Thioflavin-T assay revealed that incubation of β-amyloid (Aβ1-42) peptide without the polysaccharides showed high fluorescence intensity compared to Aβ1-42 treated with PMNP, PKPM and PURL which showed drastic reduction in fluorescence intensity. Electron micrographs also revealed continuous aggregation of amyloid fibrils in the control (Aβ1-42 alone). However, Sulfated polysaccharides samples incubated with Aβ1-42 showed disappearance of Aβ1-42 fibrils which connotes disaggregation and inhibition of aggregation of the fibrils. These results suggest that the sulfated polysaccharide samples exhibit antioxidant and neuroprotective potentials which could be explored as nutraceuticals for the management of AD.

Vitamin D-binding protein-loaded PLGA nanoparticles suppress Alzheimer's disease-related pathology in 5XFAD mice

The aggregation and accumulation of amyloid beta (Aβ) peptide is believed to be the primary cause of Alzheimer's disease (AD) pathogenesis. Vitamin D-binding protein (DBP) can attenuate Aβ aggregation and accumulation. A biocompatible polymer poly (D,L-lactic acid-co-glycolic acid) (PLGA) can be loaded with therapeutic agents and control the rate of their release. In the present study, a PLGA-based drug delivery system was used to examine the therapeutic effects of DBP-PLGA nanoparticles in Aβ-overexpressing (5XFAD) mice. DBP was loaded into PLGA nanoparticles and the characteristics of the DBP-PLGA nanoparticles were analyzed. Using a thioflavin-T assay, we observed that DBP-PLGA nanoparticles significantly inhibited Aβ aggregation in vitro. In addition, we found that intravenous injection of DBP-PLGA nanoparticles significantly attenuated the Aβ accumulation, neuroinflammation, neuronal loss and cognitive dysfunction in the 5XFAD mice. Collectively, our results suggest that DBP-PLGA nanoparticles could be a promising therapeutic candidate for the treatment of AD.

Unravelling the Role of S100A9 in the Development of Neurodegenerative Disease

S100A9 is a pro-inflammatory calcium binding protein released by neutrophils in response to neuroinflammatory events. These events can be triggered by a range of conditions including traumatic brain injury and bacterial infection but are also observed in a range of neurodegenerative conditions including Alzheimer's Disease and Parkinson's Disease. S100A9 is intrinsically amyloidogenic self-aggregating to form organised fibrils both in vitro and in vivo. These S100A9 fibrils congregate with amyloid-β to seed and accelerate the growth of amyloid-β fibrils, as observed in in vitro studies. S100A9 possesses two Ca2+ binding sites which when vacant increase its propensity to aggregate into an ordered fibrillar state. Inversely, increasing calcium concentration inhibits S100A9 fibril formation as verified by Thioflavin-T assay. Our solution-state NMR studies indicate that in the absence of calcium the protein is largely unfolded adopts a number of interconverting states which become more structured upon calcium binding. Interestingly complementary synchrotron radiation circular dichroism measurements suggest a high alpha-helical content irrespective of calcium levels, suggesting that these interconverting species are rich in short-lived helical states. To determine the structural transition that occur upon fibril formation, solid-state NMR investigations have been utilised. These studies demonstrate that S100A9 fibrils formed in vitro have a single well-ordered structure with mobile regions limited to 15-25 residues in size. Analysis of the chemical shifts indicate the protein exists as a single polymorph with a rigid protein core that is predominantly β-strand in structure. Employing paramagnetic relaxation assisted data collection (PAC) progress is being made towards a complete assignment permitting a detailed analysis of the conformation of S100A9 within amyloid fibres.

Transthyretin Amyloid Fibril Disrupting Activities of Extracts and Fractions from Juglans mandshurica Maxim. var. cordiformis (Makino) Kitam.

