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

Özet

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 Lewy bodies³. α-synuclein may contribute to the pathogenesis of PD in several ways. In general, it is thought that its abnormal, soluble oligomeric forms called protofibrils are toxic species that cause neuronal cell death by affecting various cellular targets, including synaptic function³.

The biological models used to study neurodegenerative diseases must be relevant to humans with respect to their genome and cellular biology. The best models would be human neuronal cell lines. However, these cell lines are associated with several technical issues, such as difficulties in the maintenance of cultures, low efficiency of transfection, and high expense⁴. For these reasons, an easy and reliable tool is required to accelerate the progress in this research area. Importantly, the tool should be easy to use for analyzing the collected data. From these perspectives, various model organisms have been widely used, including Drosophila, Caenorhabditis elegans, Danio rerio, yeast, and rodents⁵. Among them, yeast is the best model organism because genetic manipulation is easy, and it is cheaper than the other model organisms. Most importantly, yeast has high similarities to human cells, such as 60% sequence homology to human orthologs and 25% close homology with human disease-related genes⁶, and they also share fundamental eukaryotic cell biology. Yeast contains many proteins with similar sequences and analogous functions to those in human cells⁷. Indeed, yeast expressing human genes has been widely used as a model system to elucidate cellular processes⁸. This yeast strain is called humanized yeast and is a useful tool for exploring the function of human genes⁹. Humanized yeast has merit for studying genetic interactions because genetic manipulation is well established in yeast.

In this study, we used the yeast Saccharomyces cerevisiae as a model organism to study the pathogenesis of PD, particularly for investigating α-synuclein aggregate formation and cytotoxicity¹⁰. For the expression of α-synuclein in the budding yeast, the W303a strain was used for transformation with plasmids encoding for the wild-type and familial PD-associated variants of α-synuclein. As the W303a strain has an auxotrophic mutation on URA3, it is applicable for the selection of cells containing plasmids with URA3. The expression of α-synuclein encoded in a plasmid is regulated under the GAL1 promoter. Thus, the expression level of α-synuclein can be controlled. In addition, the fusion of green fluorescent protein (GFP) at the C-terminal region of α-synuclein allows the monitoring of α-synuclein foci formation. To understand the characteristics of the familial PD-associated variants of α-synuclein, we also expressed these variants in yeast and examined their cellular effects. This system is a straightforward tool for screening compounds or genes exhibiting protective roles against the cytotoxicity of α-synuclein.

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% raffinose, 5 mL of 20% glucose, and 100 mL of 10x -Ura drop-out before use.
   4. To make SD-Ura agar medium, dissolve 6.7 g of yeast nitrogen base with ammonium sulfate and without amino acids and 20 g of agar in 800 mL of dH₂O. Autoclave in a flask with a volume over 2 L. Add 100 mL of 20% glucose and 100 mL of 10x -Ura drop-out. Mix well and pour onto Petri dishes. Store at 4 ˚C.
   5. To make SC with galactose (SG)-Ura agar medium, dissolve 6.7 g of yeast nitrogen base with ammonium sulfate and without amino acids and 20 g of agar in 800 mL of dH₂O. Autoclave in a flask with a volume over 2 L. Add 100 mL of 20% galactose and 100 mL of 10x -Ura drop-out. Mix well and pour on Petri dishes. Store at 4 ˚C.
2. Stock solutions to use with SD media
   1. To make a 20% carbon source (glucose, galactose, and raffinose), dissolve 100 g of glucose, galactose, or raffinose in dH₂O to make a final volume of 500 mL. Autoclave and store at RT.
   2. To make 10x yeast synthetic drop-out medium supplements without uracil (10x-Ura drop-out), dissolve 19.2 g of "yeast synthetic drop-out medium supplements without uracil" in dH₂O to make a final volume of 1 L. Autoclave and store at RT.
      ​NOTE: For the preparation of a medium containing agar, add a magnetic stir bar to the flask to mix all the solutions well before pouring onto Petri dishes.

2. Yeast transformation

NOTE: The pRS426 plasmid was used to clone the α-synuclein gene and contained the URA3 gene as a selectable marker. There are familial PD-associated variants of α-synuclein that have severe disease phenotypes (e.g., cytotoxicity). These variants were also used here to monitor their cytotoxicity and aggregated foci formation. Use the same colony of yeast transformants for all the experiments to get consistent results.

