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

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