Efficient inhibition of amyloid fibrillation and cytotoxicity of α-synuclein and human insulin using biosynthesized silver nanoparticles decorated by green tea polyphenols.

Green tea polyphenols (GTPs), particularly epigallocatechin-3-gallate, stand out among natural small molecules screened for their ability to target protein aggregates due to their potent anti-amyloidogenic and neuroprotective activities against various disease-related peptides and proteins. However, the clinical applications of GTPs in amyloid-related diseases have been greatly limited by drawbacks such as poor chemical stability and low bioavailability. To address these limitations, this study utilized an Iranian green tea polyphenolic extract as a reducing agent to neutralize silver ions and facilitate the formation of silver nanoparticle capped by GTPs (GTPs-capped AgNPs). The results obtained from this study demonstrate that GTPs-capped AgNPs are more effective than free GTPs at inhibiting amyloid fibrillation and reducing cytotoxicity induced by amyloid fibrils of human insulin and α-synuclein (α-syn). This improved efficacy is attributed to the increased surface/volume ratio of GTPs-capped AgNPs, which can enhance their binding affinity to amyloidogenic species and boosts their antioxidant activity. The mechanism by which GTPs-capped AgNPs inhibit amyloid fibrillation appears to vary depending on the target protein. For structured protein human insulin, GTPs-capped AgNPs hinder fibrillation by constraining the protein in its native-like state. In contrast, GTPs-capped AgNPs modulate fibrillation of intrinsically disordered proteins like α-syn by redirecting the aggregation pathway towards the formation of non-toxic off-pathway oligomers or amorphous aggregates. These findings highlight polyphenol-functionalized nanoparticles as a promising strategy for targeting protein aggregates associated with neurodegenerative diseases.

Cell-Free and In Vivo Characterization of the Inhibitory Activity of Lavado Cocoa Flavanols on the Amyloid Protein Ataxin-3: Toward New Approaches against Spinocerebellar Ataxia Type 3.

Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder characterized by ataxia and other neurological manifestations, with a poor prognosis and a lack of effective therapies. The amyloid aggregation of the ataxin-3 protein is a hallmark of SCA3 and one of the main biochemical events prompting its onset, making it a prominent target for the development of preventive and therapeutic interventions. Here, we tested the efficacy of an aqueous Lavado cocoa extract and its polyphenolic components against ataxin-3 aggregation and neurotoxicity. The combination of biochemical assays and atomic force microscopy morphological analysis provided clear evidence of cocoa flavanols' ability to hinder ATX3 amyloid aggregation through direct physical interaction, as assessed by NMR spectroscopy. The chemical identity of the flavanols was investigated by ultraperformance liquid chromatography-high-resolution mass spectrometry. The use of the preclinical model Caenorhabditis elegans allowed us to demonstrate cocoa flavanols' ability to ameliorate ataxic phenotypes in vivo. To the best of our knowledge, Lavado cocoa is the first natural source whose extract is able to directly interfere with ATX3 aggregation, leading to the formation of off-pathway species.

Branched-Chain Amino Acid Assembly into Amyloid-like Fibrils Provides a New Paradigm for Maple Syrup Urine Disease Pathology.

Inborn error of metabolism disorders (IEMs) are a family of diseases resulting from single-gene mutations that lead to the accumulation of metabolites that are usually toxic or interfere with normal cell function. The etiological link between metabolic alteration and the symptoms of IEMs is still elusive. Several metabolites, which accumulate in IEMs, were shown to self-assemble to form ordered structures. These structures display the same biophysical, biochemical, and biological characteristics as proteinaceous amyloid fibrils. Here, we have demonstrated, for the first time, the ability of each of the branched-chain amino acids (BCAAs) that accumulate in maple syrup urine disease (MSUD) to self-assemble into amyloid-like fibrils depicted by characteristic morphology, binding to indicative amyloid-specific dyes and dose-dependent cytotoxicity by a late apoptosis mechanism. We could also detect the presence of the assemblies in living cells. In addition, by employing several in vitro techniques, we demonstrated the ability of known polyphenols to inhibit the formation of the BCAA fibrils. Our study implies that BCAAs possess a pathological role in MSUD, extends the paradigm-shifting concept regarding the toxicity of metabolite amyloid-like structures, and suggests new pathological targets that may lead to highly needed novel therapeutic opportunities for this orphan disease.

