• Aggregation kinetics
• Amyloid toxicity
• Biophysics
• Cellular interactions
• Diagnostics
• Drug discovery
• Fibril fragmentation
• Lecanemab
• Protofibrils
• Secondary nucleation
• Therapeutics

• Humans
• Amyloid/metabolism
• Parkinson Disease/metabolism
• Proteostasis Deficiencies
• Amyloid beta-Peptides

• SARI DTRA
• Project PRAMA Regione Toscana
• Centre for Misfolding Diseases
• DEVCOM-ARL

Alzheimer's disease is characterized by the accumulation in the brain of the amyloid β (Aβ) peptide in the form of senile plaques. According to the amyloid hypothesis, the aggregation process of Aβ also generates smaller soluble misfolded oligomers that contribute to disease progression. One of the mechanisms of Aβ oligomer cytotoxicity is the aberrant interaction of these species with the phospholipid bilayer of cell membranes, with a consequent increase in cytosolic Ca²⁺ levels, flowing from the extracellular space, and production of reactive oxygen species (ROS). Here we investigated the relationship between the increase in Ca²⁺ and ROS levels immediately after the exposure to misfolded protein oligomers, asking whether they are simultaneous or instead one precedes the other. Using Aβ₄₂-derived diffusible ligands (ADDLs) and type A HypF-N model oligomers (OAs), we followed the kinetics of ROS production and Ca²⁺ influx in human neuroblastoma SH-SY5Y cells and rat primary cortical neurons in a variety of conditions. In all cases we found a faster increase of intracellular Ca²⁺ than ROS levels, and a lag phase in the latter process. A Ca²⁺-deprived cell medium prevented the increase of intracellular Ca²⁺ ions and abolished ROS production. By contrast, treatment with antioxidant agents prevented ROS formation, did not prevent the initial Ca²⁺ flux, but allowed the cells to react to the initial calcium dyshomeostasis, restoring later the normal levels of the ions. These results reveal a mechanism in which the entry of Ca²⁺ causes the production of ROS in cells challenged by aberrant protein oligomers.

• AMPA receptors
• Calcium homeostasis
• Membrane destabilization
• NMDA receptors
• Neurodegenerative diseases
• Oxidative stress
• Protein misfolding

• Alzheimer’s disease
• Parkinson’s disease
• aminosterols
• neurodegeneration
• protein misfolding
• squalamine

• Amyloid beta-Peptides
• Cholestanes
• G(M1) Ganglioside
• Lipid Bilayers
• Spermine/analogs & derivatives

• Horizon 2020 Framework Programme
• Regione Toscana
• Università degli Studi di Firenze
• Ministero dellâ??Istruzione, dellâ??Università e della Ricerca

• protein aggregation
• animal disease models

• Caenorhabditis elegans/metabolism
• Caenorhabditis elegans/drug effects
• Animals
• Phenotype
• Protein Folding
• Caenorhabditis elegans Proteins/metabolism
• Caenorhabditis elegans Proteins/genetics
• Oxidative Stress/drug effects
• Intestinal Mucosa/metabolism
• Protein Aggregates
• Protein Multimerization

• Wellcome Trust
• 094425/Z/10/Z Wellcome Trust

It has been proposed that a “common core” of pathologic pathways exists for the large family of amyloid-associated neurodegenerations, including Alzheimer’s, Parkinson’s, type II diabetes and Creutzfeldt–Jacob’s Disease. Aggregates of the involved proteins, independently from their primary sequence, induced neuron membrane permeabilization able to trigger an abnormal Ca²⁺ influx leading to synaptotoxicity, resulting in reduced expression of synaptic proteins and impaired synaptic transmission. Emerging evidence is now focusing on low-molecular-weight prefibrillar oligomers (PFOs), which mimic bacterial pore-forming toxins that form well-ordered oligomeric membrane-spanning pores. At the same time, the neuron membrane composition and its chemical microenvironment seem to play a pivotal role. In fact, the brain of AD patients contains increased fractions of anionic lipids able to favor cationic influx. However, up to now the existence of a specific “common structure” of the toxic aggregate, and a “common mechanism” by which it induces neuronal damage, synaptotoxicity and impaired synaptic transmission, is still an open hypothesis. In this review, we gathered information concerning this hypothesis, focusing on the proteins linked to several amyloid diseases. We noted commonalities in their structure and membrane activity, and their ability to induce Ca²⁺ influx, neurotoxicity, synaptotoxicity and impaired synaptic transmission.

