Roles of ionotropic glutamate receptors in early developing neurons derived from the P19 mouse cell line

We cultured a P19 mouse teratocarcinoma cell line and induced its neuronal differentiation to study the function of ionotropic glutamate receptors (GluRs) in early neuronal development. Immunocytochemical studies showed 85% neuronal population at 5 days in vitro (DIV) with microtubule-associated protein 2-positive staining. Thirty percent and 50% of the cells expressed the alpha-amino-3-hydroxy-5-methyl-4-isopropinonate (AMPA) receptor subunit, GluR2/3, and the kainate (kainic acid; KA) receptor subunit, GluR5/6/7, respectively. In Western blot analysis, the temporal expression of GluR2/3 began to appear at 3 DIV, whereas GluR5/6/7 was already expressed in the undifferentiated cells. P19-derived neurons began to respond to glutamate, AMPA and KA, but not to the metabotropic GluR agonist trans-1-aminocyclopentane-1,3-decarboxylic acid, by 5 DIV in terms of increases in intracellular calcium and phospholipase C-mediated poly-phosphoinositide turnover. Furthermore, KA reduced cell death of P19-derived neurons in both atmospheric and hypobaric conditions in a phospholipase C-dependent manner. The common AMPA/KA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, but not the AMPA receptor antagonist, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium, profoundly increased hypobaric insult-induced neurotoxicity. In a flow cytometry study, the nerve growth factor-mediated antiapoptotic effect was facilitated by AMPA, with an induction of TrkA, but not p75(NTR) expression. Therefore, AMPA and KA receptors might mediate neurotrophic functions to facilitate neurotrophic factor signaling to protect neurons against hypoxic insult in early neuronal development.     

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.     

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.     

Distribution of GluR1 is altered in the olfactory bulb following neonatal naris occlusion

The olfactory system is well suited for studies of glutamate receptor plasticity. The sensory neurons are glutamatergic, and they turn over throughout life, and the olfactory bulb neurons that process their inputs express many of the known glutamate receptor subunits. Neonatal naris occlusion alters olfactory bulb development and the expression of certain neuroactive substances and receptors, at least in part due to loss of the sensory inputs. We therefore postulated that neonatal naris occlusion might alter glutamate receptor expression during postnatal development. Single nares of newborn mice were occluded on postnatal days 1-2, and the distribution of glutamate receptor subunits was evaluated using immunoperoxidase methods. Light microscopic examination on postnatal day 6 failed to reveal adult-like staining of neuronal cell bodies in the olfactory bulbs. By day 12, cell bodies that were immunoreactive (-IR) for the GluR1 subunit were visible in the external plexiform layer (EPL) of both sides. By day 18, many of the GluR1-IR cell bodies could be identified as cell types that had previously been reported to express homomeric GluR1 receptors. Analysis of single, mid-dorsal sections from 18-25-day-old mice showed that the medial EPL of the occluded side had a significantly lower density of these cell bodies. The GluR1 staining of the adjacent mitral cell layer (MCL) was also heavier on the occluded side, but no gross differences in staining for other glutamate receptor subunits were observed. Neonatal naris occlusion therefore appears to provide a new model for studying expression of GluR1 receptors during the development of a discrete population of olfactory bulb neurons.     

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。     

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。     

Human spinal motoneurons express low relative abundance of GluR2 mRNA: an implication for excitotoxicity in ALS

AMPA receptor-mediated neurotoxicity is currently the most plausible hypothesis for the etiology of amyotrophic lateral sclerosis (ALS). The mechanism initiating this type of neuronal death is believed to be exaggerated Ca2+-influx through AMPA receptors, which is critically determined by the presence or absence of the glutamate receptor subunit 2 (GluR2) in the assembly. We have provided the first quantitative measurements of the expression profile of AMPA receptor subunits mRNAs in human single neurons by means of quantitative RT-PCR with a laser microdissector. Among the AMPA subunits, GluR2 shared the vast majority throughout the neuronal subsets and tissues examined. Furthermore, both the expression level and the proportion of GluR2 mRNA in motoneurons were the lowest among all neuronal subsets examined, whereas those in motoneurons of ALS did not differ from the control group, implying that selective reduction of the GluR2 subunit cannot be a mechanism of AMPA receptor-mediated neurotoxicity in ALS. However, the low relative abundance of GluR2 might provide spinal motoneurons with conditions that are easily affected by changes of AMPA receptor properties including deficient GluR2 mRNA editing in ALS.     

