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Tau and α-synuclein (α-syn) are prone-to-aggregate proteins that can be responsible for pathological lesions found in the brains of Alzheimer's disease (AD), Lewy body dementia (LBD), and Parkinson's disease (PD) patients. The early-stage oligomers and protofibrils of tau are believed to be strongly linked to human cognitive impairment while the toxic α-syn oligomers are associated with behavioral motor deficits. Therefore, concurrent targeting of both proteinaceous aggregates and oligomers are very challenging. Herein, rhodanine-based compounds were designed and synthesized to target the fibrils and oligomers of tau and α-syn proteins. In particular, the indole-containing rhodanines 5l and 5r displayed significantly high anti-aggregation activity towards α-syn fibrils by reducing of the thioflavin-T (ThT) fluorescence to less than 5 %. Moreover, 5r showed a remarkable decrease in the fluorescence of thioflavin-S (ThS) when incubated with the non-phosphorylated tau 0N4R and 2N4R, as well as the hyperphosphorylated tau isoform 1N4R. Transmission electron microscopy (TEM) analyses validated the powerful anti-fibrillar activity of 5l and 5r towards both protein aggregates. In addition, both 5l and 5r highly suppressed 0N4R tau and α-syn oligomer formation using the photo-induced cross-linking of unmodified protein (PICUP) assay. The fluorescence emission intensity of 5l was quenched to almost half in the presence of both protein fibrils at 510 nm. 5r showed a similar fluorescence response upon binding to 2N4R fibrils while no quenching effect was observed with α-syn aggregates. Ex vivo disaggregation assay using extracted human Aβ plaques was employed to confirm the ability of 5l and 5r to disaggregate the dense fibrils. Both inhibitors reduced the Aβ fibrils isolated from AD brains. 5l and 5r failed to show activity toward the cell-based α-syn inclusion formation. However, another indolyl derivative 5j prevented the α-syn inclusion at 5 µM. Collectively, the indolyl-rhodanine scaffold could act as a building block for further structural optimization to obtain dual targeting disease-modifying candidates for AD, LBD, and PD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Aggregation and deposition of amyloid beta-protein 1-42 (Aβ42) in the brain, primarily owing to hydrophobic interactions between Aβ42 chains, is a common pathology in all forms of Alzheimer's disease (AD). Hydrophilic oligosaccharides are widely present in the extracellular matrix and on the cytoplasmic membrane. To determine if oligosaccharides bind to Aβ42 or its aggregates and consequently affect their aggregation and cellular function, this study examined the interaction of typical functional oligosaccharides with Aβ42 or its aggregates. Isomaltooligosaccharides (IMOs), particularly isomaltotriose, panose, and isomaltotetraose, functioned as molecular chaperones for Aβ42 by binding directly to Aβ42, preserving Aβ42's active conformation and cytotrophic activity. Oral IMOs reduced total plasma Aβ level and indirectly caused a slight reduction in the load of Aβ42 spots/plaques in the brain of AD model mice (male). Another branched oligosaccharide, bianntennary core pentasaccharide (BCP), had a relatively high binding specificity for Aβ42 oligomers (Aβ42O) and acted as an antagonistic binding partner for Aβ42O. Free BCP effectively blocked/prevented further assembly of Aβ42O and their toxicity to neural and vascular endothelial cell lines. Since BCP is also a signaling component of membrane targets (glycolipids, glycoproteins or receptors), it seemed that BCP had two opposing effects on the binding of Aβ42O to target cells. This study's findings suggest that these branched oligosaccharides may be potential candidates for blocking or preventing Aβ42 aggregation and Aβ42O cytotoxicity/neurotoxicity, respectively, and that IMO-like or free BCP-like oligosaccharide deficiencies in the brain may be one of the underlying mechanisms for Aβ42 aggregation and Aβ42O cytotoxicity., Competing Interests: Declaration of competing interest There are no conflicts of interest to declare., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Deposits of aggregated TAR DNA-binding protein 43 (TDP-43) in the brain are associated with several neurodegenerative diseases. It is well established that binding of RNA/DNA to TDP-43 can prevent TDP-43 aggregation, but an understanding of the structure(s) and conformational dynamics of TDP-43, and TDP-43-RNA complexes, is lacking, including knowledge of how the solution environment modulates these properties. Here, we address this challenge using hydrogen-deuterium exchange-mass spectrometry. In the presence of RNA olignoucleotides, we observe protection from exchange in the RNA recognition motif (RRM) domains of TDP-43 and the linker region between the RRM domains, consistent with nucleic acid binding modulating interdomain interactions. Intriguingly, at elevated salt concentrations, the extent of protection from exchange is reduced in the RRM domains when bound to an RNA sequence derived from the 3' UTR of the TDP-43 mRNA (CLIP34NT) compared to when bound to a (UG)6 repeat sequence. Under these conditions, CLIP34NT is no longer able to prevent TDP-43 aggregation. This suggests that a salt-induced structural rearrangement occurs when bound to this RNA, which may play a role in facilitating aggregation. Additionally, upon RNA binding, we identify differences in exchange within the short α-helical region located in the C-terminal domain (CTD) of TDP-43. These allosterically altered regions may influence the ability of TDP-43 to aggregate and fine-tune its RNA binding repertoire. Combined, these data provide additional insights into the intricate interplay between TDP-43 aggregation and RNA binding, an understanding of which is crucial for unraveling the molecular mechanisms underlying TDP-43-associated neurodegeneration.

The aggregation of Aβ monomers into oligomers with β-sheet structures is an important cause of Alzheimer's disease (AD), while the Aβ 42 peptide is more toxic and prone to aggregate. It is of great significance to study the inhibition mechanism of Aβ 42 monomer aggregation and find excellent inhibitors for the treatment of AD. Research in recent years has focused on small molecule compounds and nanoparticles, but they all have certain limitations. As a new type of porous material, a porous organic cage (POC) has potential application feasibility in the biomedical field due to its unique physicochemical properties. In this work, molecular dynamics simulations were used for the first time to explore the interaction and conformational transformation of the Aβ 42 peptide in CC3 crystals with different morphologies (planar and spherical). The results show that the adsorption of the Aβ 42 peptide on different CC3 crystals is mainly achieved through strong van der Waals forces. During the simulations, the Aβ 42 peptide undergoes various degrees of structural changes. Compared to that in water, this binding induces more irregular structures, such as turns and 3-helices, and inhibits the production of β-sheets, while enhancing the overall backbone rigidity of the Aβ 42 peptide. The transformation analysis of peptide conformation is further complemented by free energy landscape and cluster analysis. These findings provide a strong basis for CC3 crystals as novel inhibitors to inhibit the toxicity and aggregation of the Aβ 42 peptide.

Multifunctional nitrogen-doped carbon dots (N-CDs) were synthesized, and the morphology, composition, and spectral properties of N-CDs were characterized by multiple characterization techniques. The inhibition of β-amyloid (Aβ) peptide aggregation and the destruction of the Aβ fibril structure by N-CDs were also studied. The conformational transition and morphology of Aβ 42 in the presence of N-CDs were monitored by far-UV circular dichroism (CD) spectroscopy and transmission electron microscopy (TEM). The results demonstrated that the prepared N-CDs could effectively inhibit Aβ 42 peptide aggregation and depolymerize Aβ fibrils. Furthermore, the inhibition and disaggregation mechanism of existing Aβ 42 fibrils by N-CDs was studied by electron paramagnetic resonance spectroscopy (EPR). The results showed that the modulation of reactive oxygen species (ROS) by N-CDs and multiple interactions between N-CDs and Aβ 42 fibrils played a crucial part in restraining and reducing the aggregation of Aβ 42 . Our work demonstrates the therapeutic potential of N-CDs in suppressing Aβ 42 peptide aggregation and destroying existing Aβ 42 fibrils, which provides a new perspective strategy in the treatment of Alzheimer's disease (AD).

Alzheimer's disease (AD) heavily impacts human lives and is becoming serious as societies age. Inhibiting and disaggregating β-amyloid aggregates is a possible solution for AD therapy. In this study, a novel type of nanozyme based on Ru 3+ -chelated nanoscale metal organic frameworks (Ru 3+ -NMOFs), displaying strong peroxidase-like activity, was proposed as an inhibitor and disaggregator of β-amyloid aggregates. As a high concentration of hydrogen peroxide is present at the sites of β-amyloid aggregates, Ru 3+ -NMOFs could catalyze the conversion of hydrogen peroxide to hydroxyl radicals. Thus, these hydroxyl radicals would attack the β-amyloid chain, oxidizing it to enhance its hydrophilicity, which results in a decreased hydrophobic interaction and reduced degree of aggregation. Ru 3+ -NMOFs could effectively inhibit as well as disaggregate β-amyloid fibrils both in vitro and in vivo . Additionally, the reduction of the β-amyloid aggregates and the attenuation of reactive oxygen species transfer led to lower levels of inflammatory factors, which could be beneficial in alleviating AD symptoms. In a typical treatment, Ru 3+ -NMOFs could mitigate the paralysis of C. elegans CL2120 and elevate survival rates. This study opens a new avenue for MOF-based nanozymes as potential treatment agents for AD therapy.

Protein aggregation is involved in many human diseases, but characterizing the sizes and shapes of intermediate oligomers (∼10-100 nm) that are important to the formation of macroscale aggregates like amyloid fibrils is a significant analytical challenge. Here, charge detection mass spectrometry (CDMS) is used to characterize individual conformational states of bovine serum albumin oligomers with up to ∼225 molecules (15 MDa). Elongated, partially folded, and globular conformational families for each oligomer can be readily distinguished based on the extent of charging. The abundances of individual conformers vary with changes in the monomer concentration or by adding aggregation inhibitors, such as SDS, heparin, or MgCl 2 . These results show the potential of CDMS for investigating intermediate oligomers in protein aggregation processes that are important for understanding aggregate formation and inhibition mechanisms and could accelerate formulation buffer development to prevent the aggregation of biotherapeutics.

With the recent clinical success of anti-amyloid-β (Aβ) monoclonal antibodies, there is a renewed interest in agents which target the Aβ peptide of Alzheimer's disease (AD). Metal complexes are particularly well-suited for this development, given their structural versatility and ability to form stabile interactions with soluble Aβ. In this report, a small series of ruthenium-arene complexes were evaluated for their respective ability to modulate both the aggregation and cytotoxicity of Aβ. First, the stability of the complexes was evaluated in a variety of aqueous media where the complexes demonstrated exceptional stability. Next, the ability to coordinate and modulate the Aβ peptide was evaluated using several spectroscopic methods, including thioflavin T (ThT) fluorescence, dynamic light scattering (DLS), and transmission electron microscopy (TEM). Overall, the complex RuBO consistently gave the greatest inhibitory action towards Aβ aggregation, which correlated with its ability to coordinate to Aβ in solution. Furthermore, RuBO also had the lowest affinity for serum albumin, which is a key consideration for a neurotherapeutic, as this protein does not cross the blood brain barrier. Lastly, RuBO also displayed promising neuroprotective properties, as it had the greatest inhibition of Aβ-inducted cytotoxicity.

The transformation of metabolites into amyloidogenic aggregates represent an intriguing dimension in the pathophysiology of metabolic disorders, including alkaptonuria, canavan disease, and isovaleric acidemia. Central to this phenomenon are the metabolites homogentisic acid (HA), N-acetyl aspartic acid (NAA), and isovaleric acid (IVA), which we found, weave an intricate network of self-assembled structures. Leveraging an array of microscopy techniques, we traced the morphological behavior of these assemblies that exhibit concentration and time-dependent morphological transitions from isolated globules to clustered aggregates. MD simulation studies suggest significant role of hydrogen bonding interactions in the aggregation process. While displaying strong amyloidogenic propensity in solution, these aged aggregates were significantly cytotoxic to mouse neural N2a cell lines. In vivo effect in Caenorhabditis elegans (C. elegans) nematode further validated cytotoxicity of aggregates. Our findings provide fresh insights to amyloidogenic nature of HA, NAA, and IVA aggregates and their possible role in associated metabolic disorders such as alkaptonuria, canavan disease and isovaleric acidemia., (© 2024 Wiley-VCH GmbH.)

The formation and characterization of new diamagnetic ruthenium uracil mono-imine compounds: [(η 6 -p-cymene)Ru II (L)Cl][BF 4 ] (L=H 2 urpda=5-((pyridin-2-yl)methyleneamino)-6-aminouracil) for 1, urdpy=6-amino-1,3-dimethyl-5-((pyridin-2-ylmethylene)amino)uracil) for 2 or urqda=5-((quinolin-2-yl)methyleneamino)-6-aminouracil) for 3); cis-[Ru(bipy) 2 (urpy)](BF 4 ) 2 (4) (urpy=5-((pyridin-2-yl)methyleneamino)uracil) and cis-[Ru(bipy) 2 (dapd)] (5) (H 2 dadp=5,6-diaminouracil) are described. A ruthenium(IV) uracil Schiff base compound, trans-[Ru(urpda)(PPh 3 )Cl 2 ] (6) was also formed. Various physicochemical techniques were utilized to characterize the novel ruthenium compounds. Similarly, the stabilities of 1-3 and 6 monitored in chloro-containing and the non-coordinating solvent, dichloromethane show that they are kinetically inert, whereas, in a high nucleophilic environment, the chloride co-ligands of these ruthenium complexes were rapidly substituted by DMSO. In contrast, the substitution of the labile co-ligands for these ruthenium complexes by DMSO molecules in a high chloride content was suppressed. Solution chemical reactivities of the different ruthenium complexes were rationalized by density functional theory computations. Furthermore, the binding affinities and strengths between BSA and the respective ruthenium complexes were monitored using fluorescence spectroscopy. In addition, the in vitro anti-diabetic activities of the novel metal complexes were assessed in selected skeletal muscle and liver cell lines., (© 2024 Wiley-VCH GmbH.)

Studies of the structure and dynamics of oligomeric aggregates of amyloidogenic peptides pose challenges due to their transient nature. This concept article provides a brief overview of various nucleation mechanisms with reference to the classical nucleation theory and illustrates the advantages of incubating amyloidogenic peptides in reverse micelles (RMs). The use of RMs not only facilitates size regulation of oligomeric aggregates but also provides an avenue to explore protein-protein interactions among the oligomeric aggregates of various amyloidogenic peptides. Additionally, we envision the feasibility of preparing brain tissue-derived oligomeric aggregates using RMs, potentially advancing the development of monoclonal antibodies with enhanced potency against these pathological species in vivo., (© 2024 Wiley-VCH GmbH.)