• Ca2+ influx
• amyloid
• long-term potentiation
• membrane permeabilization
• neurodegeneration
• neurotoxicity
• prefibrillar oligomers
• structure
• synaptic transmission

• Amyloid/chemistry
• Amyloid/metabolism
• Amyloid beta-Peptides/chemistry
• Amyloid beta-Peptides/metabolism
• Amyloidogenic Proteins/chemistry
• Amyloidogenic Proteins/metabolism
• Animals
• Brain/metabolism
• Brain/pathology
• Disease Susceptibility
• Humans
• Neurodegenerative Diseases/etiology
• Neurodegenerative Diseases/metabolism
• Neurodegenerative Diseases/pathology
• Protein Multimerization
• Structure-Activity Relationship

Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder that is characterized by amyloid β-protein deposition in senile plaques, neurofibrillary tangles consisting of abnormally phosphorylated tau protein, and neuronal loss leading to cognitive decline and dementia. Despite extensive research, the exact mechanisms underlying AD remain unknown and effective treatment is not available. Many hypotheses have been proposed to explain AD pathophysiology; however, there is general consensus that the abnormal aggregation of the amyloid β peptide (Aβ) is the initial event triggering a pathogenic cascade of degenerating events in cholinergic neurons. The dysregulation of calcium homeostasis has been studied considerably to clarify the mechanisms of neurodegeneration induced by Aβ. Intracellular calcium acts as a second messenger and plays a key role in the regulation of neuronal functions, such as neural growth and differentiation, action potential, and synaptic plasticity. The calcium hypothesis of AD posits that activation of the amyloidogenic pathway affects neuronal Ca²⁺ homeostasis and the mechanisms responsible for learning and memory. Aβ can disrupt Ca²⁺ signaling through several mechanisms, by increasing the influx of Ca²⁺ from the extracellular space and by activating its release from intracellular stores. Here, we review the different molecular mechanisms and receptors involved in calcium dysregulation in AD and possible therapeutic strategies for improving the treatment.

• AMPA
• NMDA
• amyloid fibrils
• amyloid β peptide (Aβ)
• glutamatergic receptors
• ionic dysregulation
• neurodegeneration
• protein aggregation
• tau protein
• toxic oligomers

• Alzheimer Disease/metabolism
• Animals
• Calcium/metabolism
• Endoplasmic Reticulum/metabolism
• Homeostasis
• Humans
• Mitochondria/metabolism
• Models, Biological

• cell signaling
• lipid peroxidation
• non-apoptotic cell death
• protein aggregation

• 18K11001 Japan Society for the Promotion of Science

• protein aggregation
• mechanisms of disease
• parkinson's disease

• Amyloid/chemistry
• Amyloid/metabolism
• Animals
• Calcium/metabolism
• Cell Line, Tumor
• Cells, Cultured
• Humans
• Inclusion Bodies/metabolism
• Kinetics
• Microscopy, Confocal
• Neurons/metabolism
• Parkinson Disease/metabolism
• Protein Aggregation, Pathological
• Protein Multimerization
• Rats, Sprague-Dawley
• alpha-Synuclein/chemistry
• alpha-Synuclein/metabolism
• Rats

• MR/N000676/1 Medical Research Council
• Ministero dell'Istruzione, dell'Università e della Ricerca (Ministry of Education, University and Research)

The conversion of otherwise soluble proteins into insoluble amyloid aggregates is associated with a range of neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases, as well as non-neuropathic conditions such as type II diabetes and systemic amyloidoses. It is increasingly evident that the most pernicious species among those forming during protein aggregation are small prefibrillar oligomers. In this review, we describe the recent progress in the characterization of the cellular and molecular interactions by toxic misfolded protein oligomers. A fundamental interaction by these aggregates involves biological membranes, resulting in two major model mechanisms at the onset of the cellular toxicity. These include the membrane disruption model, resulting in calcium imbalance, mitochondrial dysfunction and intracellular reactive oxygen species, and the direct interaction with membrane proteins, leading to the alteration of their native function. A key challenge remains in the characterization of transient interactions involving heterogeneous protein aggregates. Solving this task is crucial in the quest of identifying suitable therapeutic approaches to suppress the cellular toxicity in protein misfolding diseases.

