Neuropharmacology 93 (2015) 308e313
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Postsynaptic D2 dopamine receptor supersensitivity in the striatum of mice lacking TAAR1 Stefano Espinoza a, Valentina Ghisi b, Marco Emanuele a, Damiana Leo a, Ilya Sukhanov a, Tatiana D. Sotnikova a, c, Evelina Chieregatti a, Raul R. Gainetdinov a, c, d, * a
Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia, 16163 Genova, Italy Leibniz-Institut für Molekulare Pharmakologie (FMP), 13125 Berlin, Germany Institute of Translational Biomedicine, St. Petersburg State University, St. Petersburg 199034, Russia d Skolkovo Institute of Science and Technology (Skoltech) Skolkovo, Moscow Region 143025, Russia b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 November 2014 Received in revised form 13 February 2015 Accepted 16 February 2015 Available online 24 February 2015
Trace Amine-Associated Receptor 1 (TAAR1) is a G protein-coupled receptor (GPCR) known to modulate dopaminergic system through several mechanisms. Mice lacking this receptor show a higher sensitivity to dopaminergic stimuli, such as amphetamine; however, it is not clear whether D1 or D2 dopamine receptors and which associated intracellular signaling events are involved in this modulation. In the striatum of TAAR1 knock out (TAAR1-KO mice) we found that D2, but not D1, dopamine receptors were over-expressed, both in terms of mRNA and protein levels. Moreover, the D2 dopamine receptor-related G protein-independent AKT/GSK3 signaling pathway was selectively activated, as indicated by the decrease of phosphorylation of AKT and GSK3b. The decrease in phospho-AKT levels, suggesting an increase in D2 dopamine receptor activity in basal conditions, was associated with an increase of AKT/PP2A complex, as revealed by co-immunoprecipitation experiments. Finally, we found that the locomotor activation induced by the D2 dopamine receptor agonist quinpirole, but not by the full D1 dopamine receptor agonist SKF-82958, was increased in TAAR1-KO mice. These data demonstrate pronounced supersensitivity of postsynaptic D2 dopamine receptors in the striatum of TAAR1-KO mice and indicate that a close interaction of TAAR1 and D2 dopamine receptors at the level of postsynaptic structures has important functional consequences. © 2015 Elsevier Ltd. All rights reserved.
Keywords: TAAR1 Dopamine Striatum D2 receptor
1. Introduction Trace Amine-Associated Receptor 1 (TAAR1) is a G proteincoupled receptor (GPCR) expressed in mammalian brain in major monoaminergic regions, such as ventral tegmental area (VTA) and dorsal raphe, and their projections, including striatum, amygdala, hypothalamus and frontal cortex (Borowsky et al., 2001; Bunzow et al., 2001; Di Cara et al., 2011; Lindemann et al., 2008; Wolinsky et al., 2007). Many studies have focused on the role of TAAR1 in the modulation of dopaminergic system, and it is evident that generally TAAR1 exerts a negative control on dopaminergic activity
Abbreviations: TAAR1, Trace Amine-Associated Receptor 1; DA, dopamine; GPCR, G protein-coupled receptor; VTA, ventral tegmental area. * Corresponding author. Department of Neuroscience and Brain Technologies, Italian Institute of Technology (IIT) Via Morego 30, Genova 16163, Italy. Tel.: þ39 010 71781516; fax: þ39 010 720321. E-mail address: [email protected] (R.R. Gainetdinov). http://dx.doi.org/10.1016/j.neuropharm.2015.02.010 0028-3908/© 2015 Elsevier Ltd. All rights reserved.
(Di Cara et al., 2011; Lindemann et al., 2008; Revel et al., 2011, 2012, 2013; Sukhanov et al., 2014). TAAR1 knock out (TAAR1-KO) mice are more sensitive to the neurochemical and behavioral effect of several amphetamine derivatives (Di Cara et al., 2011; Lindemann et al., 2008; Miller, 2012; Wolinsky et al., 2007). Moreover, VTA dopaminergic neurons of TAAR1-KO mice show an increase in the firing rate (Lindemann et al., 2008) and dopamine release in the nucleus accumbens due to dysregulated function of D2 dopamine autoreceptors (Leo et al., 2014). Conversely, TAAR1 selective agonists decrease hyperdopaminergic manifestations induced by pharmacological tools or present in genetic mouse models (e.g. dopamine transporter knock out (DAT-KO) mice) (Espinoza and Gainetdinov, 2014; Revel et al., 2011, 2012, 2013). Several reports have indicated that TAAR1 is able to influence striatum-dependent behaviors, such as amphetamine-induced locomotor hyperactivity and haloperidol-induced catalepsy (Espinoza et al., 2011; Wolinsky et al., 2007). However, the mechanism by which TAAR1 alters the dopamine system, dependent in major part on the D1-and D2-
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classes of dopamine receptor function, remains unclear. It has been reported that TAAR1-KO mice have higher number of striatal D2 dopamine receptors in high affinity state (Wolinsky et al., 2007). Another study showed that in vitro TAAR1 and the long isoform of the D2 dopamine receptor were able to form an heterodimer and that the formation of this complex influences TAAR1-dependent cAMP signaling (Espinoza et al., 2011). However, it remained unclarified how D2-related signaling and functions are altered in vivo in TAAR1-KO mice. Furthermore, no information is available on the status of D1 dopamine receptor activity in TAAR1-KO mice. The aim of our study was to evaluate the functionality of D1 and D2 dopamine receptors in the striatum of TAAR1-KO mice. We found that, while D1 dopamine receptors displayed no abnormalities, D2 dopamine receptors were overexpressed and D2-related G proteinindependent signaling pathway was activated. Furthermore, these data indicating selective increase of postsynaptic D2 dopamine receptor activity in TAAR1-KO mice were directly confirmed in behavioral experiments with selective agonists. 2. Materials and methods 2.1. Animals TAAR1 knock out (TAAR1-KO) mice of mixed C57BL/6J 129Sv/J background were generated as previously described (Wolinsky et al., 2007). Animal care and treatments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (USA National Institutes of Health publication #865-23, Bethesda, MD), and the protocols were approved by the Italian Ministry of Health. All TAAR1-KO and WT littermates were obtained from heterozygous mating, and genotyping was performed in all individuals. Mice of both sexes were used in all experiments. 2.2. Western blot Western blot analyses of striatal samples to evaluate dopamine signaling were performed as previously described (Beaulieu et al., 2004). The conditions of the primary antibodies were as follows: anti phospho-AKT (Thr308) (1:3000), anti phospho-GSK3b (Ser9) (1:3000), anti b-catenin (1:1000), anti b-arrestin2 (1:2000), anti PP2A (1:5000), anti phospho-ERK1/2 (1:10,000), anti phospho-CREB (1:1000), anti phospho-DARPP32 (Thr74) (1:1000), anti-total AKT (1:5000) and anti-total GSK3b (1:5000). All antibodies were purchased from Cell Signaling (Danvers, MA, USA). Total proteins were measured by the Bio-Rad Protein Assay (Bio-Rad Laboratories, Segrate, Milan, Italy). Twenty micrograms of proteins for each sample were run on a SDS-10% polyacrylamide gel under reducing conditions and then electrophoretically transferred onto nitrocellulose membranes (GE Healthcare, Milan, Italy). Blots were blocked for 1 h at room temperature with 10% non-fat dry milk in TBS þ 0.1% Tween-20 buffer, incubated with antibodies against the phosphorylated forms of the proteins and then stripped and reprobed with antibodies against the corresponding total proteins. Immunocomplexes were visualized by chemiluminescence using the Chemidoc MP Imaging System (Bio-Rad Laboratories). Densitometric analysis was performed by normalizing the phosphoproteins level to the corresponding total form. The total form of the proteins was then normalized to the actin levels. 2.3. Immunoprecipitation Immunoprecipitations were carried out as previously described (Beaulieu et al., 2005). Briefly, tissue was homogenized at 4 C in 200 ml of lysis buffer (10 mM TriseHCl [pH 7.4], 1.0% Triton X-100) containing a cocktail of protease inhibitors (SigmaeAldrich). Immunoprecipitations were conducted overnight at 4 C with anti-AKT antibody, followed by incubation with protein G sepharose (GeHealthcare).
