谷氨酸下调培养海马神经元AMPA受体G1uR2亚单位的表达

目的 研究在癫痫发病过程中,谷氨酸对AMPA受体G1uR2亚单位表达变化的影响。方法 用RT-PCR和Western Blot方法观察谷氨酸诱导培养大鼠海马神经元AMPA受体G1uR2亚单位mRNA和蛋白的表达变化。结果 在谷氨酸刺激后2h,8h,12h,培养海马神经元G1uR2 mRNA和蛋白表达明显下降,与对照组相比,差异有显著性（P〈0．05）,而非NMDA受体拮抗剂CNQX能阻断此变化。结论 在癫痫等疾病中,谷氨酸能通过激活AMAP／KA受体下调AMPA受体G1uR2亚单位的表达,参与发病过程。     

Kainic acid-induced neurotrophic activities in developing cortical neurons

Using primary cultured cortical neurons from embryonic rat brains, we elucidated an alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainic acid (KA) receptor-mediated neuroprotective mechanism through actions of nerve growth factor (NGF) in developing neurons. Neurotoxicity of KA in early days in vitro neurons was quite low compared with the mature neurons. However, pretreatment with anti-NGF antibody or TrkA inhibitor AG-879 profoundly raised KA toxicity. Furthermore, KA stimulation resulted in an increase of TrkA expression and phosphorylation, which was blocked not only by the AMPA/KA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione and AG-879, but also by the phospholipase C inhibitor U73122 and the intracellular calcium chelator BAPTA. A study of polyphosphoinositide turnover showed that KA-stimulated phospholipase C (PLC) activity was directly triggered by the AMPA/KA receptor activity, but not by the activity of TrkA or other excitatory amino acid receptor subtypes. Sources of KA-increased intracellular calcium levels were contributed by both extracellular calcium influx and intracellular calcium release and were partially sensitive to guanosine 5'-O-(2-thiodiphosphate). These results indicate that in developing cortical neurons, activation of AMPA/KA receptors by KA may induce expression, followed by activation of TrkA via PLC signaling and intracellular calcium elevation and hence increase reception of NGF on KA-challenged neurons. A G protein-coupled AMPA/KA receptor may be involved in these metabotropic events for neuronal protection.     

Ligand binding is a critical requirement for plasma membrane expression of heteromeric kainate receptors

Intracellular trafficking of ionotropic glutamate receptors is controlled by multiple discrete determinants in receptor subunits. Most such determinants have been localized to the cytoplasmic carboxyl-terminal domain, but other domains in the subunit proteins can play roles in modulating receptor surface expression. Here we demonstrate that formation of an intact glutamate binding site also acts as an additional quality-control check for surface expression of homomeric and heteromeric kainate receptors. A key ligand-binding residue in the KA2 subunit, threonine 675, was mutated to either alanine or glutamate, which eliminated affinity for the receptor ligands kainate and glutamate. We found that plasma membrane expression of heteromeric GluR6/KA2(T675A) or GluR6/KA2(T675E) kainate receptors was markedly reduced compared with wild-type GluR6/KA2 receptors in transfected HEK 293 and COS-7 cells and in cultured neurons. Surface expression of homomeric KA2 receptors lacking a retention/retrieval determinant (KA2-R/A) was also reduced upon mutation of Thr-675 and elimination of the ligand binding site. KA2 Thr-675 mutant subunits were able to co-assemble with GluR5 and GluR6 subunits and were degraded at the same rate as wild-type KA2 subunit protein. These results suggest that glutamate binding and associated conformational changes are prerequisites for forward trafficking of intracellular kainate receptors following multimeric assembly.     

The Lurcher mutation of an alpha-amino-3-hydroxy-5-methyl- 4-isoxazolepropionic acid receptor subunit enhances potency of glutamate and converts an antagonist to an agonist

A point mutation of the GluRdelta2 (A654T) glutamate receptor subunit converts it into a functional channel, and a spontaneous mutation at this site is thought to be responsible for the neurodegeneration of neurons in the Lurcher mouse. This mutation is located in a hydrophobic region of the M3 domain of this subunit, and this alanine is conserved throughout many of the glutamate receptors. We show here that site-directed mutagenesis of the homologous alanine (A636T; GluR1-L(c)) in the GluR1 AMPA receptor subunit alters its channel properties. The apparent potencies of both kainate and glutamate were increased 85- and 2000-fold, respectively. Furthermore, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)was converted from a competitive antagonist into a potent agonist. Our results demonstrate that a single amino acid within or near the putative second transmembrane region of the GluR1 subunit is critical for the binding/gating properties of this AMPA receptor.     