• amyloid fibrils
• cellular toxicity
• membrane interaction
• protein misfolding
• receptor binding

• Protein misfolding
• calcium homeostasis
• memantine
• membrane destabilization
• neurodegenerative diseases
• senile plaques

• Alzheimer Disease
• Amyloid beta-Peptides/metabolism
• Calcium/metabolism
• Homeostasis
• Humans
• Neurons/metabolism
• Peptide Fragments/metabolism
• Receptors, N-Methyl-D-Aspartate
• alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid

α-Synuclein (αS) is a presynaptic disordered protein whose aberrant aggregation is associated with Parkinson’s disease. The functional role of αS is still debated, although it has been involved in the regulation of neurotransmitter release via the interaction with synaptic vesicles (SVs). We report here a detailed characterisation of the conformational properties of αS bound to the inner and outer leaflets of the presynaptic plasma membrane (PM), using small unilamellar vesicles. Our results suggest that αS preferentially binds the inner PM leaflet. On the basis of these studies we characterise in vitro a mechanism by which αS stabilises, in a concentration-dependent manner, the docking of SVs on the PM by establishing a dynamic link between the two membranes. The study then provides evidence that changes in the lipid composition of the PM, typically associated with neurodegenerative diseases, alter the modes of binding of αS, specifically in a segment of the sequence overlapping with the non-amyloid component region. Taken together, these results reveal how lipid composition modulates the interaction of αS with the PM and underlie its functional and pathological behaviours in vitro.

α-Synuclein is a presynaptic protein whose aberrant aggregation is associated with Parkinson’s disease. Here, the authors show how αSynuclein-induced docking of synaptic vesicles is modulated by the lipid composition changes typically observed in neurodegeneration using an in vitro system.

• aggregation mechanism
• amyloids and protein aggregation
• heterogeneous nucleation
• peptides and proteins
• surfaces and interfaces

• Cell Membrane
• Cholestanes
• Lipid Bilayers
• Spermine/analogs & derivatives
• Unilamellar Liposomes

• ion channels
• lipids
• neurochemistry
• protein folding

• Amyloid/metabolism
• Amyloid beta-Peptides/metabolism
• Carboxyl and Carbamoyl Transferases/metabolism
• Cardiolipins/metabolism
• Escherichia coli/metabolism
• Escherichia coli Proteins/metabolism
• Humans
• Hydrophobic and Hydrophilic Interactions
• Lipid Bilayers/metabolism
• Membrane Potential, Mitochondrial
• Mitochondria/physiology
• Mitochondrial Membranes/metabolism
• Neurodegenerative Diseases/metabolism
• Protein Conformation
• Protein Multimerization
• Structure-Activity Relationship
• alpha-Synuclein/metabolism
• tau Proteins/metabolism

• ADDLs
• Alzheimer’s disease
• Aβ oligomers
• prion peptide

• Alzheimer Disease/drug therapy
• Alzheimer Disease/metabolism
• Alzheimer Disease/pathology
• Amyloid beta-Peptides/antagonists & inhibitors
• Amyloid beta-Peptides/toxicity
• Calcium/metabolism
• Cell Line, Tumor
• Cell Survival/drug effects
• Humans
• Ion Transport
• Mitochondria/drug effects
• Mitochondria/metabolism
• Mitochondria/pathology
• Models, Biological
• Neurons/drug effects
• Neurons/metabolism
• Neurons/pathology
• Peptide Fragments/antagonists & inhibitors
• Peptide Fragments/toxicity
• Peptides/chemistry
• Peptides/pharmacology
• PrPC Proteins/chemistry
• PrPC Proteins/metabolism
• Protein Binding
• Reactive Oxygen Species/antagonists & inhibitors
• Reactive Oxygen Species/metabolism
• Solubility

α-Synuclein (αSyn) fibrils spread from one neuronal cell to another. This prion-like phenomenon is believed to contribute to the progression of the pathology in Parkinson’s disease and other synucleinopathies. The binding of αSyn fibrils originating from affected cells to the plasma membrane of naïve cells is key in their prion-like propagation propensity. To interfere with this process, we designed polypeptides derived from proteins we previously showed to interact with αSyn fibrils, namely the molecular chaperone Hsc70 and the sodium/potassium pump NaK-ATPase and assessed their capacity to bind αSyn fibrils and/or interfere with their take-up by cells of neuronal origin. We demonstrate here that polypeptides that coat αSyn fibrils surfaces in such a way that they are changed affect αSyn fibrils binding to the plasma membrane components and/or their take-up by cells. Altogether our observations suggest that the rationale design of αSyn fibrils polypeptide binders that interfere with their propagation between neuronal cells holds therapeutic potential.

