Schizophrenia-Linked Protein tSNARE1 Regulates Endosomal Trafficking in Cortical Neurons.

TSNARE1, which encodes the protein tSNARE1, is a high-confidence gene candidate for schizophrenia risk, but nothing is known about its cellular or physiological function. We identified the major gene products of TSNARE1 and their cytoplasmic localization and function in endosomal trafficking in cortical neurons. We validated three primary isoforms of TSNARE1 expressed in human brain, all of which encode a syntaxin-like Qa SNARE domain. RNA-sequencing data from adult and fetal human brain suggested that the majority of tSNARE1 lacks a transmembrane domain that is thought to be necessary for membrane fusion. Biochemical data demonstrate that tSNARE1 can compete with Stx12 for incorporation into an endosomal SNARE complex, supporting its possible role as an inhibitory SNARE. Live-cell imaging in cortical neurons from mice of both sexes demonstrated that brain tSNARE1 isoforms localized to the endosomal network. The most abundant brain isoform, tSNARE1c, localized most frequently to Rab7+ late endosomes, and endogenous tSNARE1 displayed a similar localization in human neural progenitor cells and neuroblastoma cells. In mature rat neurons from both sexes, tSNARE1 localized to the dendritic shaft and dendritic spines, supporting a role for tSNARE1 at the postsynapse. Expression of either tSNARE1b or tSNARE1c, which differ only in their inclusion or exclusion of an Myb-like domain, delayed the trafficking of the dendritic endosomal cargo Nsg1 into late endosomal and lysosomal compartments. These data suggest that tSNARE1 regulates endosomal trafficking in cortical neurons, likely by negatively regulating early endosomal to late endosomal trafficking.SIGNIFICANCE STATEMENT Schizophrenia is a severe and polygenic neuropsychiatric disorder. Understanding the functions of high-confidence candidate genes is critical toward understanding how their dysfunction contributes to schizophrenia pathogenesis. TSNARE1 is one of the high-confidence candidate genes for schizophrenia risk, yet nothing was known about its cellular or physiological function. Here we describe the major isoforms of TSNARE1 and their cytoplasmic localization and function in the endosomal network in cortical neurons. Our results are consistent with the hypothesis that the majority of brain tSNARE1 acts as a negative regulator to endolysosomal trafficking.

The DREADD agonist clozapine N-oxide (CNO) is reverse-metabolized to clozapine and produces clozapine-like interoceptive stimulus effects in rats and mice.

Clozapine-N-oxide (CNO) has long been the ligand of choice for selectively activating Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). However, recent studies have challenged the long-held assertion that CNO is otherwise pharmacologically inert. The present study aimed to 1) determine whether CNO is reverse-metabolized to its parent compound clozapine in mice (as has recently been reported in rats), and 2) determine whether CNO exerts clozapine-like interoceptive stimulus effects in rats and/or mice. Following administration of 10.0 mg/kg CNO, pharmacokinetic analyses replicated recent reports of back-conversion to clozapine in rats and revealed that this phenomenon also occurs in mice. In rats and mice trained to discriminate 1.25 mg/kg clozapine from vehicle, CNO (1.0-20.0 mg/kg) produced partial substitution for the clozapine stimulus on average, with full substitution being detected in some individual animals of both species at doses frequently used to activate DREADDs. The present demonstration that CNO is converted to clozapine and exerts clozapine-like behavioral effects in both mice and rats further emphasizes the need for appropriate control groups in studies employing DREADDs, and highlights the utility of the drug discrimination procedure as a tool with which to screen the off-target effects of novel DREADD agonists.

In Vitro and In Vivo Characterization of the Alkaloid Nuciferine.

