The role of group I metabotropic glutamate receptors in schizophrenia

Summary. It has been proposed that glutamatergic transmission, in particular NMDA receptor function, might be altered in schizophrenia. This hypothesis is mainly based on the observation that uncompetitive NMDA receptor antagonists, e.g. phencyclidine, evoke psychotic symptoms in healthy subjects, whereas agonists interacting at the glycine site of the NMDA receptor complex, e.g. glycine or D-serine, administered jointly with typical neuroleptics, can alleviate schizophrenic symptoms. The function of NMDA receptors may be modulated by group I mGluRs (mGluR1 and mGluR5), which have also been shown to be altered in schizophrenia. In rodents, mGluR5 antagonists, but not mGluR1 ones, potentiate the locomotor activity and the deficit of prepulse inhibition (PPI) induced by uncompetitive NMDA receptor antagonists. These antagonists (of either type) administered alone are not active in the above tests. Hence, antagonists of mGluR1 and mGluR5 may evoke different effects on the NMDA receptor antagonists-induced behavior and, possibly, on schizophrenic symptoms.     

Homer 1b regulates the trafficking of group I metabotropic glutamate receptors.

The molecular basis for glutamate receptor trafficking to the plasma membrane is not understood. In the present study, we demonstrate that Homer 1b (H1b), a constitutively expressed splice form of the immediate early gene product Homer (now termed Homer 1a) regulates the trafficking and surface expression of group I metabotropic glutamate receptors. H1b inhibits surface expression of the metabotropic glutamate receptor mGluR5 in heterologous cells, causing mGluR5 to be retained in the endoplasmic reticulum (ER). In contrast, mGluR5 alone or mGluR5 coexpressed with Homer 1a successfully travels through the secretory pathway to the plasma membrane. In addition, point mutations that disrupt mGluR5 binding to H1b eliminate ER retention of mGluR5, demonstrating that H1b affects metabotropic receptor localization via a direct protein-protein interaction. Electron microscopic analysis reveals that the group I metabotropic receptor mGluR1alpha is significantly enriched in the ER of Purkinje cells, suggesting that a similar mechanism may exist in vivo. Because H1b is found in dendritic spines of neurons, local retention of metabotropic receptors within dendritic ER provides a potential mechanism for regulating synapse-specific expression of group I metabotropic glutamate receptors.     

Expression and signaling of group I metabotropic glutamate receptors in astrocytes and microglia

Stimulation of astrocytes with the excitatory neurotransmitter glutamate leads to the formation of inositol 1,4,5-trisphosphate and the subsequent increase of intracellular calcium content. Astrocytes express both ionotropic receptors and metabotropic glutamate (mGlu) receptors, of which mGlu5 receptors are probably involved in glutamate-induced calcium signaling. The mGlu5 receptor occurs as two splice variants, mGlu5a and mGlu5b, but it was hitherto unknown which splice variant is responsible for the glutamate-induced effects in astrocytes. We report here that both mRNAs encoding mGlu5 receptor splice variants are expressed by cultured astrocytes. The expression of mGlu5a receptor mRNA is much stronger than that of mGlu5b receptor mRNA in these cells. In situ hybridization experiments reveal neuronal expression of mGlu5b receptor mRNA in adult rat forebrain but a strong neuronal expression of mGlu5a mRNA only in olfactory bulb. Signals for mGlu5a receptor mRNA in the rest of the brain were diffuse and weak but consistently above background. Activation of mGlu5 receptors in astrocytes yields increases in inositol phosphate production and transient calcium responses. It is surprising that the rank order of agonist potency [quisqualate > (2S,1 'S,2'S)-2-(carboxycyclopropyl)glycine = trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (1S,3R-ACPD) > glutamate] differs from that reported for recombinantly expressed mGlu5a receptors. The expression of mGlu5a receptor mRNA and the occurrence of 1S,3R-ACPD-induced calcium signaling were found also in cultured microglia, indicating for the first time expression of mGlu5a receptors in these macrophage-like cells.     

