Factors determining the cytotoxic nature of pathogenic amyloid proteins

Amyloid proteins feature in neurodegenerative diseases and functionally throughout many organisms. Furthermore, due to their structural properties, amyloid proteins have been developed as materials in biotechnology. This raises the question of what makes disease-related amyloid proteins toxic. β-amyloid 1-42 (Aβ42) is a self-assembling protein that goes through many structural changes before forming the extracellular plaques characteristic of Alzheimer's disease. We have studied the conformational changes of the Aβ42 peptide over time by combining a range of biophysical approaches including circular dichroism, and Thioflavin T fluorescence with Transmission Electron Microscopy. Aβ42 assembly is compared to a novel, rationally-designed, assembly-resistant Aβ42 peptide variant (vAβ42), as well as the two main Aβ42 controls, Aβ reversed (Aβ42-1)and Aβ scrambled (AβS). The vAβ42 differs in sequence by only two amino acids, however, does not self-assemble or form β-sheet structures, unlike Aβ42-1and AβS which both display a high propensity to form amyloid. All three variants of Aβ42 were non-toxic in primary hippocampal cultures, highlighting the importance of primary sequence in determining the toxic nature of an amyloid protein. Furthermore, the structure andtoxicity of the naturally functional amyloid protein, GNNQQNY,and the designedfunctional amyloid peptide, FEFKFEFKK (F9), have also been characterised. These show immediate assembly into mature fibrils, do not form intermediary species and are not cytotoxic. Together, this data suggests the ability to form oligomers and the time spent in this conformation is a requirement of amyloid toxicity. To further investigate the link between size, conformation and toxicity, we compared the cytotoxicity and internalisation of oligomeric, fibrillar and sonicated fibres of Aβ42 in primary hippocampal neurons using immunolabelling and live cell imaging. As expected, the oligomeric Aβ42 was highly neurotoxic in hippocampal cultures, however fibrillar and sonicated fibrils did not have the same effect. Finally, the necessity of internalisation in mediating cytotoxicity was investigated and showed a certain threshold of intracellular accumulation must be met to induce cytotoxicity. Overall, our data suggests primary sequence, the resultant self-assembly and intermediary species formed, and intracellular accumulation are vital in determining the pathogenic properties of amyloid proteins

Amyloidogenicity and toxicity of the reverse and scrambled variants of amyloid-β 1-42

β-amyloid 1-42 (Aβ1-42) is a self-assembling peptide that goes through many conformational and morphological changes before forming the fibrils that are deposited in extracellular plaques characteristic of Alzheimer's disease. The link between Aβ1-42 structure and toxicity is of major interest, in particular, the neurotoxic potential of oligomeric species. Many studies utilise reversed (Aβ42-1) and scrambled (AβS) forms of amyloid-β as control peptides. Here, using circular dichroism, thioflavin T fluorescence and transmission electron microscopy, we reveal that both control peptides self-assemble to form fibres within 24 h. However, oligomeric Aβ reduces cell survival of hippocampal neurons, while Aβ42-1 and Aβs have reduced effect on cellular health, which may arise from their ability to assemble rapidly to form protofibrils and fibrils

Internalisation and toxicity of amyloid-β 1-42 are influenced by its conformation and assembly state rather than size

Amyloid fibrils found in plaques in Alzheimer’s disease (AD) brains are composed of amyloid-β peptides. Oligomeric amyloid-β 1-42 (Aβ42) is thought to play a critical role in neurodegeneration in AD. Here, we determine how size and conformation affect neurotoxicity and internalisation of Aβ42 assemblies using biophysical methods, immunoblotting, toxicity assays and live-cell imaging. We report significant cytotoxicity of Aβ42 oligomers and their internalisation into neurons. In contrast, Aβ42 fibrils show reduced internalisation and no toxicity. Sonicating Aβ42 fibrils generates species similar in size to oligomers but remains nontoxic. The results suggest that Aβ42 oligomers have unique properties that underlie their neurotoxic potential. Furthermore, we show that incubating cells with Aβ42 oligomers for 24 h is sufficient to trigger irreversible neurotoxicity

