TY - JOUR
AU1 - Niu, Bing
AU2 - Liu, Peipei
AU3 - Shen, Minjie
AU4 - Liu, Cao
AU5 - Wang, Li
AU6 - Wang, Feifei
AU7 - Ma, Lan
AB - Abstract Impairments in social behaviors are features of a number of psychiatric diseases associated with subtle alterations in the medial prefrontal cortex (mPFC) circuitry. G protein-coupled receptor kinase (GRK) 5 is widely expressing in the cortex, however, its role in regulation of the mPFC activity and the development of social behaviors and psychiatric disorders is unclear. Here, we found that GRK5 dificiency in mice caused social behavior impairments. Further morphological, electrophysiological, and biochemical analyses showed abnormal postsynaptic ultrastructure, impaired excitatory synaptic transmission, the increased association of raptor with mTOR, and overactivated mTORC1-S6K signaling in the mPFC of Grk5−/− mice. Conditional knockdown of GRK5 in the mPFC caused impairments in social interaction and social novelty recognition behaviors; whereas selectively overexpressing GRK5 in the mPFC of Grk5−/− mice rescued the social novelty recognition phenotype. Inhibition of the overactivated mTORC1-S6K signaling pathway by rapamycin or mGluR5 antagonist ameliorated the deficiency of the excitatory synaptic transmission in the mPFC and the social recognition of Grk5−/− mice. These results indicate that GRK5 is critical for maintaining normal mTORC1 signaling and connectivity in mPFC, and normal social behavior. GRK5, mPFC, mTORC1, social recognition Introduction G protein-coupled receptor kinases (GRKs) phosphorylate G protein-coupled receptors upon their activation and feedback inhibit receptor signaling. GRK5 is an important member of GRKs and exerts multiple cellular functions in cell cycle, apoptosis, and cytoskeleton regulation by regulating distinct substrates (Sorriento et al. 2008; Chen et al. 2010; Chen et al. 2011; Michal et al. 2012; So et al. 2012). GRK5, as the most commonly expressed GRK in the myocardial tissue of transplanted hearts, has been proposed to be a potential therapeutic target for the treatment of heart failure (Aguero et al. 2009). Studies in human subjects have suggested GRK5 as a key player in the development of cardiac hypertrophy and heart diseases (Dzimiri et al. 2004). The expression levels of GRK5 inversely correlated with left ventricular end-systolic and diastolic diameters (Monto et al. 2012). GRK5 is also involved in muscarinic regulation of airway responses. It was reported that the airway smooth muscle relaxation resulting from adrenergic stimulation was diminished in GRK5 mutants (Walker et al. 2004). Recently, genome-wide association studies identified GRK5 as a Type 2 diabetes mellitus loci in Chinese Hans (Li et al. 2013; Xia et al. 2014). Consistent with this, animal studies indicated that GRK5 is an important regulator of adipogenesis and insulin sensitivity (Wang et al. 2012a, 2012b). Besides the peripheral system, GRK5 is expressed at high levels in various brain regions, such as the septum, the cingulate cortex, the septo-hippocampal nucleus, and the locus coeruleus (Erdtmann-Vourliotis et al. 2001), GRK5 deficiency leads to M2 muscarinic receptor hyperactivation while has no effect on the responsiveness of dopamine receptors, the μ-opioid receptor, or 5-HT1A subtype of serotonin receptors (Gainetdinov et al. 1999; Gomeza et al. 1999). Our previous work revealed that GRK5 regulates neuronal morphogenesis in the cortex and hippocampus, and ablation of Grk5 in mice impairs long-term spatial memory (Chen et al. 2011). Emerging evidence suggests that GRK5 may play a role in the development of neurological disorders. Aged Grk5−/− mice display an increased hippocampal axonal defects (Suo et al. 2007). In brains of sporadic Parkinson's disease patients, GRK5 was found to accumulate in Lewy bodies and colocalize with α-synuclein, and it may be involved in the development of Parkinson's disease, through phosphorylation of alpha-synuclein (Arawaka et al. 2006; Liu et al. 2010). Previous research revealed that GRK5 is highly expressed in pyramidal cell layers of different subregions of the cortex including the medial prefrontal cortex (mPFC), and the expression level of GRK5 in these subregions changes after administration of opiate drugs (Fan et al. 2002). Abnormality of mPFC circuitry and impairments in cognition and social behaviors are associated with a number of psychiatric diseases, such as schizophrenia, depression, drug abuse (Goldstein and Volkow, 2011), and the autism spectrum disorders (ASDs) (Korade and Mirnics, 2011). Our results showed that genetic disruption of Grk5 in mice leads to impaired social behaviors, which was associated with dysregulation of mTORC1 signaling and the synaptic transmission in the mPFC, indicating the critical role of GRK5 in maintaining the normal function of mPFC circuits and social behaviors. Materials and Methods Animals Grk5−/− mice were provided by R.J. Lefkowitz and R.T. Premont (Duke University Medical Center). Grk5−/− mice and their wild-type littermates were obtained from self-crossing Grk5+/− (heterozygous) mice. Mice which possessed loxP sites on either side of exons 7 and 8 of Grk5 (Grk5fl/fl mice) were obtained from the Jackson laboratory (strain name: B6.129S4-Grk5tm2Rjl/J; The Jackson Laboratory) and backcrossed to C57BL/6 strain. Grk5fl/fl mice and their wild-type littermates were obtained from self-crossing Grk5+/fl (heterozygous) mice. Genotyping method was provided by Dr R.T. Premont (Gainetdinov et al. 1999). Of the case, 3 to 4 mice were housed in groups with the same gender and genotype, and maintained on a 12-h light/dark cycle with food and water available ad libitum. The experiments were conducted on dark phase. All procedures conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by Animal Care and Use Committee of School of Basic Medical Sciences of Fudan University. Social Behavior Tests Three-chamber social behavior test was carried out as previously described (Moy et al. 2004; Chao et al. 2010; Zhao et al. 2010). The social behavior testing apparatus was a 3-chambered box with dimensions of each chamber being 40 cm in length, 22 cm in width, and 23 cm in height. Dividing walls were made of clear Plexiglas containing small circular doors (7 cm in diameter) allowing access into each chamber. The procedure involved 3 phases: habituation, tests