The use of tissue-specific expression of polyglutamine fluorescent reporter in C.elegans is significant because it allows the discovery and characterization of proteostasis regulators in the context of an intact multicellular organism. The main advantage of this technique is that it allows visualization and quantification of the age-associated decline in cellular proteostasis in vivo, which is essential to gain deeper mechanistic insight into how organisms maintain proper folding and function of the proteome and the effects of aging. Elucidating mechanisms capable of preserving proteostasis will facilitate the development of targeted interventions for the treatment of aging associated diseases in which proteostasis is compromised and to promote healthy aging.
Proteostasis collapse is a massive clinic problem, as it underlies the development of protein misfolding diseases, including Alzheimer's, Parkinson's, Huntington's disease, and amyotrophic lateral sclerosis. Begin by using 6 centimeter diameter Petri plates to prepare five agar plates for each test condition. For experiments with RNAi, induce dsRNA production in transformed HT115 E.coli and use RNAi agar.
Use OP50 E.coli on standard NGM agar for experiments without RNAi. Grow the E.coli cultures overnight at 37 degrees Celsius while shaking at 220 RPM. On the next day, pellet the bacteria by centrifugation at 2, 400 G for 15 to 20 minutes, then aspirate the supernatant and re-suspend the pellet in one tenth of the starting volume of lb.
Aliquot 200 microliters of concentrated bacteria to each plate and allow the open plates to dry in a clean environment until all liquid has been absorbed. To synchronize the C.Elegans with hypochlorite treatment, wash gravid hermaphrodites twice with M9 buffer, then transfer them to a fresh tube and incubate in 5 milliliters of hypochlorite solution for five minutes, shaking them every minute. After the incubation, spin down the animals and wash them three times with M9 buffer.
Allow embryos to hatch in tubes overnight in 3 milliliters of M9 solution with rotation at 20 degrees Celsius. Calculate the density of L1 animals by dropping 10 microliters of L1 solution three times onto a 6 centimeter plate and counting the number of L1 animals. Periodically, mix the L1 solutions to prevent the animals from settling.
Seed 50 L1 one animals onto each plate, count and record the number seeded, and move the plates to a 20 degree Celsius incubator. Alternatively, to synchronize animals via egg lay, place five to ten young, gravid adult animals onto each plate for four to six hours and allow them to lay eggs until there are approximately 50 eggs per plate. Remove all gravid adults and move the plates with the eggs to a 20 degrees Celsius incubator.
Grow the animals until L4 stage, which will take approximately 40 hours at 20 degrees Celsius. Then add 50 microliters of 160 times FUDR. 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 10 X 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. To measure decline in proteostasis in neuronal tissue, mount the worms on a slide as previously described and take Z stack images of the head of the animals on a compound microscope using a 40 X magnification lens. Discard the slides after imaging.
After acquiring the images, flatten the Z stacks and use them to quantify the number of foci in neurons located on the nerve ring area. Plot the progression of YFP foci accumulation from days 4, 6, 8, and 10. On day two of adulthood, pick 10 synchronized animals from the plate and place them on a 10 microliter drop of M9 buffer on a microscope slide.
Repeat this step at least four times to get a sample of 40 or more animals. Video record the movement of the animals for a period of 30 seconds on a stereo microscope with a video capable camera. Once all the videos with the animals to be analyzed are recorded, play the video and score the body bends of each animal.
Plot the number of body bends for each animal in a column graph, where each dot represents the number of body bends in 30 seconds on the Y axis and the different conditions tested on the X axis. The polyglutamine repeat model has been instrumental for the identification of genes that regulate the proteostatic network. Muscle-specific polyQ-YFP expression results in accumulation of fluorescent foci that are easy to quantify under a simple fluorescent dissecting microscope.
The animals become paralyzed during midlife, as the proteome within the muscle collapses due to the proteotoxic effect of the reporter. The age-associated decline in neuronal proteostasis can be followed by directly quantifying aggregate formation and declines in coordinated body bends after placing animals into liquid. This method has been used to show that the homeo domain interacting protein kinase, a transcriptional cofactor, influences proteostasis during aging by regulating expression of autophagy and molecular chaperones.
The loss of HPK-1 increases the number of Q35-YFP aggregates that accumulate during aging. Control animals displayed an average of 18 aggregates, while the HPK1 null mutant and HPK1 RNAi treated animals displayed an average of 28 and 26 aggregates, respectively. By day eight of adulthood, 77 to 78%of HPK1 deficient animals were paralyzed, compared to only 50%of the control.
Additionally, overexpression of HPK1 was demonstrated to regulate protein aggregate formation and protect aging animals from Q35-YFP associated paralysis during aging. This method measures general decline of the proteome within a cell type. Many methods exist to assess changes in specific components of the prostatic network.
Together, they provide a comprehensive picture. Declining proteostasis is a hallmark of aging. This approach allows researchers to quantify this decline.
When combined with genetic analysis, it's a powerful tool for discovery.