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[3H]-Spiperone and [3H]-Raclopride selective D2 antagonists were used. In the case of [3H]-Spiperone, membranes (150 ml; 0.5 mg/ml final proteins concentration) were incubated with six increasing doses of this radioactive ligand (50 ml; range: 0.03e1 nM), while for [3H]-Raclopride, membranes (50 ml; 1.2 mg/ml final proteins concentration) were incubated with six increasing doses of [3H]-Raclopride (100 ml; range: 1.8e60 nM). The different reactions were carried out at RT for 1 h in the case of [3H]-SCH23390 and [3H]-Raclopride in a total volume of 200 ml of assay buffer and at RT for 2 h in the case of [3H]-Spiperone in a total volume of 250 ml of assay buffer. Nonspecific binding was measured using nonradioactive flupenthixol (10 mM) for [3H]-SCH23390 binding or haloperidol (6 mM for [3H]-Spiperone binding, 50 mM for [3H]-Raclopride binding) in parallel assay tubes and was subtracted from total binding to obtain respectively specific [3H]-SCH23390 or [3H]Spiperone/[3H]-Raclopride binding. The incubations were terminated by rapid filtration over Brandel GF/C glass fiber filters washed with ice-cold assay buffer. The filters were incubated overnight in 5 ml high flash point scintillation cocktail (LefkoFluor) before their radioactivity content was counted by a liquid scintillation counter. 2.5. Real time PCR WT and TAAR1-KO mice of 3 months were sacrificed, striata were dissected and striatal tissue was dissociated for 15 min at 37 C with Pronase enzyme (Sigma) in Hank's Balanced Salt Solution (HBSS, Invitrogen). Brain samples were triturated with three glass pipettes of decreasing tip diameter and centrifuged at 900 rpm at room temperature for 5 min. To remove excess debris, cell pellets were resuspended in HBSS and filtered through a 70 mm mesh (BD Falcon, #352350). Cells-to-CT kit (Life Technology) was used to produce DNase I digested cell lysates and perform cDNA synthesis, according to manufacturer's instructions. cDNA were used for Taqman singleplex PCR. All reagents were supplied by Applied Biosystems. PCR master mix contained 1x Taqman Universal PCR Master Mix, 1x Gene Expression Assay mix, and 1 ml cDNA for a total volume of 20 ml. The following Gene Expression Assays were used: Drd2 (Assay ID Mm00438545_m1), Drd1a (Assay ID Mm01353211_m1), Gapdh (Assay ID Mm99999915_g1) and Hprt (Assay ID Mm00446968_m1). Samples were run in three replicates for each Gene Expression Assay. PCR reactions were carried out on a 7900 Thermal Cycler (Applied Biosystems) with 40 cycles of 95 C for 15 s and 60 C for 1 min. CT values for each gene were normalized to CT values for GAPDH and HPRT to obtain a relative expression level for each replicate and the five replicates were averaged together. Gene expression data were normalized by the multiple internal control gene method with GeNorm algorithm available in qBasePlus software (Biogazelle). 2.6. Locomotor activity Locomotor activity after drug or saline treatment was assessed as previously described (Gainetdinov et al., 2003). Locomotion was evaluated in an automated Omnitech Digiscan apparatus (AccuScan Instruments, Columbus, OH) under illuminated conditions. Apparatus included 4 open field monitors. Each Open Field monitor consisted of sets of 16 light beams arrayed in the horizontal X and Y axes. The hardware detected beams broken by the animal so that the software can determine the location of the mouse within the cage. Cages were divided on 4 compartments (20 cm 20 cm). Animals were tested individually for defined periods with 5-min intervals. Horizontal activities were expressed in terms of the number of beam breaks. Briefly, WT or mutant animals were injected i.p. with either saline, 2 mg/kg quinpirole or 0.5 mg/kg SKF-82958 (Chloro-APB) and placed in the activity monitor. The experimenter conducting the behavioral test was blinded for genotype/treatment of animals. Cumulative counts for 30 min (starting 10 min after drug or saline injection to avoid handling effects) were recorded for data analysis. We used both sexes in the locomotor tests, and initially analyzed male and females separately. Since we did not observe differences in effects of drugs between males and females we pooled the data together in the final analysis.
3. Results
2.4. D1 and D2 dopamine receptor saturation binding assay on striatal membranes
3.1. Lack of TAAR1 modulates D2 dopamine receptor expression in the striatum
Binding experiments were conducted according to Ghisi et al. (2009); Striatal tissues from wild-type, DAT-KO and DAT-tg mice were rapidly dissected and immediately homogenized, using a Teflonglass homogenizer, in 2 ml of lysis buffer containing 50 mM TriseHCl (pH 7.4), 120 mM NaCl, 1 mM EDTA and a cocktail of protease inhibitors at 1:1000 dilution. The homogenate was centrifuged at 1000 rpm for 10 min at 4 C to remove tissue debris and nuclei and the resulting supernatant centrifuged twice at 40,000 g for 20 min at 4 C. The final pellet was suspended in assay buffer containing 50 mM TriseHCl (pH 7.4), 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2. For D1 receptor saturation experiments, membranes (50 ml; 1.2 mg/ml final proteins concentration) were incubated with six increasing concentrations of [3H]-SCH23390 (50 ml; range: 0.5e16 nM) and 50 ml of ketanserin (100 nM), a specific antagonist of serotonin receptor, in order to prevent the binding of [3H]-SCH23390 to these receptors. For D2 receptor saturation experiments both
Previous studies have documented TAAR1 expression in several brain regions, including the striatum, with some discrepancies reported likely due to the low level of TAAR1 expression in most areas (Di Cara et al., 2011; Lindemann et al., 2008). Thus, we first confirmed TAAR1 expression in the striatum. Using RT-PCR, we documented that TAAR1 is expressed in the striatum with no detection of transcript in TAAR1-KO mice (Fig. 1A). As a first step to evaluate the consequences of TAAR1 deletion on dopamine transmission in this area, we evaluated the levels of D1 and D2 dopamine receptor mRNA expression in the striatum of mutant mice. We
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Fig. 1. D2 but not D1 dopamine receptor expression is increased in the striatum of TAAR1-KO mice. (A) mRNA levels of TAAR1 in striatum of WT and KO mice relative to the housekeeping genes GAPDH and HPRT as measured by RT-PCR. (B) mRNA levels measured with RT-PCR from striatal extracts. (C) D1 and D2 dopamine receptor levels were measured by saturation binding experiments. [3H]-SCH23390 and [3H]-Spiperone were used to quantify D1 and D2 dopamine receptors in the striatal membranes. Levels are expressed as a percentage compared to the control. Data were analyzed by GraphPad Prism software using a one site binding (hyperbola) equation. N ¼ 5 mice per group; data represent means ± SEM. *, P < 0.05. Student's unpaired two-tailed t-test.
