Martilias S. Farrell

Evaluating historical candidate genes for schizophrenia

Prior to the genome-wide association era, candidate gene studies were a major approach in schizophrenia genetics. In this invited review, we consider the current status of 25 historical candidate genes for schizophrenia (for example, COMT, DISC1, DTNBP1 and NRG1). The initial study for 24 of these genes explicitly evaluated common variant hypotheses about schizophrenia. Our evaluation included a meta-analysis of the candidate gene literature, incorporation of the results of the largest genomic study yet published for schizophrenia, ratings from informed researchers who have published on these genes, and ratings from 24 schizophrenia geneticists. On the basis of current empirical evidence and mostly consensual assessments of informed opinion, it appears that the historical candidate gene literature did not yield clear insights into the genetic basis of schizophrenia. A likely reason why historical candidate gene studies did not achieve their primary aims is inadequate statistical power. However, the considerable efforts embodied in these early studies unquestionably set the stage for current successes in genomic approaches to schizophrenia.

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.

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 clozapines 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 clozapines 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 haloperidols efficacy was unaffected. These results show that clozapines ability to normalize NMDA-receptor hypofunction, which is characteristic of schizophrenia, depends on an intact presynaptic serotonergic neuronal system.

ANTAGONIST FUNCTIONAL SELECTIVITY: 5-HT2A SEROTONIN RECEPTOR ANTAGONISTS DIFFERENTIALLY REGULATE 5-HT2A RECEPTOR PROTEIN LEVEL IN VIVO

Dysregulation of the 5-HT2A receptor is implicated in both the etiology and treatment of schizophrenia. Although the essential role of 5-HT2A receptors in atypical antipsychotic drug actions is widely accepted, the contribution of 5-HT2A down-regulation to their efficacy is not known. We hypothesized that down-regulation of cortical 5-HT2A 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-HT2A 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-HT2A 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-HT2A antagonists tested, only ketanserin caused significant receptor protein down-regulation, whereas SR46349B up-regulated 5-HT2A receptors and potentiated PCP-hyperlocomotion; the other 5-HT2A receptor antagonists were without effect. The significance of these findings with respect to atypical antipsychotic drug action is discussed.

A chemical-genetic approach for precise spatio-temporal control of cellular signaling.

Recently we have perfected a chemical-genetic approach to gain precise spatio-temporal control of cellular signaling. This approach entails the cell-type specific expression of mutant G-protein coupled receptors which have been evolved to be activated by the pharmacologically inert drug-like small molecule clozapine N-oxide. We have named these mutant GPCRs DREADDs (Designer Receptors Exclusively Activated by Designer Drugs). In this paper we will first review recent applications of this technology for the remote control of neuronal and non-neuronal signaling. Next, we will also introduce new variants which could be useful for the control of cellular signaling in discrete cellular compartments. Finally, we will suggest future basic science and therapeutic applications of this general technology.

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.

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-HT2C receptor, which can be edited at five distinct sites. Methods used to date to quantify 5-HT2C receptor editing are labor-intensive, expensive and provide limited information regarding the relative abundance of 5-HT2C receptor editing variants. Here, we present a novel, ultra high-throughput method to quantify 5-HT2C 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-HT2C editing. We conclude that this new method is powerful and economical, and we provide evidence that alterations in 5-HT2C editing appear to be a result of regional changes in brain activity, rather than a mechanism to normalize 5-HT2C signaling.

Schizophrenia risk gene CAV1 is both pro-psychotic and required for atypical antipsychotic drug actions in vivo.

Caveolin-1 (Cav-1) is a scaffolding protein important for regulating receptor signaling cascades by partitioning signaling molecules into membrane microdomains. Disruption of the CAV1 gene has recently been identified as a rare structural variant associated with schizophrenia. Although Cav-1 knockout (KO) mice displayed no baseline behavioral disruptions, Cav-1 KO mice, similar to schizophrenic individuals, exhibited increased sensitivity to the psychotomimetic N-methyl-D-aspartate receptor antagonist phencyclidine (PCP). Thus, PCP disruption of prepulse inhibition (PPI) and PCP-induced mouse locomotor activity were both enhanced by genetic deletion of Cav-1. Interestingly, genetic deletion of Cav-1 rendered the atypical antipsychotics clozapine and olanzapine and the 5-HT2A-selective antagonist M100907 ineffective at normalizing PCP-induced disruption of PPI. We also discovered that genetic deletion of Cav-1 attenuated 5-HT2A-induced c-Fos and egr-1 expression in mouse frontal cortex and also reduced 5-HT2A-mediated Ca2+ mobilization in primary cortical neuronal cultures. The behavioral effects of the 5-HT2A agonist (2,5-dimethoxy-4-iodoamphetamine) including head twitch responses and disruption of PPI were also attenuated by genetic deletion of Cav-1, indicating that Cav-1 is required for both inverse agonist (that is, atypical antipsychotic drug) and agonist actions at 5-HT2A receptors. This study demonstrates that disruption of the CAV1 gene—a rare structural variant associated with schizophrenia—is not only pro-psychotic but also attenuates atypical antipsychotic drug actions.

