Properties of iron-induced alpha-synuclein oligomers determined on a single particle level

Formation of fibrillar protein aggregates is a key event in several neurodegenerative diseases including Parkinson's disease, which is characterized by Lewy bodies and degeneration of dopaminergic neurons. Several lines of evidence implicate involvement of alpha-synuclein oligomers as the principal toxic aggregate species as well as iron ions in the disease progression in Parkinson's disease. Using three independent single particle-based methods we could recently analyze aggregation pathways and oligomer formation of alpha-synuclein. Biophysical and structural characterization was performed by confocal single molecule fluorescence techniques and atomic force microscopy. Functional characterization included single pore electrophysiology in a lipid bilayer set-up. We characterized two different oligomer species. Organic solvents were used to trigger aggregation, which resulted in small oligomers („intermediate I“). Under these conditions, additional Fe3+- ions at low micromolar concentrations dramatically increased aggregation and induced formation of larger oligomers („intermediate II“). Both oligomer species were on-pathway to amyloid fibrils and could seed amyloid formation [1]. Notably, only Fe3+-induced oligomers were SDS-resistant. In contrast to monomers and intermediate I, only Fe3+-induced intermediate II species were able to bind to unilamelar vesicles composed of POPC (palmitoyl-oleoyl-phosphatidylcholine). Deferoxamine and Vitamin C were able to dissolute the aggregates formed in presence of Fe³+. Interestingly, Deferoxamine shows positive effects in an animal model of PD. In regard to toxicity, we could show that the intermediate II species were able to form ion-permeable pores in a planar lipid bilayer.

Finally, we developed a high-throughput confocal single-particle alpha-synuclein aggregation assay and identified Baicalein and N((prime))-benzylidene-benzohydrazide derivates inhibiting oligomer formation. Our results elucidate the role and biochemical properties of toxic oligomer species in neurodegenerative diseases and provide new approaches for drug-development.

References: 1. Kostka M, Högen T, Danzer KM, Levin J, et al., Single-particle characterization of iron-induced pore-forming alpha -synuclein Oligomers. J Biol Chem. 2008 Feb 7 [Epub ahead of print].