Measuring RAN Peptide Toxicity in C. elegans

Summary

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

Abstract

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington’s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Introduction

The inappropriate expansion of DNA repeat sequences is the genetic basis for several neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Huntington’s disease (HD)¹. While there are established cellular and animal models for these diseases, mechanisms underlying these conditions are not well defined. For example, HD is caused by the expansions of a CAG repeat sequence in the coding sequence for the Huntingtin protein Htt². Because CAG encodes the amino acid glutamine, the CAG repeat expansion results in the insertion of a polyGlutamine, or polyQ, sequence within Htt. Expanded polyQ proteins form length- and age-dependent protein aggregates that are associated with toxicity^3,4. Surprisingly, two recent studies suggest that the length of the polyQ sequence is not the main driver of HD disease onset, suggesting that polyQ-independent factors may also contribute to the disease^5,6.

One possible polyQ-independent mechanism involves a newly discovered type of protein translation termed Repeat Associated Non-AUG-dependent (RAN) translation⁷. As its name implies, RAN translation only occurs when an expanded repeat sequence is present and does not require a canonical start codon. Therefore, RAN translation occurs in all three reading frames of the repeat to produce three distinct polypeptides. In addition, because many genes also produce an antisense transcript that contains the reverse complement of the expanded repeat sequence, RAN translation also occurs in all three reading frames of the antisense transcript. Together, RAN translation expands the number of proteins produced from an expanded repeat-containing DNA sequence from one peptide to six peptides. To date, RAN translation has been observed in at least eight different repeat expansion disorders⁸. RAN peptides are observed in postmortem patient samples and only in cases where the patient carries an expanded repeat^9,10. While these peptides are clearly present in patient cells, their contribution to disease pathophysiology is unclear.

To better define the potential toxicity associated with RAN peptides, several groups have expressed each peptide in various model systems, such as yeast, flies, mice, and tissue culture cells^{11,12,13,14,15,16}. Rather than utilizing the repeat sequence for expression, these models employ a codon-variation approach in which the repeat sequence is eliminated but the amino acid sequence is preserved. Translation initiation occurs through a canonical ATG and the peptide is typically fused to a fluorescent protein at either the N- or C-terminus, neither of which appears to interfere with RAN peptide toxicity. Therefore, each construct overexpresses a single RAN peptide. Modeling the different RAN products in a multicellular organism with simple assays to measure RAN peptide toxicity is vitally important to understand how the different RAN products from each disease-causing repeat expansion contribute to cellular dysfunction and neurodegeneration.

Like other model systems, C. elegans provides a flexible and efficient experimental platform that enables studies of new disease mechanisms, such as RAN peptide toxicity. Worms offer several unique experimental attributes that are not currently available in other models of RAN peptide toxicity. First, C. elegans are optically transparent from birth until death. This allows for simple visualization of RAN peptide expression and localization, as well as in vivo analysis of neurodegeneration in live animals. Second, transgenic methods for generating RAN peptide expression models are inexpensive and fast. Given the short three-day life cycle of C. elegans, stable transgenic lines expressing any given RAN peptide in a cell-type specific manner can be produced in under a week. Third, simple phenotypic outputs can be combined with genetic screening methods, such as chemical mutagenesis or RNAi screening, to rapidly identify genes essential for RAN peptide toxicity. Finally, the short lifespan of C. elegans (~20 days) allows investigators to determine how aging, which is the greatest risk factor for most repeat expansion diseases, influences RAN peptide toxicity. Together, this combination of experimental attributes is unmatched in any other model system and offers a powerful platform for the study of RAN peptide toxicity.

Here we describe several assays that leverage the experimental advantages of C. elegans to measure the toxicity of RAN peptides and to identify genetic modifiers of this toxicity. The codon-varied ATG-initiated RAN peptides are tagged with GFP and expressed individually in either muscle cells under the myo-3 promoter or in GABAergic motor neurons under the unc-47 promoter. For expression in muscle cells, it is important that toxic RAN peptides are tagged with green fluorescent protein (GFP), or other fluorescent protein (FP) tag that can be targeted with an RNAi feeding vector. This is because toxic RAN peptide expression usually blocks growth, rendering such strains nonviable. The use of gfp(RNAi) conditionally inactivates RAN peptide expression and allows strain maintenance, genetic crosses, etc. For assays, these animals are removed from gfp(RNAi), which allows expression of the RAN peptide and the resulting phenotypes. In addition to the molecular strategy for designing codon-varied RAN peptide expression constructs, we describe assays for measuring developmental toxicity (larval motility and growth assay), post-developmental age-associated toxicity (paralysis assay), and neuron morphological defects (commissure assay).

