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.
C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington’s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.
The inappropriate expansion of DNA repeat sequences is the genetic basis for several neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Huntington’s disease (HD)1. While there are established cellular and animal models for these diseases, mechanisms underlying these conditions are not well defined. For example, HD is caused by the expansions of a CAG repeat sequence in the coding sequence for the Huntingtin protein Htt2. Because CAG encodes the amino acid glutamine, the CAG repeat expansion results in the insertion of a polyGlutamine, or polyQ, sequence within Htt. Expanded polyQ proteins form length- and age-dependent protein aggregates that are associated with 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).
1. Generating codon-varied RAN peptide expression constructs
2. Measuring the developmental toxicity of RAN peptides following RNAi-based gene knockdown: Video speed analysis protocol
3. Measuring developmental toxicity of RAN peptide: Growth assay
4. Post-developmental RAN peptide paralysis assay
5. Measuring neuron pathology: Commissure assay
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 in several genes suppressed PR developmental toxicity from 12–94% (Figure 1A).
We also measured the effect of specific gene knockdowns on the developmental motility of PR50-expressing animals using the video speed analysis method. As expected, gfp(RNAi) resulted in a large increase in motility compared to empty vector(RNAi) due to inhibition of PR50-GFP expression. We also discovered that RNAi against the proteasome subunit rpn-7 resulted in a significant increase in PR50 motility (Figure 1B).
ALS, like many neurodegenerative diseases, occurs in adults. Therefore, we analyzed adult phenotypes using the age-dependent paralysis assay. PR50-GFP exhibited up to 80% paralysis by 5 days of age. However, RNAi directed at the gene cul-6 significantly delayed paralysis, suggesting that cul-6 is required for PR50-GFP toxicity (Figure 1C). This effect was specific to cul-6(RNAi) because cul-1(RNAi) did not alter PR50-GFP toxicity.
Neurodegenerative proteins, such as toxic RAN peptides, are commonly modeled in the body wall of C. elegans4,20,21,22. However, it is also important to determine if RAN proteins cause neuropathology when expressed in C. elegans neurons, because neuron-specific toxicity is a common feature of many neurodegenerative diseases. We examined neuron-specific toxicity using the commissure assay. In day 2 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 that delays the toxic properties of several neurodegenerative proteins23 (Figure 2B).
Figure 1: Assays to evaluate the toxicity of muscle-expressed RAN peptides. (A) RAN peptide growth assay. The indicated mutants were crossed into the drIs34 (myo-3p::PR50-GFP) genetic background under gfp(RNAi) conditions. Numbers above each genotype indicate the number of progeny scored for growth. All genotypes have p < 0.05 using the Fisher’s exact test as compared to wild type. (B) Video speed analysis to measure RAN peptide toxicity during development. drIs34 (myo-3p::PR50-GFP) animals were grown under gfp(RNAi), empty vector(RNAi), or rpn-7(RNAi) conditions and then scored for motility as described. Speed was normalized to gfp(RNAi) treated animals. Data shown are mean ± SD, n = 40 for each genotype. **-p < 0.01, ***- p < 0.001, one-way ANOVA with Tukey post-test. (C) Paralysis assay to measure age onset toxicity. drIs34 (myo-3p::PR50-GFP) animals were grown on empty vector(RNAi) (‘WT’), cul-1(RNAi), or cul-6(RNAi) and paralysis was scored as described every 24 h. ***-p < 0.001, log-rank test with Bonferroni post-test correction vs. WT. n = 100 animals per genotype. Please click here to view a larger version of this figure.
Figure 2: Assay for measuring neuropathology of motor neuron expressed RAN peptides. (A) Images of adult C. elegans expressing the unc-47p::GFP motor neuron reporter in wild type or unc-47p::PR50-GFP expressing animals. The images for wild type illustrate normal commissure morphology. Images represent flattened Z-series stacks obtained by widefield fluorescence microscopy. PR50 animals demonstrate examples of commissure breakage and blebbing. (B) Effect of daf-2(e1370) on PR50 commissure blebbing. Points represent the percent blebbing commissures from a single animal, with the mean and SD shown. n = 20 animals per genotype **-p < 0.01, one-way ANOVA with Dunn’s post-hoc test. Please click here to view a larger version of this figure.
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 especially important given that most precise animal models of disease have either no phenotype or weak phenotypes not suitable for unbiased screening approaches24,25,26. Our C. elegans RAN peptide models and assays represent a powerful, complementary approach to other RAN peptide model systems, such as yeast and Drosophila, for understanding the cellular pathways important for the toxicity of these newly described proteins.
