Modulation of alpha-synuclein toxicity in yeast using a novel microfluidic-based gradient generator.

João Tiago S Fernandes,Tiago F Outeiro,Virginia Chu,João P Conde,Sandra Tenreiro,Andreia Gameiro

doi:10.1039/c4lc00756e

Abstract

Parkinson's disease (PD) is a common age-associated neurodegenerative disorder. The protein α-synuclein (aSyn) is a key factor in PD both due to its association with familial and sporadic cases and because it is the main component of the pathological protein aggregates known as Lewy bodies. However, the precise cellular effects of aSyn aggregation are still elusive. Here, we developed an elastomeric microfluidic device equipped with a chemical gradient generator and 9 chambers containing cell traps to study aSyn production and aggregation in Saccharomyces cerevisiae. This study involved capturing single cells, exposing them to specific chemical environments and imaging the expression of aSyn by means of a GFP fusion (aSyn-GFP). Using a galactose (GAL) gradient we modulated aSyn expression and, surprisingly, by tracking the behavior of single cells, we found that the response of individual cells in a population to a given stimulus can differ widely. To study the combined effect of environmental factors and aSyn expression levels, we exposed cells to a gradient of FeCl3. We found a dramatic increase in the percentage of cells displaying aSyn inclusions from 27% to 96%. Finally, we studied the effects of ascorbic acid, an antioxidant, on aSyn aggregation and found a significant reduction in the percentage of cells bearing aSyn inclusions from 87% to 37%. In summary, the device developed here offers a powerful way of studying aSyn biology with single-cell resolution and high throughput using genetically modified yeast cells.

Highlights

IntroductionThe budding yeast Saccharomyces cerevisiae has been widely explored as a model organism to investigate the molecular underpinnings of several human diseases.[1,2,3] This yeast is the best-characterized eukaryotic cell, is easy to grow under defined conditions, and can be genetically manipulated.[4,5]
The budding yeast Saccharomyces cerevisiae has been widely explored as a model organism to investigate the molecular underpinnings of several human diseases.[1,2,3] This yeast is the best-characterized eukaryotic cell, is easy to grow under defined conditions, and can be genetically manipulated.[4,5]To investigate the basic molecular effects of aSyn in the context of a living cell, human aSyn was expressed in yeast and found to induce dose-dependent cytotoxicity.[12]
The microfluidic device relies on two connected modules: a chemical gradient generator and a set of 9 chambers each containing hydrodynamic traps for yeast cells (Fig. 1A–C)

Summary

Introduction

The budding yeast Saccharomyces cerevisiae has been widely explored as a model organism to investigate the molecular underpinnings of several human diseases.[1,2,3] This yeast is the best-characterized eukaryotic cell, is easy to grow under defined conditions, and can be genetically manipulated.[4,5]. To investigate the basic molecular effects of aSyn in the context of a living cell, human aSyn was expressed in yeast and found to induce dose-dependent cytotoxicity.[12] Using GFP as a reporter and the galactose (GAL)-inducible promoter, aSyn-GFP fusions were found to initially associate with the plasma membrane and to accumulate in cytoplasmic inclusions reminiscent of protein aggregates.[12] Several additional studies followed, exploiting this yeast model for high-throughput analyses that proved instrumental in informing on the molecular mechanisms involved in aSyn toxicity.[13,14,15,16]. Microfluidic devices offer the possibility of performing biological experiments with small amounts of reagents while still being able to parallel process tens or hundreds of experiments in a very small area.[17,18] Since in microfluidic channels liquids

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