of social interaction, and social novelty recognition. Mouse was first placed in the middle compartment and allowed to explore all 3 chambers for 10 min (Chao et al. 2010). After habituation, a novel (unfamiliar) mouse (stimulus mouse; age and sex matched to the test mouse) was placed into one side of the compartments (constrained by a round wire cage that permitted nose contact between the bars but prevented extensive physical contact; stimulus mice were habituated to small wire cages for 5–10 min each for 2 days before testing), whereas the opposite side compartment only contained an empty wire cage. For evaluation of the social interaction, the test mouse was returned to the apparatus for 10 min. The time of exploration (approach, sniffing, and rearing within 2 cm from the round cage) of the conspecific and the empty wire cage by the test mouse was recorded and scored offline by a trained tester blind to genotype. For the social novelty recognition test, one side compartment of the apparatus contained the familiar mouse (from the previous social interaction phase), the other side contained a novel unfamiliar mouse. Active exploration of the familiar and the unfamiliar mouse during the 10-min period was recorded. Another social recognition procedure was performed as previously described (Jin et al. 2007; Bozdagi et al. 2010; Scattoni et al. 2011). Male mice were individual housed for 5 days to permit establishment of a home-cage territory. At the fifth day, a stimulus female was introduced into the home cage with the male mouse for a 1-min interaction for the first trial. At the end of the 1-min trial, the stimulus animal was returned to an individual holding cage. After a 10-min inter-exposure interval, the same female was introduced into the same home cage of the male mouse for 1 min. These trials were repeated and performed 4 times in all. Of the note, 10 min after the fourth trial, the fifth trial (dishabituation trial) was carried out by introducing a different stimulus female to the same male mouse for 1 min. The exploration time of the female by the male mouse was recorded, and all the data were recorded and scored offline by EthoVision XT 8.5 video tracking program. Electron Microscopy Mice were anesthetized with choral hydrate (400 mg/kg, i.p.), and perfused transcardially with 4% paraformaldehyde. The mPFC and striatum were isolated and post-fixed in 4% paraformaldehyde. Electron microscopy was performed as previously described (Peca et al. 2011). The images were taken by transmission electron microscope (TEM) (FEI/PHILIPS CM120 TEM, Philips Electron Optics B.V.). Electrophysiology Coronal sections (300 μm) containing mPFC were cut from wild-type and Grk5−/− littermates. For juvenile mice (P13-15), slices were cut on a vibratome in the cutting solution (120 mM choline chloride, 2.6 mM KCl, 0.5 mM CaCl2, 7 mM MgCl2, 26 mM NaHCO3, 1.25 mM NaH2PO4, 15 mM D-glucose, and 1.3 mM ascorbate acid), incubated in ACSF contains 126 mM NaCl, 26 mM NaHCO3, 1.2 mM NaH2PO4, 3 mM KCl, 2.4 mM CaCl2, 1.3 mM MgCl2, 10 mM D-glucose, and allowed to recover for at least half an hour at room temperature. For adult mice (6–8 weeks), slices were prepared as previously described (Yue et al. 2011). Briefly, the mice were deeply anesthetized by intraperitoneal injection of chloral hydrate (400 mg/kg, i.p.) and then transcardially perfused with cold protective ACSF (92 mM N-methyl-d-glucamine (NMDG), 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM D-glucose, 2 mM thiourea, 5 mM Na-ascorbate, 3 mM Na-pyruvate, 0.5 mM CaCl2, and 10 mM MgCl2) and then initially recovered at 32–34 °C for 10–15 min. The slices were transferred into a holding chamber containing room temperature carbogenated ACSF (119 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 12.5 mM glucose, 2 mM CaCl2, 2 mM MgCl2, 2 mM thiourea, 5 mM Na-ascorbate, and 3 mM Na-pyruvate), and slices were stored for 0.5–3 hours prior to transfer to the recording chamber for use. All solutions were saturated with 95% O2 and 5% CO2, and the slices were used within 6 h after preparation. Whole-cell voltage clamp recordings were performed in ACSF at room temperature from layer II/III pyramidal neurons in mPFC with an EPC-10 amplifier and Pulse v8.78 software (HEKA Elektronik). Intracellular solution composition was 126 mM K-gluconate, 4 mM KCl, 10 mM HEPES, 4 mM ATP-Mg, 0.3 mM GTP-Na2, 10 mM creatine phosphate (pH 7.2, 290–300 mOsm). Spontaneous miniature excitatory postsynaptic current (mEPSC) events were recorded in presence of ACSF containing 2 μM TTX and 50 μM bicuculline (Tocris Bioscience) at a holding potential of −70 mV. Spontaneous miniature inhibitory postsynaptic current (mIPSC) events were recorded in the presence of 2 μM TTX, 10 μM APV, and 10 μM NBQX disodium salt (Tocris Bioscience). In the mGluR5 blockade experiments, brain slices from wild-type and Grk5−/− mice were incubated in standard ACSF containing 40 μM MPEP (Tocris Bioscience) for at least an hour before and throughout the recording. In rapamycin experiments, the slices were pre-treated with 20 nM rapamycin (Sigma-Aldrich) or DMSO for at least 30 min before and throughout the recording. Data were filtered at 300 Hz and were analyzed by Mini Analysis Program (Synaptosoft Inc.). All electrophysiological recordings were performed and analyzed blind to genotype. Stereotaxic Surgery Male mice of 6–8-week age anesthetized with choral hydrate (400 mg/kg, i.p.) were placed in a stereotactic instrument. Microinjections were performed using custom-made injection needles (33-gauge) connected to a 10 μL Hamilton syringe. Each mPFC was injected with 0.5 μL of purified and concentrated AAV (1012 IU/mL) encoding CAG –eGFP-T2A-Cre alone, or GRK5-eGFP and eGFP under the control of the “Synapsin” promoter. The intended stereotaxic coordinates were AP + 2.0 mm; ML ± 0.3 mm (with an angle of 14° from the middle to the lateral); DV − 2.2 mm. All mice were given at least 14–21 days to recover. Immunostaining Mice were perfused transcardially with 4% paraformaldehyde. Brains were fixed in 4% paraformaldehyde overnight and transferred to 30% sucrose for 72 h before slicing 30 μm per slice of mPFC. Anti-NeuN antibody (Millipore), anti-GRK5 antibody (Abcam) or phospho-p44/42 MAPK (Erk 1/2) (Cell Signaling Technology) were diluted in blocking solution containing 0.3% Triton X-100 (Sigma-Aldrich) and 3% goat serum (Jackson Immunoresearch). The brain sections in this dilution were incubated at 4 °C overnight. For secondary antibody staining, brain sections were incubated with Cy3 anti-mouse or Alex 488 anti-rabbit IgG antibodies (Jackson Immunoresearch) for 1 h at room temperature, and then incubated with DAPI (Invitrogen) for 10 min and mounted with aqueous mounting medium (Thermo Scientific). Images were acquired on a scanning laser confocal microscope using a ×20 air objective or a ×40 oil immersion objective lens (Zeiss 510; Carl Zeiss). Cell number was counted using Image-Pro Plus software. Western blotting Brain tissues were lysed in RIPA buffer. Western blotting were carried out as described before (Wu et al. 2012). Blots were incubated with IRDye 800CW-conjugated or 700CW-conjugated antibody (Rockland Biosciences) and infrared fluorescence images were obtained with the Odyssey infrared imaging system (Li-Cor Bioscience). Antibody of synapsin was from Abcam, antibodies of GluR1, GluR2, NR2A, and NR2B were from Millipore, antibody of Homer was from Santa Cruz, antibodies of PSD-95, β-tubulin, and β-actin were from Sigma-Aldrich, antibodies of p70 S6K, phospho-p70 S6K (T389), S6, phospho-S6 (S240/244), Akt, phospho-Akt (T308), phospho-Akt (S473), 4E-BP1 and phospho-4E-BP1(T37/46) were from Cell Signaling Technology. Co-Immunoprecipitation mPFC was disassociated and lysed in Co-Immunoprecipitation buffer (20 mM Tris, pH 7.6, 150 mM NaCl, 1 mM EDTA, 1 mM NaF, 0.5% Nonidet P-40 and protease inhibitors), and protein was tumbled overnight at 4 °C with antibody to Raptor (Cell Signaling Technology). Protein A agarose bead slurry (Thermo Scientific) was added for 4 h, and the beads were then washed with Co-Immunoprecipitation buffer. Western blotting was performed with antibodies to Raptor and mTOR (Cell Signaling Technology). Statistics All data are presented as mean ± SEM. Student's t-test, two-way analysis of variance (Two-way ANOVA), or two-way repeated-measures analysis of variance (Two-way RM ANOVA) was used for statistical analysis. Bonferroni post hoc analysis was subsequently performed after Two-way ANOVA or Two-way RM ANOVA within different factors. Two-sample Kolmogorov–Smirnov test was used for cumulative frequency plot analysis. *P < 0.05, **P < 0.01, and ***P < 0.001. Results GRK5 Deficiency Leads to Social Behavior Deficit To explore the potential role of GRK5 in brain physiology and the development of psychiatric disorders, the performance of Grk5−/− mice were evaluated in a series of behavioral tests. In the open field test, Grk5−/− mice showed no significant difference in the total distance traveled or the numbers of entries into center as compared with their WT littermates (Supplementary Fig. S1A and B). In the prepulse inhibition of the startle reflex tests, Grk5−/− mice showed comparable performance with the WT mice (Supplementary Fig. S1C and D), indicating normal spontaneous activities and sensorimotor gating processes. However, Grk5−/− mice spent more time in the center zone than their WT littermates in the open field test (Supplementary Fig. S1E), and they also spent more time in the open arms than WT mice in the elevated plus maze test (Supplementary Fig. S1F and G); however, the number of entries into the open arms and the total number of entries, indicators of general exploratory activity, were not different from those of WT mice (Supplementary Fig. S1H and I). We next assessed the depression level of Grk5−/− mice in tail suspension test and sucrose preference test. The percentage of sucrose preference and the time of immobility were comparable between WT and Grk5−/− mice (Fig. 1A,B). These results indicated that the depression level of Grk5−/− mice was normal. Figure 1. View largeDownload slide GRK5 ablation leads to abnormal social recognition. Grk5−/− mice of 8–12-week old and their WT littermates were subjected to a set of behavior tests. (A) Sucrose Preference Test. Two-way RM ANOVA: Fgenotype × time (2, 44) = 1.069, P = 0.352. (B) Tail suspension test. Student's t-test: P = 0.555. (C–F) Three-chamber social behavior tests. (C) Schematic representation of the 3-chamber social behavioral test. (D) Time spent in exploring a stranger mouse (S1) and an empty cage. Two-way RM ANOVA followed by Bonferroni post hoc analysis: Fgenotype × target (1, 45) = 3.424, P = 0.071; ***P < 0.001 versus S1 within each genotype. (E) Time spent in exploring the novel mouse (S2) and the familiar mouse (S1). Fgenotype × target (1, 44) = 4.476, *P = 0.040; ***P < 0.001 versus S1 within each genotype. (F) Time spent in exploring a stranger (S3) and a novel object control (O). Fgenotype × target (1, 21) = 0.774, P = 0.389; ***P < 0.001, **P < 0.01 versus S1 within each genotype. (G and H) Home cage social recognition test. (G) Schematic representation. (H) Quantification. Two-way RM ANOVA followed by Bonferroni post hoc analysis: Fgenotype × trial (4, 76) = 2.916, *P = 0.027; Fgenotype (1, 76) = 4.738, P = 0.042. *P < 0.05, **P < 0.01 versus WT within different trials. (I and J) Two-trial Y-maze. WT versus Grk5−/− mice, time spent in novel arm (I): P = 0.673, novel arm percent (J): P = 0.566, Student's t-test versus random choice probability of 33.33%: WT, *P = 0.035, Grk5−/− mice: *P = 0.027, One-sample t-test. Data are presented as mean ± SEM. Figure 1. View largeDownload slide GRK5 ablation leads to abnormal social recognition. Grk5−/− mice of 8–12-week old and their WT littermates were subjected to a set of behavior tests. (A) Sucrose Preference Test. Two-way RM ANOVA: Fgenotype × time (2, 44) = 1.069, P = 0.352. (B) Tail suspension test. Student's t-test: P = 0.555. (C–F) Three-chamber social behavior tests. (C) Schematic representation of the 3-chamber social behavioral test. (D) Time spent in exploring a stranger mouse (S1) and an empty cage. Two-way RM ANOVA followed by Bonferroni post hoc analysis: Fgenotype × target (1, 45) = 3.424, P = 0.071; ***P < 0.001 versus S1 within each genotype. (E) Time spent in exploring the novel mouse (S2) and the familiar mouse (S1). Fgenotype × target (1, 44) = 4.476, *P = 0.040; ***P < 0.001 versus S1 within each genotype. (F) Time spent in exploring a stranger (S3) and a novel object control (O). Fgenotype × target (1, 21) = 0.774, P = 0.389; ***P < 0.001, **P < 0.01 versus S1 within each genotype. (G