Name: measuring ran peptide toxicity in c. elegans
Uploaded: 2020-04-30T15:00:00
Description: Watch this Scientific Journal Video about Measuring RAN Peptide Toxicity in C. elegans at JoVE.com

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. e...

<ol>
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F., Brignull, H. R., Weyers, J. J., Morimoto, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+threshold+for+polyglutamine-expansion+protein+aggregation+and+cellular+toxicity+is+dynamic+and+influenced+by+aging+in+Caenorhabditis+elegans.">The threshold for polyglutamine-expansion protein aggregation and cellular toxicity is dynamic and influenced by aging in Caenorhabditis elegans.</a> Proceedings of the National Academy of Sciences U S A. 99 (16), 10417-10422 (2002).</li><li>Genetic Modifiers of Huntington's Disease Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electronic+address,+g.+h.+m.+h.+e.,+Genetic+Modifiers+of+Huntington's+Disease,+C.+CAG+Repeat+Not+Polyglutamine+Length+Determines+Timing+of+Huntington's+Disease+Onset.">Electronic address, g. h. m. h. e., Genetic Modifiers of Huntington's Disease, C. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeat-associated+non-AUG+(RAN)+translation:+insights+from+pathology.">Repeat-associated non-AUG (RAN) translation: insights from pathology.</a> Laboratory Investigation. 99 (7), 929-942 (2019).</li><li>Banez-Coronel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RAN+Translation+in+Huntington+Disease.">RAN Translation in Huntington Disease.</a> Neuron. 88 (4), 667-677 (2015).</li><li>Ash, P. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unconventional+translation+of+C9ORF72+GGGGCC+expansion+generates+insoluble+polypeptides+specific+to+c9FTD/ALS.">Unconventional translation of C9ORF72 GGGGCC expansion generates insoluble polypeptides specific to c9FTD/ALS.</a> Neuron. 77 (4), 639-646 (2013).</li><li>Kramer, N. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas9+screens+in+human+cells+and+primary+neurons+identify+modifiers+of+C9ORF72+dipeptide-repeat-protein+toxicity.">CRISPR-Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity.</a> Nature Genetics. 50 (4), 603-612 (2018).</li><li>Boeynaems, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+screen+connects+nuclear+transport+genes+to+DPR+pathology+in+c9ALS/FTD.">Drosophila screen connects nuclear transport genes to DPR pathology in c9ALS/FTD.</a> Scientific Reports. 6, 20877 (2016).</li><li>Jovicic, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modifiers+of+C9orf72+dipeptide+repeat+toxicity+connect+nucleocytoplasmic+transport+defects+to+FTD/ALS.">Modifiers of C9orf72 dipeptide repeat toxicity connect nucleocytoplasmic transport defects to FTD/ALS.</a> Nature Neuroscience. 18 (9), 1226-1229 (2015).</li><li>Boeynaems, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+Separation+of+C9orf72+Dipeptide+Repeats+Perturbs+Stress+Granule+Dynamics.">Phase Separation of C9orf72 Dipeptide Repeats Perturbs Stress Granule Dynamics.</a> Molecular Cell. 65 (6), 1044-1055 (2017).</li><li>Lee, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C9orf72+Dipeptide+Repeats+Impair+the+Assembly,+Dynamics,+and+Function+of+Membrane-Less+Organelles.">C9orf72 Dipeptide Repeats Impair the Assembly, Dynamics, and Function of Membrane-Less Organelles.</a> Cell. 167 (3), 717-788 (2016).</li><li>Hao, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+dysfunction+and+neurodegeneration+in+a+C9orf72+mouse+line+expressing+poly-PR.">Motor dysfunction and neurodegeneration in a C9orf72 mouse line expressing poly-PR.</a> Nature Communications. 10 (1), 2906 (2019).</li><li>Scior, A., Preissler, S., Koch, M., Deuerling, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+PCR-free+engineering+of+highly+repetitive+DNA+sequences.">Directed PCR-free engineering of highly repetitive DNA sequences.</a> BMC Biotechnology. 11, 87 (2011).</li><li>Mello, C., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+transformation.">DNA transformation.</a> Methods in Cell Biology. 48, 451-482 (1995).</li><li>Rudich, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+localized+C9orf72-associated+arginine-containing+dipeptides+exhibit+age-dependent+toxicity+in+C.+elegans.">Nuclear localized C9orf72-associated arginine-containing dipeptides exhibit age-dependent toxicity in C. elegans.</a> Human Molecular Genetics. 26 (24), 4916-4928 (2017).</li><li>Gidalevitz, T., Krupinski, T., Garcia, S., Morimoto, R. 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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...

Watch this Scientific Journal Video about Measuring RAN Peptide Toxicity in C. elegans at JoVE.com

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 ...

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.

Research

JoVE Journal

Biology

Generation of Stable Transgenic C. elegans Using Microinjection

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Generation of Transgenic C. elegans by Biolistic Transformation

Generation of Transgenic C. elegans by Biolistic Transformation

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Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

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An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

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