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

摘要

Aggregation of a-synuclein and formation of inclusionsare hallmarks of Parkinson’s disease (PD).Aggregate formation is affected by cellular environment,but it has been studied almost exclusively incell-free systems. We quantitatively analyzed a-synucleininclusion formation and clearance in a yeast cellmodel of PD expressing either wild-type (WT) a-synucleinor the disease-associated A53T mutant from thegalactose (Gal)-inducible promoter. A computercontrolledmicrofluidics device regulated a-synucleinin cells by means of closed-loop feedback control.We demonstrated that inclusion formation is strictlyconcentration dependent and that the aggregationthreshold of the A53T mutant is about half of the WTa-synuclein (56%). We chemically modulated theproteasomal and autophagic pathways and demonstratedthat autophagy is the main determinant ofA53T a-synuclein inclusions’ clearance. In additionto proposing a technology to overcome current limitationsin dynamically regulating protein expressionlevels, our results contribute to the biology of PDand have relevance for therapeutic applications.