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

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.

Microfluidic chips for the study of cell migration under the effect of chemicals

“…The key advantages of MFCs are the possibility to configure fluid flows and to create and stabilize the concentration gradient of chemicals, low consumption in reagents, and setting of high-performance analyzes [10]. The chemoattractant concentrations formed in MFCs can vary from a few tenths to hundreds of nanograms per milliliter [11,12].…”

From the baker to the bedside: yeast models of Parkinson's disease

“…Ascorbic acid, a natural antioxidant, was found to promote a significant reduction in the percentage of yeast cells bearing aSyn inclusions 189. Remarkably, this study was performed using a new microfluidic device designed to validate compounds in yeast.…”

“…Remarkably, this study was performed using a new microfluidic device designed to validate compounds in yeast. Additional advantages are achieved by using this device, since it offers a powerful way for studying aSyn biology with single-cell resolution and high-throughput, using genetically modified yeast cells 189.…”

A microfluidic platform for physical entrapment of yeast cells with continuous production of invertase

“…The greater control over the local chemical and physical conditions inside microfluidic devices paves the way for the development of microfluidic bioreactors. Microreactors have the potential to reduce the time necessary for a biological process to go from the laboratory scale to the industrial scale by enabling high throughput cell and culture condition screening assays . Health care can also benefit from microreactor development through the creation of ‘organ‐on‐a‐chip’ devices, which can be very powerful tools for speeding up drug and therapy development .…”

“…Research groups working in the area of microfluidics have made significant advances in the past decade on microbioreactors applying them to the analysis of cell kinetics, fermentation, and cell cultivation . Reports of the use of microreactors for screening applications include work on the study of oxygen uptake, monitoring microalgae culturing and stress conditions and the utilization of a gradient generator to study the behavior of yeast under different conditions . Instrumentation has also made it possible to precisely control the conditions in the microchannel allowing for steady‐state or dynamically controlled environments.…”