Activation and Desensitization Mechanism of AMPA Receptor-TARP Complex by Cryo-EM.

AMPA receptors mediate fast excitatory neurotransmission in the mammalian brain and transduce the binding of presynaptically released glutamate to the opening of a transmembrane cation channel. Within the postsynaptic density, however, AMPA receptors coassemble with transmembrane AMPA receptor regulatory proteins (TARPs), yielding a receptor complex with altered gating kinetics, pharmacology, and pore properties. Here, we elucidate structures of the GluA2-TARP γ2 complex in the presence of the partial agonist kainate or the full agonist quisqualate together with a positive allosteric modulator or with quisqualate alone. We show how TARPs sculpt the ligand-binding domain gating ring, enhancing kainate potency and diminishing the ensemble of desensitized states. TARPs encircle the receptor ion channel, stabilizing M2 helices and pore loops, illustrating how TARPs alter receptor pore properties. Structural and computational analysis suggests the full agonist and modulator complex harbors an ion-permeable channel gate, providing the first view of an activated AMPA receptor.

Novel Functional Properties of Drosophila CNS Glutamate Receptors.

Phylogenetic analysis reveals AMPA, kainate, and NMDA receptor families in insect genomes, suggesting conserved functional properties corresponding to their vertebrate counterparts. However, heterologous expression of the Drosophila kainate receptor DKaiR1D and the AMPA receptor DGluR1A revealed novel ligand selectivity at odds with the classification used for vertebrate glutamate receptor ion channels (iGluRs). DKaiR1D forms a rapidly activating and desensitizing receptor that is inhibited by both NMDA and the NMDA receptor antagonist AP5; crystallization of the KaiR1D ligand-binding domain reveals that these ligands stabilize open cleft conformations, explaining their action as antagonists. Surprisingly, the AMPA receptor DGluR1A shows weak activation by its namesake agonist AMPA and also by quisqualate. Crystallization of the DGluR1A ligand-binding domain reveals amino acid exchanges that interfere with binding of these ligands. The unexpected ligand-binding profiles of insect iGluRs allows classical tools to be used in novel approaches for the study of synaptic regulation. VIDEO ABSTRACT.

Atypical signaling of metabotropic glutamate receptor 1 in human melanoma cells.

The metabotropic glutamate 1 (mGlu1) receptor has emerged as a novel target for the treatment of metastatic melanoma and various other cancers. Our laboratory has demonstrated that a selective, non-competitive mGlu1 receptor antagonist slows human melanoma growth in vitro and in vivo. In this study, we sought to determine if the activation of a canonical G protein-dependent signal transduction cascade, which is often used as an output of mGlu1 receptor activity in neuronal cells, correlated with mGlu1 receptor-mediated melanoma cell viability. Glutamate, the endogenous ligand of mGlu1 receptors, significantly increased melanoma cell viability, but did not stimulate phosphoinositide (PI) hydrolysis in several human melanoma cell lines. In contrast, melanoma cell viability was not increased by quisqualate, a highly potent mGlu1 receptor agonist, or DHPG, a selective group I mGlu receptor agonist. Similarly to glutamate, quisqualate also failed to stimulate PI hydrolysis in mGlu1 receptor-expressing melanoma cells. These results suggest that the canonical G protein-dependent signal transduction cascade is not coupled to mGlu1 receptors in all human melanoma cells. On the other hand, dynamin inhibition selectively decreased viability of mGlu1 receptor-expressing melanoma cells, suggesting that a mechanism requiring internalization may control melanoma cell viability. Taken together, these data demonstrate that the approaches commonly used to study mGlu1 receptor function and signaling in other systems may be inappropriate for studying mGlu1 receptor-mediated melanoma cell viability.

Anti-cancer effect of metabotropic glutamate receptor 1 inhibition in human glioma U87 cells: involvement of PI3K/Akt/mTOR pathway.

