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 6.7 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.</li>
		<li>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 &#730;C.</li>
		<li>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 &#730;C.</li>
	</ol>
	</li>
	<li>Stock solutions to use with SD media
	<ol>
		<li>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.</li>
		<li>To make 10x yeast synthetic drop-out medium supplements without uracil (10x-Ura drop-out), dissolve 19.2 g of &#34;yeast synthetic drop-out medium supplements without uracil&#34; in dH2O to make a final volume of 1 L. Autoclave and store at RT. 
		&#8203;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.</li>
	</ol>
	</li>
</ol>
2. Yeast transformation
NOTE: The pRS426 plasmid was used to clone the &#945;-synuclein gene and contained the URA3 gene as a selectable marker. There are familial PD-associated variants of &#945;-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.
<ol>
	<li>For the transformation of the yeast expression plasmids, use an empty vector (pRS426) as the control and &#945;-synuclein-containing plasmids. Referring to the standard lithium acetate (LiAc) method11, prepare six competent cells and 0.5 &#181;g of each plasmid, and perform the transformation.</li>
	<li>To select for yeast transformants containing&#160;plasmids, use the synthetic defined medium (SD medium) without uracil (SD-Ura), and incubate the plates at 30 &#730;C for 2-3 days.</li>
	<li>Patch three single colonies onto an SD-Ura agar plate for the selection of cells containing plasmids for further experiments. 
	&#8203;NOTE: To generate accurate and reliable results, we examined at least three biological replicates per strain.</li>
</ol>
3. Spotting assay
NOTE: The expression of GFP-tagged &#945;-synuclein in high-copy number plasmids is known to be associated with cytotoxicity in yeast10.
<ol>
	<li>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 &#945;-synuclein under the GAL1 promoter and 0.1% glucose. Incubate the cell culture at 30 &#176;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.</li>
	<li>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 &#176;C under agitation at 200 rpm.&#160;</li>
	<li>When the OD600 reaches 0.6-0.8, dilute the cells in 96-well plates for the spotting assay as follows.
	<ol>
		<li>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 &#181;L) in lane 1 of a 96-well plate to equalize the number of cells in each culture.</li>
		<li>Perform fivefold serial dilutions of the yeast cells by adding 160 &#181;L of sterile dH2O to the next five lanes. Ensure a total volume of 40 &#181;L 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.</li>
	</ol>
	</li>
	<li>Use a multichannel pipette to transfer 10 &#181;L 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.</li>
	<li>Incubate the spotted plates upside down at 30 &#176;C for 4 days.</li>
	<li>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.</li>
</ol>
4. Measurement of yeast growth using a microplate reader
<ol>
	<li>Inoculate the three patched yeast transformants per strain into 3 mL of SRd-Ura, and incubate at 30 &#176;C under agitation at 200 rpm overnight. 
	NOTE: To understand each colony phenotype of the humanized yeast strain expressing &#945;-synuclein, use the same transformants for all the experiments.</li>
	<li>The next day, inoculate the overnight cultured cells into a total of 200 &#181;L of fresh SD-Ura and SG-Ura in 96-well plates. Adjust the final volume, including the cells, to 200 &#181;L, and adjust the initial cell OD600 to 0.05.
	<ol>
		<li>Adjust the Program settings in the microtiter plate as follows: set the target temperature to 30 &#730; C, set the shaking (Linear) duration to 890 s, set the shaking (Linear) amplitude to 5 mm, set the interval time to 00:15:00, and set the measurement as absorbance at 600 nm.</li>
		<li>Incubate the plate for 2-3 days for all the strains to reach the stationary phase in a microplate reader.</li>
	</ol>
	</li>
	<li>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).</li>
</ol>
5. Fluorescence microscopy
<ol>
	<li>Inoculate the patched yeast transformants into 3 mL of SRd-Ura, and culture them overnight at 30 &#176;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.</li>
	<li>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 &#176;C under agitation at 200 rpm (~18 h).</li>
	<li>When an OD600 of 0.2 is achieved, add 300 &#181;L of 20% galactose, and then incubate for another 6 h at 30 &#176;C under agitation at 200 rpm.</li>
	<li>Collect the cells by centrifugation at 800 x g for 5 min, and discard the supernatant.</li>
	<li>Resuspend the cell pellet gently in 30 &#181;L of distilled water.</li>
	<li>Load 5 &#956;L 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.</li>
	<li>Perform microscopic imaging with a 100x objective using both brightfield and GFP-fluorescence filters.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Image many cells from multiple points rather than just one.</li>
	</ol>
	</li>
	<li>Analyze the images by counting the percentage of cells containing foci of &#945;-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 &#945;-synuclein. 
	NOTE: Foci formation will be observed with the wild-type, E46K, and A53T variants of &#945;-synuclein.&#160;</li>
</ol>

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The authors have no conflicts of interest to disclose.