RATIONALE: The sacred lotus (Nelumbo nucifera) contains many phytochemicals and has a history of human use. To determine which compounds may be responsible for reported psychotropic effects, we used in silico predictions of the identified phytochemicals. Nuciferine, an alkaloid component of Nelumbo nucifera and Nymphaea caerulea, had a predicted molecular profile similar to antipsychotic compounds. Our study characterizes nuciferine using in vitro and in vivo pharmacological assays. METHODS: Nuciferine was first characterized in silico using the similarity ensemble approach, and was followed by further characterization and validation using the Psychoactive Drug Screening Program of the National Institute of Mental Health. Nuciferine was then tested in vivo in the head-twitch response, pre-pulse inhibition, hyperlocomotor activity, and drug discrimination paradigms. RESULTS: Nuciferine shares a receptor profile similar to aripiprazole-like antipsychotic drugs. Nuciferine was an antagonist at 5-HT2A, 5-HT2C, and 5-HT2B, an inverse agonist at 5-HT7, a partial agonist at D2, D5 and 5-HT6, an agonist at 5-HT1A and D4 receptors, and inhibited the dopamine transporter. In rodent models relevant to antipsychotic drug action, nuciferine blocked head-twitch responses and discriminative stimulus effects of a 5-HT2A agonist, substituted for clozapine discriminative stimulus, enhanced amphetamine induced locomotor activity, inhibited phencyclidine (PCP)-induced locomotor activity, and rescued PCP-induced disruption of prepulse inhibition without induction of catalepsy. CONCLUSIONS: The molecular profile of nuciferine was similar but not identical to that shared with several approved antipsychotic drugs suggesting that nuciferine has atypical antipsychotic-like actions.

Elucidation of The Behavioral Program and Neuronal Network Encoded by Dorsal Raphe Serotonergic Neurons.

Elucidating how the brain's serotonergic network mediates diverse behavioral actions over both relatively short (minutes-hours) and long period of time (days-weeks) remains a major challenge for neuroscience. Our relative ignorance is largely due to the lack of technologies with robustness, reversibility, and spatio-temporal control. Recently, we have demonstrated that our chemogenetic approach (eg, Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)) provides a reliable and robust tool for controlling genetically defined neural populations. Here we show how short- and long-term activation of dorsal raphe nucleus (DRN) serotonergic neurons induces robust behavioral responses. We found that both short- and long-term activation of DRN serotonergic neurons induce antidepressant-like behavioral responses. However, only short-term activation induces anxiogenic-like behaviors. In parallel, these behavioral phenotypes were associated with a metabolic map of whole brain network activity via a recently developed non-invasive imaging technology DREAMM (DREADD Associated Metabolic Mapping). Our findings reveal a previously unappreciated brain network elicited by selective activation of DRN serotonin neurons and illuminate potential therapeutic and adverse effects of drugs targeting DRN neurons.

Allosteric ligands for the pharmacologically dark receptors GPR68 and GPR65.

At least 120 non-olfactory G-protein-coupled receptors in the human genome are 'orphans' for which endogenous ligands are unknown, and many have no selective ligands, hindering the determination of their biological functions and clinical relevance. Among these is GPR68, a proton receptor that lacks small molecule modulators for probing its biology. Using yeast-based screens against GPR68, here we identify the benzodiazepine drug lorazepam as a non-selective GPR68 positive allosteric modulator. More than 3,000 GPR68 homology models were refined to recognize lorazepam in a putative allosteric site. Docking 3.1 million molecules predicted new GPR68 modulators, many of which were confirmed in functional assays. One potent GPR68 modulator, ogerin, suppressed recall in fear conditioning in wild-type but not in GPR68-knockout mice. The same approach led to the discovery of allosteric agonists and negative allosteric modulators for GPR65. Combining physical and structure-based screening may be broadly useful for ligand discovery for understudied and orphan GPCRs.

A Gαs DREADD mouse for selective modulation of cAMP production in striatopallidal neurons.

