Abstract
Assembly of amyloid fibrils and small globular oligomers is associated with a significant number of human disorders that include Alzheimer’s disease, senile systemic amyloidosis, and type II diabetes. Recent findings implicate small amyloid oligomers as the dominant aggregate species mediating the toxic effects in these disorders. However, validation of this hypothesis has been hampered by the dearth of experimental techniques to detect, quantify, and discriminate oligomeric intermediates from late-stage fibrils, in vitro and in vivo. We have shown that the onset of significant oligomer formation is associated with a transition in thioflavin T kinetics from sigmoidal to biphasic kinetics. Here we showed that this transition can be exploited for screening fluorophores for preferential responses to oligomer over fibril formation. This assay identified crystal violet as a strongly selective oligomer-indicator dye for lysozyme. Simultaneous recordings of amyloid kinetics with thioflavin T and crystal violet enabled us to separate the combined signals into their underlying oligomeric and fibrillar components. We provided further evidence that this screening assay could be extended to amyloid-β peptides under physiological conditions. Identification of oligomer-selective dyes not only holds the promise of biomedical applications but provides new approaches for unraveling the mechanisms underlying oligomer versus fibril formation in amyloid assembly.
Highlights
Deposits of amyloid protein fibrils with their characteristic cross-β sheet architecture are the dominant pathological feature associated with a wide variety of age-related human disorders [1,2,3,4,5,6,7]
We have previously shown that amyloid assembly of hen egg-white lysozyme and of a dimeric Aβ40 construct, as recorded with the indicator dye thioflavin T (ThT), undergo a sharp transition from essentially oligomer-free sigmoidal to oligomer-dominated biphasic kinetics, upon crossing a threshold [41,42]
In order to separate the dye responses into their globular oligomers (gOs)/curvilinear fibrils (CFs) versus Rigid fibrils (RFs) components, a clear temporal separation of the two growth phases is highly desirable. These findings with Aβ40 and Aβ42 are consistent with the switch from oligomer-free sigmoidal to oligomer-dominated biphasic growth that we observed for dimeric Aβ40 construct (dimAβ) and hen egg-white lysozyme (hewL)
Summary
Deposits of amyloid protein fibrils with their characteristic cross-β sheet architecture are the dominant pathological feature associated with a wide variety of age-related human disorders [1,2,3,4,5,6,7]. With the exception of anti-oligomer antibodies, none of the aforementioned techniques lend themselves for oligomer detection ex vivo, let alone in vivo They all suffer from poor temporal resolution, which is a serious obstacle for kinetics studies to reveal the role of oligomers in fibril formation.
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