How ATP suppresses the fibrillation of amyloid peptides: analysis of the free-energy contributions.

Recent experiments have revealed that adenosine triphosphate (ATP) suppresses the fibrillation of amyloid peptides - a process closely linked to neurodegenerative diseases such as Alzheimer's and Parkinson's. Apart from the adsorption of ATP onto amyloid peptides, the molecular understanding is still limited, leaving the underlying mechanism for the fibrillation suppression by ATP largely unclear, especially in regards to the molecular energetics. Here we provide an explanation at the molecular scale by quantifying the free energies using all-atom molecular dynamics simulations. We found that the changes of the free energies due to the addition of ATP lead to a significant equilibrium shift towards monomeric peptides in agreement with experiments. Despite ATP being a highly charged species, the decomposition of the free energies reveals that the van der Waals interactions with the peptide are decisive in determining the relative stabilization of the monomeric state. While the phosphate moiety exhibits strong electrostatic interactions, the compensation by the water solvent results in a minor, overall Coulomb contribution. Our quantitative analysis of the free energies identifies which intermolecular interactions are responsible for the suppression of the amyloid fibril formation by ATP and offers a promising method to analyze the roles of similarly complex cosolvents in aggregation processes.

Specific Binding of Alzheimer's Aß Peptides to Extracellular Vesicles.

Alzheimer's disease (AD) is the fifth leading cause of death among adults aged 65 and older, yet the onset and progression of the disease is poorly understood. What is known is that the presence of amyloid, particularly polymerized Aß42, defines when people are on the AD continuum. Interestingly, as AD progresses, less Aß42 is detectable in the plasma, a phenomenon thought to result from Aß becoming more aggregated in the brain and less Aß42 and Aß40 being transported from the brain to the plasma via the CSF. We propose that extracellular vesicles (EVs) play a role in this transport. EVs are found in bodily fluids such as blood, urine, and cerebrospinal fluid and carry diverse "cargos" of bioactive molecules (e.g., proteins, nucleic acids, lipids, metabolites) that dynamically reflect changes in the cells from which they are secreted. While Aß42 and Aß40 have been reported to be present in EVs, it is not known whether this interaction is specific for these peptides and thus whether amyloid-carrying EVs play a role in AD and/or serve as brain-specific biomarkers of the AD process. To determine if there is a specific interaction between Aß and EVs, we used isothermal titration calorimetry (ITC) and discovered that Aß42 and Aß40 bind to EVs in a manner that is sequence specific, saturable, and endothermic. In addition, Aß incubation with EVs overnight yielded larger amounts of bound Aß peptide that was fibrillar in structure. These findings point to a specific amyloid-EV interaction, a potential role for EVs in the transport of amyloid from the brain to the blood, and a role for this amyloid pool in the AD process.

Alzheimer's Amyloid Hypothesis and Antibody Therapy: Melting Glaciers?

The amyloid cascade hypothesis for Alzheimer's disease is still alive, although heavily challenged. Effective anti-amyloid immunotherapy would confirm the hypothesis' claim that the protein amyloid-beta is the cause of the disease. Two antibodies, aducanumab and lecanemab, have been approved by the U.S. Food and Drug Administration, while a third, donanemab, is under review. The main argument for the FDA approvals is a presumed therapy-induced removal of cerebral amyloid deposits. Lecanemab and donanemab are also thought to cause some statistical delay in the determination of cognitive decline. However, clinical efficacy that is less than with conventional treatment, selection of amyloid-positive trial patients with non-specific amyloid-PET imaging, and uncertain therapy-induced removal of cerebral amyloids in clinical trials cast doubt on this anti-Alzheimer's antibody therapy and hence on the amyloid hypothesis, calling for a more thorough investigation of the negative impact of this type of therapy on the brain.

Amyloid-Forming Corpora Amylacea and Spheroid-Type Amyloid Deposition: Comprehensive Analysis Using Immunohistochemistry, Proteomics, and a Literature Review.