The Green Tea Polyphenol Epigallocatechin-Gallate (EGCG) Interferes with Microcin E492 Amyloid Formation.

Microcin E492 (MccE492) is an antimicrobial peptide and proposed virulence factor produced by some Klebsiella pneumoniae strains, which, under certain conditions, form amyloid fibers, leading to the loss of its antibacterial activity. Although this protein has been characterized as a model functional amyloid, the secondary structure transitions behind its formation, and the possible effect of molecules that inhibit this process, have not been investigated. In this study, we examined the ability of the green tea flavonoid epigallocatechin gallate (EGCG) to interfere with MccE492 amyloid formation. Aggregation kinetics followed by thioflavin T binding were used to monitor amyloid formation in the presence or absence of EGCG. Additionally, synchrotron radiation circular dichroism (SRCD) and transmission electron microscopy (TEM) were used to study the secondary structure, thermal stability, and morphology of microcin E492 fibers. Our results showed that EGCG significantly inhibited the formation of the MccE492 amyloid, resulting in mainly amorphous aggregates and small oligomers. However, these aggregates retained part of the ß-sheet SRCD signal and a high resistance to heat denaturation, suggesting that the aggregation process is sequestered or deviated at some stage but not completely prevented. Thus, EGCG is an interesting inhibitor of the amyloid formation of MccE492 and other bacterial amyloids.

Unsaturated fatty acids uniquely alter aggregation rate of α-synuclein and insulin and change the secondary structure and toxicity of amyloid aggregates formed in their presence.

Docosahexaenoic (DHA) and arachidonic acids (ARA) are omega-3 and omega-6 long-chain polyunsaturated fatty acids (LCPUFAs). These molecules constitute a substantial portion of phospholipids in plasma membranes. Therefore, both DHA and ARA are essential diet components. Once consumed, DHA and ARA can interact with a large variety of biomolecules, including proteins such as insulin and α-synuclein (α-Syn). Under pathological conditions known as injection amyloidosis and Parkinson's disease, these proteins aggregate forming amyloid oligomers and fibrils, toxic species that exert high cell toxicity. In this study, we investigate the role of DHA and ARA in the aggregation properties of α-Syn and insulin. We found that the presence of both DHA and ARA at the equimolar concentrations strongly accelerated aggregation rates of α-Syn and insulin. Furthermore, LCPUFAs substantially altered the secondary structure of protein aggregates, whereas no noticeable changes in the fibril morphology were observed. Nanoscale Infrared analysis of α-Syn and insulin fibrils grown in the presence of both DHA and ARA revealed the presence of LCPUFAs in these aggregates. We also found that such LCPUFAs-rich α-Syn and insulin fibrils exerted significantly greater toxicities compared to the aggregates grown in the LCPUFAs-free environment. These findings show that interactions between amyloid-associated proteins and LCPUFAs can be the underlying molecular cause of neurodegenerative diseases.

Green tea leaf constituents inhibit the formation of lysozyme amyloid aggregates: An effect of mutual interactions.

Amyloidoses represent a group of pathological conditions characterized by amyloid fibrils accumulating in the form of deposits in intra- or extracellular space, leading to tissue damage. The lysozyme from hen egg-white (HEWL) is often used as a universal model protein to study the anti-amyloid effects of small molecules. The in vitro anti-amyloid activity and mutual interactions of green tea leaf constituents: (-)-epigallocatechin gallate (EGCG), (-)-epicatechin (EC), gallic acid (GA), caffeine (CF) and their equimolar mixtures were studied. The inhibition of HEWL amyloid aggregation was monitored by a Thioflavin T fluorescence assay and atomic force microscopy (AFM). The interactions of the analyzed molecules with HEWL were interpreted by ATR-FTIR and protein-small ligand docking studies. EGCG was the only substance efficiently inhibiting amyloid formation (IC50 âˆ¼193 µM), slowing the aggregation process, reducing the number of fibrils and partially stabilizing the secondary structure of HEWL. Compared to EGCG alone, EGCG-containing mixtures displayed lower overall anti-amyloid efficacy. The decrease in efficiency results from (a) the spatial interference of GA, CF and EC with EGCG while binding to HEWL, (b) the propensity of CF to form a less active adduct with EGCG, which participates in interactions with HEWL in parallel with pure EGCG. This study confirms the importance of interaction studies, revealing the possible antagonistic behavior of molecules when combined.

