We thank James Bardwell and Tiago F. Outeiro for kindly sharing the plasmids containing &#945;-synuclein. Changhan Lee received funding from the National Research Foundation of Korea (NRF) funded by the Korean government (MSIT) (grant 2021R1C1C1011690), the Basic Science Research Program through the NRF funded by the Ministry of Education (grant 2021R1A6A1A10044950), and the new faculty research fund of Ajou University.

1. Preparation of media and solutions
<ol>
	<li>Media preparation
	<ol>
		<li>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).</li>
		<li>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 &#730;C.</li>
		<li>To make SC with raffinose (SRd)-Ura medium, dissolve 6....

<ol>
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F., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+cells+provide+insight+into+alpha-synuclein+biology+and+pathobiology.">Yeast cells provide insight into alpha-synuclein biology and pathobiology.</a> Science. 302 (5651), 1772-1775 (2003).</li><li>Dunham, M., Gartenberg, M., Brown, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Yeast Genetics and Genomics. , (2015).</li><li>Skinner, S. O., Sep&#250;lveda, L. A., Xu, H., Golding, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+mRNA+copy+number+in+individual+Escherichia+coli+cells+using+single-molecule+fluorescent+in+situ+hybridization.">Measuring mRNA copy number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization.</a> Nature Protocols. 8 (6), 1100-1113 (2013).</li><li>Tenreiro, S., Munder, M. C., Alberti, S., Outeiro, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+the+power+of+yeast+to+unravel+the+molecular+basis+of+neurodegeneration.">Harnessing the power of yeast to unravel the molecular basis of neurodegeneration.</a> Journal of Neurochemistry. 127 (4), 438-452 (2013).</li><li>Nielsen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+systems+biology:+Model+organism+and+cell+factory.">Yeast systems biology: Model organism and cell factory.</a> Biotechnology Journal. 14 (9), 1800421 (2019).</li><li>Eleutherio, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+and+aging:+learning+from+yeast+lessons.">Oxidative stress and aging: learning from yeast lessons.</a> Fungal Biology. 122 (6), 514-525 (2018).</li><li>Rencus-Lazar, S., DeRowe, Y., Adsi, H., Gazit, E., Laor, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+models+for+the+study+of+amyloid-associated+disorders+and+development+of+future+therapy.">Yeast models for the study of amyloid-associated disorders and development of future therapy.</a> Frontiers in Molecular Biosciences. 6, 15 (2019).</li><li>Franco, R., Rivas-Santisteban, R., Navarro, G., Pinna, A., Reyes-Resina, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+implicated+in+familial+Parkinson's+disease+provide+a+dual+picture+of+nigral+dopaminergic+neurodegeneration+with+mitochondria+taking+center+stage.">Genes implicated in familial Parkinson's disease provide a dual picture of nigral dopaminergic neurodegeneration with mitochondria taking center stage.</a> International Journal of Molecular Sciences. 22 (9), 4643 (2021).</li><li>Wan, O. W., Chung, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+alpha-synuclein+oligomerization+and+aggregation+in+cellular+and+animal+models+of+Parkinson's+disease.">The role of alpha-synuclein oligomerization and aggregation in cellular and animal models of Parkinson's disease.</a> PLoS One. 7 (6), 38545 (2012).</li><li>Xu, L., Pu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-synuclein+in+Parkinson's+disease:+From+pathogenetic+dysfunction+to+potential+clinical+application.">Alpha-synuclein in Parkinson's disease: From pathogenetic dysfunction to potential clinical application.</a> Parkinson's Disease. 2016, 1720621 (2016).</li><li>Gitler, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Parkinson's+disease+protein+&#945;-synuclein+disrupts+cellular+Rab+homeostasis.">The Parkinson's disease protein &#945;-synuclein disrupts cellular Rab homeostasis.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (1), 145-150 (2008).</li><li>Sampson, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gut+bacterial+amyloid+promotes+&#945;-synuclein+aggregation+and+motor+impairment+in+mice.">A gut bacterial amyloid promotes &#945;-synuclein aggregation and motor impairment in mice.</a> Elife. 9, 53111 (2020).</li><li>Evans, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bacterial+curli+system+possesses+a+potent+and+selective+inhibitor+of+amyloid+formation.">The bacterial curli system possesses a potent and selective inhibitor of amyloid formation.</a> Molecular Cell. 57 (3), 445-455 (2015).</li><li>Su, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compounds+from+an+unbiased+chemical+screen+reverse+both+ER-to-Golgi+trafficking+defects+and+mitochondrial+dysfunction+in+Parkinson's+disease+models.">Compounds from an unbiased chemical screen reverse both ER-to-Golgi trafficking defects and mitochondrial dysfunction in Parkinson's disease models.</a> Disease Models &amp; Mechanisms. 3 (3-4), 194-208 (2010).</li></ol>

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 processes8,13. Moreover, given the...