Regulation of the intracellular distribution, cell surface expression, and protein levels of AMPA receptor GluR2 subunits by the monocarboxylate transporter MCT2 in neuronal cells

The neuronal monocarboxylate transporter, MCT2, is not only an energy substrate carrier but it is also purported to be a binding partner for the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR2 subunit. To unravel a putative role of MCT2 in the regulation of GluR2 subcellular distribution, Neuro2A cells and primary cultures of mouse cortical neurons were co-transfected with plasmids containing sequences to express the fluorescent proteins mStrawberry (mStb)-fused MCT2 and Venus-fused GluR2. Subsequently, their subcellular distribution was visualized by fluorescence microscopy. GluR2 was led to form perinuclear and dendritic clusters together with MCT2 when co-transfected in Neuro2A cells or in neurons, following the original distribution of MCT2. MCT2 co-transfection had no effect on the intracellular distribution of several other post-synaptic proteins, although it partially affected the intracellular distribution of GluR1 similarly to GluR2. Both cell surface and total protein expression levels of GluR2 were significantly reduced by co-expression with MCT2. Finally, partial perinuclear and dendritic co-localization between MCT2 and Rab8, a member of the small GTPase family involved in membrane trafficking of AMPA receptors, was also observed in co-transfected neurons. These results suggest that MCT2 could influence AMPA receptor trafficking within neurons by modulating GluR2 sorting between different subcellular compartments.     

Differential trafficking of GluR7 kainate receptor subunit splice variants

Kainate receptors (KARs) are heteromeric ionotropic glutamate receptors that play a variety of roles in the regulation of synaptic network activity. The function of glutamate receptors (GluRs) is highly dependent on their surface density in specific neuronal domains. Alternative splicing is known to regulate surface expression of GluR5 and GluR6 subunits. The KAR subunit GluR7 exists under different splice variant isoforms in the C-terminal domain (GluR7a and GluR7b). Here we have studied the trafficking of GluR7 splice variants in cultured hippocampal neurons from wild-type and KAR mutant mice. We have found that alternative splicing regulates surface expression of GluR7-containing KARs. GluR7a and GluR7b differentially traffic from the ER to the plasma membrane. GluR7a is highly expressed at the plasma membrane, and its trafficking is dependent on a stretch of positively charged amino acids also found in GluR6a. In contrast, GluR7b is detected at the plasma membrane at a low level and retained mostly in the endoplasmic reticulum (ER). The RXR motif of GluR7b does not act as an ER retention motif, at variance with other receptors and ion channels, but might be involved during the assembly process. Like GluR6a, GluR7a promotes surface expression of ER-retained subunit splice variants when assembled in heteromeric KARs. However, our results also suggest that this positive regulation of KAR trafficking is limited by the ability of different combinations of subunits to form heteromeric receptor assemblies. These data further define the complex rules that govern membrane delivery and subcellular distribution of KARs.     

Autoantibodies to neuronal glutamate receptors in patients with paraneoplastic neurodegenerative syndrome enhance receptor activation.

BACKGROUND: Paraneoplastic syndromes are "remote" complications of cancer characterized clinically by neurological disease. The sera and cerebrospinal fluid (CSF) from patients with paraneoplastic neurological syndromes (PNS) frequently contain autoantibodies to ill-defined neuronal antigens. We report here that neuronal glutamate receptors are targets for autoantibodies found in the serum from some patients with well-characterized PNS. MATERIALS AND METHODS: We have analyzed the serum from seven patients with well-characterized PNS for the presence of autoreactive antibodies to non-NMDA glutamate receptor subunits. Autoantibodies were assessed using Western blot, immunohistochemistry, and immunocytochemistry. Whole-cell electrophysiological recordings were used to examine the effect of antibodies on glutamate receptors expressed by cortical neurons in culture. RESULTS: Six of seven patients' serum contained autoantibodies to the non-NMDA glutamate receptor (GluR) subunits GluR1, GluR4, and/or GluR5/6. No patient had autoantibodies to GluR2, and only one patient exhibited weak immunoreactivity to GluR3. Electrophysiological analysis demonstrated that the serum from four of the six GluR-antibody-positive patients enhanced glutamate-elicited currents on cultured cortical neurons but had no effect on receptor function alone. Enhancement of glutamate-elicited currents was also produced by affinity-purified antibody to GluR5. CONCLUSIONS: The occurrence of autoantibodies to specific neuronal neurotransmitter subunits in the sera of patients with PNS and the ability of these autoantibodies to modulate glutaminergic receptor function suggest that some paraneoplastic neurological injury could result from glutamate-mediated excitotoxicity.     