Protocol

1. Generating codon-varied RAN peptide expression constructs

1. Design the individual RAN peptide coding sequence utilizing synonymous codons to eliminate the fundamental repetitive DNA/RNA structure but preserve the overlying amino acid sequence.
2. Order the custom codon sequences commercially at the repeat lengths needed for the studies (typically 5–100 repeats). Include a HindIII restriction site at the 5' end and a BamHI restriction site at the 3' end to facilitate cloning into C. elegans expression vectors, such as pPD95.79.
   NOTE: If synthesis of larger constructs proves difficult, smaller building block sequences can be synthesized and then built into larger repeats using a directional ligation strategy¹⁷.
3. Subclone the peptide sequence into a C. elegans expression vector using standard T4 ligation methods.
4. Subclone a cell type-specific promoter sequence generated by PCR in front of the RAN peptide sequence to drive tissue-specific RAN peptide-GFP expression.
5. Microinject the RAN peptide construct into the gonad of wild type C. elegans to generate transgenic strains containing extrachromosomal arrays using standard methods¹⁸. For a co-injection marker, the muscle-specific RFP reporter (myo-3p::RFP (pCFJ104)) was utilized, although other markers can be utilized as well.
6. Integrate a stable transgene into the genome using standard C. elegans integration screening methods¹⁹.
7. If the RAN peptide is toxic and transgenic animals do not survive, feed animals gfp(RNAi) expressing bacteria pre- and post-injection to silence the expression of the toxic protein.
   NOTE: Removal of animals from gfp(RNAi) enables RAN peptide expression for phenotypic analyses using the assays described below.

2. Measuring the developmental toxicity of RAN peptides following RNAi-based gene knockdown: Video speed analysis protocol

1. Pour standard nematode growth medium (NGM) RNAi 24 well plates with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and 25 µg/mL carbenicillin.
2. Streak out RNAi-feeding clones in HT115 bacteria to be tested onto Luria broth (LB) + carbenicillin (25 µg/mL) agar plates and grow bacteria at 37 °C for 24 h. Include empty vector(RNAi) as the positive control for toxicity and gfp(RNAi) as the negative control.
3. For each clone, pick a single colony into 1 mL of LB + carbenicillin liquid media and grow bacteria overnight for 18 hours at 37 °C while shaking at 250 rotations per minute (RPM).
4. Spot four individual wells of the NGM RNAI 24 wells with 20 µL of the following overnight bacterial cultures: column 1 wells = gfp(RNAi); column 2 wells = (EV)RNAi; columns 3–6 wells = RNAi against genes to be tested. Allow RNAi bacteria to induce dsRNA production overnight at room temperature (RT).
5. Isolate eggs from day one adult C. elegans expressing the integrated RAN peptide transgene using the standard hypochlorite method and seed ~30 C. elegans eggs into each well of the NGM RNAi 24 well plate.
6. Incubate the 24 wells at 20 °C for 7 days.
   NOTE: This incubation time is for strains expressing toxic C9orf72 RAN dipeptides, such as PR50 or GR50. Other strains may require shorter incubation times to prevent exhaustion of the bacterial food source and subsequent starvation.
7. Acquire transmitted light videos using a Leica MZ16FA stereo dissecting microscope fitted with a DFC345FX monochrome camera connected to a Windows-compatible PC running Leica AF video acquisition software (version 2.6.0.7266).
   1. Acquire two videos from two separate wells for each RNAi condition using consistent video acquisition settings between wells. For each video, acquire 10 seconds at 800 x 600 pixel resolution and 13.16 frames per second (time voxel dimension = 0.076 seconds) using 18.6X magnification (1.49 zoom setting in AF software). Set the image exposure time to 4 milliseconds. Label each video immediately with the strain, RNAi condition, and well number.
8. Blind the experimenter to the genotype associated with each video file by randomizing the order of the videos and changing the name of the videos to numbers. Save this group of videos as a new file. Unblind the experimenter after the analysis is complete.
9. Measure the 'distance traveled' and the length of each animal for 20 different animals from each video, examining a total of ~40 worms for each RNAi condition.
   1. 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 as a number. Advance the video to the end while visually tracking the movement path of the worm. In last frame, click on a point in the center of the worm being measured and mark the next corresponding number. Then, using the draw scalebar tool, draw a line from the first number to the second number to record the "distance traveled."
   2. 'Distance traveled' is the distance between the two centroid points. This distance is shown adjacent to the measurement line. Record this measurement in a spreadsheet. Repeat this measurement for 19 other worms in the video while preserving each individual measurement in the analysis window.
10. Make measurements from animals exhibiting the most robust movement to avoid the selection of animals exhibiting no movement and biasing the data towards paralysis. 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.
11. Analyze the data using a four column spreadsheet. Column one is the worm "identifier", column two is the distance traveled / time (speed).
    1. The time of each video can be found by right clicking on the video under the experiment and going to the properties of the video.
12. Normalize the speed measurement by finding the length of each animal by selecting Quantify, then Statistics on the software. Clicking on the annotation tool icon on the video display should allow for the use of the "Draw Polyline" tool. This tool can then be used to freely trace the C. elegans from the video from the tip of the head to the tip of the tail. The length of the line will be recorded under statistics.
    1. Take a snapshot of the final video frame illustrating all of the polyline's used for sizing the C. elegans so that the data can be traced back to the animal from which they originated.
13. Analyze the data through adding two additional columns to the spreadsheet. Column three is the "Animal Length", and column four is the "Normalized Speed" (speed/animal length). Analyze the normalized speed data using a One-way ANOVA with post-hoc testing versus empty vector(RNAi).