Chickpea Peptide: A Nutraceutical Molecule Corroborating Neurodegenerative and ACE-I Inhibition.

Chickpea seeds are the source of proteins in human nutrition and attribute some nutraceutical properties. Herein, we report the effects of chickpea seed bioactive peptide on albumin, insulin, lactoglobulin and lysozyme amyloid fibril formation. Employing thioflavin T (ThT) assays and circular dichroism (CD), amyloid structural binding transition was experimented to analyze the inhibition of amyloid fibril formation. The purified active peptide with a molecular mass of 934.53 Da was evaluated in vitro for its ACE-I inhibitory, antibacterial, antifungal and antidiabetic activities. Further, in vivo animal studies were carried out in wistar rats for blood pressure lowering action. In hypertensive rats, chickpea peptide decreased 131 ± 3.57 mm of Hg for systolic blood pressure and 86 ± 1.5 mm of Hg for diastolic blood pressure after 8 h intraperitoneal administration. Additionally, the peptide suppressed the fibrillation of amyloid and destabilized the preformed mature fibrils. Data emphasize efficacy of chickpea peptide vis-a-vis ACE-Inhibitory, antibacterial, antifungal, antidiabetic and anti-amyloidogenic activities, allowing us to propose this novel peptide as a suitable candidate for nutraceutical-based drugs and seems the first kind of its nature.

Anti-Amyloid Drug Screening Methods Using Bacterial Inclusion Bodies.

Amyloid aggregation is linked to a number of human disorders that range from non-neurological illnesses such as type 2 diabetes to neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. The formation of insoluble protein aggregates with amyloid conformation inside bacteria, namely, in bacterial inclusion bodies, offers the possibility to use bacteria as simple models to study amyloid aggregation processes and potential effects of both anti-amyloid drugs and/or pro-aggregative compounds. This chapter describes fast, simple, inexpensive, highly reproducible, and tunable in vitro and in cellulo methods that use bacterial inclusion bodies as preliminary screening tools for anti-amyloid drugs.

Sappanwood-derived polyphenolic antidote of amyloidal toxins achieved detoxification via inhibition/reversion of amyloidal fibrillation.

The formidable virulence of methicillin-resistant staphylococcus aureus (MRSA) have thrown great challenges to biomedicine, which mainly derives from their autocrine phenol-soluble modulins (PSMs) toxins, especially the most toxic member termed phenol-soluble modulins α3 (PSMα3). PSMα3 cytotoxicity is attributed to its amyloidal fibrillation and subsequent formation of cross-α sheet fibrils. Inspired by the multiple biological activity of Sappanwood, herein, we adopted brazilin, a natural polyphenolic compound originated from Caesalpinia sappan, as a potential antidote of PSMα3 toxins, and attempted to prove that the regulation of PSMα3 fibrillation was an effective alexipharmic way for MRSA infections. In vitro results revealed that brazilin suppressed PSMα3 fibrillation and disassembled preformed amyloidal fibrils in a dose-dependent manner, in which molar ratio (brazilin: PSMα3) of efficient inhibition and disassembly were both 1:1. These desired regulations dominated by brazilin benefited from its bonding to core fibrils-forming residues of PSMα3 monomers urged by hydrogen bonding and pi-pi stacking, and such binding modes facilitated brazilin-mediated inhibition or disruption of interactions between neighboring PSMα3 monomers. In this context, these inhibited and disassembled PSMα3 assemblies could not easily insert into cell membrane and subsequent penetration, and thus alleviating the membrane disruption, cytoplasmic leakage, and reactive oxygen species (ROS) generation in normal cells. As such, brazilin dramatically decreased the cytotoxicity borne by toxic PSMα3 fibrils. In addition, in vivo experiments affirmed that brazilin relieved the toxicity of PSMα3 toxins and thus promoted the skin wound healing of mice. This study provides a new antidote of PSMα3 toxins, and also confirms the feasibility of the assembly-regulation strategy in development of antidotes against supramolecular fibrillation-dependent toxins.

