Glutamate,Glutamate Receptors,and Downstream Signaling Pathways

Glutamate is a nonessential amino acid, a major bioenergetic substrate for proliferating normal and neoplastic cells, and an excitatory neurotransmitter that is actively involved in biosynthetic, bioenergetic, metabolic, and oncogenic signaling pathways. Glutamate signaling activates a family of receptors consisting of metabotropic glutamate receptors (mGluRs) and ionotropic glutamate receptors (iGluRs), both of which have been implicated in chronic disabling brain disorders such as Schizophrenia and neurodegenerative diseases like Alzheimer''s, Parkinson''s, and multiple sclerosis. In this review, we discuss the structural and functional relationship of mGluRs and iGluRs and their downstream signaling pathways. The three groups of mGluRs, the associated second messenger systems, and subsequent activation of PI3K/Akt, MAPK, NFkB, PLC, and Ca/CaM signaling systems will be discussed in detail. The current state of human mGluR1a as one of the most important isoforms of Group I-mGluRs will be highlighted. The lack of studies on the human orthologues of mGluRs family will be outlined. We conclude that upon further study, human glutamate-initiated signaling pathways may provide novel therapeutic opportunities for a variety of non-malignant and malignant human diseases.     

Glutamatergic signaling in cellular transformation

The role of the glutamatergic system in cancer cell homeostasis has expanded exponentially over the last decade. Once thought to participate only in synaptic transmission and neuronal excitability, the presence of functional glutamate receptors has since been demonstrated in peripheral tissues. Most notable is the implication of glutamate receptors in the pathophysiology of various human malignancies. We previously described the oncogenic properties of metabotropic glutamate receptor 1 (Grm1), a G-protein-coupled receptor in melanoma development in vivo. TG-3, a transgenic mouse line, developed spontaneous melanoma with 100% penetrance in the absence of any known stimuli. Stable Grm1-mouse melanocytic clones display transformed phenotypes in vitro and were aggressively tumorigenic in vivo. Recent reports from other groups implicate two additional members of the metabotropic glutamate receptor family in melanomagenesis, overexpression of mGluR5 and activating mutations in GRM3. These findings highlight a previously underappreciated link between the glutamate signaling pathway and oncogenesis in melanoma biology, raising exciting possibilities in elucidating mechanisms in melanocyte transformation and exploring glutamate receptors as novel therapeutic targets. Here we further consider the potential mechanisms by which glutamate receptors can function as an oncogene leading to malignant transformation.     

Molecular mechanisms underlying specificity of excitotoxic signaling in neurons

The central role of glutamate receptors in mediating excitotoxic neuronal death in stroke, epilepsy and trauma has been well established. Glutamate is the major excitatory amino acid transmitter within the CNS and it's signaling is mediated by a number of postsynaptic ionotropic and metabotropic receptors. Although calcium ions are considered key regulators of excitotoxicity, new evidence suggests that specific second messenger pathways rather than total Ca(2+) load, are responsible for mediating neuronal degeneration. Glutamate receptors are found localized at the synapse within electron dense structures known as the postsynaptic density (PSD). Localization at the PSD is mediated by binding of glutamate receptors to submembrane proteins such as actin and PDZ containing proteins. PDZ domains are conserved motifs that mediate protein-protein interactions and self-association. In addition to glutamate receptors PDZ-containing proteins bind a multitude of intracellular signal molecules including nitric oxide synthase. In this way PDZ proteins provide a mechanism for clustering glutamate receptors at the synapse together with their corresponding signal transduction proteins. PSD organization may thus facilitate the individual neurotoxic signal mechanisms downstream of receptors during glutamate overactivity. Evidence exists showing that inhibiting signals downstream of glutamate receptors, such as nitric oxide and PARP-1 can reduce excitotoxic insult. Furthermore we have shown that uncoupling the interaction between specific glutamate receptors from their PDZ proteins protects neurons against glutamate-mediated excitotoxicity. These findings have significant implications for the treatment of neurodegenerative diseases using therapeutics that specifically target intracellular protein-protein interactions.     

