Name: Saccharomyces cerevisiae Models of Alzheimers Disease to Screen Genes, Mutations, and Chemicals Affecting Amyloid Beta Production by γ-Secretase (Video) | JoVE Journal
Uploaded: 2025-06-24T12:50:00.000+00:00
Duration: 11 min 57 s
Description: Learn to reconstitute gamma-secretase in Saccharomyces cerevisiae to screen genes, mutations, and chemicals affecting amyloid beta production. This method enables rapid functional screening of Alzheimer's disease modulators.

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5 minutes

This work was supported by JSPS KAKENHI Grant Number 22K06121. The authors wish to acknowledge support from the members of the Laboratory of Enzymology at Tohoku University.

1. Measurement of &#947;-secretase activity using a yeast reporter system
<ol>
	<li>Transform the plasmids (Table 1) into the yeast PJ-69-4A strain (Table 2) using the standard lithium acetate method to express &#947;-secretase and its substrate14. To reconstitute the cleavage reaction, transform three plasmids: two for &#947;-secretase and one for the substrate. Select transformants on SC agar medium lacking Leu, Trp, and Ura (SC-LWU).</li>
	<li>To assess substrate cleavage by growth, streak yeast strains onto SC-LWU medium also lacking histidine and adenine (SC-LWHUAde) and incubate at 30 &#176;C for 3 days.</li>
	<li>Use colony size to assess the expression levels of HIS3 and ADE2. Evaluate colony size as follows: +++ represents wild-type growth with colonies larger than 1 mm, - represents no growth, and ++ or + represents partial growth with colonies larger than 0.5 mm or smaller than 0.5 mm, respectively. Expression of these genes is an indirect measurement of &#947;-secretase activity (Figure 1).</li>
	<li>To assess substrate cleavage by &#946;-galactosidase activity, i.e., the expression level of lacZ (Figure 1), grow strains in SC-LWU medium at 30 &#176;C until the culture reaches a concentration of 1 x 107 cells/mL, corresponding to OD600 of 0.8-1.0.
	<ol>
		<li>Harvest cells from a 1.5 mL culture by centrifugation in 1.5 mL tube at 21,100 x g for 3 min at 4 &#176;C, wash with 100 &#181;L of ice-cold water and resuspend in 30 &#181;L of D-buffer (20 mM Tris/Cl (pH 8.0), 10 mM MgCl2, 50 mM KCl, 1 mM EDTA, 5% glycerol) with 1 mM dithiothreitol (DTT) and protease inhibitor cocktails PIs/water (1000x solution: 1 M p-aminobenzamidine (pABA), 1 mg/mL antipain, 1 mg/mL aprotinin, 1 mg/mL leupeptin) and PIs/DMSO (1000x solution: 1 M phenylmethylsulfonyl fluoride (PMSF), 0.1 M N-p-Tosl-L-phenylalanine chloromethyl ketone (TPCK), 1 mg/mL Chymostatin, 1 mg/mL Pepstatin).</li>
		<li>Add an acid-washed glass bead just below the surface of the solution and lyse the cells by vortexing the mixture 10x at maximum speed for 1 min while placing it on ice at 1 min intervals.</li>
		<li>Centrifuge lysed cells at 21,100 x g for 10 min at 4 &#176;C and collect the supernatant as crude lysate.</li>
		<li>Prepare the reaction mixture with 5-10 &#181;L of crude lysate and 0.4 mL of Z-buffer (40 mM NaH2PO4, 60 mM Na2HPO4, 10 mM KCl, 1 mM MgSO4). Start the reaction by adding 80 &#181;L of 0.4% o-nitrophenyl-&#946;-D-galactopyranoside (ONPG) solution, then incubate at 37 &#176;C until the solution turns pale-yellow due to the formation of nitrophenol.</li>
		<li>Stop the reaction by adding 0.2 mL of 1 M Na2CO3 solution and measure the absorbance at 420 nm to calculate &#946;-galactosidase activity.</li>
		<li>Quantify the protein concentrations in crude lysates by using a protein assay kit, based on the method of Bradford using bovine serum albumin (BSA) as a standard. Prepare the dye reagent by diluting 1 part of the dye stock with 4 parts of Double-distilled deionized (DDI) water.</li>
		<li>Mix 1-5 &#181;L of lysate or BSA solution with 100 &#181;L of diluted dye in a microtiter plate well. Incubate at room temperature for 5 min and measure absorbance at 595 nm. Determine the protein concentration from the BSA standard curve. 
		&#946;-galactosidase unit = (1,000 x A420) / (t x c x V) 
		&#8203;where t = time of incubation, c = protein concentration (mg/mL), V = volume of lysate in the assay (mL). One unit of &#946;-galactosidase is defined as the amount of enzyme required to hydrolyze 1 &#181;mol of ONPG to produce o-nitrophenol and D-galactose per min per mg of protein.</li>
	</ol>
	</li>
	<li>To confirm the expression of &#947;-secretase subunits and the substrates, grow the strains in SC-LWU medium at 30 &#176;C until the culture reaches OD600 0.8-1.0.
	<ol>
		<li>Harvest cells from a 10 mL culture by centrifugation in a 50 mL tube at 2,100 x g for 10 min at 4 &#176;C, wash with 1 mL of ice-cold water followed by centrifugation in a 1.5 mL tube at 21,100 x g for 3 min at 4 &#176;C, and resuspend in 100 &#181;L of B88-buffer (20 mM Hepes/KOH (pH 6.8), 250 mM Sorbitol, 150 mM KOAc, 5 mM Mg(OAc)2) with 1 mM DTT and the protease inhibitor cocktails, PIs/water and PIs/DMSO.</li>
		<li>Add glass beads just below the surface of the solution and lyse the cell by vortexing 10x at maximum speed for 1 min while keeping the mixture on ice for 1 min intervals.</li>
		<li>Centrifuge lysates at 845 x g for 2 min at 4 &#176;C and collect the supernatant as total lysate which contains the membrane fraction.</li>