BACKGROUND: Metabotropic glutamate receptors (mGluRs) are G-protein-coupled receptors that mediate neuronal excitability and synaptic plasticity in the central nervous system, and emerging evidence suggests a role of mGluRs in the biology of cancer. Previous studies showed that mGluR1 was a potential therapeutic target for the treatment of breast cancer and melanoma, but its role in human glioma has not been determined. METHODS: In the present study, we investigated the effects of mGluR1 inhibition in human glioma U87 cells using specific targeted small interfering RNA (siRNA) or selective antagonists Riluzole and BAY36-7620. The anti-cancer effects of mGluR1 inhibition were measured by cell viability, lactate dehydrogenase (LDH) release, TUNEL staining, cell cycle assay, cell invasion and migration assays in vitro, and also examined in a U87 xenograft model in vivo. RESULTS: Inhibition of mGluR1 significantly decreased the cell viability but increased the LDH release in a dose-dependent fashion in U87 cells. These effects were accompanied with the induction of caspase-dependent apoptosis and G0/G1 cell cycle arrest. In addition, the results of Matrigel invasion and cell tracking assays showed that inhibition of mGluR1 apparently attenuated cell invasion and migration in U87 cells. All these anti-cancer effects were ablated by the mGluR1 agonist L-quisqualic acid. The results of western blot analysis showed that mGluR1 inhibition overtly decreased the phosphorylation of PI3K, Akt, mTOR and P70S6K, indicating the mitigated activation of PI3K/Akt/mTOR pathway. Moreover, the anti-tumor activity of mGluR1 inhibition in vivo was also demonstrated in a U87 xenograft glioma model in athymic nude mice. CONCLUSION: The remarkable efficiency of mGluR1 inhibition to induce cell death in U87 cells may find therapeutic application for the treatment of glioma patients.

Biphasic modulation by mGlu5 receptors of TRPV1-mediated intracellular calcium elevation in sensory neurons contributes to heat sensitivity.

BACKGROUND AND PURPOSE: Elevation of glutamate, an excitatory amino acid, during inflammation and injury plays a crucial role in the reception and transmission of sensory information via ionotropic and metabotropic receptors. This study aimed to investigate the mechanisms underlying the biphasic effects of metabotropic glutamate mGlu5 receptor activation on responses to noxious heat. EXPERIMENTAL APPROACH: We assessed the effects of intraplantar quisqualate, a non-selective glutamate receptor agonist, on heat and mechanical pain behaviours in mice. In addition, the effects of quisqualate on the intracellular calcium response and on membrane currents mediated by TRPV1 channels, were examined in cultured dorsal root ganglion neurons from mice. KEY RESULTS: Activation of mGlu5 receptors in hind paw transiently increased, then decreased, the response to noxious heat. In sensory neurons, activation of mGlu5 receptors potentiated TRPV1-mediated intracellular calcium elevation, while terminating activation of mGlu5 receptors depressed it. TRPV1-induced currents were potentiated by activation of mGlu5 receptors under voltage clamp conditions and these disappeared after washout. However, voltage-gated calcium currents were inhibited by the mGlu5 receptor agonist, even after washout. CONCLUSIONS AND IMPLICATIONS: These results suggest that, in sensory neurons, mGlu5 receptors biphasically modulate TRPV1-mediated intracellular calcium response via transient potentiation of TRPV1 channel-induced currents and persistent inhibition of voltage-gated calcium currents, contributing to heat hyper- and hypoalgesia.

Enteral supplements of a carbon monoxide donor CORM-A1 protect against cerebrovascular dysfunction caused by neonatal seizures.

Cerebral blood flow dysregulation caused by oxidative stress contributes to adverse neurologic outcome of seizures. A carbon monoxide (CO) donor CORM-A1 has antioxidant and cytoprotective properties. We investigated whether enteral supplements of CORM-A1 can improve cerebrovascular outcome of bicuculline-induced seizures in newborn piglets. CORM-A1 (2 mg/kg) was given to piglets via an oral gastric tube 10 minutes before or 20 minutes after seizure onset. Enteral CORM-A1 elevated CO in periarachnoid cerebrospinal fluid and produced a dilation of pial arterioles. Postictal cerebral vascular responses to endothelium-, astrocyte-, and vascular smooth muscle-dependent vasodilators were tested 48 hours after seizures by intravital microscopy. The postictal responses of pial arterioles to bradykinin, glutamate, the AMPA receptor agonist quisqualic acid, ADP, and heme were greatly reduced, suggesting that seizures cause injury to endothelial and astrocyte components of the neurovascular unit. In contrast, in the two groups of piglets receiving enteral CORM-A1, the postictal cerebral vascular responsiveness to these dilators was improved. Overall, enteral supplements of CORM-A1 before or during seizures offer a novel effective therapeutic option to deliver cytoprotective mediator CO to the brain, reduce injury to endothelial and astrocyte components of cerebral blood flow regulation and to improve the cerebrovascular outcome of neonatal seizures.

