Arscott-Barber, Alexander ORCID: https://orcid.org/0009-0001-2477-3230
(2025)
Pathological Aggregation of Cystatin C and α-Synuclein in Neurodegenerative Disease.
PhD thesis, University of Sheffield.
Abstract
Neurodegenerative diseases are chronic conditions that progressive dysfunction in cognition, muscle control, and speech, driven by neuronal death and malfunction due to protein misfolding and aggregation. Current treatments are limited, and understanding the aggregation pathways involved is key to developing more effective therapies. This thesis investigates two such aggregation systems with strong associations to disease.
The first part focuses on cystatin C aggregation in Bunina bodies, observed in amyotrophic lateral sclerosis (ALS). Extensive attempts at optimising expression and purification of recombinant cystatin C from E. coli are shown, followed by in vitro aggregation assays. Cystatin C aggregation is induced in conditions similar to cytosol, where Bunina bodies form, by disrupting two structurally critical disulfide bonds. Kinetic analysis suggests a proline-isomerisation step during the aggregation process, as seen in other cystatins. Additionally, preliminary nuclear magnetic resonance (NMR) data hints at a potentially unreported binding interaction between cystatin C dimers and RNA. In vivo
overexpression of fluorescently-tagged cystatin C in mammalian cells produces Bunina body-like inclusions, which appear to be actively transported toward the nucleus via the microtubule network.
The second part explores α-synuclein aggregation on POPG lipid membranes, modelling Lewy body formation in Parkinson’s disease. This system has previously informed Parkinson’s drug development, including an ongoing phase II clinical trial, which heavily supports its disease relevance. This study shows that fully helical α-synuclein remodels POPG membranes into long cylindrical micelles, which form an inter-connected network, spaced 10-20 nm apart, consistent with a double-anchoring mechanism for α-synuclein reported in the literature. After prolonged incubation, a rapid structural transition from α-helix to β-sheet occurs, supporting a hypothesis of amyloid structural propagation along the micelle surface, providing a mechanism for accelerated aggregation and lipid sequestration by α-synuclein amyloids in Parkinson’s disease.
Metadata
Supervisors: | Staniforth, Rosemary and Parnell, Andrew and Highley, Robin |
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Keywords: | ALS; Amyotrophic Lateral Sclerosis; Parkinson's; cystatin C; α-synuclein; POPG |
Awarding institution: | University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Science (Sheffield) > School of Biosciences (Sheffield) |
Depositing User: | Mr Alexander Arscott-Barber |
Date Deposited: | 25 Jun 2025 10:06 |
Last Modified: | 25 Jun 2025 10:06 |
Open Archives Initiative ID (OAI ID): | oai:etheses.whiterose.ac.uk:37063 |
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