Here, we describe a newly generated transgenic mouse in which the Gs DREADD (rM3Ds), an engineered G protein-coupled receptor, is selectively expressed in striatopallidal medium spiny neurons (MSNs). We first show that in vitro, rM3Ds can couple to Gαolf and induce cAMP accumulation in cultured neurons and HEK-T cells. The rM3Ds was then selectively and stably expressed in striatopallidal neurons by creating a transgenic mouse in which an adenosine2A (adora2a) receptor-containing bacterial artificial chromosome was employed to drive rM3Ds expression. In the adora2A-rM3Ds mouse, activation of rM3Ds by clozapine-N-oxide (CNO) induces DARPP-32 phosphorylation, consistent with the known consequence of activation of endogenous striatal Gαs-coupled GPCRs. We then tested whether CNO administration would produce behavioral responses associated with striatopallidal Gs signaling and in this regard CNO dose-dependently decreases spontaneous locomotor activity and inhibits novelty induced locomotor activity. Last, we show that CNO prevented behavioral sensitization to amphetamine and increased AMPAR/NMDAR ratios in transgene-expressing neurons of the nucleus accumbens shell. These studies demonstrate the utility of adora2a-rM3Ds transgenic mice for the selective and noninvasive modulation of Gαs signaling in specific neuronal populations in vivo.This unique tool provides a new resource for elucidating the roles of striatopallidal MSN Gαs signaling in other neurobehavioral contexts.

Pharmacosynthetics: Reimagining the pharmacogenetic approach.

Pharmacology, in its broadest interpretation, is defined as the study of the interaction between physiological entities and drugs. In modern neuropsychopharmacology, this interaction is viewed as the drug itself on one side and signal transducer (receptor), the signal transduction cascade (effector proteins, second messengers), the cellular response (transcriptional regulation, activity modulation), the organ response (brain circuitry modulation), and, finally, the whole organism response (behavior) on the other. In other words, pharmacology has structured itself around the idea that the exogenous molecule (the drug) encodes a "signal" leading to everything on the other side including, in extreme renditions, a physiological response. The inference is that engaging a particular signal transduction pathway in a defined cell type leads inexorably to a prototypic physiological response. Thus, for instance, serotonergic activation of 5-HT(2A) receptors in rat aortic smooth muscle cells leads to an increase in intracellular Ca(++) (via IP3 release) and smooth muscle contraction (Roth et al., 1986). Here, we suggest that the invention of synthetic ligand--GPCR pairs (aka DREADDs, RASSLS, 'pharmacogenetics') permits the study of pharmacology using a shifted equation: more of the signal transduction elements moved to the left and, subsequently, under experimental control. For the purposes of disambiguation and to clarify this new interpretation as a creation of pharmacological manipulation, we present the term pharmacosynthetics to describe what has heretofore been called pharmacogenetics or chemicogenetics. This review discusses this new interpretation and reviews recent applications of the technology and considerations of the approach. This article is part of a Special Issue entitled Optogenetics (7th BRES).

Antagonist functional selectivity: 5-HT2A serotonin receptor antagonists differentially regulate 5-HT2A receptor protein level in vivo.

Dysregulation of the 5-HT(2A) receptor is implicated in both the etiology and treatment of schizophrenia. Although the essential role of 5-HT(2A) receptors in atypical antipsychotic drug actions is widely accepted, the contribution of 5-HT(2A) down-regulation to their efficacy is not known. We hypothesized that down-regulation of cortical 5-HT(2A) receptors contributes to the therapeutic action of atypical antipsychotic drugs. To test this hypothesis, we assessed the effect of chronically administered antipsychotics (clozapine, olanzapine, and haloperidol) and several 5-HT(2A) antagonists [ketanserin, altanserin, α-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidinemethanol (M100907), α-phenyl-1-(2-phenylethyl)-4-piperidinemethano (M11939), 4-[(2Z)-3-{[2-(dimethylamino)ethoxy]amino}-3-(2-fluorophenyl)prop-2-en-1-ylidene]cyclohexa-2,5-dien-1-one (SR46349B), and pimavanserin], on the phencyclidine (PCP)-induced hyperlocomotor response and cortical 5-HT(2A) receptor levels in C57BL/6J mice. Clozapine and olanzapine, but not haloperidol, induced receptor down-regulation and attenuated PCP-induced locomotor responses. Of the selective 5-HT(2A) antagonists tested, only ketanserin caused significant receptor protein down-regulation, whereas SR46349B up-regulated 5-HT(2A) receptors and potentiated PCP-hyperlocomotion; the other 5-HT(2A) receptor antagonists were without effect. The significance of these findings with respect to atypical antipsychotic drug action is discussed.