and H) Home cage social recognition test. (G) Schematic representation. (H) Quantification. Two-way RM ANOVA followed by Bonferroni post hoc analysis: Fgenotype × trial (4, 76) = 2.916, *P = 0.027; Fgenotype (1, 76) = 4.738, P = 0.042. *P < 0.05, **P < 0.01 versus WT within different trials. (I and J) Two-trial Y-maze. WT versus Grk5−/− mice, time spent in novel arm (I): P = 0.673, novel arm percent (J): P = 0.566, Student's t-test versus random choice probability of 33.33%: WT, *P = 0.035, Grk5−/− mice: *P = 0.027, One-sample t-test. Data are presented as mean ± SEM. Recently, systematic evaluation of genes differentially expressed between patients of ASDs and normal control subjects allocated GRK5 to the top disease and disorder categories associated with ASDs (Ghahramani Seno et al. 2011; Wittkowski et al. 2014). To investigate whether GRK5 is involved in regulation of social behavior, the performance of Grk5−/− mice in 3-chamber social interaction assay was evaluated. As shown in Figure 1C,D, both WT and Grk5−/− mice were able to initiate social interaction voluntarily, as they spent longer time exploring the novel (unfamiliar) mouse (Stranger 1, S1) than the empty cage (E), However, in the subsequent social novelty recognition session, WT mice spent more time exploring the novel mouse (Stranger 2, S2) than the familiar one (Stranger 1 in the previous experiment), but Grk5−/− mice showed no preference (Fig. 1E), suggesting that GRK5 is required for social novelty recognition. Additionally, both genotypes spent more time exploring the novel mouse (Stranger 3) than the novel object (O), and there was no significant difference in the exploring time on the novel object (Fig. 1F) between the Grk5−/− and the WT mice, indicating that the reduced exploration of Grk5–/– mice for the novel mice was not due to decreased novelty exploration. The potential role of GRK5 in social novelty recognition was also assessed in the home-cage social recognition test. Grk5−/− or WT males were allowed to explore the same female mouse introduced into their home-cage during each of the four 1-min trials (Fig. 1G). Although WT and Grk5−/− mice both showed a marked habituation (decreased durations of exploring) to the same female mouse (S1) in the 4 sequential trials, and a striking dishabituation (increased exploration) upon the presentation of a novel female (S2), Grk5−/− mice showed a slower decreasing rate than WT mice (Fig. 1H). Our previous study has demonstrated that Grk5−/− mice possess normal short-term memory of the novel object recognition (Chen et al. 2011), and we also showed that Grk5−/− and WT mice spent comparable time and percentage visiting the novel arm in the Y-maze test (Fig. 1I,J), suggesting that the impairment of Grk5−/− mice in social novelty recognition was unlikely caused by a general defect of short-term memory for the object or space, whereas was due to the impaired social memory of a stranger mouse. GRK5 Is Important for Maintaining the Normal Structure and Excitatory Transmission of Synapse in the mPFC The mPFC is a key region for social cognition and interaction (Frith and Frith, 2006). Patients with mPFC lesions exhibit severely impaired social behaviors (Anderson et al. 1999). To determine whether GRK5 regulates synaptic plasticity in mPFC, we examined the spine morphology and the ultrastructure of postsynaptic densities (PSDs) at mPFC in Grk5−/− mice and WT mice. Although the total spine numbers of the mPFC pyramidal neurons from WT and Grk5−/− were comparable, the percentage of the immature spine (thin spine) was significantly increased in Grk5−/− mice (Supplementary Fig. S2A and B). The ultrastructure of the synapse showed a significant reduction in the thickness of PSDs in the mPFC of Grk5−/− mice (Fig. 2A–C), whereas the length of the PSDs in the mPFC between Grk5−/− and the WT mice was not significantly different (Fig. 2D,E), suggesting that GRK5 deficiency leads to the abnormal synaptic structure. The number of docked vesicles and the total numbers of vesicles in mPFC synapses had no significant difference between WT and Grk5−/− mice (Fig. 2F,G), indicating a normal amount of presynaptic neurotransmitter in Grk5−/− mice. Figure 2. View largeDownload slide GRK5 deficiency leads to decreased thickness of PSDs and the impaired excitatory currents in the mPFC. (A) Examples of electron micrographs depicting the mPFC synaptic contacts with presynaptic vesicles (arrowheads), PSDs (arrow), and dendritic spine (asterisk) from 8–12-week-old Grk5−/− mice and the WT littermates; scale bar, 100 nm. (B–E) Quantification of the thickness and length of the mPFC PSDs. Two-sample Kolmogorov–Smirnov test, ***P < 0.001, 200–220 mPFC PSDs from 3 WT mice and 3 Grk5−/− mice). (F and G) Quantification of the docking vesicles (F) and the total vesicles number (G) of cortical synapses (vesicles docked: P = 0.707, total vesicles: P = 0.711, n = 54 PSDs from 3 WT mice and 52 PSDs from 3 Grk5−/− mice, Student's t-test). (H, K) Representative traces of mEPSCs (H) or mIPSCs (K) recorded from layer II/III pyramidal neurons in the mPFC slices from P14 mice. (I–L) Cumulative probability distribution and average interevent intervals of mEPSC (I, n = 16 cells from each group) or mIPSC (L, n = 18 cells from each group). (J–M) Cumulative probability distribution and average amplitude of mEPSC (J, n = 16 cells from each group) or mIPSC (M, n = 18 cells from each group) amplitudes. Two-sample Kolmogorov–Smirnov test for cumulative probability distribution, ***P < 0.001. Student's t-test for the average interevent intervals and amplitude, **P < 0.01. Data are presented as mean ± SEM. Figure 2. View largeDownload slide GRK5 deficiency leads to decreased thickness of PSDs and the impaired excitatory currents in the mPFC. (A) Examples of electron micrographs depicting the mPFC synaptic contacts with presynaptic vesicles (arrowheads), PSDs (arrow), and dendritic spine (asterisk) from 8–12-week-old Grk5−/− mice and the WT littermates; scale bar, 100 nm. (B–E) Quantification of the thickness and length of the mPFC PSDs. Two-sample Kolmogorov–Smirnov test, ***P < 0.001, 200–220 mPFC PSDs from 3 WT mice and 3 Grk5−/− mice). (F and G) Quantification of the docking vesicles (F) and the total vesicles number (G) of cortical synapses (vesicles docked: P = 0.707, total vesicles: P = 0.711, n = 54 PSDs from 3 WT