Glutamate release promotes growth of malignant gliomas

Glutamate neurotoxicity has been implicated in stroke, head trauma, multiple sclerosis and neurodegenerative diseases. Although recent data show that cultured glioma cells secrete glutamate, the growth potential of brain tumors has not yet been linked to an excitotoxic mechanism. Using bioluminescence detection of glutamate release from freshly prepared brain slices, we show that implanted glioma cells continue to secrete glutamate. Moreover, gliomas with high glutamate release have a distinct growth advantage in host brain that is not present in vitro. Treatment with the NMDA receptor antagonists MK801 or memantine slowed the growth of glutamate-secreting tumors in situ, suggesting that activation of NMDA receptors facilitates tumor expansion. These findings support a new approach for therapy of brain tumors, based upon antagonizing glutamate secretion or its target receptors.     

Glutamate: a truly functional amino acid

Glutamate is one of the most abundant of the amino acids. In addition to its role in protein structure, it plays critical roles in nutrition, metabolism and signaling. Post-translational carboxylation of glutamyl residues increases their affinity for calcium and plays a major role in hemostasis. Glutamate is of fundamental importance to amino acid metabolism, yet the great bulk of dietary glutamate is catabolyzed within the intestine. It is necessary for the synthesis of key molecules, such as glutathione and the polyglutamated folate cofactors. It plays a major role in signaling. Within the central nervous system, glutamate is the major excitatory neurotransmitter and its product, GABA, the major inhibitory neurotransmitter. Glutamate interaction with specific taste cells in the tongue is a major component of umami taste. The finding of glutamate receptors throughout the gastrointestinal tract has opened up a new vista in glutamate function. Glutamate is truly a functional amino acid.     

Glutamate-induced Toxicity in Hippocampal Slices Involves Apoptotic Features and p38MAPK Signaling

Glutamate excitotoxicity may culminate with neuronal and glial cell death. Glutamate induces apoptosis in vivo and in cell cultures. However, glutamate-induced apoptosis and the signaling pathways related to glutamate-induced cell death in acute hippocampal slices remain elusive. Hippocampal slices exposed to 1 or 10 mM glutamate for 1 h and evaluated after 6 h, showed reduced cell viability, without altering membrane permeability. This action of glutamate was accompanied by cytochrome c release, caspase-3 activation and DNA fragmentation. Glutamate at low concentration (10 μM) induced caspase-3 activation and DNA fragmentation, but it did not cause cytochrome c release and, it did not alter the viability of slices. Glutamate-induced impairment of hippocampal cell viability was completely blocked by MK-801 (non-competitive antagonist of NMDA receptors) and GAMS (antagonist of KA/AMPA glutamate receptors). Regarding intracellular signaling pathways, glutamate-induced cell death was not altered by a MEK1 inhibitor, PD98059. However, the p38^MAPK inhibitor, SB203580, prevented glutamate-induced cell damage. In the present study we have shown that glutamate induces apoptosis in hippocampal slices and it causes an impairment of cell viability that was dependent of ionotropic and metabotropic receptors activation and, may involve the activation of p38^MAPK pathway.     

CAL与mGluR1相互作用抑制mGluR1过度激活诱导的细胞毒性作用

代谢型谷氨酸受体1(mGluR1)过度激活介导的谷氨酸兴奋性毒性是帕金森病(PD)的主要发病机制之一。在临床试验中应用mGluRs的负性变构调节剂缓解PD病人的运动障碍已有报道,但由于精确调控mGluRs表达或活性的局限性,目前,在PD的治疗中仍存在一些问题。因此,寻找能够精确调控mGluR1表达及活性的小分子药物或内源性基因,将有可能成为解决目前PD治疗中存在问题的有效方法。本文通过体内和体外实验,对囊性纤维跨膜调节器相关配体(CAL)在mGluR1过度激活诱导的细胞毒性中的作用和机制进行研究。研究结果显示,在工具细胞HEK293中,应用mGluR1的激动剂激活受体,CAL与mGluR1的相互作用明显增强(P< 0.05),且CAL通过与mGluR1相互作用,抑制mGluR1过度激活诱导的细胞凋亡及其下游信号通路的激活。在鱼藤酮诱导的PD大鼠模型中,过表达CAL通过抑制mGluR1下游通路的激活,减少鱼藤酮引起的神经损伤(P< 0.001)。本文揭示了一种调控mGluR1活性的新机制,希望为神经系统疾病的治疗和相关研究提供新思路。     

