Novel pharmacological modulation of dystonic phenotypes caused by a gain-of-function mutation in the Na+ leak-current channel.

The Na leak-current channel (NALCN) regulates the resting membrane potential in excitable cells, thus determining the likelihood of depolarization in response to incoming signals. Gain-of-function (gf) mutations in this channel are associated with severe dystonic movement disorders in man. Currently, there are no known pharmacological antagonists or selective modulators of this important channel. A gain-of-function mutation in NALCN of C. elegans [known as unc-77(e625)] causes uncoordinated, hyperactive locomotion. We hypothesized that this hyperactive phenotype can be rescued with pharmacological modulators. Here, we summarize the results of targeted drug screening aimed at identification of drugs that corrected locomotion deficits in unc-77(e625) animals. To assay hyperactive locomotion, animals were acutely removed from food and characteristic foraging movements were quantified. Drug screening revealed that 2-aminoethoxydiphenyl borate (2-ABP), nifedipine, nimodipine, flunarizine and ethoxzolamide significantly decreased abnormal movements in unc-77(e625) animals. 2-APB also corrected egg release and coiling deficits in this strain. In addition, serotonin and dopamine both reduced hyperactive locomotion, consistent with regulatory interactions between these systems and the NALCN. 2-APB induced movement phenotypes in wild-type animals that faithfully mimicked those observed in NALCN knockout strains, which suggested that this drug may directly block the channel. Moreover, 2-APB and flunarizine showed significant structural similarities suggestive of overlap in their mode of action. Together, these studies have revealed new insights into regulation of NALCN function and led to the discovery of a potential pharmacological antagonist of the NALCN.

Akinesia and freezing caused by Na+ leak-current channel (NALCN) deficiency corrected by pharmacological inhibition of K+ channels and gap junctions.

The Na+ leak-current channel (NALCN) regulates locomotion, respiration, and intellectual development. Previous work highlighted striking similarities between characteristic movement phenotypes of NALCN-deficient animals (Drosophila and Caenorhabditis elegans) and the major symptoms of Parkinson's disease and primary progressive freezing gait. We have discovered novel physiological connections between the NALCN, K+ channels, and gap junctions that mediate regulation of locomotion in C. elegans. Drugs that block K+ channels and gap junctions or that activate Ca++ channels significantly improve movement of NALCN-deficient animals. Loss-of-function of the NALCN creates an imbalance in ions, including K+ and Ca++ , that interferes with normal cycles of depolarization-repolarization. This work suggests new therapeutic strategies for certain human movement disorders. J. Comp. Neurol. 525:1109-1121, 2017. © 2016 Wiley Periodicals, Inc.

Social feeding in Caenorhabditis elegans is modulated by antipsychotic drugs and calmodulin and may serve as a protophenotype for asociality.

Here, we define a protophenotype as an endophenotype that has been conserved during evolution. Social feeding in Caenorhabditis elegans may be an example of a protophenotype related to asociality in schizophrenia. It is regulated by the highly conserved neuropeptide Y receptor, NPR-1, and we speculated that social feeding should be affected by antipsychotic drugs. The social feeding strain, npr-1(g320), was exposed to antipsychotic drugs, dopamine or calmodulin antagonists on plates with bacterial lawns, and the number of aggregates on the plates was counted as a measure of social feeding. First-generation antipsychotics, chlorpromazine, trifluoperazine, fluphenazine, and haloperidol, and the second-generation drug, olanzapine, inhibited social feeding. Dopamine accelerated aggregation, whereas selective D2 dopamine receptor antagonists, sulpiride and raclopride, were inhibitory. Calmodulin antagonists effectively inhibited social feeding, as did RNAi knockdown of calmodulin (cmd-1) expression. In addition, gap junction inhibitors prevented aggregation, which is consistent with the hub-and-spoke arrangement of neurons that regulate social feeding via functional gap junctions. The studies described here revealed novel connections between dopaminergic signaling, the NPY receptor, calmodulin, and gap junctions in the regulation of social behavior in C. elegans. These pathways are evolutionarily-conserved, and have also been implicated in the pathogenesis of schizophrenia.

Insulin/IGF-1 signaling, including class II/III PI3Ks, ß-arrestin and SGK-1, is required in C. elegans to maintain pharyngeal muscle performance during starvation.