extracted total RNA from dissected striata of WT and TAAR1-KO mice, and using qRT-PCR, we measured D1 and D2 dopamine receptor mRNA levels. As shown in Fig. 1B, D2 dopamine receptor mRNA levels were significantly increased, while D1 dopamine receptor mRNA levels were not altered. To test if dopamine receptor levels were also affected, we directly measured the number of D1 and D2 dopamine receptors by using radioligand binding. [3H]SCH23390 was utilized to selectively detect the levels of D1 dopamine receptors, while [3H]-Spiperone was used to quantify D2 dopamine receptors. As shown in Fig. 1C, similar to what was observed with mRNA, TAAR1-KO mice had a 2-fold increase in the levels of D2 dopamine receptors in the striatum, while there was no alteration in D1 dopamine receptor density. 3.2. Striatal D2 dopamine receptor signaling is enhanced in TAAR1KO mice Evidence in the literature indicates a higher proportion of striatal D2 dopamine receptors in the high affinity state in TAAR1KO mice compared to WT mice (Wolinsky et al., 2007). An increased D2 high affinity state and an increase in amphetamine sensitivity were also found in animal models of psychosis, as well as in clinical studies of schizophrenic patients (Seeman et al., 2005). These studies, together with the present observations showing selective up-regulation of striatal D2 dopamine receptors in TAAR1-KO mice, led us to analyze the status of D2 dopamine receptor-related signaling in TAAR1-KO mice. It is well known that D2 dopamine receptors signal through the Gi proteins to reduce cAMP levels. D2 dopamine receptors can also signal in a G-protein independent manner through the multifunctional protein b-arrestin2, leading to the dephosphorylation of AKT and the subsequent activation of GSK3b (Beaulieu and Gainetdinov, 2011). Both of these pathways have been shown to be important for dopamine-mediated behaviors (Beaulieu and Gainetdinov, 2011). Thus, by using western blot, we analyzed the basal phosphorylation state of proteins associated with D1 and D2 dopamine receptor activation. We observed a basal decrease in the phosphorylation of AKT and GSK3b in the striatum of TAAR1-KO mice (Fig. 2A, B), two downstream targets of barrestin2-dependent D2 dopamine receptor signaling (Beaulieu et al., 2005) with no alterations in the levels of the proteins (Fig. 2F). It has been demonstrated that D2 dopamine receptor upon its activation forms a complex with b-arrestin2, AKT and PP2A, leading to the dephosphorylation of AKT and the subsequent dephosphorylation of GSK3b thereby increasing GSK3b activity (Beaulieu et al., 2009, 2005, 2004). In agreement, we observed that the levels of b-catenin, a target of GSK3b that is degraded by the
increased GSK3 activity, were also decreased in mutant mice (Fig. 2C). Interestingly, we observed no alterations for other effectors, such as pERK1/2, pCREB and pDARPP32 (Fig. 2F), which are regulated by both the D1 and D2 dopamine receptors via G proteinrelated PKA/cAMP signaling cascade. Thus, the activation of the AKT/GSK3 pathway correlates well with the enhanced D2 dopamine receptor function that was found in TAAR1-KO mice by our group and others. To unveil the putative mechanism for the activation of this pathway, we also evaluated the levels of PP2A that is known to form the complex with AKT and the D2 receptor upon its activation. No alterations were observed in the total levels of this protein in TAAR1-KO mice (Fig. 2D). However, immunoprecipitation experiments have revealed an increase in the formation of complex between AKT and PP2A in the striatal tissue from TAAR1KO mice (Fig. 2E), suggesting that the increased interaction between these proteins may underlie the increased dephosphorylation of AKT and GSK3b (Beaulieu et al., 2005) observed in TAAR1-KO mice. 3.3. TAAR1-KO mice are hypersensitive to the locomotor effects of D2 but not D1 dopamine receptor agonist To test whether the locomotor supersensitivity to dopaminergic stimulation observed in TAAR1-KO mice is related to D2 receptor supersensitivity, we assessed the locomotor activity of animals following treatment with the selective D2-class dopamine receptor agonist quinpirole at a high “postsynaptic” dose, i.e., 2 mg/kg, i.p. (Usiello et al., 2000). At this dose, quinpirole activates not only presynaptic autoreceptors, leading to a decrease in DA transmission and global locomotor suppression, but also provides sufficient stimulation of post-synaptic D2 dopamine receptors, inducing a minor increase in forward locomotion over the levels caused by presynaptic effects but not achieving the levels observed in salinecontrols (Gainetdinov et al., 2003). It has been previously documented that mice with an enhanced D2 dopamine receptor sensitivity due to lack of GPCR kinase GRK-6 display a higher levels of locomotor activity in response to this dose of quinpirole (Gainetdinov et al., 2003). Similarly, as shown in Fig. 3A, locomotor activity following quinpirole treatment was significantly higher in TAAR1-KO compared to WT mice, directly demonstrating the increased D2 dopamine receptor functionality. By comparison, the locomotor stimulating effect of the selective D1 dopamine receptor agonist SKF-82958 at 1 mg/kg was not altered in mutant mice compared to WT animals (Fig. 3B). Taken together, these data demonstrate a significant over-activity of post-synaptic D2 receptor-mediated transmission in the striatum of TAAR1-KO mice.