		<li>Mix 100 &#181;L of total lysate with 96 mg of urea and 40 &#181;L of 6x SDS-sample buffer (0.4 M Tris/Cl (pH 6.8), 12% SDS, 0.6M DTT, 0.05% Bromophenol Blue, 50% glycerol) and incubate at 65 &#176;C for 10 min. The addition of urea is necessary to prevent the aggregation of presenilin.</li>
		<li>Analyze the expression of &#947;-secretase and its substrates by running samples on 12.5% polyacrylamide Tris/Glycine gels and immunoblotting.</li>
		<li>Detect &#947;-secretase and its substrates by antibodies against presenilin, nicastrin, Aph1, Gal4, FLAG, HA, or Pgk1 (loading control), and the horseradish peroxidase (HRP)-conjugated secondary antibody against mouse IgG (for FLAG, HA, and Pgk1), rabbit IgG (for presenilin, Aph1, and Gal4), or guinea pig IgG (for nicastrin).</li>
		<li>For chemiluminescent detection, mix solution A (50 mM Tris/Cl (pH 8.5), 1.25 mM luminol, 0.02 mM p-coumaric acid) with solution B (50 mM Tris/Cl (pH 8.5), 0.009% H2O2) immediately before use. Incubate the blot in a sufficient volume of the mixture for 1 min. The required volume depends on the blot size; for a 10 cm x 6 cm blot, 4 mL is used. Develop the blot and quantify the signal intensity using the luminescent image analyzer.</li>
	</ol>
	</li>
</ol>
2. Measurement of A&#946; production by in vitro microsome assay
<ol>
	<li>Transform APP C55, APP C99, or &#947;-secretase plasmids (Table 1) into yeast strain PJ-69-4A pep4&#916; as described in step 1.1. to express the &#947;-secretase and its substrate separately (Table 2; &#947;311, &#947;312, and &#947;313 strains)14. Select the C55 and C99 strains on the SC-U agar medium and the &#947;-secretase strain on the SC-LW agar medium.</li>
	<li>Isolate microsomes from yeast strains expressing APP C55, APP C99, or &#947;-secretase according to the standard protocol11. Separate microsomes by sucrose density gradient centrifugation and recover at 1.2 M-1.5 M sucrose interface using a disposable plastic pipette. Determine the protein concentration in microsomes by measuring absorbance at 260 nm after solubilizing them in 0.2% SDS.</li>
	<li>Prepare the reaction mixture with two microsomes, i.e., APPC55 or &#947;-secretase, containing 40 &#181;g of protein each and &#947;-buffer (50 mM PIPES/KOH (pH 7.0), 250 mM Sucrose, 1 mM EGTA) in a final volume of 12.5 &#181;L. Solubilize by mixing with an equal volume (12.5 &#181;L) of 2% CHAPSO in &#947;-buffer and incubate in 1% CHAPSO for 1 h.</li>
	<li>To start the reaction, dilute the mixtures four-fold to a final volume of 100 &#181;L using &#947;-buffer containing 1x protease inhibitor (PI) mix: 50 &#181;M diisopropyl fluorophosphate (DIFP), 50 &#181;M PMSF, 0.1 &#181;g/mL N&#945;-p-tosyl-L-lysine chloromethyl ketone (TLCK), 0.1 &#181;g/mL antipain, 0.1 &#181;g/mL leupeptin, 0.1 mM EGTA, 1 mM thiolphan, and 5 mM phenanthroline (PNT) to 0.25% CHAPSO with 400 &#181;g/mL proteins. Incubate the reaction mixture at 37 &#176;C for 24 h. If necessary, add 0.1% phosphatidylcholine (PC), 10 &#181;M L685,458, or DAPT (&#947;-secretase inhibitors).</li>
	<li>Stop the reaction by adding 500 &#181;L of chloroform/methanol (2:1) and incubate for 1 h with vortexing intermittently. Add 900 &#181;L of methanol, mix, and centrifuge at 21,100 x g for 15 min to collect the A&#946; peptides in the pellet.</li>
	<li>Wash the pellet with 500 &#181;L of chloroform/methanol/water (1:2:0.8), allow it to air-dry, suspend it in 40 &#181;L of SDS-sample buffer, and boil the mixture for 10 min. Analyze the A&#946; production by running samples on a 16.5% Tris/Tricine polyacrylamide gel. To identify the A&#946; forms, use a 10% Tris/Tricine polyacrylamide gel with 8 M urea (pH 8.45).</li>
	<li>Detect A&#946; by immunoblotting using the anti-antibody 82E1 specific to the Asp-1 site of human A&#946; and the HRP-conjugated secondary antibody against mouse IgG. For chemiluminescent detection, mix equal amounts of solution A and B immediately before use as described in step 1.5.7, and incubate the blot in a sufficient volume of the mixture for 1 min. The required volume depends on the blot size; for a 10 cm x 6 cm blot, 4 mL is used.</li>
	<li>Develop the blot and quantify signal intensity using the luminescent image analyzer.</li>
</ol>
3. Screening for &#947;-secretase or APP mutations with modulatory effect
<ol>
	<li>To generate random mutations by error-prone PCR, design primers to incorporate 40 bp overlapping regions of the gene of interest at the 5&#39; end of each primer.</li>
	<li>Prepare 50 &#181;L of error-prone PCR reaction in 1x Taq buffer with 50 ng of template DNA, 0.2 mM dGTP, 1 mM dATP/dTTP/dCTP, 3 mM MgCl2, 0.5 mM MnCl2, 1.25 U Taq polymerase, and 400 nM primers. Use the following PCR protocol: 94 &#176;C for 5 min, followed by 30 cycles of 94 &#176;C for 30 s, 55 &#176;C for 30 s, and 72 &#176;C for 120 s, ending with 72 &#176;C for 10 min. This setting produces an approximate mutation rate of 1.9 mutations per kilobase10.</li>
	<li>Transform the mutagenized DNA fragments (4 &#181;g) with linearized plasmid DNA (4 &#181;g) into PJ69-4A yeast strain containing FAD APP or PS1 mutations14. The 40 bp overlap in primers facilitates homologous recombination between the fragments in yeast, leading to the reconstruction of the plasmid. Select transformants on SC-LWU plates as described in step 1.1.</li>