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><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 &amp;micro;L</td><td>gilson</td><td>FA10011</td><td></td></tr><tr><td>multichannel pipette 2-20 &amp;micro;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. &amp;amp; Hieter, P. Multifunctional yeast high-copy-number shuttle vectors. Gene. 110 (1), 119-122 (1992).</td></tr><tr><td>pRS426 &lt;em&gt;GAL1 &lt;/em&gt;promoter &amp;alpha;-synuclein A30P</td><td></td><td></td><td>Outeiro, T. F. &amp;amp; Lindquist, S. Yeast cells provide insight into alpha-synuclein biology and pathobiology. Science. 302 (5651), 1772-1775 (2003)</td></tr><tr><td>pRS426 &lt;em&gt;GAL1&lt;/em&gt; promoter &amp;alpha;-synuclein A53T</td><td></td><td></td><td>Outeiro, T. F. &amp;amp; Lindquist, S. Yeast cells provide insight into alpha-synuclein biology and pathobiology. Science. 302 (5651), 1772-1775 (2003)</td></tr><tr><td>pRS426 &lt;em&gt;GAL1 &lt;/em&gt;promoter &amp;alpha;-synuclein E46K</td><td></td><td></td><td>Outeiro, T. F. &amp;amp; Lindquist, S. Yeast cells provide insight into alpha-synuclein biology and pathobiology. Science. 302 (5651), 1772-1775 (2003)</td></tr><tr><td>pRS426 &lt;em&gt;GAL1 &lt;/em&gt;promoter &amp;alpha;-synuclein WT</td><td></td><td></td><td>Outeiro, T. F. &amp;amp; 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...

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A Method to Study &#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.

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

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Research

JoVE Journal

Biology

2.1K Views.  Ajou University. Dit is een eenvoudige methode om cellulaire fenotypen en kenmerken van R-pass-kernen te controleren. Het kan ook vaak worden teruggevonden in genoombrede studie om de genetische factoren te vinden die de stabiliteit van R-pass-kernen beïnvloeden. Omdat dit een goed ingeburgerd motorisch organisme is, is deze techniek eenvoudig en vereist het niet veel tijd en moeite in vergelijking met het gebruik van menselijke cellijnen of diermodellen.De aggregatie van R-pass kernen is nauw verbond...

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

Mating-based Overexpression Library Screening in Yeast

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool commonly used in yeast studies. The expression of a number of neurodegenerative disease-associated proteins in yeast causes cytotoxicity and aggregate formation, recapitulating findings seen in patients with these disorders. Here, we describe a method for screening a yeast model of the Amyotrophic Lateral Sclerosis-associated protein FUS for modifiers of its toxicity. Instead of using transformation, this new screening platform relies on the mating of yeast to introduce an arrayed library of plasmids into the yeast model. The mating method has two clear advantages: first, it is highly efficient; second, the pre-transformed arrayed library of plasmids can be stored for long-term as a glycerol stock, and quickly applied to other screens without the labor-intensive step of transformation into the yeast model each time. We demonstrate how this method can successfully be used to screen for genes that modify the toxicity of FUS.

This article presents a mating-based method to facilitate overexpression screening in budding yeast using an arrayed plasmid library.

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool ...

Genetics

Evaluation of the Impact of Protein Aggregation on Cellular Oxidative Stress in Yeast

Protein misfolding and aggregation into amyloid conformations have been related to the onset and progression of several neurodegenerative diseases. However, there is still little information about how insoluble protein aggregates exert their toxic effects in vivo. Simple prokaryotic and eukaryotic model organisms, such as bacteria and yeast, have contributed significantly to our present understanding of the mechanisms behind the intracellular amyloid formation, aggregates propagation, and toxicity. In this protocol, the use of yeast is described as a model to dissect the relationship between the formation of protein aggregates and their impact on cellular oxidative stress. The method combines the detection of the intracellular soluble/aggregated state of an amyloidogenic protein with the quantification of the cellular oxidative damage resulting from its expression using flow cytometry (FC). This approach is simple, fast, and quantitative. The study illustrates the technique by correlating the cellular oxidative stress caused by a large set of amyloid-&#946; peptide variants with their respective intrinsic aggregation propensities.

Protein aggregation elicits cellular oxidative stress. This protocol describes a method for monitoring the intracellular states of amyloidogenic proteins and the oxidative stress associated with them, using flow cytometry. The approach is used to study the behavior of soluble and aggregation-prone variants of the amyloid-&#946; peptide.

Protein misfolding and aggregation into amyloid conformations have been related to the onset and progression of several neurodegenerative diseases. ...

