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

Highlights • In silico feedback control enables regulation of α-synuclein expression in yeast • α-Synuclein inclusion formation is strictly concentration, but not time, dependent • The aggregation threshold of the α-synuclein A53T mutant is 56% of the wild-type • Autophagy induction speeds up inclusion clearance in the A53T α-synuclein strain Summary Aggregation 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