Absence of the GPR37/PAEL receptor impairs ... - The FASEB Journal

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Absence of the GPR37/PAEL receptor impairs striatal Akt and ERK2 phosphorylation, ⌬FosB expression, and conditioned place preference to amphetamine and cocaine Daniela Marazziti, Chiara Di Pietro, Silvia Mandillo, Elisabetta Golini, Rafaele Matteoni, and Glauco P. Tocchini-Valentini1 Istituto di Biologia Cellulare–Consiglio Nazionale delle Ricerche, Campus A. Buzzati-Traverso, Monterotondo Scalo, Rome, Italy ABSTRACT

The orphan G-protein-coupled receptor 37 (GPR37) colocalizes with the dopamine (DA) transporter (DAT) in mouse nigrostriatal presynaptic membranes, and its genetic ablation in homozygous null-mutant (GPR37-KO) mice provokes the marked increase of plasma membrane expression of DAT, alteration of psychostimulant-induced locomotor activity, and reduction of catalepsy induced by DAreceptor antagonists. We report that extracts from GPR37-KO mice displayed biochemical alterations of the nigrostriatal signaling pathways mediated by D1 and D2 dopaminergic receptors. Null-mutant mice showed an increase of the basal phosphorylation level of the D2-regulated Akt kinase. The basal phosphorylation of the D1-activated ERK2 kinase was not altered, but acute treatments with amphetamine or cocaine failed to produce its specific increase, as detected in samples from wild-type littermates. Furthermore, the chronic administration of cocaine to GPR37-KO mice did not increase the expression of the ⌬FosB transcription factor isoforms. Consistently, behavioral analysis showed that null-mutant animals did not respond to the incentive properties of amphetamine or cocaine, in conditioned place preference tests. Thus, the lack of GPR37 affects both ERK2- and Akt-mediated striatal signaling pathways, impairing the biochemical and behavioral responses typically induced by acute and chronic administration of psychostimulant drugs.—Marazziti, D., Di Pietro, C., Mandillo, S., Golini, E., Matteoni, R., and TocchiniValentini, G. P. Absence of the GPR37/PAEL receptor impairs striatal Akt and ERK2 phosphorylation, ⌬FosB expression, and conditioned place preference to amphetamine and cocaine. FASEB J. 25, 2071–2081 (2011). www.fasebj.org

is highly expressed in vertebrate oligodendrocytes and certain neuronal subsets, including the dopaminergic neurons of substantia nigra (SN) pars compacta (1–3). The absence of the receptor protein in homozygous Gpr37-null mutant mice, improves the survival of dopaminergic neurons on treatment with the Parkinson’s disease (PD)-inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and is associated with a reduction in striatal dopamine (DA) content and mild motor defects (4). GPR37 colocalizes with the DA transporter (DAT) in mouse striatal presynaptic membranes, and the receptor and transporter specifically interact in transfected cell membranes. This interaction plays a specific functional role, as the genetic ablation of GPR37 in homozygous null-mutant mice results in the marked augmentation of striatal, DAT-mediated DA uptake, with an increased expression of DAT at the synaptic plasma membrane and a significant reduction of cocaine-induced locomotor activity and catalepsy induced by DAreceptor antagonists (5). These results show the specific role of GPR37 in modulating the plasma membrane expression of DAT and thus participating in the regulation of DAT-mediated dopaminergic signaling and of functional responses to psychostimulant drugs. The neurotransmitter DA has been shown to regulate a variety of physiological functions, including control of movements but also emotion, reward, and affect (6 –9). Consequently, dysfunctions in the DA systems have been implicated in several neurological and psychiatric conditions, including PD, drug addiction, depression, and attention deficit hyperactivity disorder (for a review, see ref. 10). In nigrostriatal neurons, dopaminergic signals are mainly mediated

Key Words: DA 䡠 DAT 䡠 D1 receptor 䡠 D2 receptor 䡠 cellular signaling pathways

1 Correspondence: Istituto di Biologia Cellulare- Consiglio Nazionale delle Ricerche, Campus A. Buzzati-Traverso, Via E. Ramarini 32, I-00015 Monterotondo Scalo, Rome, Italy. Email: [email protected] doi: 10.1096/fj.10-175737 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information.

The orphan G-protein-coupled receptor 37 (GPR37) or parkin-associated endothelin B-like receptor (PAEL-R) 0892-6638/11/0025-2071 © FASEB