1. For the transformation of the yeast expression plasmids, use an empty vector (pRS426) as the control and α-synuclein-containing plasmids. Referring to the standard lithium acetate (LiAc) method¹¹, prepare six competent cells and 0.5 µg of each plasmid, and perform the transformation.
2. To select for yeast transformants containing plasmids, use the synthetic defined medium (SD medium) without uracil (SD-Ura), and incubate the plates at 30 ˚C for 2-3 days.
3. Patch three single colonies onto an SD-Ura agar plate for the selection of cells containing plasmids for further experiments.
   ​NOTE: To generate accurate and reliable results, we examined at least three biological replicates per strain.

3. Spotting assay

NOTE: The expression of GFP-tagged α-synuclein in high-copy number plasmids is known to be associated with cytotoxicity in yeast¹⁰.

1. Inoculate the three patched yeast transformants per strain into 3 mL of SRd-Ura for the selective growth of yeast containing the plasmids with 2% raffinose for inhibition of the induction of α-synuclein under the GAL1 promoter and 0.1% glucose. Incubate the cell culture at 30 °C under agitation at 200 rpm.
   NOTE: Repeating all the experiments with different colonies is important. Obtaining the same results from over three independent colonies means the result is reliable.
2. The next day, inoculate the overnight cultured cells into 3 mL of fresh SRd-Ura to an OD₆₀₀ of 0.2, and incubate for 6 h at 30 °C under agitation at 200 rpm. 
3. When the OD₆₀₀ reaches 0.6-0.8, dilute the cells in 96-well plates for the spotting assay as follows.
   1. Measure the OD₆₀₀ of all the samples, and dilute the samples to an OD₆₀₀ of 0.5 with sterile dH₂O (the mixed volume is 100 µL) in lane 1 of a 96-well plate to equalize the number of cells in each culture.
   2. Perform fivefold serial dilutions of the yeast cells by adding 160 µL of sterile dH₂O to the next five lanes. Ensure a total volume of 40 µL when serially diluting the cells in each lane.
      NOTE: Low dilution factors can produce larger differences between the samples. Sufficient pipetting is required to ensure uniform mixing when performing the serial dilutions. The pipette tip should be replaced at every dilution point. Otherwise, the cells attached to the surface of the tip could be transferred to the next well and affect the cell number.
4. Use a multichannel pipette to transfer 10 µL from each well to spot the samples on SD-Ura agar plates for the controls and SG-Ura agar plates to monitor toxicity.
   NOTE: The plates should be dried until the spotted samples are fully absorbed onto the plates.
5. Incubate the spotted plates upside down at 30 °C for 4 days.
6. Take images of the plates every 24 h until day 4, and then analyze the data by comparing the growth differences between strains spotted by eye. Count the individual colonies in the most diluted sample, calculate the number of viable cells in the original culture for each strain, or compare the size of the single colonies.

4. Measurement of yeast growth using a microplate reader

1. Inoculate the three patched yeast transformants per strain into 3 mL of SRd-Ura, and incubate at 30 °C under agitation at 200 rpm overnight.
   NOTE: To understand each colony phenotype of the humanized yeast strain expressing α-synuclein, use the same transformants for all the experiments.
2. The next day, inoculate the overnight cultured cells into a total of 200 µL of fresh SD-Ura and SG-Ura in 96-well plates. Adjust the final volume, including the cells, to 200 µL, and adjust the initial cell OD₆₀₀ to 0.05.
   1. Adjust the Program settings in the microtiter plate as follows: set the target temperature için 30 ˚ C, set the shaking (Linear) duration için 890 s, set the shaking (Linear) amplitude için 5 mm, set the interval time için 00:15:00, and set the measurement as absorbance at 600 nm.
   2. Incubate the plate for 2-3 days for all the strains to reach the stationary phase in a microplate reader.
3. Analyze the data by plotting the growth curve (e.g., using Workbook). Acquire the data points by averaging the values of absorbance from the three biological replicates, and indicate the error bars. Use the value of absorbance from only media as a control, and subtract this from the values from cultured cells (optional).

