NIH R21NS107797

1. Generating codon-varied RAN peptide expression constructs
<ol>
	<li>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.</li>
	<li>Order the custom codon sequences commercially at the repeat lengths needed for the studies (typically 5&#8211;100 repeats). Include a HindIII restriction site at the 5&#39; end and a BamHI restriction site at the 3&#39; 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.</li>
	<li>Subclone the peptide sequence into a C. elegans expression vector using standard T4 ligation methods.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Integrate a stable transgene into the genome using standard C. elegans integration screening methods19.</li>
	<li>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.</li>
</ol>
2. Measuring the developmental toxicity of RAN peptides following RNAi-based gene knockdown: Video speed analysis protocol
<ol>
	<li>Pour standard nematode growth medium (NGM) RNAi 24 well plates with 1 mM isopropyl &#946;-D-1-thiogalactopyranoside (IPTG) and 25 &#181;g/mL carbenicillin.</li>
	<li>Streak out RNAi-feeding clones in HT115 bacteria to be tested onto Luria broth (LB) + carbenicillin (25 &#181;g/mL) agar plates and grow bacteria at 37 &#176;C for 24 h. Include empty vector(RNAi) as the positive control for toxicity and gfp(RNAi) as the negative control.</li>
	<li>For each clone, pick a single colony into 1 mL of LB + carbenicillin liquid media and grow bacteria overnight for 18 hours at 37 &#176;C while shaking at 250 rotations per minute (RPM).</li>
	<li>Spot four individual wells of the NGM RNAI 24 wells with 20 &#181;L of the following overnight bacterial cultures: column 1 wells = gfp(RNAi); column 2 wells = (EV)RNAi; columns 3&#8211;6 wells = RNAi against genes to be tested. Allow RNAi bacteria to induce dsRNA production overnight at room temperature (RT).</li>
	<li>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.</li>
	<li>Incubate the 24 wells at 20 &#176;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.</li>
	<li>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).
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Measure the &#39;distance traveled&#39; and the length of each animal for 20 different animals from each video, examining a total of ~40 worms for each RNAi condition.
	<ol>
		<li>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 &#34;distance traveled.&#34;</li>
		<li>&#39;Distance traveled&#39; 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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Analyze the data using a four column spreadsheet. Column one is the worm &#34;identifier&#34;, column two is the distance traveled / time (speed).
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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 &#34;Draw Polyline&#34; 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.
	<ol>
		<li>Take a snapshot of the final video frame illustrating all of the polyline&#39;s used for sizing the C. elegans so that the data can be traced back to the animal from which they originated.</li>
	</ol>
	</li>
	<li>Analyze the data through adding two additional columns to the spreadsheet. Column three is the &#34;Animal Length&#34;, and column four is the &#34;Normalized Speed&#34; (speed/animal length). Analyze the normalized speed data using a One-way ANOVA with post-hoc testing versus empty vector(RNAi).</li>
</ol>
3. Measuring developmental toxicity of RAN peptide: Growth assay
<ol>
	<li>Pick ~30 gravid animals into a 50 &#181;L 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.</li>
	<li>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 &#176;C to grow and reproduce.</li>
	<li>After 24&#8211;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 &#176;C incubator.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Quantify the &#34;Percent Growth&#34; as the percentage of animals that reach the L4 or older stage out of the total brood size.</li>
	<li>Perform a 2 x 2 Fisher&#39;s exact test versus empty vector(RNAi) to determine if the percent growth is statistically significant. The categories for comparison are &#34;Growth&#34; (# of animals that reached the L4 or older stage in 72 h) and &#34;No Growth&#34; (total brood size minus the number of animals reaching L4 or older stage in 72 h).</li>
</ol>
4. Post-developmental RAN peptide paralysis assay
<ol>
	<li>Maintain integrated C. elegans transgenic strains expressing RAN peptide-GFP in the muscle on gfp(RNAi) by placing 4&#8211;6 transgenic L4&#39;s on a 6 cm gfp(RNAi) plate and grow the worms at 20 &#176;C.</li>
	<li>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).</li>
	<li>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 &#176;C for 48 h.</li>
	<li>Pick 10 sets of 10 transgenic L4&#39;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&#39;s selected for the assay all have superficially normal motility. Place the plates within a zipper storage bag to retain moisture.</li>
	<li>Place the bagged plates with L4&#39;s in the 25 &#176;C incubator.</li>
	<li>Remove the animals 24 h later from the 25 &#176;C incubator one strain at a time to minimize the amount of time they are out at RT.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Score the animals for paralysis every day for 5&#8211;7 days as described in steps 4.6&#8211;4.9.</li>
	<li>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 &#34;Alive&#34;, paralyzed worms are scored as &#34;Dead&#34;, and dead, bagged, gut extruded, desiccated, burrowed, or otherwise lost worms are scored as &#34;Censored&#34;. 
	NOTE: In this analysis, the &#34;Percent Alive&#34; number indicates the &#34;Percent Moving&#34; animals. The inverse represents the &#34;Percent Paralyzed&#34;. Use the online analysis tool OASIS (<a href="https://sbi.postech.ac.kr/oasis2/" target="_blank">https://sbi.postech.ac.kr/oasis2/</a>) to perform the log-rank statistical analyses.</li>
</ol>
5. Measuring neuron pathology: Commissure assay
<ol>
	<li>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.</li>
	<li>Pick 50 transgenic L4 animals and place them on a 6 cm OP50 plate at 25 &#176;C for 24 h.</li>
	<li>Transfer the 50 transgenic animals onto a new 6 cm OP50 plate at 25 &#176;C for 24 h.</li>
	<li>Make agarose pads for imaging the animals under widefield microscopy.
	<ol>
		<li>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.</li>
		<li>Dispense ~100 &#181;L (1 drop from a disposable plastic Pasteur pipette) of 3% molten agarose onto the middle slide with a disposable sterile transfer pipette.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Remove the transgenic C. elegans from the 25 &#176;C incubator and pick 10 transgenic C. elegans into a 100 &#181;L drop of 10 mM levamisole in a glass depression slide. Incubate for 10 min or until animals are paralyzed.</li>
	<li>Remove a slide pair from the plastic bag and carefully separate them. Label the slide with the agarose with the genotype and add 2 &#181;L 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.</li>
	<li>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).
	<ol>
		<li>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).</li>
	</ol>
	</li>
	<li>Image the worms within 45 min of placing the coverslip on the worms to minimize toxicity from immobilization.</li>
	<li>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.</li>
	<li>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.</li>
	<li>For each category, calculate the mean &#177; SD and statistically analyze with either a Student&#39;s t-test for comparison between two populations, or a one-way ANOVA for comparisons between 3 or more populations.</li>
</ol>