Glutamate levels control HT22 murine hippocampal cell death by regulating biphasic patterns of Erk1/2 activation: role of metabolic glutamate receptor 5

Extracellular glutamate concentration is a critical determinant of neuronal cell fate. We recently demonstrated that HT22 murine hippocampal cell viability was reduced by exposure to high concentrations of glutamate, whereas low concentrations promoted cell survival. Extracellular signal-regulated kinase (Erk)1/2 activation by glutamate is important for both glutamate-induced cell death and survival. In this study, we investigated the role of glutamate-induced or hydrogen peroxide (H₂O₂)-induced Erk1/2 activation in HT22 cell fate determination. Glutamate and H₂O₂ treatment similarly induced early (<1 h) Erk1/2 phosphorylation regardless of concentration. On the other hand, persistent Erk1/2 phosphorylation (16–24 h) was observed only in the presence of excess glutamate. Only the latter contributed to glutamate-induced cell death, which involved metabolic glutamate receptor 5. Our findings suggest that glutamate concentration modulates two distinct phases of Erk1/2 activation, which can explain the glutamate concentration-dependent determination of HT22 cell fate.     

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.     

Metabotropic Glutamate Receptors in Glial Cells

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS) and exerts its actions via a number of ionotropic glutamate receptors/channels and metabotropic glutamate (mGlu) receptors. In addition to being expressed in neurons, glutamate receptors are expressed in different types of glial cells including astrocytes, oligodendrocytes, and microglia. Astrocytes are now recognized as dynamic signaling elements actively integrating neuronal inputs. Synaptic activity can evoke calcium signals in astrocytes, resulting in the release of gliotransmitters, such as glutamate, ATP, and d-serine, which in turn modulate neuronal excitability and synaptic transmission. In addition, astrocytes, and microglia may play an important role in pathology such as brain trauma and neurodegeneration, limiting or amplifying the pathologic process leading to neuronal death. The present review will focus on recent advances on the role of mGlu receptors expressed in glial cells under physiologic and pathologic conditions. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

Glutamate excitotoxicity in a model of multiple sclerosis

Glutamate excitotoxicity mediated by the AMPA/kainate type of glutamate receptors damages not only neurons but also the myelin-producing cell of the central nervous system, the oligodendrocyte. In multiple sclerosis, myelin, oligodendrocytes and some axons are lost as a result of an inflammatory attack on the central nervous system. Because glutamate is released in large quantities by activated immune cells, we expected that during inflammation in MS, glutamate excitotoxicity might contribute to the lesion. We addressed this by using the AMPA/kainate antagonist NBQX to treat mice sensitized for experimental autoimmune encephalomyelitis, a demyelinating model that mimics many of the clinical and pathologic features of multiple sclerosis. Treatment resulted in substantial amelioration of disease, increased oligodendrocyte survival and reduced dephosphorylation of neurofilament H, an indicator of axonal damage. Despite the clinical differences, treatment with NBQX had no effect on lesion size and did not reduce the degree of central nervous system inflammation. In addition, NBQX did not alter the proliferative activity of antigen-primed T cells in vitro, further indicating a lack of effect on the immune system. Thus, glutamate excitotoxicity seems to be an important mechanism in autoimmune demyelination, and its prevention with AMPA/kainate antagonists may prove to be an effective therapy for multiple sclerosis.     

