Alpha-Synuclein FRET Biosensors Reveal Early Alpha-Synuclein Aggregation in the Endoplasmic Reticulum.

Endoplasmic reticulum (ER) dysfunction is important for alpha-synuclein (?S) acquired toxicity. When targeted to the ER in SH-SY5Y cells, transient or stable expression of ?S resulted in the formation of compact ?S-positive structures in a small subpopulation of cells, resembling ?S inclusions. Thus, because of the limitations of immunofluorescence, we developed a set of ?S FRET biosensors (AFBs) able to track ?S conformation in cells. In native conditions, expression in i36, a stable cell line for ER ?S, of intermolecular AFBs, reporters in which CFP or YFP has been fused with the C-terminal of ?S (?S-CFP/?S-YFP), resulted in no Förster resonance energy transfer (FRET), whereas expression of the intramolecular AFB, a probe obtained by fusing YFP and CFP with ?S N- or C- termini (YFP-?S-CFP), showed a positive FRET signal. These data confirmed that ?S has a predominantly globular, monomeric conformation in native conditions. Differently, under pro-aggregating conditions, the intermolecular AFB was able to sense significantly formation of ?S oligomers, when AFB was expressed in the ER rather than ubiquitously, suggesting that the ER can favor changes in ?S conformation when aggregation is stimulated. These results show the potential of AFBs as a new, valuable tool to track ?S conformational changes in vivo.