Slow and selective death of spinal motor neurons in vivo by intrathecal infusion of kainic acid: implications for AMPA receptor-mediated excitotoxicity in ALS

Excitotoxicity mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors has been proposed to play a major role in the selective death of motor neurons in sporadic amyotrophic lateral sclerosis (ALS), and motor neurons are more vulnerable to AMPA receptor-mediated excitotoxicity than are other neuronal subclasses. On the basis of the above evidence, we aimed to develop a rat model of ALS by the long-term activation of AMPA receptors through continuous infusion of kainic acid (KA), an AMPA receptor agonist, into the spinal subarachnoid space. These rats displayed a progressive motor-selective behavioral deficit with delayed loss of spinal motor neurons, mimicking the clinicopathological characteristics of ALS. These changes were significantly ameliorated by co-infusion with 6-nitro-7-sulfamobenso(f)quinoxaline-2,3-dione (NBQX), but not with d(-)-2-amino-5-phosphonovaleric acid (APV), and were exacerbated by co-infusion with cyclothiazide, indicative of an AMPA receptor-mediated mechanism. Among the four AMPA receptor subunits, expression of GluR3 mRNA was selectively up-regulated in motor neurons but not in dorsal horn neurons of the KA-infused rats. The up-regulation of GluR3 mRNA in this model may cause a molecular change that induces the selective vulnerability of motor neurons to KA by increasing the proportion of GluR2-lacking (i.e. calcium-permeable) AMPA receptors. This rat model may be useful in investigating ALS etiology.     

Tetrazolyl isoxazole amino acids as ionotropic glutamate receptor antagonists: synthesis, modelling and molecular pharmacology

Two 3-(5-tetrazolylmethoxy) analogues, 1a and 1b, of (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA), a selective AMPA receptor agonist, and (RS)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA), a GluR5-preferring agonist, were synthesized. Compounds 1a and 1b were pharmacologically characterized in receptor binding assays, and electrophysiologically on homomeric AMPA receptors (GluR1-4), homomeric (GluR5 and GluR6) and heteromeric (GluR6/KA2) kainic acid receptors, using two-electrode voltage-clamped Xenopus laevis oocytes expressing these receptors. Both analogues proved to be antagonists at all AMPA receptor subtypes, showing potencies (Kb=38-161 microM) similar to that of the AMPA receptor antagonist (RS)-2-amino-3-[3-(carboxymethoxy)-5-methyl-4-isoxazolyl]propionic acid (AMOA) (Kb=43-76 microM). Furthermore, the AMOA analogue, 1a, blocked two kainic acid receptor subtypes (GluR5 and GluR6/KA2), showing sevenfold preference for GluR6/KA2 (Kb=19 microM). Unlike the iGluR antagonist (S)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid [(S)-ATPO], the corresponding tetrazolyl analogue, 1b, lacks kainic acid receptor effects. On the basis of docking to a crystal structure of the isolated extracellular ligand-binding core of the AMPA receptor subunit GluR2 and a homology model of the kainic acid receptor subunit GluR5, we were able to rationalize the observed structure-activity relationships.     

Basic Fibroblast Growth Factor Selectively Increases AMPA-Receptor Subunit GluR1 Protein Level and Differentially Modulates Ca2+ Responses to AMPA and NMDA in Hippocampal Neurons

Abstract: The excitatory neurotransmitter glutamate is believed to play important roles in development, synaptic plasticity, and neurodegenerative conditions. Recent studies have shown that neurotrophic factors can modulate neuronal excitability and survival and neurite outgrowth responses to glutamate, but the mechanisms are unknown. The present study tested the hypothesis that neurotrophic factors modulate responses to glutamate by affecting the expression of specific glutamate-receptor proteins. Exposure of cultured embryonic rat hippocampal cells to basic fibroblast growth factor (bFGF) resulted in a concentration-dependent increase in levels of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-receptor subunit GluR1 protein as determined by western blot, dot-blot, and immunocytochemical analyses. In contrast, bFGF did not alter levels of GluP2/3, GluR4, or the NMDA-receptor subunit NR1. Nerve growth factor did not affect GluR1 levels. Calcium-imaging studies revealed that elevation of [Ca²⁺]_i, resulting from selective AMPA-receptor activation, was enhanced in bFGF-pretreated neurons. On the other hand, [Ca²⁺]_i responses to NMDA-receptor activation were suppressed in bFGF-treated neurons, consistent with previous studies showing that bFGF can protect neurons against NMDA toxicity. Moreover, neurons pretreated with bFGF were relatively resistant to the toxicities of glutamate and AMPA, both of which were shown to be mediated by NMDA receptors. These data suggest that differential regulation of the expression of specific glutamate-receptor subunits may be an important mechanism whereby neurotrophic factors modulate activity-dependent neuronal plasticity and vulnerability to excitotoxicity.     