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Fig. 2. D2R-related AKT/GSK3 signaling pathway is activated in TAAR1-KO mice. Densitometric western blot analysis of phosphoprotein relative levels in striatal extracts from WT and TAAR1-KO mice. Antibodies against p-Thr-308-AKT (A), p-Ser-9-GSK3b (B), b-catenin (C), PP2A (D, E) and pERK1/2, pDARPP-32, pCREB, totAKT and totGSK3 (F) were used. Antibodies for the total form of AKT and GSK3b were used as the loading controls for densitometric analysis. Immunoprecipitation of AKT revealed coimmunoprecipitation of the catalytic subunit C of PP2A from the striatal extracts of TAAR1-KO mice (E, right panel). Quantification of the interaction between AKT and PP2A, as measured by densitometric analysis (E, left panel). Average ratios of PP2A were normalized to AKT total levels and were labeled 1 in WT animals. Data represent means ± SEM. N ¼ 10e15 per group. *, p < 0.05; **, p < 0.01. Student's unpaired two-tailed t-test and repeated t-test for (F).
4. Discussion In this study, we provided the molecular mechanism responsible for the striatal dopamine hypersensitivity that has been observed in TAAR1-KO mice (Espinoza et al., 2011; Lindemann et al., 2008; Sotnikova et al., 2010; Wolinsky et al., 2007). Specifically, we demonstrated that in the striatum of TAAR1-KO mice, D2 dopamine receptors are up-regulated, and the related G protein-independent pathway (AKT/GSK3) is activated with a concomitantly increased D2-dependent locomotor activation. Taken together, these data provide further support for the close functional TAAR1-D2 dopamine receptor interaction mediated likely via receptor heterodimerization (Espinoza et al., 2011). TAAR1 function has been characterized extensively in previous years, with particular attention paid to its role in the modulation of
the dopamine system and its possible implications in psychiatric diseases (Borowsky et al., 2001; Bunzow et al., 2001; Espinoza and Gainetdinov, 2014; Lindemann and Hoener, 2005; Sotnikova et al., 2009). On one side, TAAR1-KO mice display a higher sensitivity to dopaminergic stimuli such as amphetamine and other psychostimulants (Lindemann et al., 2008; Wolinsky et al., 2007) and generally have a supersensitive dopaminergic system (Di Cara et al., 2011; Lindemann et al., 2008; Wolinsky et al., 2007) making them an interesting model relevant for schizophrenia (Wolinsky et al., 2007). On the other side, the recently developed TAAR1 selective agonists reduce manifestations of excessive dopaminergic tone caused by either pharmacological (amphetamine, cocaine) or genetic (DAT-KO mice) manipulations, further supporting the idea that TAAR1 could represent a new target for psychiatric diseases (Sotnikova et al., 2009).
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Fig. 3. D2 dopamine receptor agonist quinpirole induces an increased locomotor response in TAAR1-KO mice. Locomotor activity following 2 mg/kg quinpirole (A) and 1 mg/kg SKF-82958 (B) in WT and TAAR1-KO mice. Cumulative total distance traveled during the session (30 min, 10e40 min after injection) was calculated. Time course of quinpirole effect on locomotor activity of WT and TAAR1-KO mice is shown in the insert. Data represent means ± SEM. N ¼ 10 per group. **, p < 0.01, ***, p < 0.001. Oneway ANOVA with Dunnett's post hoc test was used.
Here, we extended these observations to an in vivo model by showing that postsynaptic D2, but not D1, dopamine receptors are up-regulated in the striatum of TAAR1-KO mice. This evidence, together with the previously reported increase in the proportion of
D2 dopamine receptors in high affinity states (Wolinsky et al., 2007), suggests that D2 dopamine receptor-mediated signaling and behavior could be markedly altered by the absence of TAAR1. To test this possibility directly, we evaluated the phosphorylation state of several proteins that are regulated by D1 and D2 receptors. It is well known that D2 dopamine receptors can signal through two major signaling pathways, the G protein-dependent, cAMP-mediated pathway and the G protein-independent, b-arrestin2dependent pathway (Beaulieu et al., 2014; Beaulieu and Gainetdinov, 2011), with both pathways being important for dopamine-related functions and behaviors (Beaulieu et al., 2008, 2005, 2004). We found that the levels of pAKT and pGSK3b were decreased, revealing the activation of the G protein-independent, b-arrestin2-dependent pathway. It has been demonstrated previously that this pathway is activated following treatment with amphetamine and in drug-naïve hyperdopaminergic DAT-KO mice in a D2 dopamine receptoredependent manner (Beaulieu et al., 2009, 2004). Moreover, we observed an increased level of the PP2A/AKT complex in the striatum of TAAR1-KO mice, suggesting that the increased proportion of PP2A, which is complexed with AKT in response to D2 dopamine receptor activation, may be responsible for AKT dephosphorylation (Beaulieu et al., 2008). The decrease in GSK3b phosphorylation typically leads to an increase in GSK3b activity, and accordingly, the expression level of a GSK3b substrate b-catenin was reduced in TAAR1-KO mice. To directly test the functionality of D1 and D2 dopamine receptors in vivo, we measured the locomotor response to selective D1 and D2 dopamine receptors agonists in mutant and control mice. In agreement with the enhanced D2-related signaling in striatum, quinpirole at high “post-synaptic” dose induced an increased locomotor response in TAAR1-KO mice, while the selective D1 dopamine receptor agonist SKF-82958 effects showed no difference between genotypes. Quinpirole is known to cause a biphasic locomotor response, by stimulating mainly presynaptic D2 dopamine receptors and inhibiting locomotor activity at low doses, and stimulating also postsynaptic receptors and promoting locomotor activity at high doses (Anzalone et al., 2012; Usiello et al., 2000). At relatively high dose affecting both the presynaptic and postsynaptic dopamine D2 receptors quinpirole enhanced locomotor stimulation in TAAR1-KO mice. Importantly, similar responses to quinpirole were observed in mice with D2 dopamine receptor supersensitivity due to GRK6 or norepinephrine transporter deficiency (Gainetdinov et al., 2003; Manago et al., 2012; Xu et al., 2000). It should be noted, that TAAR1 is known to modulate presynaptic D2-like dopamine receptors autoreceptors, generally producing an enhancement of autoreceptor regulation (Leo et al., 2014). It seems that in case of postsynaptic receptors we have an opposite regulation revealed by an enhanced sensitivity of postsynaptic D2-like receptors in the absence of TAAR1. Furthermore, as in the case of presynaptic regulation (Leo et al., 2014), a potential role of postsynaptic D3 dopamine receptors in the effects observed in this study cannot be excluded and future work will be necessary to address this question. Taken together, these observations strongly support the hypothesis that TAAR1-KO mice have a supersensitive dopamine system not only due to deficient D2 dopamine autoreceptor regulation (Leo et al., 2014) but also in part via an up-regulation of postsynaptic striatal D2 dopamine receptor levels and functions. Acknowledgment This study was supported in part by the research award from F. Hoffmann La-Roche, Basel, Switzerland and by the Russian Science
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Foundation (project N14-25-00065). We are grateful to Lundbeck A/G and Lundbeck USA for generously providing TAAR1 knockout mice. We thank Dr. M. Morini, D. Cantatore and F. Piccardi for their excellent technical assistance.