Interaction between glutamate signalling and immune attack in damaging oligodendrocytes

Glutamate is the principal excitatory neurotransmitter in the CNS, but it is also a potent neurotoxin that can kill nerve cells. Glutamate damages oligodendrocytes, like neurons, by excitotoxicity which is caused by sustained activation of AMPA, kainate and NMDA receptors. Glutamate excitotoxicity depends entirely on Ca(2+) overload of the cytoplasm and can be initiated by disruption of glutamate homeostasis. Thus, inhibition of glutamate uptake in isolated oligodendrocytes in vitro and in the optic nerve in vivo, is sufficient to trigger cell death which is prevented by glutamate receptor antagonists. In turn, activated, but not resting microglia, can compromise glutamate homeostasis and induce oligodendrocyte excitotoxicity, which is attenuated either by AMPA/kainate antagonists or by the blockade of the system x(c)- antiporter present in microglia. By contrast, non-lethal, brief, activation of glutamate receptors in oligodendrocytes rapidly sensitizes these cells to complement attack. Intriguingly, these effects are exclusively mediated by kainate receptors which induce Ca(2+) overload of the cytosol and the generation of reactive oxygen species. In conjunction, these observations reveal novel mechanisms by which neuroinflammation alters glutamate homeostasis and triggers oligodendrocyte death. Conversely, they also show how glutamate signaling in oligodendrocytes might induce immune attack. In both instances direct activation of glutamate receptors present in oligodendrocytes plays a pivotal role in either initiating or executing death signals, which might be relevant to the pathogenesis of white matter disorders.     

Molecular and cellular mechanisms of excitotoxic neuronal death

Glutamate receptor-mediated excitatory neurotransmission plays a key role in neural development, differentiation and synaptic plasticity. However, excessive stimulation of glutamate receptors induces neurotoxicity, a process that has been defined as excitotoxicity. Excitotoxicity is considered to be a major mechanism of cell death in a number of central nervous system diseases including stroke, brain trauma, epilepsy and chronic neurodegenerative disorders. Unfortunately clinical trials with glutamate receptor antagonists, that would logically prevent the effects of excessive receptor activation, have been associated with untoward side effects or little clinical benefit. Therefore, uncovering molecular pathways involved in excitotoxic neuronal death is of critical importance to future development of clinical treatment of many neurodegenerative disorders where excitotoxicity has been implicated. This review discusses the current understanding of the molecular and cellular mechanisms of excitotoxicity and their roles in the pathogenesis of diseases of the central nervous system.     

Glutamate Stimulates Local Protein Synthesis in the Axons of Rat Cortical Neurons by Activating α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors and Metabotropic Glutamate Receptors

Glutamate is the principal excitatory neurotransmitter in the mammalian CNS. By analyzing the metabolic incorporation of azidohomoalanine, a methionine analogue, in newly synthesized proteins, we find that glutamate treatments up-regulate protein translation not only in intact rat cortical neurons in culture but also in the axons emitting from cortical neurons before making synapses with target cells. The process by which glutamate stimulates local translation in axons begins with the binding of glutamate to the ionotropic AMPA receptors and metabotropic glutamate receptor 1 and members of group 2 metabotropic glutamate receptors on the plasma membrane. Subsequently, the activated mammalian target of rapamycin (mTOR) signaling pathway and the rise in Ca²⁺, resulting from Ca²⁺ influxes through calcium-permeable AMPA receptors, voltage-gated Ca²⁺ channels, and transient receptor potential canonical channels, in axons stimulate the local translation machinery. For comparison, the enhancement effects of brain-derived neurotrophic factor (BDNF) on the local protein synthesis in cortical axons were also studied. The results indicate that Ca²⁺ influxes via transient receptor potential canonical channels and activated the mTOR pathway in axons also mediate BDNF stimulation to local protein synthesis. However, glutamate- and BDNF-induced enhancements of translation in axons exhibit different kinetics. Moreover, Ca²⁺ and mTOR signaling appear to play roles carrying different weights, respectively, in transducing glutamate- and BDNF-induced enhancements of axonal translation. Thus, our results indicate that exposure to transient increases of glutamate and more lasting increases of BDNF would stimulate local protein synthesis in migrating axons en route to their targets in the developing brain.     

