Oligodendroglial metabotropic glutamate receptors are developmentally regulated and involved in the prevention of apoptosis

Oligodendrocytes (OLs) are responsible for axon myelination and are the principal cells targeted in preterm white matter injury. The cellular and molecular mechanisms involved in white matter development and immature OL injury are incompletely understood. Metabotropic glutamate receptors (mGluRs) modulate neuronal development and survival, and have recently been identified in oligodendrocyte progenitor cells (OPCs). Using the highly homogeneous CG-4 OPC line and O4 marker-immunoselected primary OLs, we established the differentiation stage-specific expression profile of mGluR3 and mGluR5 mRNAs and proteins in the oligodendroglial lineage and type-2-astrocytes (ASTs). Our quantitative analysis indicated no changes in mGluR3, but a significant down-regulation of mGluR5a mRNA and protein expression during differentiation of OPCs into OLs or ASTs. The down-regulation of mGluR5a had functional consequences, with significantly fewer OLs and ASTs than OPCs responding to the group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine with intracellular Ca(2+) concentration oscillations. Neither stimulation nor inhibition of mGluR3 or mGluR5 altered OPC migration, suggesting that these receptors do not play prominent roles in the regulation of OPC motility. The activation of mGluR5 completely protected OPCs and substantially reduced staurosporine-induced apoptosis in OLs. This suggests that the down-regulation of mGluR5 in premyelinating OLs is likely to contribute to their increased vulnerability, and that the targeting of mGluR5 may be a potential therapeutic strategy for future development.     

Group I metabotropic glutamate receptors are expressed in the chicken retina and by cultured retinal amacrine cells

Glutamate is well established as an excitatory neurotransmitter in the vertebrate retina. Its role as a modulator of retinal function, however, is poorly understood. We used immunocytochemistry and calcium imaging techniques to investigate whether metabotropic glutamate receptors are expressed in the chicken retina and by identified GABAergic amacrine cells in culture. Antibody labeling for both metabotropic glutamate receptors 1 and 5 in the retina was consistent with their expression by amacrine cells as well as by other retinal cell types. In double-labeling experiments, most metabotropic glutamate receptor 1-positive cell bodies in the inner nuclear layer also label with anti-GABA antibodies. GABAergic amacrine cells in culture were also labeled by metabotropic glutamate receptor 1 and 5 antibodies. Metabotropic glutamate receptor agonists elicited Ca(2+) elevations in cultured amacrine cells, indicating that these receptors were functionally expressed. Cytosolic Ca(2+) elevations were enhanced by metabotropic glutamate receptor 1-selective antagonists, suggesting that metabotropic glutamate receptor 1 activity might normally inhibit the Ca(2+) signaling activity of metabotropic glutamate receptor 5. These results demonstrate expression of group I metabotropic glutamate receptors in the avian retina and suggest that glutamate released from bipolar cells onto amacrine cells might act to modulate the function of these cells.     

Group III metabotropic glutamate receptor activation suppresses self-replication of undifferentiated neocortical progenitor cells

We evaluated the possible functional expression of metabotropic glutamate receptors (mGluRs) by neural progenitors from embryonic mouse neocortex. Constitutive expression was seen with group I, II, and III mGluRs in undifferentiated cells and neurospheres formed by clustered cells during culture with epidermal growth factor. The group III mGluR agonist, l -2-amino-4-phosphonobutyrate, drastically reduced proliferation activity at 1–100 μM without inducing cell death, with group I and group II mGluR agonists being ineffective, in these neurospheres. Both forskolin and a group III mGluR antagonist significantly increased the proliferation alone, but significantly prevented the suppression by l -2-amino-4-phosphonobutyrate. Activation of group III mGluR significantly decreased mRNA expression of the cell cycle regulator cyclinD1, in addition to inhibiting the transactivation mediated by cAMP of cyclinD1 gene in the pluripotent P19 progenitor cells. Prior activation of group III mGluR led to a significant decrease in the number of cells immunoreactive for a neuronal marker, with an increase in that for an astroglial marker irrespective of differentiation inducers. These results suggest that group III mGluR may be functionally expressed to suppress self-renewal capacity through a mechanism related to cAMP formation with promotion of subsequent differentiation into astroglial lineage in neural progenitors.     