In C. elegans, pharyngeal pumping is regulated by the presence of bacteria. In response to food deprivation, the pumping rate rapidly declines by about 50-60%, but then recovers gradually to baseline levels on food after 24 hr. We used this system to study the role of insulin/IGF-1 signaling (IIS) in the recovery of pharyngeal pumping during starvation. Mutant strains with reduced function in the insulin/IGF-1 receptor, DAF-2, various insulins (INS-1 and INS-18), and molecules that regulate insulin release (UNC-64 and NCA-1; NCA-2) failed to recover normal pumping rates after food deprivation. Similarly, reduction or loss of function in downstream signaling molecules (e.g., ARR-1, AKT-1, and SGK-1) and effectors (e.g., CCA-1 and UNC-68) impaired pumping recovery. Pharmacological studies with kinase and metabolic inhibitors implicated class II/III phosphatidylinositol 3-kinases (PI3Ks) and glucose metabolism in the recovery response. Interestingly, both over- and under-activity in IIS was associated with poorer recovery kinetics. Taken together, the data suggest that optimum levels of IIS are required to maintain high levels of pharyngeal pumping during starvation. This work may ultimately provide insights into the connections between IIS, nutritional status and sarcopenia, a hallmark feature of aging in muscle.

Clozapine and lithium require Caenorhabditis elegans ß-arrestin and serum- and glucocorticoid-inducible kinase to affect Daf-16 (FOXO) localization.

Numerous studies have implicated low levels of signaling in the Akt network with psychotic illnesses, and a growing body of literature has shown that all classes of antipsychotic drugs increase Akt signaling. The most clinically effective antipsychotic drug is clozapine. With Caenorhabditis elegans as a model system, this study demonstrates that clozapine is unique among antipsychotic drugs because it requires ß-arrestin and serum and glucocorticoid-inducible kinase (SGK) in addition to Akt to suppress the nuclear localization of DAF-16 (Forkhead box O [FOXO]). Lithium, a mood stabilizer often used to treat psychosis, also requires ß-arrestin and SGK to suppress the nuclear localization of DAF-16.

A C. elegans eIF4E-family member upregulates translation at elevated temperatures of mRNAs encoding MSH-5 and other meiotic crossover proteins.

Caenorhabditis elegans expresses five family members of the translation initiation factor eIF4E whose individual physiological roles are only partially understood. We report a specific role for IFE-2 in a conserved temperature-sensitive meiotic process. ife-2 deletion mutants have severe temperature-sensitive chromosome-segregation defects. Mutant germ cells contain the normal six bivalents at diakinesis at 20 degrees C but 12 univalents at 25 degrees C, indicating a defect in crossover formation. Analysis of chromosome pairing in ife-2 mutants at the permissive and restrictive temperatures reveals no defects. The presence of RAD-51-marked early recombination intermediates and 12 well condensed univalents indicate that IFE-2 is not essential for formation of meiotic double-strand breaks or their repair through homologous recombination but is required for crossover formation. However, RAD-51 foci in ife-2 mutants persist into inappropriately late stages of meiotic prophase at 25 degrees C, similar to mutants defective in MSH-4/HIM-14 and MSH-5, which stabilize a critical intermediate in crossover formation. In wild-type worms, mRNAs for msh-4/him-14 and msh-5 shift from free messenger ribonucleoproteins to polysomes at 25 degrees C but not in ife-2 mutants, suggesting that IFE-2 translationally upregulates synthesis of MSH-4/HIM-14 and MSH-5 at elevated temperatures to stabilize Holliday junctions. This is confirmed by an IFE-2-dependent increase in MSH-5 protein levels.

Antipsychotic drugs activate the C. elegans akt pathway via the DAF-2 insulin/IGF-1 receptor.

The molecular modes of action of antipsychotic drugs are poorly understood beyond their effects at the dopamine D2 receptor. Previous studies have placed Akt signaling downstream of D2 dopamine receptors, and recent data have suggested an association between psychotic illnesses and defective Akt signaling. To characterize the effect of antipsychotic drugs on the Akt pathway, we used the model organism C. elegans, a simple system where the Akt/forkhead box O transcription factor (FOXO) pathway has been well characterized. All major classes of antipsychotic drugs increased signaling through the insulin/Akt/FOXO pathway, whereas four other drugs that are known to affect the central nervous system did not. The antipsychotic drugs inhibited dauer formation, dauer recovery, and shortened lifespan, three biological processes affected by Akt signaling. Genetic analysis showed that AKT-1 and the insulin and insulin-like growth factor receptor, DAF-2, were required for the antipsychotic drugs to increase signaling. Serotonin synthesis was partially involved, whereas the mitogen activated protein kinase (MAPK), SEK-1 is a MAP kinase kinase (MAPKK), and calcineurin were not involved. This is the first example of a common but specific molecular effect produced by all presently known antipsychotic drugs in any biological system. Because untreated schizophrenics have been reported to have low levels of Akt signaling, increased Akt signaling might contribute to the therapeutic actions of antipsychotic drugs.