Regulation of glutamate carboxypeptidase II hydrolysis of N-acetylaspartylglutamate (NAAG) in crayfish nervous tissue is mediated by glial glutamate and acetylcholine receptors

Glutamate carboxypeptidase II (GCPII), a glial ectoenzyme, is responsible for N-acetylaspartylglutamate (NAAG) hydrolysis. Its regulation in crayfish nervous tissue was investigated by examining uptake of [3H]glutamate derived from N-acetylaspartyl-[3H]glutamate ([3H]NAAG) to measure GCPII activity. Electrical stimulation (100 Hz, 10 min) during 30 min incubation with [3H]NAAG increased tissue [3H]glutamate tenfold. This was prevented by 2-(phosphonomethyl)-pentanedioic acid (2-PMPA), a GCPII inhibitor, suggesting that stimulation increased the hydrolysis of [3H]NAAG and metabolic recycling of [3H]glutamate. Antagonists of glial group II metabotropic glutamate receptors (mGLURII), NMDA receptors and acetylcholine (ACh) receptors that mediate axon-glia signaling in crayfish nerve fibers decreased the effect of stimulation by 58-83%, suggesting that glial receptor activation leads to stimulation of GCPII activity. In combination, they reduced [3H]NAAG hydrolysis during stimulation to unstimulated control levels. Agonist stimulation of mGLURII mimicked the effect of electrical stimulation, and was prevented by antagonists of GCPII or mGLURII. Raising extracellular K+ to three times the normal level stimulated [3H]NAAG release and GCPII activity. These effects were also blocked by antagonists of GCPII and mGLUR(II). No receptor antagonist or agonist tested or 2-PMPA affected uptake of [3H]glutamate. We conclude that NAAG released from stimulated nerve fibers activates its own hydrolysis via stimulation of GCPII activity mediated through glial mGLURII, NMDA and ACh receptors.     

Chronic excitotoxin-induced axon degeneration in a compartmented neuronal culture model

Glutamate excitotoxicity is a major pathogenic process implicated in many neurodegenerative conditions, including AD (Alzheimer''s disease) and following traumatic brain injury. Occurring predominantly from over-stimulation of ionotropic glutamate receptors located along dendrites, excitotoxic axonal degeneration may also occur in white matter tracts. Recent identification of axonal glutamate receptor subunits within axonal nanocomplexes raises the possibility of direct excitotoxic effects on axons. Individual neuronal responses to excitotoxicity are highly dependent on the complement of glutamate receptors expressed by the cell, and the localization of the functional receptors. To enable isolation of distal axons and targeted excitotoxicity, murine cortical neuron cultures were prepared in compartmented microfluidic devices, such that distal axons were isolated from neuronal cell bodies. Within the compartmented culture system, cortical neurons developed to relative maturity at 11 DIV (days in vitro) as demonstrated by the formation of dendritic spines and clustering of the presynaptic protein synaptophysin. The isolated distal axons retained growth cone structures in the absence of synaptic targets, and expressed glutamate receptor subunits. Glutamate treatment (100 μM) to the cell body chamber resulted in widespread degeneration within this chamber and degeneration of distal axons in the other chamber. Glutamate application to the distal axon chamber triggered a lesser degree of axonal degeneration without degenerative changes in the untreated somal chamber. These data indicate that in addition to current mechanisms of indirect axonal excitotoxicity, the distal axon may be a primary target for excitotoxicity in neurodegenerative conditions.     

Targeting the lipids LPA and S1P and their signalling pathways to inhibit tumour progression

The bioactive lipids lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), the enzymes that generate and degrade them, and the receptors that receive their signals are all potential therapeutic targets in cancer. LPA and S1P signalling pathways can modulate a range of cellular processes that contribute to tumourigenesis, such as proliferation and motility, and components of the signalling pathways often show aberrant expression and altered activity upon malignant transformation. This article reviews LPA- and S1P-mediated activities that might contribute to the aetiology of cancer, and examines the potential of the many antagonists that have been developed to inhibit LPA and S1P signalling pathways. In addition, the outcomes of various clinical trials using LPA- and S1P-associated targets in cancer and other diseases are described, and future directions are discussed.