Quantitative Characterization of α-Synuclein Aggregation in Living Cells through Automated Microfluidics Feedback Control

Giansimone Perrino,Cathal Wilson,Marco Santorelli,Diego Di Bernardo

doi:10.1016/j.celrep.2019.03.081

Giansimone Perrino, Cathal Wilson + Show 2 more

Open Access

https://doi.org/10.1016/j.celrep.2019.03.081

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Abstract

SummaryAggregation of α-synuclein and formation of inclusions are hallmarks of Parkinson’s disease (PD). Aggregate formation is affected by cellular environment, but it has been studied almost exclusively in cell-free systems. We quantitatively analyzed α-synuclein inclusion formation and clearance in a yeast cell model of PD expressing either wild-type (WT) α-synuclein or the disease-associated A53T mutant from the galactose (Gal)-inducible promoter. A computer-controlled microfluidics device regulated α-synuclein in cells by means of closed-loop feedback control. We demonstrated that inclusion formation is strictly concentration dependent and that the aggregation threshold of the A53T mutant is about half of the WT α-synuclein (56%). We chemically modulated the proteasomal and autophagic pathways and demonstrated that autophagy is the main determinant of A53T α-synuclein inclusions’ clearance. In addition to proposing a technology to overcome current limitations in dynamically regulating protein expression levels, our results contribute to the biology of PD and have relevance for therapeutic applications.

Highlights

Cell-free, cellular, and animal models of Parkinson’s disease (PD) have been developed to study the formation of inclusions (Visanji et al, 2016; Koprich et al, 2017; Lazaro et al, 2017)
We demonstrated that inclusion formation is strictly concentration dependent and that the aggregation threshold of the A53T mutant is about half of the WT a-synuclein (56%)
In addition to proposing a technology to overcome current limitations in dynamically regulating protein expression levels, our results contribute to the biology of PD and have relevance for therapeutic applications

Summary

Introduction

Cell-free, cellular, and animal models of PD have been developed to study the formation of inclusions (Visanji et al, 2016; Koprich et al, 2017; Lazaro et al, 2017). Subsequent in vitro studies, building on these previous works, have precisely quantified the molecular steps of a-synuclein fibril formation and rate constants of associated reactions, greatly contributing to current understanding of a-synuclein pathobiology (Giehm et al, 2011; Cohen et al, 2011, 2012; Buell et al, 2014; Garcia et al, 2014; Lorenzen et al, 2014; Galvagnion et al, 2015, 2016; Flagmeier et al, 2016; Iljina et al, 2016) These in vitro studies have shown that a-synuclein aggregation kinetics are strongly affected by the presence of lipid vesicles, highlighting the importance of studying such processes in whole cells, because the cellular environment is much more complex than the commonly used in vitro conditions (Flagmeier et al, 2016; Galvagnion et al, 2015)

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