Abstract
The aggregation of amyloid-β peptides into protein fibres is one of the main neuropathological features of Alzheimer's disease (AD). While imaging of amyloid-β aggregate morphology in vitro is extremely important for understanding AD pathology and in the development of aggregation inhibitors, unfortunately, potentially highly toxic, early aggregates are difficult to observe by current electron microscopy and atomic force microscopy (AFM) methods, due to low contrast and variability of peptide attachment to the substrate. Here, we use a poly-L-Lysine (PLL) surface that captures all protein components from monomers to fully formed fibres, followed by nanomechanical mapping via ultrasonic force microscopy (UFM), which marries high spatial resolution and nanomechanical contrast with the non-destructive nature of tapping mode AFM. For the main putative AD pathogenic component, Aβ1-42, the PLL-UFM approach reveals the morphology of oligomers, protofibrils and mature fibres, and finds that a fraction of small oligomers is still present at later stages of fibril assembly.
Highlights
The aggregation of amyloid-b peptides into protein fibres is one of the main neuropathological features of Alzheimer’s disease (AD)
For the main putative AD pathogenic component, Ab1-42, the PLL-ultrasonic force microscopy (UFM) approach reveals the morphology of oligomers, protofibrils and mature fibres, and finds that a fraction of small oligomers is still present at later stages of fibril assembly
AD is one of the most widely studied of the amyloidoses, a group of diseases in which the central pathogenic feature is the accumulation of insoluble amyloid fibrils, which are typically 7–10 nm in width, with a high b-pleated sheet content, and an ability to bind to the dyes Congo red or thioflavin T (ThT)[3,4]
Summary
The aggregation of amyloid-b peptides into protein fibres is one of the main neuropathological features of Alzheimer’s disease (AD). Multiple methods are currently used to study the dynamics of Ab aggregation, including ThT and Congo red binding[11], size exclusion chromatography[8], light scattering[7,8], mass-spectroscopy[8] and immunoassays[8,9] These techniques provide specific information on one particular aspect of amyloid aggregation, such as b-sheet content, particle size or the availability of epitope binding sites. They do not allow for detailed study of overall morphology, or any alterations in morphology resulting from the structural transitions undergone during the pathway(s) leading from monomeric peptide, through oligomeric assemblies, to PF and MF structures. AFM needs atomically flat, or at minimum, nanoscaleflat, rigid substrates, somewhat limiting its applicability to some studies, compared to TEM, where free standing membranes are widely used on ‘‘holey silicon nitride’’ or ‘‘holey carbon’’ TEM grids
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