	<li>Count transformants on SC-LWU plates and replicate them onto SC-LWHUAde plates using velvet. To replicate, press the transformant plate onto a velvet-covered cylindrical block, then press a fresh SC-LWHUAde plate onto the velvet to transfer the colonies. Strains expressing APP or PS1 FAD mutants grow poorly on SC-LWHUAde, while those with modulatory mutations exhibit robust growth and form colonies.</li>
	<li>To recover plasmid DNA from positive clones, culture the strains in 3 mL of SC-LWU medium overnight at 30 &#176;C until OD600 &#62;1.0. Centrifuge the cells in a 15 mL tube at 2,100 x g for 10 min at 4 &#176;C, wash with 0.8 mL ice-cold water followed by centrifugation in a 1.5 mL tube at 21,100 x g for 3 min at 4 &#176;C, and then resuspend in 0.3 mL of lysis solution (10 mM Tris/Cl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 2% Triton X-100, 1% SDS).</li>
	<li>Lyse cells by adding 0.3 mL of phenol:chloroform:isoamyl alcohol (25:24:1) and 0.3 g glass beads. Vortex at maximum speed for 10 min, then centrifuge at 21,100 x g for 10 min at room temperature.</li>
	<li>After centrifugation, the sample separates into an upper aqueous phase and a lower phenol phase. Recover the aqueous phase and mix with 30 &#181;L of 3 M sodium acetate and 700 &#181;L of 100% ethanol. Avoid taking materials on the interphase.</li>
	<li>Centrifuge the solution at 21,100 x g for 10 min at 4 &#176;C, wash the pellet with 70% ethanol, air-dry, and resuspend in 15 &#181;L of TE buffer.</li>
	<li>Since the concentration of plasmid DNA is typically too low for further analysis, it is necessary to amplify it in E. coli. Transform the plasmid DNA into E. coli DH5&#945; by high-efficiency transformation methods15 and purify for further analysis. Identify mutations by DNA sequencing using the Sanger method16.</li>
	<li>This method is applicable to mutagenized plasmids prepared by different methods (as in steps 3.1 and 3.2). If targeting specific protein regions, design inverse PCR primers with random nucleotides. After PCR and ligation to circularize the plasmid DNA, a library of mutagenized plasmids is obtained. Introduce specific mutations using the site-specific mutagenesis kit.</li>
</ol>
4. Screening for &#947;-secretase inhibitor (GSI) in yeast
<ol>
	<li>Use the high-throughput chemical screening of GSI developed based on yeast growth or &#946;-galactosidase assay, as described below.</li>
	<li>Conduct GSI screening with yeast strains, &#947;124, &#947;126, and &#947;190 (Table 2). Select GSI candidates as compounds that reduce the growth or &#946;-galactosidase activity of wild-type &#947;-secretase on APP (&#947;124) and/or Notch (&#947;126) (Table 3).</li>
	<li>Select substrate-specific APP-sparing or Notch-sparing GSIs. Exclude toxic compounds by counter-screening with the soluble Gal4 expression. GSI candidates should not affect the growth or &#946;-galactosidase activity of the Gal4 control strain (&#947;190) (Table 3).</li>
	<li>For the growth assay, grow strains in SC-LWHUAde medium containing potential GSIs in 384-well plates from OD600 of 0.03. Incubate overnight at 30 &#176;C without shaking. Monitor growth after 24 h by OD600.</li>
	<li>For &#946;-galactosidase assays in a 384-well format, grow strains to a concentration of 1 x 107 cells/mL in SC-LWU medium containing potential GSIs. Lyse the cells using the Beta-Glo assay system and measure &#946;-galactosidase activity using 6-o-&#946;-galactopyranosyl-luciferin as a substrate according to the manufacturer&#39;s protocol.</li>
	<li>For GSI candidates, measure the A&#946; production by in vitro microsome assay to confirm the inhibitory effect.</li>
</ol>
5. Screening for &#947;-secretase modulator (GSM) in yeast
<ol>
	<li>Use the high-throughput chemical screening of GSM, which has been developed based on yeast growth or &#946;-galactosidase assay, as described below.</li>
	<li>Conduct GSM screening with yeast strains, &#947;106, &#947;112, and &#947;5 (Table 2). Select GSM candidates as compounds that enhance the growth or &#946;-galactosidase activity of FAD PS1 (&#947;106 and &#947;112) or FAD APP mutant strains (Table 4). Exclude non-specific reporter activator chemicals by counter-screening with the strain without &#947; secretase catalytic subunit. GSM candidates should not affect the growth or &#946;-galactosidase activity of the control strain without PS1 (&#947;5) (Table 4).</li>
	<li>For the growth assay, grow strains in SC-LWHUAde medium containing potential GSMs in 384-well plates from OD600 of 0.03. Incubate overnight at 30 &#176;C without shaking. Monitor growth after 48 h by OD600.</li>
	<li>For &#946;-galactosidase assays in a 384-well format, grow strains to a concentration of 1 x 107 cells/mL in SC-LWU medium containing potential GSMs. Lyse the cells using the Beta-Glo assay system and measure &#946;-galactosidase activity using 6-o-&#946;-galactopyranosyl-luciferin as a substrate according to the manufacturer&#39;s protocol.</li>
	<li>For GSM candidates, measure the A&#946; production by in vitro microsome assay to confirm the modulatory effect, i.e., the reduction of toxic A&#946; forms A&#946;42 and A&#946;43.</li>
</ol>