3. Measuring developmental toxicity of RAN peptide: Growth assay

1. Pick ~30 gravid animals into a 50 µL spot of hypochlorite solution (10 mL of bleach, 2.5 mL of 10 N NaOH, 37.5 mL of dH₂O) on either a gfp(RNAi), (EV)RNAi, or (gene-specific) 6 cm RNAi plate.
2. After 24 h, move six transgenic progeny that have crawled out of the hypochlorite solution spot to a new 6 cm RNAi plate identical to the RNAi conditions on the plate on which they were subjected to the hypochlorite solution treatment. Put these progeny (F1) at 20 °C to grow and reproduce.
3. After 24–48 h, pick 10 transgenic L4 animals onto 6 cm RNAi matching the RNAi conditions that the animals have been growing on. Allow the animals to pulse lay eggs for 24 h in a 20 °C incubator.
4. After 24 h, remove the adult animals and discard them. Count the number of eggs and L1 larvae laid during the 24 h period using a mechanical handheld counter. This is the total brood size.
5. Over the next 72 h, count the number of animals that reach the L4 or older stage. As each animal is counted, remove it from the plate.
6. Quantify the "Percent Growth" as the percentage of animals that reach the L4 or older stage out of the total brood size.
7. Perform a 2 x 2 Fisher's exact test versus empty vector(RNAi) to determine if the percent growth is statistically significant. The categories for comparison are "Growth" (# of animals that reached the L4 or older stage in 72 h) and "No Growth" (total brood size minus the number of animals reaching L4 or older stage in 72 h).