Parkinson&#39;s disease (PD) is a serious problem for aging societies worldwide. The aggregation of &#945;-synuclein is closely associated with PD, and protein aggregates of &#945;-synuclein are widely used as a molecular biomarker for diagnosing the disease1. &#945;-synuclein is a small acidic protein (140 amino acids in length) with three domains, namely the N-terminal lipid-binding &#945;-helix, the amyloid-binding central domain (NAC), and the C-terminal acidic tail2. The misfolding of &#945;-synuclein can occur spontaneously and eventually leads to the formation of amyloid aggregates called Lewy bodies3. &#945;-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 &#945;-synuclein aggregate formation and cytotoxicity10. For the expression of &#945;-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 &#945;-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 &#945;-synuclein encoded in a plasmid is regulated under the GAL1 promoter. Thus, the expression level of &#945;-synuclein can be controlled. In addition, the fusion of green fluorescent protein (GFP) at the C-terminal region of &#945;-synuclein allows the monitoring of &#945;-synuclein foci formation. To understand the characteristics of the familial PD-associated variants of &#945;-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 &#945;-synuclein.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96 well plate</td>
			<td>SPL</td>
			<td>30096</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>TAESHIN</td>
			<td>0158</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>BD Difco</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Breathe-easy</td>
			<td>diversified biotech</td>
			<td>BEM-1</td>
			<td>Gas permeable sealing membrane for microtiter plates</td>
		</tr>
		<tr>
			<td>cover glasses</td>
			<td>Marienfeld</td>
			<td></td>
			<td>24 x 60 mm</td>
		</tr>
		<tr>
			<td>Culture tube</td>
			<td>SPL</td>
			<td>40014</td>
			<td></td>
		</tr>
		<tr>
			<td>Cuvette</td>
			<td>ratiolab</td>
			<td>2712120</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Galactose</td>
			<td>sigma</td>
			<td>G0625</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Raffinose pentahydrate</td>
			<td>Daejung</td>
			<td>6638-4105</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator (shaking)</td>
			<td>Labtron</td>
			<td></td>
			<td>model: SHI1</td>
		</tr>
		<tr>
			<td>Incubator (static)</td>
			<td>Vision scientific</td>
			<td></td>
			<td>model: VS-1203PV-O</td>
		</tr>
		<tr>
			<td>LiAc</td>
			<td>sigma</td>
			<td>L6883</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Tecan</td>
			<td>30050303 01</td>
			<td>Model: Infinite 200 pro</td>
		</tr>
		<tr>
			<td>multichannel pipette 20-200 &#181;L</td>
			<td>gilson</td>
			<td>FA10011</td>
			<td></td>
		</tr>
		<tr>
			<td>multichannel pipette 2-20 &#181;L</td>
			<td>gilson</td>
			<td>FA10009</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus microscope</td>
			<td>Olympus</td>
			<td>IX-53</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG</td>
			<td>sigma</td>
			<td>P4338</td>
			<td>average mol wt 3,350</td>
		</tr>
		<tr>
			<td>Petridish</td>
			<td>SPL</td>
			<td>10090</td>
			<td></td>
		</tr>
		<tr>
			<td>pRS426</td>
			<td></td>
			<td></td>
			<td>Christianson, T. W., Sikorski, R. S., Dante, M., Shero, J. H. &#38; Hieter, P. Multifunctional yeast high-copy-number shuttle vectors. Gene. 110 (1), 119-122 (1992).</td>
		</tr>
		<tr>
			<td>pRS426 GAL1 promoter &#945;-synuclein A30P</td>
			<td></td>
			<td></td>
			<td>Outeiro, T. F. &#38; Lindquist, S. Yeast cells provide insight into alpha-synuclein biology and pathobiology. Science. 302 (5651), 1772-1775 (2003)</td>
		</tr>
		<tr>
			<td>pRS426 GAL1 promoter &#945;-synuclein A53T</td>
			<td></td>
			<td></td>
			<td>Outeiro, T. F. &#38; Lindquist, S. Yeast cells provide insight into alpha-synuclein biology and pathobiology. Science. 302 (5651), 1772-1775 (2003)</td>
		</tr>
		<tr>
			<td>pRS426 GAL1 promoter &#945;-synuclein E46K</td>
			<td></td>
			<td></td>
			<td>Outeiro, T. F. &#38; Lindquist, S. Yeast cells provide insight into alpha-synuclein biology and pathobiology. Science. 302 (5651), 1772-1775 (2003)</td>
		</tr>
		<tr>
			<td>pRS426 GAL1 promoter &#945;-synuclein WT</td>
			<td></td>
			<td></td>
			<td>Outeiro, T. F. &#38; Lindquist, S. Yeast cells provide insight into alpha-synuclein biology and pathobiology. Science. 302 (5651), 1772-1775 (2003)</td>
		</tr>
		<tr>
			<td>Reservoir</td>
			<td>SPL</td>
			<td>23050</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>eppendorf</td>
			<td>6131 05560</td>
			<td></td>
		</tr>
		<tr>
			<td>W303a</td>
			<td></td>
			<td></td>
			<td>Present from James Bardwell</td>
		</tr>
		<tr>
			<td>Yeast nitrogen base w/o amino acids</td>
			<td>Difco</td>
			<td>291940</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast synthetic drop-out medium supplements without uracil</td>
			<td>sigma</td>
			<td>Y1501</td>
			<td></td>
		</tr>
		<tr>
			<td>YPD</td>
			<td>Condalab</td>
			<td>1547.00</td>
			<td></td>
		</tr>
	</tbody>
</table>