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by 2 classes of G-protein-coupled receptors (GPCRs), D1 and D2 receptors (11). Stimulation of striatal D2-class receptors by DA leads to specific dephosphorylation and inactivation of the serine/threonine kinase Akt on its regulatory Thr308 residue (12–14). DA also stimulates the phosphorylation of extracellular signal-regulated kinase (ERK), through multiple signaling mechanisms that involve D1 and/or D2 dopaminergic receptors (15–18). The balanced modulation of the striatal Akt- and ERK-mediated signaling pathways ultimately regulates the motor and psychobehavioral responses to DA stimulation, and its alteration is critically involved in several behavioral disorders, including drug addiction (19 –21). DAT-mediated uptake plays a fundamental role in regulating the DA concentration at nigrostriatal synapses and, therefore, the intensity and duration of dopaminergic signaling (22–24). The crucial involvement of DAT has been exemplified by experiments with genetically modified mouse strains carrying null deletions or expressing additional copies of the DAT gene (25, 26). Psychostimulant drugs of abuse, including cocaine, amphetamine, and derivatives, bind DAT, alter its synaptic membrane trafficking, and block or reverse DA uptake, thus increasing the synaptic DA concentration and the sustained activation of striatal dopaminergic signaling pathways (27, 28). In rodents and primates, the expression of certain components of the activator protein 1 (AP-1) complex, including the c-Fos, FosB, and JunB transcription factors, has been shown to be required for the induction of neuronal transcriptional responses to a variety of stimuli, including physiological and pathological dopaminergic signaling (29 –31). Similar mechanisms are involved in the specific alterations of gene expression profiles, which are elicited by repeated pharmacological stimulation and are associated with the development of druginduced reward, behavioral sensitization, and addiction (32–34). In particular, the chronic administration of various drugs of abuse, including cocaine, opiates, and nicotine, has been shown to induce the expression and nuclear accumulation of C-terminal truncated 35- to 37-kDa isoforms of ⌬FosB in distinct regions of the brain, including the nucleus accumbens and dorsal striatum (34 –36). This study investigated whether the absence of the GPR37 receptor, which is associated with increased DAT-mediated uptake and hypodopaminergia at nigrostriatal synapses, could alter the regulation of striatal dopaminergic signaling. The level of activation of D1and D2-receptor-regulated ERK2 and Akt protein kinases was analyzed and compared, in Gpr37-null mutant mice and wild-type (WT) littermates, under basal conditions and in response to acute or chronic amphetamine or cocaine administration. The striatal expression of ⌬FosB was studied, as a specific marker of gene expression modulation, following chronic cocaine treatment (37). Behavioral analysis was also carried out to assay drug-induced conditioned place preference (CPP) and behavioral sensitization, as specific experi2072

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mental paradigms for measuring reinforcing effects and addictive properties (29). The analysis of striatal extracts from GPR37-knockout (KO) mice showed a significant increase of the basal phosphorylation level of D2-receptor-regulated Akt kinase. The basal phosphorylation of D1-receptor-activated ERK2 protein was not altered, and it was not specifically augmented by acute treatments with amphetamine or cocaine. Furthermore, the chronic administration of cocaine did not increase the expression of ⌬FosB. Consistently, behavioral studies showed that null-mutant animals did not respond to the incentive properties of amphetamine or cocaine, in an unbiased CPP paradigm, and they also did not exhibit any significant drug-induced behavioral sensitization. These findings indicate that the lack of GPR37 affects the striatal signaling pathways that are mediated by ERK2 and Akt protein kinases, impairing, in particular, the biochemical and behavioral responses typically induced by acute and chronic administration of psychostimulant drugs.

MATERIALS AND METHODS Reagents Okadaic acid, cocaine hydrochloride, and d-amphetamine sulfate were obtained from Sigma-Aldrich (St. Louis, MO, USA). The anti-phospho-ERK1/2 (ERK1: phospho-Thr202/ pospho-Tyr204; ERK2: pospho-Thr185/pospho-Tyr187), antitotal-ERK, anti-total-Akt, and anti-total DARPP32 were obtained from Cell Signaling Technology (Beverly, MA, USA); the anti-phospho-Akt (phospho-Thr308) and anti-FosB (H237) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); the anti-phospho-DARPP32 (phospho-Thr34) was from PhosphoSolutions (Aurora, CO, USA); the anti-␣-tubulin (clone DM1A) was from Sigma-Aldrich. Horseradish peroxidase-linked anti-mouse secondary antibody was from Amersham Biosciences–GE Healthcare (Piscataway, NJ, USA), and horseradish peroxidase-linked anti-rabbit secondary antibody was from Cell Signaling Technology. Experimental animals Homozygous GPR37-KO male mice and their WT littermates (10 –16 wk old for drug treatments and Western blot analysis; 8 mo old for CPP experiments) were used (4). All mice were bred from heterozygous crossings (mixed genetic background: 75% C57BL/6J; 25% 129P2/OlaHsd). The individual mouse genotype was determined by PCR analysis of tail genomic DNA, as described previously (4). After weaning, mice were housed by litter of the same sex, 3–5 mice/cage in individually ventilated caging racks, and maintained in a temperature-controlled room at 21 ⫾ 1°C, on a 12-h lightdark cycle (lights on at 7:00 AM) with food and water available ad libitum. Each cage was equipped with sawdust contained in nest paper bags (Scobis Uno bags; Mucedola, Settimo Milanese, Italy) and a red transparent igloo house (Tecniplast, Buguggiate, Italy), as forms of environmental enrichment. All animals were born and bred in a specific pathogen-free facility and were subjected to experimental protocols approved by the Veterinary Department of the Italian Ministry of Health, according to the ethical and safety rules and

