Nuclear localized C9orf72-associated arginine-containing dipeptides exhibit age-dependent toxicity in C. elegans

Abstract

A hexanucleotide repeat expansion mutation in the C9orf72 gene represents a prevalent genetic cause of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Non-canonical translation of this repeat gives rise to several distinct dipeptide protein species that could play pathological roles in disease. Here, we show in the model system Caenorhabditis elegans that expression of the arginine-containing dipeptides, but not alanine-containing dipeptides, produces toxic phenotypes in multiple cellular contexts, including motor neurons. Expression of either (PR)50 or (GR)50 during development caused a highly penetrant developmental arrest, while post-developmental expression caused age-onset paralysis. Both (PR)50- and (GR)50-green fluorescent protein tagged dipeptides were present in the nucleus and nuclear localization was necessary and sufficient for their toxicity. Using an inducible expression system, we discovered that age-onset phenotypes caused by (PR)50 required both continual (PR)50 expression and an aged cellular environment. The toxicity of (PR)50 was modified by genetic mutations that uncouple physiological aging from chronological aging. However, these same mutations failed to modify the toxicity of (GR)50, suggesting that (PR)50 and (GR)50 exert their toxicity through partially distinct mechanism(s). Changing the rate of physiological aging also mitigates toxicity in other C. elegans models of ALS, suggesting that the (PR)50 dipeptide might engage similar toxicity mechanisms as other ALS disease-causing proteins.

Figure 1.

Arginine-containing DPRs are toxic in motor neurons. (A) Molecular strategy for expression of codon-varied dipeptide-repeat proteins in C. elegans. (B) Liquid thrashing quantification of transgenic animals expressing the indicated DPR under the motor neuron-specific unc-47 promoter. N = 20 animals per genotype. Each symbol represents quantification for one animal, horizontal line indicates population median. ***P < 0.001 versus GFP control (one-way non-parametric ANOVA with Dunn’s post hoc test). (C) Representative images of unc-47+ motor neurons in animals expressing the indicated DPR. Arrow points to an example of membrane blebbing. Arrowhead points to examples of commissure breaks. Scale bar = 10 μm. (D) Quantification of membrane blebbing and commissure breaks in each of the indicated strains. For each animal, we counted the total number of commissures with blebbing or breaks and divided by the total number of detectable commissures. N = 20 animals per genotype. Each symbol represents quantification for one animal expressed as a percentage. The horizontal line indicates population median with interquartile range. ****P < 0.0001 versus GFP control (one-way non-parametric ANOVA with Dunn’s post hoc test). N = 20 animals per genotype.

Figure 2.

Muscle expressed arginine-containing dipeptides are toxic in C. elegans. (A) Brood size for worms expressing each integrated dipeptide protein in the presence or absence of gfp(RNAi) at 25 °C. Data for each strain are normalized to the mean brood size of animals grown on gfp(RNAi). ***P < 0.001 (one-way ANOVA with post hoc Tukey’s test). Raw brood size data for 25 °C are included in Supplementary Material, Table S1. (B) Paralysis assay for adult animals raised in the absence of gfp(RNAi). N ≥ 43–49 animals per genotype. ***P < 0.001 (Log-rank test with Bonferroni adjusted P-value). (C) Fluorescent microscopy of Day 1 adult hermaphrodites expressing (PR)₅-GFP, (PR)₁₅-GFP, (PR)₂₅-GFP or (PR)₅₀-GFP (green) in the muscle. Muscle DNA (marked by a myo-3p::his-58-mCherry reporter) is in red. Arrows point to sites of nuclear GFP puncta. Scale bar = 10 μm. All images were acquired using identical exposure settings. The row labeled ‘myo-3p::(PR)₅₀ (enhanced)’ was adjusted for brightness and contrast in the GFP channel differently from the other images so that the (PR)₅₀-GFP signal is observable. (D) Quantification of the GFP levels from the indicated PR repeat animals. Each point shows the measured value for a single nucleus, horizontal line indicates population median with interquartile range. **P < 0.01, ****P < 0.0001 versus GFP control (one-way non-parametric ANOVA with Dunn’s post hoc test). (E) Paralysis assay of animals expressing the indicated number of (PR) repeats under the control of the myo-3 promoter. ‘Day 0’ animals were isolated as L4 stage animals. N = 48–50 animals per genotype. ***P < 0.001 versus GFP control (Log-rank test with Bonferroni adjusted P-value).

Figure 3.

(PR)₅₀ and (GR)₅₀ are localized to the nucleolus in C. elegans muscle cells (A) Imaging of live, anesthetized Day 1 adult C. elegans hermaphrodites expressing the indicated dipeptide. Green shows dipeptide-GFP localization expressed in muscle, red shows histone-tagRFP within the muscle nucleus. Arrowhead points to the histone-free nucleolar region. Scale bar=10 μm. (B) Quantification of DPR–GFP signal that is observed in nucleoplasm, nucleolus, and non-nuclear cellular compartments. N = 55–78 nuclei from five to six animals. Data shown are means ± S.D.

Figure 4.

Arginine-containing DPRs are not aggregates. (A) Representative images from FRAP analysis of subcellular localized DPR proteins expressed in muscle. Dashed outline indicates site of photobleaching and post-bleaching quantification. Recovery images are 150 s post-bleach. Scale bar = 2 μm. (B) Quantification of FRAP imaging. Data shown are mean ± S.E.M. from 9 to 10 datasets per genotype. (C) Average equilibrium fluorescence recovery after 60 s. Data shown are mean ± S.D. from 9 to 10 datasets per genotype. n.s. versus FIB-1-GFP (one-way ANOVA with Dunn’s multiple comparison test).