mice and 52 PSDs from 3 Grk5−/− mice, Student's t-test). (H, K) Representative traces of mEPSCs (H) or mIPSCs (K) recorded from layer II/III pyramidal neurons in the mPFC slices from P14 mice. (I–L) Cumulative probability distribution and average interevent intervals of mEPSC (I, n = 16 cells from each group) or mIPSC (L, n = 18 cells from each group). (J–M) Cumulative probability distribution and average amplitude of mEPSC (J, n = 16 cells from each group) or mIPSC (M, n = 18 cells from each group) amplitudes. Two-sample Kolmogorov–Smirnov test for cumulative probability distribution, ***P < 0.001. Student's t-test for the average interevent intervals and amplitude, **P < 0.01. Data are presented as mean ± SEM. To assess the function of GRK5 in mPFC synaptic transmission, pharmacologically isolated spontaneous mEPSCs of the mPFC pyramidal neurons were recorded at postnatal day 14. We found no significant difference in the frequency of mEPSCs obtained from the mPFC neurons between WT and Grk5−/− mice; however, the amplitude of mEPSCs recorded from the pyramidal neurons of Grk5−/− mice was significantly decreased, as compared with those from the WT mice (Fig. 2H–J). Similar results were obtained with mPFC layer II/III neurons in older (6–8 weeks) WT and Grk5−/− mice (Supplementary Fig. S2C–F). These results suggest that GRK5 regulates excitatory synaptic strength, and the loss of GRK5 may cause a decrease in either the excitatory vesicle neurotransmitter release or number of postsynaptic excitatory receptors. The analysis of the spontaneous mIPSCs showed GRK5 deficiency did not change the average frequency or the amplitude of mIPSC events (Fig. 2K–M). Significant difference in the cumulative probability distribution, but not the average interevent interval of IPSC was observed between WT and Grk5−/− mice, suggesting a minor increase in inhibitory presynaptic release probability and altered inhibitory synaptic transmission in Grk5−/− mice. GRK5 in the mPFC Plays a Key Role in Regulating the Social Behavior To examine if the effect of ablation of GRK5 gene on social behavior is a result of abnormal brain development or dysregulation of signaling in adult brain, region-specific conditional knockdown of GRK5 was done by injection of adeno-associated virus AAV-CAG-eGFP-T2A-Cre (Supplementary Fig. S3A) into the mPFC of adult Grk5fl/fl mice (Figure 3A,B; Supplementary Fig. S3B and C). Selective knockdown of GRK5 in the mPFC of adult WT mice had no effect on their performance in open field (Supplementary Fig. S3D and E) and EPM (Supplementary Fig. S3F) tests, but impaired social behavior, as indicated by the loss of preferential exploration in tests for the social interaction (Fig. 3C) and social novelty recognition (Fig. 3D). The conditional knockdown of GRK5 in the mPFC of adult mice mediated by viral infection appeared to produce more dramatic effect on social interaction, as compared with that observed in Grk5−/− mice. We speculate that in the Grk5−/− mice, signaling pathways parallel to GRK5 may be activated or enhanced during the development to compensate for the effect of GRK5 gene ablation, resulting in less severe behavioral phenotypes. Figure 3. View largeDownload slide The effects of knockdown or restore GRK5 expression in the mPFC on social behavior. (A–D) WT or Grk5fl/fl mice were infected with AAV-CAG-eGFP-T2A-Cre in the mPFC. (A) AAV-CAG-eGFP-T2A-Cre infection in the mPFC. Scale bar, 1 cm. (B) Enlarged immunostaining images of AAV-CAG-eGFP-T2A-Cre infected mPFC cells. Scale bar, 100 μm. (C and D) 3-chamber social tests of mice infected with AAV-CAG-eGFP-T2A-Cre in the mPFC (C) The time spent in exploring a stranger (S1) and an empty cage (E): Fgenotype × target (1, 16) = 5.576, *P = 0.031; (D) Time spent in exploring the novel mouse (S2) and the familiar mouse (S1). Fgenotype × target (1, 14) = 6.558, *P = 0.023; (E–I) WT or Grk5−/− mice were infected with AAV-Syn-GRK5-eGFP or AAV-Syn-eGFP in the mPFC. (E) Representative images of AAV-Syn-GRK5-eGFP or AAV-Syn-eGFP infected mPFC neurons in the WT mice. Scale bar, 100 μm. (F–I) 3-chamber social behavior tests of the WT (F and G) and Grk5−/− mice (H and I) injected with the AAV-Syn-GRK5-eGFP or AAV-Syn-eGFP virus. (F) Ftreatment × target (1, 20) = 3.417, P = 0.079; (G) Ftreatment × target (1, 20) = 0.309, P = 0.584; (H) Time spent in exploring a stranger (S1) and an empty cage (E) Ftreatment × target (1, 24) = 2.608, P = 0.119; (I) F treatment × target (1, 19) = 7.976, *P = 0.011; Two-way RM-ANOVA. *P < 0.05, **P < 0.01, and ***P < 0.001 by Bonferroni post hoc analysis. Data are presented as mean ± SEM. Figure 3. View largeDownload slide The effects of knockdown or restore GRK5 expression in the mPFC on social behavior. (A–D) WT or Grk5fl/fl mice were infected with AAV-CAG-eGFP-T2A-Cre in the mPFC. (A) AAV-CAG-eGFP-T2A-Cre infection in the mPFC. Scale bar, 1 cm. (B) Enlarged immunostaining images of AAV-CAG-eGFP-T2A-Cre infected mPFC cells. Scale bar, 100 μm. (C and D) 3-chamber social tests of mice infected with AAV-CAG-eGFP-T2A-Cre in the mPFC (C) The time spent in exploring a stranger (S1) and an empty cage (E): Fgenotype × target (1, 16) = 5.576, *P = 0.031; (D) Time spent in exploring the novel mouse (S2) and the familiar mouse (S1). Fgenotype × target (1, 14) = 6.558, *P = 0.023; (E–I) WT or Grk5−/− mice were infected with AAV-Syn-GRK5-eGFP or AAV-Syn-eGFP in the mPFC. (E) Representative images of AAV-Syn-GRK5-eGFP or AAV-Syn-eGFP infected mPFC neurons in the WT mice. Scale bar, 100 μm. (F–I) 3-chamber social behavior tests of the WT (F and G) and Grk5−/− mice (H and I) injected with the AAV-Syn-GRK5-eGFP or AAV-Syn-eGFP virus. (F) Ftreatment × target (1, 20) = 3.417, P = 0.079; (G) Ftreatment × target (1, 20) = 0.309, P = 0.584; (H) Time spent in exploring a stranger (S1) and an empty cage (E) Ftreatment × target (1, 24) = 2.608, P = 0.119; (I) F treatment × target (1, 19) = 7.976, *P = 0.011; Two-way RM-ANOVA. *P < 0.05, **P < 0.01, and ***P < 0.001 by Bonferroni post hoc analysis. Data are presented as mean ± SEM. Next, we overexpressed GRK5 in the mPFC neurons of the adult WT and Grk5−/− mice, via injection of AAV-Syn-GRK5-eGFP (Fig. 3E, Supplementary Fig. S3G and H). Overexpressing GRK5 in the mPFC of WT mice brought increased significance in the time spent on exploring S1 mice versus empty cage and exploration