Prions are infectious agents that cause fatal neurodegenerative diseases in the brain. The wide-accepted protein-only hypothesis states that the misfolded form of prion protein (PrP) is the sole constituent of prions, and the self-propagating process of PrP is considered to play a central role in prion pathogenesis. Prions are believed to propagate when a PrP assembly enters a cell and replicates to produce two or more fibrils, leading to an exponential increase in PrP aggregate number with time. However, the molecular basis of this process has not yet been established in detail. This prion-like replication is also suggested to be the mechanism in the development of other notorious neurodegenerative disorders, such as Alzheimer's and Parkinson's disease. In this thesis, I use single-aggregate imaging to study fibril fragmentation and elongation of individual murine PrP aggregates from seeded aggregation in vitro. From fluorescence imaging of individual PrP aggregates on the coverslip surface, elongation and fragmentation of the PrP assemblies have been directly observed. PrP elongation occurs via a structural conversion from a proteinase K (PK)-sensitive to PK-resistant conformer. Fibril fragmentation was found to be length-dependent and resulted in the formation of PK-sensitive fragments. To gain more insights into the mechanism of the spread of PrP, the quantified kinetic profiles allows the determination of the rate constants for these processes through the use of kinetic modelling. This enables the estimation of a simple framework for aggregate propagation through the brain, assuming that doubling of the aggregate number is rate-limiting. In contrast, the same method was applied to measurement for α-Synuclein (αS) aggregation, which has been suggested to be prion-like and is associated with Parkinson's disease. While αS aggregated by the same mechanism, it showed significantly slower elongation and fragmentation rate constants than PrP, leading to much slower replication rate. Furthermore, the measurements in αS aggregation has been extended to the cellular environment, I use super-resolution imaging to study the amplification of endogenous αS aggregation in cells and the transcellular spread of αS. Endogenous αS showed a clear amplification in number of aggregates with time after seed transduction, and the newly-formed αS aggregates are likely to spread through cell-to-cell transmission. The proteasome was demonstrated to possess a novel disaggregase function for αS fibrils and thus produce more seeds for further replication. It partially explains that αS aggregation in cells was found to replicate at a substantially faster rate than that in vitro. Determining the nature of the oligomers formed during aggregation has been experimentally difficult due to the lack of suitable methods capable of detecting and characterising the low level of oligomers. To address this problem, I have studied the early formation of PrP oligomers formed during aggregation in vitro using various single-molecule methods. The early aggregation of PrP is observed to form a thioflavin T (ThT)-inactive and two ThT-active species of oligomers, which differ in size and temporal evolution. The ThT-active oligomers undergo a structural conversion from a PK-sensitive to PK-resistant conformer, while a fraction of which grow into mature fibrils. These results also enable the establishment of a kinetic framework for elucidating temporal evolution of PrP aggregation and the relationship between oligomers and fibrils. Overall, my research identifies fibril elongation with fragmentation are the key molecular processes leading to PrP and αS aggregate replication, an important concept in prion biology, and provides a simple framework to estimate the rate of prion and prion-like spreading in animals. The results also show that a diverse range of oligomers is formed and co-exist during PrP aggregation which differ both in their structure and properties and provides mechanistic insights into a prion aggregation. The work provides a new quantitative approach to describe the prion-like property in neurodegenerative diseases from a kinetic perspective that can be verified in extending studies in other proteins or in cells.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:763856 |
| Date | January 2019 |
| Creators | Sang, Chieh |
| Contributors | Klenerman, David |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.repository.cam.ac.uk/handle/1810/287929 |
Page generated in 0.0016 seconds