ABSTRACT:

Alpha-synuclein is a 140-residue natively unfolded protein, whose aggregation is implicated in the development of Parkinson;s Disease. It is thought that the cytotoxic species is not the mature fibril, but rather a prefibrillar aggregate which has membrane-permeabilizing properties. We have used Small Angle X-ray Scattering (SAXS) to determine the low-resolution structures of the different species formed during alpha-synuclein fibrillation in a non-invasive fashion. In addition to the starting monomer-dimer equilibrium and two bona fide fibril types accumulating towards the end of the aggregation process, we have identified a wreath-shaped oligomeric state which has a very distinct central hole. Both its structure and the kinetics of its formation are consistent with an on-pathway role, while its membrane-permeabilizing properties identify it as a putative cytotoxic species. We have also used SAXS to monitor the fibrillation of alpha-synuclein in the presence of the surfactant SDS and find that the fibrillar aggregates grow in a continuous fashion, forming beads on a string where the individual beads are stabilized by intermolecular a-synuclein contacts. The high reproducibility of this aggregative behaviour has formed the basis for a high-throughput screening assay involving 746.000 compounds that has allowed us to identify a significant number of compounds with the ability to inhibit early-stage aggregation of alpha-synuclein. This distinguishes the assay from previous assays that have focused mainly on the ability to prevent formation of alpha-synuclein fibrils. The hits from our assay may form the basis for a therapeutic intervention against Parkinson’s Disease.