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Article

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

by
Fabiana Miraglia
1,2,
Verdiana Valvano
1,
Lucia Rota
1,
Cristina Di Primio
1,
Valentina Quercioli
1,
Laura Betti
2,
Gino Giannaccini
2,
Antonino Cattaneo
1,3 and
Emanuela Colla
1,*
1
Bio@SNS Laboratory, Scuola Normale Superiore, 56126 Pisa, Italy
2
Department of Pharmacy, University of Pisa, 56126 Pisa, Italy
3
Neurotrophins and Neurodegenerative Diseases Laboratory, Rita Levi-Montalcini European Brain Research Institute, 00161 Rome, Italy
*
Author to whom correspondence should be addressed.
Life 2020, 10(8), 147; https://doi.org/10.3390/life10080147
Submission received: 26 June 2020 / Revised: 6 August 2020 / Accepted: 7 August 2020 / Published: 11 August 2020
(This article belongs to the Collection Function, Regulation, and Dysfunction of Intrinsically Disordered Proteins)
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Abstract

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

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MDPI and ACS Style

Miraglia, F.; Valvano, V.; Rota, L.; Di Primio, C.; Quercioli, V.; Betti, L.; Giannaccini, G.; Cattaneo, A.; Colla, E. Alpha-Synuclein FRET Biosensors Reveal Early Alpha-Synuclein Aggregation in the Endoplasmic Reticulum. Life 2020, 10, 147. https://doi.org/10.3390/life10080147

AMA Style

Miraglia F, Valvano V, Rota L, Di Primio C, Quercioli V, Betti L, Giannaccini G, Cattaneo A, Colla E. Alpha-Synuclein FRET Biosensors Reveal Early Alpha-Synuclein Aggregation in the Endoplasmic Reticulum. Life. 2020; 10(8):147. https://doi.org/10.3390/life10080147

Chicago/Turabian Style

Miraglia, Fabiana, Verdiana Valvano, Lucia Rota, Cristina Di Primio, Valentina Quercioli, Laura Betti, Gino Giannaccini, Antonino Cattaneo, and Emanuela Colla. 2020. "Alpha-Synuclein FRET Biosensors Reveal Early Alpha-Synuclein Aggregation in the Endoplasmic Reticulum" Life 10, no. 8: 147. https://doi.org/10.3390/life10080147

APA Style

Miraglia, F., Valvano, V., Rota, L., Di Primio, C., Quercioli, V., Betti, L., Giannaccini, G., Cattaneo, A., & Colla, E. (2020). Alpha-Synuclein FRET Biosensors Reveal Early Alpha-Synuclein Aggregation in the Endoplasmic Reticulum. Life, 10(8), 147. https://doi.org/10.3390/life10080147

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