5. Fluorescence microscopy

1. Inoculate the patched yeast transformants into 3 mL of SRd-Ura, and culture them overnight at 30 °C under agitation at 200 rpm.
   NOTE: To determine the correlation of the phenotype with the cytotoxicity and foci formation, use the same colonies for the experiment.
2. The next day, reinoculate the overnight cultured cells into 3 mL of fresh SRd-Ura to an OD₆₀₀ of 0.003. Incubate the culture until the OD₆₀₀ reaches 0.2 at 30 °C under agitation at 200 rpm (~18 h).
3. When an OD₆₀₀ of 0.2 is achieved, add 300 µL of 20% galactose, and then incubate for another 6 h at 30 °C under agitation at 200 rpm.
4. Collect the cells by centrifugation at 800 x g for 5 min, and discard the supernatant.
5. Resuspend the cell pellet gently in 30 µL of distilled water.
6. Load 5 μL of the resuspended cells onto a slide glass. Prepare an agarose gel pad¹² and put it onto the loaded cells to make it spread evenly.
7. Perform microscopic imaging with a 100x objective using both brightfield and GFP-fluorescence filters.
   1. First, use brightfield to focus the cell with a 100x objective using immersion oil. Use the screen capture option for generating the images using the program.
   2. Switch to a GFP-fluorescence filter. Adjust the image exposure time to between 50 ms and 300 ms to achieve a clear image by balancing the signal-to-noise ratio. Screen capture the image using the program.
   3. Image many cells from multiple points rather than just one.
8. Analyze the images by counting the percentage of cells containing foci of α-synuclein aggregates using analytical software. Observe the differences between the soluble GFP protein signal and the foci-formed GFP signal, as well as the diffusion of GFP with the A30P variant form of α-synuclein.
   NOTE: Foci formation will be observed with the wild-type, E46K, and A53T variants of α-synuclein. 

Representative 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).

The α-synuclein expression under the GAL1 promoter in the pRS426 vector showed significant growth retardation on the agar plate containing galactose as an inducer (Figure 2). The difference in growth by α-synuclein expression was also observed in liquid cultures (Figure 3). In both culture conditions, wild-type α-synuclein and variants with E46K or A53T mutations showed growth defects due to α-synuclein toxicity. However, the α-synuclein A30P variant, which does not form aggregates, showed a non-toxic phenotype.

To understand the relationship between cytotoxicity and the aggregation of α-synuclein, a method to monitor the status of α-synuclein in yeast is necessary. The α-synuclein was tagged by a GFP protein at the C-terminus to monitor the status of α-synuclein in live yeast cells by detecting the GFP signal using fluorescence microscopy. The strains exhibiting severe cytotoxicity showed foci of α-synuclein aggregates, but in the A30P variant, a less toxic form of α-synuclein was diffused throughout the cells (Figure 4).

Figure 1: α-synuclein domains and constructs used in this study. (A) The structure of human α-synuclein with the three distinct domains-the N-terminal domain, the NAC domain, and the C-terminal domain. The amino acid residues are indicated at the bottom. The orange lines indicate the mutated sites. (B) The recombinant plasmid constructs used in this study. The pRS426 plasmid was used as a backbone. α-synuclein was fused with a GFP tag at the C-terminus, and its expression was controlled by the GAL1 promoter. Abbreviations: NAC = non-amyloid-β component; GFP = green fluorescent protein. Please click here to view a larger version of this figure.

Figure 2: Cytotoxicity of α-synuclein on agar due to the expression of high-copy number plasmids in yeast. Yeast cells expressing GAL1-driven α-synuclein-GFP variants in pRS426 plasmids were spotted in fivefold dilutions on a selective yeast medium containing 2% glucose or 2% galactose. The cells were incubated for 3 days at 30 °C, and the plates were photographed. Abbreviations: GFP = green fluorescent protein; EV = empty vector; WT = wild-type; α-Syn = α-synuclein. Please click here to view a larger version of this figure.

