Spectral Heterogeneity of Thioflavin T Binding to Aβ42:Aβ40 Mixed Fibrils: Implications for Alzheimer's Disease Screening.

Abstract

In Alzheimer's disease (AD), the amyloid β (Aβ) protein self-assembles, whereby Aβ40 and Aβ42 peptides interact, forming a mixed fibrillar assembly. Evaluating local Aβ40:Aβ42 mixed fibril conformations remains challenging, requiring a simple method to compare microscopic (molecular-scale) and macroscopic (plaque-scale) findings. The aim of the current study was to design a method to analyze Aβ fibril formation in a single sample without drying via fluorescent thioflavin T (ThT) labeling. The analysis revealed spectral heterogeneity associated with the ThT-binding mixed fibrils. Although the fluorescence wavelength associated with higher Aβ42:Aβ40 fibril ratios remained relatively unchanged, those associated with lower Aβ42:Aβ40 fibril ratios exhibited significant heterogeneity. This suggests that the local β-sheet structure exhibits significant variability at lower Aβ42:Aβ40 ratios. This specific feature can be attributed to differences in the shape of the "funnel" in the energy landscape during Aβ assembly. Thus, our protocol facilitates rapid and efficient screening of fibril conformational alterations compared to conventional techniques. Cumulatively, our results demonstrate that comparing the spectral features of ThT with the kinetic and morphological characteristics of a single sample provides specific molecular insights related to the origin of Aβ42:Aβ40 ratio-dependent molecular mechanism-insights that cannot be detected through conventional kinetic and morphological analyses alone.