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guidelines for the use of animals in biomedical research provided by the relevant Italian laws and European Union directives (86/609/EC and subsequent). Drug administration Cocaine hydrochloride and d-amphetamine sulfate were dissolved in 0.9% NaCl vehicle solution (2 mg/ml and 0.4 mg/ml, respectively). For acute treatments, mice were injected i.p. with amphetamine (4 mg/kg body weight; WT: n⫽14; GPR37-KO: n⫽16) or saline (WT: n⫽16; GPR37-KO: n⫽15) and sacrificed at 10, 30, or 60 min after injection. Another group of mice was injected i.p. with cocaine (20 mg/kg body weight; WT: n⫽16; GPR37-KO: n⫽16) or saline (WT: n⫽14; GPR37-KO: n⫽15) and sacrificed at 10, 20, or 40 min after injection. For chronic cocaine treatment, mice were injected with saline (WT: n⫽6; GPR37KO: n⫽5) or cocaine (20 mg/kg; WT: n⫽5; GPR37-KO: n⫽5), 1⫻/d for 7 d, and sacrificed 18 –24 h after the last injection (29, 38). For repeated administration during the CPP procedure, mice were injected i.p. with either saline, 4 mg/kg/d amphetamine, or 20 mg/kg/d cocaine for 8 d, on alternate days (Fig. 5A). In a different experiment, a lower dose of amphetamine (0.5 mg/kg/d) was also tested, using the same CPP procedure (Supplemental Fig. S3). Western blot analysis Mice were sacrificed by cervical dislocation, and the whole left striatum was rapidly dissected out, as described previously (39). Tissue samples were homogenized in boiling 10 mg/ml SDS solution, supplemented with 2 ␮M okadaic acid, as previously reported (13). All protein extracts were quantified using a detergent-compatible protein assay (DC protein assay; Bio-Rad Laboratories, Hercules, CA, USA), resolved by SDS/PAGE (20 –50 ␮g total protein/sample), and analyzed by Western blot. Protein antigens were labeled with primary antibodies (dilution ratios: anti-phospho-Akt, 1:200; anti-total-Akt,, 1:1000; anti-phospho-DARPP32, 1:1000; anti-total-DARPP32, 1:1000; anti-phospho-ERK1–2, 1:1000; antitotal-ERK1–2, 1:1000; anti-FosB, 1:1000; and anti-␣-tubulin, 1:1000), and horseradish peroxidase-conjugated secondary antibodies, according to the producer’s instructions. Blotted membranes were processed for enhanced chemiluminescence detection onto Hyperfilm ECL radiographic film (Amersham Biosciences–GE Healthcare). The chemiluminescent signal of immunoreactive bands was imaged and quantified with a Kodak Gel Logic 2200 Imager and Molecular Imaging software system (Carestream Health, Rochester, NY, USA). Average relative levels of phosphorylated protein kinases and DARPP32 were calculated as ratios to respective mean levels of total protein. Average relative levels of total ⌬FosB and DARPP32 proteins were calculated as ratios to respective mean levels of ␣-tubulin. The mean levels of total Akt and ERK2 proteins did not vary during the 60-min time period studied, following the acute administration of psychostimulant drugs (Supplemental Fig. S2). When appropriate, results were normalized to mean values obtained from control groups, injected with saline only, as indicated in figure legends. CPP test Place conditioning was performed in a 3-compartment apparatus (40) made of light gray polyvinyl chloride. Two custom-made chambers (15⫻20⫻40 cm) were connected by an alley (9⫻20 cm) that had 2 openings (5⫻5 cm) with IMPAIRED STRIATAL SIGNALING IN GPR37-KO MICE

sliding doors. In each chamber, 2 triangular solid shapes were arranged to form 2 different patterns (always covering the same surface of the chamber) that served as conditioned stimuli. The following procedure was followed (Fig. 5A): on d 1, mice were placed in the central compartment and left free to explore the entire apparatus for 20 min (pretest phase). From d 2 to 9, mice underwent a conditioning procedure. On d 2, 4, 6, and 8, they were administered i.p. either saline, 0.5 or 4 mg/kg amphetamine, or 20 mg/kg cocaine, as detailed above, and they were then confined for 40 min in the preassigned drug-paired side (randomly either A or B, unbiased procedure); on d 3, 5, 7, and 9, all mice received a saline injection and were left for 40 min in the unpaired side (either B or A). On d 10 (test), mice were placed in the apparatus for 20 min and were free to explore all 3 compartments. Locomotor activity and time spent in each compartment were recorded and automatically measured by a camera placed on the ceiling and connected to a video-tracking system (Viewpoint, Lyon, France). Behavioral sensitization to the psychomotor effects of amphetamine and cocaine was also assessed, by measuring the distance traveled during each 40-min session of conditioning (d 2–9), and data were analyzed for the first 10-min period each day. Mice that scored ⫾ 2 sd from their group mean for time spent in each of the 3 compartments during the pretest, or that scored ⬎⫾400 s in the difference between compartment A and B, thereby showing a preference for one of the compartments, were excluded from the analysis. CPP was evaluated by measuring percentage of time spent during the test in the drug-paired compartment vs. total time spent in paired and unpaired compartments (% preference), and time spent in the drug-paired compartment during pretest and test phases. Statistical analysis ANOVA and t tests of behavioral and Western blot data were performed with StatView 5.0 (SAS Institute, Cary, NC, USA) and Prism 5.0 (GraphPad Software, La Jolla, CA, USA). Independent variables were genotype, treatment group, or time after injection. Post hoc tests were applied when appropriate. Level of significance was set at P ⬍ 0.05.