Pharmacological characterization of metabotropic glutamate receptors in cultured cerebellar granule cells

A detailed pharmacological characterization of metabotropic glutamate receptors (mGluR) was performed in primary cultures of cerebellar granule cells at 6 days in vitro (DIV). The rank order of agonists induced polyphosphoinositide (PPI) hydrolysis (after correcting for the ionotropic component in the response) was as follows: in terms of efficiency, Glu>quisqualate (quis)=ibotenate (ibo)>(1_S,3_R)-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD)>-methyl-amino-l-alanine (BMAA) and in terms of potency, quis>ACPD>Glu>ibo=BMAA. Ionotropic excitatory amino acid (EAA) receptor agonists, such as -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) were relatively inactive (in the presence of Mg²⁺). Quis and ACPD-induced PPI hydrolysis was unaffected by ionotropic Glu receptor antagonists, but was inhibited, in part by L-2-amino-3-phosphonopropionate (AP3). In contrast, Glu-or ibo- induced PPI hydrolysis was reduced, in part, by both AP3 and NMDA receptor antagonists. Characteristic interactions involving different transmitter receptors were noted. PPI hydrolysis evoked by quis and 1_S,3_R-ACPD was not additive. In contrast, PPI hydrolysis stimulated by quis/ACPD and carbamylcholine was additive (indicating different receptors/transduction pathways). In the presence of Mg²⁺, the metabotropic response to quis/AMPA and NMDA was synergistic (this being consistent with AMPA receptor-induced depolarization activating NMDA receptor). On the other hand, in Mg²⁺-free buffer the effects of quis and NMDA, at concentrations causing maximal PPI hydrolysis, were additive (indicating that PPI hydrolysis was effected by two different mechanisms). Thus, in cerebellar granule cells EAAs elicit PPI hydrolysis by acting at two distinct receptor types: (i) metabotropic Glu receptors (mGluR), with pharmacological characteristics suggesting the expression of a unique mGluR receptor that shows certain similarities to those observed for the mGluR1 subtype (Aramori and Nakanishi, 1992) and (ii) NMDA receptors. The physiological agonist, Glu, is able to stimulate both receptor classes.Abbreviations ACPD (1_S,3_R)-1-amino-cyclopentane-1,3-dicarboxylic acid - AMPA -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid - AP₃ L-2-amino-3-phosphono-propionate - AP₅ D-2-amino-5-phosphonopentenoate - BMAA -methyl-amino-L-alanine - DIV days in vitro - DNOX 6,7-dinitroouinoxoline-2,3-dione - EAA excitatory amino acids - Glu glutamate - InsP inositol monophosphate - mGluR metabotropic glutamate receptors - MK-801 (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohept-5,10-imine hydrogen maleate - NMDA N-methyl-D-aspartate - PPI polyphosphoinositide - quis quisqualate     

代谢型谷氨酸受体在突触可塑性中的作用

突触可塑性是近几年神经科学研究的热点之一,因为它对于理解神经系统的学习、学习和记忆、多咱神经疾病等许多过程有着重要的意义。除了离子型谷氨酸受体外,代谢型谷氨酸受体也参与了一些脑区中不同形式的突触可塑性变化。本文就代谢型谷氨酸受体选择性激动剂和拮抗剂对长时程增强和长时程抑制的作用进行了综述,以助于人们进一步理解突触可塑性的细胞和分子机制。     

Inhibition of microtubule formation by metabotropic glutamate receptors

Activation of glutamate receptors is known to alter the biophysical state of the cytoskeleton of neurons in the developing brain. In this study, we examined the ability of G protein-coupled metabotropic glutamate receptors (mGluRs) to inhibit the formation of processes induced by the expression of the microtubule-associated protein MAP2c. The infection of insect MG-1 cells with a recombinant baculovirus (BV) encoding MAP2c induced the formation of fine filamentous processes. The binding of MAPs to tubulin promotes tubulin polymerization and the formation of microtubules. Co-infection with BVs for the phosphoinositide (PI)-linked mGluR1a or mGluR1b receptor subtypes inhibited the formation of processes induced by MAP2c, whereas co-infection with BVs encoding the mGluR4a or mGluR4b subtypes that couple to adenylyl cyclase did not inhibit the formation of processes. The biochemical pathways responsible for producing the inhibitory effect of mGluR1 were investigated. Inhibitors of protein kinase C, calcium/calmodulin-dependent kinase, and protein tyrosine kinases did not block the inhibitory effect of mGluR1a. The calcium chelator BAPTA and the calcium depletor thapsigargin also did not affect the ability of mGluR1a to inhibit process formation. In contrast, inhibitors of phospholipase C reversed the effect of mGluR1 on process formation, suggesting that one or more metabolites in the PI pathway were responsible for the inhibitory effect. These findings indicate that PIs generated by activation of mGluRs inhibit the binding of MAPs to tubulin and reduce tubulin polymerization and microtubule stability.     