Protein aggregation, particularly the formation of amyloid fibrils, is associated with numerous human disorders, including Parkinson's disease. This neurodegenerative condition is characterised by the accumulation of α-Synuclein amyloid fibrils within intraneuronal deposits known as Lewy bodies or neurites. C-terminally truncated forms of α-Synuclein are frequently observed in these inclusions in the brains of patients, and their increased aggregation propensity suggests a role in the disease's pathogenesis. This study demonstrates that the small molecule ZPD-2 acts as a potent inhibitor of both the spontaneous and seeded amyloid polimerisation of C-terminally truncated α-Synuclein by interfering with early aggregation intermediates. This dual activity positions this molecule as a promising candidate for therapeutic development in treating synucleinopathies., (© 2024 The Author(s). The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Protein misfolding is a biological process that leads to protein aggregation. Anomalous misfolding and aggregation of human superoxide dismutase (hSOD1) into amyloid aggregates is a characteristic feature of amyotrophic lateral sclerosis (ALS), a neurodegenerative illness. Thus, focusing on the L38R mutant may be a wise decision to comprehend the SOD1 disease process in ALS. We suggest that Bisdemethoxycurcumin (BDMC) may be a strong anti-amyloidogenic polyphenol against L38R mutant aggregation. Protein stability, hydrophobicity, and flexibility were altered when BDMC was bound to the L38R mutant, as shown by molecular dynamic (MD) simulations and molecular docking. FTIR data shows α-Helix dominance in BDMC-containing samples, with reduced β-sheet and disordered peaks, indicating the decrease of aggregate species. ThT aggregation kinetics curves show BDMC reduces L38R mutant aggregation dose-dependently, with higher BDMC concentrations yielding greater reductions. TEM images showed various quantities of amorphous aggregates, but notably, 60 μM BDMC markedly reduced aggregate density, underscoring BDMC's inhibitory effect. Hemolysis tests revealed aggregate species in BDMC-treated samples were less toxic than in L38R mutant samples alone at the same concentrations and exposure times. Overall, BDMC has substantial potential to develop highly effective inhibitors that mitigate the risk of fatal ALS., Competing Interests: Declaration of competing interest The authors declare that no conflict of interest exists for the present work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The stabilization of protein therapeutics against aggregation is crucial for maintaining their efficacy and safety. This study investigated the synergistic effects of cyclodextrins (CDs) and electrolytes at high concentrations on the stabilization of immunoglobulin G (IgG), insulin, and adeno-associated virus (AAV) vectors. The effects of 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) combined with various electrolytes were evaluated using human plasma-derived IgG as a model protein. The HP-β-CD and L(+)-arginine hydrochloride combination synergistically increased the onset temperature of protein aggregation and inhibited the formation of soluble and insoluble aggregates during long-term storage. Notably, this synergistic effect was not observed when sucrose was used instead of HP-β-CD. Similar synergistic effects were observed with insulin and AAV vectors. The findings suggest that the stabilization mechanism could potentially involve enhanced interactions between HP-β-CD and IgG, preventing protein-protein interactions. However, the combination did not synergistically improve the solubility of free aromatic amino acids, including tyrosine and tryptophan. This study highlights the potential of using the combination of CDs and electrolytes as a promising formulation strategy for stabilizing complex protein therapeutics. Further studies are needed to elucidate the underlying mechanisms and generalize the approach to other proteins with varying physicochemical properties., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work presented in this study., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The complex nature of Alzheimer's disease (AD) etiopathology is among the principal hurdles to developing effective anti-Alzheimer agents. Tau pathology and Amyloid-β (Aβ) accumulation are hallmarks and validated therapeutic strategies of AD. GSK-3β is a serine/threonine kinase involved in tau phosphorylation. Its excessive activity also contributes to the production of Aβ plaques, making GSK-3β an attractive AD target. Taking this into account, In this article, we outline the design, synthesis, and biological validation of a focused library of 1,2,3,4-tetrahydropyrimidine based derivatives as inhibitors of GSK-3β, tau phosphorylation, and Aβ accumulation. The inhibitory activity of forty nine synthetic compounds was tested against GSK-3β and other AD-relevant kinases. The kinetic experiments revealed the mode of GSK-3β inhibition by the most potent compound 44. The in- vitro drug metabolism and pharmacokinetic studies were thereafter performed. The anti-aggregation activity of the most potent GSK-3β inhibitor was tested using AD transgenic Caenorhabditis elegans (C. elegans) strain CL2006 for quantification of Aβ plaques and BR5706 C. elegans strain for tau pathology evaluation. We then evaluated the blood-brain barrier permeability and got promising results. Therefore, we present compound 44 as a potential ATP-competitive GSK-3β inhibitor with good metabolism and pharmacokinetic profile, anti-aggregation properties for amyloid beta protein, and reduction in tau-phosphorylation levels. We recommend more investigation into compound 44-based small molecules as possible targets for AD disease-modifying treatments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the selective loss of motor neurons. A bidirectional communication system known as the "microbiota-gut-brain" axis has a regulatory function in neurodegenerative disorders. The impact of probiotics on ALS through the "microbiota-gut-brain" axis remains uncertain. A longitudinal investigation was conducted to examine the alterations in the structure of the ileum and colon in mutant superoxide dismutase 1 (SOD1 G93A ) transgenic mice models of ALS by using immunofluorescence and Western blotting. Subsequently, the mice were administered a multistrain probiotic mixture (LBE) or vehicle orally, starting from 60 days of age until the terminal stage of the disease. The effects of these agents on the behavior, gut microbiota, microbial metabolites, and pathological processes of the spinal and intestine of SOD1 G93A mice were analyzed, with a focus on exploring potential protective mechanisms. SOD1 G93A mice exhibit various structural abnormalities in the intestine. Oral administration of LBE improved the proinflammatory response, reduced aberrant superoxide dismutase 1 (SOD1) aggregation, and protected neuronal cells in the intestine and spinal cord of SOD1 G93A mice. Furthermore, LBE treatment resulted in a change in intestinal microbiota, an increase in short-chain fatty acid levels, and an enhancement in autophagy flux. SOD1 G93A mice exhibited various structural abnormalities in the intestine. LBE can improve the proinflammatory response, reduce aberrant SOD1 aggregation, and protect neuronal cells in the spinal cord and intestine of SOD1 G93A mice. The positive effect of LBE can be attributed to increased short-chain fatty acids and enhanced autophagy flux., Competing Interests: Declarations. Ethics Approval: All animal procedures were performed in accordance with the Regulations for the Administration of Laboratory Animals set forth by the Ministry of Science and Technology of the People’s Republic of China. All experiments were approved by the Research Ethics Committee of the Second Hospital of Hebei Medical University (Shijiazhuang, Hebei, P.R. China, protocol code: 2022AE269, date of approval: 2022.3.14). Consent to Participate: Not applicable. Consent for Publication: Not applicable. Competing Interests: The authors declare no competing interests., (© 2024. The Author(s).)

Protein aggregation is undesirable for cells due to its possible toxicity, and is also undesirable in biotechnology and pharmaceuticals. Polyamines are known to be capable of both suppressing and stimulating protein aggregation. In the present work polyamines (spermidine, putrescine) have been shown to alter the pathway of α-lactalbumin aggregation induced by dithiothreitol, leading to the formation of larger protein particles during the initial stages of aggregation and promoting the later stage of sticking of aggregates. According to the aggregation kinetics data, polyamines accelerate protein aggregation in a concentration-dependent manner, with a maximum at 50 mM spermidine and 100 mM putrescine. With a further increase in polyamines concentration the effect of aggregation acceleration decreased, thus, the modulation of the aggregation rate by polyamines was shown. A comparison of the aggregation kinetics and hydrodynamic radii growth data registered by dynamic light scattering with the data obtained by asymmetric flow field-flow fractionation and analytical ultracentrifugation allowed us to describe the early stages of aggregation and formation of initial α-lactalbumin clusters. Our results provide a deeper insight into the mechanism of amorphous aggregation of α-lactalbumin and polyamines action on protein aggregation and protein-protein interaction in general., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

The photo-oxidation of amyloid-β (Aβ) protein catalyzed by Aβ-targeting photosensitizers shows high potential in treating Alzheimer's disease (AD). Herein, we report the first example of photosensitizers based on the rofecoxib scaffold, in which rational introduction of the electron-absorbing pyridinium/quinolinium moiety to the skeleton of rofecoxib could not only extend the absorption and emission wavelengths but also increase the efficiency of singlet oxygen ( 1 O 2 ) production. The emission wavelengths of R-S-MP , R-S-MC , and R-S-MQ are red-shifted to 860 nm, which might benefit the NIR imaging of Aβ aggregates with low photoscattering and autofluorescence. In addition, R-S-MP can identify Aβ plaques in brain sections of AD mice and detect abnormal viscosity environments, facilitating the pathological study of Alzheimer's disease. Most importantly, upon complexation with Aβ plaques, R-S-MP and R-S-MC could produce high singlet oxygen ( 1 O 2 ) under light irradiation, which can achieve the specific photo-oxidation of Aβ protein. Our optimized photosensitizers could change the conformation of β-rich Aβ protein and enhance its clearance through the lysosomal pathway, leading to the reduction of the Aβ-mediated neurotoxicity. All these excellent characteristics of our dual-functional photosensitizers for simultaneous imaging and photo-oxidation of Aβ aggregates suggest their promising prospects in pathological research in AD.

Aβ42 aggregation was implicated in the pathogenesis of Alzheimer's disease (AD) without effective treatment available currently. Future efforts in clinical trials should instead focus on applying those antiamyloid treatment strategies to the preclinical stage and "the earlier, the better". How to identify and inhibit Aβ42 oligomers in the different stages of aggregation is therefore becoming the key to controlling primary aggregation and consequent AD development. Aggregation-induced emission probe DNTPH was demonstrated recently, enabling detection of amyloid at wavelengths up to 710 nm and exhibiting strong inhibitory effects on Aβ fibrosis at low dose. However, the detection and inhibition mechanisms of Aβ oligomers at various early stages of aggregation remain unknown. To this end, we built four different morphologies of Aβ42 pentamers characterized by products in monomeric aggregate (P M ), primary nucleation (P P ), secondary nucleation (P S ), and fibril stages (P F ) to explore the distinguishable ability and inhibition mechanisms of DNTPH with different concentrations upon binding. The results showcased that DNTPH does detect the four different Aβ42 oligomers with conspicuous fluorescence (λ P M = 657 nm, λ P P = 639 nm, λ P S = 630 nm, and λ P F = 648 nm) but fails to distinguish them, indicating that additional improvements are required further for the probe to achieve it. The inhibition mechanisms of DNTPH on the four Aβ42 aggregation are however of amazing differences. For P M and P P , aggregation was inhibited by altering the secondary structural composition, i.e., by decreasing the β-sheet and toxic turn (residues 22-23) probabilities, respectively. For P S , inhibition was achieved by segregating and keeping the two disordered monomeric species (P SM ) away from the ordered secondary seed species (P SF ) and consequently blocking further growth of the P SF seed. The inhibition mechanism for P S is first probed and proposed so far, as far as we know, and the corresponding aggregation stage of P S is the most important one among the four stages. The inhibition of P F was triggered by distorting the fibril chains, disrupting the ordered fibril surface for the contact of monomers. In addition, the optimal inhibitory concentrations of DNTPH for P M , P P , and P F were determined to be 1:3, while for P S , it was 1:5. This outcome offers a novel perspective for designing drugs targeting Aβ42 oligomers at different aggregation stages.

