This study investigates the decline of proteostasis in aging using transgenic Caenorhabditis elegans. The accumulation of misfolded polyglutamine proteins in muscle cells leads to cellular dysfunction and paralysis.
Cellular protein homeostasis, or proteostasis, involves the regulation of protein folding and function, along with degradation of misfolded proteins. During aging, proteostasis machinery declines, causing misfolded proteins to accumulate and further aggregate, resulting in cellular dysfunction.
To determine age-associated proteostasis decline, begin with developmentally synchronized, adult transgenic Caenorhabditis elegans.
The muscle cells of these worms express proteins with abnormally-expanded polyglutamine, or polyQ, repeats, fused with a fluorescent tag. PolyQ proteins are prone to misfolding, and hence upon accumulation, can form aggregates.
Transfer the worms onto a microscope slide comprising an agarose pad and immobilize with suitable chemical for live-cell imaging. Using a fluorescence microscope, visualize the worms' body wall muscles over a period of time to determine the bright, fluorescent foci signals, corresponding to the fluorescent polyQ aggregates.
Within days, owing to age-related proteostasis decline, progressive polyQ aggregate accumulation occurs, leading to higher number of fluorescent foci. The increased polyQ aggregates in muscle cells cause toxicity and disrupt muscle cell function, resulting in paralysis.
Under a microscope, record the number of live, paralyzed worms lacking movement upon mechanical stimuli. Increased worm paralysis rate suggests age-associated proteostasis decline in muscle tissue in the worms.
To measure decline in proteostasis in muscle tissue, pick 20 animals, and mount them on a microscope slide with a 3% agarose pad and a 5-microliter drop of 10-millimolar sodium azide.
After all worms are immobilized, image the whole bodies of the animals using a 10x magnification lens. Use a FITC or YFP filter, and the same exposure, for every animal. When finished, discard the slides.
Count the number of foci in the body wall muscles of the whole animal. Foci are brighter, punctuated signals that can be differentiated from the dimmer soluble signal in the background.
On scoring days, look at the plates with the animals, and record the number of paralyzed animals. Then, remove paralyzed animals from the plate.
At the completion of the experiment, calculate the paralysis rate for each condition, and plot the paralysis progression.
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