Amyloid-ß oligomers have a profound detergent-like effect on lipid membrane bilayers, imaged by atomic force and electron microscopy.

The ability of amyloid-ß peptide (Aß) to disrupt membrane integrity and cellular homeostasis is believed to be central to Alzheimer's disease pathology. Aß is reported to have various impacts on the lipid bilayer, but a clearer picture of Aß influence on membranes is required. Here, we use atomic force and transmission electron microscopies to image the impact of different isolated Aß assembly types on lipid bilayers. We show that only oligomeric Aß can profoundly disrupt the bilayer, visualized as widespread lipid extraction and subsequent deposition, which can be likened to an effect expected from the action of a detergent. We further show that Aß oligomers cause widespread curvature and discontinuities within lipid vesicle membranes. In contrast, this detergent-like effect was not observed for Aß monomers and fibers, although Aß fibers did laterally associate and embed into the upper leaflet of the bilayer. The marked impact of Aß oligomers on membrane integrity identified here reveals a mechanism by which these oligomers may be cytotoxic.

Copper Redox Cycling Inhibits Aß Fibre Formation and Promotes Fibre Fragmentation, while Generating a Dityrosine Aß Dimer.

Oxidative stress and the formation of plaques which contain amyloid-ß (Aß) peptides are two key hallmarks of Alzheimer's disease (AD). Dityrosine is found in the plaques of AD patients and Aß dimers have been linked to neurotoxicity. Here we investigate the formation of Aß dityrosine dimers promoted by Cu2+/+ Fenton reactions. Using fluorescence measurements and UV absorbance, we show that dityrosine can be formed aerobically when Aß is incubated with Cu2+ and hydrogen-peroxide, or in a Cu2+ and ascorbate redox mixture. The dityrosine cross-linking can occur for both monomeric and fibrillar forms of Aß. We show that oxidative modification of Aß impedes the ability for Aß monomer to form fibres, as indicated by the amyloid specific dye Thioflavin T (ThT). Transmission electron microscopy (TEM) indicates the limited amyloid assemblies that form have a marked reduction in fibre length for Aß(1-40). Importantly, the addition of Cu2+ and a reductant to preformed Aß(1-40) fibers causes their widespread fragmentation, reducing median fibre lengths from 800 nm to 150 nm upon oxidation. The processes of covalent cross-linking of Aß fibres, dimer formation, and fibre fragmentation within plaques are likely to have a significant impact on Aß clearance and neurotoxicity.

Serum Albumin's Protective Inhibition of Amyloid-ß Fiber Formation Is Suppressed by Cholesterol, Fatty Acids and Warfarin.

Central to Alzheimer's disease (AD) pathology is the assembly of monomeric amyloid-ß peptide (Aß) into oligomers and fibers. The most abundant protein in the blood plasma and cerebrospinal fluid is human serum albumin. Albumin can bind to Aß and is capable of inhibiting the fibrillization of Aß at physiological (µM) concentrations. The ability of albumin to bind Aß has recently been exploited in a phase II clinical trial, which showed a reduction in cognitive decline in AD patients undergoing albumin-plasma exchange. Here we explore the equilibrium between Aß monomer, oligomer and fiber in the presence of albumin. Using transmission electron microscopy and thioflavin-T fluorescent dye, we have shown that albumin traps Aß as oligomers, 9 nm in diameter. We show that albumin-trapped Aß oligomeric assemblies are not capable of forming ion channels, which suggests a mechanism by which albumin is protective in Aß-exposed neuronal cells. In vivo albumin binds a variety of endogenous and therapeutic exogenous hydrophobic molecules, including cholesterol, fatty acids and warfarin. We show that these molecules bind to albumin and suppress its ability to inhibit Aß fiber formation. The interplay between Aß, albumin and endogenous hydrophobic molecules impacts Aß assembly; thus, changes in cholesterol and fatty acid levels in vivo may impact Aß fibrillization, by altering the capacity of albumin to bind Aß. These observations are particularly intriguing given that high cholesterol or fatty acid diets are well-established risk factors for late-onset AD.

Ion Channel Formation by Amyloid-ß42 Oligomers but Not Amyloid-ß40 in Cellular Membranes.

A central hallmark of Alzheimer's disease is the presence of extracellular amyloid plaques chiefly consisting of amyloid-ß (Aß) peptides in the brain interstitium. Aß largely exists in two isoforms, 40 and 42 amino acids long, but a large body of evidence points to Aß(1-42) rather than Aß(1-40) as the cytotoxic form. One proposed mechanism by which Aß exerts toxicity is the formation of ion channel pores that disrupt intracellular Ca2+ homeostasis. However, previous studies using membrane mimetics have not identified any notable difference in the channel forming properties between Aß(1-40) and Aß(1-42). Here, we tested whether a more physiological environment, membranes excised from HEK293 cells of neuronal origin, would reveal differences in the relative channel forming ability of monomeric, oligomeric, and fibrillar forms of both Aß(1-40) and Aß(1-42). Aß preparations were characterized with transmission electron microscopy and thioflavin T fluorescence. Aß was then exposed to the extracellular face of excised membranes, and transmembrane currents were monitored using patch clamp. Our data indicated that Aß(1-42) assemblies in oligomeric preparations form voltage-independent, non-selective ion channels. In contrast, Aß(1-40) oligomers, fibers, and monomers did not form channels. Ion channel conductance results suggested that Aß(1-42) oligomers, but not monomers and fibers, formed three distinct pore structures with 1.7-, 2.1-, and 2.4-nm pore diameters. Our findings demonstrate that only Aß(1-42) contains unique structural features that facilitate membrane insertion and channel formation, now aligning ion channel formation with the differential neurotoxic effect of Aß(1-40) and Aß(1-42) in Alzheimer's disease.