A Method to Study α-Synuclein Toxicity and Aggregation Using a Humanized Yeast Model

Summary

An in vivo physiological model of α-synuclein is required to study and understand the pathogenesis of Parkinson's disease. We describe a method to monitor the cytotoxicity and aggregate formation of α-synuclein using a humanized yeast model.

Abstract

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by progressive cell death caused by the formation of Lewy bodies containing misfolded and aggregated α-synuclein. α-synuclein is an abundant presynaptic protein that regulates synaptic vesicle trafficking, but the accumulation of its proteinaceous inclusions results in neurotoxicity. Recent studies have revealed that various genetic factors, including bacterial chaperones, could reduce the formation of α-synuclein aggregates in vitro. However, it is also important to monitor the anti-aggregation effect in the cell to apply this as a potential treatment for the patients. It would be ideal to use neuronal cells, but these cells are difficult to handle and take a long time to exhibit the anti-aggregation phenotype. Therefore, a quick and effective in vivo tool is required for the further evaluation of in vivo anti-aggregation activity. The method described here was used to monitor and analyze the anti-aggregation phenotype in the humanized yeast Saccharomyces cerevisiae, which expressed human α-synuclein. This protocol demonstrates in vivo tools that could be used for monitoring α-synuclein-induced cellular toxicity, as well as the formation of α-synuclein aggregates in cells.

Introduction

Parkinson's disease (PD) is a serious problem for aging societies worldwide. The aggregation of α-synuclein is closely associated with PD, and protein aggregates of α-synuclein are widely used as a molecular biomarker for diagnosing the disease¹. α-synuclein is a small acidic protein (140 amino acids in length) with three domains, namely the N-terminal lipid-binding α-helix, the amyloid-binding central domain (NAC), and the C-terminal acidic tail². The misfolding of α-synuclein can occur spontaneously and eventually leads to the formation of amyloid aggregates called Lew....

Protocol

1. Preparation of media and solutions

1. Media preparation
   1. To prepare YPD medium, dissolve 50 g of YPD powder in dH₂O to make a final volume of 1 L. Autoclave for sterilization. Store at room temperature (RT).
   2. To make YPD agar medium, dissolve 50 g of YPD powder and 20 g of agar in 1 L of dH₂O. Autoclave for sterilization. After cooling down, pour onto Petri dishes. Store at 4 ˚C.
   3. To make SC with raffinose (SRd)-Ura medium, dissolve 6.7 g of yeast nitrogen base with ammonium sulfate and without amino acids in 795 mL of dH₂O. Autoclave for sterilization. Add 100 mL of 20% ....

Results

The high expression of α-synuclein is known to be linked to neuronal cell death and PD in model systems of PD. This study describes three methods to monitor the cytotoxicity of α-synuclein and the foci formation of aggregated α-synuclein in yeast. Here, the α-synuclein was overexpressed in yeast, and the phenotypes of wild-type α-synuclein and three variants of α-synuclein known as familial mutants of PD were examined (Figure 1 and the Table of Materials).

Discussion

Given the complexity of various cellular systems in humans, it is advantageous to use yeast as a model for studying human neurodegenerative diseases. Although it is nearly impossible to investigate the complex cellular interactions of the human brain using yeast, from a single-cell perspective, yeast cells have a high level of similarity to human cells in terms of the genomic sequence homology and fundamental eukaryotic cellular processes^8,13. Moreover, given the.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96 well plate	SPL	30096
Agarose	TAESHIN	0158
Bacto Agar	BD Difco	214010
Breathe-easy	diversified biotech	BEM-1	Gas permeable sealing membrane for microtiter plates
cover glasses	Marienfeld		24 x 60 mm
Culture tube	SPL	40014
Cuvette	ratiolab	2712120
D-(+)-Galactose	sigma	G0625
D-(+)-Glucose	sigma	G8270
D-(+)-Raffinose pentahydrate	Daejung	6638-4105
Incubator (shaking)	Labtron		model: SHI1
Incubator (static)	Vision scientific		model: VS-1203PV-O
LiAc	sigma	L6883
Microplate reader	Tecan	30050303 01	Model: Infinite 200 pro
multichannel pipette 20-200 µL	gilson	FA10011
multichannel pipette 2-20 µL	gilson	FA10009
Olympus microscope	Olympus	IX-53
PEG	sigma	P4338	average mol wt 3,350
Petridish	SPL	10090
pRS426			Christianson, T. W., Sikorski, R. S., Dante, M., Shero, J. H. & Hieter, P. Multifunctional yeast high-copy-number shuttle vectors. Gene. 110 (1), 119-122 (1992).
pRS426 GAL1 promoter α-synuclein A30P			Outeiro, T. F. & Lindquist, S. Yeast cells provide insight into alpha-synuclein biology and pathobiology. Science. 302 (5651), 1772-1775 (2003)
pRS426 GAL1 promoter α-synuclein A53T			Outeiro, T. F. & Lindquist, S. Yeast cells provide insight into alpha-synuclein biology and pathobiology. Science. 302 (5651), 1772-1775 (2003)
pRS426 GAL1 promoter α-synuclein E46K			Outeiro, T. F. & Lindquist, S. Yeast cells provide insight into alpha-synuclein biology and pathobiology. Science. 302 (5651), 1772-1775 (2003)
pRS426 GAL1 promoter α-synuclein WT			Outeiro, T. F. & Lindquist, S. Yeast cells provide insight into alpha-synuclein biology and pathobiology. Science. 302 (5651), 1772-1775 (2003)
Reservoir	SPL	23050
Spectrophotometer	eppendorf	6131 05560
W303a			Present from James Bardwell
Yeast nitrogen base w/o amino acids	Difco	291940
Yeast synthetic drop-out medium supplements without uracil	sigma	Y1501
YPD	Condalab	1547.00