RESULTS GPR37-KO mice show increased levels of Akt (Thr308) phosphorylation under basal conditions Gpr37-null mutant mice show an increased expression of DAT at the striatal synaptic plasma membrane, with an enhancement of DAT-mediated uptake of DA and a moderate reduction of its striatal levels (4, 5, 41). Related results have been reported for transgenic mice overexpressing the DAT protein, with marked hypodopaminergia and specific alterations of the D2-receptor-mediated Akt/GSK3 signaling cascade (26, 39). We studied the alterations of D1- and D2-receptormediated signaling pathways in GPR37-KO mice, by measuring the relative phosphorylation levels of ERK2 and Akt protein kinases under basal conditions. Whole striatum samples from untreated WT and GPR37-KO adult male littermates were rapidly 2073

phetamine results in a further increase of locomotor activity, while acute cocaine treatment produces lower motor effects compared to WT littermates (4, 5). Concurring data have been reported for DAT-overexpressing transgenic mice (26, 39). To characterize signaling events underlying these behaviors, we examined the effects of acute amphetamine or cocaine treatments on the modulation of Akt and ERK2 phosphorylation, in WT and GPR37-KO mice. The average relative levels of phosphorylated proteins were measured by Western blot analysis of striatal extracts obtained from littermates of the two genotypes, at different time points following the acute drug treatment. All values were normalized to those obtained from corresponding control groups, injected with the saline vehicle only (Fig. 2 and 3). The samples from amphetamine-treated (4 mg/kg) WT animals Figure 1. Relative levels of striatal phospho-Akt (Thr308) and phospho-ERK2 in WT and GPR37-KO mice under basal conditions. Western blots of immunolabeled Akt (A) or ERK2 (B) were used to measure and compare phospho-protein kinase levels in the whole striatum of Gpr37-null mutant mice (⫺/⫺) and their WT littermates (⫹/⫹) bred under basal conditions. Average relative levels of phosphorylated Akt (p-Akt; A) or ERK2 (p-ERK2; B) were calculated as ratios to mean levels of respective total protein. Graphs represent average ⫾ se values (n⫽5– 6 mice/group). **P ⬍ 0.01 vs. ⫹/⫹; t test.

dissected, homogenized, and subjected to Western blot with antibodies specific for the total protein kinases or their phosphorylated protein residues (Akt: phospho-Thr308; ERK2: phospho-Thr185 and phospho-Tyr187), as reported previously (13). The average relative level of phospho-Akt was found to be significantly increased in the samples from Gpr37null mutants (unpaired t test: t(10)⫽⫺3.41, P⬍0.01; Fig. 1A), while the phospho-ERK2 level was not appreciably changed (unpaired t test: t(8)⫽0.43, n.s.; Fig. 1B), when compared to the corresponding values obtained from WT control samples. These results indicate the existence of a basal alteration of Akt signaling in animals lacking the GPR37 receptor. The average phosphorylation level of the Thr34 residue of the DA- and cAMP-regulated 32-kDa protein (DARPP32), a regulator of striatal dopaminergic signaling in postsynaptic medium spiny neurons (42), was also significantly reduced in the samples from GPR37-KO mice, with no appreciable change of its total level (unpaired t test: t(11)⫽3.22, P⬍0.01; Supplemental Fig. S1). GPR37-KO mice show altered levels of Akt (Thr308) phosphorylation following acute amphetamine stimulation Dopaminergic psychostimulant drugs are potent inducers of hyperactivity in rodents, and they activate DAreceptor-mediated pathways, inducing specific modifications of Akt and ERK2 phosphorylation levels (41). In GPR37-KO mutants the acute administration of am2074

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Figure 2. Striatal phosphorylation of Akt (Thr308) and ERK2 after acute amphetamine treatment are affected in GPR37-KO mice. Time-course study of striatal phospho-Akt (A) and phospho-ERK2 (B) levels, following an acute i.p. injection of amphetamine (4 mg/kg). Whole left striatum of Gpr37-null mutant mice (⫺/⫺) and their WT littermates (⫹/⫹) was collected at 10, 30, or 60 min after injection, and protein extracts were used for Western blot analysis. Average relative levels of phosphorylated Akt (p-Akt) and ERK2 (p-ERK2) were calculated as ratios to mean levels of total Akt or ERK2 proteins. Results are represented in arbitrary units normalized to average phospho-Akt or phospho-ERK2 levels observed in saline vehicle-treated mice of the same genotype. Graphs represent average ⫾ se values (n⫽3–5 mice/group). A) *P ⬍ 0.01, **P ⬍ 0.001 vs. ⫹/⫹; Fisher’s PLSD post hoc test. B) *P ⬍ 0.05, ***P ⬍ 0.0005 vs. ⫹/⫹; ##P ⬍ 0.005, ###P ⬍ 0.0001 vs. 10 min; Fisher’s PLSD post hoc test.


