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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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Suppression by acr-7(lf) is specific.A series of 22 nAChR mutants were tested for suppression of clozapine-induced larval arrest, and only acr-7(lf) produced robust suppression.
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pgen-1003313-g005: Suppression by acr-7(lf) is specific.A series of 22 nAChR mutants were tested for suppression of clozapine-induced larval arrest, and only acr-7(lf) produced robust suppression.

Mentions: The acr-7(tm863) allele is a 625 bp out-of-frame deletion with a 7 bp insertion, which removes exons 6–7 and most of exons 5 and 8 (Figure 3B) and lacks all four transmembrane domains (Figure 3C). Therefore, acr-7(tm863) is predicted to be a allele. We backcrossed the acr-7(tm863) allele to the wild-type strain six times to generate the strain EAB200, which, like animals exposed to acr-7(RNAi) (Figure 1B), displayed normal development and normal adult morphology. On 320 µM clozapine, 95% of acr-7(tm863) mutant animals reached the L4 and young adult (YA) stages by day 3, compared to 0% of N2 animals (Figure 1B and Figure 4A). Therefore, acr-7(tm863) partially suppressed clozapine-induced developmental delay, confirming the knockdown results from our RNAi screen. Transgenic strain acr-7(tm863) II mchEx40, which expresses a full-length translational Pacr-7::acr-7::GFP (Green Fluorescent Protein) fusion construct, was rescued for the Scla phenotype. On 320 µM clozapine, 100% of these transgenic animals were arrested at early larval stages (L1, L2, and L3) on day 3, comparable to the delay seen in N2 animals (Figure 4A). We investigated the specificity of acr-7(lf) suppression by testing mutations in 21 other C. elegans nAChR subunits and found that only acr-7(lf) suppressed clozapine-induced developmental delay (Figure 5).


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)

Suppression by acr-7(lf) is specific.A series of 22 nAChR mutants were tested for suppression of clozapine-induced larval arrest, and only acr-7(lf) produced robust suppression.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585123&req=5

pgen-1003313-g005: Suppression by acr-7(lf) is specific.A series of 22 nAChR mutants were tested for suppression of clozapine-induced larval arrest, and only acr-7(lf) produced robust suppression.
Mentions: The acr-7(tm863) allele is a 625 bp out-of-frame deletion with a 7 bp insertion, which removes exons 6–7 and most of exons 5 and 8 (Figure 3B) and lacks all four transmembrane domains (Figure 3C). Therefore, acr-7(tm863) is predicted to be a allele. We backcrossed the acr-7(tm863) allele to the wild-type strain six times to generate the strain EAB200, which, like animals exposed to acr-7(RNAi) (Figure 1B), displayed normal development and normal adult morphology. On 320 µM clozapine, 95% of acr-7(tm863) mutant animals reached the L4 and young adult (YA) stages by day 3, compared to 0% of N2 animals (Figure 1B and Figure 4A). Therefore, acr-7(tm863) partially suppressed clozapine-induced developmental delay, confirming the knockdown results from our RNAi screen. Transgenic strain acr-7(tm863) II mchEx40, which expresses a full-length translational Pacr-7::acr-7::GFP (Green Fluorescent Protein) fusion construct, was rescued for the Scla phenotype. On 320 µM clozapine, 100% of these transgenic animals were arrested at early larval stages (L1, L2, and L3) on day 3, comparable to the delay seen in N2 animals (Figure 4A). We investigated the specificity of acr-7(lf) suppression by testing mutations in 21 other C. elegans nAChR subunits and found that only acr-7(lf) suppressed clozapine-induced developmental delay (Figure 5).

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