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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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ACR-7 protein sequence alignment and acr-7 gene structure.(A) The C. elegans ACR-7 protein and human α7-nAChR subunit CHRNA7 were aligned using ClustalW. The four putative transmembrane domains, as predicted for the human CHRNA7 protein, were boxed. nAChRs are members of the cys-loop family of ionotropic neurotransmitter receptors, and this conserved di-cysteine loop is dotted line-boxed. α-nAChR subunits contain a pair of vicinal cysteines within the Ach binding site, and these residues are marked with asterisks. The C. elegans and human proteins share 36% identity overall and 41% identity within the pore-lining transmembrane domain II (M2). (B) The acr-7 gene contains 13 exons, and the region deleted in the tm863 allele is marked with a line. tm863 is an out-of-frame deletion which removes exons 6–7 and most of exons 5 and 8. (C) The tm863 knockout is predicted to lack all four transmembrane domains and to be a  allele.
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pgen-1003313-g003: ACR-7 protein sequence alignment and acr-7 gene structure.(A) The C. elegans ACR-7 protein and human α7-nAChR subunit CHRNA7 were aligned using ClustalW. The four putative transmembrane domains, as predicted for the human CHRNA7 protein, were boxed. nAChRs are members of the cys-loop family of ionotropic neurotransmitter receptors, and this conserved di-cysteine loop is dotted line-boxed. α-nAChR subunits contain a pair of vicinal cysteines within the Ach binding site, and these residues are marked with asterisks. The C. elegans and human proteins share 36% identity overall and 41% identity within the pore-lining transmembrane domain II (M2). (B) The acr-7 gene contains 13 exons, and the region deleted in the tm863 allele is marked with a line. tm863 is an out-of-frame deletion which removes exons 6–7 and most of exons 5 and 8. (C) The tm863 knockout is predicted to lack all four transmembrane domains and to be a allele.

Mentions: We sought to understand the molecular mechanisms of action of the RNAi suppressors in detail and chose to focus initially on acr-7. The rationale for focusing on acr-7, as opposed to other new candidate APD targets, included potential relevance to psychotic disorders [15]–[17], probability of a human ortholog [28], [29], and potential for physiological characterization [30]. Blasting the ACR-7 protein sequence against the human genome using Wormbase BLAT revealed that ACR-7 is homologous to a number of human α-like nAChRs, but which human gene is the true acr-7 ortholog is not known. Homology comparisons with the human α7-nAChR showed 36% identity overall and greater identity within the first two predicted transmembrane domains of the ion channel (Figure 3A). The pore-lining transmembrane domain II (M2) and neighboring residues play an important role in the ion selectivity filter of nAChRs. Specifically, substitution of the glutamate residue immediately preceding M2 or the glutamate residue at the extracellular mouth of the pore significantly reduces the calcium permeability of various nAChR subunits [31]. We noted that both glutamate residues are conserved in ACR-7, suggesting that, like the human α7-nAChR, ACR-7 is highly permeable to calcium. Our attempts to obtain voltage-clamp recordings of ACR-7 expressed in heterologous systems, including Xenopus laevis oocytes and cultured HEK293T and PC12 cells, failed, consistent with the possibility that ACR-7 is a subunit of a heteromeric channel [32], [33].


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)

ACR-7 protein sequence alignment and acr-7 gene structure.(A) The C. elegans ACR-7 protein and human α7-nAChR subunit CHRNA7 were aligned using ClustalW. The four putative transmembrane domains, as predicted for the human CHRNA7 protein, were boxed. nAChRs are members of the cys-loop family of ionotropic neurotransmitter receptors, and this conserved di-cysteine loop is dotted line-boxed. α-nAChR subunits contain a pair of vicinal cysteines within the Ach binding site, and these residues are marked with asterisks. The C. elegans and human proteins share 36% identity overall and 41% identity within the pore-lining transmembrane domain II (M2). (B) The acr-7 gene contains 13 exons, and the region deleted in the tm863 allele is marked with a line. tm863 is an out-of-frame deletion which removes exons 6–7 and most of exons 5 and 8. (C) The tm863 knockout is predicted to lack all four transmembrane domains and to be a  allele.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585123&req=5

pgen-1003313-g003: ACR-7 protein sequence alignment and acr-7 gene structure.(A) The C. elegans ACR-7 protein and human α7-nAChR subunit CHRNA7 were aligned using ClustalW. The four putative transmembrane domains, as predicted for the human CHRNA7 protein, were boxed. nAChRs are members of the cys-loop family of ionotropic neurotransmitter receptors, and this conserved di-cysteine loop is dotted line-boxed. α-nAChR subunits contain a pair of vicinal cysteines within the Ach binding site, and these residues are marked with asterisks. The C. elegans and human proteins share 36% identity overall and 41% identity within the pore-lining transmembrane domain II (M2). (B) The acr-7 gene contains 13 exons, and the region deleted in the tm863 allele is marked with a line. tm863 is an out-of-frame deletion which removes exons 6–7 and most of exons 5 and 8. (C) The tm863 knockout is predicted to lack all four transmembrane domains and to be a allele.
Mentions: We sought to understand the molecular mechanisms of action of the RNAi suppressors in detail and chose to focus initially on acr-7. The rationale for focusing on acr-7, as opposed to other new candidate APD targets, included potential relevance to psychotic disorders [15]–[17], probability of a human ortholog [28], [29], and potential for physiological characterization [30]. Blasting the ACR-7 protein sequence against the human genome using Wormbase BLAT revealed that ACR-7 is homologous to a number of human α-like nAChRs, but which human gene is the true acr-7 ortholog is not known. Homology comparisons with the human α7-nAChR showed 36% identity overall and greater identity within the first two predicted transmembrane domains of the ion channel (Figure 3A). The pore-lining transmembrane domain II (M2) and neighboring residues play an important role in the ion selectivity filter of nAChRs. Specifically, substitution of the glutamate residue immediately preceding M2 or the glutamate residue at the extracellular mouth of the pore significantly reduces the calcium permeability of various nAChR subunits [31]. We noted that both glutamate residues are conserved in ACR-7, suggesting that, like the human α7-nAChR, ACR-7 is highly permeable to calcium. Our attempts to obtain voltage-clamp recordings of ACR-7 expressed in heterologous systems, including Xenopus laevis oocytes and cultured HEK293T and PC12 cells, failed, consistent with the possibility that ACR-7 is a subunit of a heteromeric channel [32], [33].

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