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

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.