The onset and progression of numerous protein misfolding diseases are associated with the presence of oligomers formed during the aberrant aggregation of several different proteins, including amyloid-β (Aβ) in Alzheimer’s disease and α-synuclein (αS) in Parkinson’s disease. These small, soluble aggregates are currently major targets for drug discovery. In this study, we show that trodusquemine, a naturally-occurring aminosterol, markedly reduces the cytotoxicity of αS, Aβ and HypF-N oligomers to human neuroblastoma cells by displacing the oligomers from cell membranes in the absence of any substantial morphological and structural changes to the oligomers. These results indicate that the reduced toxicity results from a mechanism that is common to oligomers from different proteins, shed light on the origin of the toxicity of the most deleterious species associated with protein aggregation and suggest that aminosterols have the therapeutically-relevant potential to protect cells from the oligomer-induced cytotoxicity associated with numerous protein misfolding diseases.

Limbocker et al. show that trodusquemine, an aminosterol, reduces the cytotoxicity of protein misfolded oligomers by displacing them from cell membranes in the absence of any overt structural/ morphological changes in them. This mechanism appears to be general, as they test it for oligomers of αS, Aβ and the model protein HypF-N to human neuroblastoma cells.

• Alzheimer’s disease
• amyloid-β
• biophysics
• protein misfolded oligomers
• rationally designed antibodies
• research tools
• structure–toxicity relationship

• Alzheimer Disease/metabolism
• Amyloid beta-Peptides/immunology
• Amyloid beta-Peptides/metabolism
• Amyloid beta-Protein Precursor/metabolism
• Antibodies/immunology
• Antibodies/metabolism
• Antibody Formation/immunology
• Brain/metabolism
• Drug Design
• Humans
• Neurons/metabolism
• Peptide Fragments/metabolism
• Plaque, Amyloid/metabolism
• Protein Aggregates/physiology
• Protein Engineering/methods
• Structure-Activity Relationship

• MR/S033947/1 Medical Research Council

Extracellular vesicles (EVs) from human Alzheimer's disease (AD) biospecimens contain amyloid beta (Aβ) peptide and tau. While AD EVs are known to affect brain disease pathobiology, their biochemical and molecular characterizations remain ill defined.

EVs were isolated from the cortical gray matter of 20 AD and 18 control brains. Tau and Aβ levels were measured by immunoassay. Differentially expressed EV proteins were assessed by quantitative proteomics and machine learning.

Levels of pS396 tau and Aβ1–42 were significantly elevated in AD EVs. High levels of neuron‐ and glia‐specific factors are detected in control and AD EVs, respectively. Machine learning identified ANXA5, VGF, GPM6A, and ACTZ in AD EV compared to controls. They distinguished AD EVs from controls in the test sets with 88% accuracy.

In addition to Aβ and tau, ANXA5, VGF, GPM6A, and ACTZ are new signature proteins in AD EVs.

Alzheimer’s disease is associated with the deposition of the amyloid-β peptide (Aβ) into extracellular senile plaques in the brain. In vitro and in vivo observations have indicated that transthyretin (TTR) acts as an Aβ scavenger in the brain, but the mechanism has not been fully resolved. We have monitored the aggregation process of Aβ₄₀ by thioflavin T fluorescence, in the presence or absence of different concentrations of preformed seed aggregates of Aβ₄₀, of wild-type tetrameric TTR (WT-TTR), and of a variant engineered to be stable as a monomer (M-TTR). Both WT-TTR and M-TTR were found to inhibit specific steps of the process of Aβ₄₀ fibril formation, which are primary and secondary nucleations, without affecting the elongation of the resulting fibrils. Moreover, the analysis shows that both WT-TTR and M-TTR bind to Aβ₄₀ oligomers formed in the aggregation reaction and inhibit their conversion into the shortest fibrils able to elongate. Using biophysical methods, TTR was found to change some aspects of its overall structure following such interactions with Aβ₄₀ oligomers, as well as with oligomers of Aβ₄₂, while maintaining its overall topology. Hence, it is likely that the predominant mechanism by which TTR exerts its protective role lies in the binding of TTR to the Aβ oligomers and in inhibiting primary and secondary nucleation processes, which limits both the toxicity of Aβ oligomers and the ability of the fibrils to proliferate.

