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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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A genome-wide RNAi screen for scla genes yielded the nAChR gene acr-7.(A) Experimental design of the feeding RNAi screen in liquid culture. On day 1, rrf-3 animals were synchronized, and bacteria was cultured from the Ahringer RNAi feeding library. Synchronized L1 animals were added to induced cultures on day 2 and were allowed to grow to adulthood for 3 days. Clozapine was added on day 5, and progeny were allowed to develop for 3 days. Progeny were scored for the Scla phenotype on day 8. (B1) N2 animals grew to adulthood within 3 days in the presence of 0.1% DMSO alone. (B2) 320 µM clozapine caused larval arrest in N2 animals exposed to feeding RNAi bacteria with empty vector alone. (B3) acr-7(RNAi) animals in 0.1% DMSO alone displayed normal development. (B4) acr-7(RNAi) suppressed developmental delay caused by 320 µM clozapine. (B5) The acr-7(tm863) knockout suppressed developmental delay caused by 320 µM clozapine. Note that experiments depicted in (B) were performed on NGM plates, not in liquid culture, to allow clear photographs.
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pgen-1003313-g001: A genome-wide RNAi screen for scla genes yielded the nAChR gene acr-7.(A) Experimental design of the feeding RNAi screen in liquid culture. On day 1, rrf-3 animals were synchronized, and bacteria was cultured from the Ahringer RNAi feeding library. Synchronized L1 animals were added to induced cultures on day 2 and were allowed to grow to adulthood for 3 days. Clozapine was added on day 5, and progeny were allowed to develop for 3 days. Progeny were scored for the Scla phenotype on day 8. (B1) N2 animals grew to adulthood within 3 days in the presence of 0.1% DMSO alone. (B2) 320 µM clozapine caused larval arrest in N2 animals exposed to feeding RNAi bacteria with empty vector alone. (B3) acr-7(RNAi) animals in 0.1% DMSO alone displayed normal development. (B4) acr-7(RNAi) suppressed developmental delay caused by 320 µM clozapine. (B5) The acr-7(tm863) knockout suppressed developmental delay caused by 320 µM clozapine. Note that experiments depicted in (B) were performed on NGM plates, not in liquid culture, to allow clear photographs.

Mentions: To identify novel molecular mechanisms of action of APDs, we conducted a genetic suppressor screen for APD targets in an animal [18]. APDs cause larval arrest or developmental delay in C. elegans (Figure 1B). APDs inhibit pharyngeal pumping [12], [19], [20], indicating that the developmental phenotype has a neuromuscular basis. To identify potential APD targets, we performed a genome-wide RNAi screen for Suppressors of Clozapine-induced Larval Arrest (scla genes). Mutants that escaped clozapine-induced larval arrest grew to adulthood and were easily detected under a dissecting microscope (Figure 1B, [21]). The experimental design is outlined in Figure 1A and involved a primary RNAi screen in liquid culture followed by triplicate testing in agar wells. To ensure adequate knockdown of potential targets, we exposed animals to two rounds of RNAi and tested progeny in the second generation. We also employed the NL4256 rrf-3(pk1426) II strain, which is hypersensitive to RNAi and which improves detection of genes with postembryonic mutant phenotypes, to maximize recovery of neuronal genes [22].


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)

A genome-wide RNAi screen for scla genes yielded the nAChR gene acr-7.(A) Experimental design of the feeding RNAi screen in liquid culture. On day 1, rrf-3 animals were synchronized, and bacteria was cultured from the Ahringer RNAi feeding library. Synchronized L1 animals were added to induced cultures on day 2 and were allowed to grow to adulthood for 3 days. Clozapine was added on day 5, and progeny were allowed to develop for 3 days. Progeny were scored for the Scla phenotype on day 8. (B1) N2 animals grew to adulthood within 3 days in the presence of 0.1% DMSO alone. (B2) 320 µM clozapine caused larval arrest in N2 animals exposed to feeding RNAi bacteria with empty vector alone. (B3) acr-7(RNAi) animals in 0.1% DMSO alone displayed normal development. (B4) acr-7(RNAi) suppressed developmental delay caused by 320 µM clozapine. (B5) The acr-7(tm863) knockout suppressed developmental delay caused by 320 µM clozapine. Note that experiments depicted in (B) were performed on NGM plates, not in liquid culture, to allow clear photographs.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003313-g001: A genome-wide RNAi screen for scla genes yielded the nAChR gene acr-7.(A) Experimental design of the feeding RNAi screen in liquid culture. On day 1, rrf-3 animals were synchronized, and bacteria was cultured from the Ahringer RNAi feeding library. Synchronized L1 animals were added to induced cultures on day 2 and were allowed to grow to adulthood for 3 days. Clozapine was added on day 5, and progeny were allowed to develop for 3 days. Progeny were scored for the Scla phenotype on day 8. (B1) N2 animals grew to adulthood within 3 days in the presence of 0.1% DMSO alone. (B2) 320 µM clozapine caused larval arrest in N2 animals exposed to feeding RNAi bacteria with empty vector alone. (B3) acr-7(RNAi) animals in 0.1% DMSO alone displayed normal development. (B4) acr-7(RNAi) suppressed developmental delay caused by 320 µM clozapine. (B5) The acr-7(tm863) knockout suppressed developmental delay caused by 320 µM clozapine. Note that experiments depicted in (B) were performed on NGM plates, not in liquid culture, to allow clear photographs.
Mentions: To identify novel molecular mechanisms of action of APDs, we conducted a genetic suppressor screen for APD targets in an animal [18]. APDs cause larval arrest or developmental delay in C. elegans (Figure 1B). APDs inhibit pharyngeal pumping [12], [19], [20], indicating that the developmental phenotype has a neuromuscular basis. To identify potential APD targets, we performed a genome-wide RNAi screen for Suppressors of Clozapine-induced Larval Arrest (scla genes). Mutants that escaped clozapine-induced larval arrest grew to adulthood and were easily detected under a dissecting microscope (Figure 1B, [21]). The experimental design is outlined in Figure 1A and involved a primary RNAi screen in liquid culture followed by triplicate testing in agar wells. To ensure adequate knockdown of potential targets, we exposed animals to two rounds of RNAi and tested progeny in the second generation. We also employed the NL4256 rrf-3(pk1426) II strain, which is hypersensitive to RNAi and which improves detection of genes with postembryonic mutant phenotypes, to maximize recovery of neuronal genes [22].

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