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A genome-wide RNAi screen in Caenorhabditis elegans identifies the nicotinic acetylcholine receptor subunit ACR-7 as an antipsychotic drug target.

Saur T, DeMarco SE, Ortiz A, Sliwoski GR, Hao L, Wang X, Cohen BM, Buttner EA - PLoS Genet. (2013)

Bottom Line: We validate the requirement for acr-7 by showing that acr-7 knockout suppresses clozapine-induced larval arrest and that expression of a full-length translational GFP fusion construct rescues this phenotype. nAChR agonists phenocopy the developmental effects of clozapine, while nAChR antagonists partially block these effects.ACR-7 is strongly expressed in the pharynx, and clozapine inhibits pharyngeal pumping. acr-7 knockout and nAChR antagonists suppress clozapine-induced inhibition of pharyngeal pumping.No APDs are known to activate nAChRs, but a number of studies indicate that α7-nAChR agonists may prove effective for the treatment of psychosis. α-like nAChR signaling is a mechanism through which clozapine may produce its therapeutic and/or toxic effects in humans, a hypothesis that could be tested following identification of the mammalian ortholog of C. elegans acr-7.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
We report a genome-wide RNA interference (RNAi) screen for Suppressors of Clozapine-induced Larval Arrest (scla genes) in Caenorhabditis elegans, the first genetic suppressor screen for antipsychotic drug (APD) targets in an animal. The screen identifies 40 suppressors, including the α-like nicotinic acetylcholine receptor (nAChR) homolog acr-7. We validate the requirement for acr-7 by showing that acr-7 knockout suppresses clozapine-induced larval arrest and that expression of a full-length translational GFP fusion construct rescues this phenotype. nAChR agonists phenocopy the developmental effects of clozapine, while nAChR antagonists partially block these effects. ACR-7 is strongly expressed in the pharynx, and clozapine inhibits pharyngeal pumping. acr-7 knockout and nAChR antagonists suppress clozapine-induced inhibition of pharyngeal pumping. These findings suggest that clozapine activates ACR-7 channels in pharyngeal muscle, leading to tetanus of pharyngeal muscle with consequent larval arrest. No APDs are known to activate nAChRs, but a number of studies indicate that α7-nAChR agonists may prove effective for the treatment of psychosis. α-like nAChR signaling is a mechanism through which clozapine may produce its therapeutic and/or toxic effects in humans, a hypothesis that could be tested following identification of the mammalian ortholog of C. elegans acr-7.

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Genetic and pharmacological characterization of clozapine-induced larval arrest.(A) Clozapine induced developmental delay in wild-type C. elegans in a concentration-dependent manner. acr-7(tm863) partially suppressed this developmental delay, and the suppression was rescued by expression of ACR-7 in the mutant background. Expression was driven by a putative acr-7 promoter in the case of acr-7(tm863) mchEx40 or by the pharyngeal muscle-specific myo-2 promoter in the case of acr-7(tm863) mchEx207. Growth was measured as the percentage of different development stages 72 hours after loading synchronized L1 animals into the drug plates. Different concentrations (80, 160, 320 µM) of clozapine were dissolved in DMSO with the maximum concentration of DMSO being 0.1%. 0.1% DMSO alone was used for control wells. (B) nAChR agonists nicotine (Nico) and levamisole (Leva) induced developmental delay in a concentration-dependent manner, mimicking the effect of clozapine. (C) Nicotine-induced developmental delay is suppressed by acr-7(tm863), and suppression was partially rescued by expression of full-length ACR-7 in the mutant background. (D) Nicotine receptor antagonists d-TC (100 and 500 µM) and DHβE (100 and 500 µM) suppressed clozapine (Cloz)-induced developmental delay. d-TC or DHβE alone did not affect the growth of the animals at 100 and 500 µM.
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pgen-1003313-g004: Genetic and pharmacological characterization of clozapine-induced larval arrest.(A) Clozapine induced developmental delay in wild-type C. elegans in a concentration-dependent manner. acr-7(tm863) partially suppressed this developmental delay, and the suppression was rescued by expression of ACR-7 in the mutant background. Expression was driven by a putative acr-7 promoter in the case of acr-7(tm863) mchEx40 or by the pharyngeal muscle-specific myo-2 promoter in the case of acr-7(tm863) mchEx207. Growth was measured as the percentage of different development stages 72 hours after loading synchronized L1 animals into the drug plates. Different concentrations (80, 160, 320 µM) of clozapine were dissolved in DMSO with the maximum concentration of DMSO being 0.1%. 0.1% DMSO alone was used for control wells. (B) nAChR agonists nicotine (Nico) and levamisole (Leva) induced developmental delay in a concentration-dependent manner, mimicking the effect of clozapine. (C) Nicotine-induced developmental delay is suppressed by acr-7(tm863), and suppression was partially rescued by expression of full-length ACR-7 in the mutant background. (D) Nicotine receptor antagonists d-TC (100 and 500 µM) and DHβE (100 and 500 µM) suppressed clozapine (Cloz)-induced developmental delay. d-TC or DHβE alone did not affect the growth of the animals at 100 and 500 µM.

Mentions: We tested growth effects over a range of clozapine concentrations (40–320 µM). N2 animals grown in the presence of 0.1% DMSO alone reached adulthood in ∼3 days (Figure 1B and Figure 4A). Consistent with our previous study [12], clozapine inhibited the development of N2 animals in a dose-dependent fashion. For example, 98% of control animals were gravid adults on day 3, compared to 36% on 80 µM clozapine and 0% on 320 µM clozapine (Figure 1B and Figure 4A).