Raman spectroscopy assisted by localized plasmon resonances generating effective hot spots at the gaps between intertwined silver nanowires is herein adopted to unravel characteristic molecular motifs on the surface of Aβ₄₂ misfolded oligomers that are critical in driving intermolecular interactions in neurodegeneration.

Unraveling characteristic structural determinants at the basis of Aβ₄₂ oligomers' neurotoxicity by a sub-molecular SERS investigation of their surface.

• Misfolded oligomers
• innate immunity
• molecular mechanisms
• neurodegenerative disease
• neuroinflammation
• protein misfolding

Biological membranes are key elements for the maintenance of cell architecture and physiology. Beyond a pure barrier separating the inner space of the cell from the outer, the plasma membrane is a scaffold and player in cell-to-cell communication and the initiation of intracellular signals among other functions. Critical to this function is the plasma membrane compartmentalization in lipid microdomains that control the localization and productive interactions of proteins involved in cell signal propagation. In addition, cells are divided into compartments limited by other membranes whose integrity and homeostasis are finely controlled, and which determine the identity and function of the different organelles. Here, we review current knowledge on membrane lipid composition in the plasma membrane and endomembrane compartments, emphasizing its role in sustaining organelle structure and function. The correct composition and structure of cell membranes define key pathophysiological aspects of cells. Therefore, we explore the therapeutic potential of manipulating membrane lipid composition with approaches like membrane lipid therapy, aiming to normalize cell functions through the modification of membrane lipid bilayers.

• 2OHOA
• composition
• endomembrane
• lipid
• membrane
• structure
• therapy

• Acrylamide (PubChem CID: 6579)
• Alzheimer's disease
• Amyloid
• Aβ(1-42) peptide
• Congo Red (PubChem CID: 11313)
• Hydroxythyrosol (PubChem: 82755) Dimethylsulfoxide (PubChem CID: 679)
• Hydroxytyrosol
• MTT (PubChem CID: 64965)
• N,N-dimethylformamide (PubChem CID: 6228)
• Oleuropein Aglycone (PubChem CID: 56842347)
• Oleuropein aglycone
• Sodium Dodecil Sulfate (PubChem CID: 3423265)

• Amyloid beta-Peptides/pharmacology
• Boron Compounds/pharmacology
• Cell Line, Tumor
• Cell Membrane/drug effects
• Cell Membrane/physiology
• Fluorescent Dyes/pharmacology
• Humans
• Neuropeptides/pharmacology
• Peptide Fragments/pharmacology
• Viscosity

• Aggregation
• Amyloid-beta
• Lipids
• Membrane
• Oligomer
• Strain
• Surfactant

• P20 GM103476 NIGMS NIH HHS
• R01 GM120634 NIGMS NIH HHS
• R15 AG046915 NIA NIH HHS

Here, we examined the effects of phosphatidylserine (PS) and cholesterol on the fibril-forming properties of the N-terminal 1‒83 fragment of an amyloidogenic G26R variant of apoA-I bound to small unilamellar vesicles. A thioflavin T fluorescence assay together with microscopic observations showed that PS significantly retards the nucleation step in fibril formation by apoA-I 1‒83/G26R, whereas cholesterol slightly enhances fibril formation. Circular dichroism analyses demonstrated that PS facilitates a structural transition from random coil to α-helix in apoA-I 1‒83/G26R with great stabilization of the α-helical structure upon lipid binding. Isothermal titration calorimetry measurements revealed that PS induces a marked increase in capacity for binding of apoA-I 1‒83/G26R to the membrane surface, perhaps due to electrostatic interactions of positively charged amino acids in apoA-I with PS. Such effects of PS to enhance lipid interactions and inhibit fibril formation of apoA-I were also observed for the amyloidogenic region-containing apoA-I 8‒33/G26R peptide. Fluorescence measurements using environment-sensitive probes indicated that PS induces a more solvent-exposed, membrane-bound conformation in the amyloidogenic region of apoA-I without affecting membrane fluidity. Since cell membranes have highly heterogeneous lipid compositions, our findings may provide a molecular basis for the preferential deposition of apoA-I amyloid fibrils in tissues and organs.