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augment of the average phospho-ERK2 level was detected in the null-mutant samples, which showed, instead, an overall decrease, below control values, at all sampled time points (Fig. 2B; ANOVA detected a significant effect of genotype ⫻ time after injection: F(1,20)⫽13.63, P⬍0.0005). These findings indicate that in GPR37-KO mice, the increased basal level of phospho-Akt may also be associated with alterations of ERK2 activity, in response to acute amphetamine administration (13, 16, 41). GPR37-KO mice do not show an increase of ERK2 phosphorylation following acute cocaine treatment

Figure 3. Striatal ERK2 phosphorylation after acute cocaine treatment is inhibited in GPR37-KO mice. Time-course study of striatal phospho-Akt (A) and phospho-ERK2 (B) levels, following an acute i.p. injection of cocaine (20 mg/kg). Whole left striatum of Gpr37-null mutant mice (⫺/⫺) and their WT littermates (⫹/⫹) was collected at 10, 20, or 40 min after injection, and protein extracts were used for Western blot analysis. Average relative levels of phosphorylated Akt (p-Akt) and ERK2 (p-ERK2) were calculated as ratios to mean levels of total Akt or ERK2 proteins. Results are represented in arbitrary units normalized to average phospho-Akt or phospho-ERK2 levels observed in saline vehicle-treated mice of the same genotype. Graphs represent average ⫾ se values (n⫽3– 6 mice/group). *P ⬍ 0.05 vs. ⫹/⫹; ##P ⬍ 0.005 vs. 10 min; Fisher’s PLSD post hoc test.

exhibited the expected variation (13, 41) of the average level of phospho-Akt, with an initial decrease below the control value (within 10 min after injection), followed by a marked increase (within 30 min after injection) and a later decrease to control levels, within 60 min after injection (Fig. 3A and Supplemental Fig. S2A). Phospho-ERK2 levels initially rose (within 10 min after injection) and then significantly decreased (within 30 min after injection) below the control value (Fig. 3B and Supplemental Fig. S2C), as reported previously (16). Samples from Gpr37-null mutants displayed significant differences in the modulation of Akt and ERK2 phosphorylation. The average phospho-Akt level was initially higher than the corresponding control value (within 10 min after injection) and it showed a subsequent decrease (within 30 min after injection) and no later significant variation (Fig. 2A, ANOVA detected a significant effect of the interaction genotype ⫻ time after injection: F(1,20)⫽27.53, P⬍0.0001). Notably, no IMPAIRED STRIATAL SIGNALING IN GPR37-KO MICE

Several studies have demonstrated the critical role of ERK2 activation in mediating cocaine-induced intracellular signaling. For instance, both repeated and acute cocaine administration is known to induce ERK2 phosphorylation in the rodent striatum (15, 16, 19, 24, 43, 44). We performed a time-course study of the modulation of Akt and ERK2 phosphorylation, in striatal samples from both Gpr37-null mutants and WT littermates, following acute cocaine injection (20 mg/kg). Both genotype groups did not show significant variations of the striatal phospho-Akt levels, as compared to saline control values (Fig. 3A and Supplemental Fig. S2B). The levels of phospho-ERK2 rapidly increased, in WT mice, (within 10 min after injection) and then gradually declined to control levels, by 40 min after injection, as reported previously (44); Fig. 3B and Supplemental Fig. S2D). Instead, no significant variation of ERK2 phosphorylation was detected in the samples obtained from the GPR37-KO animals, during the entire course of the analysis (Fig. 3B; ANOVA detected a significant effect of the interaction genotype ⫻ time after injection: F(1,20)⫽7.62, P⬍0.005). Thus, the genetic ablation of the GPR37 receptor is also associated with a substantial impairment of the typical, cocaine-induced, increase of ERK2 phosphorylation. GPR37-KO mice show decreased ⌬FosB expression on chronic cocaine treatment Chronic cocaine stimulation specifically elicits expression of a C-terminal truncated splice isoform of the FosB transcription factor (⌬FosB) in mammalian striatal tissue (35, 36, 45). We analyzed the expression of ⌬FosB in striatal samples from GPR37-KO mice and WT littermates after 1 wk chronic treatment with cocaine (20 mg/kg/d; see Materials and Methods) or saline vehicle solution. Whole striata were dissected and homogenized (18 –24 h after the end of treatment), and the relative levels of ⌬FosB protein were measured by Western blot. Values were averaged for each group and normalized to the mean of the relevant saline control group. The Gpr37-null mutants showed a significant reduction in the levels of ⌬FosB expression (unpaired t test: t(3)⫽⫺3.38, P⬍0.05; Fig. 4), whereas a marked increase was measured in WT animals (unpaired t test: t(5)⫽⫺8.34, P⬍0.0005; Fig. 4), indicating that GPR37 2075

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Figure 4. Striatal ⌬FosB expression after chronic cocaine treatment is inhibited in GPR37-KO mice. A) Representative Western blots of ⌬FosB and ␣-tubulin in whole left striatum extracts of Gpr37-null mutant mice (⫺/⫺) and their WT littermates (⫹/⫹), following chronic treatment with cocaine (20 mg/kg/d, for 7 d). Molecular mass of proteins is given at right. S, saline; CC, chronic cocaine treatment. B) Average ratio of ⌬FosB to ␣-tubulin was quantified and normalized to the mean of saline-treated mice of the same genotype (n⫽5 mice/group). *P ⬍ 0.05, ***P ⬍ 0.0005 vs. saline.