Amyloid Protein-Biofunctionalized Polydopamine Nanoparticles Demonstrate Minimal Plasma Protein Fouling and Efficient Photothermal Therapy.

Polydopamine (PDA) shows great application potential in photothermal therapy (PTT) of tumors due to its excellent photothermal performance. However, PDA rich in a large number of catechin structures, with strong adhesion, can readily attach to plasma proteins in blood to form protein corona, which greatly hinders the transfer efficiency to tumors and reduces the bioavailability. In this paper, a simple, rapid phase-transitioned albumin biomimetic nanocorona (TBSA) is used for the surface camouflage of PDA nanoparticles for minimal plasma protein fouling and efficient PTT. TBSA coating is formed by the BSA-derived amyloid through the hydrophobic aggregation near the isoelectric point and the rupture of disulfide bonds by tris(2-carboxyethyl) phosphine. The stable PDA@TBSA complexes are formed by camouflaging TBSA onto the surface of PDA through hydrophobic, electrostatic, and covalent binding between TBSA and PDA, which showed excellent anti-plasma protein adsorption properties profited from the surface charge of PDA@TBSA approaching equilibrium and the surface passivation of BSA. The plasma protein thickness of the PDA@TBSA surface is 6 times lower than that of PDA at adsorption saturation. In vitro and in vivo experiments have revealed that PDA@TBSA has an excellent photothermal antitumor effect compared to PDA. Both PDA and PDA@TBSA treatment plus 808 nm laser irradiation result in more than 70% inhibition on tumor cell proliferation. In addition, PDA@TBSA does not cause a significant inflammatory response and tissue damage. Taken together, the TBSA coating endows PDA with low-fouling functions in blood and improves the residence time of PDA in blood and enrichment in the tumor tissue. This work offers a novel and efficient strategy for the design of functional nanosystems exploiting the speciality of the biomolecular corona formation around nanomaterials.

Nutrient-sensing amyloid metastasis.

Amyloids are organized suprastructural polypeptide arrangements. The prevalence of amyloid-related processes of pathophysiological relevance has been linked to aging-related degenerative diseases. Besides the role of genetic polymorphisms on the relative risk of amyloid diseases, the contributions of nongenetic ontogenic cluster of factors remain elusive. In recent decades, mounting evidences have been suggesting the role of essential micronutrients, in particular transition metals, in the regulation of amyloidogenic processes, both directly (such as binding to amyloid proteins) or indirectly (such as regulating regulatory partners, processing enzymes, and membrane transporters). The features of transition metals as regulatory cofactors of amyloid proteins and the consequences of metal dyshomeostasis in triggering amyloidogenic processes, as well as the evidences showing amelioration of symptoms by dietary supplementation, suggest an exaptative role of metals in regulating amyloid pathways. The self- and cross-talk replicative nature of these amyloid processes along with their systemic distribution support the concept of their metastatic nature. The role of amyloidosis as nutrient sensors would act as intra- and transgenerational epigenetic metabolic programming factors determining health span and life span, viability, which could participate as an evolutive selective pressure.

Influence of the macromolecular crowder alginate in the fibrillar organization of the functional amyloid FapC from Pseudomonas aeruginosa.