A genome-wide RNAi screen in Caenorhabditis elegans identifies the nicotinic acetylcholine receptor subunit ACR-7 as an antipsychotic drug target.

Saur T, DeMarco SE, Ortiz A, Sliwoski GR, Hao L, Wang X, Cohen BM, Buttner EA - PLoS Genet. (2013)

Genetic and pharmacological characterization of clozapine-induced larval arrest.(A) Clozapine induced developmental delay in wild-type C. elegans in a concentration-dependent manner. acr-7(tm863) partially suppressed this developmental delay, and the suppression was rescued by expression of ACR-7 in the mutant background. Expression was driven by a putative acr-7 promoter in the case of acr-7(tm863) mchEx40 or by the pharyngeal muscle-specific myo-2 promoter in the case of acr-7(tm863) mchEx207. Growth was measured as the percentage of different development stages 72 hours after loading synchronized L1 animals into the drug plates. Different concentrations (80, 160, 320 µM) of clozapine were dissolved in DMSO with the maximum concentration of DMSO being 0.1%. 0.1% DMSO alone was used for control wells. (B) nAChR agonists nicotine (Nico) and levamisole (Leva) induced developmental delay in a concentration-dependent manner, mimicking the effect of clozapine. (C) Nicotine-induced developmental delay is suppressed by acr-7(tm863), and suppression was partially rescued by expression of full-length ACR-7 in the mutant background. (D) Nicotine receptor antagonists d-TC (100 and 500 µM) and DHβE (100 and 500 µM) suppressed clozapine (Cloz)-induced developmental delay. d-TC or DHβE alone did not affect the growth of the animals at 100 and 500 µM.
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pgen-1003313-g004: Genetic and pharmacological characterization of clozapine-induced larval arrest.(A) Clozapine induced developmental delay in wild-type C. elegans in a concentration-dependent manner. acr-7(tm863) partially suppressed this developmental delay, and the suppression was rescued by expression of ACR-7 in the mutant background. Expression was driven by a putative acr-7 promoter in the case of acr-7(tm863) mchEx40 or by the pharyngeal muscle-specific myo-2 promoter in the case of acr-7(tm863) mchEx207. Growth was measured as the percentage of different development stages 72 hours after loading synchronized L1 animals into the drug plates. Different concentrations (80, 160, 320 µM) of clozapine were dissolved in DMSO with the maximum concentration of DMSO being 0.1%. 0.1% DMSO alone was used for control wells. (B) nAChR agonists nicotine (Nico) and levamisole (Leva) induced developmental delay in a concentration-dependent manner, mimicking the effect of clozapine. (C) Nicotine-induced developmental delay is suppressed by acr-7(tm863), and suppression was partially rescued by expression of full-length ACR-7 in the mutant background. (D) Nicotine receptor antagonists d-TC (100 and 500 µM) and DHβE (100 and 500 µM) suppressed clozapine (Cloz)-induced developmental delay. d-TC or DHβE alone did not affect the growth of the animals at 100 and 500 µM.
Mentions: We tested growth effects over a range of clozapine concentrations (40–320 µM). N2 animals grown in the presence of 0.1% DMSO alone reached adulthood in ∼3 days (Figure 1B and Figure 4A). Consistent with our previous study [12], clozapine inhibited the development of N2 animals in a dose-dependent fashion. For example, 98% of control animals were gravid adults on day 3, compared to 36% on 80 µM clozapine and 0% on 320 µM clozapine (Figure 1B and Figure 4A).

Bottom Line: We validate the requirement for acr-7 by showing that acr-7 knockout suppresses clozapine-induced larval arrest and that expression of a full-length translational GFP fusion construct rescues this phenotype. nAChR agonists phenocopy the developmental effects of clozapine, while nAChR antagonists partially block these effects.ACR-7 is strongly expressed in the pharynx, and clozapine inhibits pharyngeal pumping. acr-7 knockout and nAChR antagonists suppress clozapine-induced inhibition of pharyngeal pumping.No APDs are known to activate nAChRs, but a number of studies indicate that α7-nAChR agonists may prove effective for the treatment of psychosis. α-like nAChR signaling is a mechanism through which clozapine may produce its therapeutic and/or toxic effects in humans, a hypothesis that could be tested following identification of the mammalian ortholog of C. elegans acr-7.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
We report a genome-wide RNA interference (RNAi) screen for Suppressors of Clozapine-induced Larval Arrest (scla genes) in Caenorhabditis elegans, the first genetic suppressor screen for antipsychotic drug (APD) targets in an animal. The screen identifies 40 suppressors, including the α-like nicotinic acetylcholine receptor (nAChR) homolog acr-7. We validate the requirement for acr-7 by showing that acr-7 knockout suppresses clozapine-induced larval arrest and that expression of a full-length translational GFP fusion construct rescues this phenotype. nAChR agonists phenocopy the developmental effects of clozapine, while nAChR antagonists partially block these effects. ACR-7 is strongly expressed in the pharynx, and clozapine inhibits pharyngeal pumping. acr-7 knockout and nAChR antagonists suppress clozapine-induced inhibition of pharyngeal pumping. These findings suggest that clozapine activates ACR-7 channels in pharyngeal muscle, leading to tetanus of pharyngeal muscle with consequent larval arrest. No APDs are known to activate nAChRs, but a number of studies indicate that α7-nAChR agonists may prove effective for the treatment of psychosis. α-like nAChR signaling is a mechanism through which clozapine may produce its therapeutic and/or toxic effects in humans, a hypothesis that could be tested following identification of the mammalian ortholog of C. elegans acr-7.

Show MeSH
Related in: MedlinePlus