Bacterial dormancy is a valuable strategy to survive stressful conditions. Toxins from chromosomal toxin-antitoxin systems have the potential to halt cell growth, induce dormancy, and eventually promote a stress-tolerant persister state. Due to their potential toxicity when overexpressed, sophisticated expression systems are needed when studying toxin genes. Here, we present a moderate expression system for toxin genes based on an artificial 5' untranslated region. We applied the system to induce expression of the toxin gene tisB from the chromosomal type I toxin-antitoxin system tisB/istR-1 in Escherichia coli . TisB is a small hydrophobic protein that targets the inner membrane, resulting in depolarization and ATP depletion. We analyzed TisB-producing cells by RNA-sequencing and revealed several genes with a role in recovery from TisB-induced dormancy, including the chaperone genes ibpAB and spy . The importance of chaperone genes suggested that TisB-producing cells are prone to protein aggregation, which was validated by an in vivo fluorescent reporter system. We moved on to show that TisB is an essential factor for protein aggregation upon DNA damage mediated by the fluoroquinolone antibiotic ciprofloxacin in E. coli wild-type cells. The occurrence of protein aggregates correlates with an extended dormancy duration, which underscores their importance for the life cycle of TisB-dependent persister cells., Importance: Protein aggregates occur in all living cells due to misfolding of proteins. In bacteria, protein aggregation is associated with cellular inactivity, which is related to dormancy and tolerance to stressful conditions, including exposure to antibiotics. In Escherichia coli , the membrane toxin TisB is an important factor for dormancy and antibiotic tolerance upon DNA damage mediated by the fluoroquinolone antibiotic ciprofloxacin. Here, we show that TisB provokes protein aggregation, which, in turn, promotes an extended state of cellular dormancy. Our study suggests that protein aggregation is a consequence of membrane toxins with the potential to affect the duration of dormancy and the outcome of antibiotic therapy., Competing Interests: The authors declare no conflict of interest.

An effective therapeutic strategy to suppress Alzheimer's disease (AD) progression is to disrupt β-sheet rich neurotoxic soluble amyloid-β (Aβ) aggregates. Previously, we identified new pentapeptides (RVVPI and RIAPA) with notably enhanced ability to block Aβ 42 aggregation as compared to Aβ 42 C-terminal derived peptide RIIGL using integrated computational protocol. In this work, the potential of RIIGL, RVVPI, and RIAPA for the structural destabilization of Aβ 42 protofibril was assessed by molecular dynamics (MD) simulations and in vitro studies. The binding free energy analysis depicts that charged residues influence Aβ 42 protofibril-pentapeptide interactions. Notably, RVVPI displays a more pronounced destabilization effect than other peptides due to higher conformational fluctuations, and disruption of salt bridge (K28-A42) interactions in Aβ 42 protofibril. RVVPI exhibited highest inhibitory activity (Inhibition=66.2 %, IC 50 =5.57±0.83 μM) against Aβ 42 aggregation consistent with computational results. Remarkably, RVVPI displayed ~4.5 fold lower IC 50 value as compared to RIIGL. ThT and TEM studies highlighted the enhanced efficiency of RVVPI (62.4 %) in the disassembly of pre-formed Aβ 42 fibrils than RIIGL and RIAPA. The combined in silico and in vitro studies identified a new peptide, RVVPI, as an efficient inhibitor of Aβ 42 fibrillation and disassembly of Aβ 42 aggregates., (© 2024 Wiley-VCH GmbH.)

A group of N-phenylbenzofuran-2-carboxamide and N-phenylbenzo[b]thiophene-2-carboxamide derivatives were designed and synthesized as a novel class of Aβ42 aggregation modulators. In the thioflavin-T based fluorescence aggregation kinetics study, compounds 4 a, 4 b, 5 a and 5 b possessing a methoxyphenol pharmacophore were able to demonstrate concentration dependent inhibition of Aβ42 aggregation with maximum inhibition of 54 % observed for compound 4 b. In contrast, incorporation of a 4-methoxyphenyl ring in compounds 4 d and 5 d led to a significant increase in Aβ42 fibrillogenesis demonstrating their ability to accelerate Aβ42 aggregation. Compound 4 d exhibited 2.7-fold increase in Aβ42 fibrillogenesis when tested at the maximum concentration of 25 μM. These results were further confirmed by electron microscopy studies which demonstrates the ability of compounds 4 a, 4 b, 4 d, 5 a, 5 b and 5 d to modulate Aβ42 fibrillogenesis. Compounds 5 a and 5 b provided significant neuroprotection to mouse hippocampal neuronal HT22 cells against Aβ42-induced cytotoxicity. Molecular docking studies suggest that the orientation of the bicyclic aromatic rings (either benzofuran or benzo[b]thiophene) plays a major role in moderating their ability to either inhibit or accelerate Aβ42 aggregation. Our findings support the application of these novel derivatives as pharmacological tools to study the mechanisms of Aβ42 aggregation., (© 2024 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

Based on the three typical gels under KCl substitution groups, the effect of partial substitution of NaCl by KCl (groups: T 1:0.6 M NaCl; T 2: 0.3 M NaCl +0.3 M KCl; T 3: 0.2 M NaCl +0.4 M KCl; T 4:0.6 M KCl) on the aggregation behavior and gel characteristics of myosin was evaluated. The significant changes in hydrophobicity and sulfhydryl content (P < 0.05) indicate KCl substitution enhances myosin aggregation through hydrophobic interactions and disulfide bonds. According to Ca 2+ -ATP, scanning electron microscopes (SEM) and the rheological results, T2 had a smoother network structure at about 75 °C. Noticeably, T3 had high water holding capacity (WHC), but its gel had some visible cavities. T4 had a gel structure with several irregular aggregates due to a greater aggregation rate. Thus, appropriate partial substitution of NaCl by KCl could enhance beef myosin gel properties and heat-induced aggregation behavior., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The main pathological mechanisms of Alzheimer's Disease (AD) are extracellular senile plaques caused by β-amyloid (Aβ) deposition and intracellular neurofibrillary tangles derived from hyperphosphorylated Tau protein (p-Tau). However, it is difficult to obtain a good curative effect because of the poor brain bioavailability of drugs, which is attributed to the blood-brain barrier (BBB) restriction and complicated brain conditions. Herein, HM-DK was proposed for synergistic therapy of AD by using hollow mesoporous manganese dioxide (HM) as a carrier to deliver an Aβ-inhibiting peptide and a Dp-peptide inhibitor of Tau-related fibril formation synergistically. Inspired by 4T1 cancer cells promoting BBB penetration during brain metastasis, a prospective biomimetic nanocarrier (HM-DK@CM) encapsulated by 4T1 cell membranes was designed. After crossing the BBB, HM-DK@CM inhibited Aβ aggregation and prevented Tau phosphorylation simultaneously. Moreover, by taking advantage of the catalase-like activity of HM, HM-DK@CM relieved oxidative stress and altered the microenvironment associated with the development of AD. Compared with the single therapeutic drug, HM-DK@CM restored nerve damage and improved AD mice's learning and memory abilities by decreasing Aβ oligomer, p-Tau protein, and inflammation through various pathways for synergistic therapy, which has broad prospects for the effective treatment of AD.

Synergy between antimicrobial peptides (AMPs) may be the key to their evolutionary success and could be exploited to develop more potent antibacterial agents. One of the factors thought to be essential for AMP potency is their conformational flexibility, but characterising the diverse conformational states of AMPs experimentally remains challenging. Here we introduce a method for characterising the conformational flexibility of AMPs and provide new insights into how the interplay between conformation and aggregation in synergistic AMP combinations yields emergent properties. We use unsupervised learning and molecular dynamics simulations to show that mixing two AMPs from the Winter Flounder family (pleurocidin (WF2) & WF1a) constrains their conformational space, reducing the number of distinct conformations adopted by the peptides, most notably for WF2. The aggregation behaviour of the peptides is also altered, favouring the formation of higher-order aggregates upon mixing. Critically, the interaction between WF1a and WF2 influences the distribution of WF2 conformations within aggregates, revealing how WF1a can modulate WF2 behaviour. Our work paves the way for deeper understanding of the synergy between AMPs, a fundamental process in nature.

