Metabotropic glutamate receptor 1 promotes cementoblast proliferation via MAP kinase signaling pathways

Purpose/aim: Glutamate is one of the signaling molecules responsible for transmission in the central nervous system. Periodontal ligament (PDL) cells were recently reported to express metabotropic glutamate receptors (mGluRs). However, the functions of mGluR signaling in PDL cells or PDL-related cells remain largely unknown. The aim of this study was to investigate the expression and function of mGluRs in PDL-related cells. Materials and methods: OCCM-30 cells, immortalized murine cementoblasts, were stimulated with l-glutamate or mGluRs antagonists. The cells’ proliferative response was evaluated using a colorimetric assay and gene expression was assessed using real-time polymerase chain reaction. The nuclear translocation of cyclin D1 was evaluated by immunohistochemistry. Results: l-Glutamate promoted the proliferation of OCCM-30 cells, which expressed mGluR1, but not mGluR5. Dihydroxyphenylglycine (DHPG), an agonist of group I mGluRs (mGluR1 and mGluR5), also promoted cell proliferation, and this was inhibited by LY456236, an mGluR1 antagonist. DHPG increased the expression of cyclin D1, a key regulator of cell proliferation, and its nuclear translocation. DHPG also increased the expression of Bcl2A1, an antiapoptotic oncogene and simultaneously reduced the expression of Bax, a pro-apoptotic marker. Furthermore, the DHPG-induced proliferation of OCCM-30 cells was reduced by pretreatment with SB203580, SP600125, and PD98059, inhibitors of p38, JNK, and ERK1/2, respectively. Conclusions: These findings indicate that activation of mGluR1 expressed by OCCM-30 cells induces cell proliferation in a manner that is dependent on mitogen-activated protein kinase pathways and that cyclin D1 and Bcl2A1/Bax may be involved. Our results provide useful information for elucidating the mechanisms underlying cementum homeostasis and regeneration.     

Metabotropic glutamate receptor dependent long-term depression in the cortex

Metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD), a type of synaptic plasticity, is characterized by a reduction in the synaptic response, mainly at the excitatory synapses of the neurons. The hippocampus and the cerebellum have been the most extensively studied regions in mGluR-dependent LTD, and Group 1 mGluR has been reported to be mainly involved in this synaptic LTD at excitatory synapses. However, mGluR-dependent LTD in other brain regions may be involved in the specific behaviors or diseases. In this paper, we focus on five cortical regions and review the literature that implicates their contribution to the pathogenesis of several behaviors and specific conditions associated with mGluR-dependent LTD.     

Cone synapses in mammalian retinal rod bipolar cells

Some mammalian rod bipolar cells (RBCs) can receive excitatory chemical synaptic inputs from both rods and cones (DBC_R2), but anatomical evidence for mammalian cone‐RBC contacts has been sparse. We examined anatomical cone‐RBC contacts using neurobiotin (NB) to visualize individual mouse cones and standard immuno‐markers to identify RBCs, cone pedicles and synapses in mouse and baboon retinas. Peanut agglutinin (PNA) stained the basal membrane of all cone pedicles, and mouse cones were positive for red/green (R/G)‐opsin, whereas baboon cones were positive for calbindin D‐28k. All synapses in the outer plexiform layer were labeled for synaptic vesicle protein 2 (SV2) and PSD (postsynaptic density)‐95, and those that coincided with PNA resided closest to bipolar cell somas. Cone‐RBC synaptic contacts were identified by: (a) RBC dendrites deeply invaginating into the center of cone pedicles (invaginating synapses), (b) RBC dendritic spines intruding into the surface of cone pedicles (superficial synapses), and (c) PKCα immunoreactivity coinciding with synaptic marker SV2, PSD‐95, mGluR6, G protein beta 5 or PNA at cone pedicles. One RBC could form 0‐1 invaginating and 1‐3 superficial contacts with cones. 20.7% and 38.9% of mouse RBCs contacted cones in the peripheral and central retina (p < .05, n = 14 samples), respectively, while 34.4% (peripheral) and 48.5% (central) of cones contacted RBCs (p > .05). In baboon retinas (n = 4 samples), cone‐RBC contacts involved 12.2% of RBCs (n = 416 cells) and 22.5% of cones (n = 225 cells). This suggests that rod and cone signals in the ON pathway are integrated in some RBCs before reaching AII amacrine cells.     

