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>
	<li>Cleary, J. D., Ranum, L. P. <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-ATG+(RAN)+translation:+new+starts+in+microsatellite+expansion+disorders.">Repeat associated non-ATG (RAN) translation: new starts in microsatellite expansion disorders.</a> Current Opinion in Genetics and Development. 26, 6-15 (2014).</li><li>The Huntington's Disease Collaborative Research Group. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+gene+containing+a+trinucleotide+repeat+that+is+expanded+and+unstable+on+Huntington's+disease+chromosomes.">A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes.</a> Cell. 72 (6), 971-983 (1993).</li><li>Scherzinger, 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=Huntingtin-encoded+polyglutamine+expansions+form+amyloid-like+protein+aggregates+in+vitro+and+in+vivo.">Huntingtin-encoded polyglutamine expansions form amyloid-like protein aggregates in vitro and in vivo.</a> Cell. 90 (3), 549-558 (1997).</li><li>Morley, J. 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. CAG Repeat Not Polyglutamine Length Determines Timing of Huntington's Disease Onset.</a> Cell. 178 (4), 887-900 (2019).</li><li>Wright, G. E. B., 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=Length+of+Uninterrupted+CAG,+Independent+of+Polyglutamine+Size,+Results+in+Increased+Somatic+Instability,+Hastening+Onset+of+Huntington+Disease.">Length of Uninterrupted CAG, Independent of Polyglutamine Size, Results in Increased Somatic Instability, Hastening Onset of Huntington Disease.</a> American Journal of Human Genetics. 104 (6), 1116-1126 (2019).</li><li>Cleary, J. D., Ranum, L. P. <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-ATG+(RAN)+translation+in+neurological+disease.">Repeat-associated non-ATG (RAN) translation in neurological disease.</a> Human Molecular Genetics. 22 (1), 45-51 (2013).</li><li>Banez-Coronel, M., Ranum, L. P. 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. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Destabilizing+protein+polymorphisms+in+the+genetic+background+direct+phenotypic+expression+of+mutant+SOD1+toxicity.">Destabilizing protein polymorphisms in the genetic background direct phenotypic expression of mutant SOD1 toxicity.</a> PLoS Genetics. 5 (3), 1000399 (2009).</li><li>Nollen, E. 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=Genome-wide+RNA+interference+screen+identifies+previously+undescribed+regulators+of+polyglutamine+aggregation.">Genome-wide RNA interference screen identifies previously undescribed regulators of polyglutamine aggregation.</a> Proceedings of the National Academy of Sciences U S A. 101 (17), 6403-6408 (2004).</li><li>Satyal, S. 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=Polyglutamine+aggregates+alter+protein+folding+homeostasis+in+Caenorhabditis+elegans.">Polyglutamine aggregates alter protein folding homeostasis in Caenorhabditis elegans.</a> Proceedings of the National Academy of Sciences U S A. 97 (11), 5750-5755 (2000).</li><li>Boccitto, M., Lamitina, T., Kalb, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Daf-2+signaling+modifies+mutant+SOD1+toxicity+in+C.+elegans.">Daf-2 signaling modifies mutant SOD1 toxicity in C. elegans.</a> PLoS One. 7 (3), 33494 (2012).</li><li>Liu, Y., 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+BAC+Mouse+Model+with+Motor+Deficits+and+Neurodegenerative+Features+of+ALS/FTD.">C9orf72 BAC Mouse Model with Motor Deficits and Neurodegenerative Features of ALS/FTD.</a> Neuron. 90 (3), 521-534 (2016).</li><li>Peters, O. 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=Human+C9ORF72+Hexanucleotide+Expansion+Reproduces+RNA+Foci+and+Dipeptide+Repeat+Proteins+but+Not+Neurodegeneration+in+BAC+Transgenic+Mice.">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><li>O'Rourke, J. G., 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+BAC+Transgenic+Mice+Display+Typical+Pathologic+Features+of+ALS/FTD.">C9orf72 BAC Transgenic Mice Display Typical Pathologic Features of ALS/FTD.</a> Neuron. 88 (5), 892-901 (2015).</li><li>Mizielinska, 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=C9orf72+repeat+expansions+cause+neurodegeneration+in+Drosophila+through+arginine-rich+proteins.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+profiling+of+Caenorhabditis+elegans+motility.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+auxin-inducible+degradation+(AID)+system+enables+versatile+conditional+protein+depletion+in+C.+elegans.">The auxin-inducible degradation (AID) system enables versatile conditional protein depletion in C. elegans.</a> Development. 142 (24), 4374-4384 (2015).</li></ol>

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.

measuring-ran-peptide-toxicity-in-c-elegans

Research

JoVE Journal

Biology

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Measuring Peptide Translocation into Large Unilamellar Vesicles

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1. Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors1-3. Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms4,5, particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes6,7. In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides1,8. A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)9-13. LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies14,15. Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II16,17. In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties18, to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating peptides are cleaved as they encounter uninhibited trypsin encapsulated with the LUVs, leading to disassociation from the LUV membrane and a drop in FRET signal. The drop in FRET signal observed for a translocating peptide is significantly greater than that observed for the same peptide when the LUVs contain both trypsin and trypsin inhibitor, or when a peptide that does not spontaneously cross lipid membranes is exposed to trypsin-containing LUVs. This change in fluorescence provides a direct quantification of peptide translocation over time.

This protocol details a method for the quantitative measure of peptide translocation into large unilamellar lipid vesicles. This method also provides information about the rate of membrane translocation and can be used to identify peptides that efficiently and spontaneously cross lipid bilayers.

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1.  Many of these are referred ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

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

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

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

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

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

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

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

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound responses and repair in this model have illuminated our understanding of the cytoskeletal and genomic responses to tissue damage. The two most commonly used methods to wound the C. elegans adult skin are pricks with microinjection needles, and local laser irradiation. Needle wounding locally disrupts the cuticle, epidermis, and associated extracellular matrix, and may also damage internal tissues. Laser irradiation results in more localized damage. Wounding triggers a succession of readily assayed responses including elevated epidermal Ca2+ (seconds-minutes), formation and closure of an actin-containing ring at the wound site (1-2 hr), elevated transcription of antimicrobial peptide genes (2-24 hr), and scar formation. Essentially all wild type adult animals survive wounding, whereas mutants defective in wound repair or other responses show decreased survival. Detailed protocols for needle and laser wounding, and assays for quantitation and visualization of wound responses and repair processes (Ca dynamics, actin dynamics, antimicrobial peptide induction, and survival) are presented.

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The adult C. elegans skin is a tractable model for studies of epithelial wound responses, including wound closure, scar formation, and innate immunity.

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound ...