An Assay to Study Effect of Test Compound against PolyQ-Mediated Neurotoxicity in Worms

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.

To prepare nematodes for the polyQ neurotoxicity assay, transfer 300 to 500 synchronized L1 larvae of HA759 nematodes to each well of a 48-well plate with 500 microliters of S medium containing OP50 strain of E. coli and 5 milligrams per milliliter of astragalan. After sealing the plates, incubate, harvest, and resuspend the nematodes in M9 buffer, as demonstrated.

Now prepare an agarose pad by adding 2 grams of agarose to 100 milliliters of M9 buffer, and heat the solution in a microwave to near boiling. Dispense 0.5 milliliters of melted agarose onto the center of a 1-millimeter thick microscopy glass slide, placed between two pieces of 2-millimeter thick glass plates, covering with another slide vertically. Gently remove the top slide once agarose cools down and is solidified.

Begin the ASH neuronal survival assay by adding a drop of 20 millimolar sodium azide onto the agarose pad, and then transferring 15 to 20 HA759 nematodes into the drop to immobilize them. Place a cover slip gently over the nematodes.

Now, keep the slide under a fluorescence microscope fitted with a digital camera. Select a 40x objective lens and FITC filter to detect GFP-positive ASH neurons in the head region of the nematodes.

Select more than 50 nematodes in each group randomly, to count the number of nematodes with GFP-labeled bilateral ASH neurons in their head region, and then, calculate the survival rate of ASH neurons.