Transthyretin-related amyloidosis is a slowly progressive disorder caused by deposition of insoluble amyloid plaques formed by fibrillization of mutant or defective transthyretin (TTR) monomers that leads to neurodegeneration and organ failure. Thus, any compound exhibiting TTR amyloid formation inhibitory activity or TTR amyloid fibril disrupting activity might be a potential candidate for the development of therapies for these disorders. Our aim in this study was the evaluation of the TTR amyloid fibril disrupting potential of extracts of leaves and immature fruits of two Juglans plants, i.e., Juglans mandshurica var. sachalinensis and Juglans mandshurica var. cordiformis. The TTR amyloid fibril disrupting activity was measured by Thioflavin-T (ThT) assay and PROTEOSTAT® Protein aggregation assay methods. A fifty percent acetone extract of the fruits of Juglans mandshurica var. cordiformis showed strong amyloid fibril disrupting activity, and was further fractionated using different solvents. Ethyl acetate and n-butanol fractions showed significant activity in both assays. Syringic acid was isolated and identified as main compound in both of these fractions; however, it did not show any activity. Furthermore, some of the previously reported compounds from Juglans plants including naphthoquinone derivatives and phenolic compounds were evaluated to identify the potential bioactive compounds. Among them, juglone, a naphthoquinone derivative showed promising activity. However, juglone also showed strong cytotoxicity in HEK293 cells. Thus, future studies should focus on the isolation and identification of naphthoquinone derivatives or other compounds from Juglans plan ts with potent bioactivity and low cytotoxicity.

On the role of synthesized hydroxylated chalcones as dual functional amyloid-β aggregation and ferroptosis inhibitors for potential treatment of Alzheimer's disease

In addition to amyloid cascade hypothesis, ferroptosis – a recently identified cell death pathway associated with the accumulation of lipid hydroperoxides – was hypothesized as one of the main forms of cell death in Alzheimer's disease. Herein, a series of hydroxylated chalcones were designed and synthesized as dual-functional inhibitors to inhibit amyloid-β peptide (Aβ) aggregation as well as ferroptosis simultaneously. Thioflavin-T assay indicated trihydroxy chalcones inhibited Aβ aggregation better. In human neuroblastoma SH-SY5Y cells, cytoprotective chalcones 14a-c with three hydroxyl substituents exhibited a significant neuroprotection against Aβ1-42 aggregation induced toxicity. In addition, chalcones 14a-c were found to be good inhibitors of ferroptosis induced by either pharmacological inhibition of the hydroperoxide-detoxifying enzyme Gpx4 using (1S, 3R)-RSL4 or cystine/glutamate antiporter system Xc− inhibition by erastin through lipid peroxidation inhibition mechanism. Trihydroxy chalcone 14a was also able to completely subvert lipid peroxidation induced by Aβ1-42 aggregation in SH-SY5Y cells indicating that they can reduce the neurotoxicity involved with oxidative stress. Compound 14a-c showed good ADMET properties and blood-brain barrier penetration in silico simulation software. From these data, a picture emerges wherein trihydroxy chalcones are potential candidates for the treatment of Alzheimer's disease by simultaneously inhibition of Aβ1-42 aggregation and ferroptosis.

Photoactive chlorin e6 is a multifunctional modulator of amyloid-β aggregation and toxicity via specific interactions with its histidine residues.

The self-assembly of Aβ to β-sheet-rich neurotoxic oligomers is a main pathological event leading to Alzheimer's disease (AD). Selective targeting of Aβ oligomers without affecting other functional proteins is therefore an attractive approach to prevent the disease and its progression. In this study, we report that photodynamic treatment of Aβ in the presence of catalytic amounts of chlorin e6 can selectively damage Aβ and inhibit its aggregation and toxicity. Chlorin e6 also reversed the amyloid aggregation process in the dark by binding its soluble and low molecular weight oligomers, as shown by thioflavin T (ThT) fluorescence and photoinduced cross-linking of unmodified protein (PICUP) methods. Using HSQC NMR spectroscopy, ThT assays, amino acid analysis, SDS/PAGE, and EPR spectroscopy, we show that catalytic amounts of photoexcited chlorin e6 selectively damage the Aβ histidine residues H6, H13, and H14, and induce Aβ cross-linking by generating singlet oxygen. In contrast, photoexcited chlorin e6 was unable to cross-link ubiquitin and α-synuclein, demonstrating its high selectivity for Aβ. By binding to the Aβ histidine residues, catalytic amounts of chlorin e6 can also inhibit the Cu2+-induced aggregation and toxicity in darkness, while at stoichiometric amounts it acts as a chelator to reduce the amount of free Cu2+. This study demonstrates the great potential of chlorin e6 as a multifunctional agent for treatment of AD, and shows that the three N-terminal Aβ histidine residues are a suitable target for Aβ-specific drugs.