This study aimed to elucidate the similarities and differences between amyloid-forming corpora amylacea (CA) in the prostate and lung, examine the nature of CAs in cystic tumors of the atrioventricular node (CTAVN), and clarify the distinctions between amyloid-forming CA and spheroid-type amyloid deposition. We conducted proteomics analyses using liquid chromatography-tandem mass spectrometry with laser microdissection and immunohistochemistry to validate the characteristics of CAs in the lung and prostate. Our findings revealed that the CAs in these organs primarily consisted of common proteins (ß2-microglobulin and lysozyme) and locally produced proteins. Moreover, we observed a discrepancy between the histopathological and proteomic analysis results in CTAVN-associated CAs. In addition, while the histopathological appearance of the amyloid-forming CAs and spheroid-type amyloid deposits were nearly identical, the latter deposition lacked ß2-microglobulin and lysozyme and exhibited evident destruction of the surrounding tissue. A literature review further supported these findings. These results suggest that amyloid-forming CAs in the lung and prostate are formed through a shared mechanism, serving as waste containers (wasteosomes) and/or storage for excess proteins (functional amyloids). In contrast, we hypothesize that while amyloid-forming CA and spheroid-type amyloid deposits are formed, in part, through common mechanisms, the latter are pathological.

Amyloid Typing in Cardiac Amyloidosis Using Western Blotting.

BACKGROUND: Cardiac amyloidosis (CA) is characterized by the extracellular deposition of misfolded protein in the heart. Precise identification of the amyloid type is often challenging, but critical, since the treatment and prognosis depend on the disease form and the type of deposited amyloid. Coexistence of clinical conditions such as old age, monoclonal gammopathy, chronic inflammation, or peripheral neuropathy in a patient with cardiomyopathy creates a differential diagnosis between the major types of CA: amyloidosis light chains (AL), amyloidosis transthyretin (ATTR) and amyloidosis A (AA). OBJECTIVES: To demonstrate the utility of the Western blotting (WB)-based amyloid typing method in patients diagnosed with cardiac amyloidosis where the type of amyloid was not obvious based on the clinical context. METHODS: Congo red positive endomyocardial biopsy specimens were studied in patients where the type of amyloid was uncertain. Amyloid proteins were extracted and identified by WB. Mass spectrometry (MS) of the electrophoretically resolved protein-in-gel bands was used for confirmation of WB data. RESULTS: WB analysis allowed differentiation between AL, AA, and ATTR in cardiac biopsies based on specific immunoreactivity of the electrophoretically separated proteins and their characteristic molecular weight. The obtained results were confirmed by MS. CONCLUSIONS: WB-based amyloid typing method is cheaper and more readily available than the complex and expensive gold standard techniques such as MS analysis or immunoelectron microscopy. Notably, it is more sensitive and specific than the commonly used immunohistochemical techniques and may provide an accessible diagnostic service to patients with amyloidosis in Israel.

Histological Typing in Patients With Cardiac Amyloidosis: JACC Review Topic of the Week.

Cardiac amyloidosis is increasingly recognized as a treatable form of heart failure. Highly effective specific therapies have recently become available for the 2 most frequent forms of cardiac amyloidosis: immunoglobulin light chain amyloidosis and transthyretin (ATTR) amyloidosis. Nevertheless, initiation of specific therapies requires recognition of cardiac amyloidosis and appropriate characterization of the amyloid type. Although noninvasive diagnosis is possible for ATTR cardiac amyloidosis, histological demonstration and typing of amyloid deposits is still required for a substantial number of patients with ATTR and in all patients with light chain amyloidosis and other rarer forms of cardiac amyloidosis. Amyloid histological typing can be performed using different techniques: mass spectrometry, immunohistochemistry, and immunoelectron microscopy. This review describes which patients require histological confirmation of cardiac amyloidosis along with when and how to type amyloid deposits in histologic specimens. Furthermore, it covers the characteristics and limitations of the different typing methods that are available in clinical practice.

Low-Dose Radiation Therapy Impacts Microglial Inflammatory Response without Modulating Amyloid Load in Female TgF344-AD Rats.

