Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: what you see is not always what you get

Abstract

AbstractBimolecular fluorescence complementation (BiFC) was introduced a decade ago as a method to monitor alpha-synuclein (α-syn) oligomerization in intact cells. Since then, several α-syn BiFC cellular assays and animal models have been developed based on the assumption that an increase in the fluorescent signal correlates with increased α-syn oligomerization or aggregation. Despite the increasing use of these assays and models in mechanistic studies, target validation and drug screening, there have been no reports that 1) validate the extent to which the BiFC fluorescent signal correlates with α-syn oligomerization at the biochemical level; 2) provide a structural characterization of the oligomers and aggregates formed by the BiFC fragments; or 3) investigate the extent to which the oligomers of the fluorescent complex resemble oligomers formed on the pathway to α-syn fibrillization. To address this knowledge gap, we first analysed the expression level and oligomerization properties of the individual constituents of α-syn-Venus, one of the most commonly used BiFC systems, in HEK-293 & SH-SY5Y cells from three different laboratories using multiple approaches, including size exclusion chromatography, semiquantitative Western blot analysis, in-cell crosslinking, immunocytochemistry and sedimentation assays. Next, we investigated the biochemical and aggregation properties of α-syn upon co-expression of both BiFC fragments. Our results show that 1) the C-terminal-Venus fused to α-syn (α-syn-Vc) is present in much lower abundance than its counterpart with N-terminal-Venus fused to α-syn (Vn-α-syn) ; 2) Vn-α-syn exhibits a high propensity to form oligomers and higher-order aggregates; and 3) the expression of either or both fragments does not result in the formation of α-syn fibrils or cellular inclusions. Furthermore, our results suggest that only a small fraction of Vn-α-syn is involved in the formation of the fluorescent BiFC complex and that some of the fluorescent signal may arise from the association or entrapment of α-syn-Vc in Vn-α-syn aggregates. The fact that the N-terminal fragment exists predominantly in an aggregated state also indicates that one must exercise caution when using this system to investigate α-syn oligomerization in cells or in vivo. Altogether, our results suggest that cellular and animal models of oligomerization, aggregation and cell-to-cell transmission that are based on the α-syn BiFC systems should be thoroughly characterized at the biochemical level to ensure that they reproduce the process of interest and measure what they are intended to measure.Graphical AbstractBimolecular fluorescence complementation (BiFC) was introduced a decade ago to monitor alpha-synuclein oligomerization in intact cells, based on the assumption that an increase in the fluorescent signal correlates with α-synuclein oligomerization and aggregation. Herein, we used several biochemical and cellular assays to characterize commonly used α-synuclein Venus BiFC assays. Our results show that one of the BiFC fragments (Vn-α-synuclein) exhibits higher expression levels and aggregation propensity than its counterpart (α-synuclein-Vc), thus complicating the interpretation of the molecular interactions that give rise to the fluorescence signal and raise concerns about their application to investigate α-syn oligomerization in cells or in vivo.