(46) is critically involved in regulating the cocainestimulated expression of ⌬FosB. GPR37-KO mice do not respond to the incentive properties of amphetamine and cocaine in a CPP test Rodents and primates can readily establish preference for an environment associated with cocaine, amphetamine, and other psychostimulants, thus allowing the use of specific CPP experimental paradigms to analyze and indirectly measure positive reinforcing effects and potentially addictive properties of drugs (47). To investigate possible alterations in GPR37-KO mice of the dopaminergic drug-induced reinforcement, genotypematched male littermates were tested for their place

preference for a compartment previously associated with the effects of amphetamine or cocaine administration during specific CPP experimental procedures (Fig. 5A). As shown in Fig. 5B, Gpr37-null mutants did not respond to the incentive properties of amphetamine and cocaine, while WT mice showed a clear preference for the compartment paired with the drugs (ANOVA effects: genotype, F(1,71)⫽1.40, n.s.; treatment, F(2,71)⫽4.90, P ⬍ 0.05; genotype ⫻ treatment, F(2,71)⫽3.31, P⬍0.05). No difference between genotypes or treatment groups was observed in the time spent in the drug-paired compartment throughout the pretest phase (Fig. 5C). During the test, WT mice spent significantly more time in the compartment associated with amphetamine or cocaine (ANOVA for treatment effect: F(2,33)⫽7.40, P⬍0.005), whereas no

Figure 5. Lack of psychostimulant-induced place preference in GPR37-KO mice. A) Timeline of CPP experimental procedure. On d 1 (pretest) and d 10 (test), Gpr37-null mutant mice (⫺/⫺) and their WT littermates (⫹/⫹) were free to explore the whole 3-compartment apparatus for 20 min. On d 2, 4, 6, and 8, mice were administered saline, amphetamine (4 mg/kg), or cocaine (20 mg/kg) and placed for 40 min in their respective drug-paired compartment. On d 3, 5, 7, and 9, all mice were administered saline and placed in the drug-unpaired compartment. B) Place preference was assessed during the test as the percentage of time spent in the drug-paired compartment over the total time spent in both drug-paired and -unpaired compartments. Dotted line indicates 50% of time spent in each compartment (i.e., no compartment preference. **P ⬍ 0.001 vs. saline; Fisher’s PLSD post hoc test. C) Time spent in the drug-paired compartment before and after conditioning (i.e., during pretest and test; n⫽8 –17 mice/group). *P ⬍ 0.05, **P ⬍ 0.001 vs. ⫹/⫹ saline and pretest; t test. 2076

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difference between treatments was observed in GPR37-KO mice (ANOVA for treatment effect: F(2,38)⫽0.32, n.s.). An additional lower dose of amphetamine was also tested in the CPP procedure. Neither the WT nor the GPR37-KO animals showed a significant preference for the compartment paired with 0.5 mg/kg dose of amphetamine (Supplemental Fig. S3A). However, a trend to spend more time in the drug-paired compartment was observed for both genotypes (Supplemental Fig. S3B). During the CPP procedure, it was possible to monitor for 40 min the locomotor activity after each saline or drug injection (Fig. 5A), thus allowing the assessment of behavioral sensitization to the psychomotor effects of amphetamine and cocaine. GPR37-KO mice did not show a significant increase of locomotor activity over 4 d of daily repeated drug administration (repeated measures ANOVA significant effects: treatment, F(2,85)⫽109.70, P⬍0.0001; day, F(3,255)⫽11.28, P⬍0.0001; day ⫻ genotype, F(3,255)⫽8.59, P⬍0.0001; day ⫻ treatment, F(6,255)⫽12.27, P⬍0.0001; day ⫻ genotype ⫻ treatment, F(6,255)⫽2.36, P⬍0.05; Fig. 6A). Only WT mice showed the development and expression (Fig. 6) of behavioral sensitization to the psychomotor effects of amphetamine and cocaine (ANOVA effects: genotype, F(1,86)⫽13.81, P⬍0.005; treatment, F(2,86)⫽7.73, P⬍0.001, genotype ⫻ treatment, F (2,86) ⫽4.02, P⬍0.05; Fig. 6B). Interestingly, the analysis of the locomotion on d 2 confirmed that GPR37-KO mice show a significantly enhanced activity after an acute administration of amphetamine, at either low (0.5

mg/kg; Supplemental Figure S3C) or high (4 mg/kg; Fig. 6A) dose.

