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

Abstract

Abbreviations:a-syn: a-synuclein; a-syn-V: a-synuclein-full length Venus; Vn-a-syn: n-terminal fragment of Venus fused to the amino-terminus of a-syn; a-syn-Vc: c-terminal fragment of Venus fused to the carboxy-terminus of a-syn; BiFC: bimolecular fluorescent complementation; PD: Parkinson´s disease; SEC: size exclusion chromatography Abstract Bimolecular 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, including mouse, rat and Drosophila models, based on the assumption that an increase in the fluorescence 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 by the fluorescent complex resemble oligomers that form on the pathway to α-syn fibrillization. To address this knowledge gap, we first analyzed 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 cells 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 two BiFC fragments are not expressed at the same level since C-terminal-Venus fused to α-syn (a-syn-Vc) was present in very low abundance; 2) the fragment with Nterminal-Venus fused to α-syn (Vn-a-syn) exhibited a high propensity to form oligomers and higher-order aggregates; 3) the expression of either or both fragments does not result in the formation of α-syn fibrils, cellular inclusions or the accumulation of pS129 immunoreactive αsyn aggregates. Furthermore, our results suggest that only a small fraction of Vn-a-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 a-syn-Vc in Vn-a-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 oligomerization, aggregation and cell-tocell transmission assays and model systems based on a-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.

Studies on alpha-synuclein and islet amyloid polypeptide interaction

“…The intensive dot-like fluorescence that developed in cells expressing Aβ/IAPP could not be seen. This is in line with earlier reports on aSyn-aSyn interaction studies using BiFC where no amyloid formation has been detected (Frey et al, 2021). The results argue against the formation of amyloid composed of IAPP and aSyn.…”

FRET‐based assay for intracellular evaluation of α‐synuclein aggregation inhibitors

“…FRET approach allows to detect the interaction between αSyn monomers labeled with two different fluorophores. As fluorescent protein tags strongly interfere with αSyn fibrillization (Afitska et al, 2017; Frey et al, 2021), we selected relatively small organic fluorescent labels, Atto565 and Atto647. When αSyn is in fibrillar form, the labels are close to each other (<5 nm) and hence may undergo FRET, i.e.…”