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Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm
Postsynaptic D2 dopamine receptor supersensitivity in the striatum of mice lacking TAAR1 Stefano Espinoza a, Valentina Ghisi b, Marco Emanuele a, Damiana Leo a, Ilya Sukhanov a, Tatiana D. Sotnikova a, c, Evelina Chieregatti a, Raul R. Gainetdinov a, c, d, * a
Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia, 16163 Genova, Italy Leibniz-Institut für Molekulare Pharmakologie (FMP), 13125 Berlin, Germany Institute of Translational Biomedicine, St. Petersburg State University, St. Petersburg 199034, Russia d Skolkovo Institute of Science and Technology (Skoltech) Skolkovo, Moscow Region 143025, Russia b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 November 2014 Received in revised form 13 February 2015 Accepted 16 February 2015 Available online 24 February 2015
Trace Amine-Associated Receptor 1 (TAAR1) is a G protein-coupled receptor (GPCR) known to modulate dopaminergic system through several mechanisms. Mice lacking this receptor show a higher sensitivity to dopaminergic stimuli, such as amphetamine; however, it is not clear whether D1 or D2 dopamine receptors and which associated intracellular signaling events are involved in this modulation. In the striatum of TAAR1 knock out (TAAR1-KO mice) we found that D2, but not D1, dopamine receptors were over-expressed, both in terms of mRNA and protein levels. Moreover, the D2 dopamine receptor-related G protein-independent AKT/GSK3 signaling pathway was selectively activated, as indicated by the decrease of phosphorylation of AKT and GSK3b. The decrease in phospho-AKT levels, suggesting an increase in D2 dopamine receptor activity in basal conditions, was associated with an increase of AKT/PP2A complex, as revealed by co-immunoprecipitation experiments. Finally, we found that the locomotor activation induced by the D2 dopamine receptor agonist quinpirole, but not by the full D1 dopamine receptor agonist SKF-82958, was increased in TAAR1-KO mice. These data demonstrate pronounced supersensitivity of postsynaptic D2 dopamine receptors in the striatum of TAAR1-KO mice and indicate that a close interaction of TAAR1 and D2 dopamine receptors at the level of postsynaptic structures has important functional consequences. © 2015 Elsevier Ltd. All rights reserved.
Keywords: TAAR1 Dopamine Striatum D2 receptor
1. Introduction Trace Amine-Associated Receptor 1 (TAAR1) is a G proteincoupled receptor (GPCR) expressed in mammalian brain in major monoaminergic regions, such as ventral tegmental area (VTA) and dorsal raphe, and their projections, including striatum, amygdala, hypothalamus and frontal cortex (Borowsky et al., 2001; Bunzow et al., 2001; Di Cara et al., 2011; Lindemann et al., 2008; Wolinsky et al., 2007). Many studies have focused on the role of TAAR1 in the modulation of dopaminergic system, and it is evident that generally TAAR1 exerts a negative control on dopaminergic activity
Abbreviations: TAAR1, Trace Amine-Associated Receptor 1; DA, dopamine; GPCR, G protein-coupled receptor; VTA, ventral tegmental area. * Corresponding author. Department of Neuroscience and Brain Technologies, Italian Institute of Technology (IIT) Via Morego 30, Genova 16163, Italy. Tel.: þ39 010 71781516; fax: þ39 010 720321. E-mail address: [email protected] (R.R. Gainetdinov). http://dx.doi.org/10.1016/j.neuropharm.2015.02.010 0028-3908/© 2015 Elsevier Ltd. All rights reserved.
(Di Cara et al., 2011; Lindemann et al., 2008; Revel et al., 2011, 2012, 2013; Sukhanov et al., 2014). TAAR1 knock out (TAAR1-KO) mice are more sensitive to the neurochemical and behavioral effect of several amphetamine derivatives (Di Cara et al., 2011; Lindemann et al., 2008; Miller, 2012; Wolinsky et al., 2007). Moreover, VTA dopaminergic neurons of TAAR1-KO mice show an increase in the firing rate (Lindemann et al., 2008) and dopamine release in the nucleus accumbens due to dysregulated function of D2 dopamine autoreceptors (Leo et al., 2014). Conversely, TAAR1 selective agonists decrease hyperdopaminergic manifestations induced by pharmacological tools or present in genetic mouse models (e.g. dopamine transporter knock out (DAT-KO) mice) (Espinoza and Gainetdinov, 2014; Revel et al., 2011, 2012, 2013). Several reports have indicated that TAAR1 is able to influence striatum-dependent behaviors, such as amphetamine-induced locomotor hyperactivity and haloperidol-induced catalepsy (Espinoza et al., 2011; Wolinsky et al., 2007). However, the mechanism by which TAAR1 alters the dopamine system, dependent in major part on the D1-and D2-
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classes of dopamine receptor function, remains unclear. It has been reported that TAAR1-KO mice have higher number of striatal D2 dopamine receptors in high affinity state (Wolinsky et al., 2007). Another study showed that in vitro TAAR1 and the long isoform of the D2 dopamine receptor were able to form an heterodimer and that the formation of this complex influences TAAR1-dependent cAMP signaling (Espinoza et al., 2011). However, it remained unclarified how D2-related signaling and functions are altered in vivo in TAAR1-KO mice. Furthermore, no information is available on the status of D1 dopamine receptor activity in TAAR1-KO mice. The aim of our study was to evaluate the functionality of D1 and D2 dopamine receptors in the striatum of TAAR1-KO mice. We found that, while D1 dopamine receptors displayed no abnormalities, D2 dopamine receptors were overexpressed and D2-related G proteinindependent signaling pathway was activated. Furthermore, these data indicating selective increase of postsynaptic D2 dopamine receptor activity in TAAR1-KO mice were directly confirmed in behavioral experiments with selective agonists. 2. Materials and methods 2.1. Animals TAAR1 knock out (TAAR1-KO) mice of mixed C57BL/6J 129Sv/J background were generated as previously described (Wolinsky et al., 2007). Animal care and treatments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (USA National Institutes of Health publication #865-23, Bethesda, MD), and the protocols were approved by the Italian Ministry of Health. All TAAR1-KO and WT littermates were obtained from heterozygous mating, and genotyping was performed in all individuals. Mice of both sexes were used in all experiments. 2.2. Western blot Western blot analyses of striatal samples to evaluate dopamine signaling were performed as previously described (Beaulieu et al., 2004). The conditions of the primary antibodies were as follows: anti phospho-AKT (Thr308) (1:3000), anti phospho-GSK3b (Ser9) (1:3000), anti b-catenin (1:1000), anti b-arrestin2 (1:2000), anti PP2A (1:5000), anti phospho-ERK1/2 (1:10,000), anti phospho-CREB (1:1000), anti phospho-DARPP32 (Thr74) (1:1000), anti-total AKT (1:5000) and anti-total GSK3b (1:5000). All antibodies were purchased from Cell Signaling (Danvers, MA, USA). Total proteins were measured by the Bio-Rad Protein Assay (Bio-Rad Laboratories, Segrate, Milan, Italy). Twenty micrograms of proteins for each sample were run on a SDS-10% polyacrylamide gel under reducing conditions and then electrophoretically transferred onto nitrocellulose membranes (GE Healthcare, Milan, Italy). Blots were blocked for 1 h at room temperature with 10% non-fat dry milk in TBS þ 0.1% Tween-20 buffer, incubated with antibodies against the phosphorylated forms of the proteins and then stripped and reprobed with antibodies against the corresponding total proteins. Immunocomplexes were visualized by chemiluminescence using the Chemidoc MP Imaging System (Bio-Rad Laboratories). Densitometric analysis was performed by normalizing the phosphoproteins level to the corresponding total form. The total form of the proteins was then normalized to the actin levels. 2.3. Immunoprecipitation Immunoprecipitations were carried out as previously described (Beaulieu et al., 2005). Briefly, tissue was homogenized at 4 C in 200 ml of lysis buffer (10 mM TriseHCl [pH 7.4], 1.0% Triton X-100) containing a cocktail of protease inhibitors (SigmaeAldrich). Immunoprecipitations were conducted overnight at 4 C with anti-AKT antibody, followed by incubation with protein G sepharose (GeHealthcare).