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<li>Peters, O. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+C9ORF72+Hexanucleotide+Expansion+Reproduces+RNA+Foci+and+Dipeptide+Repeat+Proteins+but+Not+Neurodegeneration+in+BAC+Transgenic+Mice%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Human C9ORF72 Hexanucleotide Expansion Reproduces RNA Foci and Dipeptide Repeat Proteins but Not Neurodegeneration in BAC Transgenic Mice.</a> Neuron. 88 (5), 902-909 (2015).</li>
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<li>Mizielinska, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((C9orf72+repeat+expansions+cause+neurodegeneration+in+Drosophila+through+arginine-rich+proteins%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins.</a> Science. 345 (6201), 1192-1194 (2014).</li>
<li>Krajacic, P., Shen, X., Purohit, P. K., Arratia, P., Lamitina, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biomechanical+profiling+of+Caenorhabditis+elegans+motility%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Biomechanical profiling of Caenorhabditis elegans motility.</a> Genetics. 191 (3), 1015-1021 (2012).</li>
<li>Zhang, L., Ward, J. D., Cheng, Z., Dernburg, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+auxin-inducible+degradation+%28AID%29+system+enables+versatile+conditional+protein+depletion+in+C.+elegans%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">The auxin-inducible degradation (AID) system enables versatile conditional protein depletion in C. elegans.</a> Development. 142 (24), 4374-4384 (2015).</li>
</ol>

The authors have nothing to disclose.

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...

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&#8217;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 toxicity3,4. 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 disease5,6.
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 repeat9,10. 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 cells11,12,13,14,15,16. 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).

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measuring ran peptide toxicity in c. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington&#8217;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.

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...

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Measuring RAN Peptide Toxicity in C. elegans

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.

Measuring RAN Peptide Toxicity in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...

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Research

JoVE Journal

Biology

6.6K Views.  University of Pittsburgh. RAN peptiden zijn een kenmerk van herhaalde expansie neurodegeneratieve aandoeningen zoals de ZvH, amyotrofische laterale sclerose en frontotemporale dementie. Deze protocollen bieden een belangrijke gestandaardiseerde aanpak voor het kwantificeren van RAN peptide toxiciteit in de C.elegans model systeem. De beschreven technieken zijn eenvoudig te leren en goedkoop, kunnen snel worden uitgevoerd en maken gebruik van de reproduceerbare kenmerken van het C.elegans-systeem, zoals beweging en rep...

Video: Measuring RAN Peptide Toxicity in C. elegans

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

Assaying &#946;-amyloid Toxicity using a Transgenic C. elegans Model

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

Neuroscience

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and maintenance issues. On the contrary, the nematode Caenorhabditis elegans (C. elegans) has advantages to make it an ideal choice for toxicity testing: a short lifespan, easy cultivation, and efficient reproduction. Here, we describe a protocol for the automatic phenotypic profiling of C. elegans in a 384-well plate. The nematode worms are cultured in a 384-well plate with liquid medium and chemical treatment, and videos are taken of each well to quantify the chemical influence on 33 worm features. Experimental results demonstrate that the quantified phenotype features can classify and predict the acute toxicity for different chemical compounds and establish a priority list for further traditional chemical toxicity assessment tests in a rodent model.

