PolyQ Aggregation Assay to Identify Neuroprotective Effect of a Compound in Worms
Transcripción
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.