a method to study &#945;-synuclein toxicity and aggregation using a humanized yeast model

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 &#945;-synuclein. &#945;-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 &#945;-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 &#945;-synuclein. This protocol demonstrates in vivo tools that could be used for monitoring &#945;-synuclein-induced cellular toxicity, as well as the formation of &#945;-synuclein aggregates in cells.

The high expression of &#945;-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 &#945;-synuclein and the foci formation of aggregated &#945;-synuclein in yeast. Here, the &#945;-synuclein was overexpressed in yeast, and the phenotypes of wild-type &#945;-synuclein and three variants of &#945;-synuclein known as familial mutants of PD were examined (Figure 1 and the Table of Materials...

Watch this Scientific Journal Video about A Method to Study Î±-Synuclein Toxicity and Aggregation Using a Humanized Yeast Model at JoVE.com

A Method to Study &amp;#945;-Synuclein Toxicity and Aggregation Using a Humanized Yeast Model

An in vivo physiological model of &#945;-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 &#945;-synuclein using a humanized yeast model.

A Method to Study &#945;-Synuclein Toxicity and Aggregation Using a Humanized Yeast Model

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by progressive cell death caused by the formation of Lewy ...

This is a straightforward method to monitor cellular phenotypes and features of R-pass nuclei. It can be also often recovered in genome-wide study to find the genetic factors affecting the stability of R-pass nuclei. Since is this a well-established motor organism, this technique is easy and does not require much time and effort compared to using human cell lines or animal models.The aggregation of R-pass nuclei is closely associated with Parkinson's disease. The proteins or chemicals which are likely affecting the stability of R-pass nuclei can be tested in this Humanized yeast model. To begin, patch three single colonies onto an SD-URA agar plate for the selection of cells containing alpha-synuclein plasmids.Then, inoculate the three-patched yeast transformants per strain into 3 milliliters of SRD-URA for the selective growth of yeast containing the plasmids with 2%raffinose for inhibition of the induction of alpha-synuclein under the Gal1 promoter and 0.1%glucose. Incubate the inoculated culture at 30 degrees celsius under agitation at 200 rotations per minute. The next day, inoculate the overnight cultured cells into 3 milliliters of fresh SRD-URA till they reach an optical density of 0.2 at 600 nanometers.Then, incubate the culture for six hours at 30 degrees celsius under agitation at 200 rotations per minute. Measure the optical density at 600 nanometer using a spectrophotometer. Next, dilute the samples to an optical density of 0.5 at 600 nanometer with sterile distilled water in lane 1 of a 96-well plate to equalize the number of cells in each culture.Perform 5-fold serial dilutions of the yeast cells by adding 160 microliters of sterile distilled water to the next five lanes and ensure a total volume of 40 microliters when serially diluting the cells in each lane. Use a multichannel pipette to transfer 10 microliters from each well to spot the samples on SD-URA agar plates for the controls and SG-URA agar plates to monitor toxicity. Incubate the spotted plates upside down at 30 degrees celsius for four days.Take images of the plates every 24 hours until day four and then analyze the data by comparing the growth differences between strains spotted by eye. Inoculate the three patched yeast transformants per strain into three milliliters of SRD-URA and incubate at 30 degrees celsius under agitation at 200 rotations