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<li>Han, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((OCIAD2+activates+%CE%B3-secretase+to+enhance+amyloid+%CE%B2+production+by+interacting+with+nicastrin%5BTitle%5D)+AND+%22Cell+Mol+Life+Sci%22%5BJournal%5D)">OCIAD2 activates γ-secretase to enhance amyloid β production by interacting with nicastrin.</a> Cell Mol Life Sci. 71 (13), 2561-2576 (2014).</li>
<li>Self, W. K., Holtzman, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Emerging+diagnostics+and+therapeutics+for+Alzheimer%27s+disease%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">Emerging diagnostics and therapeutics for Alzheimer's disease.</a> Nat Med. 29, 2187-2199 (2023).</li>
<li>Solopova, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fatal+iatrogenic+cerebral+%CE%B2-amyloid-related+arteritis+in+a+woman+treated+with+lecanemab+for+Alzheimer%27s+disease%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Fatal iatrogenic cerebral β-amyloid-related arteritis in a woman treated with lecanemab for Alzheimer's disease.</a> Nat Commun. 14 (1), 8220(2023).</li>
<li>Zhang, Y., Chen, H., Li, R., Sterling, K., Song, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amyloid+%CE%B2-based+therapy+for+Alzheimer%27s+disease%3A+challenges%2C+successes+and+future%5BTitle%5D)+AND+%22Signal+Transduct+Target+Ther%22%5BJournal%5D)">Amyloid β-based therapy for Alzheimer's disease: challenges, successes and future.</a> Signal Transduct Target Ther. 8 (1), 248(2023).</li>
<li>Xia, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%CE%B3-Secretase+and+its+modulators%3A+twenty+years+and+beyond%5BTitle%5D)+AND+%22Neurosci+Lett%22%5BJournal%5D)">γ-Secretase and its modulators: twenty years and beyond.</a> Neurosci Lett. 701, 162-169 (2019).</li>
<li>Muttenthaler, M., King, G. F., Adams, D. J., Alewood, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Trends+in+peptide+drug+discovery%5BTitle%5D)+AND+%22Nat+Rev+Drug+Discov%22%5BJournal%5D)">Trends in peptide drug discovery.</a> Nat Rev Drug Discov. 20 (4), 309-325 (2021).</li>
<li>Souza, J. M., Goncalves, B. D. C., Gomez, M. V., Vieira, L. B., Ribeiro, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+toxins+as+therapeutic+tools+to+treat+neurodegenerative+disease%5BTitle%5D)+AND+%22Front+Pharmacol%22%5BJournal%5D)">Animal toxins as therapeutic tools to treat neurodegenerative disease.</a> Front Pharmacol. 9, 145(2018).</li>
</ol>