Extracellular calcium modulates actions of orthosteric and allosteric ligands on metabotropic glutamate receptor 1α.

Metabotropic glutamate receptor 1α (mGluR1α), a member of the family C G protein-coupled receptors, is emerging as a potential drug target for various disorders, including chronic neuronal degenerative diseases. In addition to being activated by glutamate, mGluR1α is also modulated by extracellular Ca(2+). However, the underlying mechanism is unknown. Moreover, it has long been challenging to develop receptor-specific agonists due to homologies within the mGluR family, and the Ca(2+)-binding site(s) on mGluR1α may provide an opportunity for receptor-selective targeting by therapeutics. In the present study, we show that our previously predicted Ca(2+)-binding site in the hinge region of mGluR1α is adjacent to the site where orthosteric agonists and antagonists bind on the extracellular domain of the receptor. Moreover, we found that extracellular Ca(2+) enhanced mGluR1α-mediated intracellular Ca(2+) responses evoked by the orthosteric agonist l-quisqualate. Conversely, extracellular Ca(2+) diminished the inhibitory effect of the mGluR1α orthosteric antagonist (S)-α-methyl-4-carboxyphenylglycine. In addition, selective positive (Ro 67-4853) and negative (7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester) allosteric modulators of mGluR1α potentiated and inhibited responses to extracellular Ca(2+), respectively, in a manner similar to their effects on the response of mGluR1α to glutamate. Mutations at residues predicted to be involved in Ca(2+) binding, including E325I, had significant effects on the modulation of responses to the orthosteric agonist l-quisqualate and the allosteric modulator Ro 67-4853 by extracellular Ca(2+). These studies reveal that binding of extracellular Ca(2+) to the predicted Ca(2+)-binding site in the extracellular domain of mGluR1α modulates not only glutamate-evoked signaling but also the actions of both orthosteric ligands and allosteric modulators on mGluR1α.

Anxiolytic properties of Valeriana officinalis in the zebrafish: a possible role for metabotropic glutamate receptors.

Valerian extract is used in complementary and alternative medicine for its anxiolytic and sedative properties. Our previous research demonstrated valerian interactions with glutamate receptors. The purpose of this study was to determine if valerian anxiolytic properties are mediated by metabotropic glutamate receptors (mGluR) such as mGluR (1/5) (mGluR I) and mGluR (2/3) (mGluR II). Adult wild-type zebrafish (Danio rerio) prefer the black compartment and avoid the white compartment in the dark/light preference task. Zebrafish exposed to 1 mg/mL of valerian extract or 0.00117 mg/mL valerenic acid increased their residence time in the white side by 84.61 ± 6.55 % and 58.30 ± 8.97 %, respectively. LAP3 (mGluR I antagonist) and EGLU (mGluR II antagonist) significantly inhibited the effects of valerian and valerenic acid. These results demonstrated that valerian and valerenic acid have anxiolytic properties in the zebrafish. Moreover, the selective interaction of valerian with mGluR I and II represent an alternative explanation for the anxiolytic properties of this plant and support the role of mGluR in anxiety.

Orthosteric and allosteric drug binding sites in the Caenorhabditis elegans mgl-2 metabotropic glutamate receptor.

The metabotropic glutamate receptors (mGluRs) are evolutionarily conserved from nematodes to vertebrates. The Caenorhabditis elegans (C. elegans) genome contains three mGluR genes referred to as mgl-1, mgl-2, and mgl-3. The aim of this study was to characterize the pharmacological profiles of orthosteric and allosteric mGluR ligands on mgl-2. A phylogenetic analysis revealed that mgl-2 is closely associated with the mammalian Group 1 mGluRs (mGluR1 and mGluR5) and is distinct from Group 2 and 3 mGluRs. The ligand binding domain of mgl-2 displayed higher homology to the rat Group 1 mGluRs binding domains compared to the level of homology in the heptahelical transmembrane domain regions. We found that, when transiently expressed in human embryonic kidney 293 cells, mgl-2 can be activated by glutamate and couples to human G-proteins to induce the release of intracellular calcium. Dose-response analyses revealed that mgl-2 has approximately a 15-20-fold lower affinity for glutamate and quisqualate compared to rat mGluR5. In contrast to orthosteric agonists, Group 1 negative allosteric modulators that target the transmembrane domain were ineffective at mgl-2. Surprisingly, CDPPB, an mGluR5 positive allosteric modulator, potentiated glutamate mediated activation of mgl-2, although MPEP and fenobam, two mGluR5 antagonists that share similar binding residues with CDPPB were ineffective at mgl-2. These findings indicate that selective pressures on mGluR protein structures have resulted in conservation of the glutamate binding site, whereas the allosteric modulator sites have been subjected to greater divergent evolutionary changes.