Neuroprotective role of PrPC against kainate-induced epileptic seizures and cell death depends on the modulation of JNK3 activation by GluR6/7-PSD-95 binding

Cellular prion protein (PrP(C)) is a glycosyl-phosphatidylinositol-anchored glycoprotein. When mutated or misfolded, the pathogenic form (PrP(SC)) induces transmissible spongiform encephalopathies. In contrast, PrP(C) has a number of physiological functions in several neural processes. Several lines of evidence implicate PrP(C) in synaptic transmission and neuroprotection since its absence results in an increase in neuronal excitability and enhanced excitotoxicity in vitro and in vivo. Furthermore, PrP(C) has been implicated in the inhibition of N-methyl-d-aspartic acid (NMDA)-mediated neurotransmission, and prion protein gene (Prnp) knockout mice show enhanced neuronal death in response to NMDA and kainate (KA). In this study, we demonstrate that neurotoxicity induced by KA in Prnp knockout mice depends on the c-Jun N-terminal kinase 3 (JNK3) pathway since Prnp(o/o)Jnk3(o/o) mice were not affected by KA. Pharmacological blockage of JNK3 activity impaired PrP(C)-dependent neurotoxicity. Furthermore, our results indicate that JNK3 activation depends on the interaction of PrP(C) with postsynaptic density 95 protein (PSD-95) and glutamate receptor 6/7 (GluR6/7). Indeed, GluR6-PSD-95 interaction after KA injections was favored by the absence of PrP(C). Finally, neurotoxicity in Prnp knockout mice was reversed by an AMPA/KA inhibitor (6,7-dinitroquinoxaline-2,3-dione) and the GluR6 antagonist NS-102. We conclude that the protection afforded by PrP(C) against KA is due to its ability to modulate GluR6/7-mediated neurotransmission and hence JNK3 activation.     

谷氨酸下调培养海马神经元AMPA 受体GluR2亚单位的表达

目的研究在癫痫发病过程中,谷氨酸对AMPA受体Glu R2亚单位表达变化的影响。方法用RT-PCR和Western Blot方法观察谷氨酸诱导培养大鼠海马神经元AMPA受体Glu R2亚单位mRNA和蛋白的表达变化。结果在谷氨酸刺激后2h,8h,12h,培养海马神经元Glu R2mRNA和蛋白表达明显下降,与对照组相比,差异有显著性(P<0.05),而非NMDA受体拮抗剂CNQX能阻断此变化。结论在癫痫等疾病中,谷氨酸能通过激活AMAP/KA受体下调AMPA受体GluR2亚单位的表达,参与发病过程。     

AMPA receptor subunit GluR2 gates injurious signals in ischemic stroke

Ischemic stroke, or a brain attack, is the third leading cause of death in developed countries. A critical feature of the disease is a highly selective pattern of neuronal loss; certain identifiable subsets of neurons--particularly CA1 pyramidal neurons in the hippocampus are severely damaged, whereas others remain intact. A key step in this selective neuronal injury is Ca2+/Zn2+ entry into vulnerable neurons through alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor channels, a principle subtype of glutamate receptors. AMPA receptor channels are assembled from glutamate receptor (GluR)1, -2, -3, and -4 subunits. Circumstance data have indicated that the GluR2 subunits dictate Ca2+/Zn2+ permeability of AMPA receptor channels and gate injurious Ca2+/Zn2+ signals in vulnerable neurons. Therefore, targeting to the AMPA receptor subunit GluR2 can be considered a practical strategy for stroke therapy.     