Biochemistry

Interview: Protein Folding and Studies of Neurodegenerative Diseases

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Growth Assays to Assess Polyglutamine Toxicity in Yeast

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's disease, and Huntington's disease 1. Huntington's disease (HD) is caused by the abnormal expansion of a polyglutamine (polyQ) region within the protein huntingtin. The polyQ-expanded huntingtin protein attains an aberrant conformation (i.e. it misfolds) and causes cellular toxicity 2. At least eight further neurodegenerative diseases are caused by polyQ-expansions, including the Spinocerebellar Ataxias and Kennedy&#x2019;s disease 3.
The model organism yeast has facilitated significant insights into the cellular and molecular basis of polyQ-toxicity, including the impact of intra- and inter-molecular factors of polyQ-toxicity, and the identification of cellular pathways that are impaired in cells expressing polyQ-expansion proteins 3-8. Importantly, many aspects of polyQ-toxicity that were found in yeast were reproduced in other experimental systems and to some extent in samples from HD patients, thus demonstrating the significance of the yeast model for the discovery of basic mechanisms underpinning polyQ-toxicity. 
A direct and relatively simple way to determine polyQ-toxicity in yeast is to measure growth defects of yeast cells expressing polyQ-expansion proteins. This manuscript describes three complementary experimental approaches to determine polyQ-toxicity in yeast by measuring the growth of yeast cells expressing polyQ-expansion proteins. The first two experimental approaches monitor yeast growth on plates, the third approach monitors the growth of liquid yeast cultures using the BioscreenC instrument. 
Furthermore, this manuscript describes experimental difficulties that can occur when handling yeast polyQ models and outlines strategies that will help to avoid or minimize these difficulties. The protocols described here can be used to identify and to characterize genetic pathways and small molecules that modulate polyQ-toxicity. Moreover, the described assays may serve as templates for accurate analyses of the toxicity caused by other disease-associated misfolded proteins in yeast models.

This manuscript describes three complementary protocols for assessing the toxicity of polyglutamine (polyQ)-expansion proteins in the yeast Saccharomyces cerevisiae. These protocols can easily be modified to monitor the toxicity of other misfolded proteins in yeast.

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

Neuroscience

An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). Although mouse models of tauopathy have provided a valuable resource for investigating the neurotoxic mechanisms of aggregated tau, it is becoming increasingly apparent that, due to interspecies differences in neurophysiology, the mouse brain is unsuitable for modeling the human condition. Advances in cell culture methods have made human neuronal cultures accessible for experimental use in vitro and have aided in the development of neurotherapeutics. However, despite the adaptation of human neuronal cell cultures, in vitro models of human tauopathy are not yet widely available. This protocol describes a cellular model of tau aggregation in which human neurons are transduced with lentiviral-derived vectors that code for pathogenically mutated tau fused to a yellow fluorescent protein (YFP) reporter. Transduced cultures produce tau aggregates that stain positively for thioflavin and display markers of neurotoxicity, such as decreased axonal length and increased lysosomal volume. This procedure may be a useful and cost-effective model for studying human tauopathies.

This protocol details a procedure in which human neuronal cultures are transduced with lentiviral constructs coding for mutant human tau. Transduced cultures display tau aggregates and associated pathologies.

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). ...

Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on &#945;-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson&#8217;s disease, Lewy bodies (LB), is mimicked by the addition of &#945;-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated &#945;-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing &#945;-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-&#945;-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated &#945;-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of &#945;-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.

Here, we present a detailed protocol to study neuronal &#945;-synuclein accumulation in primary mouse dopamine neurons. Phosphorylated &#945;-synuclein aggregates in neurons are induced with pre-formed &#945;-synuclein fibrils. Automated imaging of immunofluorescently labeled cells and unbiased image analysis make this robust protocol suitable for medium-to-high throughput screening of drugs that inhibit &#945;-synuclein accumulation.

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with ...

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic lateral sclerosis, and &#945;-synuclein, implicated in Parkinson&#8217;s disease, are toxic and form cytoplasmic aggregates in yeast. These features recapitulate protein pathologies observed in patients with these disorders. Thus, yeast are an ideal platform for isolating toxicity suppressors from libraries of protein variants. We are interested in applying protein disaggregases to eliminate misfolded toxic protein conformers. Specifically, we are engineering Hsp104, a hexameric AAA+ protein from yeast that is uniquely capable of solubilizing both disordered aggregates and amyloid and returning the proteins to their native conformations. While Hsp104 is highly conserved in eukaryotes and eubacteria, it has no known metazoan homologue. Hsp104 has only limited ability to eliminate disordered aggregates and amyloid fibers implicated in human disease. Thus, we aim to engineer Hsp104 variants to reverse the protein misfolding implicated in neurodegenerative disorders. We have developed methods to screen large libraries of Hsp104 variants for suppression of proteotoxicity in yeast. As yeast are prone to spontaneous nonspecific suppression of toxicity, a two-step screening process has been developed to eliminate false positives. Using these methods, we have identified a series of potentiated Hsp104 variants that potently suppress the toxicity and aggregation of TDP-43, FUS, and &#945;-synuclein. Here, we describe this optimized protocol, which could be adapted to screen libraries constructed using any protein backbone for suppression of toxicity of any protein that is toxic in yeast.

Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic ...

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

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