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[3H]-Spiperone and [3H]-Raclopride selective D2 antagonists were used. In the case of [3H]-Spiperone, membranes (150 ml; 0.5 mg/ml final proteins concentration) were incubated with six increasing doses of this radioactive ligand (50 ml; range: 0.03e1 nM), while for [3H]-Raclopride, membranes (50 ml; 1.2 mg/ml final proteins concentration) were incubated with six increasing doses of [3H]-Raclopride (100 ml; range: 1.8e60 nM). The different reactions were carried out at RT for 1 h in the case of [3H]-SCH23390 and [3H]-Raclopride in a total volume of 200 ml of assay buffer and at RT for 2 h in the case of [3H]-Spiperone in a total volume of 250 ml of assay buffer. Nonspecific binding was measured using nonradioactive flupenthixol (10 mM) for [3H]-SCH23390 binding or haloperidol (6 mM for [3H]-Spiperone binding, 50 mM for [3H]-Raclopride binding) in parallel assay tubes and was subtracted from total binding to obtain respectively specific [3H]-SCH23390 or [3H]Spiperone/[3H]-Raclopride binding. The incubations were terminated by rapid filtration over Brandel GF/C glass fiber filters washed with ice-cold assay buffer. The filters were incubated overnight in 5 ml high flash point scintillation cocktail (LefkoFluor) before their radioactivity content was counted by a liquid scintillation counter. 2.5. Real time PCR WT and TAAR1-KO mice of 3 months were sacrificed, striata were dissected and striatal tissue was dissociated for 15 min at 37 C with Pronase enzyme (Sigma) in Hank's Balanced Salt Solution (HBSS, Invitrogen). Brain samples were triturated with three glass pipettes of decreasing tip diameter and centrifuged at 900 rpm at room temperature for 5 min. To remove excess debris, cell pellets were resuspended in HBSS and filtered through a 70 mm mesh (BD Falcon, #352350). Cells-to-CT kit (Life Technology) was used to produce DNase I digested cell lysates and perform cDNA synthesis, according to manufacturer's instructions. cDNA were used for Taqman singleplex PCR. All reagents were supplied by Applied Biosystems. PCR master mix contained 1x Taqman Universal PCR Master Mix, 1x Gene Expression Assay mix, and 1 ml cDNA for a total volume of 20 ml. The following Gene Expression Assays were used: Drd2 (Assay ID Mm00438545_m1), Drd1a (Assay ID Mm01353211_m1), Gapdh (Assay ID Mm99999915_g1) and Hprt (Assay ID Mm00446968_m1). Samples were run in three replicates for each Gene Expression Assay. PCR reactions were carried out on a 7900 Thermal Cycler (Applied Biosystems) with 40 cycles of 95 C for 15 s and 60 C for 1 min. CT values for each gene were normalized to CT values for GAPDH and HPRT to obtain a relative expression level for each replicate and the five replicates were averaged together. Gene expression data were normalized by the multiple internal control gene method with GeNorm algorithm available in qBasePlus software (Biogazelle). 2.6. Locomotor activity Locomotor activity after drug or saline treatment was assessed as previously described (Gainetdinov et al., 2003). Locomotion was evaluated in an automated Omnitech Digiscan apparatus (AccuScan Instruments, Columbus, OH) under illuminated conditions. Apparatus included 4 open field monitors. Each Open Field monitor consisted of sets of 16 light beams arrayed in the horizontal X and Y axes. The hardware detected beams broken by the animal so that the software can determine the location of the mouse within the cage. Cages were divided on 4 compartments (20 cm 20 cm). Animals were tested individually for defined periods with 5-min intervals. Horizontal activities were expressed in terms of the number of beam breaks. Briefly, WT or mutant animals were injected i.p. with either saline, 2 mg/kg quinpirole or 0.5 mg/kg SKF-82958 (Chloro-APB) and placed in the activity monitor. The experimenter conducting the behavioral test was blinded for genotype/treatment of animals. Cumulative counts for 30 min (starting 10 min after drug or saline injection to avoid handling effects) were recorded for data analysis. We used both sexes in the locomotor tests, and initially analyzed male and females separately. Since we did not observe differences in effects of drugs between males and females we pooled the data together in the final analysis.
3. Results
2.4. D1 and D2 dopamine receptor saturation binding assay on striatal membranes
3.1. Lack of TAAR1 modulates D2 dopamine receptor expression in the striatum
Binding experiments were conducted according to Ghisi et al. (2009); Striatal tissues from wild-type, DAT-KO and DAT-tg mice were rapidly dissected and immediately homogenized, using a Teflonglass homogenizer, in 2 ml of lysis buffer containing 50 mM TriseHCl (pH 7.4), 120 mM NaCl, 1 mM EDTA and a cocktail of protease inhibitors at 1:1000 dilution. The homogenate was centrifuged at 1000 rpm for 10 min at 4 C to remove tissue debris and nuclei and the resulting supernatant centrifuged twice at 40,000 g for 20 min at 4 C. The final pellet was suspended in assay buffer containing 50 mM TriseHCl (pH 7.4), 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2. For D1 receptor saturation experiments, membranes (50 ml; 1.2 mg/ml final proteins concentration) were incubated with six increasing concentrations of [3H]-SCH23390 (50 ml; range: 0.5e16 nM) and 50 ml of ketanserin (100 nM), a specific antagonist of serotonin receptor, in order to prevent the binding of [3H]-SCH23390 to these receptors. For D2 receptor saturation experiments both
Previous studies have documented TAAR1 expression in several brain regions, including the striatum, with some discrepancies reported likely due to the low level of TAAR1 expression in most areas (Di Cara et al., 2011; Lindemann et al., 2008). Thus, we first confirmed TAAR1 expression in the striatum. Using RT-PCR, we documented that TAAR1 is expressed in the striatum with no detection of transcript in TAAR1-KO mice (Fig. 1A). As a first step to evaluate the consequences of TAAR1 deletion on dopamine transmission in this area, we evaluated the levels of D1 and D2 dopamine receptor mRNA expression in the striatum of mutant mice. We
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Fig. 1. D2 but not D1 dopamine receptor expression is increased in the striatum of TAAR1-KO mice. (A) mRNA levels of TAAR1 in striatum of WT and KO mice relative to the housekeeping genes GAPDH and HPRT as measured by RT-PCR. (B) mRNA levels measured with RT-PCR from striatal extracts. (C) D1 and D2 dopamine receptor levels were measured by saturation binding experiments. [3H]-SCH23390 and [3H]-Spiperone were used to quantify D1 and D2 dopamine receptors in the striatal membranes. Levels are expressed as a percentage compared to the control. Data were analyzed by GraphPad Prism software using a one site binding (hyperbola) equation. N ¼ 5 mice per group; data represent means ± SEM. *, P < 0.05. Student's unpaired two-tailed t-test.