Alzheimer's disease-like paired helical filament assembly from truncated tau protein is independent of disulphide cross-linking

Abstract Alzheimer's disease is characterised by the self-assembly of tau and amyloid β proteins into oligomers and fibrils. Tau protein assembles into paired helical filaments (PHFs) that constitute the neurofibrillary tangles observed in neuronal cell bodies in individuals with Alzheimer's disease. The mechanism of initiation of tau assembly into {PHFs} is not well understood. Here we report that a truncated 95-amino acid tau fragment (corresponding to residues 297-391 of full-length tau) assembles into PHF-like fibrils in vitro without the need for other additives to initiate or template the process. Using electron microscopy, circular dichroism and X-ray fibre diffraction, we have characterised the structure of the fibrils formed from truncated tau for the first time. To explore the contribution of disulphide formation to fibril formation, we have compared the assembly of tau(297-391) under reduced and non-reducing conditions and for truncated tau carrying a {C322A} substitution. We show that disulphide bond formation inhibits assembly and that the {C322A} variant rapidly forms long and highly ordered PHFs

Europium as an inhibitor of Amyloid-β(1-42) induced membrane permeation

Soluble Amyloid-beta (Aβ) oligomers are a source of cytotoxicity in Alzheimer's disease (AD). The toxicity of Aβ oligomers may arise from their ability to interact with and disrupt cellular membranes mediated by GM1 ganglioside receptors within these membranes. Therefore, inhibition of Aβ–membrane interactions could provide a means of preventing the toxicity associated with Aβ. Here, using Surface Plasmon field-enhanced Fluorescence Spectroscopy, we determine that the lanthanide, Europium III chloride (Eu3+), strongly binds to GM1 ganglioside-containing membranes and prevents the interaction with Aβ42 leading to a loss of the peptides ability to cause membrane permeation. Here we discuss the molecular mechanism by which Eu3+ inhibits Aβ42-membrane interactions and this may lead to protection of membrane integrity against Aβ42 induced toxicity

Misfolded amyloid-ß-42 impairs the endosomal–lysosomal pathway

Misfolding and aggregation of proteins is strongly linked to several neurodegenerative diseases, but how such species bring about their cytotoxic actions remains poorly understood. Here we used specifically-designed optical reporter probes and live fluorescence imaging in primary hippocampal neurons to characterise the mechanism by which prefibrillar, oligomeric forms of the Alzheimer’s-associated peptide, Aß42, exert their detrimental effects. We used a pH-sensitive reporter, Aß42-CypHer, to track Aß internalisation in real-time, demonstrating that oligomers are rapidly taken up into cells in a dynamin-dependent manner, and trafficked via the endo-lysosomal pathway resulting in accumulation in lysosomes. In contrast, a non-assembling variant of Aß42 (vAß42) assayed in the same way is not internalised. Tracking ovalbumin uptake into cells using CypHer or Alexa Fluor tags shows that preincubation with Aß42 disrupts protein uptake. Our results identify a potential mechanism by which amyloidogenic aggregates impair cellular function through disruption of the endosomal–lysosomal pathway

Structure dependent effects of Amyloid-ß on long-term memory in Lymnaea stagnalis

Amyloid-ß (Aß) peptides are implicated in the causation of memory loss, neuronal impairment, and neurodegeneration in Alzheimer's disease. Our recent work revealed that Aß 1–42 and Aß 25–35 inhibit long-term memory (LTM) recall in Lymnaea stagnalis (pond snail) in the absence of cell death. Here, we report the characterization of the active species prepared under different conditions, describe which Aß species is present in brain tissue during the behavioral recall time point and relate the sequence and structure of the oligomeric species to the resulting neuronal properties and effect on LTM. Our results suggest that oligomers are the key toxic Aß1–42 structures, which likely affect LTM through synaptic plasticity pathways, and that Aß 1–42 and Aß 25–35 cannot be used as interchangeable peptides