Bacterial biofilms are an alternative lifestyle in which communities of bacteria are embedded in an extracellular matrix manly composed by polysaccharides, nucleic acids and proteins, being the hallmark of bacterial survival in a variety of ecological niches. Amyloid fibrils are one of the proteinaceous components of such extracellular crowded environments. FapC is the main component of the functional amyloid recently discovered in Pseudomonas species, including the opportunistic pathogen P. aeruginosa, which is a major cause of nosocomial infections and contamination of medical devices. Considering that several functional roles have been attributed to this bacterial amyloid, FapC emerged as a novel target to control Pseudomonas biofilm formation and to design new treatments against chronic infections. In this study, we used complementary biophysical techniques to evaluate conformational signatures of FapC amyloids formed in the presence of alginate, the major exopolysaccharide associated with the mucoid phenotype of P. aeruginosa strains isolated from cystic fibrosis patients. We found that the this naturally occurring macromolecular crowder leads to morphological similar yet polymorphic FapC fibrils, highlighting the importance of considering the complexity of the extracellular matrix in order to improve our understanding of microbial functional amyloids.

A Negative Energy Balance Is Associated with Metabolic Dysfunctions in the Hypothalamus of a Humanized Preclinical Model of Alzheimer's Disease, the 5XFAD Mouse.

Increasing evidence links metabolic disorders with neurodegenerative processes including Alzheimer's disease (AD). Late AD is associated with amyloid (Aß) plaque accumulation, neuroinflammation, and central insulin resistance. Here, a humanized AD model, the 5xFAD mouse model, was used to further explore food intake, energy expenditure, neuroinflammation, and neuroendocrine signaling in the hypothalamus. Experiments were performed on 6-month-old male and female full transgenic (Tg5xFAD/5xFAD), heterozygous (Tg5xFAD/-), and non-transgenic (Non-Tg) littermates. Although histological analysis showed absence of Aß plaques in the hypothalamus of 5xFAD mice, this brain region displayed increased protein levels of GFAP and IBA1 in both Tg5xFAD/- and Tg5xFAD/5xFAD mice and increased expression of IL-1ß in Tg5xFAD/5xFAD mice, suggesting neuroinflammation. This condition was accompanied by decreased body weight, food intake, and energy expenditure in both Tg5xFAD/- and Tg5xFAD/5xFAD mice. Negative energy balance was associated with altered circulating levels of insulin, GLP-1, GIP, ghrelin, and resistin; decreased insulin and leptin hypothalamic signaling; dysregulation in main metabolic sensors (phosphorylated IRS1, STAT5, AMPK, mTOR, ERK2); and neuropeptides controlling energy balance (NPY, AgRP, orexin, MCH). These results suggest that glial activation and metabolic dysfunctions in the hypothalamus of a mouse model of AD likely result in negative energy balance, which may contribute to AD pathogenesis development.

Decoding the Inter-Relationship between Sleep Disorders and Alzheimer's Disease Pathogenesis.

Alzheimer's Disease (AD), characterized by abnormally phosphorylated tau, Paired Helical Filaments (PHFs), Neurofibrillary Tangles (NFTs), deregulated mammalian Target Of Rapamycin (mTOR), and Aß deposits, is a multifactorial disease with sleep disorders being one of the causative agents. Therefore, we have reviewed the literature and have tried to decode the existence of positive feedback, reciprocal and a bidirectional relationship allying between sleep disturbances and AD. Much light has been thrown on the role of tau pathology and amyloid pathology in sleep pathology and its association with AD pathology. We have also discussed the role of melatonin in regulating sleep disorders and AD. The neuroprotective effect of melatonin via inhibiting tau hyperphosphorylation and Aß deposition has also been discussed. Moreover, astrocytes involvement in aggravating AD has also been highlighted in this review. Several therapeutic approaches aimed at improving both sleep disorders and AD have been duly discussed such as administration of antidepressants and antihistamines, immunotherapy, metal chelators, melatonin supplementation, light therapy and physical activity. Despite consistent efforts, the complete etiology concerning sleep disorder and AD is still unclear. Therefore, further research is needed to unravel the mechanism involved and also to develop strategies that may help in obstructing AD in its preclinical stage.

Computational Study of Natural Compounds for the Clearance of Amyloid-Βeta: A Potential Therapeutic Management Strategy for Alzheimer's Disease.