The mechanism of presynaptic long-term depression mediated by group I metabotropic glutamate receptors

1. Metabotropic glutamate receptors (mGluRs) are known to play a role in synaptic plasticity. In a study of rat hippocampal brain slices, we find that a brief perfusion of a group I mGluR agonist, (S)-3,5-dihydroxyphenylglycine (DHPG), induced a robust long-term depression (DHPG-LTD) in area CA1.2. The action was accompanied by an enhancement of the paired-pulse facilitation (PPF) ratio.3. At the same time DHPG enhanced ionophoretic responses to alpha-amino-3- hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA), kainic acid (KA), and N-methyl-D-aspartate (NMDA) in CA1 pyramidal neurons. This was only partially reversed by washing.4. These observations indicate that DHPG exerts two opposing actions, suppression of the synaptic transmission and facilitation of postsynaptic responses. However, the presynaptic action dominates, since the net effect of monosynaptic activation is a reduction of response.5. Perfusion of DHPG reduced three calcium-dependent responses in CA3 pyramidal neurons, which are presynaptic to CA1 neurons. These are calcium spike width and amplitude, after-hyperpolarization (AHP), and spike frequency adaptation (SFA).6. These results suggest that the DHPG-LTD results from modulation of the presynaptic calcium currents by group l mGluRs.     

Direct interaction enables cross-talk between ionotropic and group I metabotropic glutamate receptors

Functional interplay between ionotropic and metabotropic receptors frequently involves complex intracellular signaling cascades. The group I metabotropic glutamate receptor mGlu5a co-clusters with the ionotropic N-methyl-d-aspartate (NMDA) receptor in hippocampal neurons. In this study, we report that a more direct cross-talk can exist between these types of receptors. Using bioluminescence resonance energy transfer in living HEK293 cells, we demonstrate that mGlu5a and NMDA receptor clustering reflects the existence of direct physical interactions. Consequently, the mGlu5a receptor decreased NMDA receptor current, and reciprocally, the NMDA receptor strongly reduced the ability of the mGlu5a receptor to release intracellular calcium. We show that deletion of the C terminus of the mGlu5a receptor abolished both its interaction with the NMDA receptor and reciprocal inhibition of the receptors. This direct functional interaction implies a higher degree of target-effector specificity, timing, and subcellular localization of signaling than could ever be predicted with complex signaling pathways.     

Mechanism of regulation of casein kinase I activity by group I metabotropic glutamate receptors

Previously, we reported that (S)-3,5-dihydroxypenylglycine (DHPG), an agonist for group I metabotropic glutamate receptors (mGluRs), stimulates CK1 and Cdk5 kinase activities in neostriatal neurons, leading to enhanced phosphorylation, respectively, of Ser-137 and Thr-75 of DARPP-32 (dopamine and cAMP-regulated phosphoprotein, 32 kDa). We have now investigated the signaling pathway that leads from mGluRs to casein kinase 1 (CK1) activation. In mouse neostriatal slices, the effect of DHPG on phosphorylation of Ser-137 or Thr-75 of DARPP-32 was blocked by the phospholipase Cbeta inhibitor, the Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA/AM), and the calcineurin inhibitor cyclosporin A. In neuroblastoma N2a cells, the effect of DHPG on the activity of transfected HA-tagged CK1(epsilon) was blocked by BAPTA/AM and cyclosporin A. In neostriatal slices, the effect of DHPG on Cdk5 activity was also abolished by BAPTA/AM and cyclosporin A, presumably through blocking activation of CK1. Metabolic labeling studies and phosphopeptide mapping revealed that a set of C-terminal sites in HA-CK1epsilon were transiently dephosphorylated in N2a cells upon treatment with DHPG, and this was blocked by cyclosporin A. A mutant CK1epsilon with a nonphosphorylatable C-terminal domain was not activated by DHPG. Together, these studies suggest that DHPG activates CK1(epsilon) via Ca(2+)-dependent stimulation of calcineurin and subsequent dephosphorylation of inhibitory C-terminal autophosphorylation sites.     

Group I metabotropic glutamate receptors mediate a dual role of glutamate in T cell activation

Metabotropic glutamate receptors (mGluR) are present in cells of the nervous system, where they are activated by one of the main neurotransmitters, glutamate. They are also expressed in cells outside the nervous system. We identified and characterized two receptors belonging to group I mGluR, mGlu1R and mGlu5R, in human cell lines of lymphoid origin and in resting and activated lymphocytes from human peripheral blood. Both are highly expressed in the human Jurkat T cell line, whereas mGlu5R is expressed only in the human B cell line SKW6.4. In blood lymphocytes, mGlu5R is expressed constitutively, whereas mGlu1R is expressed only upon activation via the T cell receptor-CD3 complex. Group I receptors in the central nervous system are coupled to phospholipase C, whereas in blood lymphocytes, activation of mGlu5R does not trigger this signaling pathway, but instead activates adenylate cyclase. On the other hand, mGlu5R does not mediate ERK1/2 activation, whereas mGlu1R, which is coupled neither to phospholipase C nor to calcium channels and whose activation does not increase cAMP, activates the mitogen-activated protein kinase cascade. The differential expression of mGluR in resting and activated lymphocytes and the different signaling pathways that are triggered when mGlu1Rs or mGlu5Rs are activated point to a key role of glutamate in the regulation of T cell physiological function. The study of the signaling pathways (cAMP production and ERK1/2 phosphorylation) and the proliferative response obtained in the presence of glutamate analogs suggests that mGlu1R and mGlu5R have distinct functions. mGlu5R mediates the reported inhibition of cell proliferation evoked by glutamate, which is reverted by the activation of inducible mGlu1R. This is a novel non-inhibitory action mechanism for glutamate in lymphocyte activation. mGlu1R and mGlu5R thus mediate opposite glutamate effects in human lymphocytes.     

