Measuring RAN Peptide Toxicity in C. elegans

RAN peptides are a feature of repeat expansion neurodegenerative disorders such as Huntington's disease, amyotrophic lateral sclerosis, and frontotemporal dementia. These protocols provide an important standardized approach for quantifying RAN peptide toxicity in the C.elegans model system. The techniques described are simple to learn and inexpensive, can be performed quickly, and leverage the reproducible features of the C.elegans system such as movement and reproduction.

When attempting this protocol, the major challenge is obtaining consistent RAN peptide toxicity and expression. The major factors to control include drug temperature, timing of temperature shifts and preparation of assay plates. It is important for experimenters to consistently classify the phenotypes of the animals in this age-dependent process assay.

Visual demonstration of this technique shows their classification criteria. Begin by pouring standard nematode growth medium with one millimolar IPTG and 25 micrograms per milliliter carbenicillin into 24-well plates. Streak out RNAi feeding clones in HT115 bacteria onto LB carbenicillin agar and grow them at 37 degrees Celsius for 24 hours.

On the next day, pick a single colony into one milliliter of LB carbenicillin liquid media and grow the bacteria for 18 hours at 37 degrees Celsius while shaking at 250 rpm. Spot four individual wells of the NGM RNAi 24-well plate with 20 microliters of overnight bacterial cultures as described in the text manuscript and allow RNAi bacteria to induce double-stranded RNA production overnight. Use the standard hypochlorite method to isolate eggs from day one adult C.elegans expressing the integrated RAN peptide transgene.

Then seed about 30 eggs into each well of the 24-well plate and incubate the plate at 20 degrees Celsius for seven days. To perform video speed analysis, acquire two videos from two separate wells for each RNAi condition using a stereo dissecting microscope fitted with a monochrome camera. Make sure to use consistent acquisition settings between wells.

For each video, set the duration for 10 seconds and the time interval to 76 milliseconds. Set the image exposure time to four milliseconds and use the 1.49 zoom setting. Then select two by two binning.

Verify that the resolution is 800 by 600 pixels and start recording. After acquisition, label each video immediately with the strain, RNAi condition and well number. Then measure the distance traveled and the length of each animal for 20 different animals per video, examining a total of about 40 worms for each RNAi condition.

In the software, select the annotation tools button. Then the draw text tool. Click on a point in the center of the worm being measured in the first frame of the video and mark it with a number.

Advance the video to the end while visually tracking the worm. Then click on it and mark it with the next corresponding number. Use the draw scale bar tool to draw a line from the first number to the second number and record the distance traveled in a spreadsheet.

Repeat this measurement for 19 other worms in the video while preserving each individual measurement in the analysis window. Once the measurements are complete, take a snapshot of the final video frame illustrating all of the measurement lines so that the data can be traced back to the animal from which it originated. Next, analyze the data using a four-column spreadsheet where column one is the worm identifier and column two is the speed.

The time of each video can be found by right-clicking on the video under the experiment and going to the properties. To normalize the speed measurement, find the length of each animal. Use the draw polyline tool to freely trace the C.elegans from the tip of the head to the tip of the tail.

Then take a snapshot of the final video frame illustrating all the polylines. The length of the lines will be recorded under statistics. Add two columns to the spreadsheet, one for animal length and one for normalized speed.

Finally, analyze the normalized speed data using a one-way ANOVA with post-hoc testing versus empty vector RNAi. Place four to six transgenic L4 C.elegans expressing RAN peptide GFP on a six centimeter GFP RNAi plate and grow them at 20 degrees Celsius. If the experiment is utilizing RNAi to test for genetic effects on RAN peptide toxicity, move 10 transgenic gravid adults to each of two empty vector RNAi GFP RNAi or gene-specific RNAi plates.

If mutants are being used to analyze genetic effects on RAN peptide toxicity, place 10 gravid wild type or mutant animals expressing the same RAN transgene on each of two empty vector RNAi or GFP RNAi plates. Grow them at 20 degrees Celsius for 48 hours. Pick 10 sets of 10 transgenic L4s from the RNAi plates and place each set on a three centimeter RNAi plate.

Make sure that the worms selected for the assay all have superficially normal motility. Place the plates in a plastic bag to retain moisture and incubate them at 25 degrees Celsius. After 24 hours, analyze the animals for mobility by tapping the plates on the dissecting microscope and checking for movement.

If a worm moves more than a body length, count it as mobile and transfer it to a new three centimeter RNAi plate. Use a platinum pick to tap the remaining worms on the head and tail. If the animal moves more than a body length, count the animal as mobile and transfer it to a new three centimeter RNAi plate.

If significant paralysis is not observed in the empty vector control by day two, terminate the assay and start over. Group all nonmoving worms together so that the movement of more than a body length can be easily detected. Count all paralyzed, bagged, or dead worms and discard the plate.

Continue scoring the animals for paralysis everyday for five to seven days. The growth assay was used to measure the effect of gene inhibitions on the toxicity of RAN dipeptides that are found in ALS patients with a G4C2 repeat expansion. Expression of PR50 GFP alone resulted in a complete growth arrest, but knockdown of several genes suppressed the developmental toxicity.

The effect of specific gene knockdowns on the developmental motility of PR50 expressing animals was measured using the video speed analysis method. As expected, inhibition of PR50 GFP expression resulted in a large increase in motility compared to empty vector RNAi. Furthermore, the RNAi against the proteasome subunit RPN7 resulted in the significant increase in PR50 motility.

When adult phenotypes were analyzed with the age-dependent paralysis assay, PR50 GFP exhibited up to 80%paralysis by five days of age. However, RNAi directed at the gene cul-6 significantly delayed paralysis suggesting that cul-6 is required for PR50 GFP toxicity. To determine if RAN proteins cause neuropathology when expressed in C.elegans neurons, neuron-specific toxicity was examined using the commissure assay.

In day two adults, PR50 GFP expression in the motor neurons led to a significant increase in motor neuron blebbing. This neuropathology was significantly suppressed by a mutation in the insulin IGF receptor gene homolog DAF-2 which delays the toxic properties of several neurodegenerative proteins. For the behavioral assay, it is important that the RAN peptides produce a highly penetrative phenotype.

Such genes or drugs could provide new biomarkers or therapeutic options for RAN peptide diseases. Using these assays, we have performed a genome-wide RNAi screen for suppressors of the C9ORF72 associated RAN peptide proline-arginine. Genes identified in this screen include many new and conserved genes that will be the subject of future mechanistic studies.