The authors have nothing to disclose.

Critical steps in the protocol and troubleshooting 
The reconstitution of &#947;-secretase in yeast using genetic and biochemical methods is relatively straightforward, but there are some critical steps to ensure success.
During yeast transformation (steps 1.1, 2.1, and 3.3), multiple plasmids should be introduced: two for the &#947;-secretase components and one for the substrates. Sequential transformation (one at a time) can also be performed, but it is time-consuming. ...

&#947;-Secretase is responsible for the intramembrane cleavage of over 150 type-I transmembrane proteins, including the amyloid precursor protein (APP) and Notch1. The significance of intramembrane cleavage includes not only protein turnover but also signal transduction through the generated fragments. For example, the processing of APP produces small amyloid &#946; peptides (A&#946;). Although A&#946; should be removed as a degradation product, in Alzheimer&#39;s disease, it accumulates in the brain to form senile plaques2. On the other hand, the processing of Notch is essential for NOTCH signaling3, which is crucial for development. &#947;-Secretase consists of four transmembrane proteins: presenilin (PS1 or PS2), nicastrin (NCT), anterior pharynx-1 (Aph-1a or 1b), and presenilin enhancer 2 (Pen2)4. These proteins have a total of 19 transmembrane domains: there are nine in PS, seven in Aph1, two in Pen2, and one in NCT. PS serves as the catalytic subunit and contains two aspartic acid residues located in transmembrane domains TM6 and TM75. During maturation of the complex, PS undergoes endoproteolytic cleavage within its hydrophilic loop HL6, leading to the formation of amino-terminal and carboxyl-terminal fragments4. Mutations associated with Familial Alzheimer&#39;s disease (FAD) are found in both the APP and PS genes2. These pathogenic missense mutations adversely affect &#947;-secretase activity, reducing its ability to effectively process APP. This leads to increased production of the aggregation-prone A&#946;42 forms rather than the common A&#946;406. The exact mechanisms by which these mutations disrupt the cleavage remain unclear. &#947;-Secretase modulators (GSMs), which can alter the ratio of A&#946;42 to other A&#946; forms, show promise as potential therapeutic agents for the treatment of Alzheimer&#39;s disease by lowering A&#946;42 levels7.
The budding yeast Saccharomyces cerevisiae has long served as a model organism to investigate key functions in eukaryotic cells, such as cell division, protein transport, and autophagy. Researchers have employed functional complementation to study human homologs, including cell division cycle&#160;(CDC), secretion&#160;(SEC), and autophagy-related&#160;(ATG) genes. Transforming yeast mutants with human genes allows researchers to analyze cellular processes from a functional perspective. Of the five known families of intramembrane proteases, yeast possesses only three: signal peptide peptidase (Ypf1), rhomboids (Pcp1 and Rbd2), and Ras-converting enzyme (Rce1). However, there are no homologs of &#947;-secretase or site-2 protease in yeast8, making the complementation approach not suitable. Despite the absence of yeast counterparts for these proteases, S. cerevisiae remains valuable for exploring human proteins. Techniques such as yeast one-hybrid, two-hybrid, and three-hybrid assays, which use the yeast transcriptional activator Gal4, are employed to detect interactions between DNA, transcription factors, proteins, or RNA9. We have modified the Gal4 system to monitor intramembrane proteolysis mediated by &#947;-secretase.
We have successfully reconstituted the &#947;-secretase cleavage reaction in yeast by introducing human &#947;-secretase along with artificial substrates fused to Gal4 (Figure 1)10. The cleavage reaction was tracked through the Gal reporter system, either by monitoring yeast growth or measuring &#946;-galactosidase activity. Additionally, we have identified various A&#946; forms (A&#946;38, A&#946;40, A&#946;42, A&#946;43, and A&#946;45) released in yeast microsomes by immunoblotting (Figure 1)11.
When FAD-related PS1 or APP mutants were introduced into this system, variable degrees of loss-of-function activity were observed12. We conducted a growth-based screen to identify suppressor mutations that restore the impaired function of these FAD mutants (Figure 2). These suppressors not only rescued the defect but also independently enhanced the trimming activity of &#947;-secretase. Specifically, they reduced the levels of longer forms of A&#946;, particularly A&#946;42, in a manner similar to A&#946;42-lowering &#947;-secretase modulators (GSMs). The yeast reconstitution system allows screening of mutations, genes, and compounds that regulate activity, including &#947;-secretase inhibitor (GSI) and modulator (GSM)13.
The goal of this protocol is to accomplish two main objectives: i) to establish a yeast-based &#947;-secretase screening system and ii) to provide an example of reconstitution techniques for human enzymatic reactions in yeast, emphasizing the essence of reconstitution methods. We outline the methods in three key procedures with the following essential steps: 1) reconstituting &#947;-secretase in yeast, 2) performing an in vitro &#947;-secretase assay using yeast microsomes, and 3-5) screening &#947;-secretase mutations and chemical regulators in yeast.

