Single-molecule detection and characterisation of alpha-synuclein aggregates
Aberrant protein aggregation is a predominant feature of many neurodegenerative disorders. It has long been recognised that aggregates of alpha-synuclein (α-syn) drive pathogenesis in Parkinson’s Disease (PD), and it is widely accepted that small α-syn oligomers are the key cytotoxic species in PD. Notably, however, these oligomeric species are difficult to characterise using traditional biochemical ensemble methods due to their high level of heterogeneity and low abundance. Single-molecule fluorescence microscopy techniques have emerged as a suitable approach to circumventing this problem, enabling the detection of individual aggregates amongst monomeric protein and thus facilitating the identification, quantification, and characterisation of rare oligomeric species. However, cellular mechanisms of α-syn aggregation are poorly understood. Furthermore, there remains some limitations to the singlemolecule techniques currently available. This thesis describes the work completed to address some of these issues. Chapter 1 provides the contextual background for the work presented in this thesis, detailing the biological aspects of α-syn, its aggregation, and its implications in PD, as well as outlining the single-molecule techniques used to investigate aggregate species. Chapter 2 describes the methodologies undertaken in this thesis, and chapters 3 to 5 describe the findings made using the single-molecule techniques which were utilised and developed in this work. One primary approach for studying species in single-molecule experiments involves directly labelling biomolecules of interest with a suitable fluorophore. Early steps in α-syn aggregation have previously been identified using fluorescently tagged α-syn and single-molecule Förster resonance energy transfer (smFRET) in vitro; however, the characterisation of early aggregate formation in cells has thus far been difficult to achieve. Chapter 3 describes the use of duallabelled α-syn to detect and characterise aggregates formed both intracellularly and in vitro via smFRET, using both single-molecule confocal microscopy coupled with microfluidics and iii total internal reflection fluorescence microscopy (TIRFM) to determine both the sizes and structures of the oligomers formed. This work reveals the presence of distinct oligomeric species in vitro and in neurons resulting from structural conversion during early aggregate formation. The approach taken in Chapter 3 is highly suitable for investigating aggregate formation resulting from the addition of exogenous α-syn to samples of interest. However, such an approach is not ideal for the detection and characterisation of endogenous aggregates due to issues with the covalent labelling of cellular protein. Extrinsic amyloid dyes are typically used as an alternative approach to labelled protein; however, such dyes are non-protein-specific and bind to the common amyloid beta-sheet motif. As an alternative, the work presented in Chapter 4 describes a novel single-molecule method to specifically detect and characterise α-syn aggregates with high sensitivity, making use of a high-affinity antibody labelled with orthogonal fluorophores which is combined with fast-flow microfluidics and single-molecule confocal microscopy. This enables the quantification and size approximation of α-syn aggregates at picomolar concentrations, both in vitro and in biological samples. Although the kinetics of α-syn aggregation have been studied extensively, much of our current knowledge stems from ensemble averaging techniques which are associated with high levels of variability and are not conducive to detecting the earliest steps in aggregate formation. In addition, there remains uncertainty surrounding the effect of familial variants and posttranslational modifications (PTM) on aggregation. Chapter 5 encompasses the study of the effects of the ubiquitous N-terminal acetylation PTM, in addition to the familial, rapid-onset G51D mutation, on α-syn aggregation, using the novel detection method developed in Chapter 4. This is used in conjunction with single-molecule detection with thioflavin-T (ThT) to reveal new insights into the aggregation of α-syn variants. Overall, the work presented here provides new insights into the aggregation of α-syn via the use and development of single-molecule techniques. The advancements made have added to the current understanding of the molecular mechanisms of α-syn aggregation, both in vitro and in neurons, and have also been used to develop a novel single-molecule detection method for α-syn aggregates. The work presented in this thesis has resulted in two published papers, ’Pathological structural conversion of alpha-synuclein at the mitochondria induces neuronal toxicity’ in Nature Neuroscience, and ’Single-molecule two-color coincidence detection of unlabeled alpha-synuclein aggregates’ in Angewandte Chemie International Edition. Furthermore, the novel detection method presented here holds promise for measuring α-syn oligomeric load in clinical samples due to its high sensitivity and specificity for α-syn aggregates. This may therefore be used in future studies for identifying, detecting, and studying potential biomarkers in PD, with potential use in disease diagnosis. It is therefore expected that the work from this thesis will be used to aid researchers towards better understanding the mechanisms of α-syn aggregation, both in vitro and in clinical samples.