A striking issue is the scarcity of imaging probes for the early diagnosis of Alzheimer's disease. For the development of Aβ biomarkers, a mitochondria targeting, de novo designed, aggregation-induced emission (AIE) probe Cou-AIE-TPP + is constructed by engineering the aromatic coumarin framework into the bridge of electron donor-acceptor-donor tethered with a lipophilic cationic triphenylphosphonium (TPP + ) group. The synthesized Cou-AIE-TPP + probe exhibits biocompatibility, noncytotoxicity, and a huge Stokes shift (124 nm in PBS). Cou-AIE-TPP + has respectable fluorescence augmentation inside the aggregated Aβ40 in comparison with monomeric Aβ40 with a high binding affinity ( K d = 83 nM) to Aβ40 aggregates, is capable of detecting the kinetics of amyloid aggregation, and is superior to the gold standard probe thioflavin T. Fluorescence lifetime and brightness are also augmented when the probe Cou-AIE-TPP + binds with Aβ aggregates in PBS. Cou-AIE-TPP + ( λ em 604 nm) selectively targets and images neuronal cell mitochondria, is useful to monitor mitochondrial morphology alteration and damage during Aβ40-induced neurotoxicity, recognizes neurotoxic Aβ fibrils, and is highly colocalized with thioflavin T, showing a decent Pearson correlation coefficient of 0.91 in the human neuroblastoma SH-SY5Y cell line. These findings indicate that the mitochondria targeting, de novo designed, functional AIE-based solvatofluorochromic Cou-AIE-TPP + probe is a promising switch on biomarkers for fluorescence imaging of Aβ aggregates and to monitor mitochondrial morphology change and dysfunction during Aβ-induced neurotoxicity, which may offer imperative direction for the advancement of compelling AIE biomarkers for targeted early stage Aβ diagnosis in the future.

The aggregation of amyloid β peptide (Aβ) in the presence of elevated levels of transition-metal ions, e.g. , Fe 3+ , Cu 2+ , Zn 2+ , is accountable for enhanced cellular toxicity in Alzheimer's disease. Many strategies are reported to inhibit either Cu 2+ , Zn 2+ , or Fe 3+ -induced Aβ fibrillation, focused on one metal. Herein, a taurine-containing adaptable metal sequestering peptide (AMSP) has been developed as the modulator of any of the cited metal-induced Aβ-aggregation in vitro . We designed the peptide conjugate comprising VFFA as a recognition motif and a taurine moiety coupled with a pendant chain of glutamic acid such that the -SO 3 H groups of taurine lie nearby 13 His and 14 His of Aβ, and sequester such metal ions that construct the salt bridge preponderantly via 13 His-metal- 14 His composition as well as bridges with 6 His of Aβ. We checked the modulation of fibrillar aggregates of Aβ in the presence of metal ions by monitoring the fibril accumulation using several biophysical methods. The results established that non-aggregating AMSP sequesters Zn 2+ preferably, along with Fe 3+ and Cu 2+ ions from the metal-Aβ complex at the physiological condition, efficiently inhibiting Aβ aggregation. While such adaptable metal binders that can chelate various metals are new, AMSP inhibits aggregation through selective recognition and metal scavenging.

Type 2 diabetes mellitus and Alzheimer's disease, are two closely related pathological situations that are connected at the molecular level. In recent years, amylin, which is co-secreted with insulin, has been proposed for being a main actor in this context due to its capacity to form aggregates in a β-sheet-like structure. In a diabetic milieu, there is an increase in the production and secretion of insulin and amylin. We have analysed the role of resveratrol on aggregate formation and in the production of extracellular vesicles with amylin in its interior and in pancreatic β cells overexpressing human amylin (INS1E-hIAPP). Furthermore, we have explored the consequences of the exposition of the conditioned medium derived from INS1E-hIAPP in the hippocampal cell line HT-22 and the role of resveratrol in this cell line. Hippocampal cells were exposed to conditioned media obtained from rat insulinoma 1E overexpressing human amylin in the presence or in the absence of resveratrol. When we exposed HT-22 cells to the conditioned media of INS1E-hIAPP we observed amylin-aggregates inside HT-22 cells. Resveratrol was able to alleviate this effect not only in HT-22 but also in pancreatic β cells. Furthermore, resveratrol decreased the average exosome size produced by the INS1E-hIAPP stimulated with high glucose, diminishing the toxic effect of these exosomes in HT-22 cells. We have uncovered that resveratrol inhibits the aggregation capacity of amylin and it can diminish the deleterious spreading of the toxic protein, to other cell types such as the hippocampal neuron cells, HT-22., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

A hallmark of Parkinson disease (PD) is a progressive degeneration of neurons in the substantia nigra pars compacta, hypothalamus, and thalamus. Although the exact etiology of irreversible neuronal degeneration is unclear, a growing body of experimental evidence indicates that PD could be triggered by the abrupt aggregation of α-synuclein (α-Syn), a small membrane protein that is responsible for cell vesicle trafficking. Phospholipids uniquely alter the rate of α-Syn aggregation and, consequently, change the cytotoxicity of α-Syn oligomers and fibrils. However, the role of cholesterol in the aggregation of α-Syn remains unclear. In this study, we used Caenorhabditis elegans that overexpressed α-Syn to investigate the effect of low (15%), normal (30%), and high (60%) concentrations of cholesterol on α-Syn aggregation. We found that an increase in the concentration of cholesterol in diets substantially shortened the lifespan of C. elegans . Using biophysical methods, we also investigated the extent to which large unilamellar vesicles (LUVs) with low, normal, and high concentrations of cholesterol altered the rate of α-Syn aggregation. We found that only lipid membranes with a 60% concentration of cholesterol substantially accelerated the rate of protein aggregation. Cell assays revealed that α-Syn fibrils formed in the presence of LUVs with different concentrations of cholesterol exerted very similar levels of cytotoxicity to rat dopaminergic neurons. These results suggest that changes in the concentration of cholesterol in the plasma membrane, which in turn could be caused by nutritional preferences, could accelerate the onset and progression of PD.

Alzheimer's disease (AD) and Parkinson's disease (PD) are multifactorial, chronic diseases involving neurodegeneration. According to recent studies, it is hypothesized that the intraneuronal and postsynaptic accumulation of misfolded proteins such as α-synuclein (α-syn) and tau, responsible for Lewy bodies (LB) and tangles, respectively, disrupts neuron functions. Considering the co-occurrence of α-syn and tau inclusions in the brains of patients afflicted with subtypes of dementia and LB disorders, the discovery and development of small molecules for the inhibition of α-syn and tau aggregation can be a potentially effective strategy to delay neurodegeneration. Urea is a chaotropic agent that alters protein solubilization and hydrophobic interactions and inhibits protein aggregation and precipitation. The presence of three hetero atoms (O/S and N) in proximity can coordinate with neutral, mono, or dianionic groups to form stable complexes in the biological system. Therefore, in this study, we evaluated urea and thiourea linkers with various substitutions on either side of the carbamide or thiocarbamide functionality to compare the aggregation inhibition of α-syn and tau. A thioflavin-T (ThT) fluorescence assay was used to evaluate the level of fibril formation and monitor the anti-aggregation effect of the different compounds. We opted for transmission electron microscopy (TEM) as a direct means to confirm the anti-fibrillar effect. The oligomer formation was monitored via the photoinduced cross-linking of unmodified proteins (PICUP). The anti-inclusion and anti-seeding activities of the best compounds were evaluated using M17D intracellular inclusion and biosensor cell-based assays, respectively. Disaggregation experiments were performed with amyloid plaques extracted from AD brains. The analogues with indole, benzothiazole, or N , N -dimethylphenyl on one side with halo-substituted aromatic moieties had shown less than 15% cutoff fluorescence obtained with the ThT assay. Our lead molecules 6T and 14T reduced α-syn oligomerization dose-dependently based on the PICUP assays but failed at inhibiting tau oligomer formation. The anti-inclusion effect of our lead compounds was confirmed using the M17D neuroblastoma cell model. Compounds 6T and 14T exhibited an anti-seeding effect on tau using biosensor cells. In contrast to the control, disaggregation experiments showed fewer Aβ plaques with our lead molecules (compounds 6T and 14T ). Pharmacokinetics (PK) mice studies demonstrated that these two thiourea-based small molecules have the potential to cross the blood-brain barrier in rodents. Urea and thiourea linkers could be further improved for their PK parameters and studied for the anti-inclusion, anti-seeding, and disaggregation effects using transgenic mice models of neurodegenerative diseases.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that currently lacks effective therapy. Given the heterogeneity of clinical and molecular profiles of ALS patients, personalized diagnostics and pathological characterization represent a powerful strategy to optimize patient stratification, thereby enabling personalized treatment. Immortalized lymphocytes from sporadic and genetic ALS patients recapitulate some pathological hallmarks of the disease, facilitating the fundamental task of drug screening. However, the molecular aggregation of ALS has not been characterized in this patient-derived cellular model. Indeed, protein aggregation is one of the most prominent features of neurodegenerative diseases, and therefore, models to test drugs against personalized pathological aggregation could help discover improved therapies. With this work, we aimed to characterize the aggregation profile of ALS immortalized lymphocytes and test several drug candidates with different mechanisms of action. In addition, we have evaluated the molecular aggregation in motor neurons derived from two hiPSC cell lines corresponding to ALS patients with different mutations in TARDBP . The results provide valuable insight into the different characterization of sporadic and genetic ALS patients' immortalized lymphocytes, their differential response to drug treatment, and the usefulness of proteome homeostasis characterization in patients' cells.

