Detecting and Tracking β-Amyloid Oligomeric Forms and Dynamics In Vitro by a High-Sensitivity Fluorescent-Based Assay.

Aggregation of β-amyloid protein is a hallmark pathology of the neurodegenerative disorder Alzheimer's disease and proceeds from monomers to insoluble misfolded fibril forms via soluble and highly toxic oligomeric intermediates. Given the dual feature of being the most toxic form of the Aβ aggregate proteome and an early marker of pathogenesis, there is a need for sensitive methods that can be used to detect Aβ oligomers and investigate the dynamics of aggregation. Herein, we describe a method based on the application of an oligomer-sensitive fluorescent chemical probe pTP-TFE combined with the use of a QIAD (Quantitative determination of Interference with Aβ Aggregate Size Distribution) assay to correctly identify Aβ oligomers in high sensitivity.

The inhibitory action of the chaperone BRICHOS against the α-Synuclein secondary nucleation pathway.

The complex kinetics of disease-related amyloid aggregation of proteins such as α-Synuclein (α-Syn) in Parkinson's disease and Aβ42 in Alzheimer's disease include primary nucleation, amyloid fibril elongation and secondary nucleation. The latter can be a key accelerator of the aggregation process. It has been demonstrated that the chaperone domain BRICHOS can interfere with the secondary nucleation process of Aβ42.

Single-molecule digital sizing of proteins in solution.

The physical characterization of proteins in terms of their sizes, interactions, and assembly states is key to understanding their biological function and dysfunction. However, this has remained a difficult task because proteins are often highly polydisperse and present as multicomponent mixtures. Here, we address this challenge by introducing single-molecule microfluidic diffusional sizing (smMDS).

α-Synuclein oligomers form by secondary nucleation.

Oligomeric species arising during the aggregation of α-synuclein are implicated as a major source of toxicity in Parkinson's disease, and thus a major potential drug target. However, both their mechanism of formation and role in aggregation are largely unresolved. Here we show that, at physiological pH and in the absence of lipid membranes, α-synuclein aggregates form by secondary nucleation, rather than simple primary nucleation, and that this process is enhanced by agitation.

The role of shear forces in primary and secondary nucleation of amyloid fibrils.

Shear forces affect self-assembly processes ranging from crystallization to fiber formation. Here, the effect of mild agitation on amyloid fibril formation was explored for four peptides and investigated in detail for A[Formula: see text]42, which is associated with Alzheimer's disease. To gain mechanistic insights into the effect of mild agitation, nonseeded and seeded aggregation reactions were set up at various peptide concentrations with and without an inhibitor.