The role of striatal metabotropic glutamate receptors in Parkinson's disease

The primary cause of Parkinson's disease is a loss of dopamine in the corpus striatum. It has been postulated that this effect leads to disinhibition of the striopallidal pathway and secondarily, to a functional shift towards glutamatergic stimulation. The aim of the present study was to find out whether inhibition of glutamatergic transmission at a level of metabotropic glutamate receptors (mGluRs) in the striatum may alleviate parkinsonian-like symptoms in rats. The non-competitive antagonist of receptor subtype 5 (mGluR5), MPEP (1.0-10 mg/kg ip), or the agonist of group II mGluRs, LY354,740 (5-10 mg/kg ip), reduced haloperidol-induced muscle rigidity and catalepsy. Intrastriatal injections of the mGluR1 antagonist, (RS) AIDA (7.5-15 microg/0.5 microl), but not of the agonist of group II mGluRs, 2R,4R-APDC (7.5-15 microg/0.5 microl), inhibited the muscle rigidity induced by haloperidol. In order to search for an influence of mGluRs on the striopallidal pathway, the effect of MPEP or of the agonist of group II mGluRs, DCG-IV, on the proenkephalin (PENK) mRNA expression in the dorso-lateral striatum was examined by an in situ hybridization. Repeated MPEP (6 x 10 mg/kg ip) administration did not influence PENK expression in na?ve rats, but diminished that increased by haloperidol. In contrast, repeated DCG-IV (3 x 1 nmol/4 microl icv) injections enhanced both the control and the haloperidol-increased levels of PENK expression. The obtained results suggest that blockade of group I mGluRs, or stimulation of group II mGluRs may be important to ameliorate parkinsonian symptoms. Striatal mGluRs may contribute to at least some of these effects.     

Phosphorylation of the homer-binding domain of group I metabotropic glutamate receptors by cyclin-dependent kinase 5

Phosphorylation of neurotransmitter receptors can modify their activity and regulate neuronal excitability. Cyclin-dependent kinase 5 (cdk5) is a proline-directed serine/threonine kinase involved not only in neuronal development, but also in synaptic function and plasticity. Here we demonstrate that group I metabotropic glutamate receptors (mGluRs), which modulate post-synaptic signaling by coupling to intracellular signal transduction pathways, are phosphorylated by cdk5. In vitro kinase assays reveal that cdk5 phosphorylates mGluR5 within the domain of the receptor that interacts with the scaffolding protein homer. Using a novel phosphospecific mGluR antibody, we show that the homer-binding domain of both mGluR1 and mGluR5 are phosphorylated in vivo , and that inhibition of cdk5 with siRNA decreases the amount of phosphorylated receptor. Furthermore, kinetic binding analysis, by surface plasmon resonance, indicates that phosphorylation of mGluR5 enhances its association with homer. Homer protein complexes in the post-synaptic density, and their disruption by an activity-dependent short homer 1a isoform, have been shown to regulate the trafficking and signaling of the mGluRs and impact many neuroadaptive processes. Phosphorylation of the mGluR homer-binding domain, in contrast to homer 1a induction, provides a novel mechanism for potentially regulating a subset of homer interactions.     

Role of group I metabotropic glutamate receptors and NMDA receptors in homocysteine-evoked acute neurodegeneration of cultured cerebellar granule neurones