The formation of neurofibrillary tangles (NFTs), composed of tau protein aggregates, is a hallmark of neurodegenerative diseases known as tauopathies, including Alzheimer's disease (AD). NFTs consist of paired helical filaments (PHFs) of tau protein with a dominant β-sheet secondary structure. Within these PHFs, the PHF6 hexapeptide (Val 306 -Gln-Ile-Val-Tyr-Lys 311 ) has been commonly highlighted as a key site for tau protein nucleation. Palmatine chloride (PC) has been identified as an inhibitor of PHF6 aggregation, capable of reducing aggregation propensity at submicromolar concentrations. In pursuit of novel anti-AD drugs targeting early tau aggregation stages, we conducted an in silico study to elucidate PC's mechanism of action during PHF6 aggregation. Our observations suggest that while PHF6 can still initiate self-aggregation in the presence of PC, PC molecules subtly influence PHF6 aggregation dynamics, favoring smaller aggregates over larger complexes. The study underlined the key roles of aromatic rings in PC binding to different PHF6 aggregates by interacting through π-π stacking with the PHF6 Tyr310 side chain. The presence of aromatic rings in compounds to be able to inhibit the earlier complexation phase seems to be essential. These in silico findings lay a foundation for the design of compounds that could intervene in resolving the neurotoxicity of protein aggregates in AD.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the aggregation of α-synuclein into toxic amyloid fibrils. Recent research suggests that bile acids altered in PD may influence their aggregation. This study investigates the effects of lithocholic acid (LCA) and deoxycholic acid (DCA) on α-synuclein aggregation and toxicity. LCA significantly accelerates aggregation, reducing the lag phase by 75%, while DCA has a milder impact, decreasing the lag phase by 30%. Binding studies show that LCA interacts with the NAC region and DCA with the N-terminal region of α-synuclein. Aggregation assays and electrophoresis reveal that LCA promotes the formation of toxic, SDS-resistant oligomers more effectively than DCA. Cytotoxicity assays confirm a lower cell viability in LCA-treated samples. Additionally, combined LCA and DCA treatment results in enhanced aggregation and toxicity, indicating a synergistic effect. These findings highlight the role of bile acids in α-synuclein aggregation and PD pathogenesis, suggesting that targeting bile acid metabolism could be a therapeutic strategy for PD.

It has been shown recently, without an explanation of the possible molecular mechanisms involved, that 4-(2-hydroxyethyl)-1-piperazinepropanesulphonic (EPPS) acid effectively protects from the neurotoxicity induced by oligomers and plaques formed by the protein amyloid-β protein. Here we report the same protective effect, obtained in vitro (HT22-diff cell line) and ex vivo (hippocampal slices) models, against amyloid neurotoxicity induced by oligomers of salmon Calcitonin (sCT), which has been shown to be a good model for the study of neurodegenerative diseases. Based on biophysical studies focusing on the protein aggregation kinetic and the interaction of the aggregates with model membranes, we propose a possible molecular mechanism underlying the protective effects. Taken together, our results indicate that EPPS is able to counteract the direct association (primary aggregation) of harmless low-molecular weight aggregates (dimers and trimers) or their aggregation catalysed by surfaces present in the solution (secondary aggregation). Thus, EPPS stabilizes harmless aggregates and hinders the formation of toxic and metastable prefibrillar oligomers. Overall, our data demonstrate that EPPS is an excellent drug candidate for the treatment of neurodegeneration due to misfolded proteins, such as Alzheimer's or Parkinson's disease., (© 2024. The Author(s).)

This study explores the effect of different theaflavins (TFs) concentrations (0, 100, 300, 600 and 900 mg/L) on the structure, aggregation behavior and gelation properties of pork myofibrillar protein (MP). The protein structure and aggregation behavior were characterized by free sulfhydryl groups, surface hydrophobicity, fluorescence emission spectra, particle size and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The gel properties of samples were characterized by gel strength, cooking loss, microstructure and gel supernatant SDS-PAGE. The results showed a significant decrease in free thiol content with increasing TFs concentration, suggesting thiol-quinone covalent interaction between TFs and thiol group of MP. Intrinsic fluorescence spectroscopy confirmed a static quenching between TFs and MP. And TFs reduced the particle size of MP suspension and caused no protein aggregation bond in SDS-PAGE. For gel properties, TFs caused a decrease of gel strength from 96.77 g to 21.91 g and an increase in cooking loss from 40.34 % to 71.15 %. The bond of protein aggregates in gel supernatants SDS-PAGE revealed that some protein aggregates formed by disulfide bonding were not involve in gel formation with TFs addition. In conclusion, TFs cause thiol loss of MP and impaired MP gelling ability by interfering with disulfide bond formation during gelation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Preventing β-amyloid (Aβ) peptide aggregation by Au nanoparticles (NPs) is a promising strategy for the treatment of Alzheimer's disease. However, construction of Au nanostructures with easy preparation and high therapeutic efficiency is still a challenge. Herein, one-step pulsed laser ablation in water is used to fabricate epigallocatechin-3-gallate (EGCG) modified Au (Au-EGCG) NPs with uniform size. The as-obtained Au-EGCG NPs can effectively inhibit β-amyloid (1-42) peptide (Aβ 42 ) aggregation by the interaction with peptides, which is confirmed by transmission electron microscopy (TEM), fluorescence spectroscopy (thioflavin T (ThT), tyrosine and 8-anilinonaphthalene-1-sulfonic acid (ANS) assays), and Fourier transform infrared (FT-IR) spectroscopy. Besides, they can also effectively attenuate Aβ 42 -induced cytotoxicity based on the cell viability experiments. This work provides a facile approach to synthesize the surface-functionalized Au NPs for enhanced inhibition of Aβ aggregation and amelioration of Aβ-induced cytotoxicity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Parkinson's disease (PD) is primarily characterized by loss of dopaminergic neurons in the substantia nigra pars compacta region of the brain and accumulation of aggregated forms of alpha-synuclein (α-Syn), an intrinsically disordered protein, in the form of Lewy Bodies and Lewy Neurites. Substantial evidences point to the aggregated/fibrillar forms of α-Syn as a central event in PD pathogenesis, underscoring the modulation of α-Syn aggregation as a promising strategy for PD treatment. Consequently, numerous anti-aggregation agents, spanning from small molecules to polymers, have been scrutinized for their potential to mitigate α-Syn aggregation and its associated toxicity. Among these, small molecule modulators like osmoprotectants, polyphenols, cellular metabolites, metals, and peptides have emerged as promising candidates with significant potential in PD management. This article offers a comprehensive overview of the effects of these small molecule modulators on the aggregation propensity and associated toxicity of α-Syn and its PD-associated mutants. It serves as a valuable resource for identifying and developing potent, non-invasive, non-toxic, and highly specific small molecule-based therapeutic arsenal for combating PD. Additionally, it raises pertinent questions aimed at guiding future research endeavours in the field of α-Syn aggregation remodelling., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing financial interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Introduction: As aggregation underpins Tau toxicity, aggregation inhibitor peptides may have disease-modifying potential. They are therefore currently being designed and target either the 306 VQIVYK 311 aggregation-promoting hotspot found in all Tau isoforms or the 275 VQIINK 280 aggregation-promoting hotspot found in 4R isoforms. However, for any Tau aggregation inhibitor to potentially be clinically relevant for other tauopathies, it should target both hotspots to suppress aggregation of Tau isoforms, be stable, cross the blood-brain barrier, and rescue aggregation-dependent Tau phenotypes in vivo., Methods: We developed a retro-inverso, stable D-amino peptide, RI-AG03 [Ac-rrrrrrrrGpkyk(ac)iqvGr-NH2], based on the 306 VQIVYK 311 hotspots which exhibit these disease-relevant attributes., Results: Unlike other aggregation inhibitors, RI-AG03 effectively suppresses aggregation of multiple Tau species containing both hotspots in vitro and in vivo, is non-toxic, and suppresses aggregation-dependent neurodegenerative and behavioral phenotypes., Discussion: RI-AG03 therefore meets many clinically relevant requirements for an anti-aggregation Tau therapeutic and should be explored further for its disease-modifying potential for Tauopathies., Highlights: Our manuscript describes the development of a novel peptide inhibitor of Tau aggregation, a retro-inverso, stable D-amino peptide called RI-AG03 that displays many clinically relevant attributes. We show its efficacy in preventing Tau aggregation in both in vitro and in vivo experimental models while being non-toxic to cells. RI-AG03 also rescues a biosensor cell line that stably expresses Tau repeat domains with the P301S mutation fused to Cer/Clo and rescues aggregation-dependent phenotypes in vivo, suppressing neurodegeneration and extending lifespan. Collectively our data describe several properties and attributes of RI-AG03 that make it a promising disease-modifying candidate to explore for reducing pathogenic Tau aggregation in Tauopathies such as Alzheimer's disease. Given the real interest in reducing Tau aggregation and the potential clinical benefit of using such agents in clinical practice, RI-AG03 should be investigated further for the treatment of Tauopathies after validation in mammalian models. Tau aggregation inhibitors are the obvious first choice as Tau-based therapies as much of Tau-mediated toxicity is aggregation dependent. Indeed, there are many research efforts focusing on this therapeutic strategy with aggregation inhibitors being designed against one of the two aggregation-promoting hotspots of the Tau protein. To our knowledge, RI-AG03 is the only peptide aggregation inhibitor that inhibits aggregation of Tau by targeting both aggregation-promoting hotspot motifs simultaneously. As such, we believe that our study will have a significant impact on drug discovery efforts in this arena., (© 2024 The Author(s). Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

