Parkinson's disease in the spotlight: unraveling nanoscale α-Synuclein oligomers using ultrasensitive single-molecule spectroscopy

During the last 15 years, we have witnessed a major shift in the research focus to understand the cause of amyloid diseases. The attention has shifted from the fully matured amyloid fibrils to the nanometer sized aggregation intermediates called oligomers as the potentially cytotoxic species that are at the basis of these diseases. In this thesis, we focused on alpha-Synuclein oligomers, potentially key players in Parkinson’s disease. Ever since the first realization that the alpha-Synuclein oligomers are cytotoxic and might be responsible for Parkinson's disease, much effort has been devoted to: fully characterize these oligomers in terms of structure, morphology, and aggregation number, that is, the number of monomers forming one oligomer. This information is essential for understanding the disease process and to identify specific targets for pharmaceutical intervention. However, even information on the aggregation numbers of the different oligomers is lacking, simply because standard techniques struggle to provide reliable result. Therefore, a more direct approach is needed that avoids these problems. We developed a new approach to determine the aggregation number of protein aggregates that combines single-molecule photobleaching and sub-stoichiometric labeling. By counting the number of discrete photobleaching steps in the intensity time traces for a large number of distinct oligomers and by applying a statistical analysis on the histogram of bleaching steps, the aggregation number can be determined. We used this approach to study the aggregation numbers of alpha-Synuclein oligomers formed under several different aggregation conditions. Interestingly, in contrast to earlier reports, our findings show that alpha-Synuclein forms oligomers of a defined number of monomers and not of a wide distribution in the number of monomers per oligomer. Depending on the conditions, oligomers of different aggregation number are formed. These findings of species of distinct aggregation number imply that oligomers organize in a stable structure, but that the structure depends on the aggregation conditions. Linking the well-defined aggregation number and specific structure of the oligomers to their cytotoxicity may allow insights into the cause of the disease and provide specific targets for pharmaceutical intervention.