Novel methods to study amyloid oligomers

Amyloid protein deposits are the hallmark of neurodegenerative diseases such as Alzheimer’s, Parkinson’s, or diabetes type II. Although their connection to the diseases is not fully understood, many studies point at intermediate species called oligomers as being the main responsible for the toxicity of these proteins. This makes them strong candidates as therapeutic targets. However, due to their low relative concentration, heterogenicity and transient nature, methods to study and characterize oligomers are lacking. First, we optimized the cross-linking method photo-induced crosslinking of unmodified proteins (PICUP). By cross-linking monomers within an oligomer together, the otherwise transient interaction can be turned into covalent ones, allowing one to study oligomers with great ease. Our optimized method has allowed us to observe cross-linking of alpha-synuclein (αSyn) as quickly as with 1 ms reaction, and we have proven this method to be a valuable tool for oligomer studies. Moreover, we have developed a mass-spectrometry analysis protocol to gain structural insight into the oligomers, allowing us to understand the behavior of αSyn oligomers during aggregation, their binding to lipid vesicles, and their interaction with chaperone DNJB6. Second, we have made use of a custom made confocal fluorescence set-up to perform microfluidic free flow electrophoresis on amyloid-beta (Aβ₄₂). This method allows us to separate oligomers of different electrophoretic mobility from each other and perform single-molecule fluorescence measurements to count the amount and size of every species in a single sample. With the help of this method, we have studied differences in oligomer population between idle and agitated aggregation, synthetic and recombinant Aβ₄₂ and even the ability of fibrils to catalyze oligomer dissociation.