Fibrinogen (Fg), an essential plasma glycoprotein involved in the coagulation cascade, undergoes structural alterations upon exposure to various chemicals, impacting its functionality and contributing to pathological conditions. This research article explored the effects of 4-Chloro-o-phenylenediamine (4-Cl-o-PD), a common hair dye component (IUPAC = 1-Chloro-3,4-diaminobenzene), on human fibrinogen through comprehensive computational, biophysical, and biochemical approaches. The formation of a stable ligand-protein complex is confirmed through molecular docking and molecular dynamics simulations, revealing possible interaction having a favorable -4.8 kcal/mol binding energy. Biophysical results, including UV-vis and fluorescence spectroscopies, corroborated with the computational findings, whereas Fourier transform infrared spectroscopy (FT-IR) and circular dichroism spectroscopy (CD) provide insights into the alterations of secondary structures upon interaction with 4-Cl-o-PD. Anilinonaphthalene-sulfonic acid (ANS) fluorescence showed a partially unfolded protein, with enhanced α to β-sheet transition as evidenced by thioflavin T (ThT) spectroscopy and microscopy. Moreover, biochemical assays confirmed the formation of carbonyl compounds that may be responsible for the oxidation of methionine residues in fibrinogen. Electrophoresis and electron microscopy confirmed the formation of aggregates. Our findings elucidate the interaction pattern of 4-Cl-o-PD with Fg, leading to structural perturbation, which may have potential implications for fibrinogen misfolding or its aggregation. Protein aggregation or its misfolded products affect peripheral tissues and the central nervous system. Many chronic progressive diseases, like type II diabetes mellitus, Alzheimer's disease, Parkison's disease, and Creutzfeldt-Jakob disease are associated with intrinsically aberrant disordered proteins. Understanding these interactions may offer new perspectives on the safety and biocompatibility of dye compounds, which may contribute to developing improved strategies for acquired amyloidogenesis., Competing Interests: Declaration of competing interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Misfolding and aggregation of specific proteins are associated with liquid-liquid phase separation (LLPS), and these protein aggregates can interfere with normal cellular functions and even lead to cell death, possibly affecting gene expression regulation and cell proliferation. Therefore, understanding the role of LLPS in disease may help to identify new mechanisms or therapeutic targets and provide new strategies for disease treatment. There are several ways to disrupt LLPS, including screening small molecules or small molecule drugs to target the upstream signaling pathways that regulate the LLPS process, selectively dissolve and destroy RNA droplets or protein aggregates, regulate the conformation of mutant protein, activate the protein degradation pathway to remove harmful protein aggregates. Furthermore, harnessing the mechanism of LLPS can improve drug development, including preparing different kinds of drug delivery carriers (microneedles, nanodrugs, imprints), regulating drug internalization and penetration behaviors, screening more drugs to overcome drug resistance and enhance receptor signaling. This review initially explores the correlation between aberrant LLPS and disease, highlighting the pivotal role of LLPS in preparing drug development. Ultimately, a comprehensive investigation into drug-mediated regulation of LLPS processes holds significant scientific promise for disease management., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The aggregation of the protein α-synuclein into amyloid deposits is associated with multiple neurological disorders, including Parkinson's disease. Soluble amyloid oligomers are reported to exhibit higher toxicity than insoluble amyloid fibrils, with dimers being the smallest toxic oligomer. Small molecule drugs, such as fasudil, have shown potential in targeting α-synuclein aggregation and reducing its toxicity. In this study, we use atomistic molecular dynamics simulations to demonstrate how fasudil affects the earliest stage of aggregation, namely dimerization. Our results show that the presence of fasudil reduces the propensity for intermolecular contact formation between protein chains. Consistent with previous reports, our analysis confirms that fasudil predominantly interacts with the negatively charged C-terminal region of α-synuclein. However, we also observe transient interactions with residues in the charged N-terminal and hydrophobic NAC regions. Our simulations indicate that while fasudil prominently interacts with the C-terminal region, it is the transient interactions with residues in the N-terminal and NAC regions that effectively block the formation of intermolecular contacts between protein chains and prevent early dimerization of this disordered protein., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Amyloid plaque formation in the brain is mainly responsible for the onset of Alzheimer's disease (AD). Structure-based peptides have gained importance in recent years, and rational design of the peptide sequences for the prevention of Aβ-aggregation and related toxicity is imperative. In this study, we investigate the structural modification of tetrapeptides derived from the hydrophobic C-terminal region of Aβ 42 "VVIA-NH 2 " and its retro-sequence "AIVV-NH 2 ." A preliminary screening of synthesized peptides through an MTT cell viability assay followed by a ThT fluorescence assay revealed a peptide 13 (Ala-Ile-Aib-Val-NH 2 ) that showed protection against Aβ-aggregation and associated neurotoxicity. The presence of the α-helix inducer "Aib" in peptide 13 manifested the conformational transition from cross-β-sheets to α-helical content in Aβ 42 . The absence of fibrils in electron microscopic analysis suggested the inhibitory potential of peptide 13. The HRMS, DLS, and ANS studies further confirmed the inhibitory activity of 13, and no cytotoxicity was observed. The structure-based peptide described herein is a promising amyloid-β inhibitor and provides a new lead for the development of AD therapeutics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Serum amyloid A (SAA) is a small protein consisting of 104 residues and, under physiological conditions, exists mainly in hexameric form. It belongs to the positive acute-phase proteins, which means that its plasma concentration increases rapidly in response to injury, inflammation, and infection. The accumulation of SAA molecules promotes the formation of amyloid aggregates, which deposit extracellularly in many organs, causing their dysfunction. In our previous work, we successfully designed a peptidomimetic that inhibited the aggregation of amyloidogenic SAA fragments. In the present paper, we show how the same inhibitor, named saa3Dip, affects the oligomerization and aggregation processes of MetSAA1.1 protein. The thioflavin T assay showed that saa3Dip inhibited its fibrillization. The measurement of the internal fluorophore fluorescence (Trp) showed differences that occurred in the tertiary structure of MetSAA1.1 in the presence of the inhibitor, which was also confirmed by CD spectra in the aromatic range. FTIR results suggested that saa3Dip could stabilize some fragments of the native structure of MetSAA1.1, which was confirmed by determining the melting temperature (Tm) of the protein-inhibitor complex. AFM images demonstrated that the presence of saa3Dip prevented the formation of large SAA aggregates. Our results suggest that saa3Dip stabilizes the native conformation of MetSAA1.1., Competing Interests: The authors declare no conflict of interest.