The presynaptic component of the serotonergic system is required for clozapine's efficacy.

Clozapine, by virtue of its absence of extrapyramidal side effects and greater efficacy, revolutionized the treatment of schizophrenia, although the mechanisms underlying this exceptional activity remain controversial. Combining an unbiased cheminformatics and physical screening approach, we evaluated clozapine's activity at >2350 distinct molecular targets. Clozapine, and the closely related atypical antipsychotic drug olanzapine, interacted potently with a unique spectrum of molecular targets. This distinct pattern, which was not shared with the typical antipsychotic drug haloperidol, suggested that the serotonergic neuronal system was a key determinant of clozapine's actions. To test this hypothesis, we used pet1(-/-) mice, which are deficient in serotonergic presynaptic markers. We discovered that the antipsychotic-like properties of the atypical antipsychotic drugs clozapine and olanzapine were abolished in a pharmacological model that mimics NMDA-receptor hypofunction in pet1(-/-) mice, whereas haloperidol's efficacy was unaffected. These results show that clozapine's ability to normalize NMDA-receptor hypofunction, which is characteristic of schizophrenia, depends on an intact presynaptic serotonergic neuronal system.

Assessing serotonin receptor mRNA editing frequency by a novel ultra high-throughput sequencing method.

RNA editing is a post-transcriptional modification of pre-mRNA that results in increased diversity in transcriptomes and proteomes. It occurs in a wide variety of eukaryotic organisms and in some viruses. One of the most common forms of pre-mRNA editing is A-to-I editing, in which adenosine is deaminated to inosine, which is read as guanosine during translation. This phenomenon has been observed in numerous transcripts, including the mammalian 5-HT(2C) receptor, which can be edited at five distinct sites. Methods used to date to quantify 5-HT(2C) receptor editing are labor-intensive, expensive and provide limited information regarding the relative abundance of 5-HT(2C) receptor editing variants. Here, we present a novel, ultra high-throughput method to quantify 5-HT(2C) receptor editing, compare it to a more conventional method, and use it to assess the effect of a range of genetic and pharmacologic manipulations on 5-HT(2C) editing. We conclude that this new method is powerful and economical, and we provide evidence that alterations in 5-HT(2C) editing appear to be a result of regional changes in brain activity, rather than a mechanism to normalize 5-HT(2C) signaling.

Behavioral characterization of serotonergic activation in the flatworm Planaria.

Serotonin (5-hydroxytryptamine, 5-HT) receptors have been identified in Planaria, a model used for studying the pharmacology of behavioral phenomena. This study characterized the behavioral and locomotor effects of 5-HT, a 5-HT1A agonist, a 5-HT1B/2C agonist, and a 5-HT1A antagonist to examine the role of 5-HT receptor activation in this species. Planarians were video recorded individually in a clear plastic cube containing drug solution or vehicle. To quantify locomotor velocity (pLMV), planarians were placed individually into a dish containing drug solution or vehicle and the rate of gridline crossings was recorded. For the antagonist experiments, four conditions were studied: water alone, agonist alone, antagonist alone, and agonist plus antagonist. The decrease in pLMV induced by the5-HT1A agonist (8-OH-DPAT), and the 5-HT1B/2C agonist (mCPP), was antagonized by pretreatment with the 5-HT1A antagonist (WAY-100635) at a dose that had no effect of its own on pLMV. At a higher concentration of WAY-100635, further decreases in pLMV induced by 8-OH-DPAT were observed. Each agonist produced increased occurrences of 'C-like position' and 'screw-like hyperkinesia', 5-HT and mCPP produced 'writhing', and only mCPP produced a significant increase in duration of 'headswing' behavior. The results demonstrate that the 5-HT1A receptor identified in Planaria mediates behavioral responses to 5-HT receptor ligands, supporting the notion that planarians possess functional 5-HT receptors and might serve as a simple model for their study.