GluR2 knockdown reveals a dissociation between [Ca2+]i surge and neurotoxicity

Reduction in GluR2 subunit expression and subsequent increases in AMPA receptor mediated Ca(2+) currents were postulated to exacerbate glutamate neurotoxicity following seizures or global ischemia. To directly test the effects of shifting the GluR1/GluR2 subunit ratio on excitotoxicity, GluR2 antisense deoxyoligonucleotides (AS-ODNs) were applied to dissociated hippocampal cultures for 1-8 days. The GluR1/GluR2 protein ratio was examined immunohistochemically and by Western blotting. [Ca(2+)](i) concentrations were determined by ratiometric imaging of Fura 2-loaded cells. The cultures were exposed to glutamate, AMPA, NMDA or kainic acid (KA) 3 days after GluR2 knockdown and cell viability was determined 1 day later by MTT reduction assay or Trypan blue exclusion. Although GluR2 AS-ODNs increased the GluR1/GluR2 protein ratio in a time dependent manner, neurons and glia appeared healthy and MTT reduction values were similar to untreated and sense controls. Basal [Ca(2+)](i) levels were unchanged but [Ca(2+)](i) was selectively increased by agonist stimulation of AMPA receptors. Unexpectedly, delayed neurotoxicity was attenuated at saturating doses of glutamate while little difference in cell viability was observed at lower doses or with the other excitotoxins at any concentration. Therefore, there was a dissociation between rises in AMPA receptor-mediated Ca(2+) influx and neurotoxicity despite marked decreases in GluR2 but not GluR1 immunoreactivity. It is proposed that a modification of AMPA receptor stochiometry that raises agonist-stimulated Ca(2+) influx during an excitotoxic insult may have eventual neuroprotective effects.     

Altered mRNA editing and expression of ionotropic glutamate receptors after kainic acid exposure in cyclooxygenase-2 deficient mice

Kainic acid (KA) binds to the AMPA/KA receptors and induces seizures that result in inflammation, oxidative damage and neuronal death. We previously showed that cyclooxygenase-2 deficient (COX-2(-/-)) mice are more vulnerable to KA-induced excitotoxicity. Here, we investigated whether the increased susceptibility of COX-2(-/-) mice to KA is associated with altered mRNA expression and editing of glutamate receptors. The expression of AMPA GluR2, GluR3 and KA GluR6 was increased in vehicle-injected COX-2(-/-) mice compared to wild type (WT) mice in hippocampus and cortex, whereas gene expression of NMDA receptors was decreased. KA treatment decreased the expression of AMPA, KA and NMDA receptors in the hippocampus, with a significant effect in COX-2(-/-) mice. Furthermore, we analyzed RNA editing levels and found that the level of GluR3 R/G editing site was selectively increased in the hippocampus and decreased in the cortex in COX-2(-/-) compared with WT mice. After KA, GluR4 R/G editing site, flip form, was increased in the hippocampus of COX-2(-/-) mice. Treatment of WT mice with the COX-2 inhibitor celecoxib for two weeks decreased the expression of AMPA/KA and NMDAR subunits after KA, as observed in COX-2(-/-) mice. After KA exposure, COX-2(-/-) mice showed increased mRNA expression of markers of inflammation and oxidative stress, such as cytokines (TNF-α, IL-1β and IL-6), inducible nitric oxide synthase (iNOS), microglia (CD11b) and astrocyte (GFAP). Thus, COX-2 gene deletion can exacerbate the inflammatory response to KA. We suggest that COX-2 plays a role in attenuating glutamate excitotoxicity by modulating RNA editing of AMPA/KA and mRNA expression of all ionotropic glutamate receptor subunits and, in turn, neuronal excitability. These changes may contribute to the increased vulnerability of COX-2(-/-) mice to KA. The overstimulation of glutamate receptors as a consequence of COX-2 gene deletion suggests a functional coupling between COX-2 and the glutamatergic system.     

Glutamate-induced cobalt uptake elicited by kainate receptors in rat taste bud cells

Glutamate-induced cobalt uptake reveals non-N-methyl-D-aspartate (non-NMDA) glutamate receptors (GluRs) in rat taste bud cells. However, it is not known which type of non-NMDA glutamate receptors is involved. We used a cobalt staining technique combined with pharmacological tests for kainate or alpha-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) receptors and/or immunohistochemistry against subunits of GluRs to examine the presence of non-NMDA receptors in rat foliate tastebud cells. Cobalt uptake into taste cells was elicited by treating taste buds with glutamate, kainate or SYM 2081, a kainate receptor agonist. Treating taste buds with AMPA or fluorowillardiine did not stimulate significant cobalt uptake. Moreover, 6-cyano-7-nitro-quinoxaline-2, 3-dione significantly reduced cobalt staining elicited by glutamate or kainate receptor agonists, but SYM 2206, an AMPA receptor antagonist, did not. Immunohistochemistry against subunits of GluRs reveals GluR6 and KA1-like immunoreactivity. Moreover, most glutamate-induced cobalt-stained cells showed GluR6 and KA1-like immunoreactivity. These results suggest that glutamate-induced cobalt uptake in taste cells occurs mainly via kainate type GluRs.     

Calcium influx through subunits GluR1/GluR3 of kainate/AMPA receptor channels is regulated by cAMP dependent protein kinase.