Conditional toxicity of muscle-expressed RAN peptides
Measuring RAN peptide toxicity requires a chase period for elimination of the effects of gfp(RNAi). However, the time between removal from gfp(RNAi) and the emergence of phenotypes can be inconsistent if care is not taken to precisely time the initiation of experiments, selection of staged animals, temperature shifts, etc. As we have become more experienced with these assays, the timing and penetrance of RAN peptide phenotypes has become relatively consistent. One of the most critical steps in these assays is the proper shift of animals to 25 °C. Without this shift, RAN peptides exhibit significantly weaker toxicity. However, animals cannot be continually grown at 25 °C because they do not grow and reproduce, presumably due to higher baseline expression of the RAN peptide. This is not unlike the situation in yeast or Drosophila where strains expressing toxic RAN peptides are kept under permissive conditions (i.e., low temperature) but then acutely shifted to restrictive conditions (i.e., higher temperature) prior to the assay13,15 to enhance peptide expression. In such conditional expression systems, genetic conditions that enhance or suppress toxicity could result from changes in peptide expression levels. To determine if genetic modifiers of RAN peptide toxicity act via alterations in transgene expression levels, we take two approaches. First, many of our transgenic strains express a second RFP reporter under the control of the same promoter used to drive expression of the RAN peptide. Changes in promoter activity that cause reduced or increased RAN peptide expression cause similar reductions or increases in RFP levels. We consider mutants or RNAi conditions that significantly alter RFP levels to act nonspecifically. Second, we perform quantitative PCR and Western blotting to determine if the levels of RAN peptide mRNA or protein are altered between conditions. However, Western-based detection of RAN peptides can sometimes prove difficult or impossible, as we and others have observed with the C9orf72-derived RAN peptide PR50-GFP. In such cases, we quantify in vivo GFP levels to determine if protein expression is altered between the conditions being compared.
Developmental toxicity: Advantages and limitations
Forced overexpression of toxic RAN peptides in muscle often leads to developmental arrest in C. elegans. While this clearly does not mimic human disease pathology, it provides a powerful starting point for genetic suppressor screens, because RAN toxicity suppressors are easily identified as animals that grow and reproduce in the absence of gfp(RNAi). Some suppressors will facilitate growth due to reduction of transgene expression. To differentiate between these possibilities, we generally include a second RFP reporter driven by the same promoter in our RAN peptide transgenic strain. Suppressors that reduce or silence transgene expression will also exhibit reduced RFP expression levels. On the other hand, suppressors that exhibit normal or elevated RFP levels are unlikely to function via reduction of transgene expression. Much like the use of yeast growth or Drosophila eye morphology13,15,27, these approaches, while not precisely modeling aspects of the disease in humans, provide a powerful screening tool for the initial identification of RAN peptide modifiers.
Post-developmental toxicity (paralysis assay): Advantages and limitations
The advantage of the paralysis assay is that a relative large number of animals (50–100) can be measured with tools present in any worm lab. Paralysis is also a severe phenotype that is easily detected. The main limitation of this assay is that is insensitive to movement defects that do not cause complete paralysis. Such defects may require more sensitive and quantitative approaches to measure, such as the thrashing assays or kinematic movement assays28. Paralysis assays should be performed blinded to genotype if possible and care should be taken to ensure that worms are not undergoing any other type of stress (e.g., contamination, starvation), that could significantly impact the assay results. C. elegans expressing toxic RAN peptides sometimes fail to exhibit a robust paralysis phenotype. This is likely due to persistent effects of gfp(RNAi) continuing to suppress RAN peptide expression. In these cases, if we fail to observe significant paralysis (>10%) in the empty vector(RNAi) control animals after day 2, we typically terminate the assay and initiate another replicate. One modification of this assay could involve the use of alternative inducible RAN peptide expression systems. The only other commonly used inducible expression system for C. elegans is the heat shock promoter. However, the required heat shock can cause stress responses that might affect the toxicity of the proteins. The future application of other conditional expression systems, such as the auxin-inducible degron (AID) system29, could significantly enhance our ability to study RAN peptide toxicity.
Neurodegeneration/commissure assay: Advantages and limitations
Motor neuron commissures in C. elegans represent the axon of one motor neuron passing between the ventral and dorsal nervous cords. Blebbing and breakage can be easily detected in the isolated commissures, allowing neurodegeneration to be quickly quantified in live animals (Figure 2). While examining the animals, it is important they do not incubate in levamisole for an extended period of time as this can cause premature animal death, which leads to neuronal blebbing and breakage in the absence of any toxic RAN peptide. In some cases, commissures are completely degenerated and no longer visible. Therefore, they cannot be scored for neuropathological features because our assay measures the percentages of commissures broken or blebbing out of the total number of observed commissures. In these cases, the commissure assay might underestimate the effect of the RAN peptide.
In conclusion, the assays described in this paper are useful for measuring toxicity caused by RAN peptides in C. elegans. Using gfp(RNAi) to regulate expression of the proteins allows for post-developmental phenotypes to be observed. Our approaches can be easily adapted to perform large-scale genetic screens for suppressors or enhancers of toxicity. Secondary assays, such as the paralysis assay and commissure assay can confirm that toxicity is suppressed post-developmentally and test if the mechanism of suppression is conserved in neurons.
The authors have nothing to disclose.
NIH R21NS107797
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