<table><tbody><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>anti Amyloid&amp;beta; (N) (82E1) antibody</td><td>IBL</td><td>Cat# 10323</td><td></td></tr><tr><td>anti Aph1a, 245-265 (C terminus) antibody</td><td>BioLegend</td><td>Cat# 823101</td><td></td></tr><tr><td>anti FLAG (M2) antibody</td><td>Sigma</td><td>Cat# F3165</td><td></td></tr><tr><td>anti Gal4 antibody</td><td>Sigma</td><td>Cat# G9293</td><td></td></tr><tr><td>anti HA (12CA5) antibody</td><td>Sigma</td><td>Cat# 11 583 816 001</td><td></td></tr><tr><td>anti Nicastrin antibody</td><td>Chemicon</td><td>Cat# AB5890</td><td></td></tr><tr><td>anti PGK1 (22C5D8) antibody</td><td>abcam</td><td>Cat# ab197960</td><td></td></tr><tr><td>anti PS1 N-terminus (G1Nr) or loop (GIL) antibodies</td><td>Takasugi et.al., J. Biol. Chem., 277, 50198-50205</td><td></td><td></td></tr><tr><td>anti rabbit&amp;nbsp; IgG, HRP-linked antibody</td><td>Cell Signaling</td><td>Cat# 7074</td><td></td></tr><tr><td>anti-guinea pig IgG-HRP</td><td>Santa Cruz Biotechnology</td><td>Cat# sc-2438</td><td></td></tr><tr><td>anti-mouse IgG, HRP-linked antibody</td><td>Cell Signaling</td><td>Cat# 7076</td><td></td></tr><tr><td>&lt;strong&gt;Bacterial and yeast strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; DH5&amp;alpha;</td><td>Invitrogen</td><td>Cat# 18265017</td><td></td></tr><tr><td>&lt;em&gt;S. cerevisiae &lt;/em&gt;PJ-69-4A</td><td>James P. et. al., Genetics 144:1425-1436 (1996)</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Chemicals, peptides, recombinant proteins&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>(3,5-Difluorophenylacetyl)-Ala-Phg-OBu&lt;sup&gt;t &lt;/sup&gt;(DAPT)</td><td>Peptide Institute</td><td>Cat# 3219-v</td><td></td></tr><tr><td>Amyloid &amp;beta;-protein (Human, 1-38)</td><td>Peptide Institute</td><td>Cat# 4484-v</td><td></td></tr><tr><td>Amyloid &amp;beta;-protein (Human, 1-40)</td><td>Peptide Institute</td><td>Cat# 4307-v</td><td></td></tr><tr><td>Amyloid &amp;beta;-protein (Human, 1-42)</td><td>Peptide Institute</td><td>Cat# 4349-v</td><td></td></tr><tr><td>Amyloid &amp;beta;-protein (Human, 1-43)</td><td>Peptide Institute</td><td>Cat# 4370-v</td><td></td></tr><tr><td>antipain, dihydrochloride</td><td>Peptide Institute</td><td>Cat# 4062</td><td></td></tr><tr><td>aprotinin</td><td>Sigma</td><td>Cat# A1153</td><td></td></tr><tr><td>CHAPSO</td><td>Dojindo</td><td>Cat# 343-05163</td><td></td></tr><tr><td>Chymostatin</td><td>Sigma</td><td>Cat# C7268</td><td></td></tr><tr><td>Diisopropyl fluorophosphate (DIFP)</td><td>Wako chemical</td><td>Cat# 049-18921</td><td></td></tr><tr><td>L685,458</td><td>Peptide Institute</td><td>Cat# 4394-v</td><td></td></tr><tr><td>leupeptin</td><td>Peptide Institute</td><td>Cat# 4041</td><td></td></tr><tr><td>N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK)</td><td>Sigma</td><td>Cat# T4376</td><td></td></tr><tr><td>N&lt;sup&gt;&amp;alpha;&lt;/sup&gt;-p-tosyl-L-lysine chloromethyl ketone (TLCK)</td><td>Sigma</td><td>Cat# T7254</td><td></td></tr><tr><td>o-nitrophenyl-&amp;beta;-D-galactopyranoside (ONPG)</td><td>Sigma</td><td>Cat# N1127</td><td></td></tr><tr><td>p-aminobenzamidine (pABA), dihydrochloride</td><td>Sigma</td><td>Cat# 857661</td><td></td></tr><tr><td>Pepstatin A</td><td>Sigma</td><td>Cat# P5318</td><td></td></tr><tr><td>Phenanthroline (PNT)</td><td>Sigma</td><td>Cat# P9375</td><td></td></tr><tr><td>phenylmethylsulfonyl fluoride (PMSF)</td><td>Sigma</td><td>Cat# P7626</td><td></td></tr><tr><td>phosphatidyl choline (PC), egg yolk, Type XVI-E</td><td>Sigma</td><td>Cat# P3556</td><td></td></tr><tr><td>Taq DNA polymerase</td><td>Takara</td><td>Cat# R001A</td><td></td></tr><tr><td>thiolphan</td><td>Sigma</td><td>Cat# T6031</td><td></td></tr><tr><td>&lt;strong&gt;Commercial assay kits, other materials, and Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Beta-Glo Assay System</td><td>Promega</td><td>Cat# E4720</td><td></td></tr><tr><td>Bio-Rad Protein Assay kit</td><td>Bio-Rad</td><td>Cat# 5000001</td><td></td></tr><tr><td>Glass beads, acid-washed (425-600 &amp;mu;m)</td><td>Sigma</td><td>Cat# G8772</td><td></td></tr><tr><td>ImageQuant LAS4000</td><td>GE Healthcare</td><td>ImageQuant LAS4000</td><td></td></tr><tr><td>QuikChange Site-Directed Mutagenesis Kit</td><td>Agilent</td><td>Cat# 200518</td><td></td></tr></tbody></table>

saccharomyces cerevisiae models of alzheimer's disease to screen genes, mutations, and chemicals affecting amyloid beta production by &#947;-secretase

Saccharomyces cerevisiae Models of Alzheimer's Disease to Screen Genes, Mutations, and Chemicals Affecting Amyloid Beta Production by &#947;-Secretase

&#947;-Secretase is a protease complex embedded in the cell membrane, consisting of the catalytic presenilin subunits (PS1 or PS2) and three additional co-factors: nicastrin, Aph-1, and Pen2. It cleaves the transmembrane domains of type-I transmembrane proteins, such as the amyloid precursor protein (APP) and Notch. The cleavage of APP generates the amyloid &#946; peptides (A&#946;), which accumulate in patients with Alzheimer's disease. Despite significant research, the exact mechanism of this unique proteolysis, which occurs within the lipid bilayers, is still not fully understood. To study the enzymatic properties of &#947;-secretase, we have established a yeast reporter system using artificial &#947;-secretase substrates containing APP or Notch fragments fused to the transcriptional activator Gal4. The &#947;-secretase activity was evaluated by transcriptional activation of reporter genes upon Gal4 release from the membrane-bound substrates, as assessed by the growth of yeast or &#946;-galactosidase activity. Furthermore, we have developed an in vitro assay to identify the different forms of A&#946; produced from yeast microsomes. These yeast models provide a platform to screen mutations, genes, and compounds that affect &#947;-secretase function. By studying the loss of function properties of PS1 familial Alzheimer's disease (FAD) mutants, it is possible to screen for FAD suppressor mutations and &#947;-secretase modulators (GSMs), in addition to &#947;-secretase inhibitors (GSIs). In this report, we describe the genetic and biochemical methods used to study &#947;-secretase activity in the yeast system with the essential steps of the protocol for the video.

Using the techniques described so far, we have analyzed genes, mutations, chemicals, and peptides as factors that affect the activity of γ-secretase10,11,12,13,17,18,19. Growth-based screening of genes and mutations allows testing of a large number of clones (100,000-1,000,000). The large si...

Here, we describe a yeast system reconstituting human &#947;-secretase. This system allows for the identification and study of mutations affecting activity and for screening of &#947;-secretase inhibitors (GSIs). Utilizing the loss of function properties of familial Alzheimer's disease mutants, it is possible to screen for &#947;-secretase modulators (GSMs).

&#947;-Secretase is a protease complex embedded in the cell membrane, consisting of the catalytic presenilin subunits (PS1 or PS2) and three additional ...

We are reconstituting gamma-secretase in yeast to investigate how FAD-linked mutations impact its activity and suppressor mutations or chemical modulators restore its function and reduce pathogenic Abeta 42 levels. We combine functional analysis using mammalian cell and yeast system with structure analysis via cryo-EM to study mechanism of gamma-secretase modulation. We analyzed FAD mutations using gamma-secretase reconstitution system in yeast and identified the suppressors that could restore activity of gamma-secretase FAD mutations, and also reduce the level of Abeta 42, mimicking the effect of gamma-secretase modulators.Our protocol enables rapid scalable functional screening of gamma-secretase activity in yeast, allowing analysis of human mutations and modulators without relying on complex mammalian systems. We will focus on identifying new genetic or chemical modulators and inhibitors of gamma-secretase A, illustrating mechanism of Abeta 42 production and expanding yeast-based reconstruction systems whole as a human intramembrane proteolysis. To begin, transform the plasmids into the yeast PJ69-4A strain using the standard lithium acetate method to express gamma-secretase and its substrate.Streak the transformed yeast strains onto SC-LWHUA medium, also lacking histidine and adenine, to assess substrate cleavage based on growth. Incubate the plates at 30 degrees Celsius for three days. Evaluate colony size to assess expression of HIS3 and ADE2 genes.To assess substrate cleavage via beta lactase activity, grow the yeast strains in SC-LWU liquid medium at 30 degrees Celsius until the culture reaches a concentration of 10 to the power of seven cells per milliliter. Pipette out 1.5 milliliters of the culture into centrifuge tubes, then centrifuge the tubes at 21, 100 G for three minutes at four degrees Celsius in a 1.5 milliliter tube. Wash the pellet with 100 microliters of ice cold water and re-suspend it in 30 microliters of debuffer containing one millimolar Dithiothreitol and protease inhibitor cocktails made with water and dimethyl sulfoxide.Now add an acid washed glass bead so that it is just below the surface of the solution. Vortex the mixture 10 times for one minute each at maximum speed. With a hot 25 gauge needle, make a hole in the bottom of the tube.Centrifuge the lysed cells again at 865G for two minutes, then again 21, 100 G for 10 minutes at four degrees Celsius. Collect the supernatant as the crude lysate. Next, combine five to 10 microliters of crude lysate with 0.4 milliliters of Z buffer.Then add 80 microliters of 0.4%o-nitrophenyl-beta-D-galactoside solution and vortex the mixture. Incubate at 37 degrees Celsius until the solution turns pale yellow due to nitrophenol formation. Stop the reaction by adding 0.2 milliliters of one molar sodium carbonate solution.Measure absorbance at 420 nanometers to determine beta-galactosidase activity. Quantify protein concentrations in crude lysates using a protein assay kit based on the Bradford Method with bovine serum albumin as a standard. Prepare the dye reagent by diluting one part of the stock with four parts of double distilled deionized water.Mix one to five microliters of each lysate or standard with 100 microliters of diluted dye in a microtiter plate well. After incubating the plate at room temperature for five minutes, measure absorbance at 595 nanometers to determine protein concentration using the standard curve. Start by combining two microsomes, each containing 40 to 80 micrograms of protein with gamma buffer in a final volume of 12.5 microliters.Pipette an equal volume of 2%CHAPSO in gamma buffer before incubating for one hour. To start the reaction, dilute the mixture fourfold to a final volume of 100 microliters with gamma buffer containing protease inhibitor mix. Incubate the reaction at 37 degrees Celsius for 24 hours.Pipette 500 microliters of chloroform methanol mixture in a two to one ratio to stop the reaction, and incubate for one hour, vortexing intermittently. Next, add 900 microliters of methanol and mix thoroughly. Centrifuge the tubes at 21, 100 G for 15 minutes to pellet amyloid beta peptides.Wash the pellet with 500 microliters of chloroform methanol water mixture in a one to two to 0.8 ratio, and centrifuge again. After air drying the pellet, suspend it in 40 microliters of SDS sample buffer, then boil for 10 minutes. Now run the samples on a 16.5%Tris-Tricine polyacrylamide gel to analyze amyloid beta production.Immunoblot with the 82E1 primary antibody specific to the aspartic acid 1 site of human amyloid-beta. Use horseradish peroxidase conjugated secondary antibody against mouse immunoglobulin G for detection. For chemiluminescent detection, prepare the substrate by mixing equal amounts of solution A and B, and incubate the membrane in the prepared solution for one minute.Develop the blot and quantify the signal intensity using a luminescent image analyzer. Design primers for error prone polymerase chain reaction that incorporate 40 base pair overlapping sequences from the gene of interest at the five prime ends of both forward and reverse primers. Now prepare a 50 microliter error-prone polymerase chain reaction.Run the thermal cycler for the specified number of cycles. Transform four micrograms of mutagen DNA fragments along with four micrograms of linearized plasmid into the PJ69-4A yeast strain, expressing familial Alzheimer's disease APP or PS1 mutations. Then select transformants on SC-LWU plates.Count the colonies on SC-LWU plates. Replicate them by pressing the original plate onto a velvet covered block. Then press a fresh SC-LWHU aid plate medium lacking histidine and adenine plate to transfer colonies.To recover plasmid DNA from positive clones, inoculate the strains in three milliliters of SC-LWU medium. Incubate overnight at 30 degrees Celsius until optical density at 600 nanometers exceeds one. Centrifuge the cells in a 15 milliliter tube at 2, 100 G for 10 minutes at four degrees Celsius.Then wash the pellet with 0.8 milliliters of ice cold water. Transfer the suspension into a new 1.5 milliliter tube before centrifuging again for three minutes. Re-suspend the pellet in 0.3 milliliters of lysis solution.Add 0.3 milliliters of phenol-chloroform-isoamyl alcohol mixture in a 25 to 24 to one ratio and 0.3 grams of glass beads to lyse the cells. Vortex the mixture at maximum speed for 10 minutes, then centrifuge. Transfer the upper aqueous phase to a new tube.Mix it with 30 microliters of three molar sodium acetate and 700 microliters of 100%ethanol to precipitate DNA. After centrifuging the DNA pellet as before, wash with 70%ethanol, air dry. Add 15 microliters of TE buffer and vortex to re-suspend the DNA in TE buffer.Transform the purified plasmid DNA into Escherichia coli DH5-alpha using high efficiency transformation techniques. The double mutant F411Y/S438P supported robust yeast growth in the absence of nicastrin, forming colonies larger than one millimeter, and showed full beta-galactosidase activity comparable to wild type PS1. S438P showed weak growth, while F411Y showed none, and S438P showed low beta-galactosidase activity, while F411Y showed no activity.Rescreening cells from the PS1 F411Y mutant reconfirmed F411Y/S438P as the dominant active clone, with 40 positive hits, while only one additional clone showed alternative triple mutations. Screening from the S438P mutant revealed multiple combinations with low clone counts, except for V236M/S438P, which appeared in 13 positive clones, indicating S438P's dominant role over other mutations. The L30F/T164A Aph-1a mutant increased total amyloid beta production under both phosphatidylcholine positive and phosphatidylcholine negative conditions, particularly enhancing aggregation prone Abeta 42 and a Abeta 43 species in the phosphatidylcholine negative condition.Phosphatidylcholine enhanced amyloid beta production for both wild type and mutant forms, while gamma-secretase inhibitor L685, 458 blocked Abeta generation completely.