Excitatory synaptic transmission in the central nervous system (CNS) is mediated by three major classes of glutamate receptors, namely the ionotropic NMDA (N-Methyl-D-Aspartate) and KA/AMPA (kainate/alpha-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid) receptors and the metabotropic receptor type. Among the ionotropic receptors, NMDA receptors are thought to mediate their physiological response mainly through the influx of extracellular calcium, while KA/AMPA receptor channels are mainly thought to carry the influx of monovalent cations. Recently, we have challenged this view by showing that cloned KA/AMPA receptor subunits GluR1 and GluR3 form ion channels which are permeable to calcium. We now directly demonstrate large increases in intracellular calcium concentrations induced by calcium fluxes through KA/AMPA receptor channels in solutions with physiological calcium concentrations. Calcium fluxes were observed through glutamate receptor channels composed of the subunits GluR1 and GluR3, which are both abundantly present in various types of central neurones. The calcium influx was fluorometrically monitored in Xenopus oocytes injected with the calcium indicator dye fura-2. Bath application of the membrane permeable analogue of adenosine cyclic monophosphate (cAMP) potentiated the current and also the flux of calcium through open KA/AMPA receptor channels. Further pharmacological experiments suggested that this effect was mediated by the activation of protein kinase A. Our results provide a molecular interpretation for the function of calcium permeable KA/AMPA receptor channels in neurones and identify two of the subunits of the KA/AMPA receptor channel which are regulated by the cAMP dependent second messenger system.     

Laminar segregation of the cortical plate during corticogenesis is accompanied by changes in glutamate receptor expression

We tested the hypothesis that subtypes of glutamate receptors (GluRs) are differentially expressed during corticogenesis. The neocortex of fetal sheep (term = approximately 145 days) was evaluated by immunoblotting and immunohistochemistry to determine the protein expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (GluR1, GluR2/GluR3 [GluR2/3], and GluR4), kainate (KA) receptors (GluR6/GluR7 [GluR6/7]), and a metabotropic GluR (mGluR5). AMPA/KA receptors and mGluR5 were expressed in neocortex by midgestation. GluR1 and mGluR5 expression increased progressively, with expression being maximal just before birth and then decreasing postnatally. GluR2/3 and GluR6/7 levels increased progressively during corticogenesis to reach adult levels near term. GluR4 was expressed at low levels during corticogenesis and in adult neocortex. The localizations of GluRs in the developing neocortex were distinct. Each GluR had a differential localization within the marginal zone, cortical plate, and subplate. GluR subtypes were expressed in laminar patterns before major cytoarchitectonic segregation occurred based on Nissl staining, although connectional patterns were emergent by midgestation based on labeling of corticostriatal projections with DiI. The GluR localizations changed during cortical plate segregation, resulting in highly differential distributions in the neocortex at term. AMPA/KA receptors were expressed transiently in proliferative zones and in developing white matter. Oligodendrocytes in fetal brain expressed AMPA receptors. The expression of ion channel and metabotropic GluR subtypes is dynamic during corticogenesis, with subtype- and subunit-specific regulation occurring during the laminar segregation of the cortical plate and differentiation of the neocortex.     

Regulation of Kainate Receptor Subunit mRNA by Stress and Corticosteroids in the Rat Hippocampus

Kainate receptors are a class of ionotropic glutamate receptors that have a role in the modulation of glutamate release and synaptic plasticity in the hippocampal formation. Previous studies have implicated corticosteroids in the regulation of these receptors and recent clinical work has shown that polymorphisms in kainate receptor subunit genes are associated with susceptibility to major depression and response to anti-depressant treatment. In the present study we sought to examine the effects of chronic stress and corticosteroid treatments upon the expression of the mRNA of kainate receptor subunits GluR5-7 and KA1-2. Our results show that, after 7 days, adrenalectomy results in increased expression of hippocampal KA1, GluR6 and GluR7 mRNAs, an effect which is reversed by treatment with corticosterone in the case of KA1 and GluR7 and by aldosterone treatment in the case of GluR6. 21 days of chronic restraint stress (CRS) elevated the expression of the KA1 subunit, but had no effect on the expression of the other subunits. Similarly, 21 days of treatment with a moderate dose of corticosterone also increased KA1 mRNA in the dentate gyrus, whereas a high corticosterone dose has no effect. Our results suggest an interaction between hippocampal kainate receptor composition and the hypothalamic-pituitary-adrenal (HPA) axis and show a selective chronic stress induced modulation of the KA1 subunit in the dentate gyrus and CA3 that has implications for stress-induced adaptive structural plasticity.