Background: Low-dose radiation therapy (LD-RT) has demonstrated in preclinical and clinical studies interesting properties in the perspective of targeting Alzheimer's disease (AD), including anti-amyloid and anti-inflammatory effects. Nevertheless, studies were highly heterogenous with respect to total doses, fractionation protocols, sex, age at the time of treatment and delay post treatment. Recently, we demonstrated that LD-RT reduced amyloid peptides and inflammatory markers in 9-month-old TgF344-AD (TgAD) males. Objective: As multiple studies demonstrated a sex effect in AD, we wanted to validate that LD-RT benefits are also observed in TgAD females analyzed at the same age. Methods: Females were bilaterally treated with 2 Gy×5 daily fractions, 2 Gy×5 weekly fractions, or 10 fractions of 1 Gy delivered twice a week. The effect of each treatment on amyloid load and inflammation was evaluated using immunohistology and biochemistry. Results: A daily treatment did not affect amyloid and reduced only microglial-mediated inflammation markers, the opposite of the results obtained in our previous male study. Moreover, altered fractionations (2 Gy×5 weekly fractions or 10 fractions of 1 Gy delivered twice a week) did not influence the amyloid load or neuroinflammatory response in females. Conclusions: A daily treatment consequently appears to be the most efficient for AD. This study also shows that the anti-amyloid and anti-inflammatory response to LD-RT are, at least partly, two distinct mechanisms. It also emphasizes the necessity to assess the sex impact when evaluating responses in ongoing pilot clinical trials testing LD-RT against AD.

gp120-derived amyloidogenic peptides form amyloid fibrils that increase HIV-1 infectivity.

Apart from mediating viral entry, the function of the free HIV-1 envelope protein (gp120) has yet to be elucidated. Our group previously showed that EP2 derived from one ß-strand in gp120 can form amyloid fibrils that increase HIV-1 infectivity. Importantly, gp120 contains ~30 ß-strands. We examined whether gp120 might serve as a precursor protein for the proteolytic release of amyloidogenic fragments that form amyloid fibrils, thereby promoting viral infection. Peptide array scanning, enzyme degradation assays, and viral infection experiments in vitro confirmed that many ß-stranded peptides derived from gp120 can indeed form amyloid fibrils that increase HIV-1 infectivity. These gp120-derived amyloidogenic peptides, or GAPs, which were confirmed to form amyloid fibrils, were termed gp120-derived enhancers of viral infection (GEVIs). GEVIs specifically capture HIV-1 virions and promote their attachment to target cells, thereby increasing HIV-1 infectivity. Different GAPs can cross-interact to form heterogeneous fibrils that retain the ability to increase HIV-1 infectivity. GEVIs even suppressed the antiviral activity of a panel of antiretroviral agents. Notably, endogenous GAPs and GEVIs were found in the lymphatic fluid, lymph nodes, and cerebrospinal fluid (CSF) of AIDS patients in vivo. Overall, gp120-derived amyloid fibrils might play a crucial role in the process of HIV-1 infectivity and thus represent novel targets for anti-HIV therapeutics.

Amyloid-induced neurodegeneration: A comprehensive review through aggregomics perception of proteins in health and pathology.

Amyloidosis of protein caused by fibrillation and aggregation are some of the most exciting new edges not only in protein sciences but also in molecular medicines. The present review discusses recent advancements in the field of neurodegenerative diseases and therapeutic applications with ongoing clinical trials, featuring new areas of protein misfolding resulting in aggregation. The endogenous accretion of protein fibrils having fibrillar morphology symbolizes the beginning of neuro-disorders. Prognostic amyloidosis is prominent in numerous degenerative infections such as Alzheimer's and Parkinson's disease, Amyotrophic lateral sclerosis (ALS), etc. However, the molecular basis determining the intracellular or extracellular evidence of aggregates, playing a significant role as a causative factor in neurodegeneration is still unclear. Structural conversions and protein self-assembly resulting in the formation of amyloid oligomers and fibrils are important events in the pathophysiology of the disease. This comprehensive review sheds light on the evolving landscape of potential treatment modalities, highlighting the ongoing clinical trials and the potential socio-economic impact of novel therapeutic interventions in the realm of neurodegenerative diseases. Furthermore, many drugs are undergoing different levels of clinical trials that would certainly help in treating these disorders and will surely improve the socio-impact of human life.

Structures and Dynamics of ß-Rich Oligomers of ATTR (105-115) Assembly.