4. Post-developmental RAN peptide paralysis assay

1. Maintain integrated C. elegans transgenic strains expressing RAN peptide-GFP in the muscle on gfp(RNAi) by placing 4–6 transgenic L4's on a 6 cm gfp(RNAi) plate and grow the worms at 20 °C.
2. If the experiment is utilizing RNAi to test for genetic effects on RAN peptide toxicity, move 10 transgenic gravid adults from an unstarved plate to each of two 6 cm empty vector(RNAi) (positive control for paralysis, negative control for genetic effects), gfp(RNAi) (negative control for paralysis, positive control for genetic effects), gene-specific(RNAi) (i.e., 10 gravid adults per 6 cm plate).
3. If mutants are being used to analyze genetic effects on RAN peptide toxicity, place 10 gravid WT or mutant animals expressing the same RAN transgene on each of two 6 cm empty vector(RNAi) or gfp(RNAi) plates. Grow the worms at 20 °C for 48 h.
4. Pick 10 sets of 10 transgenic L4's from the 6 cm RNAi plates and place each set on a 3 cm RNAi plate (i.e., n = 100 animals for each genotype). Ensure that the L4's selected for the assay all have superficially normal motility. Place the plates within a zipper storage bag to retain moisture.
5. Place the bagged plates with L4's in the 25 °C incubator.
6. Remove the animals 24 h later from the 25 °C incubator one strain at a time to minimize the amount of time they are out at RT.
7. Tap the plates on the dissecting microscope to check for movement. If the animals move more than a body length, count the animal as mobile and transfer it to a new 3 cm RNAi plate. Use a platinum pick to tap the remaining worms on the head or tail. If the animal moves more than a body length, count the animal as mobile and transfer it to a new 3 cm RNAi plate.
   NOTE: Do not exceed 10 worms per plate when transferring. Be careful to avoid moving progeny to the new RNAi plate because the assay depends on following aged animals only.
8. Group all of the nonmoving worms together so that movement of more than a body length can easily be detected. Give the animal at least one minute to move more than one body length. If it still does not move, it is paralyzed, bagged (i.e., larvae hatched inside mother), or dead.
9. Censor animals that exhibit bagging, desiccate on the sides of the plate, exhibit extruded intestines, burrow, become lost, or die from the assay at the time of detection. Paralyzed worms are counted, left on the old RNAi plate, and discarded.
10. Score the animals for paralysis every day for 5–7 days as described in steps 4.6–4.9.
11. Analyze paralysis data with a log-rank test identical to the one used to analyze lifespan. In this statistical analysis, moving worms are scored as "Alive", paralyzed worms are scored as "Dead", and dead, bagged, gut extruded, desiccated, burrowed, or otherwise lost worms are scored as "Censored".
    NOTE: In this analysis, the "Percent Alive" number indicates the "Percent Moving" animals. The inverse represents the "Percent Paralyzed". Use the online analysis tool OASIS (https://sbi.postech.ac.kr/oasis2/) to perform the log-rank statistical analyses.

5. Measuring neuron pathology: Commissure assay

1. Generate transgenic C. elegans expressing the RAN peptide of interest in the GABAergic neurons using the unc-47 promoter. Also express unc-47p::GFP or unc-47p::RFP to reveal the cellular morphology of the GABA neurons.
2. Pick 50 transgenic L4 animals and place them on a 6 cm OP50 plate at 25 °C for 24 h.
3. Transfer the 50 transgenic animals onto a new 6 cm OP50 plate at 25 °C for 24 h.
4. Make agarose pads for imaging the animals under widefield microscopy.
   1. Place two pieces of tape on top of two microscope slides. Place a cleaned slide without tape in the middle of the two taped slides.
   2. Dispense ~100 µL (1 drop from a disposable plastic Pasteur pipette) of 3% molten agarose onto the middle slide with a disposable sterile transfer pipette.
   3. Immediately place a second clean slide across the drop of molten agarose so that it rests on the taped slides and creates a thin and even layer of agarose between the slides.
   4. After the agarose has cooled and solidified, carefully remove the two slides with the layer of agarose between them, without separating them, and place them in a plastic bag with a damp piece of paper under them. Make one slide per 10 worms to be examined along with three extra slides.
5. Remove the transgenic C. elegans from the 25 °C incubator and pick 10 transgenic C. elegans into a 100 µL drop of 10 mM levamisole in a glass depression slide. Incubate for 10 min or until animals are paralyzed.
6. Remove a slide pair from the plastic bag and carefully separate them. Label the slide with the agarose with the genotype and add 2 µL of 10 mM levamisole to the middle of the agarose. Move the 10 animals into the levamisole on the agarose pad. Cover the animals with a #1 thickness coverslip.
7. Image animals in which the vulva is oriented such that it is on the right side of the animal. If the vulva is on the left side of the head, the commissures of the motor neurons will be underneath the animals and not as clearly visible, making accurate quantification difficult. Omit quantification from these animals. In this orientation, the commissures of the motor neurons are nearest to the coverslip and are clearly visible on an inverted widefield fluorescence microscope. Visualize the neuron commissures expressing GFP with a Leica DMI4000B inverted widefield fluorescence microscope with a 63X 1.4X NA oil immersion lens and a Leica L5 GFP filter set (Ex480/40nm;Em527/30nm). If the neuron commissures express RFP instead of GFP, utilize a Leica TX2 RFP filter set (Ex560/40nm;Em645/75nm).
   1. Visualize the neuron commissures expressing GFP with an inverted widefield fluorescence microscope with a 63x 1.4x NA oil immersion lens and a GFP filter set (Ex480/40 nm; Em527/30 nm). If the neuron commissures express RFP instead of GFP, utilize a red fluorescent protein (RFP) filter set (Ex560/40 nm; Em 645/75 nm).
8. Image the worms within 45 min of placing the coverslip on the worms to minimize toxicity from immobilization.
9. For each worm, count the number of visible commissures. There are 16 visible commissures in wild type animals. Also count the number of commissures that have large beads (i.e., blebs) in the commissures as well as the number of commissures that are broken. To confirm the commissure is broken, follow the commissure from the dorsal cord to the ventral cord by adjusting the focal plane.
   NOTE: Commissures in which the unc-47p::GFP fluorescence exhibits a gap are considered broken. A broken commissure typically has a bleb on either side of the break, helping show that the commissures is broken. Count the amount of blebbing and breaks for 20 worms.
10. Calculate the fraction of commissures with blebs or breaks over the total number of observed commissures for each animal. The absolute number of commissures counted per animal (including wild type, blebbed, and broken events) can also be measured.
11. For each category, calculate the mean ± SD and statistically analyze with either a Student's t-test for comparison between two populations, or a one-way ANOVA for comparisons between 3 or more populations.

