Rakesh Karmacharya et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Pharmacogenomic Studies in C. elegans Used to Identify Novel Targets of Clozapine"

Rakesh Karmacharya, Bruce Cohen, Ned Buttner, Miriam Lundy

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

We used C. elegans for pharmacogenomic studies to identify possible targets for psychiatric drugs. Schizophrenia, a psychiatric disorder characterized by delusions, hallucinations and cognitive deficits, is treated with a variety of antipsychotic medications including haloperidol and clozapine. Clozapine stands out among the antipsychotic medications for its efficacy in treatment-refractory schizophrenia and the production of minimal extrapyramidal symptoms that are seen with haloperidol. The molecular basis of clozapine"s superior efficacy and its side effects are unknown. We tested a range of phenotypes in C. elegans in the presence and absence of these drugs. Our experiments identified behavioral and developmental effects that were specific for clozapine vis-à-vis haloperidol as well as effects shared by both drugs. We observed behavioral effects of these drugs on locomotion, egg-laying and pharyngeal pumping. We conducted additional experiments with mutants deficient in dopamine and serotonin biosynthesis. We found that the clozapine-specific effects did not require the existence of dopaminergic or serotonergic pathways. Mutants lacking octopamine partially rescued the suppression of pharyngeal pumping caused by both clozapine and haloperidol. This suggests that trace amine pathways may play a role in the effects of clozapine and haloperidol. We also observed a developmental phenotype of larval arrest in worms exposed to clozapine, but not to haloperidol. We identified mutants that suppressed clozapine-induced larval arrest, which point to novel interactions between clozapine and the insulin signaling pathway. Hence, we have identified a unique novel target for clozapine and another novel target shared by clozapine and haloperidol. These findings have implications for understanding the unique therapeutic and toxic effects of different antipsychotic medications.

Gregory Sliwoski et al. (2008) C.elegans Neuronal Development Meeting "Identification of Clozapine Effects on the Insulin Signaling Pathway in Caenorhabditis elegans"

Rakesh Karmacharya, Gregory Sliwoski, Edgar Buttner, Bruce Cohen

[C.elegans Neuronal Development Meeting, 2008]

Clozapine is an atypical antipsychotic drug that displays unique efficacy in treatment-refractory schizophrenia and that produces minimal extrapyramidal side effects. The molecular mechanisms underlying clozapine''s profile of therapeutic and toxic effects remain unknown. Taking a genetic approach to this problem, we have characterized a range of clozapine-induced developmental and behavioral phenotypes in C. elegans. Here we report studies of clozapine''s developmental phenotype, a complete arrest at the L1 and L2 larval stages. Larval arrest was also seen with the active clozapine metabolite N-desmethyl clozapine but was not observed with other atypical or typical antipsychotic drugs. Using a candidate gene approach, we discovered a role for the the insulin signaling pathway in clozapine-induced larval arrest. Loss-of-function mutations in age-1 produce a strong suppression of arrest, while daf-2(lf) and lin-14(lf) produce weaker but significant suppression. We found that the rate of pharyngeal pumping in clozapine-arrested larvae is severely reduced and that this reduction is suppressed in age-1(lf) mutants. Consistent with the notion that clozapine activates the insulin signaling pathway, we found that DAF-16::GFP is cytoplasmically localized in clozapine-arrested larvae. In addition, we found that clozapine exposure induces expression of AGE-1::GFP. Our findings demonstrate drug-specific interaction between clozapine and the insulin signaling pathway in C. elegans and hold implications for understanding the unique effects of clozapine in humans.

Pant A et al. (2016) Exp Gerontol "Corrigendum to "Beta-caryophyllene modulates expression of stress response genes and mediates longevity in Caenorhabditis elegans" [Exp. Gerontol. 57 (2014) 81-95]."

Saikia SK, Pant A, Shukla V, Asthana J, Pandey R, Akhoon BA

[Exp Gerontol, 2016]

The authors would like to acknowledge the contribution of Vikas Mishra for this article.Therefore the correct authorship of this paper at the time should have read as follows:Aakanksha Pant*, Vikas Mishra*, Shilpi K. Saikia, Virendra Shukla, Jyotsna Asthana, Bashir A. Akhoon, Rakesh Pandey.(* authors with equal contribution).The authors would like to apologize for any inconvenience caused.