Alzheimer's disease (AD) is a widespread dynamic neurodegenerative malady. Its etiology is still not clear. One of the foremost pathological features is the extracellular deposits of Amyloid-beta (Aß) peptides in senile plaques. The interaction of Aß and the receptor for advanced glycation end products at the blood-brain barrier is also observed in AD, which not only causes the neurovascular anxiety and articulation of proinflammatory cytokines, but also directs reduction of cerebral bloodstream by upgrading the emission of endothelin-1 to induce vasoconstriction. In this process, RAGE is deemed responsible for the influx of Aß into the brain through BBB. In the current study, we predicted the interaction potential of the natural compounds vincamine, ajmalicine and emetine with the Aß peptide concerned in the treatment of AD against the standard control, curcumin, to validate the Aß peptide-compounds results. Protein-protein interaction studies have also been carried out to see their potential to inhibit the binding process of Aß and RAGE. Moreover, the current study verifies that ligands are more capable inhibitors of a selected target compared to positive control with reference to ΔG values. The inhibition of Aß and its interaction with RAGE may be valuable in proposing the next round of lead compounds for effective Alzheimer's disease treatment.

An automated microliter-scale high-throughput screening system (MSHTS) for real-time monitoring of protein aggregation using quantum-dot nanoprobes.

Protein aggregation is the principal component of numerous protein misfolding pathologies termed proteinopathies, such as Alzheimer's disease, Parkinson's disease, prion disease, and AA amyloidosis with unmet treatment needs. Protein aggregation inhibitors have great potential for the prevention and treatment of proteinopathies. Here we report the development of an automated real-time microliter-scale high throughput screening (MSHTS) system for amyloid aggregation inhibitors using quantum-dot nanoprobes. Screening 504 crude extracts and 134 low molecular weight aromatic compounds revealed the relationship of amyloid-ß (Aß) aggregation inhibitory activities of plant extracts using a plant-based classification. Within the eudicots, rosids, Geraniales and Myrtales showed higher activity. Screening low molecular weight aromatic compounds demonstrated that the structure of tropolone endows it with potential Aß aggregation inhibitory activity. The activity of the most active tropolone derivative was higher than that of rosmarinic acid. MSHTS also identified three chaperone molecules as tau aggregation inhibitors. These results demonstrate that our automated MSHTS system is a novel and robust tool that can be adapted to a wide range of compounds and aggregation-prone polypeptides.

Effects of folic acid supplementation on cognitive function and Aß-related biomarkers in mild cognitive impairment: a randomized controlled trial.

PURPOSE: Observational studies have frequently reported that low blood folate concentrations are associated with poor cognitive performance. Our previous studies have shown the potential beneficial effect on the metabolite levels of methionine cycle and peripheral blood inflammatory cytokines from 6- and 12-month folic acid supplementation on cognitive function in mild cognitive impairment (MCI). This study aims to continue exploring the effect of 24-month folic acid supplementation on cognitive function and pathological mechanism in MCI. METHODS: 180 individuals with MCI were identified and randomly divided into intervention (folic acid 400 µg/day, n = 90) and convention (n = 90) groups. Cognitive function (WAIS-RC) and blood Aß-related biomarkers were measured at baseline and at 6, 12, 18, and 24 months. Data were analyzed using generalized estimating equation. This trial has been registered with Trial Number: ChiCTR-TRC-13003227. RESULTS: During the follow-up, scores of full scale IQ, verbal IQ, and subdomains of Information and Digit Span were significantly higher in the intervention group than those in the convention group (P < 0.05). In the intervention group, blood homocysteine, S-adenosylhomocysteine (SAH), Aß-42, and the expression of APP-mRNA were decreased (P < 0.05), while S-adenosylmethionine (SAM), SAM/SAH ratio, and the expression of DNA methyltransferase mRNA were increased (P < 0.05). CONCLUSION: Folic acid supplementation appears to improve cognitive function and reduce blood levels of Aß-related biomarkers in MCI. Larger-scale double-blind placebo-controlled randomized trials of longer duration are needed.

Effective suppression of the modified PHF6 peptide/1N4R Tau amyloid aggregation by intact curcumin, not its degradation products: Another evidence for the pigment as preventive/therapeutic "functional food".