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

A quantitative method has been developed to identify and predict the acute toxicity of chemicals by automatically analyzing the phenotypic profiling of Caenorhabditis elegans. This protocol describes how to treat worms with chemicals in a 384-well plate, capture videos, and quantify toxicological related phenotypes.

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and ...

Environment

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

Measuring Caenorhabditis elegans Sensitivity to the Acetylcholine Receptor Agonist Levamisole

At the neuromuscular junction (NMJ), the binding of the excitatory neurotransmitter acetylcholine (ACh) to postsynaptic receptors leads to muscle contraction. As in vertebrate skeletal muscle, cholinergic signaling in the body wall muscles of the model organism Caenorhabditis elegans is required for locomotion. Exposure to levamisole, a pharmacological agonist of one class of ACh receptors on the body wall muscles, causes time-dependent paralysis of wild-type animals. Altered sensitivity to levamisole suggests defects in signaling at the NMJ or muscle function. Here, a protocol for a liquid levamisole assay performed on C. elegans grown in 24-well plates is presented. Vigorous swimming of the animals in liquid allows for the assessment and quantitation of levamisole-induced paralysis in hundreds of worms over a one-hour time period without requiring physical manipulation. This procedure can be used with both wild-type and mutants that have altered sensitivity to levamisole to demonstrate the functional consequences of altered signaling at the NMJ.

Measuring Caenorhabditis elegans Sensitivity to the Acetylcholine Receptor Agonist Levamisole

The present protocol describes an assay to determine response to levamisole, a pharmacological agonist of one class of Caenorhabditis elegans acetylcholine receptors. In this liquid levamisole swim assay, researchers visually observe and quantitate the time-dependent paralysis of animals cultivated in 24-well plates.

At the neuromuscular junction (NMJ), the binding of the excitatory neurotransmitter acetylcholine (ACh) to postsynaptic receptors leads to muscle ...

Genetics

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A non-mammalian soil round worm, Caenorhabditis elegans, is a valuable model organism for toxicology studies due to its convenience and lack of animal ethics issues compared with mammalian animal systems. In this protocol, a detailed procedure of toxicological evaluation of chemicals in C. elegans is described. A clinical anticancer drug, etoposide, which targets human topoisomerase II and inhibits DNA replication of human cancer cells, was selected as a model testing chemical. Age-synchronized C. elegans eggs were exposed to either dimethyl sulfoxide (DMSO) or etoposide, and then the growth of C. elegans was monitored every day for 4 days by the stereo microscope observation. The total number of eggs laid from C. elegans treated with DMSO or etoposide was also counted by using the stereo microscope. Etoposide treatment significantly affected the growth and reproduction of C. elegans. By comparison of the total number of eggs laid from worms with different treatment periods of chemicals, it can be decided that the reproductive toxicity of chemicals on C. elegans reproduction is reversible or irreversible. These protocols may be helpful for both the development of various drugs and risk assessment of environmental toxicants.

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

A basic protocol to evaluate the toxicity of chemicals in a model animal, Caenorhabditis elegans, is described. The method is convenient and useful for the development of pharmaceuticals as well as for the risk assessment of various environmental pollutants.

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A ...

Medicine

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s disease (HD) is principally driven by a CAG nucleotide repeat that encodes an expanded glutamine tract in huntingtin protein. Thus, the inhibition of polyglutamine (polyQ) aggregation and, in particular, aggregation-associated neurotoxicity is a useful strategy for the prevention of HD and other polyQ-associated conditions. This paper introduces generalized experimental protocols to assess the neuroprotective capacity of test compounds against HD using established polyQ transgenic Caenorhabditis elegans models. The AM141 strain is chosen for the polyQ aggregation assay as an age-associated phenotype of discrete fluorescent aggregates can be easily observed in its body wall at the adult stage due to muscle-specific expression of polyQ::YFP fusion proteins. In contrast, the HA759 model with strong expression of polyQ-expanded tracts in ASH neurons is used to examine neuronal death and chemoavoidance behavior. To comprehensively evaluate the neuroprotective capacity of target compounds, the above test results are ultimately presented as a radar chart with profiling of multiple phenotypes in a manner of direct comparison and direct viewing.

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Here, we present a protocol to assess the neuroprotective activities of test compounds in Caenorhabditis elegans, including polyglutamine aggregation, neuronal death, and chemoavoidance behavior, as well as an exemplary integration of multiple phenotypes.

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s ...

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer&#8217;s disease (AD), Parkinson&#8217;s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for ...

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

Measuring RAN Peptide Toxicity in C. elegans

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