DISCUSSION This study revealed that the genetic ablation of GPR37 induces specific alterations of the nigrostriatal dopaminergic signaling pathways in the mouse brain and affects the neurochemical and behavioral responses that are elicited by acute and chronic administration of dopaminergic drugs. Under basal conditions, striatal extracts from Gpr37-null mutants display an increased phosphorylation of the D2-receptor-regulated Akt kinase, while the phosphorylation of the D1-activated ERK2 kinase is not significantly altered (Fig. 1). The null-mutant animals also show a basal dephosphorylation of DARPP32 (Supplemental Fig. S1), a crucial postsynaptic activator of D1- and D2-receptor-mediated signaling, which is specifically expressed in striatal medium spiny neurons (42). These findings are consistent with the results reported for DAT-KO mice (48) and a recently produced DAT-overexpressing transgenic strain (39). In addition, the latter model exhibits a strong increase of the plasma membrane expression of DAT and of the DATmediated DA uptake at nigrostriatal synapses, and these alterations are also present, although on a reduced scale, in the GPR37-KO animals (5). It is possible to hypothesize that GPR37 can directly or indirectly participate in the regulation of the trafficking and expres-

Figure 6. Lack of behavioral sensitization to psychostimulants in GPR37-KO mice. A) Locomotor activity in the first 10 min, during conditioning days (d 2–9), after administration of saline, amphetamine, or cocaine, as described in Fig. 5. No effect of genotype or treatment on locomotor activity was observed during the days of saline administration. *P ⬍ 0.05, **P ⬍ 0.005, ***P ⬍ 0.0001 vs. d 2; °P ⬍ 0.05, °°P ⬍ 0.0005 vs. d 4; ##P ⬍ 0.005 vs. ⫹/⫹ d 2; t test. B) Difference between d 8 and 2 in distance traveled in the first 10 min after amphetamine or cocaine administration (n⫽11–18 mice/group). **P ⬍ 0.0005, ***P ⬍ 0.0001 vs. saline; #P ⬍ 0.05, ##P ⬍ 0.01 vs. ⫹/⫹; Fisher’s PLSD post hoc test. IMPAIRED STRIATAL SIGNALING IN GPR37-KO MICE

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sion of DAT at the synaptic plasma membrane and, consequently, of the level of activation of striatal dopaminergic signaling (5). In transgenic mice, the overexpression of DAT in SN neurons provokes marked hypodopaminergia, up-regulation of striatal D1 and D2 receptors, and hyperphosphorylation of Akt (39). The reduction in the DA level that is detected in GPR37-KO animals is linked to decreased antagonist affinity and a limited increase in the number of dopaminergic receptors (4, 5, 49), but it is still associated with an augment of the basal phosphorylation of Akt (Fig. 1), which can ultimately be responsible for the reported reduction of certain locomotor activities (4, 49). In GPR37-KO mice, the basal reduction of nigrostriatal dopaminergic input could also induce an impaired gating of glutamatergic drive by medium spiny neurons, leading to an overall alteration of motor responses (50). The occurrence of a basal alteration of Akt phosphorylation may also hamper the normal biochemical response to acute amphetamine administration (Fig. 2A and Supplemental Fig. S2A), in agreement with the reported persistence of the drug-induced stimulation of locomotor activity in GPR37-KO mice, compared to WT littermates (4). These findings are consistent with the data obtained from DAT-overexpressing mice (26) and support the hypothesis that the regulation of the phospho-Akt level is more sensitive to variations of the synaptic DA tone than of the D2-receptor number (39). Notably, GPR37-KO mutants also exhibit a marked inhibition of D1-receptor-mediated ERK2 phosphorylation in response to acute treatment with amphetamine or cocaine (Figs. 2B and 3B and Supplemental Fig. S2), indicating that the basal hyperphosphorylation of Akt may also be associated with specific alterations of ERK2 signaling, which is normally elicited by psychostimulants (13, 16). The ERK pathway plays a crucial role in the establishment and development of long-lasting behavioral effects of psychostimulants and other drugs of abuse (51). ERK-mediated signaling regulates the time course of neuronal expression and activation of components of the AP-1 complex, including the c-Fos, FosB, and JunB transcription factors, which are required for the transcriptional responses to a variety of stimuli, including physiological and pathological dopaminergic signaling (29 –31). In WT mice, chronic exposure to cocaine results in increased expression and gradual long-term accumulation of ⌬FosB (33, 35). This effect is significantly reduced in Gpr37-null mutants (Fig. 4), confirming the relevance of the D1-receptor/ERK2 pathway in modulating the drug-induced striatal accumulation of specific transcription complex components. The analysis of behavioral data additionally supports the hypothesis that the absence of GPR37 may strongly alter D1-receptor-mediated signaling, in response to acute and chronic drug treatment. In fact, in GPR37-KO animals, the inhibition of striatal 2078

