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

Fabiana Miraglia,Gino Giannaccini,Valentina Quercioli,Emanuela Colla,Laura Betti,Lucia Rota,Antonino Cattaneo,Verdiana Valvano,Cristina Di Primio

doi:10.3390/life10080147

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.

Highlights

In the last decade, the necessity to investigate alpha-synuclein conformational changes in vivo and the pathological consequences of its misfolding on cell function, gave rise to the development of fluorescent molecular tools, able to track the formation of αS high molecular weight (HMW) species [1,2,3,4,5].αS, the dominant component of Lewy bodies, proteinaceous inclusions found in Parkinson’s disease (PD)patients, can aggregate in highly ordered protofibrils, according to a nucleation reaction, with different intermediate states of oligomer assembly [6]
Biosensors (AFBs) with the aim to detect a variety of αS structures in cells and we used them to investigate αS’s link to the endoplasmic reticulum (ER), a cellular organelle that has been shown to be affected by αS toxicity [17]
Immunofluorescence analysis confirmed the presence of αS-positive structures in about 20–25% of cells, no clear sign of cellular toxicity was associated with prolonged induction of ER-αS or with formation of ER-αS

Summary

Introduction

The necessity to investigate alpha-synuclein (αS) conformational changes in vivo and the pathological consequences of its misfolding on cell function, gave rise to the development of fluorescent molecular tools, able to track the formation of αS high molecular weight (HMW) species [1,2,3,4,5].αS, the dominant component of Lewy bodies, proteinaceous inclusions found in Parkinson’s disease (PD)patients, can aggregate in highly ordered protofibrils, according to a nucleation reaction, with different intermediate states of oligomer assembly [6]. Development of FRET reporters based on the fusion of αS with YFP and/or CFP provided insights on αS native conformation in cells and its ability to propagate [1,16]. With this in mind, in the present work, we developed a set of αS FRET biosensors (AFBs) with the aim to detect a variety of αS structures in cells and we used them to investigate αS’s link to the endoplasmic reticulum (ER), a cellular organelle that has been shown to be affected by αS toxicity [17]. Comparison of the ability to FRET of AFBs when expressed in the ER or ubiquitously in native or pro-aggregating conditions, suggests that αS compartmentalization in the ER can favor formation of HMW species

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