Strategies for the identification of allosteric modulators of G-protein-coupled receptors

Once considered a pharmacological curiosity, allosteric modulation of seven transmembrane domain G-protein-coupled receptors (GPCRs) has emerged as a potentially powerful means to affect receptor function for therapeutic purposes. Allosteric modulators, which interact with binding sites topologically distinct from the orthosteric ligand binding sites, can potentially provide improved selectivity and safety, along with maintenance of spatial and temporal aspects of GPCR signaling. Accordingly, drug discovery efforts for GPCRs have increasingly focused on the identification of allosteric modulators. This review is devoted to an examination of the strategies, challenges, and opportunities for high-throughput screening for allosteric modulators of GPCRs, with particular focus on the identification of positive allosteric modulators.     

Metabotropic Glutamate Receptor 5 Upregulation in Children with Autism is Associated with Underexpression of Both Fragile X Mental Retardation Protein and GABAA Receptor Beta 3 in Adults with Autism

Recent work has demonstrated the impact of dysfunction of the GABAergic signaling system in brain and the resultant behavioral pathologies in subjects with autism. In animal models, altered expression of Fragile X mental retardation protein (FMRP) has been linked to downregulation of GABA receptors. Interestingly, the autistic phenotype is also observed in individuals with Fragile X syndrome. This study was undertaken to test previous theories relating abnormalities in levels of FMRP to GABA_A receptor underexpression. We observed a significant reduction in levels of FMRP in the vermis of adults with autism. Additionally, we found that levels of metabotropic glutamate receptor 5 (mGluR5) protein were significantly increased in vermis of children with autism versus age and postmortem interval matched controls. There was also a significant decrease in level of GABA_A receptor beta 3 (GABRβ3) protein in vermis of adult subjects with autism. Finally, we found significant increases in glial fibrillary acidic protein in vermis of both children and adults with autism when compared with controls. Taken together, our results provide further evidence that altered FMRP expression and increased mGluR5 protein production potentially lead to altered expression of GABA_A receptors. Anat Rec,, 2011. © 2011 Wiley‐Liss, Inc.     

The mGluR7 allosteric agonist AMN082 produces antidepressant-like effects by modulating glutamatergic signaling

Currently prescribed antidepressants affect the reuptake and/or metabolism of biogenic amines. Unfortunately for patients, these treatments require several weeks to produce significant symptom remission. However, recently it has been found that ketamine, a dissociative anesthetic agent that noncompetitively antagonizes NMDA (N-Methyl-d-aspartic acid) receptors, has rapid antidepressant effects at sub-anesthetic doses in clinically depressed patients. These findings indicate that modulation of the glutamatergic system could be an efficient way to achieve antidepressant activity. For this reason, other mechanisms influencing glutamatergic functioning have gained interest. For example, the metabotropic glutamate receptor 7 (mGluR7) allosteric agonist AMN082 (N,N′-dibenzyhydryl-ethane-1,2-diamine dihydrochloride) has been shown to be effective in the forced swim and tail-suspension test, behavioral assays sensitive to antidepressants. Here we extend the characterization of AMN082 by demonstrating its effects on differential reinforcement of low rates of responding (DRL)-30, another assay sensitive to antidepressants. Furthermore, we show the engagement of glutamatergic signaling by demonstrating the ability of the selective AMPA (2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid) receptor antagonist NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione) to reverse the effects of AMN082 in the tail suspension test. In contrast, NBQX failed to reverse the effects of imipramine in the same behavioral test. Finally, we report that behaviorally efficacious doses of AMN082 modulate phosphorylation of AMPA and NMDA receptor subunits in the hippocampus. These results suggest that the antidepressant-like effects of AMN082 are, at least in part, due to modulation of AMPA and NMDA receptor activity. Therefore, our findings confirm the hypothesis that mGluR7 could represent a novel target for treating depression.     

The mTOR signaling pathway in the prefrontal cortex is compromised in major depressive disorder

Recent studies demonstrate that rapid antidepressant response to ketamine is mediated by activation of the mammalian target of rapamycin (mTOR) signaling pathway, leading to increased synaptic proteins in the prefrontal cortex (PFC) of rats. Our postmortem studies indicate robust deficits in prominent postsynaptic proteins including N-methyl-d-aspartate (NMDA) receptor subunits (NR2A, NR2B), metabotropic glutamate receptor subtype 5 (mGluR5) and postsynaptic density protein 95 kDa (PSD-95) in the PFC in major depressive disorder (MDD). We hypothesize that deficits in the mTOR-dependent translation initiation pathway contribute to the molecular pathology seen in the PFC of MDD subjects, and that a rapid reversal of these abnormalities may underlie antidepressant activity. The majority of known translational regulation occurs at the level of initiation. mTOR regulates translation initiation via its downstream components: p70-kDa ribosomal protein S6 kinase (p70S6K), and eukaryotic initiation factors 4E and 4B (eIF4E and eIF4B). In this study, we examined the expression of mTOR and its core downstream signaling targets: p70S6K, eIF4E, and eIF4B in the PFC of 12 depressed subjects and 12 psychiatrically healthy controls using Western blot. Levels of eIF4E phosphorylated at serine 209 (p-eIF4E-Ser209) and eIF4B phosphorylated at serine 504 (p-eIF4B-Ser504) were also examined. Adjacent cortical tissue samples from both cohorts of subjects were used in our previous postmortem analyses. There was a significant reduction in mTOR, p70S6K, eIF4B and p-eIF4B protein expression in MDD subjects relative to controls. No group differences were observed in eIF4E, p-eIF4E or actin levels. Our findings show deficits in mTOR-dependent translation initiation in MDD particularly via the p70S6K/eIF4B pathway, and indicate a potential association between marked deficits in synaptic proteins and dysregulation of mTOR signaling in MDD.     

