Development of the Amyloid Fibril Characterisation Toolbox - New use for old dyes

Amyloid fibrils are self-assembled protein homopolymers that playcentral roles in the pathology of several human diseases, most notably in progressiveneurodegenerative diseases such as Alzheimer’s disease (AD). In AD, fibrils areformed by the amyloid-β (Aβ) peptide, an enzymatic cleavage product that is naturallyproduced in human brain tissue. The fibrillar Aβ accumulates in deposits known assenile plaques in the diseased AD brain, and the processes by which this occurs arepostulated to be primary causes of the neuronal loss that is associated with ADprogression. As such, it is of great importance to understand how Aβ fibrils areformed, and why they exert neurotoxic effects in AD patients.In pursuit of this goal, there is a need for more effective and precise methods ofanalysing amyloid material. In this work, the aim was to improve the read outs oftechniques that are currently used, and to develop novel methods for amyloiddetection. The main focus has been on small molecules that work as light-switchmolecules in presence of amyloids, i.e. they exhibit greatly enhanced fluorescenceintensity upon binding to amyloid fibrils. I have characterised the classic amyloid stainthioflavin-T (ThT), and showed how it can differentiate between different amyloidsamples based on their morphological attributes, particularly upon detection of itsfluorescence lifetime. I have also characterised the binding and fluorescent propertiesof YOYO-1; a traditional DNA stain that proved to be an attractive alternative to ThTand that therefore may function as a novel amyloid stain, extending the amyloidrecognition toolbox.The Thesis also outlines the development of an existing protocol for expression andpurification of monomeric seed-free amyloid-β(1-42) that has been setup in ourlaboratory in order to facilitate reproducible investigation into the formation kineticsof Aβ(1-42) fibrils.This work is a stepping stone towards studies of amyloid-β formation kinetics andneurotoxicity. I intend to utilise the fluorescence techniques described herein, togetherwith the developed protocol for Aβ production, to explore different stages of Aβaggregation; how they exert their neurotoxic effects, and if these properties aremodulated by lipid membranes and molecular crowding effects