Inhibition of non-vesicular glutamate release by group III metabotropic glutamate receptors in the nucleus accumbens

Previous in vitro studies have shown that group III metabotropic glutamate receptors (mGluRs) regulate synaptic glutamate release. The present study used microdialysis to characterize this regulation in vivo in rat nucleus accumbens. Reverse dialysis of the group III mGluR agonist l-(+)-2-amino-4-phosphonobutyric acid (L-AP4) decreased, whereas the antagonist (R,S)-alpha-methylserine-O-phosphate (MSOP) increased the extracellular level of glutamate. The decrease by L-AP4 or the increase by MSOP was antagonized by co-administration of MSOP or L-AP4, respectively. Activation of mGluR4a by (1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid or mGluR6 by 2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid had no effect on extracellular glutamate. (R,S)-4-Phosphonophenylglycine (PPG), another group III agonist with high affinity for mGluR4/6/8, reduced extracellular glutamate only at high concentrations capable of binding to mGluR7. The increase in extracellular glutamate by MSOP was tetrodotoxin-independent, and resistant to both the L-type and N-type Ca2+ channel blockers. L-AP4 failed to block 30 mm K+-induced vesicular glutamate release. Blockade of glutamate uptake by d,l-threo-beta-benzyloxyaspartate caused a Ca2+-independent elevation in extracellular glutamate that was reversed by L-AP4. Finally, (S)-4-carboxyphenylglycine, an inhibitor of cystine-glutamate antiporters, attenuated the L-AP4-induced reduction in extracellular glutamate. Together, these data indicate that group III mGluRs regulate in vivo extracellular glutamate in the nucleus accumbens by inhibiting non-vesicular glutamate release.     

Functional and molecular characterization of metabotropic glutamate receptors expressed in rat striatal cholinergic interneurones

In the present study we have used single-cell RT-PCR in conjunction with electrophysiology to examine the expression and functional properties of metabotropic glutamate receptors (mGluRs) expressed within biochemically identified cholinergic interneurones in the rat striatum. Using single-cell RT-PCR, it was possible to demonstrate the presence of mGluR1, mGluR2, mGluR3, mGluR5 and mGluR7 mRNAs within single cholinergic interneurones. Bath application of the non-selective mGluR agonist (1 S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1 S,3R-ACPD) or the group-I mGluR agonist 3,5-dihydroxyphenylglycine (DHPG) depolarized all cholinergic neurones tested by activation of an inward current at -60 mV. The effects of DHPG were partially inhibited by the mGluR5 selective antagonist 6-methyl-2-(pherazo)-3-pyridinol and by the non-selective group-I antagonist alpha-methyl-4-carboxyphenylglycine but were not mimicked by the group-II and group-III selective mGluR agonists 2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) and L-2-amino-4-phosphonobutanoate (L-AP4), respectively. Intrastriatal stimulation evoked an excitatory postsynaptic current within cholinergic neurones that was reversibly inhibited by bath application of the group-II and group-III selective mGluR agonists DCG-IV and L-AP4, respectively, via presynaptic actions. In summary, we have identified the mGluRs expressed by striatal cholinergic interneurones and demonstrated that their activation produces modulatory effects via both pre- and postsynaptic mechanisms.     

Agonist-induced internalization of the metabotropic glutamate receptor 1a is arrestin- and dynamin-dependent

At present, little is known regarding the mechanism of metabotropic glutamate receptor (mGluR) trafficking. To facilitate this characterization we inserted a haemagglutinin (HA) epitope tag in the extracellular N-terminal domain of the rat mGluR1a. In human embryonic kidney cells (HEK293), transiently transfected with HA-mGluR1a, the epitope-tagged receptor was primarily localized to the cell surface prior to agonist stimulation. Following stimulation with glutamate (10 microM; 30 min) the HA-mGluR1a underwent internalization to endosomes. Further quantification of receptor internalization was provided by ELISA experiments which showed rapid agonist-induced internalization of the HA-mGluR1a. To determine whether agonist-induced mGluR1a internalization is an arrestin- and dynamin-dependent process, cells were cotransfected with HA-mGluR1a and either of these dynamin-K44A or arrestin-2 (319-418). Expression of either dominant negative mutant constructs with receptor strongly inhibited glutamate-induced (10 microM; 30 min) HA-mGluR1a internalization. In addition, wild-type arrestin-2-green fluorescent protein (arrestin-2-GFP) or arrestin-3-GFP underwent agonist-induced translocation from cytosol to membrane in HEK293 cells coexpressing HA-mGluR1a. Taken together our observations demonstrate that agonist-induced internalization of mGluR1a is an arrestin- and dynamin-dependent process.     

