An In Vivo Assay to Detect Mitophagy in Transgenic Nematodes

1. Detection of Mitophagy in C. elegans

NOTE: The nematode C. elegans provides a platform to assay mitophagy at the organismal level. Two strains can be used to monitor mitophagy: (1) mitochondria-targeted Rosella (mtRosella) or (2) mitophagy receptor DCT-1 fused with GFP along with autophagosomal marker LGG-1 fused with Discosoma sp. red fluorescent protein (DsRed).

1. Mitophagy monitoring using Rosella biosensor
   NOTE: Rosella is a molecular biosensor that combines a pH-insensitive DsRed fused to a pH-sensitive GFP variant. The Rosella biosensor takes advantage of the pH differences between the acidic lysosome (pH ~4.5) and other cellular compartments. This biosensor has been used to successfully monitor mitophagy in Saccharomyces cerevisiae and several mammalian cell lines, such as HeLa, HEK293, and HCT116. We adapted this versatile dual-fluorescent reporter and generated transgenic C. elegans nematodes expressing mitochondria-targeted Rosella (mtRosella) in body-wall muscle cells. Mitophagy induction is indicated by a reduced GFP/DsRed ratio of mtRosella fluorescence.
   1. Day 1: Pick L4 larvae of worms expressing mitochondria-targeted Rosella (mtRosella) in body-wall muscle cells onto a Nematode Growth Media (NGM) plate seeded with E. coli (OP50) using a dissection microscope. Place 10-20 worms/plate on at least three 3.5 cm plates. Incubate the nematodes at the standard temperature of 20 °C.
      NOTE: See reference for the worm anatomy, including identification of L4 larvae, and the way to make a pick which is used to transfer worms from one location to another.
   2. Day 5: Synchronize nematodes by selecting 15-20 L4 transgenic larvae and transferring them onto fresh OP50 seeded NGM plate. Use at least five plates per experimental condition.
   3. Day 7: Prepare vehicle plates for mitophagy-affecting drugs of interest or positive controls.
      1. Kill E. coli (OP50) bacteria seeded by exposing NGM plates for 15 min with 222 µW/cm² (intensity) of UV light to ensure that mitophagy-inducing compounds are not metabolized by bacteria. Plates must be exposed with 2,000 J/m².
         ​NOTE: Seconds of exposure needed = 2,000/((intensity in µW/cm2)*100).
      2. Add compound(s) of interest to the top of seeded NGM plates. Add an equivalent amount of compound vehicle (solvent used to dissolve compound, such as dimethyl sulfoxide (DMSO)) to the vehicle plates as a negative control. For positive control of mitophagy induction, a mitochondrial toxicant, paraquat (8 mM final concentration) was used. Add a solution containing 10 µL of 8 M paraquat stock with 190 µL of ddH₂O on the top of the agar plate for the treatment group. For the vehicle group, add a solution containing 10 µL of DMSO with 190 µL of ddH₂O on the top of the agar plate.
         NOTE: The paraquat-working solution may be kept for 1 month at 4 °C.
      3. Gently swirl the plates until the drug or vehicle coats the entire surface. Allow the plates to dry with the lids closed at room temperature for at least 1 h before transferring the worms.
         NOTE: Drug plates can also be prepared by adding drugs in the liquid NGM before it solidifies.
   4. Day 7: Transfer 10-20 of 2-day-old adult transgenic animals prepared in step 2.1.2 to plates containing either paraquat, compound(s) of interest, or vehicle (DMSO). Incubate the transgenic animals at 20 °C for 2 days.
   5. Day 9: Prepare 2% agarose pads. Then add one droplet (10 µL) of 20 mM levamisole in M9 per pad. Immobilize the transgenic animals for imaging, by placing them in the M9-levamisole drop. Gently place a coverslip on the top.
   6. Capture single transgenic animals using a camera attached to an epifluorescence microscope. Acquire fluorescent images of whole transgenic nematodes expressing mtRosella in body-wall muscle cells at 10X magnification. Save the collected images.
   7. Process the acquired images with ImageJ software. Analyze the body wall muscle cells located in the head of the worm to avoid autofluorescence in the intestinal tissue. Measure the average pixel intensity value and total area for each fluorescent image only of the head region of each animal. To analyze the specific area of interest:
      1. Select 'split channel' under the 'image' and 'color' drop-down menu to convert images .
      2. Utilize the 'freehand selection' tool to capture the fluorescent area of interest.
      3. Select the 'measurement' command under the 'analyze' drop-down menu and perform pixel intensity analysis.
      4. Pixel intensity should be normalized by dividing intensity by the total area analyzed. Upon normalization, calculate the GFP to DsRed ratio.
   8. Use statistical analysis software to either conduct student t-test (comparison between two groups) or ANOVA (comparison among multiple groups) for statistical analysis with p < 0.05 as significant.
2. Assessing mitophagy using co-localization between DCT-1 and LGG-1
   NOTE: DCT-1 is an outer mitochondrial membrane protein that acts as a mitophagy receptor in response to stress conditions, similar to its putative orthologues BNIP3 and NIX/BNIP3L in mammals. Thus, mitophagy initiation is assessed by co-localization between DCT-1 mitophagy receptor and autophagosomal membrane protein LGG-1 (homolog of the mammalian LC3).
   1. Day 1: Pick L4 larvae of transgenic animals expressing both DCT-1::GFP and DsRed::LGG-1 in body-wall muscle cells5 onto an OP50 seeded NGM plate. Place 5-10 worms/3.5 cm plate, and use at least three plates. Incubate the nematodes at 20 °C.
      NOTE: The transgenes are located on separate plasmids and they are not integrated in the genome. Pick up nematodes carrying both rol-6(su1006) and pmyo-2 GFP contransformation markers. Only transgenic worms with both contransformation markers express both DCT-1::GFP and DsRed::LGG-1 in body-wall muscle cells.
   2. Follow the steps from 1.1.2-1.1.5.
   3. Image single body-wall muscle cells using a camera attached to a confocal microscope. Detect the borders of entire body-wall muscle cells and take z-stack images under 63x magnification. Higher magnification can also be used.
   4. Open and process images acquired with the confocal software. Initiation of mitophagy is indicated by co-localization of the mitophagy receptor (DCT-1::GFP) to the autophagosomal marker (DsRed::LGG-1). Manually measure co-localization events in each stack of body-wall muscle cell. It is essential to keep all microscope and camera settings (lens and magnifier, filters, exposure time, resolution, laser intensity, gain, etc.) consistent throughout experiments. All settings should be noted.
   5. Use statistical analysis software to either conduct student t-test (comparison between two groups) or ANOVA (comparison among multiple groups) for statistical analysis with p < 0.05 as significant. For two-way comparisons use the Student's t-test (p < 0.05 is considered significant). For multiple comparisons use the single factor (ANOVA) variance analysis, corrected by the post hoc Bonferroni test. We recommend analyzing at least 50-70 animals or 30-50 body-wall muscle cells for each experimental condition. Repeat the experiment at least three times.