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

Licentiate thesis, 2015

Amyloid fibrils are self-assembled protein homopolymers that play central roles in the pathology of several human diseases, most notably in progressive neurodegenerative diseases such as Alzheimer’s disease (AD). In AD, fibrils are formed by the amyloid-β (Aβ) peptide, an enzymatic cleavage product that is naturally produced in human brain tissue. The fibrillar Aβ accumulates in deposits known as senile plaques in the diseased AD brain, and the processes by which this occurs are postulated to be primary causes of the neuronal loss that is associated with AD progression. As such, it is of great importance to understand how Aβ fibrils are formed, and why they exert neurotoxic effects in AD patients. In pursuit of this goal, there is a need for more effective and precise methods of analysing amyloid material. In this work, the aim was to improve the read outs of techniques that are currently used, and to develop novel methods for amyloid detection. The main focus has been on small molecules that work as light-switch molecules in presence of amyloids, i.e. they exhibit greatly enhanced fluorescence intensity upon binding to amyloid fibrils. I have characterised the classic amyloid stain thioflavin-T (ThT), and showed how it can differentiate between different amyloid samples based on their morphological attributes, particularly upon detection of its fluorescence lifetime. I have also characterised the binding and fluorescent properties of YOYO-1; a traditional DNA stain that proved to be an attractive alternative to ThT and that therefore may function as a novel amyloid stain, extending the amyloid recognition toolbox. The Thesis also outlines the development of an existing protocol for expression and purification of monomeric seed-free amyloid-β(1-42) that has been setup in our laboratory in order to facilitate reproducible investigation into the formation kinetics of Aβ(1-42) fibrils. This work is a stepping stone towards studies of amyloid-β formation kinetics and neurotoxicity. I intend to utilise the fluorescence techniques described herein, together with the developed protocol for Aβ production, to explore different stages of Aβ aggregation; how they exert their neurotoxic effects, and if these properties are modulated by lipid membranes and molecular crowding effects.