Clozapine Interaction with Phosphatidyl Inositol 3-Kinase (PI3K)/Insulin-Signaling Pathway in Caenorhabditis elegans

Abstract

Clozapine has superior and unique effects as an antipsychotic agent, but the mediators of these effects are not known. We studied behavioral and developmental effects of clozapine in Caenorhabditis elegans, as a model system to identify previously undiscovered mechanisms of drug action. Clozapine induced early larval arrest, a phenotype that was also seen with the clozapine metabolite N-desmethyl clozapine but not with any other typical or atypical antipsychotic drug tested. Mutations in the insulin receptor/daf-2 and the phosphatidyl inositol 3-kinase (PI3K)/age-1 suppressed clozapine-induced larval arrest, suggesting that clozapine may activate the insulin signaling pathway. Consistent with this notion, clozapine also increased expression of an age-1::GFP reporter. Activation of the insulin signaling pathway leads to cytoplasmic localization of the fork head transcription factor FOXO/daf-16. Clozapine produced cytoplasmic localization of DAF-16::GFP in arrested L1 larvae, in contrast to stressors such as starvation or high temperature which produce nuclear localization of DAF-16::GFP in arrested L1 larvae. Clozapine also inhibited pharyngeal pumping in C. elegans, an effect that may contribute to but did not explain clozapine-induced larval arrest. Our findings demonstrate a drug-specific interaction between clozapine and the PI3K/insulin signaling pathway in C. elegans. As this pathway is conserved across species, the results may have implications for understanding the unique effects of clozapine in humans.

Antipsychotic Drugs Activate the C. elegans Akt Pathway via the DAF-2 Insulin/IGF-1 Receptor

“…Interestingly, clozapine, but not olanzapine or trifluoperazine, required both AKT-1 and Figure 6B and C). Clozapine was recently reported to activate PI3K in C. elegans (44), which is consistent with our results. We previously showed that antipsychotic drugs increase serotonin production in C. elegans (24), and drug-induced cytoplasmic retention of DAF-16::GFP was modestly reduced in a serotonin-absent tph-1 mutant ( Figure 6D).…”

Clozapine Modulates Glucosylceramide, Clears Aggregated Proteins, and Enhances ATG8/LC3 in Caenorhabditis elegans

“…To address differences among worm strains, we adjusted concentrations of the drug, for example, for some clozapine-sensitive mutants, the drug concentrations were lowered. These procedures follow observations published in previous manuscripts (Karmacharya et al, 2009(Karmacharya et al, , 2011.…”

“…It also alters worm behavior, including decreasing the rate of pharyngeal pumping, inducing faster egg laying, and increasing the speed of locomotion (Donohoe et al, 2006;Karmacharya et al, 2011). The insulin/PKT pathway (which is altered in schizophrenia) has a role in clozapine-induced development delay/lethality, but it does not fully explain the effects of clozapine (Karmacharya et al, 2009). Clozapine is also involved in modulating serotonin-controlled behavior (Donohoe et al, 2009).…”

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in <em>Caenorhabditis elegans</em>

“…APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping 1,2 . Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics.…”

“…Thus, C. elegans neurogenetics and neurobiology offer methods for discovering novel molecular mechanisms of action of APDs. In nematodes, APD exposure early in development produces developmental delay, and at higher concentrations, lethality 2,6 . APD exposure during adulthood produces behavioral phenotypes.…”