An Assay to Study Effect of Test Compound against PolyQ-Mediated Neurotoxicity in Worms
Transcrição
Certain inherited neurodegenerative disorders involve proteins with a long string of glutamine residues, called polyglutamine, polyQ, tract. These structurally unstable, misfolding-prone proteins eventually accumulate to form toxic aggregates, negatively affecting neuronal function and survival.
To assess a test compound's protective potential on neuronal survival against polyQ-mediated neurotoxicity, begin with a suspension of transgenic Caenorhabditis elegans larvae expressing fluorescent and polyQ proteins in ASH — paired chemosensory — neurons.
Transfer into wells of a multi-well plate containing media and bacterial food source. Add the test compound to one set of wells. Seal the plate, minimizing media dehydration and contamination. Incubate.
During worm development, polyQ proteins form aggregates in the ASH neurons, leading to aggregation-associated neurotoxicity and impact neuronal survival.
If present, the test compound enters the neurons and, based on its neuroprotective ability, may suppress polyQ protein aggregation, mitigating associated neurotoxicity and improving neuronal survival.
Collect the adult worms and centrifuge. Resuspend the worms in a suitable buffer and transfer onto an agarose pad with an appropriate chemical for immobilization.
Using a fluorescence microscope, visualize the ASH neurons to detect fluorescent proteins, an indicator of neuron survival.
Higher fluorescence in treatment than control wells suggests increased neuronal survival rate, indicating the neuroprotective potential of the test compound in suppressing polyQ aggregation and associated neurotoxicity.