Protein misfolded oligomers are considered the most toxic species amongst those formed in the process of amyloid formation and the molecular basis of their toxicity, although not completely understood, is thought to originate from the interaction with the cellular membrane. Here, we sought to highlight the molecular determinants of oligomer-membrane interaction by atomic force microscopy. We monitored the interaction between multiphase supported lipid bilayers and two types of HypF-N oligomers displaying different structural features and cytotoxicities. By our approach we imaged with unprecedented resolution the ordered and disordered lipid phases of the bilayer and different oligomer structures interacting with either phase. We identified the oligomers and lipids responsible for toxicity and, more generally, we established the importance of the membrane lipid component in mediating oligomer toxicity. Our findings support the importance of GM1 ganglioside in mediating the oligomer-bilayer interaction and support a mechanism of oligomer cytotoxicity involving bilayer destabilization by globular oligomers within GM1-rich ordered raft regions rather than by annular oligomers in the surrounding disordered membrane domains.

• AFM
• amyloid oligomers
• ganglioside GM1
• protein aggregation
• protein misfolding

• CD spectroscopy
• ESR spectroscopy
• Nucleophosmin C-terminal domain
• Protein-membrane interaction

Amyloids were first identified in association with amyloidoses, human diseases in which proteins and peptides misfold into amyloid fibrils. Subsequent studies have identified an array of functional amyloid fibrils that perform physiological roles in humans. Given the potential for the production of toxic species in amyloid assembly reactions, it is remarkable that cells can produce these functional amyloids without suffering any obvious ill effect. Although the precise mechanisms are unclear, there are a number of ways in which amyloid toxicity may be prevented. These include regulating the level of the amyloidogenic peptides and proteins, minimising the production of prefibrillar oligomers in amyloid assembly reactions, sequestrating amyloids within membrane bound organelles, controlling amyloid assembly by other molecules, and disassembling the fibrils under physiological conditions. Crucially, a better understanding of how toxicity is avoided in the production of functional amyloids may provide insights into the prevention of amyloid toxicity in amyloidoses.

• amyloids
• fibril
• functional amyloid
• oligomers
• toxicity

• Amyloid/chemistry
• Amyloid/metabolism
• Amyloidogenic Proteins/chemistry
• Amyloidogenic Proteins/metabolism
• Amyloidosis/metabolism
• Amyloidosis/pathology
• Humans
• Peptides/chemistry
• Peptides/metabolism

• Wellcome Trust
• 092896 Wellcome Trust

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is an extremely abundant protein in the brain where, remarkably, it is estimated to make up 1–5% of total neuronal protein. Although it comprises only 223 amino acids it has one of the most complicated 3D knotted structures yet discovered. Beyond its expression in neurons UCH-L1 has only very limited expression in other healthy tissues but it is highly expressed in several forms of cancer. Although UCH-L1 is classed as a deubiquitinating enzyme (DUB) the direct functions of UCH-L1 remain enigmatic and a wide array of alternative functions has been proposed. UCH-L1 is not essential for neuronal development but it is absolutely required for the maintenance of axonal integrity and UCH-L1 dysfunction is implicated in neurodegenerative disease. Here we review the properties of UCH-L1, and how understanding its complex structure can provide new insights into its roles in neuronal function and pathology.

• axon
• neurites
• ubiquitin C-terminal hydrolase L1 (UCH-L1)
• ubiquitin ligases
• ubiquitin proteasome system

• 25‐hydroxycholesterol
• T cells
• amyloid beta‐42
• cholera toxin B subunit
• intracellular transport

• Animals
• Carboxyl and Carbamoyl Transferases/chemistry
• Carboxyl and Carbamoyl Transferases/metabolism
• Cell Line, Tumor
• Clusterin/chemistry
• Clusterin/genetics
• Clusterin/metabolism
• Escherichia coli Proteins/chemistry
• Escherichia coli Proteins/metabolism
• Humans
• Mice
• Prealbumin/chemistry
• Prealbumin/genetics
• Prealbumin/metabolism
• Protein Aggregates
• Protein Folding
• Recombinant Proteins/chemistry
• Recombinant Proteins/genetics
• alpha-Crystallin B Chain/chemistry
• alpha-Crystallin B Chain/genetics
• alpha-Crystallin B Chain/metabolism