Transthyretin (TTR) is a tetrameric homologous protein that can dissociate into monomers. Misfolding and aggregation of TTR can lead to amyloid transthyretin amyloidosis (ATTR), which can cause many diseases (e.g., senile systemic amyloidosis, familial amyloid cardiomyopathy, and familial amyloid polyneuropathy). Despite growing evidence indicating that small oligomers play a critical role in regulating cytotoxicity, the structures of these oligomeric intermediates and their conformational transformations are still unclear, impeding our understanding of neurodegenerative mechanisms and the development of therapeutics targeting early aggregation species. The TTR monomer protein consists of various fragments prone to self-aggregation, including the residue 105-115 sequence. Therefore, our study investigated the assembly progress of ATTR (105-115) peptides using all-atom molecular dynamics simulations. The findings indicate that the probability of ß-sheet content increases with increasing numbers of peptides. Additionally, interactions between hydrophobic residues L110 and L111 are crucial for the formation of a ß-rich oligomer formation. These ß-rich oligomers may adopt ß-barrel conformations, potentially toxic oligomer species. Free-energy analysis reveals that ß-barrel conformations serve as intermediates for these ß-rich oligomers. Our insights into the structural ensemble dynamics of ATTR (105-115) contribute to understanding the physical mechanisms underlying the ß-barrel oligomers of ATTR. These findings may shed light on the pathological role of ATTR in neurodegenerative diseases and offer potential therapeutic targets.

Investigating a Novel Neurodegenerative Disease Toxic Mechanism Involving Lipid Binding Specificity of Amyloid Oligomers.

Exploring the mechanisms underlying the toxicity of amyloid oligomers (AOs) presents a significant opportunity for discovering cures and developing treatments for neurodegenerative diseases. Recently, using a combination of ion mobility spectrometry-mass spectrometry (IMS-MS) and X-ray crystallography (XRC), we showed that the peptide KVKVLWDVIEV, which is the G95W mutant of αB-Crystallin (90-100) and abbreviated as G6W, self-assembles up to a dodecamer that structurally resembles lipid transport proteins. The glycine to tryptophan mutation promotes not only larger oligomers and enhanced cytotoxicity in brain slices than the wild type but also a narrow hydrophobic cavity suitable for fatty acid or phospholipid binding. Here, we determine the plausibility of a novel cytotoxic mechanism where the G6W's structural motif could perturb lipid homeostasis by determining its lipid binding selectivity and specificity. We show that the G6W oligomers have a strong affinity toward unsaturated phospholipids with a preference toward phospholipids containing 16-C alkyl chains. Molecular dynamics simulations demonstrate how an unsaturated, 16-C phospholipid fits tightly inside and outside G6W's hydrophobic cavity. This binding is exclusive to the G6W peptide, as other amyloid oligomers with different atomic structures, including its wildtype αB-Crystallin (90-100) and several superoxide dismutase 1 (SOD1) peptides that are known to self-assemble into amyloid oligomers (SOD1P28K and SOD1WG-GW), do not experience the same strong binding affinity. While the existing chaperone-lipid hypothesis on amyloid toxicity suggests amyloid-lipid complexes perforate cell membranes, our work provides a new outlook, indicating that soluble amyloid oligomers disrupt lipid homeostasis via selective protein-ligand interactions. The toxic mechanisms may arise from the formation of unique amyloid oligomer structures assisted by lipid ligands or impaired lipid transports.

Metal Ions Can Modulate the Self-Assembly and Activity of Catalytic Peptide Amyloids.

Rational design of peptides has become a powerful tool to produce self-assembled nanostructures with the ability to catalyze different chemical reactions, paving the way to develop minimalistic enzyme-like nanomaterials. Catalytic amyloid-like assemblies have emerged among the most versatile and active, but they often require additional factors for activity. Elucidating how these factors influence the structure and activity is key for the design. Here, we showed that biologically relevant metal ions can guide and modulate the self-assembly of a small peptide into diverse amyloid architectures. The morphology and catalytic activity of the resulting fibrils were tuned by the specific metal ion decorating the surface, whereas X-ray structural analysis of the amyloids showed ion-dependent shape sizes. Molecular dynamics simulations showed that the metals can strongly affect the local conformational space, which can trigger major rearrangements of the fibrils. Our results demonstrate that the conformational landscape of catalytic amyloids is broad and tunable by external factors, which can be critical for future design strategies.