The abnormal aggregation of Aβ proteins, inflammatory responses, and mitochondrial dysfunction have been reported as major targets in Alzheimer's disease (AD). Photooxygenation of the amyloid-β peptide (Aβ) is viewed as a promising therapeutic intervention for AD treatment. However, the limitations of the depth of the external light source passing through the brain and the toxic side effects on healthy tissues are two significant challenges in the photooxidation of Aβ aggregates. We proposed a method to initiate the chemical stimulation of Aβ 1-42 aggregate oxidation through H 2 O 2 and correct the abnormal microenvironment of the lesions by eliminating the cascading reactions of oxidative stress. The degradable G-poly(thioacetal) undergoes cascade release of cinnamaldehyde (CA) and thioacetal triggered by endogenous H 2 O 2 , with CA in turn amplifying degradation by generating more H 2 O 2 through mitochondrial dysfunction. A series of novel photosensitizers have been prepared and synthesized for use in the photodynamic oxidation of Aβ 1-42 aggregates under white light activation. The nanoparticles (BD-6-QM/NPs) self-assembled from BD-6-QM, bis[2,4,5-trichloro-6-(pentoxycarbonyl) phenyl] ester (CPPO), and G-poly(thioacetal) not only exhibit H 2 O 2 -stimulated controlled release but also can be chemically triggered by H 2 O 2 to generate singlet oxygen to inhibit Aβ 1-42 aggregates, reducing the Aβ 1-42 -induced neurotoxicity.

Triosephosphate isomerase (TPI) dysfunction is a critical factor in diverse pathological conditions. Deficiencies in TPI lead to the accumulation of toxic methylglyoxal (MGO), which induces non-enzymatic post-translational modifications, thus compromising protein stability and leading to misfolding. This study investigates how specific TPI mutations (E104D, N16D, and C217K) affect the enzyme's structural stability when exposed to its substrate glyceraldehyde 3-phosphate (G3P) and MGO. We employed circular dichroism, intrinsic fluorescence, native gel electrophoresis, and Western blotting to assess the structural alterations and aggregation propensity of these TPI mutants. Our findings indicate that these mutations markedly increase TPI's susceptibility to MGO-induced damage, leading to accelerated loss of enzymatic activity and enhanced protein aggregation. Additionally, we observed the formation of MGO-induced adducts, such as argpyrimidine (ARGp), that contribute to enzyme inactivation and aggregation. Importantly, the application of MGO-scavenging molecules partially mitigated these deleterious effects, highlighting potential therapeutic strategies to counteract MGO-induced damage in TPI-related disorders.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder, characterized by the accumulation of amyloid-beta (Aβ) plaques and neuroinflammation. This study investigates the protective effects of noradrenaline (NA) on human microglial cells exposed to lipopolysaccharides (LPS) and Aβ aggregates-major contributors to inflammation and cellular damage in AD. The reduced Aβ aggregation in the HMC3 human microglial cells co-treated with Aβ and NA was confirmed by thioflavin T (ThT) assay, fluorescent ThT staining, and immunocytochemistry (ICC). The significantly increased viability of HMC3 cells after 48 h of incubation with NA at 50 µM, 25 µM, and 10 µM, exposed to IC50 LPS and IC50 Aβ, was confirmed by XTT and LDH assays. Moreover, we found that NA treatment at 25 μM and 50 μM concentrations in HMC3 cells exposed to IC50 LPS or IC50 Aβ results in an increased proliferation of HMC3 cells, their return to normal morphology, decreased levels of DNA damage, reduced caspase-3 activity, decreased expression of pro-apoptotic DDIT3 and BAX, and increased expression of anti-apoptotic BCL-2 genes and proteins, leading to enhanced cell survival, when compared to that of the HMC3 cells treated only with IC50 LPS or IC50 Aβ. Furthermore, we showed that NA induces the degradation of both extracellular and intracellular Aβ deposits and downregulates hypoxia-inducible factor 1α (HIF-1α), which is linked to impaired Aβ clearance and AD progression. These findings indicate that NA holds promise as a therapeutic target to address microglial dysfunction and potentially slow the progression of AD. Its neuroprotective effects, particularly in reducing inflammation and regulating microglial activity, warrant further investigation into its broader role in mitigating neuroinflammation and preserving microglial function in AD.

Fused in sarcoma (FUS), a multifunctional deoxyribonucleic acid (DNA)/ribonucleic acid (RNA)-binding protein, has been implicated in various cancer types, including sarcoma and leukemia. Despite its association with these diseases, there has been limited exploration of FUS as a cancer therapy target, primarily because its dynamic nature makes it difficult to target specifically. In this study, we explored a kind of β-sheet peptide foldamer, named β 4 -TAT, to influence FUS aggregation by targeting its RNA recognition motifs (RRM). This approach leverages the noncovalent interaction characteristics of peptide self-assembly processes. The β 4 sequence, derived from the FUS RRM β-sheet, in combination with TAT, a peptide known for its nuclear targeting capability, enables β 4 -TAT to bind specifically to the analogous β 4 sequence within FUS. Notably, β 4 -TAT effectively induces FUS aggregation within cells, leading to the death of cancer cells. Our work developed a novel peptide foldamer-based strategy for inducing protein aggregation, paving the way for innovative therapeutic approaches in targeting FUS-associated cancers.

Amyloid β aggregation is an important factor in Alzheimer's disease. Since calcium homeostasis plays an important role in amyloid β aggregation, it is crucial to study the interaction between calcium ions and amyloid β directly at the surface of the lipid membrane. With supercritical angle techniques, the signal of interest at the surface is easily separated from the bulk solution, making them a powerful tool for aggregation study. In this work, the influence of calcium ions on amyloid β aggregation over different aggregation time periods is investigated with supercritical angle Raman and fluorescence spectroscopy and microscopy. Note that calcium ions have a larger influence on amyloid β 1-42 than on the 40 amino acid variant. We found that a small layer of calcium ions significantly protects the lipid membrane against the protein insertion process.

Human calcitonin (hCT) is an endogenous polypeptide commonly employed in treating bone resorption-related illnesses, but its clinical application is limited due to its high aggregation tendency. Metalloporphyrins are effective in suppressing amyloid fibrillation, positioning them as potential drug candidates for amyloidogenic disorders like Alzheimer's and type 2 diabetes. In this work, we investigated the effects of Fe(III) meso -tetra(4-sulfonatophenyl)porphine chloride (FeTPPS), a highly efficient ONOO - decomposition catalyst, on hCT aggregation. Our findings reveal that FeTPPS effectively precludes hCT fibrillation by stabilizing the monomers and delaying the structural transition from α-helix bundles to β-sheet-rich aggregates. The macrocyclic ring of FeTPPS plays a significant role in disrupting hCT self-associations. Among various porphyrin analogs, those with an iron center and negatively charged peripheral substituents exhibit a stronger inhibitory effect on hCT aggregation. Spectroscopic analyses and computational simulations indicate that FeTPPS binds to hCT's core aggregation region via complexation with His20 in a 1 : 1 molar ratio. Hydrophobic interaction, hydrogen bonding, and π-π stacking with the residues involving Tyr12, Phe19, and Ala26 also contribute to the interactions. Collectively, our study provides a promising approach for developing novel hCT drug formulations and offers theoretical guidance for designing metalloporphyrin-based inhibitors for various amyloidosis conditions.

Protein aggregation is associated with various diseases caused by protein misfolding. Among them, amylin deposition is a prominent feature of type 2 diabetes. At present, the mechanism of amylin aggregation remains unclear, and this has hindered the treatment of type 2 diabetes. In this study, we analyzed the aggregation process of amylin using the quantum dot (QD) imaging method. QD fluorescence imaging revealed that in the presence of 100 μM amylin, aggregates appeared after 12 h of incubation, while a large number of aggregates formed after 24 h of incubation, with a standard deviation (SD) value of 5.435. In contrast, 50 μM amylin did not induce the formation of aggregates after 12 h of incubation, although a large number of aggregates were observed after 24 h of incubation, with an SD value of 2.883. Confocal laser microscopy observations revealed that these aggregates were deposited in three dimensions. Transmission electron microscopy revealed that amylin existed as misfolded fibrils in vitro and that QDs were uniformly bound to the amylin fibrils. In addition, using a microliter-scale high-throughput screening (MSHTS) system, we found that rosmarinic acid, a polyphenol, inhibited amylin aggregation at a half-maximal effective concentration of 852.8 μM. These results demonstrate that the MSHTS system is a powerful tool for evaluating the inhibitory activity of amylin aggregation. Our findings will contribute to the understanding of the pathogenesis of amylin-related diseases and the discovery of compounds that may be useful in the treatment and prevention of these diseases.