A novel constitutively active mutation in the second cytoplasmic loop of metabotropic glutamate receptor

G protein-coupled receptors have a common structural motif of seven transmembrane alpha-helices and are classified into different families showing no sequence similarity. Extensive studies have been conducted on the structure-function relationship in family 1 receptors, but those in other families have not been well studied. In this study, to investigate the molecular basis leading to the G protein activation by metabotropic glutamate receptor (mGluR), the member of family 3, we searched for the amino acid residues responsible for the G protein activation in the second cytoplasmic loop, which was thought to be the main G protein binding region. Analyses of the systematical mutations of Gi/Go-coupled mGluR8 revealed the presence of a constitutively active mutation in the C-terminal region of the second loop. The corresponding mutation in the second loop of Gq-coupled mGluR1 also exhibited high agonist-independent activity. These results indicate that there is a common constitutive active mutation site regardless of mGluR subtypes, suggesting that the structural change of the junction between the second cytoplasmic loop and helix IV is strongly linked to the formation of the active state.     

Regulated release of BDNF by cortical oligodendrocytes is mediated through metabotropic glutamate receptors and the PLC pathway

A number of studies suggest that OLGs (oligodendrocytes), the myelinating cells of the central nervous system, are also a source of trophic molecules, such as neurotrophins that may influence survival of proximate neurons. What is less clear is how the release of these molecules may be regulated. The present study investigated the effects of BDNF (brain-derived neurotrophic factor) derived from cortical OLGs on proximate neurons, as well as regulatory mechanisms mediating BDNF release. Initial work determined that BDNF derived from cortical OLGs increased the numbers of VGLUT1 (vesicular glutamate transporter 1)-positive glutamatergic cortical neurons. Furthermore, glutamate acting through metabotropic, and not AMPA/kainate or NMDA (N-methyl-d-aspartate), receptors increased BDNF release. The PLC (phospholipase C) pathway is a key mediator of metabotropic actions to release BDNF in astrocytes and neurons. Treatment of OLGs with the PLC activator m-3M3FBS [N-(3-trifluoromethylphenyl)-2,4,6-trimethylbenzenesulfonamide] induced robust release of BDNF. Moreover, release elicited by the metabotropic receptor agonist ACPD [trans-(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid] was inhibited by the PLC antagonist {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, the IP₃ (inositol triphosphate 3) receptor inhibitor 2-APB (2-aminoethoxydiphenylborane) and the intracellular calcium chelator BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis(acetoxymethyl ester)]. Taken together, these results suggest that OLG lineage cells release BDNF, a molecule trophic for proximate neurons. BDNF release is regulated by glutamate acting through mGluRs (metabotropic glutamate receptors) and the PLC pathway. Thus glutamate and BDNF may be molecules that support neuron–OLG interactions in the cortex.     

突触前代谢型谷氨酸受体调节神经递质的释放

谷氨酸通过激活离子型受体（iGluR)介导快速兴奋性突触传递,参与脑内几乎所有生理过程。谷氨酸过量释放可导致与脑缺血,缺氧及变性疾病有关的兴奋毒作用,最终引起神经元的死亡。代谢型谷氨酸受体（mGluRs)是一个与G－蛋白偶联的受体家族,分三型共八个亚型。其中Ⅱ和Ⅲ型mGluRs主要位于突触前,发挥对谷氨酸释放的负反馈调节。Ⅲ型mGluRs中的mGluR7位于谷氨酸能末梢突触前膜的活性区,发挥自身受体的作用,对正常情况下突触传递过程的谷氨酸释放进行负反馈调节;而属于Ⅱ型的mGluR2及属于Ⅲ型的mGluR4和mGluR8,则位于远离突有膜活性区的外突触区,因而正常突触传递过程中释放的谷氨酸量不能激活它们。只有在突触传递增强的情况下才被激活,抑制递质的释放。国外,mGluRs还分布在GABA能纤维末梢,通过突触前机制抑制GABA的释放。对突触前膜受体尤其是位于外突触区的mGluRs受体的研究,将有可能开发出理想的工具药,从而预防和阻止谷氨酸过量释放引起的神经毒及神经元的死亡。     

Adenosine A2A receptors and metabotropic glutamate 5 receptors are co-localized and functionally interact in the hippocampus: a possible key mechanism in the modulation of N-methyl-D-aspartate effects