AMYGNN: A Graph Convolutional Neural Network-Based Approach for Predicting Amyloid Formation from Polypeptides.

There has been an increasing interest in the use of amyloids for constructing various functional materials. The design of amyloid-associated functional materials requires the identification of the core peptide sequences as the fundamental building block. The existing computational methods are limited in terms of delineating polypeptides, the typical non-Euclidean structural data, and they fail to capture the dynamic interactions between amino acids due to ignoring the contextual information from surrounding amino acids. Here, we first propose the use of a state-of-the-art graph convolutional neural network for predicting the trends of amyloid formation from specific peptide sequences (AMYGNN) by abstracting each polypeptide as a graph, in which the constituting amino acids are viewed as nodes and edges characterizing the connections between pairs of amino acids are established when they meet a given distance threshold (Cα-Cα ≤ 5 Å). Our model achieves high performance with accuracy (0.9208), G-mean (0.9203), MCC (0.8417), and F1 (0.9235) in determining the characteristic peptide sequences to form amyloid. 32 of 534 crucial amino acid properties that greatly contribute to the formation of amyloids are ascertained, and the ß-folding-like graph structure of a polypeptide is believed to be essential for the formation of amyloid. Our model enables the mapping of polypeptides with underlying interactions between amino acids and provides a quick and precise predictive framework for directing the construction of amyloid-associated functional materials.

Sodium oligomannate alters gut microbiota, reduces cerebral amyloidosis and reactive microglia in a sex-specific manner.

It has recently become well-established that there is a connection between Alzheimer's disease pathology and gut microbiome dysbiosis. We have previously demonstrated that antibiotic-mediated gut microbiota perturbations lead to attenuation of Aß deposition, phosphorylated tau accumulation, and disease-associated glial cell phenotypes in a sex-dependent manner. In this regard, we were intrigued by the finding that a marine-derived oligosaccharide, GV-971, was reported to alter gut microbiota and reduce Aß amyloidosis in the 5XFAD mouse model that were treated at a point when Aß burden was near plateau levels. Utilizing comparable methodologies, but with distinct technical and temporal features, we now report on the impact of GV-971 on gut microbiota, Aß amyloidosis and microglial phenotypes in the APPPS1-21 model, studies performed at the University of Chicago, and independently in the 5X FAD model, studies performed at Washington University, St. Louis.Methods To comprehensively characterize the effects of GV-971 on the microbiota-microglia-amyloid axis, we conducted two separate investigations at independent institutions. There was no coordination of the experimental design or execution between the two laboratories. Indeed, the two laboratories were not aware of each other's experiments until the studies were completed. Male and female APPPS1-21 mice were treated daily with 40, 80, or 160 mg/kg of GV-971 from 8, when Aß burden was detectable upto 12 weeks of age when Aß burden was near maximal levels. In parallel, and to corroborate existing published studies and further investigate sex-related differences, male and female 5XFAD mice were treated daily with 100 mg/kg of GV-971 from 7 to 9 months of age when Aß burden was near peak levels. Subsequently, the two laboratories independently assessed amyloid-ß deposition, metagenomic, and neuroinflammatory profiles. Finally, studies were initiated at the University of Chicago to evaluate the metabolites in cecal tissue from vehicle and GV-971-treated 5XFAD mice.Results These studies showed that independent of the procedural differences (dosage, timing and duration of treatment) between the two laboratories, cerebral amyloidosis was reduced primarily in male mice, independent of strain. We also observed sex-specific microbiota differences following GV-971 treatment. Interestingly, GV-971 significantly altered multiple overlapping bacterial species at both institutions. Moreover, we discovered that GV-971 significantly impacted microbiome metabolism, particularly by elevating amino acid production and influencing the tryptophan pathway. The metagenomics and metabolomics changes correspond with notable reductions in peripheral pro-inflammatory cytokine and chemokine profiles. Furthermore, GV-971 treatment dampened astrocyte and microglia activation, significantly decreasing plaque-associated reactive microglia while concurrently increasing homeostatic microglia only in male mice. Bulk RNAseq analysis unveiled sex-specific changes in cerebral cortex transcriptome profiles, but most importantly, the transcriptome changes in the GV-971-treated male group revealed the involvement of microglia and inflammatory responses.Conclusions In conclusion, these studies demonstrate the connection between the gut microbiome, neuroinflammation, and Alzheimer's disease pathology while highlighting the potential therapeutic effect of GV-971. GV-971 targets the microbiota-microglia-amyloid axis, leading to the lowering of plaque pathology and neuroinflammatory signatures in a sex-dependent manner when given at the onset of Aß deposition or when given after Aß deposition is already at higher levels.