time for a novel mouse versus the familiar mouse (Fig. 3F,G), as compared with the mice infected with AAV-Syn-eGFP. Similarly, the Grk5−/− mice infected with AAV-Syn-GRK5-eGFP also exhibited increased statistical significance in exploration time for S1 mice versus empty cage (Fig. 3H). Notably, the selective expression of GRK5 in mPFC neurons resulted in a significant increase in time spent exploring the novel (S2) mice over the familiar (S1) mouse, indicating the rescue of the social novelty recognition phenotype of Grk5−/−mice (Fig. 3I). Taken together, these data suggest that GRK5 in the mPFC of mice plays an important role in regulating the social behavior. GRK5 Is Critical for Maintaining the Normal mTORC1 Signaling in mPFC The decreased mEPSC amplitude observed in Grk5−/− mPFC may be induced by a decrease in excitatory vesicle release or postsynaptic excitatory receptors. Since we have shown that the release of presynaptic neurotransmitter was not impaired in Grk5−/− mice (Fig. 2F and G), we investigated the role of GRK5 in regulation of the expression of synaptic proteins next. The mRNA levels of various PSD proteins in purified synaptosome samples of WT and Grk5−/− mPFC were examined. No significant difference in transcript levels of most glutamatergic and GABAergic postsynaptic or presynaptic markers including Gad1, Gad2, and Nlgn3, except for only slight decreases in Gabbr1 and Gabrb2 (GABAergic postsynaptic markers), were detected (Supplementary Fig. S4A). Consistently, no significant differences were found in the protein levels of the synaptic and structural markers (Supplementary Fig. S4B and C). Dysregulation of mTORC1 and its translational control pathways have been shown in several psychiatric syndromes including schizophrenia, depression, and bipolar disorders (de Lacy and King, 2013). We found that the phosphorylation level of ribosomal protein S6 kinase (S6K), a downstream effector of the mTORC1, was significantly increased in Grk5−/− mPFC (Fig. 4A). The phosphorylation of S6, a substrate of S6K, was also increased in Grk5−/− mice (Fig. 4B). The immunofluorescent assay (Fig. 4C) showed that the expression of p-S6 in NeuN positive Cells was also dramatically increased in the mPFC of Grk5−/− mice, indicative of the upregulated S6K-S6 signaling in the mPFC neurons. Figure 4. View largeDownload slide Loss of GRK5 leads to upregulation of mTORC1 signaling, (A and B) Lysates from WT and Grk5−/− mPFC were subjected to Western analysis with indicated antibodies. Representative immunoblots (upper panel) and quantification results (lower panel) of 3 independent experiments are shown. (C) Immunostaining of mPFC sections of WT and Grk5−/− mice. Quantification results of the percentage of p-S6+ cells in total NeuN+ cells are shown. Scale bar, 50 μm. (D) Western blots of WT and Grk5−/− mPFC lysates probed with antibody against p-Akt (T308/S473) or Akt. (E and F) WT and Grk5−/− mice were treated with DHPG (50 μM, 1 μL, i.c.v.) 30 min before transcardial perfusion. (E) The immunostaining of the mPFC sections. Scale bar, 50 μm. Arrows indicate the cells that are positive for both p-ERK (green) and NeuN (red). (F) Quantification results of the percentage of p-ERK+ cells in total NeuN+ cells. Student's t-test or Two-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001. Data are presented as mean ± SEM. Figure 4. View largeDownload slide Loss of GRK5 leads to upregulation of mTORC1 signaling, (A and B) Lysates from WT and Grk5−/− mPFC were subjected to Western analysis with indicated antibodies. Representative immunoblots (upper panel) and quantification results (lower panel) of 3 independent experiments are shown. (C) Immunostaining of mPFC sections of WT and Grk5−/− mice. Quantification results of the percentage of p-S6+ cells in total NeuN+ cells are shown. Scale bar, 50 μm. (D) Western blots of WT and Grk5−/− mPFC lysates probed with antibody against p-Akt (T308/S473) or Akt. (E and F) WT and Grk5−/− mice were treated with DHPG (50 μM, 1 μL, i.c.v.) 30 min before transcardial perfusion. (E) The immunostaining of the mPFC sections. Scale bar, 50 μm. Arrows indicate the cells that are positive for both p-ERK (green) and NeuN (red). (F) Quantification results of the percentage of p-ERK+ cells in total NeuN+ cells. Student's t-test or Two-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001. Data are presented as mean ± SEM. mGluRs and NMDARs mediate changes in synaptic plasticity through PI3K-Akt-mTORC1 and MEK-ERK pathways to activate S6K, and increase cap-dependent protein translation (Klann and Dever 2004; Darnell and Klann 2013). We thus examined the activation of Akt and ERK signaling in the mPFC of Grk5−/− and WT mice. The phosphorylation levels of Akt (T308 and S473) in mPFC of Grk5−/− mice were not different from those of WT mice at quiescent state (Fig. 4D). Moreover, group I mGluR agonist DHPG induced comparable increase in ERK phosphorylation in mPFC of WT and Grk5−/− mice (Fig. 4E,F). These data suggest that GRK5 may regulate S6K signaling through an effector downstream of Akt or ERK, or via a pathway independent of Akt or ERK. Previous research has found that GRK5 can directly interact with mTORC1 complex through its binding to regulatory associated protein of mTOR (Raptor) in HEK293 cells (Burkhalter et al. 2013). Consistently, although the total protein level of Raptor and mTORC1 in mPFC was not significantly different in the WT and Grk5−/− mice (Fig. 5A), an increased Raptor-mTOR association was detected in Grk5−/− mice (Fig. 5B), indicating that GRK5 may hinder association of Raptor with mTOR, and inhibit the activation of mTORC1 to prevent overactivation of Raptor-dependent mTOR signaling. Figure 5. View largeDownload slide Loss of GRK5 leads to the increased binding of raptor and mTOR and rapamycin treatment reinstated the dysregulated mTORC1 signaling pathway in mPFC of Grk5−/− mice. The mPFC lysates were subjected to Western or co-immunoprecipitation analysis with the indicated antibodies. (A) Representative immunoblots (upper panel) and quantification results (lower panel) of 3 independent experiments are shown. (B) Co-immunoprecipitation with a Raptor antibody. Student's t-test, *P < 0.05. (C–F) WT and Grk5−/− mice were treated with vehicle, rapamycin (10 mg/kg, i.p.), or MPEP (10 mg/kg, i.p.). The mPFC sections were removed 30 min later and