Elevated level of metabotropic glutamate receptor 2/3 in the prefrontal cortex in major depression

Clinical, postmortem and preclinical research strongly implicates dysregulation of glutamatergic neurotransmission in major depressive disorder (MDD). Recently, metabotropic glutamate receptors (mGluRs) have been proposed as attractive targets for the discovery of novel therapeutic approaches against depression. The aim of this study was to examine mGluR2/3 protein levels in the prefrontal cortex (PFC) from depressed subjects. In addition, to test whether antidepressants influence mGluR2/3 expression we also studied levels of mGluR2/3 in fluoxetine-treated monkeys. Postmortem human prefrontal samples containing Brodmann's area 10 (BA10) were obtained from 11 depressed and 11 psychiatrically healthy controls. Male rhesus monkeys were treated chronically with fluoxetine (dose escalated to 3 mg/kg, p.o.; n = 7) or placebo (n = 6) for 39 weeks. The mGluR2/3 immunoreactivity was investigated using Western blot method. There was a robust (+ 67%) increase in the expression of the mGlu2/3 protein in the PFC of depressed subjects relative to healthy controls. The expression of mGlu2/3 was unchanged in the PFC of monkeys treated with fluoxetine. Our findings provide the first evidence that mGluR2/3 is elevated in the PFC in MDD. This observation is consistent with reports showing that mGluR2/3 antagonists exhibit antidepressant-like activity in animal models and demonstrates that these receptors are promising targets for the discovery of novel antidepressants.     

ATP敏感性钾通道(KATP)开放剂对脑缺血/再灌注大鼠谷氨酸受体1a和谷氨酸转运体1的影响

目的 研究ATP敏感性钾通道(KATP)开放剂克罗卡林对大鼠脑缺血/再灌注后脑梗死体积、代谢型谷氨酸受体1α(mGluRlα)和谷氨酸转运体1(GLT-1或者EAAT-2)的影响.方法 雄性Wistar大鼠按序号随机分为假手术组(A组)、缺血/再灌注脑缺血对照组(middle cerebral artery occlusion MCAOB,B组)、MCAO+ATP敏感性钾通道(KATP)开放剂克罗卡林组(C组).应用线栓法建立大鼠大脑中动脉闭塞再灌注(middle cerebral artery occlusion,MCAO)模型,大鼠脑缺血/再灌注24 h后,Bederson法评价动物的神经行为功能,氯化三苯基四氮唑(TTC)染色观察脑梗死体积,免疫组化测定谷氨酸受体1α(mGluR1α)和GLT-1.结果 脑缺血/再灌注24 h后,动物均表现神经行为功能障碍,大鼠神经行为功能评分C组(2.23±0.11)较B组(2.61±0.19)明显改善,差异有统计学意义(P＜0.05);缺血侧出现脑梗塞病灶,脑梗死体积C组(138.3±29.9)较B组(182.5±34.5)显著减小,差异具有统计学意义(P＜0.05);B组mGluR1α吸光度(0.293±0.009)与A组mGluR1α吸光度(0.183±0.008)相比,吸光度明显增加,差异有统计学意义(P＜0.05)、B组GLT-吸光度(0.147±0.009)与A组GLT-吸光度(0.255±0.009)相比,吸光度明显减少,差异有统计学意义(P＜0.05);C组mGluR1α吸光度(0.227±0.009)与B组mGluR1α吸光度(0.293±0.009)相比,其吸光度明显减小,差异有统计学意义(P＜0.05)、C组GLT-1吸光度(0.212±0.008)与B组GLT-1吸光度(0.147±0.009)相比明显增大,差异有统计学意义(P＜0.05).结论 预防性应用克罗卡林可能通过减少mGluR1α表达,增强GLT-1表达,减少谷氨酸堆积,减小脑梗死体积,从而减轻脑组织缺血/再灌注损伤.