Angelica Ortiz et al. (2008) C.elegans Neuronal Development Meeting "Identifying Pathways Modulated by Clozapine in Caenorhabditis elegans Behavior"

Sarah Demarco, Miriam Lundy, Edgar Buttner, Bruce Cohen, Rakesh Karmacharya, Angelica Ortiz

[C.elegans Neuronal Development Meeting, 2008]

The atypical antipsychotic clozapine effectively treats refractory schizophrenia. It also presents serious side-effects, such as agranulocytosis and metabolic syndrome. However, the molecular basis of this drug''s unique therapeutic profile remains unknown. Previously, we characterized clozapine-induced behavioral phenotypes in C. elegans and reported clozapine-specific effects in comparison to the typical antipsychotic haloperidol. The results showed that clozapine enhanced egg laying without the involvement of dopaminergic systems. We also observed that exposure to clozapine decreased the rate of pharyngeal pumping and the rate of locomotion in well-fed worms. We went on to test olanzapine, an atypical antipsychotic that has a structure similar to clozapine but that lacks clozapine''s unique efficacy in treating schizophrenia. Interestingly, we found that olanzapine failed to recapitulate clozapine''s effect on egg laying. By testing a panel of C. elegans mutants with defects in biogenic amine neurotransmitter systems, we aimed to identify novel pathways underlying clozapine-induced behaviors. We found that tdc-1, a gene required for tyramine synthesis, and ser-4, which encodes a serotonin-octopamine receptor, mediate the clozapine-induced increase in egg laying. Trace amine signaling also appears to be necessary for clozapine''s inhibition of pharyngeal pumping. Our observations suggest that the clozapine-induced decrease in pharyngeal pumping requires the presence of two distinct tyramine receptors: tyra-2, a G protein-coupled tyramine receptor, and sho-1, a tyramine-gated ion channel. We also found that tbh-1, a gene encoding tyramine beta-hydroxylase for octopamine synthesis, is needed for clozapine to decrease pharyngeal pumping rates. Clozapine''s inhibition of locomotion rate requires both the trace amine system and the biosynthesis of serotonin and dopamine. Interestingly, we found that worms lacking sho-1 did not demonstrate clozapine-induced reduction in locomotion rate. Similarly, mutations in bas-1, a gene encoding an aromatic amino acid decaboxylase necessary for serotonin and dopamine synthesis, did not show a change in locomotion rate after clozapine exposure. These results demonstrate that clozapine may be acting through a variety of pathway to produce its wide range of effects. Our observations may shed light on the molecular mechanisms underlying clozapine''s effects in human patients.

Harrington, Sean et al. (2017) International Worm Meeting "The discovery of neuroactive small molecule tools using a high-throughput egg laying screening pipeline."

Yip, Chris, Harrington, Sean, Roy, Peter, Giuliani, Maximiliano, Au, Aaron

[International Worm Meeting, 2017]

The neuromuscular circuit that governs C. elegans egg-laying is well understood and the majority of regulatory elements within it are highly conserved. The egg-laying system has been exploited previously to probe the polypharmacology of established neuromodulators such as clozapine (Karmacharya et al. (2011), Brain Res., 1393(1), 91-99) and imipramine (Weinshenker et al. (1999), J of Neurosci., 19(22), 9831-9840). Findings from these studies translated to a mouse model demonstrating the conserved nature of components of the egg-laying system. Here, we aim to identify novel small-molecule modulators of neuromuscular function by screening for those that modulate egg-laying behaviour in the worm. To do so, we have developed a high-throughput screening pipeline that takes advantage of the amenability of the egg-laying phenotype to computational counting. Using this screening pipeline, we have screened through 4642 molecules and identified 83 molecules that either stimulate or suppress egg laying behaviour at a concentration of 60 M or less. Many of these molecules have no reported relevant bioactivities in the literature. Several of our hits are bioactive in a vertebrate model (Danio rerio) suggesting the possibility of a conserved mechanism of action (Burns et al. (2015), Nature Comm., 6(1), doi:10.1038/ncomms8485). We are currently characterizing the mechanism of action of these molecules using four parallel approaches: 1) testing their interaction with a panel of human neuronal targets (NIH Psychoactive Drug Screening Project - Besnard et al. (2012), Nature, 492(7428), 215-220); 2) thermal proteome profiling using quantitative mass spectrometry to identify candidate targets in worms (Franken et al. (2015), Nature Protoc., 10(10), 1567-1593); 3) forward genetics screens to identify candidate targets (Kwok et al. (2006), Nature, 441(7089), 91-95); & 4) understanding their interaction with a panel of C. elegans egg laying mutants. We hope that our hits will become useful tools for future biological investigations, and that a subset might have therapeutic potential to modulate neuromuscular activity in more complex systems.

Wang, X. et al. (2011) International Worm Meeting "scla-1 Encodes a Nonreceptor Tyrosine Kinase Required for Antipsychotic Drug-induced Developmental Delay and Lethality in C. elegans."

Buttner, E. A., Wang, X., Cohen, B. M., Hao, L. M.