Figure 5.

Nuclear localization of muscle expressed arginine-containing DPRs is necessary and sufficient for toxicity. (A) Representative images of the indicated soluble or membrane anchored DPR (green), the HIS-58-mCherry nuclear marker (red), and a merged image. Arrows point to nuclear regions defined by the HIS-58 mCherry signal. Coincident green and red signals in the ‘TM-(PR)₅₀ image is intestinal autofluorescence. Scale bar = 10 μm. (B) Paralysis assay comparing animals expressing soluble or transmembrane localized (GR)₅₀ (top) or (PR)₅₀ (bottom). N = 48–50 animals per genotype, ***P < 0.001, **P < 0.01, *P < 0.05 (Log-rank test with Bonferroni adjusted P-value). (C) Representative images of HIS-58 anchored (PR)₅₀ (green; top) or unanchored (PR)₅₀ (green; bottom), HIS-58-mCherry nuclear marker (red) and a merged image. Scale bar = 5 μm. (D) Paralysis assay comparing animals expressing nuclear localized GFP or (PR)_50.n ≥ 30 animals per genotype, **P < 0.01 (Log-rank test with Bonferroni adjusted P-value). (E) Paralysis assay comparing soluble (PR)₅₀ with his-58-(PR)₅₀. n≥ 50 animals [(PR)₅₀] or 80 animals [his-58-(PR)₅₀], ***P < 0.001 (Log-rank test with Bonferroni adjusted P-value).

Figure 6.

Age-onset toxicity requires continuous DPR expression. (A) Images of worms expressing the indicated DPR under the control of a heat shock inducible promoter. Red fluorescence shows the myo-3p::mCherry transgenic marker. Green fluorescence shows the hsp-16.2p::DPR–GFP signal. All DPRs are shown 2–4 h after heat shock. Insets show an enlarged view of the region identified by the dashed boxes. Arrows in the (GA)₅₀ inset point to putative protein aggregates, suggesting that (GA)₅₀ rapidly aggregates after synthesis. Arrows in the (PR)₅₀ inset point to sites of nuclear localization in the intestine. No such nuclear localization was observed in the (GR)₅₀line after heat shock. (B) Developmental toxicity of transgenic embryos expressing the indicated hsp-16.2p::DPR–GFP. Embryos were heat shocked at 35 °C for 30 min and then returned to 20 °C. The number that reached ≥L4 after 48 h were scored as ‘growth’ while the remaining animals were scored as ‘no growth’. N ≥ 81 transgenic animals per condition. ***P < 0.001 (Fisher’s exact test versus GFP + H.S.). (C) Adult toxicity of animals expressing either hsp-16.2p::GFP or hsp-16.2p:(PR)₅₀-GFP. Animals were subjected to either a heat shock (30 min, 35 °C) on Day 1, Day 4 or on Days 1, 2, 3 and 4. Every 24 h, animals were scored as either mobile, censored or affected (paralyzed + dead = ‘% with phenotype’). Heat shock induction of (GA)₅₀, (PA)₅₀ and (GR)₅₀ produced phenotypes identical to that of GFP. N ≥ 60 animals per genotype. ***P < 0.001 versus GFP; Days 1–4 H.S (Log-rank test with Bonferroni adjusted P-value).

Figure 7.

Altering the rate of aging affects (PR)₅₀ but not (GR)₅₀ toxicity. (A) Paralysis assay of animals expressing GFP, (GR)₅₀ or (PR)₅₀ under the control of the myo-3 promoter in the wild-type or daf-2(e1370) background. ‘Day 0’ animals were isolated as L4 stage animals. N = 48–50 animals per genotype. n.s., ‘not significant’; **P < 0.01 versus GFP control (Log-rank test with Bonferroni adjusted P-value). (B) Paralysis assay of animals expressing (PR)₅₀ under the control of the myo-3 promoter in the wild-type, daf-16(mu86), or daf-2(e1370); daf-16(mu86) background. N = 48–50 animals per genotype. n.s., ‘not significant’; ***P < 0.001 versus (PR)₅₀ (Log-rank test with Bonferroni adjusted P-value). (C) Paralysis assay of animals expressing (PR)₅₀ under the control of the myo-3 promoter in the wild-type or hsf-1(sy441) mutant background. N = 48–50 animals per genotype. ***P < 0.001 versus wild-type (Log-rank test with Bonferroni adjusted P-value). (D) Fluorescent microscopy images of day 1 adult worms expressing either (GR)₅₀ or (PR)₅₀ (green) and soluble muscle mCherry (red) in the wild-type or daf-2(e1370) mutant background. Arrow points to site of nuclear DPR accumulation. Scale bar=10 μm. (E) Fluorescent microscopy images of Day 1 adult worms expressing (PR)₅₀ (green) and soluble muscle mCherry (red) in the daf-16(mu86) or daf-2(e1370); daf-16(mu86) mutant background. Arrow points to site of nuclear DPR accumulation. Scale bar=10 μm . (F) Fluorescent microscopy images of day 1 adult worms expressing (PR)₅₀ (green) and soluble muscle mCherry (red) in the wild-type or hsf-1(sy441) mutant background. Arrow points to site of nuclear DPR accumulation. Scale bar=10 μm.