the lysates were subjected to Western analysis with the indicated antibodies. (C) Western blot analysis of phospho-S6k and total S6K in mPFC. (D) The quantification results of 3–4 independent experiments. (E) Western analysis of S6 and phospho-S6 (S240/244) in mPFC. (F) Western blot analysis of 4E-BP1 and phospho-4E-BP1 (T37/46) in mPFC. Two-way ANOVA, *P < 0.05, **P < 0.01, and ***P < 0.001 by Bonferroni post hoc analysis. Data are presented as mean ± SEM. Figure 5. View largeDownload slide Loss of GRK5 leads to the increased binding of raptor and mTOR and rapamycin treatment reinstated the dysregulated mTORC1 signaling pathway in mPFC of Grk5−/− mice. The mPFC lysates were subjected to Western or co-immunoprecipitation analysis with the indicated antibodies. (A) Representative immunoblots (upper panel) and quantification results (lower panel) of 3 independent experiments are shown. (B) Co-immunoprecipitation with a Raptor antibody. Student's t-test, *P < 0.05. (C–F) WT and Grk5−/− mice were treated with vehicle, rapamycin (10 mg/kg, i.p.), or MPEP (10 mg/kg, i.p.). The mPFC sections were removed 30 min later and the lysates were subjected to Western analysis with the indicated antibodies. (C) Western blot analysis of phospho-S6k and total S6K in mPFC. (D) The quantification results of 3–4 independent experiments. (E) Western analysis of S6 and phospho-S6 (S240/244) in mPFC. (F) Western blot analysis of 4E-BP1 and phospho-4E-BP1 (T37/46) in mPFC. Two-way ANOVA, *P < 0.05, **P < 0.01, and ***P < 0.001 by Bonferroni post hoc analysis. Data are presented as mean ± SEM. Inhibition of mTORC1 Signaling Ameliorates the Impaired Synaptic Transmission in mPFC and Social Behavior Phenotype Caused by GRK5 Deficiency We found that the increased phosphorylation of S6K1 and S6 in GRK5 deficient mPFC could be blocked by mGluR5 antagonist 2-Methyl-6-(phenylethynyl) pyridine (MPEP) or mTORC1 inhibitor rapamycin (Fig. 5C–E). Whereas, the phosphorylation level of 4E-BP1, another mTORC1-targeted ribosome protein was not changed (Fig. 5C,F). To investigate whether the decreased excitatory synaptic currents in mPFC observed in Grk5−/− mice is due to hyperactivation of mTORC1 signaling, we examined the effects of rapamycin on mEPSCs of mPFC neurons in Grk5−/− mice. An acute treatment with 20 nM rapamycin restored the amplitude of excitatory synaptic currents in the pyramidal neurons of the Grk5−/− mice to a level comparable to that of WT slices, whereas had no obvious effect on mEPSC events in the WT mice (Fig. 6A–C). Similarly, 40 μM MPEP increased the amplitude of mEPSCs in Grk5−/− pyramidal neurons and restored it to a normal level (Fig. 6D–F). In the 3-chamber tests, intraperitoneal injection of 10 mg/kg rapamycin or MPEP did not have significant effects on the social behaviors of the WT mice (Fig. 6G,H), whereas it increased the exploration time toward the novel mice (stanger S2) and rescued the social novelty recognition impairment in Grk5−/− mice (Fig. 6I,J). In the home-cage social recognition test, the impaired habituation to the same female (S1) of Grk5−/− mice was also rescued by the rapamycin and MPEP treatment (Fig. 6K). These data suggest that inhibiting the dysregulated mTORC1 signaling ameliorates the impaired synaptic transmission in the mPFC and social novelty recognition caused by GRK5 deficiency. Figure 6. View largeDownload slide Inhibition of mTORC1 signaling in Grk5−/− mice ameliorates the mPFC excitatory current and the impaired social novelty recognition (A) Representative traces of the mEPSCs recorded from layer II/III pyramidal neurons in acute mPFC slices incubated with the mTORC1 inhibitor rapamycin. (B and C) The mEPSC interevent intervals (B) and amplitude (C) of the WT and Grk5−/− pyramidal neurons treated with 20 nM Rapamycin. Two-way ANOVA: for IEI: Fgenotype × treatment (1, 67) = 0.00298, P = 0.957, for amplitude: Fgenotype × treatment (1, 67) = 21.874, ***P < 0.001. (D) Representative traces of the mEPSCs recorded from layer II/III pyramidal neurons in the acute mPFC slices incubated with the mGluR5 antagonist MPEP. (E and F) The effect of 40 μM MPEP treatment on the interevent intervals (E) and amplitude (F) of mEPSCs (For IEI, Fgenotype × treatment (1, 63) = 0.0564, P = 0.813. For amplitude, Fgenotype× treatment (1, 63) = 5.653, *P = 0.020. (G–J) 3-chamber social behavior tests of the WT (G and H) and Grk5−/− mice (I and J). Mice were injected with the MPEP (10 mg/kg, i.p.) 30 min or rapamycin (10 mg/kg, i.p.) for 2 days before behavior tests. (G) Ftreatment × target (2, 55) = 0.507, P = 0.605. (H) Ftreatment × target (2, 55) = 0.00645, P = 0.994. (I) Ftreatment × target (2, 50) = 0.202, P = 0.818. (J) Ftreatment × target (2, 45) = 3.253, P = 0.048. Two-way RM ANOVA, *P < 0.05, **P < 0.01, and ***P < 0.001 by Bonferroni post hoc analysis. (K) Home-cage social recognition test of the WT and Grk5−/− mice injected with the MPEP (10 mg/kg, i.p.) or rapamycin. Two-way RM ANOVA: Ftreatment x trial (12, 152) = 3.402 (P < 0.001), Ftreatment (3, 152) = 3.574 (P = 0.023). Bonferroni post hoc analysis: KO-Vehicle versus WT-Vehicle across trials: P = 0.048, *P < 0.05, **P < 0.01 KO-Vehicle versus WT-Vehicle at each trial. Data are presented as mean ± SEM. Figure 6. View largeDownload slide Inhibition of mTORC1 signaling in Grk5−/− mice ameliorates the mPFC excitatory current and the impaired social novelty recognition (A) Representative traces of the mEPSCs recorded from layer II/III pyramidal neurons in acute mPFC slices incubated with the mTORC1 inhibitor rapamycin. (B and C) The mEPSC interevent intervals (B) and amplitude (C) of the WT and Grk5−/− pyramidal neurons treated with 20 nM Rapamycin. Two-way ANOVA: for IEI: Fgenotype × treatment (1, 67) = 0.00298, P = 0.957, for amplitude: Fgenotype × treatment (1, 67) = 21.874, ***P < 0.001. (D) Representative traces of the mEPSCs recorded from layer II/III pyramidal neurons in the acute mPFC slices incubated with the mGluR5 antagonist MPEP. (E and F) The effect of 40 μM MPEP treatment on the interevent intervals (E) and amplitude (F) of mEPSCs (For IEI, Fgenotype × treatment (1, 63) = 0.0564, P = 0.813. For amplitude, Fgenotype× treatment (1, 63) = 5.653, *P = 0.020. (G–J) 3-chamber social behavior tests of the WT (G and H) and Grk5−/− mice (I and J). Mice were injected with the MPEP (10 mg/kg, i.p.) 30 min or rapamycin (10 mg/kg, i.p.) for 2 days before behavior tests. (G) Ftreatment × target (2, 55) = 0.507, P = 0.605. (H) Ftreatment × target (2, 55) = 0.00645, P = 0.994. (I) Ftreatment × target (2, 50) = 0.202, P = 0.818. (J) Ftreatment × target (2, 45) = 3.253, P = 0.048. Two-way RM ANOVA, *P < 0.05, **P < 0.01, and ***P < 0.001 by Bonferroni post hoc analysis. (K) Home-cage social recognition test of the WT and Grk5−/− mice injected with the MPEP (10 mg/kg, i.p.) or rapamycin. Two-way RM ANOVA: Ftreatment x trial (12, 152) = 3.402 (P < 0.001), Ftreatment (3, 152) = 3.574 (P = 0.023). Bonferroni post hoc analysis: KO-Vehicle versus WT-Vehicle across trials: P = 0.048, *P < 0.05, **P < 0.01 KO-Vehicle versus WT-Vehicle at each trial. Data are presented as mean ± SEM. Discussion Multiple studies have suggested that GRK5 plays a key role in the development of cardiac hypertrophy and heart diseases and GRK5 has been proposed to be a potential therapeutic target for the treatment of heart failure (Dzimiri et al. 2004; Aguero et al. 2009). Studies in the central nerve system revealed that GRK5 is highly expressed in different subregions of the cortex and has been implicated in the development of neurological disorders. Impairments in social behaviors are features of a number of psychiatric diseases associated with subtle alterations in mPFC circuitry. Grk5−/− mice showed impaired social recognition, as well as dysregulated mTORC1 signaling and synaptic dysfunction in the mPFC, indicating that GRK5 modulates the physiological function of the mPFC and exerts an important role in social behaviors. Our results revealed the association of GRK5 with dysfunction of social behavior and its potential involvement in the development of psychiatric diseases, such as schizophrenia, depression, and ASD. In this study, we found that Grk5−/− mice showed overactivated mTORC1 signaling and reduced mEPSC amplitude in mPFC. The dysfunction of mEPSCs and the dysregulation of mTORC1 signaling exist in several mental diseases models (Cohen et al. 2011; Peca et al. 2011; Wan et al. 2011; Yizhar et al. 2011). Several previous studies have shown that mTORC1 activation positively regulates excitatory synaptic function. TSC1-cKO and Pten-cKO hippocampal neurons showed hyperactive mTORC1 signaling including increased phosphorylation levels of S6 and 4E-BP, and an increased soma size; the hippocampal excitatory transmission of Tsc1-KO neurons was increased (Tavazoie et al. 2005; Bateup et al. 2011, 2013) or not different from control (Weston et al. 2014). Tsai et al. found no significant difference in synaptic inputs to TSC1-cKO cerebellar Purkinje cells (Tsai et al. 2012), whereas Yang et al. found that hypothalamic POMC neurons with upregulated mTOR signaling were electronically silent (Yang et al. 2012), indicating the different roles of mTORC1 signaling in the excitatory transmission in different brain regions. FKBP12-deficient mice showed increased basal mTOR-Raptor interactions but enhanced hippocampal LTP (Hoeffer et al. 2008), indicating the complexity of mTORC1 activation and its psychological functions. The frequency of inhibitory synaptic transmission was minor altered in Grk5−/− mice and the transcription levels of GABAergic receptors such as Gabbr1 and Gabrb2 were slightly decreased in Grk5−/− mice, suggesting that GRK5 may be required for the regulation of excitatory synaptic transmission, and have minor contribution to the inhibitory synaptic transmission. The increased phosphorylation of S6K and its substrate S6 was detected in Grk5−/− mice, indicating that GRK5 is likely involved in the regulation of mTORC1-S6K pathway. Burkhalter et al. recently demonstrated that GRK5 regulates mTORC1 signaling through direct interaction with Raptor (Burkhalter et al. 2013), which is associated with mTOR to form mTORC1 complex (Hoeffer and Klann 2010). In the current study, we found an increased mTOR-raptor association in the mPFC of Grk5−/− mice. GRK5 is likely to inhibit mTORC1 signaling through its competitive binding to Raptor. When GRK5 level is low, increased Raptor-mTOR binding may lead to overactivation of mTORC1 signaling. Additionally, both rapamycin and MPEP can downregulate the overactivated mTORC1 signaling pathway and ameliorate the impaired social behaviors of Grk5−/−mice, supporting the notion that GRK5 regulates social behavior via an mTORC1 mechanism. Our results indicate a critical role of GRK5 in maintaining normal mTORC1 signaling, cortical connectivity, and social recognition, thus reveal that GRK5 may be a potential target for treatment of psychiatric disorders, such as schizophrenia and ASDs. Supplementary Material Supplementary material is available at Cerebral Cortex online. Authors’ Contribution B.N., F.W., and L.M. planned the animal experiments and analysed the data. B.N. and P.L. contributed to the acquisition of the behavior tests and the biological data. F.W. carried out the stereotaxic surgery and biochemistry experiments. M.S. carried out the electrophysiology experiments. P.L. and L.W. carried out the immunohistochemical experiments. C.L. constructed the viral vectors. B.N. drafted the manuscript. F.W. and L.M. revised the manuscript. Funding This research was supported by grants from the Natural Science Foundation of China (31430033, 31421091, 91232307, 31270027, 31671042, and 91632307) and the Ministry of Science and Technology (2014CB942801, 2015CB553501, 2013CB835102). Notes We thank Drs. R.J. Lefkowitz and R.T. Premont for Grk5−/− mice. Conflict of Interest: None declared. References Aguero J, Almenar L, D'Ocon P, Oliver E, Monto F, Rueda J, Vicente D, Martinez-Dolz L, Salvador A. 2009. Myocardial and peripheral lymphocytic transcriptomic dissociation of beta-adrenoceptors and G protein-coupled receptor kinases in heart transplantation. J Heart Lung Transplant . 28: 1166– 1171. Google Scholar CrossRef Search ADS PubMed  Anderson SW, Bechara A, Damasio H, Tranel D, Damasio AR. 1999. 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TI - GRK5 Regulates Social Behavior Via Suppression of mTORC1 Signaling in Medial Prefrontal Cortex
JF - Cerebral Cortex
DO - 10.1093/cercor/bhw364
DA - 2018-02-01
UR - https://www.deepdyve.com/lp/oxford-university-press/grk5-regulates-social-behavior-via-suppression-of-mtorc1-signaling-in-UL87OTWn00
SP - 421
EP - 432
VL - 28
IS - 2
DP - DeepDyve
ER -