A dominant mutation in a neuronal acetylcholine receptor subunit leads to motor neuron degeneration in Caenorhabditis elegans

Abstract

Inappropriate or excessive activation of ionotropic receptors can have dramatic consequences for neuronal function and, in many instances, leads to cell death. In Caenorhabditis elegans, nicotinic acetylcholine receptor (nAChR) subunits are highly expressed in a neural circuit that controls movement. Here, we show that heteromeric nAChRs containing the acr-2 subunit are diffusely localized in the processes of excitatory motor neurons and act to modulate motor neuron activity. Excessive signaling through these receptors leads to cell-autonomous degeneration of cholinergic motor neurons and paralysis. C. elegans double mutants lacking calreticulin and calnexin-two genes previously implicated in the cellular events leading to necrotic-like cell death (Xu et al. 2001)-are resistant to nAChR-mediated toxicity and possess normal numbers of motor neuron cell bodies. Nonetheless, excess nAChR activation leads to progressive destabilization of the motor neuron processes and, ultimately, paralysis in these animals. Our results provide new evidence that chronic activation of ionotropic receptors can have devastating degenerative effects in neurons and reveal that ion channel-mediated toxicity may have distinct consequences in neuronal cell bodies and processes.

Figure 1.

acr-2 is expressed in cholinergic motor neurons and modulates motor neuron activity. A, Confocal image of the posterior ventral nerve cord of an adult animal coexpressing a GABA-specific marker (Punc-47::mCherry) and an ACR-2-specific marker (Pacr-2::GFP). No overlap is observed between GFP-expressing and mCherry-expressing neurons. The animal is oriented with the posterior (tail) on the right. B, Quantification of movement on a food-free agar plate. Average number of body bends per minute for wild type, acr-2(ok1887) mutants, and acr-2(ok1887) mutants expressing full-length ACR-2::GFP (ufIs42) over a 5 min period are shown. Data represents mean ± SEM of at least 10 trials; **p < 0.01 compared to wild type. C, Time course of paralysis in the presence of the cholinesterase inhibitor aldicarb (1 mm) for wild type, acr-2(ok1887) mutants, and acr-2(ok1887) mutants expressing full-length ACR-2::GFP (ufIs42). The percentage of immobilized animals calculated every 15 min over a time course of 2 h is shown. Each data point represents the mean ± SEM of at least four trials. *p < 0.001, two-way ANOVA.

Figure 2.

Transgenic animals expressing the dominant ACR-2(L/S) transgene are severely uncoordinated. A, B, Still image of a wild-type animal (A) and a transgenic animal expressing the ACR-2(L/S) transgene (B). Note the coiled posture and reduced size that occurs as a result of ACR-2(L/S) expression. C, Quantification of movement on a food-free agar plate. Average number of body bends per minute for wild type, acr-2(ok1887) mutants, and transgenic animals expressing full-length ACR-2(L/S) (ufIs25) counted over a 5 min period are shown. Data represent mean ± SEM. of at least 10 trials; *p < 0.02; **p < 0.0001.

Figure 3.

Transgenic expression of ACR-2(L/S) leads to a loss of cholinergic motor neurons. A, A1, DIC images of an adult wild type animal (A) and an adult transgenic animal expressing ACR-2(L/S) (A1). Triangles denote lesions observed along the ventral nerve cord of transgenic animals expressing ACR-2(L/S). Images show a region directly posterior of the vulva and are oriented with the ventral surface facing up and the anterior of the animal to the left. B, B1, Wide-field epifluorescent images of a transgenic animal expressing the cholinergic neuron marker Punc-17::GFP (vsIs48) (B) and a transgenic animal coexpressing ACR-2(L/S) with Punc-17::GFP (B1). The few Punc-17::GFP-labeled motor neurons that remain in transgenic ACR-2(L/S) animals include the six VCs (indicated) and a more variable group of ∼10–12 neurons (arrowheads). C, C1, Wide-field epifluorescent images of a transgenic animal expressing Punc-47::mCherry (C) and a transgenic animal coexpressing Punc-47::mCherry with ACR-2(L/S) (C1). The full complement of Punc-47::mCherry-labeled motor neurons remains in transgenic ACR-2(L/S) animals and is indicated. D, Quantification of the total number of motor neurons in wild-type (gray) and ACR-2(L/S) animals (black); *p < 0.01. For all images, animals are positioned with the head on the left side of the image.

Figure 4.

ACR-2(L/S)-induced motor neuron cell death is initiated before hatch. A, B, Wide-field epifluorescent images of wild-type (A) and transgenic ACR-2(L/S) (B) animals expressing Punc-17::GFP imaged 16, 28, 38, and 48 h after bleach synchronization of embryos. C, D, Confocal images of first larval stage wild-type (C) and transgenic ACR-2(L/S) (D) animals expressing Punc-17::GFP. Swollen or dying neuronal cell bodies are indicated (arrowheads). E, Quantification of the average number of cell bodies at the time points indicated for wild type (black bars) and transgenic ACR-2(L/S) animals (gray bars). Bars represent the mean ± SEM for 5–8 animals at each time point.

Figure 5.