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.

Re-Engineering Therapeutic Anti-Aß Monoclonal Antibody to Target Amyloid Light Chain.

Amyloidosis involves the deposition of misfolded proteins. Even though it is caused by different pathogenic mechanisms, in aggregate, it shares similar features. Here, we tested and confirmed a hypothesis that an amyloid antibody can be engineered by a few mutations to target a different species. Amyloid light chain (AL) and ß-amyloid peptide (Aß) are two therapeutic targets that are implicated in amyloid light chain amyloidosis and Alzheimer's disease, respectively. Though crenezumab, an anti-Aß antibody, is currently unsuccessful, we chose it as a model to computationally design and prepare crenezumab variants, aiming to discover a novel antibody with high affinity to AL fibrils and to establish a technology platform for repurposing amyloid monoclonal antibodies. We successfully re-engineered crenezumab to bind both Aß42 oligomers and AL fibrils with high binding affinities. It is capable of reversing Aß42-oligomers-induced cytotoxicity, decreasing the formation of AL fibrils, and alleviating AL-fibrils-induced cytotoxicity in vitro. Our research demonstrated that an amyloid antibody could be engineered by a few mutations to bind new amyloid sequences, providing an efficient way to reposition a therapeutic antibody to target different amyloid diseases.

On the importance of being amidated: Analysis of the role of the conserved C-terminal amide of amylin in amyloid formation and cytotoxicity.

The polypeptide hormone Amylin (also known as islet amyloid polypeptide) plays a role in regulation of glucose metabolism, but forms pancreatic islet amyloid deposits in type 2 diabetes. The process of islet amyloid formation contributes to ß-cell dysfunction and the development of the disease. Amylin is produced as a pro-from and undergoes processing prior to secretion. The mature hormone contains an amidated C-terminus. Analysis of an alignment of vertebrate amylin sequences reveals that the processing signal for amidation is strictly conserved. Furthermore, the enzyme responsible for C-terminal amidation is found in all of these organisms. Comparison of the physiologically relevant amidated form to a variant with a free C-terminus (Amylin-COO-) shows that replacement of the C-terminal amide with a carboxylate slows, but does not prevent amyloid formation. Pre-fibrillar species produced by both variants are toxic to cultured ß-cells, although hAmylin-COO- is moderately less so. Amyloid fibrils produced by either peptide are not toxic. Prior work (ACS Pharmacol. Translational. Sci. 1, 132-49 (2018)) shows that Amylin- COO- exhibits a 58-fold reduction in activation of the Amylin1 receptor and 20-fold reduction in activation of the Amylin3 receptor. Thus, hAmylin-COO- exhibits significant toxicity, but significantly reduced activity and offers a reagent for studies which aim to decouple hAmylin's toxic effects from its activity. The different behaviours of free and C-terminal amidated Amylin should be considered when designing systems to produce the polypeptide recombinantly.

Early Diagnosis of Neurodegenerative Diseases: What Has Been Undertaken to Promote the Transition from PET to Fluorescence Tracers.