The loss of an electrostatic contact unique to AMPA receptor ligand binding domain 2 slows channel activation.

Ligand-gated ion channels undergo conformational changes that transfer the energy of agonist binding to channel opening. Within ionotropic glutamate receptor (iGluR) subunits, this process is initiated in their bilobate ligand binding domain (LBD) where agonist binding to lobe 1 favors closure of lobe 2 around the agonist and allows formation of interlobe hydrogen bonds. AMPA receptors (GluAs) differ from other iGluRs because glutamate binding causes an aspartate-serine peptide bond in a flexible part of lobe 2 to rotate 180° (flipped conformation), allowing these residues to form cross-cleft H-bonds with tyrosine and glycine in lobe 1. This aspartate also contacts the side chain of a lysine residue in the hydrophobic core of lobe 2 by a salt bridge. We investigated how the peptide flip and electrostatic contact (D655-K660) in GluA3 contribute to receptor function by examining pharmacological and structural properties with an antagonist (CNQX), a partial agonist (kainate), and two full agonists (glutamate and quisqualate) in the wildtype and two mutant receptors. Alanine substitution decreased the agonist potency of GluA3(i)-D655A and GluA3(i)-K660A receptor channels expressed in HEK293 cells and differentially affected agonist binding affinity for isolated LBDs without changing CNQX affinity. Correlations observed in the crystal structures of the mutant LBDs included the loss of the D655-K660 electrostatic contact, agonist-dependent differences in lobe 1 and lobe 2 closure, and unflipped D(A)655-S656 bonds. Glutamate-stimulated activation was slower for both mutants, suggesting that efficient energy transfer of agonist binding within the LBD of AMPA receptors requires an intact tether between the flexible peptide flip domain and the rigid hydrophobic core of lobe 2.

Selective and sustained α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor activation in cerebellum induces dystonia in mice.

Dystonia is a neurological disorder characterized by involuntary muscle contractions that cause twisting movements and abnormal postures. Functional imaging consistently reveals cerebellar overactivity in dystonic patients regardless of the type or etiology of the disorder. To explore mechanisms that might explain the basis for the cerebellar overactivity in dystonia, normal mice were challenged with intracerebellar application of a variety of agents that induce hyperexcitability. A nonspecific increase in cerebellar excitability, such as that produced by picrotoxin, was not associated with dystonia. Instead, glutamate receptor activation, specifically AMPA receptor activation, was necessary to evoke dystonia. AMPA receptor agonists induced dystonia, and AMPA receptor antagonists reduced the dystonia induced by glutamate receptor agonists. AMPA receptor antagonists also ameliorated the dystonia exhibited by the dystonic mouse mutant tottering, suggesting that AMPA receptors may play a role in some other genetic models of dystonia. Furthermore, AMPA receptor desensitization mediated the expression of dystonia. Preventing AMPA receptor desensitization with cyclothiazide or the nondesensitizing agonist kainic acid exacerbated the dystonic response. These results suggest the novel hypothesis that the cerebellar overactivity observed in neuroimaging studies of patients with dystonia may be an indirect reflection of abnormal glutamate signaling. In addition, these results imply that reducing AMPA receptor activation by blocking AMPA receptors and promoting AMPA receptor desensitization or negative allosteric modulators may prove to be beneficial for treating dystonia.

Metabotropic glutamate receptor-1: a potential therapeutic target for the treatment of breast cancer.