Mutations in nicotinic acetylcholine receptor subunits suppress ACR-2(L/S)-induced paralysis. A, Quantification of the average number of body bends per minute for wild-type animals, acr-12(ok367), unc-63(ok1075), unc-38(e264), unc-74(e883), and unc-50(e306) mutants in the absence (gray bars) or presence (black bars) of the ACR-2(L/S) transgene. Animals were placed on a food-free agar plate, and the average number of body bends per minute was quantified over a 5 min period. Data represent the mean ± SEM. of at least 10 animals for each genotype. B, Schematic of the membrane topology of ACR-12 with approximate location, allele names, and molecular nature of loss-of-function mutations that suppress ACR-2(L/S) toxicity indicated. C, Quantification of the average number of body bends per minute for the following genotypes: wild type, acr-12(ok367), transgenic ACR-2(L/S), acr-12 mutants expressing ACR-2(L/S), acr-12 mutants expressing ACR-2(L/S) together with an extrachromosomal array containing Punc-47::ACR-12, and acr-12 mutants expressing ACR-2(L/S) together with an extrachromosomal array containing the Pacr-2::ACR-12 cDNA. Data represent the mean ± SEM. for 5–10 animals. D–H, Still images of adult animals on NGM plates without food for the genotypes indicated. “GABA” and “ACh” refer to specific expression of the acr-12 cDNA in GABAergic or cholinergic neurons using the unc-47 or acr-2 promoters, respectively.

Figure 6.

ACR-2(L/S)-mediated motor neuron loss is completely prevented in animals doubly mutant for calnexin and calreticulin. A–F, Representative wide-field images of Punc-17::GFP fluorescence in the ventral nerve cord of adult animals for the genotypes indicated. For each image the head is oriented to the left. The alleles used were ced-3(ok2734), cnx-1(ok2234), and crt-1(ok948). G, Quantification of the average number of Punc-17::GFP-labeled cell bodies present in the ventral nerve cord for the genotypes indicated. Data represent the mean ± SEM for at least 10 animals per genotype. * p < 0.01, **p < 0.01, compared to transgenic ACR-2(L/S) animals.

Figure 7.

Progressive destabilization of motor neuron processes in cnx-1;crt-1;ACR-2(L/S) animals. A, B, Frames showing the movement of L1 transgenic ACR-2(L/S) (A) or cnx-1;crt-1;ACR-2(L/S) (B) animals 30 s (t = 0), 60 s (+30), or 90 s (+60) after transfer to an agar plate. The black arrow marks the starting positions of the worms in each frame. The transgenic ACR-2(L/S) animal does not move from where it was placed on the plate. The dashed line shows the movement path of the L1 cnx-1;crt-1;ACR-2(L/S) animal over the course of 60 s. The additional tracks on the plate show the path of the animal in an ∼30 s period before imaging began. C, D, Confocal images of a first larval stage (C) or adult cnx-1;crt-1;ACR-2(L/S) animals expressing an integrated Punc-17::GFP (D). Images show Z-projections of 15 confocal planes (0.5 μm/slice) (C) or 16 confocal planes (0.5 μm/slice) (D). Arrows (D) indicate positions of commissures. Dashed box (D) indicates area with multiple neuronal defects. E–G, Confocal images of cnx-1;crt-1;ACR-2(L/S) animals taken at hatch (E), 24 h posthatch (F), and adulthood (G). In each case, a region immediately posterior of the vulva was imaged and the ventral nerve cord is positioned at the top. Arrows indicate commissural processes; arrowheads indicate the ventral nerve cord. Images show Z-projections of 12 confocal planes (E), 23 confocal planes (F), or 33 confocal planes (G) (0.5 μm/slice). Scale bars represent 10 μm. H, Quantification of percentage of animals moving at hatch in ACR-2(L/S) and cnx-1;crt-1;ACR-2(L/S) animals. Data represent the mean number of animals ± SEM making more than two consecutive body bends during a 3 min period. I, Quantification of the percentage of cnx-1;crt-1 and cnx-1;crt-1;ACR-2(L/S) animals with defective neuronal process morphology. Observed defects include defasciculation of the ventral nerve cord, wandering commissural processes, or ectopic branching.

Figure 8.

Perturbation of intracellular calcium and reduced ACR-2::GFP levels in cnx-1;crt-1 double mutants suggest dual mechanisms for cell death suppression. A–C, Quantification of the average number of Punc-17::GFP-labeled cell bodies present in the ventral nerve cord in adult animals treated with dantrolene (A), L1 animals treated with EGTA (B), and adult unc-68(e540) mutant animals (C). Data represent the mean ± SEM. for at least eight animals. **p < 0.01, ***p < 0.01, compared to untreated ACR-2(L/S) animals. D, E, Confocal images of adult wild type (D) or cnx-1;crt-1 (E) animals expressing ACR-2::GFP. Images are taken immediately posterior to the vulva and show Z-projections of seven confocal planes (0.5 μm/slice). F, G, Quantification of fluorescence levels in cell bodies (F) and ventral nerve cord processes (G) of wild-type and ACR-2(L/S) animals transgenically expressing ACR-2::GFP. Data represent the mean ± SEM. for at least 10 animals. ***p < 0.01, compared to ACR-2(L/S) animals.