[International Worm Meeting, 2011]

Pharmacogenomic studies in model systems have the potential to define previously unknown mechanisms of action of medications used in human illness and to discover new genes and signaling pathways in the model system, itself. We initiated pharmacogenomic studies in C. elegans to identify novel signal transduction pathways through which antipsychotic drugs (APDs), and in particular, the most effective APD, clozapine, exert their biological effects. These drugs have both growth stimulating and toxic effects in humans. Many of these drugs cause larval arrest and lethality in C. elegans. We took advantage of these phenotypes by performing a genome-wide feeding RNAi screen for suppressors of clozapine-induced larval arrest (Scla). The screen yielded 42 suppressors, one of which is F22B3.8, a gene we have named scla-1. scla-1 shares 31% identity with the nonreceptor tyrosine kinase IL-2-inducible T cell kinase (ITK), which is critical for the development, function and differentiation of T cells. We validated our RNAi result by backcrossing two deletions of scla-1 six times and then confirming the Scla phenotype of each strain. In addition to suppression of clozapine-induced larval arrest, we found that these mutants suppress both developmental delay and lethality induced by other APDs, such as chlorpromazine and fluphenazine. We also found that clozapine induces expression of scla-1 as assayed by q-RT-PCR. We are currently generating transgenic worms carrying transcriptional and translational GFP reporters for scla-1. We will test our translational construct for rescue of the Scla phenotype of our two knockouts. Previous work by our laboratory1 and by Weeks et al.2 showed that APDs activate the insulin/IGF-1 signaling (IIS) in C. elegans. In mammals, activation of ITK depends on phosphatidylinositol-3-kinase3, which is a known target of the IIS effectors. Therefore, we are constructing an scla-1;daf-16::gfp animal to test the potential interaction of these pathways in C. elegans. The effects of APDs are likely determined by actions at multiple sites, and by defining the downstream interactions of pathways modulated by APDs, we may better understand their therapeutic and toxic effects. This knowledge may lead to improved treatments. 1. Karmacharya et al (2009) Neuropsychopharmacology 34, 1968-1978. 2. Weeks et al (2010) ACS Chem Neurosci 1, 463-473. 3. Fernandes et al (2009) MBC 20, 3690-3699.

Hao, L. et al. (2011) International Worm Meeting "Antipsychotic drug-induced developmental delay and lethality require the sphingomyelin synthase gene sms-1."

Buttner, E., Cohen, B., Hao, L.

[International Worm Meeting, 2011]

We use C. elegans for pharmacogenomic studies to identify novel genes underlying the mechanisms of action of antipsychotic drugs (APDs). Many APDs delay worm development or cause lethality in a dose-dependent manner, but clozapine, which is uniquely effective clinically, also has unique effects on growth and behavior in worms. A genome-wide RNAi screen for suppressors of clozapine-induced larval arrest (Scla) yielded 42 genes, including the sphingomyelin synthase gene sms-1. Sphingomyelin synthases are critical for cell growth and survival, since they regulate important signaling molecules via conversion of ceramide and phosphatidylcholine to sphingomyelin and diacylglycerol. We validated our RNAi result by outcrossing the sms-1 deletion ok2399 six times and then confirming the Scla phenotype. In addition, sms-1(ok2399) suppresses the developmental effects of other APDs, such as chlorpromazine and fluphenazine. In contrast, deletions of the sphingomyelin synthase genes sms-2 and sms-3 fail to produce the Scla phenotype. sms-1(ok2399) is also slow-growing and uncoordinated (Unc). Since sms-1 encodes six isoforms, we made three transcriptional and three translational GFP fusion constructs of these isoforms in order to generate transgenic animals for analysis of their gene expression patterns and protein localization. We will test our translational constructs for rescue of the Scla phenotype. We will also test a variety of mutants involved in ceramide and diacylglycerol metabolism and signaling for the Scla phenotype. sms-1(lf) is expected to increase ceramide levels, and in other systems ceramide has been shown to antagonize PKB/Akt activity. Previous work by our laboratory1 and by Weeks et al.2 showed that APDs activate the insulin signaling pathway in C. elegans. Therefore, we are constructing an sms-1;daf-16::gfp animal to test the potential interaction of these pathways. The clinical effects of APDs arise slowly, probably through modulation of multiple cell signaling pathways. While many of the direct and immediate effects of APDs have been identified, their downstream effects on signaling pathways have not been defined. Because many of the genes in these signaling pathways are conserved across species, our studies may illuminate new pathways by which APDs produce their effects in humans, leading to improved or novel treatments for psychotic disorders. References: 1.Karmacharya, R. et al. Clozapine interaction with phosphatidyl inositol 3-kinase (PI3K)/insulin-signaling pathway in Caenorhabditis elegans. Neuropsychopharmacol 34, 1968-1978 (2009). 2.Weeks, K.R. et al. Antipsychotic drugs acivate the C. elegans Akt pathway vis the DAF-2/Insulin/IGF-1 receptor. ACS Chem Neurosci 1, 463-473 (2010).