Metabotropic glutamate receptors are G-protein-coupled receptors normally expressed in the central nervous system where they mediate neuronal excitability, synaptic plasticity, and feedback inhibition of neurotransmitter release. However, recent data suggest that these receptors are also expressed and functional in some cancers, most notably melanoma. We detected the expression of metabotropic glutamate receptor-1 (gene: GRM1; protein: mGluR1) in triple negative breast cancer cells and evaluated its role in regulating the pro-proliferative phenotype of these cells. mGluR1 inhibitors (Riluzole or BAY36-7620) inhibited the proliferation of triple negative breast cancer cells in a time- and dose-dependent manner and this inhibition correlated with increased apoptosis as demonstrated by increase in PARP cleavage products and Annexin V staining. mGluR1 knockdown using Lentiviral constructs expressing shRNA targeting GRM1 also inhibited proliferation compared to non-silencing controls. In addition, treatment of mice bearing MDA-MB-231 xenografts with Riluzole or BAY36-7620, by intraperitoneal injection, resulted in a significant reduction in tumor volume of up to 80%. Moreover, Riluzole was effective against triple negative breast cancer xenografts in mice at doses equivalent to those currently being used in humans for the treatment of amyotrophic lateral sclerosis. Our observations implicate mGluR1 and glutamate signaling as a promising new molecular target for the treatment of breast cancer. Even more promising, Riluzole, because it is an oral drug that can be administered with low toxicity, represents a promising approach in the treatment of triple negative breast cancer.

Evaluation of cannabinoid receptor 2 and metabotropic glutamate receptor 1 functional responses using a cell impedance-based technology.

Recently, new technologies based on biosensors and called label free have been developed. These technologies eliminate the need for using markers and dyes. The authors applied one of these technologies, based on measurement of cell impedance variation, to study the pharmacological profiles of ligands for the cannabinoid receptor 2 (CB2), a Gi-coupled receptor, and for the metabopotropic glutamate receptor 1 (mGluR1), a Gq-coupled receptor. Reference agonists and antagonists/inverse agonists for the 2 receptors were applied to recombinant cell lines and impedance monitored over time. Agonists (JWH133 and CP55940 for CB2; quisqualate, glutamate, 1S-3R-ACPD, and S-3,5-DHPG for mGluR1) triggered a variation of impedance consistent in both potency and efficacy with data obtained using classical assays measuring cAMP or Ca(2+) levels. This effect was not present in the parental nontransfected cell line, confirming specific receptor-mediated response. Application of antagonists (AM630 for CB2; YM298198, SCH1014222, J&J16259685, and CPCCOEt for mGluR1) reduced agonist-induced impedance changes. The only exception was the mGluR1 antagonist BAY367620 that, while active in the Ca(2+) assay, was inactive in the impedance assay. Overall, these results confirm the possibility of using cell impedance-based technology to study the pharmacological profile of ligands acting at G-protein-coupled receptors coupled to different downstream signaling pathways.

Lateral diffusion of inositol 1,4,5-trisphosphate receptor type 1 in Purkinje cells is regulated by calcium and actin filaments.

Inositol 1,4,5-trisphosphate receptor type 1 (IP(3) R1) is an intracellular Ca(2+) release channel that plays crucial roles in the functions of Purkinje cells. The dynamics of IP(3) R1 on the endoplasmic reticulum membrane and the distribution of IP(3) R1 in neurons are thought to be important for the spatial regulation of Ca(2+) release. In this study, we analyzed the lateral diffusion of IP(3) R1 in Purkinje cells in cerebellar slice cultures using fluorescence recovery after photobleaching. In the dendrites of Purkinje cells, IP(3) R1 showed lateral diffusion with an effective diffusion constant of approximately 0.30 µm(2) /s, and the diffusion of IP(3) R1 was negatively regulated by actin filaments. We found that actin filaments were also involved in the regulation of IP(3) R1 diffusion in the spine of Purkinje cells. Glutamate or quisqualic acid stimulation, which activates glutamate receptors and leads to a Ca(2+) transient in Purkinje cells, decreased the diffusion of IP(3) R1 and increased the density of actin in spines. These findings indicate that the neuronal activity-dependent augmentation of actin contributes to the stabilization of IP(3) R1 in spines.

Effects of TRH and its analogues on primary cortical neuronal cell damage induced by various excitotoxic, necrotic and apoptotic agents.