The conversion of peptides or proteins from their soluble native states into intractable amyloid deposits is associated with a wide range of human disorders. Misfolded protein oligomers formed during the process of aggregation have been identified as the primary pathogenic agents in many such conditions. Here, we show the existence of a quantitative relationship between the degree of binding to neuronal cells of different types of oligomers formed from a model protein, HypF-N, and the GM1 content of the plasma membranes. In addition, remarkably similar behavior is observed for oligomers of the Aβ₄₂ peptide associated with Alzheimer’s disease. Further analysis has revealed the existence of a linear correlation between the level of the influx of Ca²⁺ across neuronal membranes that triggers cellular damage, and the fraction of oligomeric species bound to the membrane. Our findings indicate that the susceptibility of neuronal cells to different types of misfolded oligomeric assemblies is directly related to the extent of binding of such oligomers to the cellular membrane.

The first genetic variant of β₂‐microglobulin (b2M) associated with a familial form of systemic amyloidosis has been recently described. The mutated protein, carrying a substitution of Asp at position 76 with an Asn (D76N b2M), exhibits a strongly enhanced amyloidogenic tendency to aggregate with respect to the wild‐type protein. In this study, we characterized the D76N b2M aggregation path and performed an unprecedented analysis of the biochemical mechanisms underlying aggregate cytotoxicity. We showed that, contrarily to what expected from other amyloid studies, early aggregates of the mutant are not the most toxic species, despite their higher surface hydrophobicity. By modulating ganglioside GM1 content in cell membrane or synthetic lipid bilayers, we confirmed the pivotal role of this lipid as aggregate recruiter favouring their cytotoxicity. We finally observed that the aggregates bind to the cell membrane inducing an alteration of its elasticity (with possible functional unbalance and cytotoxicity) in GM1‐enriched domains only, thus establishing a link between aggregate‐membrane contact and cell damage.

• GM1 ganglioside
• amyloid cytotoxicity
• membrane bilayers
• protein misfolding
• systemic amyloidosis

We have explored amyloid formation using poly(amino acid) model systems in which differences in peptide secondary structure and hydrophobicity can be introduced in a controlled manner. We show that an environmentally sensitive fluorescent dye, dapoxyl, is able to identify β-sheet structure and hydrophobic surfaces, structural features likely to be related to toxicity, as a result of changes in its excitation and emission profiles and its relative quantum yield. These results show that dapoxyl is a multidimensional probe of the time dependence of amyloid aggregation, which provides information about the presence and nature of metastable aggregation intermediates that is inaccessible to the conventional probes that rely on changes in quantum yield alone.

Fibrillar aggregates involved in neurodegenerative diseases have the ability to spread from one cell to another in a prion-like manner. The underlying molecular mechanisms, in particular the binding mode of the fibrils to cell membranes, are poorly understood. In this work we decipher the modality by which aggregates bind to the cellular membrane, one of the obligatory steps of the propagation cycle. By characterizing the binding properties of aggregates made of α-synuclein or huntingtin exon 1 protein displaying similar composition and structure but different lengths to mammalian cells we demonstrate that in both cases aggregates bind laterally to the cellular membrane, with aggregates extremities displaying little or no role in membrane binding. Lateral binding to artificial liposomes was also observed by transmission electron microscopy. In addition we show that although α-synuclein and huntingtin exon 1 fibrils bind both laterally to the cellular membrane, their mechanisms of interaction differ. Our findings have important implications for the development of future therapeutic tools that aim to block protein aggregates propagation in the brain.

• Alzheimer's disease
• Aβ oligomers
• GM1 ganglioside
• Single molecule tracking

• Amyloid aggregation
• Amyloid β-peptide
• Lipid raft
• Membrane heterogeneity
• Model membrane

• Amyloid/metabolism
• Animals
• Diabetes Mellitus, Type 2/metabolism
• Humans
• Islet Amyloid Polypeptide/biosynthesis
• Islets of Langerhans/metabolism