Temporal and Spatial Transcriptional Fingerprints by Antipsychotic or Propsychotic Drugs in Mouse Brain

<div><p>Various types of antipsychotics have been developed for the treatment of schizophrenia since the accidental discovery of the antipsychotic activity of chlorpromazine. Although all clinically effective antipsychotic agents have common properties to interact with the dopamine D2 receptor (D2R) activation, their precise mechanisms of action remain elusive. Antipsychotics are well known to induce transcriptional changes of immediate early genes (IEGs), raising the possibility that gene expressions play an essential role to improve psychiatric symptoms. Here, we report that while different classes of antipsychotics have complex pharmacological profiles against D2R, they share common transcriptome fingerprint (TFP) profile of IEGs in the murine brain <i>in vivo</i> by quantitative real-time PCR (qPCR). Our data showed that various types of antipsychotics with a profound interaction of D2R including haloperidol (antagonist), olanzapine (antagonist), and aripiprazole (partial agonist) all share common spatial TFPs closely homologous to those of D2R antagonist sulpiride, and elicited greater transcriptional responses in the striatum than in the nucleus accumbens. Meanwhile, D2R agonist quinpirole and propsychotic NMDA antagonists such as MK-801 and phencyclidine (PCP) exhibited the contrasting TFP profiles. Clozapine and propsychotic drug methamphetamine (MAP) displayed peculiar TFPs that reflect their unique pharmacological property. Our results suggest that transcriptional responses are conserved across various types of antipsychotics clinically effective in positive symptoms of schizophrenia and also show that temporal and spatial TFPs may reflect the pharmacological features of the drugs. Thus, we propose that a TFP approach is beneficial to evaluate novel drug candidates for antipsychotic development.</p></div

D2R antagonist shows spatial TFPs homologous to those by antipsychotics, while D2 agonist and propsychotic agents exhibit their peculiar TFPs.

<p>TFPs of seven IEGs over time induced by D2R antagonist/agonist and propsychotic drugs in mouse brain; (a) Selective D2R antagonist sulpiride (100 mg/kg, i.p.) and agonist quinpirole (10 mg/kg, i.p.), and (b) Propsychotic agents MAP (3 mg/kg, s.c.), PCP (10 mg/kg, s.c.) and MK-801 (1 mg/kg, s.c.). The bar depicted in the bottom right corner represents the expression level of the gene searched in a TFP. Red represents high expression, green represents vehicle-control level expression, and blue represents low expression.</p

TFPs of seven IEGs by treatment of propsychotic agents showing dose-response pattern in mouse brain.

<p>Significant reduction of <i>Egr2</i> in nucleus accumbens, striatum and prefrontal cortex, and increases of <i>c-fos</i> and <i>Ccn1</i> in all tested regions are characteristic of NMDA antagonists, whilst MAP serves robust IEGs induction in all four regions studied here. The bar depicted in the bottom right corner represents the expression level of the gene searched in a TFP. Red represents high expression, green represents vehicle-control level expression, and blue represents low expression. Propsychotic agents; MAP (1 hr treatment, 1 mg/kg to 10 mg/kg, s.c.), PCP (1 hr treatment, 1 mg/kg to 10 mg/kg, s.c.), MK-801 (1hr treatment, 0.1 mg/kg to 3 mg/kg, s.c.).</p

Antipsychotics induce distinctive IEG expression changes in nucleus accumbens and striatum, mainly due to their prominent D2R antagonistic activity.

<p>(a) Time courses of seven IEG expressions induced by 0.3 mg/kg (p.o.) of haloperidol. Data are shown as mean± SEM (n = 5). Filled circles, p<0.05 in unpaired t-test with Welch’s correction, compound (n = 5) versus vehicle (n = 5) at the same time point; open circles, not significant. The changes of expression levels are shown as fold change over vehicle. (b) TFP of seven IEGs over time induced by antipsychotic agents in mouse brain. Antipsychotics; haloperidol (0.3 mg/kg, p.o.), aripiprazole (3 mg/kg, p.o.), olanzapine (10 mg/kg, p.o.) and clozapine (30 mg/kg, p.o.). The bar depicted in the bottom right corner represents the expression level of the gene searched in a TFP. Red represents high expression, green represents vehicle-control level expression, and blue represents low expression.</p

Antipsychotics treatment except clozapine commonly induce three IEGs, <i>c-fos</i>, <i>Arc</i> and <i>Egr2</i> in a dose-dependent manner in nucleus accumbens and striatum.

<p>(a) Dose-dependent effects of haloperidol (1 hr treatment, 0.03 mg/kg to 1 mg/kg, p.o.). Note that four IEGs (<i>c-fos</i>, <i>Arc</i>, <i>Egr1</i> and <i>Egr2</i>) are dose-dependently elevated in nucleus accumbens and striatum. The dashed horizontal line indicates baseline (the value of 1).Data are shown as mean± SEM (n = 4~5). *p<0.025, significantly different from vehicle at a dose of 0 mg/kg in Williams’ test. (b) TFPs of seven IEGs showing dose-response pattern by treatment of four antipsychotics in mouse brain. Significant induction for three IEGs (<i>c-fos</i>, <i>Arc</i> and <i>Egr2</i>) were commonly observed in nucleus accumbens and striatum among three antipsychotics, haloperidol, aripiprazole and olanzapine. Antipsychotics; haloperidol (1 hr treatment, 0.03 mg/kg to 1 mg/kg, p.o.), aripiprazole (2 hrs treatment, 0.1 mg/kg to 3 mg/kg, p.o.), olanzapine (1 hr treatment, 0.3 mg/kg to 10 mg/kg, p.o.) and clozapine (1 hr treatment, 1 mg/kg to 30 mg/kg, p.o.). The bar depicted in the bottom right corner represents the expression level of the gene searched in a TFP. Red represents high expression, green represents vehicle-control level expression, and blue represents low expression.</p