The tripeptide thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2) has been shown to possess neuroprotective activity in in vitro and in vivo models. Since its potential utility is limited by relatively rapid metabolism, metabolically stabilized analogues have been constructed. In the present study we investigated the influence of TRH and its three stable analogues: Montirelin (MON, CG-3703), RGH-2202 (L-6-keto-piperidine-2carbonyl-l-leucyl-l-prolinamide) and Z-TRH (N-carbobenzyloxy-pGlutamyl-Histydyl-Proline) in various models of mouse cortical neuronal cell injury. Twenty four hour pre-treatment with TRH and its analogues in low micromolar concentrations attenuated the neuronal cell death evoked by excitatory amino acids (EAAs: glutamate, NMDA, kainate, quisqualate) and hydrogen peroxide. All the peptides showed neuroprotective action on staurosporine (St)-evoked apoptotic neuronal cell death, but this effect was caspase-3 independent. Interestingly, in mixed neuronal-glial cell preparations only MON decreased St- and glutamate-evoked neurotoxicity. None of the peptides inhibited the doxorubicin- and lactacystin-induced neuronal cortical cell death, agents acting via activation of death receptor (FAS) or inhibition of proteasome function, respectively. Furthermore, we found that neither inhibitors of PI3-K (wortmannin, LY 294002) nor MAPK/ERK1/2 (PD 098059, U 0126) were able to inhibit neuroprotective properties of TRH and MON in St model of apoptosis. The protection mediated by TRH and MON it that model was also not connected with influence of peptides on the pro-apoptotic GSK-3beta and JNK protein kinase expression and activity. Further studies showed that calpains, calcium-activated proteases were induced by Glu, but not by St in cortical neurons. Moreover, the Glu-evoked increase in spectrin alpha II cleavage product induced by calpains was blocked by TRH. The obtained data showed that the potency of TRH and its analogues in inhibiting EAAs- and H(2)O(2)-induced neuronal cell death from the highest to lowest activity was: MON>TRH>Z-TRH>RHG. Interestingly, all peptides were active against St-induced apoptosis, however, on concentration basis MON was far more potent than the other peptides. None of the peptides inhibited Dox- and LC-evoked apoptotic cell death. Additionally, the data exclude potential role of pro-survival (PI3-K/Akt and MAPK/ERK1/2) and pro-apoptotic (GSK-3beta and JNK) pathways in neuroprotective effects of TRH and its analogues on St-induced neuronal apoptosis. Moreover, the results point to involvement of the inhibition of calpains in the TRH neuroprotective effect in Glu model of neuronal cell death.

Agonist-induced internalization of mGluR1alpha is mediated by caveolin.

Agonist-induced internalization of metabotropic glutamate receptors (mGluRs) plays an important role in neuronal signaling. Although internalization of mGluRs has been reported to be mediated by clathrin-dependent pathway, studies describing clathrin-independent pathways are emerging. Here, we report that agonist-induced internalization of mGluR1alpha is mediated by caveolin. We show that two caveolin-binding motifs of mGluR1alpha interact with caveolin1/2. Using cell surface-immunoprecipitation and total internal reflection fluorescence imaging, we found that agonist-induced internalization of mGluR1alpha is regulated by caveolin-binding motifs of the receptor in heterologous cells. Moreover, in the cerebellum, group I mGluR agonist dihydroxyphenylglycol increased the interaction of phosphorylated caveolin with mGluR1alpha. This interaction was blocked by methyl-beta-cyclodextrin, known to disrupt caveolin/caveolae-dependent signaling by cholesterol depletion. Methyl-beta-cyclodextrin also blocked the agonist-induced internalization of mGluR1alpha. Thus, these findings represent the evidence for agonist-induced internalization of mGluR1alpha via caveolin and suggest that caveolin might play a role in synaptic metaplasticity by regulating internalization of mGluR1alpha in the cerebellum.

Clinical feasibility for cell therapy using human neuronal cell line to treat neuropathic behavioral hypersensitivity following spinal cord injury in rats.