Behavioral adaptation in C. elegans produced by antipsychotic drugs requires serotonin and is associated with calcium signaling and calcineurin inhibition.

Chronic administration of antipsychotic drugs produces adaptive responses at the cellular and molecular levels that may be responsible for both the main therapeutic effects and rebound psychosis, which is often observed upon discontinuation of these drugs. Here we show that some antipsychotic drugs produce significant functional changes in serotonergic neurons that directly impact feeding behavior in the model organism, Caenorhabditis elegans. In particular, antipsychotic drugs acutely suppress pharyngeal pumping, which is regulated by serotonin from the NSM neurons. By contrast, withdrawal from food and drug is accompanied by a striking recovery and overshoot in the rate of pharyngeal pumping. This rebound response is absent or diminished in mutant strains that lack tryptophan hydroxylase (TPH-1) or the serotonin receptors SER-7 and SER-1, and is blocked by serotonin antagonists, which implicates serotonergic mechanisms in this adaptive response. Consistent with this, continuous drug exposure stimulates an increase in serotonin and the number of varicosities along the NSM processes. Cyclosporin A and calcineurin mutant strains mimic the effects of the antipsychotic drugs and reveal a potential role for the calmodulin-calcineurin signaling pathway in the response of serotonergic neurons. Similar molecular and cellular changes may contribute to the long-term adaptive response to antipsychotic drugs in patients.

Antipsychotic drugs up-regulate tryptophan hydroxylase in ADF neurons of Caenorhabditis elegans: role of calcium-calmodulin-dependent protein kinase II and transient receptor potential vanilloid channel.

Antipsychotic drugs produce acute behavioral effects through antagonism of dopamine and serotonin receptors, and long-term adaptive responses that are not well understood. The goal of the study presented here was to use Caenorhabditis elegans to investigate the molecular mechanism or mechanisms that contribute to adaptive responses produced by antipsychotic drugs. First-generation antipsychotics, trifluoperazine and fluphenazine, and second-generation drugs, clozapine and olanzapine, increased the expression of tryptophan hydroxylase-1::green fluorescent protein (TPH-1::GFP) and serotonin in the ADF neurons of C. elegans. This response was absent or diminished in mutant strains lacking the transient receptor potential vanilloid channel (TRPV; osm-9) or calcium/calmodulin-dependent protein kinase II (CaMKII; unc-43). The role of calcium signaling was further implicated by the finding that a selective antagonist of calmodulin and a calcineurin inhibitor also enhanced TPH-1::GFP expression. The ADF neurons modulate foraging behavior (turns/reversals off food) through serotonin production. We found that short-term exposure to the antipsychotic drugs altered the frequency of turns/reversals off food. This response was mediated through dopamine and serotonin receptors and was abolished in serotonin-deficient mutants (tph-1) and strains lacking the SER-1 and MOD-1 serotonin receptors. Consistent with the increase in serotonin in the ADF neurons induced by the drugs, drug withdrawal after 24-hr treatment was accompanied by a rebound in the number of turns/reversals, which demonstrates behavioral adaptation in serotonergic systems. Characterization of the cellular, molecular, and behavioral adaptations to continuous exposure to antipsychotic drugs may provide insight into the long-term clinical effects of these medications.

Antipsychotic drugs alter neuronal development including ALM neuroblast migration and PLM axonal outgrowth in Caenorhabditis elegans.

Antipsychotic drugs are increasingly being prescribed for children and adolescents, and are used in pregnant women without a clear demonstration of safety in these populations. Global effects of these drugs on neurodevelopment (e.g., decreased brain size) have been reported in rats, but detailed knowledge about neuronal effects and mechanisms of action are lacking. Here we report on the evaluation of a comprehensive panel of antipsychotic drugs in a model organism (Caenorhabditis elegans) that is widely used to study neuronal development. Specifically, we examined the effects of the drugs on neuronal migration and axonal outgrowth in mechanosensory neurons visualized with green fluorescent protein expressed from the mec-3 promoter. Clozapine, fluphenazine, and haloperidol produced deficits in the development and migration of ALM neurons and axonal outgrowth in PLM neurons. The defects included failure of neuroblasts to migrate to the proper location, and excessive growth of axons past their normal termination point, together with abnormal morphological features of the processes. Although the antipsychotic drugs are potent antagonists of dopamine and serotonin receptors, the neurodevelopmental deficits were not rescued by co-incubation with serotonin or the dopaminergic agonist, quinpirole. Other antipsychotic drugs, risperidone, aripiprazole, quetiapine, trifluoperazine and olanzapine, also produced modest, but detectable, effects on neuronal development. This is the first report that antipsychotic drugs interfere with neuronal migration and axonal outgrowth in a developing nervous system.