saccharomyces-cerevisiae-models-alzheimer-s-disease-to-screen-genes

Research

JoVE Journal

Neuroscience

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human β-amyloid peptide (Aβ).  Induced expression of Aβ in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate Aβ toxicity.

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

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.

High-throughput Yeast Plasmid Overexpression Screen

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human Aβ42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

A Caenorhabditis elegans Model System for Amylopathy Study

While the transport of cell surface proteins is relatively easily studied, visualizing the trafficking of intracellular proteins is much more difficult. Here, we use constructs incorporating photoactivatable GFP and demonstrate a method to accurately follow the amyloid precursor protein from the Golgi apparatus to down-stream compartments and follow its clearance.

Imaging the Intracellular Trafficking of APP with Photoactivatable GFP

We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-β (Aβ) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer's disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of β-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

We have successfully generated two substrate-specific γ-secretase assays. Both cell-based assays presented here are designed to quantify γ-secretase enzymatic activities via the output of firefly luciferase reporters.

Quantitative Measurement of &#947;-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms

Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid β protein on brain tissues of Alzheimer's disease and cerebral amyloid angiopathy samples using MALDI-IMS.

Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

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's disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

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

This protocol illustrates a cell encapsulation method by rapid physical gelation of alginate to immobilize cells. Obtained microbeads allow controlled and sustained secretion of amyloid-β over time and can be used to study the effects of secreted amyloid-β in in vitro and in vivo models.

Fabrication of Amyloid-&#946;-Secreting Alginate Microbeads for Use in Modelling Alzheimer's Disease

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.

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

Learn to reconstitute gamma-secretase in Saccharomyces cerevisiae to screen genes, mutations, and chemicals affecting amyloid beta production. This method enables rapid functional screening of Alzheimer's disease modulators.

Saccharomyces cerevisiae Models of Alzheimers Disease to Screen Genes, Mutations, and Chemicals Affecting Amyloid Beta Production by γ-Secretase (Video) | JoVE Journal

Saccharomyces cerevisiae Models of Alzheimer's Disease to Screen Genes, Mutations, and Chemicals Affecting Amyloid Beta Production by γ-Secretase

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