per minute overnight. The next day, measure the optical density of the cells cultured overnight at 600 nanometers and inoculate the cells into a total of 200 microliters of fresh SD-URA and SG-URA in 96-well plates.Adjust the final volume including the cells to 200 microliters and adjust the initial cell optical density to 0.05 at 600 nanometers. Adjust the program settings in the microtiter plate. Set the target temperature to 30 degrees celsius, the interval time to 15 minutes, the shaking duration to 890 seconds, the shaking amplitude to 5 millimetres, and the measurement as absorbance at 600 nanometers.Incubate the plate for 2-3 days for all the strains to reach the stationary phase in a microplate reader. Analyze the data by plotting the growth curve. Inoculate the patched yeast transformants into 3 milliliters of SRD-URA and culture them overnight at 30 degrees celsius under agitation at 200 rotations per minute.The next day, re-inoculate the overnight cultured cells into 3 milliliters of fresh SRD-URA to an optical density of 0.003 at 600 nanometers, then incubate the culture at 30 degrees celsius under agitation at 200 rotations per minute, until the optical density reaches 0.2. Then add 300 microliters of 20%galactose, and then incubate for another 6 hours at 30 degrees celsius under agitation at 200 rotations per minute. Collect one milliliters of the cell culture by centrifugation at 800 g for 5 minutes and discard the supernatant.Resuspend the cell pellet gently in 30 microliters of distilled water. Prepare an agarose gel pad on a glass slide. Resuspend the cells and load five microliters of the cell resuspension on the glass slide, and cover the cells using the cut agarose pad for examination.To perform microscopic imaging with a 100x objective, use the screen capture option for generating images using the program and select brightfield to focus the cell with a 100x objective using immersion oil. Switch to a GFP fluorescence filter and adjust the image exposure time to between 50 millisecond and 300 millisecond to achieve a clear image by balancing the signal-to-noise ratio. Open the image file and analyze the data by counting the cells based on the number of foci in the cells using image analysis software.The synuclein expression under the Gal1 promoter in the PRS426 vector showed significant growth retardation on the agar plate containing galactose as an inducer. The difference in growth by synuclein expression was also observed in liquid cultures. In both culture conditions, wild-type synuclein and variants with E46K or A53T mutations showed growth defects due to synuclein toxicity.However, the synuclein A30p variant, which does not form aggregates, showed a non-toxic phenotype. The strains exhibiting severe cytotoxicity showed foci of synuclein aggregates, but in the A30p variant, a less toxic form of synuclein was diffused throughout the cells. To achieve reproducible results, it is important to use the cell in the same growth stage in each experiment, particularly when you monitor synuclein-associated phenotypes.As we have shown in fluorescent microscope data, synuclein can form larger aggregates. Therefore, it can be simply fractionated by centrifugation and can be quantified by western blotting using synuclein-specific antibody. This technique is applicable for high-throughput screening to find novel genetic factors and chemical compounds affecting the aggregation of synuclein.Thus, we hope to find new therapeutic candidates for Parkinson's disease.

a-method-to-study-synuclein-toxicity-aggregation-using-humanized

Research

JoVE Journal

Biology

2.0K ビュー.  Ajou University. これは、Rパス核の細胞表現型と特徴を監視するための簡単な方法です。また、Rパス核の安定性に影響を与える遺伝的要因を見つけるために、ゲノムワイドな研究でしばしば回収することができます。これは十分に確立された運動生物であるため、この技術は簡単で、ヒト細胞株または動物モデルを使用する場合と比較して多くの時間と労力を必要としません。Rパ?...