Alzheimer's Disease (AD) and Parkinson's Disease (PD) represent two among the most frequent neurodegenerative diseases worldwide. A common hallmark of these pathologies is the misfolding and consequent aggregation of amyloid proteins into soluble oligomers and insoluble ß-sheet-rich fibrils, which ultimately lead to neurotoxicity and cell death. After a hundred years of research on the subject, this is the only reliable histopathological feature in our hands. Since AD and PD are diagnosed only once neuronal death and the first symptoms have appeared, the early detection of these diseases is currently impossible. At present, there is no effective drug available, and patients are left with symptomatic and inconclusive therapies. Several reasons could be associated with the lack of effective therapeutic treatments. One of the most important factors is the lack of selective probes capable of detecting, as early as possible, the most toxic amyloid species involved in the onset of these pathologies. In this regard, chemical probes able to detect and distinguish among different amyloid aggregates are urgently needed. In this article, we will review and put into perspective results from ex vivo and in vivo studies performed on compounds specifically interacting with such early species. Following a general overview on the three different amyloid proteins leading to insoluble ß-sheet-rich amyloid deposits (amyloid ß1-42 peptide, Tau, and α-synuclein), a list of the advantages and disadvantages of the approaches employed to date is discussed, with particular attention paid to the translation of fluorescence imaging into clinical applications. Furthermore, we also discuss how the progress achieved in detecting the amyloids of one neurodegenerative disease could be leveraged for research into another amyloidosis. As evidenced by a critical analysis of the state of the art, substantial work still needs to be conducted. Indeed, the early diagnosis of neurodegenerative diseases is a priority, and we believe that this review could be a useful tool for better investigating this field.

The Inhibition Effect of Epigallocatechin-3-Gallate on the Co-Aggregation of Amyloid-ß and Human Islet Amyloid Polypeptide Revealed by Replica Exchange Molecular Dynamics Simulations.

Alzheimer's disease and Type 2 diabetes are two epidemiologically linked diseases which are closely associated with the misfolding and aggregation of amyloid proteins amyloid-ß (Aß) and human islet amyloid polypeptide (hIAPP), respectively. The co-aggregation of the two amyloid proteins is regarded as the fundamental molecular mechanism underlying their pathological association. The green tea extract epigallocatechin-3-gallate (EGCG) has been extensively demonstrated to inhibit the amyloid aggregation of Aß and hIAPP proteins. However, its potential role in amyloid co-aggregation has not been thoroughly investigated. In this study, we employed the enhanced-sampling replica exchange molecular dynamics simulation (REMD) method to investigate the effect of EGCG on the co-aggregation of Aß and hIAPP. We found that EGCG molecules substantially diminish the ß-sheet structures within the amyloid core regions of Aß and hIAPP in their co-aggregates. Through hydrogen-bond, π-π and cation-π interactions targeting polar and aromatic residues of Aß and hIAPP, EGCG effectively attenuates both inter-chain and intra-chain interactions within the co-aggregates. All these findings indicated that EGCG can effectively inhibit the co-aggregation of Aß and hIAPP. Our study expands the potential applications of EGCG as an anti-amyloidosis agent and provides therapeutic options for the pathological association of amyloid misfolding disorders.

Structure of Amyloid Peptide Ribbons Characterized by Electron Microscopy, Atomic Force Microscopy, and Solid-State Nuclear Magnetic Resonance.

Polypeptides often self-assemble to form amyloid fibrils, which contain cross-ß structural motifs and are typically 5-15 nm in width and micrometers in length. In many cases, short segments of longer amyloid-forming protein or peptide sequences also form cross-ß assemblies but with distinctive ribbon-like morphologies that are characterized by a well-defined thickness (on the order of 5 nm) in one lateral dimension and a variable width (typically 10-100 nm) in the other. Here, we use a novel combination of data from solid-state nuclear magnetic resonance (ssNMR), dark-field transmission electron microscopy (TEM), atomic force microscopy (AFM), and cryogenic electron microscopy (cryoEM) to investigate the structures within amyloid ribbons formed by residues 14-23 and residues 11-25 of the Alzheimer's disease-associated amyloid-ß peptide (Aß14-23 and Aß11-25). The ssNMR data indicate antiparallel ß-sheets with specific registries of intermolecular hydrogen bonds. Mass-per-area values are derived from dark-field TEM data. The ribbon thickness is determined from AFM images. For Aß14-23 ribbons, averaged cryoEM images show a periodic spacing of ß-sheets. The combined data support structures in which the amyloid ribbon growth direction is the direction of intermolecular hydrogen bonds between ß-strands, the ribbon thickness corresponds to the width of one ß-sheet (i.e., approximately the length of one molecule), and the variable ribbon width is a variable multiple of the thickness of one ß-sheet (i.e., a multiple of the repeat distance in a stack of ß-sheets). This architecture for a cross-ß assembly may generally exist within amyloid ribbons.