Disruption of the MicroRNA 137 Primary Transcript Results in Early Embryonic Lethality in Mice

To the editors: Multiple lines of evidence support a role for microRNA 137 (miR-137) in the etiology of schizophrenia (1–4) and fundamental neuronal processes (5–7). In the largest genome-wide association meta-analysis for schizophrenia,(8) the second most significant association is in MIR137HG, the gene encoding miR-137 (rs1702294, P=3.4×10−19). Prior reports indicated that genes with predicted miR-137 target sites were enriched for smaller GWAS P-values (2), raising the possibility that miR-137 regulates a gene network involved in schizophrenia. To understand more about the role of miR-137, we describe here our characterization of embryonic development, behavior, and gene expression in mice with targeted disruption of the Mir137 transcript (supplementary detailed information can be found at: http://crowley.web.unc.edu/files/2014/03/mir137.ko_.supplement.pdf). Mir137tm1Mtm mice were created in a project to generate conditional, reporter-tagged targeted mutations in 162 miRNAs (9). These mice were created using a “knockout-first” strategy (10) to produce a knockout at the RNA processing level. We purchased heterozygous (+/−) breeder mice (www.mmrrc.org/catalog/sds.php?mmrrc_id=36301). We confirmed genetic background (~75% C57BL/6J) and backcrossed twice to C57BL/6J to create animals of >95% C57BL/6J ancestry. To our knowledge, these Mir137 mutant mice have not been characterized previously. Seven heterozygous × heterozygous matings produced 35 living offspring, none of which were homozygous (−/−) for the targeted Mir137 allele (Figure 1A, P < 0.001), suggesting complete embryonic lethality in the presence of knockout of Mir137. Figure 1 Mir137 knockout mice (−/−) are embryonic lethal. (A) Heterozygous × heterozygous matings failed to yield a live born (−/−) mouse or embryo, and timed matings revealed an excess of resorbed embryos at E11.5. Heterozygous ... To confirm embryonic lethality and to determine when lethality occurred, we conducted timed matings (six heterozygous × heterozygous and four heterozygous × wildtype). On embryonic day 11.5 (E11.5), all embryos were collected (Figure 1A). Again, no homozygous (−/−) embryos were identified (P < 0.001). We observed significantly more resorbed embryos from heterozygous × heterozygous matings (P < 0.0001), indicating that embryonic lethality must occur after implantation (~E4.5) but before E11.5. Efforts to identify a Mir137 −/− mouse by genotyping resorbed embryos at E11.5 or collecting flushed embryos at E3.5 were unsuccessful, likely due to insufficient and/or impure DNA preparations from small amounts of tissue. We did not observe reduced viability of heterozygous mice (+/−) after genotyping 256 offspring (P=0.14) collected from birth to three weeks of age, indicating that one copy of Mir137 is sufficient for survival (Figure 1A). We subjected wild-type and heterozygous animals to a battery of behavioral tests (e.g., basic sensorium, activity, social behavior, learning/memory, etc.), and observed no consistent and interpretable differences. These results also suggest compensation from the remaining allele. We next examined the expression of Mir137 and the levels of mature miR-137 in the brains of heterozygous and wildtype animals (−/− embryos died too early for collection of sufficient tissue). Using allele-specific reverse transcriptase PCR, we confirmed that the targeted allele leads to complete loss of Mir137 expression downstream of the polyA sequence (Figure 1B). However, mature miR-137 levels were not significantly different between heterozygous and wild-type animals (Figure 1C), suggesting that heterozygous mice have compensatory upregulation or decreased degradation of miR-137. These expression data are consistent with the lack of a viability or behavioral phenotype in the heterozygous mice. To circumvent the embryonic lethality of Mir137 −/− mice, we attempted to make conditional knockouts (9), such that ablation of Mir137 could be limited to a particular tissue. The construct contains a combination of FRT and loxP sites intended to allow conditional mutagenesis by crossing to germline deleter Flp mice (expected to restore the wild-type allele) and then crossing to tissue-specific Cre transgenic mice (expected to generate adult brain-specific knockouts). Crosses between Mir137 +/− mice and an efficient Flp line led to several successfully recombined mice, heterozygous for the rescued allele. Repeated intercrosses of these recombined mice, however, failed to yield any homozygous offspring. Therefore, we were unable to rescue embryonic lethality. We reasoned that there were two major possibilities for the rescue failure. First, the targeting construct could have inserted into the wrong genomic position or could have sequence errors. We have likely excluded this possibility by confirming the location of the integration site and re-sequencing >95% of the construct without identifying an error. Second, Flp may have been unable to restore proper gene function due to issues inherent with the gene construct (e.g., FRT or LoxP sites in critical part of the gene). We favor this possibility because we were unable to detect any reporter gene (beta-galactosidase) activity in Mir137 +/− embryos or adult tissues, suggesting that the endogenous Mir137 promoter was inadvertently inactivated. Furthermore, bioinformatic analysis of the region (using additional data that became available after we were well into this project), suggests that the exogenous gene targeting elements may indeed interfere with endogenous functional elements, and act to prevent conditional mutagenesis (Figure 1D). The LoxP site upstream of Mir137 is in a putative splice donor sequence and the FRT site lies between highly conserved DNase hypersensitivity sites that are active in embryonic brain. In conclusion, these results suggest that at least one functional copy of Mir137 is essential for embryonic development. These data are consistent with miR-137 playing important roles in development and perhaps also in neurodevelopmental disorders like schizophrenia. It appears that miR-137’s biological pathway is capable of homeostatic compensation and, while we do not yet understand the functional impact of Mir137 schizophrenia risk variants, it is conceivable that this homeostatic capacity could be affected in some way. It is clear, however, that this genomic region is more complex than previously thought, and the design of targeted mutations in this region should incorporate all available genomic data.