The overall goal of these experiments is to evaluate levels of mitophagy in human cells, C.elegans, and mice. Mitophagy has become a very important concept in the crossroads of neuro-degeneration, metabolic diseases and aging, and insight into the methodologies that are presented here and future use of these could be helping to make advances in this important field. The main advantages of this technique are that it detects mitophagy in a robust and efficient manner and enables the detection of mitophagy both in vitro and in vivo.
This technique presented here will assist researchers to develop new insight and to develop new strategies for therapy against neuro-generative diseases, including Alzheimer's and Parkinson's, where we know there is a mitochondrial dysfunction. In addition to providing insights into the molecular mechanisms of mitophagy, these method may lead to the discovery of novel drug candidates through the screening of mitophage inducers. Visual demonstrations of these methods is critical as levels of mitophagy are very sensitive to changes in experimental conditions in both C.elegans and mice.
Seed 300, 000 to one million cells in a 35 millimeter glass bottom microwell dish, 10 millimeter microwell, so that the cells reach 70%confluency on the next day. Maintain the cells in complete cell culture medium and incubate them in an incubator at 37 degrees Celsius and 5%carbon dioxide. The next day, dilute 15 microliters of the DNA transfection reagent with 150 microliters of serum free medium according to the manufacturer's protocol.
Also, dilute two to five micrograms of the mito-Keima plasmid DNA with 150 microliters of serum free medium. Add the diluted mito-Keima plasmid to the diluted DNA transfection reagent and vortex the mixture for 10 seconds. Then incubate the mixture for 10 minutes at room temperature.
Next, add the DNA transfection reagent mixture drop-wise to the cells, gently shake the plates for five to 10 seconds to mix the solution. At this point, place the cells back into the incubator and incubate them for 24 hours. The next day, change the media to fresh complete cell culture medium and then incubate the transfected cells for an additional 24 hours.
Image the transfected cells using confocal microscopy. Randomly image 20 areas under 40 times magnification in order to document a total of 100 to 200 cells in every well. On day one, pick L4 larvae of transgenic animals expressing both DCT-1 GFP and DSRed LGG-1 in body wall muscle cells and place them onto an OP50 seeded nematode growth media plate.
Place five to 10 worms onto each 3.5 centimeter plate using at least three plates. When finished, incubate the nematodes at 20 degrees Celsius. Next, on day five, synchronize the nematodes by selecting 15 to 20 L4 transgenic larvae and transferring them onto fresh OP50 seeded plates.
Use at least five plates for each experimental condition. On day seven, prepare some of the vehicle plates for mitophagy affecting drugs of interest and some to use as positive controls. Next, expose E.coli seeded plates to UV light for 15 minutes at an intensity of 222 microwatts per centimeter squared, to ensure the mitophagy inducing compounds on the plates are not metabolized by bacteria.
Now, add the compound of interest in 10 microliters to the top of the seeded plates and add an equivalent amount of compound vehicle to the vehicle plates as a negative control. For positive control of mitophagy induction, add paraquat, a mitochondrial toxicant. Gently swirl the plates until the solution coats the entire surface, and allow the plates to dry with the lids closed, at room temperature, for at least one hour.
Once dry, transfer 10 to 20 two-day old adult transgenic animals to the plates. Incubate them at 20 degrees Celsius for two days. On day nine, prepare 2%agarose pads and add one droplet of 20 millimolar levamisole in M9, per pad.
Immobilize the transgenic animals for imaging by placing them in the M9 levamisole drop. Then, gently place a cover slip on the top of the drop. Place the sample on a confocal microscope stage and image the single body wall muscle cells by taking Z-stack images under 63 times magnification.
Place a fresh, isolated mito-Keima mouse liver on a chilled metal plate, on ice, to rapidly cool the tissue. Keep it there while processing the tissue as quickly as possible, as signal from the mito-Keima protein remains stable on ice for up to one hour. Next, use a pair of curved forceps to lift the liver sample and rinse it with five milliliters of ice cold PBS.
Finally, image the tissue sections using confocal microscopy with two sequential excitations. Using the procedure presented here, human HeLa cells were transfected with the mito-Keima plasmid. Healthy cells demonstrated a well-organized mitochondrial network with few incidences of mitophagy.
However, cells pretreated with a mitochondrial uncoupler FCCP, exhibited a profound increase in mitophagy incidence. Mitophagy was also measured using the co-localization of LAMP2 and COXII. For in vivo detection of mitophagy in C.elegans, transgenic nematodes expressing MT rosella in body wall muscle cells were treated with eight millimolars of paraquat.
The level of mitophagy induction indicated by a decrease in the pH-sensitive fluorescence ratio, was found to be significantly decreased in the nematodes treated with paraquat. Additionally, the elevated number of co-localization events between DCT-1 fused GPF and DSRed fused LGG-1, signifies mitophagy stimulation in response to oxidative stress. Finally, these images represent mito-Keima signals from the liver and cerebellum of a mito-Keima mouse.
Tissues need to be kept cold and imaged as quickly as possible, but when prepared properly, can be used to provide insight into mitophagy in normal and pathophysiological conditions. The use of this technique has paved the ways for researchers in the broad field of neuro-degeneration and mitochondria biology, to explore mitochondrial functions in different model systems including cultured cells, worms, or C.elegans, and mouse models. After watching this video, you should have a grasp of multiple methods to use in the quantitative evaluation of mitophagy levels in both C.elegans and mice.