Anatomical Transcriptome of G Protein-Coupled Receptors Leads to the Identification of a Novel Therapeutic Candidate GPR52 for Psychiatric Disorders

<div><p>Many drugs of abuse and most neuropharmacological agents regulate G protein-coupled receptors (GPCRs) in the central nervous system (CNS)_ENREF_1. The striatum, in which dopamine D1 and D2 receptors are enriched, is strongly innervated by the ventral tegmental area (VTA), which is the origin of dopaminergic cell bodies of the mesocorticolimbic dopamine system_ENREF_3 and plays a central role in the development of psychiatric disorders_ENREF_4. Here we report the comprehensive and anatomical transcript profiling of 322 non-odorant GPCRs in mouse tissue by quantitative real-time PCR (qPCR), leading to the identification of neurotherapeutic receptors exclusively expressed in the CNS, especially in the striatum. Among them, GPR6, GPR52, and GPR88, known as orphan GPCRs, were shown to co-localize either with a D2 receptor alone or with both D1 and D2 receptors in neurons of the basal ganglia. Intriguingly, we found that GPR52 was well conserved among vertebrates, is Gs-coupled and responsive to the antipsychotic drug, reserpine. We used three types of transgenic (Tg) mice employing a <i>Cre-lox</i> system under the control of the GPR52 promoter, namely, GPR52-LacZ Tg, human GPR52 (hGPR52) Tg, and hGPR52-GFP Tg mice. Detailed histological investigation suggests that GPR52 may modulate dopaminergic and glutamatergic transmission in neuronal circuits responsible for cognitive function and emotion. In support of our prediction, GPR52 knockout and transgenic mice exhibited psychosis-related and antipsychotic-like behaviors, respectively. Therefore, we propose that GPR52 has the potential of being a therapeutic psychiatric receptor. This approach may help identify potential therapeutic targets for CNS diseases.</p></div

Anatomically comprehensive profiling of mouse GPCR mRNA expression reveals the CNS specific clusters.

<p><b>A,</b> Six CNS specific clusters were highlighted by red square. <b>B,</b> Cluster 4, 11, and 12 indicated by red numbers in (<b>A</b>) were enlarged. Red and Green indicates orphan GPCRs of our interest and adhesion GPCRs, respectively. <b>C,</b> Pie charts of the numbers of orphan and ligand-known GPCRs in all and six CNS specific clusters.</p

Distributions of mGPR52 mRNA in wild type mice and LacZ signals in mGPR52-LacZ Tg mice.

<p>mGPR52 mRNA.</p><p>+++, highest; ++, moderate density; +, low density;</p><p>mGPR52-LacZ Tg mice.</p><p>○, LacZ-positive.</p

Gene expression distribution and protein localization of GPR52.

<p><b>A,</b> GPR52 mRNA is abundantly expressed in human brain. GPR52 mRNAs were quantified by qPCR throughout human tissues. Data represent the ratios of GPR52 to glyceraldehydes-3-phosphate dehydrogenase (GAPDH) mRNA. <b>B–C,</b> ISH investigation of GPR52 mRNA was performed in rat striatum and medial prefrontal cortex. Double-ISH analysis of GPR52 with DRD1/DRD2 in adult male rats. Localizations of GPR52 and DRD1/DRD2 mRNAs were shown as brown and blue signals, respectively, in striatum (<b>B</b>) and medial prefrontal cortex (<b>C</b>). Allow indicates the double-stained neurons. Bar: 25 µm. <b>D,</b> Distribution of LacZ signals in GPR52-LacZ Tg mouse brain. Serial frontal brain sections (rostral → caudal) were stained with X-Gal in GPR52-LacZ Tg mouse. Black characters on the left side of the pictures show LacZ-positive cell bodies and fibers while purple characters on the right side show the fibers. Results and abbreviations were summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090134#pone-0090134-t002" target="_blank">Table 2</a>. Bar: 1 mm. <b>E,</b> Double detections of GFP signals with DRD2 in frontal brain sections of hGPR52-GFP Tg mouse. Green and red colors show GFP and DRD2 immunopositive signals, respectively. Bar: 200 µm.</p

Expression of GPR88, GPR52, and GPR6 in DRD1 or DRD2- expressing neurons of rat basal ganglion by Double-ISH analysis.

<p>A–C, GPR88 (red) and DRD1/DRD2 (green) in striatum (A), nucleus accumbens (shell) (B), and olfactory tubecle (C). D–F, GPR52 (red) and DRD1/DRD2 (green) in striatum (D), nucleus accumbens (shell) (E), and olfactory tubecle (F). G–I, GPR6 (blue) and DRD1/DRD2 (brown) in striatum (G), nucleus accumbens (shell) (H), and olfactory tubecle (I). Bar: 100 µm.</p