extracted total RNA from dissected striata of WT and TAAR1-KO mice, and using qRT-PCR, we measured D1 and D2 dopamine receptor mRNA levels. As shown in Fig. 1B, D2 dopamine receptor mRNA levels were significantly increased, while D1 dopamine receptor mRNA levels were not altered. To test if dopamine receptor levels were also affected, we directly measured the number of D1 and D2 dopamine receptors by using radioligand binding. [3H]SCH23390 was utilized to selectively detect the levels of D1 dopamine receptors, while [3H]-Spiperone was used to quantify D2 dopamine receptors. As shown in Fig. 1C, similar to what was observed with mRNA, TAAR1-KO mice had a 2-fold increase in the levels of D2 dopamine receptors in the striatum, while there was no alteration in D1 dopamine receptor density. 3.2. Striatal D2 dopamine receptor signaling is enhanced in TAAR1KO mice Evidence in the literature indicates a higher proportion of striatal D2 dopamine receptors in the high affinity state in TAAR1KO mice compared to WT mice (Wolinsky et al., 2007). An increased D2 high affinity state and an increase in amphetamine sensitivity were also found in animal models of psychosis, as well as in clinical studies of schizophrenic patients (Seeman et al., 2005). These studies, together with the present observations showing selective up-regulation of striatal D2 dopamine receptors in TAAR1-KO mice, led us to analyze the status of D2 dopamine receptor-related signaling in TAAR1-KO mice. It is well known that D2 dopamine receptors signal through the Gi proteins to reduce cAMP levels. D2 dopamine receptors can also signal in a G-protein independent manner through the multifunctional protein b-arrestin2, leading to the dephosphorylation of AKT and the subsequent activation of GSK3b (Beaulieu and Gainetdinov, 2011). Both of these pathways have been shown to be important for dopamine-mediated behaviors (Beaulieu and Gainetdinov, 2011). Thus, by using western blot, we analyzed the basal phosphorylation state of proteins associated with D1 and D2 dopamine receptor activation. We observed a basal decrease in the phosphorylation of AKT and GSK3b in the striatum of TAAR1-KO mice (Fig. 2A, B), two downstream targets of barrestin2-dependent D2 dopamine receptor signaling (Beaulieu et al., 2005) with no alterations in the levels of the proteins (Fig. 2F). It has been demonstrated that D2 dopamine receptor upon its activation forms a complex with b-arrestin2, AKT and PP2A, leading to the dephosphorylation of AKT and the subsequent dephosphorylation of GSK3b thereby increasing GSK3b activity (Beaulieu et al., 2009, 2005, 2004). In agreement, we observed that the levels of b-catenin, a target of GSK3b that is degraded by the
increased GSK3 activity, were also decreased in mutant mice (Fig. 2C). Interestingly, we observed no alterations for other effectors, such as pERK1/2, pCREB and pDARPP32 (Fig. 2F), which are regulated by both the D1 and D2 dopamine receptors via G proteinrelated PKA/cAMP signaling cascade. Thus, the activation of the AKT/GSK3 pathway correlates well with the enhanced D2 dopamine receptor function that was found in TAAR1-KO mice by our group and others. To unveil the putative mechanism for the activation of this pathway, we also evaluated the levels of PP2A that is known to form the complex with AKT and the D2 receptor upon its activation. No alterations were observed in the total levels of this protein in TAAR1-KO mice (Fig. 2D). However, immunoprecipitation experiments have revealed an increase in the formation of complex between AKT and PP2A in the striatal tissue from TAAR1KO mice (Fig. 2E), suggesting that the increased interaction between these proteins may underlie the increased dephosphorylation of AKT and GSK3b (Beaulieu et al., 2005) observed in TAAR1-KO mice. 3.3. TAAR1-KO mice are hypersensitive to the locomotor effects of D2 but not D1 dopamine receptor agonist To test whether the locomotor supersensitivity to dopaminergic stimulation observed in TAAR1-KO mice is related to D2 receptor supersensitivity, we assessed the locomotor activity of animals following treatment with the selective D2-class dopamine receptor agonist quinpirole at a high “postsynaptic” dose, i.e., 2 mg/kg, i.p. (Usiello et al., 2000). At this dose, quinpirole activates not only presynaptic autoreceptors, leading to a decrease in DA transmission and global locomotor suppression, but also provides sufficient stimulation of post-synaptic D2 dopamine receptors, inducing a minor increase in forward locomotion over the levels caused by presynaptic effects but not achieving the levels observed in salinecontrols (Gainetdinov et al., 2003). It has been previously documented that mice with an enhanced D2 dopamine receptor sensitivity due to lack of GPCR kinase GRK-6 display a higher levels of locomotor activity in response to this dose of quinpirole (Gainetdinov et al., 2003). Similarly, as shown in Fig. 3A, locomotor activity following quinpirole treatment was significantly higher in TAAR1-KO compared to WT mice, directly demonstrating the increased D2 dopamine receptor functionality. By comparison, the locomotor stimulating effect of the selective D1 dopamine receptor agonist SKF-82958 at 1 mg/kg was not altered in mutant mice compared to WT animals (Fig. 3B). Taken together, these data demonstrate a significant over-activity of post-synaptic D2 receptor-mediated transmission in the striatum of TAAR1-KO mice.