Results

We used the assays described here to evaluate the effect of different gene inhibitions on the toxicity of RAN dipeptides that are found in ALS patients with a G₄C₂ repeat expansion. Using the growth assay to measure developmental toxicity, we analyzed the effects of several genetic knockout mutants identified in a genome-wide RNAi screen suppressors of muscle-expressed PR50-GFP toxicity. While expression of PR50-GFP alone resulted in a completely penetrant growth arrest, loss of function mutations i...

Discussion

Here we report methods that can be used to assay RAN peptide toxicity modeled in the muscle or in the neurons of C. elegans. While neurodegenerative proteins have an age onset phenotype in human patients, they can also exhibit developmentally toxicity when overexpressed in model systems. Overexpression has significant interpretive limitations, but it also provides a powerful starting point for genetic or pharmacological screens aimed at identifying genes or drugs that can reverse toxic phenotypes. This is especi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
35mm x 10mm Petri Dish, Sterile	CELLTREAT Scientific Products	50-202-036	Nematode growth plates and RNAi
AGAR GRANULATED 2KILOGRAM	BD DIAGNOSTIC SYSTEMS	DF0145070	Nematode growth plates and RNAi
AGAROSE ULTRAPURE	LIFE TECHNOLOGIES	16500500	Microinjection to generate RAN peptide transgenic strains
CARBENICILLIN 5G	THERMO SCI FAIRLAWN CHEMICALS	BP26485	Nematode growth plates and RNAi
COVER GLASSES NO 1 22MM 1OZ/PK	THERMO SCI ERIE	12542B	Imaging for commissure assay
FEMOTIPS DISPSBL MICROINJ 20CS	EPPENDORF NORTH AMERICA BIOTOOLS	E5242952008	Microinjection to generate RAN peptide transgenic strains
FF COV GLASS NO1 40X22MM 1OZPK	THERMO SCI ERIE	125485C	Microinjection to generate RAN peptide transgenic strains
Fisherbrand Superfrost Plus Microscope Slides	THERMO SCI ERIE	12-550-15	Imaging for commissure assay
Gibco Bacto Peptone	Gibco	DF0118-17-0	Nematode growth plates and RNAi
HALOCARBON OIL 700	SIGMA-ALDRICH INC	H8898-50ML	Microinjection to generate RAN peptide transgenic strains
IPTG BIOTECH 10G	THERMO SCI FAIRLAWN CHEMICALS	BP162010	Nematode growth plates and RNAi
Leica Advanced Fluorescence imaging software	Leica Microsystems	LAS-AF	Image acquisition software for video speed analysis and commissure assay
Leica Immersion type N (Oil)	W NUHSBAUM INC	NC9547002	Imaging for commissure assay
LEVAMISOLE HYDROCHLORIDE 10GR	THERMO SCI ACROS ORGANICS	AC187870100	Imaging for commissure assay
MICROLOADER TIPS 2 X 96 PCS	EPPENDORF NORTH AMERICA BIOTOOLS	E5242956003	Microinjection to generate RAN peptide transgenic strains
PETRI DISH, 60X15MM,500/CS	CORNING LIFE SCIENCES PLASTIC	FB0875713A	Nematode growth plates and RNAi
TISSUE CULT PLATE 24WEL 50/CS	CORNING LIFE SCIENCES DL	87721	Nematode growth plates and RNAi