Using leucine zipper to facilitate α-synuclein assembly

The accumulation of filamentous α-synuclein (α-S) is associated with Parkinson's disease. It remains controversial as to the mode (antiparallel or parallel) of α-S self-assembly and whether an exact alignment of the central hydrophobic region is essential. In the present study, we performed in vitro assembly using α-S with or without the attachment of artificial leucine zippers (Zips) capable of forming either parallel or antiparallel coiled coils and included a spacer in one derivative. Results showed that Zips accelerate filament assembly in both the parallel and antiparallel fashions, that a precise alignment of the central hydrophobic region is not essential, and that the antiparallel pairs displayed the highest thioflavin T signals. More importantly, cells expressing Zip-fused α-S, but not α-S alone, formed α-S immunopositive and thioflavin S-positive inclusions in 7 days. The results suggest that α-S can assemble in both parallel and antiparallel modes but have a higher tendency to assemble in the latter mode and that cells overexpressing Zip-fused α-S may be used to screen α-S assembly inhibitors due to enhanced ability to form inclusions.