Hyperhomocysteinemia is a risk factor in neurodegeneration. It has been suggested that apart from disturbances in methylation processes, the mechanisms of this effect may include excitotoxicity mediated by the N-methyl-D-aspartate (NMDA) receptors. In this study we demonstrate that apart from NMDA receptors, also group I metabotropic glutamate receptors participate in acute homocysteine (Hcy)-induced neurotoxicity in cultured rat cerebellar granule neurones. Primary neuronal cultures were incubated for 30 min in the Mg(2+)-free ionic medium containing homocysteine and other ligands, and neurodegenerative changes were assessed 24h later using propidium iodide staining. D,L-Homocysteine given alone appeared to be a weak neurotoxin, with EC(50) of 17.4mM, whereas EC(50) for L-glutamate was 0.17 mM. Addition of 50 microM glycine enhanced homocysteine neurotoxicity, and only that portion of neurotoxicity was abolished by 0.5 microM MK-801, an uncompetitive NMDA receptor antagonist. The net stimulation of 45Ca uptake by granule cells incubated in the presence of 25 mM D,L-homocysteine with 50 microM glycine was only 3% of the net uptake evoked by 1mM glutamate. Application of an antagonist of group I metabotropic glutamate receptors (mGluRs) LY367385 at 25 and 250 microM concentrations, induced a dose-dependent partial neuroprotection, whereas given together with MK-801 completely prevented neurotoxicity. In the absence of glycine, LY367385 and MK-801 given alone failed to induce neuroprotection, while applied together completely prevented homocysteine neurotoxicity. Agonist of group I mGluRs, 10 trans-azetidine-2,3-dicarboxylic acid (t-ADA) induced significant neurotoxicity. This study shows for the first time that acute homocysteine-induced neurotoxicity is mediated both by group I mGluRs and NMDA receptors, and is not accompanied by massive influx of extracellular Ca(2+) to neurones.     

Stimulation of group II metabotropic glutamate receptors or inhibition of group I ones exerts anxiolytic-like effects in rats

Using the conflict drinking Vogel test in rats as a model we examined the anxiolytic-like activity of (S)-4-carboxyphenylglycine (S-4CPG), an antagonist of group I metabotropic glutamate receptors (mGlu receptors), of (RS)-a-methylserine-O-phosphate-monophenyl ester (MSOPPE), an antagonist of group II mGlu receptors, and of (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I), an agonist of group II mGlu receptors. The obtained results indicate that intrahippocampal administration of S-4CPG and L-CCG-I, but not MSOPPE to rats produces a dose-dependent anticonflict effect, which is unrelated to the reduced perception of the stimulus or to an increased thirst drive. The hippocampus may be one of the neuroanatomical sites of the anxiolytic-like effects of either agent.     

Developmental regulation of group I metabotropic glutamate receptors in the premature brain and their protective role in a rodent model of periventricular leukomalacia

Cerebral white matter injury in premature infants, known as periventricular leukomalacia (PVL), is common after hypoxia-ischemia (HI). While ionotropic glutamate receptors (iGluRs) can mediate immature white matter injury, we have previously shown that excitotoxic injury to premyelinating oligodendrocytes (preOLs) in vitro can be attenuated by group I metabotropic glutamate receptor (mGluR) agonists. Thus, we evaluated mGluR expression in developing white matter in rat and human brain, and tested the protective efficacy of a central nervous system (CNS)-penetrating mGluR agonist on injury to developing oligodendrocytes (OLs) in vivo. Group I mGluRs (mGluR1 and mGluR5) were strongly expressed on OLs in neonatal rodent cerebral white matter throughout normal development, with highest expression early in development on preOLs. Specifically at P6, mGluR1 and mGLuR5 were most highly expressed on GalC-positive OLs compared to neurons, axons, astrocytes and microglia. Systemic administration of (1S,3R) 1-aminocyclopentane-trans-1,3,-dicarboxylic acid (ACPD) significantly attenuated the loss of myelin basic protein in the white matter following HI in P6 rats. Assessment of postmortem human tissue showed both mGluR1 and mGluR5 localized on immature OLs in white matter throughout development, with mGluR5 highest in the preterm period. These data indicate group I mGluRs are highly expressed on OLs during the peak period of vulnerability to HI and modulation of mGluRs is protective in a rodent model of PVL. Group I mGluRs may represent important therapeutic targets for protection from HI-mediated white matter injury.     

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.     

Postsynaptic scaffold protein Homer 1a protects against traumatic brain injury via regulating group I metabotropic glutamate receptors

Traumatic brain injury (TBI) produces excessive glutamate, leading to excitotoxicity via the activation of glutamate receptors. Postsynaptic density scaffold proteins have crucial roles in mediating signal transduction from glutamate receptors to their downstream mediators. Therefore, studies on the mechanisms underlying regulation of excitotoxicity by scaffold proteins can uncover new treatments for TBI. Here, we demonstrated that the postsynaptic scaffold protein Homer 1a was neuroprotective against TBI in vitro and in vivo, and this neuroprotection was associated with its effects on group I metabotropic glutamate receptors (mGluRs). Upon further study, we found that Homer 1a mainly affected neuronal injury induced by mGluR1 activation after TBI and also influenced mGluR5 function when its activity was restored. The ability of Homer 1a to disrupt mGluR-ERK signaling contributed to its ability to regulate the functions of mGluR1 and mGluR5 after traumatic injury. Intracellular Ca²⁺ and PKC were two important factors involved in the mediation of mGluR-ERK signaling by Homer 1a. These results define Homer 1a as a novel endogenous neuroprotective agent against TBI.     

