AMYLOIDS-TCCD · Early stages of amyloid fibrils formation elucidated by single-molecule fluorescence two-colour coincidence detection

Final Activity Report Summary - AMYLOIDS-TCCD (Early stages of amyloid fibrils formation elucidated by single-molecule fluorescence two-colour coincidence detection)

The amyloid diseases, including for instance Alzheimer's Crueztfeld-Jakob's and Parkinson's diseases, represent nowadays a major area of research due to the high incidence of these diseases, especially in aged population, and the mechanism by which cells are killed is not understood. To address this multi-disciplinary approaches are needed to elucidate the mechanisms of amyloid fibril formation and cytotoxicity. It is currently accepted that species formed during the early stage of the reaction most likely represent the critical pathological species in many amyloid disorders. These species are small soluble aggregates comprising several units of the misfolded protein, so-called oligomers, and they likely are building blocks of the mature fibrils. However, a full characterisation of these oligomers in the early stages is challenging because they are typically very heterogeneous, partially unstable, and present in low abundance. In this fellowship, the detection and characterisation of amyloidogenic oligomers was tackled through an innovative technique based on the direct counting of individual molecules one by one called single-molecule fluorescence. The methodology relies on attaching a fluorescent tag to the protein of interest so that the protein can be detected by exciting the tag with a laser and collecting the fluorescence emitted by single molecules. A novel two-colour single-molecule fluorescence methodology, developed in the host group, has provided the required sensitivity and specificity to detect very low populations of oligomers, but also provided quantitative information on relative populations and size heterogeneity, oligomers stability, and aggregation mechanisms. The methodology was initially validated using a model amyloidogenic SH3 domain. Nevertheless, in the final stage of the fellowship the two-colour single-molecule fluorescence technique was importantly demonstrated to be of general application, having been applied to biomedically relevant, disease-related proteins as neuroserpin, alpha-synuclein, and amyloid-beta fragments. The biomedical relevance of these systems and the unique capabilities of the single-molecule technology make the current project an important advance in the amyloid fibrils field. Importantly, our single molecule fluorescence methodology is of general application in the study of early events in the amyloids formation reaction, and is currently being used to detect and analyse oligomers from disease-related proteins such as alpha-synuclein or amyloid-beta fragments.

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