Alzheimer's disease is associated with the aggregation of amyloid-β (Aβ) peptides into oligomers and fibrils. We have explored how model lipid membranes modulate the rate and mechanisms of Aβ(1-42) self-assembly, in order to shed light on how this pathological reaction may occur in the lipid-rich environments that the peptide encounters in the brain. Using a combination of in vitro biophysical experiments and theoretical approaches, we show that zwitterionic DOPC lipid vesicles accelerate the Aβ(1-42) fibril growth rate by interacting specifically with the growing fibrils. We probe this interaction with help of a purpose-developed Förster resonance energy transfer assay that monitors the proximity between a fibril-specific dye and fluorescent lipids in the lipid vesicle membrane. To further rationalise these findings we use mathematical models to fit the aggregation kinetics of Aβ(1-42) and find that lipid vesicles alter specific mechanistic steps in the aggregation reaction; they augment monomer-dependent secondary nucleation at the surface of existing fibrils and facilitate monomer-independent catalytic processes consistent with fibril fragmentation. We further show that DOPC vesicles have no effect on primary nucleation. This finding is consistent with experiments showing that Aβ(1-42) monomers do not directly bind to the lipid bilayer. Taken together, our results show that plain lipid membranes with charge and composition that is representative of outer cell membranes can significantly augment autocatalytic steps in the self-assembly of Aβ(1-42) into fibrils. This new insight suggests that strategies to reduce fibril-lipid interactions in the brain may have therapeutic value.

Thioflavin-T (ThT) is one of the most commonly used dyes for amyloid detection, but the origin of its fluorescence enhancement is not fully understood. Herein we have characterised the ThT fluorescence response upon binding to the Aβ(1-40) and Aβ(1-42) variants of the Alzheimer's-related peptide amyloid-β, in order to explore how the photophysical properties of this dye relates to structural and morphological properties of two amyloid fibril types formed by peptides with a high degree of sequence homology. We show that the steady-state ThT fluorescence is 1.7 times more intense with Aβ(1-40) compared to Aβ(1-42) fibrils in concentration matched samples prepared under quiescent conditions. By measuring the excited state lifetime of bound ThT, we also demonstrate a distinct difference between the two fibril isoforms, with Aβ(1-42) fibrils producing a longer ThT fluorescence lifetime compared to Aβ(1-40). The substantial steady-state intensity difference is therefore not explained by differences in fluorescence quantum yield. Further, we find that the ThT fluorescence intensity, but not the fluorescence lifetime, is dependent on the fibril preparation method (quiescent versus agitated conditions). We therefore propose that the fluorescence lifetime is inherent to each isoform and sensitively reports on fibril microstructure in the protofilament whereas the total fluorescence intensity relates to the amount of exposed β-sheet in the mature Aβ fibrils and hence to differences in their morphology. Our results highlight the complexity of ThT fluorescence, and demonstrate its extended use in amyloid fibril characterisation.

Thioflavin-T binds to and detects amyloid fibrils via fluorescence enhancement. Using a combination of linear dichroism and fluorescence spectroscopies, we report that the relation between the emission intensity and binding of thioflavin-T to insulin fibrils is nonlinear and discuss this in relation to its use in kinetic assays. We demonstrate, from fluorescence lifetime recordings, that the nonlinearity is due to thioflavin-T being sensitive to self-quenching. In addition, thioflavin-T can induce fibril compaction but not alter fibril structure. Our work underscores the photophysical complexity of thioflavin-T and the necessity of calibrating the linear range of its emission response for quantitative in vitro studies.

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