This study investigates the protective capacity of test compounds against polyglutamine (polyQ) aggregation-associated proteotoxicity using transgenic Caenorhabditis elegans. The research focuses on the impact of these compounds on neuronal dysfunction and death caused by protein accumulation.
In some neurodegenerative disorders, the disease-associated protein comprises an expanded polyglutamine, polyQ, tract — string of consecutive glutamine residues — and is aberrantly conformed. The impaired conformation destabilizes protein homeostasis, causing protein accumulation and formation of toxic aggregates, leading to neuronal dysfunction and death.
To screen a test compound's protective capacity in alleviating polyQ aggregation-associated proteotoxicity, obtain a suspension of transgenic Caenorhabditis elegans larvae expressing fluorescently-tagged polyQ proteins in body wall muscle cells.
Take a multi-well plate containing media supplemented with bacterial food. Add the test compound in one set of wells. Transfer the larvae into the multi-well plate. Seal the plate to prevent media dehydration and microbial contamination. Incubate, allowing the larvae to grow into adults.
During development, as the nematode ages, polyQ proteins progressively accumulate within the muscle cells and form aggregates.
If the test compound is present in media, it enters the muscle cells and, depending on its protective capacity, may exert an anti-polyQ aggregation effect, suppressing and delaying polyQ aggregation.
Harvest the adult nematodes and centrifuge. Resuspend the pelleted nematodes. Transfer into a multi-well plate. Immobilize the nematodes using sodium azide.
Using a high-resolution fluorescence device, count the fluorescent puncta, representing polyQ aggregates, per nematode.
A lower average number of polyQ aggregates in treatment than control wells suggests a higher protective capacity of the test compound in alleviating polyQ aggregation-associated proteotoxicity.
Begin by transferring 300 to 500 synchronized L1 larvae of AM141 nematodes to each well of a 48-well plate with 500 microliters of S medium containing OP50 strain of E. coli, and five milligrams per milliliter of astragalan. Seal the plate with parafilm, and incubate it at 20 degrees Celsius and 120 rotations per minute for desired time intervals.
Transfer the nematodes in a sterile 1.5-milliliter microcentrifuge tube, and wash with M9 buffer three times by centrifugation, to remove the remaining OP50 cells. Then, resuspend the AM141 nematodes in M9 buffer.
Now, transfer 10 to 15 nematodes into each well in a 384-well plate, setting 10 replicate wells for each treatment, and add 10 microliters of 200 millimolar sodium azide to each well, to paralyze the nematodes by allowing them to settle down to the bottom.
Place the plate in a high-content imaging system to acquire fluorescent images. Open the image acquisition software, and set up the parameters mentioned in the text manuscript. Analyze the image data by opening the "Review Plate Data" window and selecting the "Test Plate" for image analysis.
Double-click on a test well to display its image. Then, select the "Count Nuclei" as the analysis method, and click on the "Configure Settings" button. Define the source image from the FITC channel and select the "Standard Algorithm."
Set the image analysis parameters as described in the text manuscript, and test them to optimize the method of analysis. Save the settings and run the analysis on all the wells. Export the "Total Nuclei" as the total number of Q40::YFP aggregates in each well.
Count the number of nematodes in each well, and calculate the average number of Q40::YFP aggregates per nematode in each group. Then, calculate the inhibition index.
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