On transient assemblies in amyloid formation: mechanistic insights and therapeutic strategies

The aberrant assembly of proteins into aggregates is a common feature in over fifty human diseases, including neurodegenerative disorders. While the accumulation of amyloid fibrils hallmarks diagnoses of these conditions, pre-fibrllar oligomers are increasingly recognised as the major toxic agents. However, these assemblies pose significant experimental difficulties due to their transient nature, structural heterogeneity, and low concentrations in the aggregation reactions, which render them invisible to conventional techniques. In this thesis, to address these challenges, I explore orthogonal approaches to studying and targeting aggregation, focusing on α-Synuclein and Amyloid-β – proteins implicated in Parkinson’s and Alzheimer’s diseases, respectively. To begin, I present a novel microfluidic platform integrating free-flow electrophoresis with complementary analytical techniques, which allows for a multifaceted characterisation of amyloid oligomers. In this approach, heterogenous oligomer mixtures are fractionated on-chip and subsequently collected for downstream analyses, enabling the direct correlation between biophysical, structural, and functional properties of distinct species, providing previously unattainable insights into their features that confer toxicity in disease pathology. I then investigate the mechanisms of oligomer formation and dissociation in protein aggregation reactions. Through a combination of kinetic assays and single-molecule spectroscopy, I demonstrate that α-Synuclein oligomers form through secondary nucleation under physiologically relevant conditions. Using Amyloid-β as a model system, I then examine the impact of mechanical forces on amyloid formation, revealing that shear accelerates aggregation by facilitating the detachment of primary and secondary nuclei from catalytic surfaces. Furthermore, this study uncovers the bidirectional role of fibrils in oligomer dynamics, with implications for their stability and therapeutic strategies.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Andrzejewska, Ewa

Advisor dc:contributor.advisor

• Knowles, Tuomas

• amyloid
• oligomers
• microfluidics

Licence

• https://www.repository.cam.ac.uk/bitstreams/7ff671a2-52c4-4bd6-954a-ba5376237ced/download
• http://purl.org/NET/rdflicense/allrightsreserved

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.118488

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/384515