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Fig. 2. D2R-related AKT/GSK3 signaling pathway is activated in TAAR1-KO mice. Densitometric western blot analysis of phosphoprotein relative levels in striatal extracts from WT and TAAR1-KO mice. Antibodies against p-Thr-308-AKT (A), p-Ser-9-GSK3b (B), b-catenin (C), PP2A (D, E) and pERK1/2, pDARPP-32, pCREB, totAKT and totGSK3 (F) were used. Antibodies for the total form of AKT and GSK3b were used as the loading controls for densitometric analysis. Immunoprecipitation of AKT revealed coimmunoprecipitation of the catalytic subunit C of PP2A from the striatal extracts of TAAR1-KO mice (E, right panel). Quantification of the interaction between AKT and PP2A, as measured by densitometric analysis (E, left panel). Average ratios of PP2A were normalized to AKT total levels and were labeled 1 in WT animals. Data represent means ± SEM. N ¼ 10e15 per group. *, p < 0.05; **, p < 0.01. Student's unpaired two-tailed t-test and repeated t-test for (F).
4. Discussion In this study, we provided the molecular mechanism responsible for the striatal dopamine hypersensitivity that has been observed in TAAR1-KO mice (Espinoza et al., 2011; Lindemann et al., 2008; Sotnikova et al., 2010; Wolinsky et al., 2007). Specifically, we demonstrated that in the striatum of TAAR1-KO mice, D2 dopamine receptors are up-regulated, and the related G protein-independent pathway (AKT/GSK3) is activated with a concomitantly increased D2-dependent locomotor activation. Taken together, these data provide further support for the close functional TAAR1-D2 dopamine receptor interaction mediated likely via receptor heterodimerization (Espinoza et al., 2011). TAAR1 function has been characterized extensively in previous years, with particular attention paid to its role in the modulation of
the dopamine system and its possible implications in psychiatric diseases (Borowsky et al., 2001; Bunzow et al., 2001; Espinoza and Gainetdinov, 2014; Lindemann and Hoener, 2005; Sotnikova et al., 2009). On one side, TAAR1-KO mice display a higher sensitivity to dopaminergic stimuli such as amphetamine and other psychostimulants (Lindemann et al., 2008; Wolinsky et al., 2007) and generally have a supersensitive dopaminergic system (Di Cara et al., 2011; Lindemann et al., 2008; Wolinsky et al., 2007) making them an interesting model relevant for schizophrenia (Wolinsky et al., 2007). On the other side, the recently developed TAAR1 selective agonists reduce manifestations of excessive dopaminergic tone caused by either pharmacological (amphetamine, cocaine) or genetic (DAT-KO mice) manipulations, further supporting the idea that TAAR1 could represent a new target for psychiatric diseases (Sotnikova et al., 2009).
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Fig. 3. D2 dopamine receptor agonist quinpirole induces an increased locomotor response in TAAR1-KO mice. Locomotor activity following 2 mg/kg quinpirole (A) and 1 mg/kg SKF-82958 (B) in WT and TAAR1-KO mice. Cumulative total distance traveled during the session (30 min, 10e40 min after injection) was calculated. Time course of quinpirole effect on locomotor activity of WT and TAAR1-KO mice is shown in the insert. Data represent means ± SEM. N ¼ 10 per group. **, p < 0.01, ***, p < 0.001. Oneway ANOVA with Dunnett's post hoc test was used.
Here, we extended these observations to an in vivo model by showing that postsynaptic D2, but not D1, dopamine receptors are up-regulated in the striatum of TAAR1-KO mice. This evidence, together with the previously reported increase in the proportion of
D2 dopamine receptors in high affinity states (Wolinsky et al., 2007), suggests that D2 dopamine receptor-mediated signaling and behavior could be markedly altered by the absence of TAAR1. To test this possibility directly, we evaluated the phosphorylation state of several proteins that are regulated by D1 and D2 receptors. It is well known that D2 dopamine receptors can signal through two major signaling pathways, the G protein-dependent, cAMP-mediated pathway and the G protein-independent, b-arrestin2dependent pathway (Beaulieu et al., 2014; Beaulieu and Gainetdinov, 2011), with both pathways being important for dopamine-related functions and behaviors (Beaulieu et al., 2008, 2005, 2004). We found that the levels of pAKT and pGSK3b were decreased, revealing the activation of the G protein-independent, b-arrestin2-dependent pathway. It has been demonstrated previously that this pathway is activated following treatment with amphetamine and in drug-naïve hyperdopaminergic DAT-KO mice in a D2 dopamine receptoredependent manner (Beaulieu et al., 2009, 2004). Moreover, we observed an increased level of the PP2A/AKT complex in the striatum of TAAR1-KO mice, suggesting that the increased proportion of PP2A, which is complexed with AKT in response to D2 dopamine receptor activation, may be responsible for AKT dephosphorylation (Beaulieu et al., 2008). The decrease in GSK3b phosphorylation typically leads to an increase in GSK3b activity, and accordingly, the expression level of a GSK3b substrate b-catenin was reduced in TAAR1-KO mice. To directly test the functionality of D1 and D2 dopamine receptors in vivo, we measured the locomotor response to selective D1 and D2 dopamine receptors agonists in mutant and control mice. In agreement with the enhanced D2-related signaling in striatum, quinpirole at high “post-synaptic” dose induced an increased locomotor response in TAAR1-KO mice, while the selective D1 dopamine receptor agonist SKF-82958 effects showed no difference between genotypes. Quinpirole is known to cause a biphasic locomotor response, by stimulating mainly presynaptic D2 dopamine receptors and inhibiting locomotor activity at low doses, and stimulating also postsynaptic receptors and promoting locomotor activity at high doses (Anzalone et al., 2012; Usiello et al., 2000). At relatively high dose affecting both the presynaptic and postsynaptic dopamine D2 receptors quinpirole enhanced locomotor stimulation in TAAR1-KO mice. Importantly, similar responses to quinpirole were observed in mice with D2 dopamine receptor supersensitivity due to GRK6 or norepinephrine transporter deficiency (Gainetdinov et al., 2003; Manago et al., 2012; Xu et al., 2000). It should be noted, that TAAR1 is known to modulate presynaptic D2-like dopamine receptors autoreceptors, generally producing an enhancement of autoreceptor regulation (Leo et al., 2014). It seems that in case of postsynaptic receptors we have an opposite regulation revealed by an enhanced sensitivity of postsynaptic D2-like receptors in the absence of TAAR1. Furthermore, as in the case of presynaptic regulation (Leo et al., 2014), a potential role of postsynaptic D3 dopamine receptors in the effects observed in this study cannot be excluded and future work will be necessary to address this question. Taken together, these observations strongly support the hypothesis that TAAR1-KO mice have a supersensitive dopamine system not only due to deficient D2 dopamine autoreceptor regulation (Leo et al., 2014) but also in part via an up-regulation of postsynaptic striatal D2 dopamine receptor levels and functions. Acknowledgment This study was supported in part by the research award from F. Hoffmann La-Roche, Basel, Switzerland and by the Russian Science
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Foundation (project N14-25-00065). We are grateful to Lundbeck A/G and Lundbeck USA for generously providing TAAR1 knockout mice. We thank Dr. M. Morini, D. Cantatore and F. Piccardi for their excellent technical assistance.
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