Size-Dependent Characterization of Alpha-Synuclein Aggregates Unveils Their Toxicity

Neurodegenerative diseases are devastating, incurable and an increasing global concern. As life-expectancy increases so does the number of people affected. By 2050, over 12 million people worldwide above the age of 65 will suffer from Parkinson's disease (PD). However, the understanding of the molecular pathways involved in this and other neurodegenerative diseases is limited. The aggregation of intrinsically disordered proteins such as α-synuclein has been associated with neuronal loss in PD. Evidence suggests that the small soluble aggregates formed during the aggregation process are likely to be the cytotoxic species. The disease relevant properties of small aggregates are challenging to study with traditional biophysical techniques because of their low abundance and heterogeneity in size, structure and stability. We approach this problem by using an array of biophysical tools at both the bulk and single-molecule regimes to correlate the structure and the subsequent toxicity of differently sized α-synuclein aggregates. We have optimised a density gradient centrifugation method to separate aggregates by size and then confirm the successful size separation using super-resolution fluorescence microscopy. We then assessed the cytotoxic potential of the sized aggregates by quantifying both the membrane disruption and the consequent influx of Ca2+ into single-liposomes and the production of TNF-α by microglia-like cells upon incubation with the aggregates. Together these results allowed us to determine the fraction containing the most toxic species. Finally, we are applying the same methods we developed for recombinant α-synuclein aggregates to study small protein aggregates present in biologically relevant samples such as PD brain tissue. This is the first time to our knowledge that this has been achieved and it will provide new insights into the failure of protein homeostasis in PD.