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⌬FosB expression and nuclear accumulation is associated with the impaired induction of CPP by cocaine or amphetamine (Fig. 5 and Supplemental Fig. S3) and of behavioral sensitization to both drugs (Fig. 6 and Supplemental Fig. S3; refs. 44, 52, 53). Consistently with the above findings, DAT-overexpressing mice show increased amphetamine-induced locomotor activity, impairment of CPP at higher doses of amphetamine, and a reduced response to natural rewards (26). Conversely, DAT-KO mice have been found more sensitive to the rewarding effects of amphetamine (54). DAToverexpressing mice also exhibit a significant preference for the compartment paired to a lower dose (0.5 mg/kg) of amphetamine (26), unlike results from the analysis of GPR37-KO mice (Supplemental Fig. S3). This could be due to the lower level of DAT overexpression at synaptic plasma membranes in the latter null-mutant strain (4), as well as to additional, unknown alterations induced by the absence of GPR37, e.g., in transducing responses to yet unidentified specific ligands. The comparison of altered cocaine reward effects, as exhibited by GPR37-KO animals, has to take into account the more controversial data obtained from different studies with DAT-null mutants. It has been reported that cocaine-induced CPP is not significantly altered (52), although an increasing trend has also been detected (55), or it has been found to decrease at certain cocaine doses (56). It has also been suggested that CPP to amphetamine and cocaine may be mediated by the combined involvement of both DAT and serotonin transporter molecules (46, 55). In the present study, the mouse locomotor activity could be monitored during the CPP procedure, thereby allowing the analysis of the amphetamine and cocaine effects after both acute (d 2) and repeated administration regimens. When compared to their WT littermates, GPR37-KO mice displayed increased locomotor activity after acute administration of amphetamine at both lower and higher doses (Fig. 6A and Supplemental Fig. S3C, d 2) and unaltered locomotion after cocaine acute administration (Fig. 6A), as previously reported (4). The null-mutant animals also exhibited a lack of behavioral sensitization to the psychomotor effects of both drugs after 4 injections on alternate days (Fig. 6). These motor effects are linked to a significantly diminished expression of striatal ⌬FosB after chronic cocaine administration (Fig. 4). A similar link was observed in FosB-KO mice showing altered cocaine-induced CPP and locomotor sensitization (29). However, it should be noted that the evaluation of the rewarding and reinforcing effects of amphetamine and cocaine in mouse mutant models can be achieved using different paradigms (self-administration, CPP, behavioral sensitization, hyperlocomotion) that do not necessarily refer to the same mechanism of action and thus the same behavioral output (57). We hypothesize that the absence of GPR37, producing the overexpression of DAT molecules on the


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presynaptic membrane (5), can result in DA level variations, which may alter the postsynaptic ERK/ FosB-mediated signaling and consequently change the psychomotor and reinforcing effects of acute and chronic amphetamine and cocaine. The above findings suggest that the genetic or biochemical inhibition of GPR37 functions may be instrumental in the development and application of novel in vivo models aimed at reducing reward, behavioral sensitization, and effects induced by psychostimulants of abuse. The experimental blocking of GPR37 could also be applied to the modeling of other drug-induced motor dysfunctions that are specifically associated with the ERK-stimulated striatal expression of ⌬FosB, such as the dyskinetic side effects of L-DOPA in the treatment of Parkinson’s disease (58, 59). The lack of GPR37 differentially affects ERK and Akt functions, and it is associated with an impairment of the biochemical and behavioral responses typically induced by acute and chronic administration of psychostimulant drugs. Further experimental analysis will be required to identify the protein components that actually interact with and are affected by GPR37, in vivo. The direct interaction between DAT and GPR37 has not yet been demonstrated, although the two proteins have been shown to colocalize at the nigrostriatal presynaptic membranes and to coimmunoprecipitate from cultured cell extracts (5). It cannot presently be ruled out that GPR37 might modulate DAT trafficking and metabolism via specific interaction with other presynaptic proteins that participate in the complex regulation of DAT function, like, for instance, the mainly presynaptic short isoform of D2 receptor, which is involved in the regulation of DA synthesis and release (60). Experimental work with transfected cells has revealed that the trafficking of GPR37 to the plasma membrane is increased when it is coexpressed with the long isoform of the D2 receptor, D2L (61). The two proteins specifically coimmunoprecipitate, and the interaction with GPR37 effectively increases the affinity of D2L for specific antagonists (5, 61). These results raise the possibility of a functional interaction in vivo of GPR37 with D2L, which is predominantly expressed in the nigrostriatal postsynaptic membranes (60). In GPR37-KO animals, the basal increase of Akt phosphorylation is associated with a significant dephosphorylation of DARPP32, with no sensible changes of its total level (Supplemental Fig. S1). DARPP32 is a specific marker of striatal medium spiny neurons, and its expression and activation are finely regulated by signaling cascades mediated by both D1 and D2 postsynaptic receptors (42). It would be important to investigate whether GPR37 may also be expressed in striatal postsynaptic neurons and so directly participate in regulating postsynaptic dopaminergic systems, as hypothesized, for instance, for the trace amine-associated receptor 1 that binds the DA metabolite 3-methoxytyramine IMPAIRED STRIATAL SIGNALING IN GPR37-KO MICE

(62, 63). Future research efforts will also be directed to functionally characterize the in vivo interactions among GPR37, DAT, and other DAT-interacting proteins and to clearly define the role of GPR37 in modulating DAT trafficking and metabolism, in response to acute and chronic administration of psychostimulant drugs. The authors are grateful to C. Gross and G. Di Segni for critical reading of the manuscript. The authors greatly thank G. Di Franco, G. D’Erasmo, and A. Ventrera for excellent technical assistance; A. Ferrara and T. Cuccurullo for secretarial work. This study was supported by Italian Ministry of Research grants (FIRB-Internazionale G. Armenise Harvard Foundation 2005, FIRB-Idee Progettuali 2005, PRIN-Cofin. 2007), and EU-Framework Programmes 6 and 7 (EURASNET, EUMODIC, and PHENOSCALE contracts).

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