Figure 3: Cytotoxicity of α-synuclein in liquid media due to the expression of high-copy number plasmids in yeast. Yeast cells expressing GAL1-driven α-synuclein-GFP variants were cultured in liquid media in a 96-well plate at 30 °C for 2 days, and the growth was monitored using a microplate reader. Abbreviations: GFP = green fluorescent protein; EV = empty vector; WT = wild-type; α-Syn = α-synuclein. Please click here to view a larger version of this figure.

Figure 4: Analysis of yeast cells expressing α-synuclein-GFP by fluorescence microscopy. α-synuclein-GFP variants were induced by adding galactose and incubating for 6 h. The confirmation of α-synuclein-GFP was done by fluorescence microscopy. Scale bar = 10 µm. Abbreviations: GFP = green fluorescent protein; EV = empty vector; WT = wild-type; α-Syn = α-synuclein. Please click here to view a larger version of this figure.

Figure 5: Summary of this method for measuring cytotoxicity and aggregate formation of α-synuclein using humanized yeast. Please click here to view a larger version of this figure.

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 ease of genetic manipulation, yeast can facilitate the study of complex and demanding human systems. Therefore, yeast has been widely used as a model system to understand eukaryotic cellular processes and the pathogenesis of various diseases^14,15,16. In particular, there are discrepancies between the in silico, in vitro, and in vivo aggregation properties of amyloidogenic proteins, including α-synuclein¹⁷. Taking into account the complexity and unpredictability of protein behavior in the cell, it is important to study protein properties in vivo. Thus, to understand what is happening in a cell with α-synuclein, it is necessary to investigate it in in vivo conditions, and yeast is thought to be a useful and powerful model system for this.

In this study, the properties of α-synuclein in yeast in a highly overexpressed condition using a 2 micron plasmid were analyzed. The α-synuclein level was controlled by an inducible GAL1 promoter. This promoter is activated when galactose is available as a nutrient, and thus, this system allows us to study phenotypes derived from specific levels of α-synuclein protein expression. The overexpression of the wild-type and α-synuclein variants, except for A30P, caused toxicity in the spotting assay, as well as in the growth curve analysis. These wild-type and α-synuclein variants were examined using fluorescence microscopy to monitor the protein aggregates of α-synuclein in the cells. From these experiments, the formation of foci derived from α-synuclein aggregation was observed in the α-synuclein wild-type and variants exhibiting cytotoxicity. These results confirm that the cellular toxicity and foci formation resulting from misfolded α-synuclein protein aggregation are linked to one of the representative phenotypes of PD in neuronal cells^18,19.

It is important to incubate the transformants for 3 days and to choose big and reddish colonies for further experiments. The W303a strain has an adenine mutation, which makes the cell turn red²⁰. Therefore, choosing a white colony results in working with the wrong contaminant. Choosing the appropriate cell stage for the induction of α-synuclein by adding galactose is important in order to observe a reproducible phenotype. For testing the effect of a specific protein on the cytotoxicity of α-synuclein, other proteins can be co-expressed by co-transformation. However, plasmids with Ura markers must be avoided as the α-synuclein plasmid is maintained with Ura.

This method is not limited to the study of the cellular characteristics of α-synuclein itself and can be a useful tool to validate candidate genes that may influence the aggregation of α-synuclein. Interestingly, various bacterial proteins (e.g., chaperones and curli) have recently been reported to affect the formation of α-synuclein in vitro^21,22. The effect of a candidate gene on α-synuclein-derived toxicity or aggregate formation can be monitored by co-expression. For example, α-synuclein maintained by the URA3 marker gene was co-expressed in cells with a candidate gene from the pRS425 plasmid, which contained a galactose-inducible promoter, and a LEU2 marker. Furthermore, this system could be used for the purpose of genetic screening using gene deletion or overexpression libraries, as well as chemical libraries, in yeast²³. However, in genome-wide screening assays that aim to uncover genetic interactions, co-overexpression with other proteins in a plasmid will not be easy to achieve in terms of transformation efficiency. In such situations, it may be better to express α-synuclein in the genome in multiple copies to manipulate the toxic form²⁰. In conclusion, this yeast model system and protocol provide a straightforward and comprehensive platform to understand the physiology of α-synuclein and evaluate genes with the potential ability to modulate α-synuclein toxicity and aggregation (Figure 5).

Materials

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