Curcumin is a natural product with multiple biological activities and numerous potential therapeutic applications. In present study, the influence of curcumin and its degradation products (DPs) on the amyloid aggregation of Tau protein and the related PHF6 peptide were investigated. We provided experimental/theoretical evidence for suppressing effects of the compounds on the amyloid formation using far-UV CD as well as AFM, XRD and docking techniques and showed that the parent curcumin displayed stronger inhibition effect against Tau fibril aggregation. The obtained results suggest that the curcumin/DPs binding sites on the Tau molecule are likely to be the same, and provide a good structural basis to explain the efficient aggregation suppressing behavior of the curcumin, compared to the DPs. So, developing more stable curcumin nanoparticle formulations with improved curcumin bioavailability are of great importance. Curcumin's multi-functionality is also highly significant for the therapeutic application of this natural compound against various human diseases.

SynAggreg: A Multifunctional High-Throughput Technology for Precision Study of Amyloid Aggregation and Systematic Discovery of Synergistic Inhibitor Compounds.

Numerous proteins can coalesce into amyloid self-assemblies, which are responsible for a class of diseases called amyloidoses, but which can also fulfill important biological functions and are of great interest for biotechnology. Amyloid aggregation is a complex multi-step process, poorly prone to detailed structural studies. Therefore, small molecules interacting with amyloids are often used as tools to probe the amyloid aggregation pathway and in some cases to treat amyloidoses as they prevent pathogenic protein aggregation. Here, we report on SynAggreg, an in vitro high-throughput (HT) platform dedicated to the precision study of amyloid aggregation and the effect of modulator compounds. SynAggreg relies on an accurate bi-fluorescent amyloid-tracer readout that overcomes some limitations of existing HT methods. It allows addressing diverse aspects of aggregation modulation that are critical for pathomechanistic studies, such as the specificity of compounds toward various amyloids and their effects on aggregation kinetics, as well as the co-assembly propensity of distinct amyloids and the influence of prion-like seeding on self-assembly. Furthermore, SynAggreg is the first HT technology that integrates tailored methodology to systematically identify synergistic compound combinations-an emerging strategy to improve fatal amyloidoses by targeting multiple steps of the aggregation pathway. To this end, we apply analytical combinatorial scores to rank the inhibition efficiency of couples of compounds and to readily detect synergism. Finally, the SynAggreg platform should be suited for the characterization of a broad class of amyloids, whether of interest for drug development purposes, for fundamental research on amyloid functions, or for biotechnological applications.

Beneficial Effects of Sideritis scardica and Cichorium spinosum against Amyloidogenic Pathway and Tau Misprocessing in Alzheimer's Disease Neuronal Cell Culture Models.

BACKGROUND: Natural products are a significantly underutilized source of potential treatments against human disease. Alzheimer's disease (AD) is a prime example of conditions that could be amenable to such treatments as suggested by recent findings. OBJECTIVE: Aiming to identify novel potentially therapeutic approaches against AD, we assessed the effects of Cichorium spinosum and Sideritis scardica extracts, both distinct components of the Mediterranean diet. METHODS/RESULTS: After the detailed characterization of the extracts' composition using LC-HRMS methods, they were evaluated on two AD neuronal cell culture models, namely the AßPP overexpressing SH-SY5Y-AßPP and the hyperphosphorylated tau expressing PC12-htau. Initially their effect on cell viability of SH-SY5Y and PC12 cells was examined, and subsequently their downstream effects on AßPP and tau processing pathways were investigated in the SH-SY5Y-AßPP and PC12-htau cells. We found that the S. scardica and C. spinosum extracts have similar effects on tau, as they both significantly decrease total tau, the activation of the GSK3ß, ERK1 and/or ERK2 kinases of tau, as well as tau hyperphosphorylation. Furthermore, both extracts appear to promote AßPP processing through the alpha, non-amyloidogenic pathway, albeit through partly different mechanisms. CONCLUSIONS: These findings suggest that C. spinosum and S. scardica could have a notable potential in the prevention and/or treatment of AD, and merit further investigations at the in vivo level.