Drug discovery based on genetic and metabolic findings in schizophrenia.

Recent progress in the genetics of schizophrenia provides the rationale for re-evaluating causative factors and therapeutic strategies for this disease. Here, we review the major candidate susceptibility genes and relate the aberrant function of these genes to defective regulation of energy metabolism in the schizophrenic brain. Disturbances in energy metabolism potentially lead to neurodevelopmental deficits, impaired function of the mature nervous system and failure to maintain neurites/dendrites and synaptic connections. Current antipsychotic drugs do not specifically address these underlying deficits; therefore, a new generation of more effective medications is urgently needed. Novel targets for future drug discovery are identified in this review. The coordinated application of structure-based drug design, systems biology and research on model organisms may greatly facilitate the search for next-generation antipsychotic drugs.

Antipsychotic drugs disrupt normal development in Caenorhabditis elegans via additional mechanisms besides dopamine and serotonin receptors.

Antipsychotic drugs may produce adverse effects during development in humans and rodents. However, the extent of these effects has not been systematically characterized nor have molecular mechanisms been identified. Consequently, we sought to evaluate the effects of an extensive panel of antipsychotic drugs in a model organism, Caenorhabditis elegans, whose development is well characterized and which offers the possibility of identifying novel molecular targets. For these studies, animals were grown from hatching in the presence of vehicle (control) or antipsychotic drugs over a range of concentrations (20-160microM) and growth was analyzed by measuring head-to-tail length at various intervals. First-generation antipsychotics (e.g., fluphenazine) generally slowed growth and maturation more than second-generation drugs such as quetiapine and olanzapine. This is consistent with in vitro effects on human neuronal cell lines. Clozapine, a second-generation drug, produced similar growth deficits as haloperidol. Converging lines of evidence, including the failure to rescue growth with high concentrations of agonists, suggested that the drug-induced delay in development was not mediated by the major neurotransmitter receptors recognized by the antipsychotic drugs. Moreover, in serotonin-deficient tph-1 mutants, the drugs dramatically slowed development and led to larval arrest (including dauer formation) and neuronal abnormalities. Evaluation of alternative targets of the antipsychotics revealed a potential role for calmodulin and underscored the significance of Ca(2+)-calmodulin signaling in development. These findings suggest that antipsychotic drugs may interfere with normal developmental processes and provide a tool for investigating the key signaling pathways involved.

Translation of a small subset of Caenorhabditis elegans mRNAs is dependent on a specific eukaryotic translation initiation factor 4E isoform.

The mRNA cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) participates in protein synthesis initiation, translational repression of specific mRNAs, and nucleocytoplasmic shuttling. Multiple isoforms of eIF4E are expressed in a variety of organisms, but their specific roles are poorly understood. We investigated one Caenorhabditis elegans isoform, IFE-4, which has homologues in plants and mammals. IFE-4::green fluorescent protein (GFP) was expressed in pharyngeal and tail neurons, body wall muscle, spermatheca, and vulva. Knockout of ife-4 by RNA interference (RNAi) or a null mutation produced a pleiotropic phenotype that included egg-laying defects. Sedimentation analysis demonstrated that IFE-4, but not IFE-1, was present in 48S initiation complexes, indicating that it participates in protein synthesis initiation. mRNAs affected by ife-4 knockout were determined by DNA microarray analysis of polysomal distribution. Polysome shifts, in the absence of total mRNA changes, were observed for only 33 of the 18,967 C. elegans mRNAs tested, of which a disproportionate number were related to egg laying and were expressed in neurons and/or muscle. Translational regulation was confirmed by reduced levels of DAF-12, EGL-15, and KIN-29. The functions of these proteins can explain some phenotypes observed in ife-4 knockout mutants. These results indicate that translation of a limited subset of mRNAs is dependent on a specific isoform of eIF4E.