Hippocampal metabotropic glutamate 5 receptors (mGlu5Rs) regulate both physiological and pathological responses to glutamate. Because mGlu5R activation enhances NMDA-mediated effects, and given the role played by NMDA receptors in synaptic plasticity and excitotoxicity, modulating mGlu5R may influence both the physiological and the pathological effects elicited by NMDA receptor stimulation. We evaluated whether adenosine A2A receptors (A(2A)Rs) modulated mGlu5R-dependent effects in the hippocampus, as they do in the striatum. Co-application of the A(2A)R agonist CGS 21680 with the mGlu5R agonist (RS)-2-chloro-s-hydroxyphenylglycine(CHPG) synergistically reduced field excitatory postsynaptic potentials in the CA1 area of rat hippocampal slices. Endogenous tone at A(2A)Rs seemed to be required to enable mGlu5R-mediated effects, as the ability of CHPG to potentiate NMDA effects was antagonized by the selective A(2A)R antagonist ZM 241385 in rat hippocampal slices and cultured hippocampal neurons, and abolished in the hippocampus of A(2A)R knockout mice. Evidence for the interaction between A(2A)Rs and mGlu5Rs was further strengthened by demonstrating their co-localization in hippocampal synapses. This is the first evidence showing that hippocampal A(2A)Rs and mGlu5Rs are co-located and act synergistically, and that A(2A)Rs play a permissive role in mGlu5R receptor-mediated potentiation of NMDA effects in the hippocampus.     

Regulatory role of C-terminus in the G-protein coupling of the metabotropic glutamate receptor 1

The signaling property of metabotropic glutamate receptor 1alpha (mGlu1alpha) is different from that of short-form splice variants. This could be caused by the exposure of a cluster of positively charged amino acid residues, RRKK, in the proximal C-tail which is thought to be masked by the long C-tail of mGlu1alpha. We found that the RRKK residues, when exposed, attenuate Gq coupling and decrease the basal activity and the surface expression of mGlu1, in agreement with previous results. Moreover, these residues abolish the Gi/o coupling of mGlu1, but do not affect glutamate-induced dimeric rearrangement and protein kinase A-dependent modulation of mGlu1. These results suggest that the RRKK residues do not inhibit the conformational change upon glutamate binding and protein accessibility to the intracellular loops where G-protein coupling occurs, but rather act as an inhibitory domain against G-protein coupling in a different manner depending on the type of G protein.     

Calcium dependence of native metabotropic glutamate receptor signaling in central neurons

Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors that are distributed throughout the brain and play important roles in regulation of synaptic efficacy. Some studies report that mGluRs heterologously expressed in nonneuronal cells are sensitive not only to glutamate but also to extracellular Ca²⁺ (Ca _o ²⁺ ). We studied the Ca _o ²⁺ -sensitivity of native mGluRs in mammalian central neurons. In cerebellar Purkinje cells that naturally express type-1 mGluR (mGluR1), physiological levels of Ca _o ²⁺ (around 2 mM) activate mGluR1-mediated intracellular Ca²⁺ mobilization. The activation of the native mGluR1 response to Ca _o ²⁺ appears to be slower than that to glutamate. Ca _o ²⁺ (2 mM) also augments glutamate analog-evoked, native mGluR1-mediated inward cation current and intracellular Ca _o ²⁺ mobilization. Detailed analysis of this effect suggests that Ca _o ²⁺ modulates the glutamate responsiveness of native and heterologously expressed mGluR1s in different manners. These findings suggest that Ca _o ²⁺ may enhance the basal level and glutamate responsiveness of neuronal mGluR signaling in vivo.     

Oncogenic activities of metabotropic glutamate receptor 1 (Grm1) in melanocyte transformation

Previously, we reported a transgenic mouse line, TG-3, that develops spontaneous melanoma with 100% penetrance. We demonstrated that ectopic expression of Grm1 in melanocytes was sufficient to induce melanoma in vivo. In this present study, the transforming properties of Grm1 in two cultured immortalized melanocytes were investigated. We showed that, in contrast to parental melanocytes, these Grm1-clones have lost their requirement of TPA supplement for proliferation and have acquired the ability to form colonies in semi-solid medium. Xenografts of these cells formed robust tumors in both immunodeficient nude and syngeneic mice with a short latency (3-5 days). The malignancy of these cells was demonstrated by angiogenesis and invasion to the muscle and the intestine. The requirement of Grm1 expression for the maintenance of transformation was demonstrated by an inducible siRNA system. Induction of expression of siRNA for Grm1 reduced the number of proliferating/viable cells in vitro and suppressed in vivo xenografted tumor growth in comparison with control. Taken together, these results showed that expression of exogeneously introduced Grm1 is sufficient to induce full transformation of immortalized melanocytes.