Antagonists of group I metabotropic glutamate receptors do not inhibit induction of ischemic tolerance in gerbil hippocampus

In this study we tested the effect of antagonists of two subtypes of the group I metabotropic glutamate receptors (mGluRs GI) on the induction of ischemic tolerance in relation to brain temperature. These experiments were prompted by indications that glutamate receptors may participate in the mechanisms of ischemic preconditioning. The role of NMDA receptors in the induction of ischemic tolerance has been debated while there is lack of information concerning the involvement of mGluRs GI in this phenomenon. The tolerance to injurious 3 min forebrain ischemia in Mongolian gerbils was induced 48 h earlier by 2 min preconditioning ischemia. Brain temperature was measured using telemetry equipment. EMQMCM and MTEP, antagonists of mGluR1 and mGluR5, respectively, were injected i.p. at a dose of 5 mg/kg. They were administered either before preconditioning ischemia in a single dose or after 2 min ischemia three times every 2 h. Both antagonists did not inhibit the induction of ischemic tolerance. Thus, our data indicate that group I metabotropic glutamate receptors do not play an essential role in the induction of ischemic tolerance.     

Localization and function of group III metabotropic glutamate receptors in rat pancreatic islets

Pancreatic islets contain ionotropic glutamate receptors that can modulate hormone secretion. The purpose of this study was to determine whether islets express functional group III metabotropic glutamate (mGlu) receptors. RT-PCR analysis showed that rat islets express the mGlu8 receptor subtype. mGlu8 receptor immunoreactivity was primarily displayed by glucagon-secreting alpha-cells and intrapancreatic neurons. By demonstrating the immunoreactivities of both glutamate and the vesicular glutamate transporter 2 (VGLUT2) in these cells, we established that alpha-cells express a glutamatergic phenotype. VGLUT2 was concentrated in the secretory granules of islet cells, suggesting that glutamate might play a role in the regulation of glucagon processing. The expression of mGlu8 by glutamatergic cells also suggests that mGlu8 may function as an autoreceptor to regulate glutamate release. Pancreatic group III mGlu receptors are functional because mGlu8 receptor agonists inhibited glucagon release and forskolin-induced accumulation of cAMP in isolated islets, and (R,S)-cyclopropyl-4-phosphonophenylglycine, a group III mGlu receptor antagonist, reduced these effects. Because excess glucagon secretion causes postprandial hyperglycemia in patients with type 2 diabetes, group III mGlu receptor agonists could be of value in the treatment of these patients.     

Ca-dependent binding of calcium-binding protein 1 to presynaptic group III metabotropic glutamate receptors and blockage by phosphorylation of the receptors

Presynaptic group III metabotropic glutamate receptors (mGluRs) and Ca²⁺ channels are the main neuronal activity-dependent regulators of synaptic vesicle release, and they use common molecules in their signaling cascades. Among these, calmodulin (CaM) and the related EF-hand Ca²⁺-binding proteins are of particular importance as sensors of presynaptic Ca²⁺, and a multiple of them are indeed utilized in the signaling of Ca²⁺ channels. However, despite its conserved structure, CaM is the only known EF-hand Ca²⁺-binding protein for signaling by presynaptic group III mGluRs. Because the mGluRs and Ca²⁺ channels reciprocally regulate each other and functionally converge on the regulation of synaptic vesicle release, the mGluRs would be expected to utilize more EF-hand Ca²⁺-binding proteins in their signaling. Here I show that calcium-binding protein 1 (CaBP1) bound to presynaptic group III mGluRs competitively with CaM in a Ca²⁺-dependent manner and that this binding was blocked by protein kinase C (PKC)-mediated phosphorylation of these receptors. As previously shown for CaM, these results indicate the importance of CaBP1 in signal cross talk at presynaptic group III mGluRs, which includes many molecules such as cAMP, Ca²⁺, PKC, G protein, and Munc18-1. However, because the functional diversity of EF-hand calcium-binding proteins is extraordinary, as exemplified by the regulation of Ca²⁺ channels, CaBP1 would provide a distinct way by which presynaptic group III mGluRs fine-tune synaptic transmission.