Management of neuropathic pain remains problematic; however, cell therapy to treat the effects of pain on the sensory system after spinal cord injury (SCI) could be a useful approach. Since many clinical trials ultimately do not succeed, use of cell therapy will require that safety and efficacy issues be addressed early in preclinical rat studies. We used the human neuronal cell line hNT2.17, which secretes the inhibitory neurotransmitters gamma-aminobutyric acid and glycine, in an excitotoxic SCI pain model after intraspinal injection of quisqualic acid into rats. One week after lumbar transplant of these cells, behavioral hypersensitivity was permanently reversed. Antinociceptive grafts displayed an optimal transplant time that included moderate effectiveness with chronic SCI and late graft placement and that required a minimal course of cyclosporine A 2 weeks after transplant for durable reversal of painlike behaviors. In addition, grafts did not need to be placed near the SCI level to be effective. These data suggest not only that these cells are safe and efficacious but also that they could be an effective clinical tool for treating SCI-associated neuropathic pain.

Subtype selective kainic acid receptor agonists: discovery and approaches to rational design.

(S)-Glutamic acid (Glu) is the major excitatory neurotransmitter in the mammalian central nervous system, activating the plethora of glutamate receptors (GluRs). In broad lines, the GluRs are divided into two major classes: the ionotropic Glu receptors (iGluRs) and the metabotropic Glu receptors (mGluRs). Within the iGluRs, five subtypes (KA1, KA2, iGluR5-7) show high affinity and express full agonist activity upon binding of the naturally occurring amino acid kainic acid (KA). Thus these receptors have been named the KA receptors. This review describes all-to our knowledge-published KA receptor agonists. In total, over 100 compounds are described by means of chemical structure and available pharmacological data. With this perspective review, it is our intention to ignite and stimulate inspiration for future design and synthesis of novel subtype selective KA receptor agonists.

Group I metabotropic glutamate receptors control proliferation, survival and differentiation of cultured neural progenitor cells isolated from the subventricular zone of adult mice.

Neural progenitor cells (NPCs) are found in the subventricular zone (SVZ) of the adult brain, a specialized neurogenic niche that might provide a substrate for brain repair after injury. The incomplete knowledge of how NPCs in the niche respond to local signals limits the use of cultured NPCs in the development of cell transplantation strategies. We show that neurospheres obtained from the SVZ of the adult mouse expressed functional mGlu1 and mGlu5 metabotropic glutamate receptors. Pharmacological blockade of mGlu5 receptors promoted the apoptotic death of progenitors undergoing differentiation into neurons (PSA/NCAM+ cells for the most part), whereas blockade of mGlu1 receptors reduced the proliferation rate of NPCs, and promoted their differentiation towards the neuronal lineage. We conclude that endogenous activation of mGlu5 receptors might support specifically the survival of neuronal-restricted precursors, whereas endogenous activation of mGlu1 receptors might sustain the proliferation of earlier progenitors. Moreover, mGlu1 receptor antagonists increased the survival of NPCs, suggesting that endogenously activated mGlu1 receptors might play a role in the natural cell loss regulating the number or the type of progenitors.

Inducible expression and pharmacological characterization of the mouse metabotropic glutamate 5b receptor.

The metabotropic glutamate receptor subtype 5 (mGlu5) and glutamatergic neurotransmission are associated with the pathophysiology of disorders such as anxiety, depression or chronic pain. Human and rat mGlu5 receptors have been cloned and characterized previously. We now describe the cloning of the mouse mGlu5b receptor gene from adult mouse brain and its expression using an ecdysone-inducible system. This subtype has an extra 96 bp sequence which is inserted to the cytoplasmic tail and is identical to the insert present in human and rat mGlu5b. Mouse mGlu5b receptor expression was induced in HEK-293EcR cells by incubation with ponasterone A, an analogue of the insect hormone ecdysone. A fluorometric calcium transient assay system was used to characterize the basic pharmacologic profile of an isolated stable cell line. Quisqualic acid was the most potent receptor agonist (EC(50) approximately 7 nM) although the cells also responded to l-glutamic acid and the Group I-selective receptor agonist, 3,5-dihydroxyphenylglycine (3,5-DHPG). The calcium transients stimulated by these agonists were potently inhibited by reference allosteric mGlu5 antagonists - 2-methyl-6-(phenylethynyl)pyridine (MPEP), 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) and 3-methoxy-5-(pyridine-2-ylethynyl)pyridine (methoxy-PEPy) (IC(50) ranges: 0.8-66 nM). The availability of this mouse mGlu5b receptor-expressing cell line will facilitate in vitro characterization of mGlu5 receptor-selective agonists or antagonists prior to in vivo pharmacologic testing.