Biochemical Characterization and Localization of a Non-N-Methyl-D-Aspartate Glutamate Receptor in Rat Brain

The structure and distribution of non-N-methyl-D-aspartate glutamate receptors in the rat brain were studied using subunit-specific antibodies that recognize the receptor subunit GluR1. The GluR1 protein, a 106-kDa glycoprotein, appears predominantly in synaptic plasma membranes, where it is highly enriched in the postsynaptic densities. When synaptic plasma membranes are solubilized with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, high-affinity alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) binding and GluR1 immunoreactivity comigrate at a native Mr of 610,000. GluR1 is enriched in the hippocampus and cerebellar cortex but is present throughout the CNS. It is found on neuronal cell bodies and processes within most regions of the brain; within the cerebellum, however, it is localized to the Bergmann glia. These data suggest that the GluR1 protein is a subunit of multimeric AMPA-preferring glutamate receptors present on neurons and on specialized glia.     

Immunocytochemical localization of neurons containing the AMPA GluR2/3 subunit in the hamster visual cortex

AMPA glutamate receptors play a crucial role in brain functions such as synaptic plasticity and development. We have studied the chemo-architecture of the AMPA glutamate receptor subtype GluR2/3 in the hamster visual cortex by immunocytochemistry and compared it with the distribution of the calcium-binding proteins, calbindin D28K and calretinin. Anti-GluR2/3-immunoreactive (IR) neurons were predominantly located in layers II/III, V, and VI, and the majority of the labeled neurons were round or oval. However, many pyramidal cells in layer V were also labeled. Two-color immunofluorescence revealed that none of the GluR2/3-IR neurons contained calbindin D28 K or calretinin. Thus specific layers of neurons express the GluR2/3 subunit and these do not correlate with expression of calbindin D28K and calretinin.     

Brain-derived neurotrophic factor regulates surface expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptors by enhancing the N-ethylmaleimide-sensitive factor/GluR2 interaction in developing neocortical neurons

In hippocampal neurons, the exocytotic process of alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)-type glutamate receptors is known to depend on activation of N-methyl-d-aspartate channels and its resultant Ca(2+) influx from extracellular spaces. Here we found that brain-derived neurotrophic factor (BDNF) induced a rapid surface translocation of AMPA receptors in an activity-independent manner in developing neocortical neurons. The receptor translocation became evident within hours as monitored by [(3)H]AMPA binding and was resistant against ionotropic glutamate receptor antagonists as evidenced with surface biotinylation assay. This process required intracellular Ca(2+) and was inhibited by the blockers of conventional exocytosis, brefeldin A, botulinum toxin B, and N-ethylmaleimide. To explore the translocation mechanism of individual AMPA receptor subunits, we utilized the human embryonic kidney (HEK) 293 cells carrying the BDNF receptor TrkB. After the single transfection of GluR2 cDNA or GluR1 cDNA into HEK/TrkB cells, BDNF triggered the translocation of GluR2 but not that of GluR1. Subsequent mutation analysis of GluR2 carboxyl-terminal region indicated that the translocation of GluR2 subunit in HEK293 cells involved its N-ethylmaleimide-sensitive factor-binding domain but not its PDZ-interacting site. Following co-transfection of GluR1 and GluR2 cDNAs, solid phase cell sorting revealed that GluR1 subunits were also able to translocate to the cell surface in response to BDNF. An immunoprecipitation assay confirmed that BDNF stimulation can enhance the interaction of GluR2 with N-ethylmaleimide-sensitive factor. These results reveal a novel role of BDNF in regulating the surface expression of AMPA receptors through a GluR2-NSF interaction.     

Distribution of AMPA glutamate receptor GluR1 subunit-immunoreactive neurons and their co-localization with calcium-binding proteins and GABA in the mouse visual cortex

The neuronal localization of alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptor (GluR) subunits is vital as they play key roles in the regulation of calcium permeability. We have examined the distribution of the calcium permeable AMPA glutamate receptor subunit GluR1 in the mouse visual cortex immunocytochemically. We compared this distribution to that of the calcium-binding proteins calbindin D28K, calretinin, and parvalbumin, and of GABA. The highest density of GluR1-immunoreactive (IR) neurons was found in layers II/III. Enucleation appeared to have no effect on the distribution of GluR1-IR neurons. The labeled neurons varied in morphology; the majority were round or oval and no pyramidal cells were labeled by the antibody. Two-color immunofluorescence revealed that 26.27%, 10.65%, and 40.31% of the GluR1-IR cells also contained, respectively, calbindin D28K, calretinin, and parvalbumin. 20.74% of the GluR1-IR neurons also expressed GABA. These results indicate that many neurons that express calcium-permeable GluR1 also express calcium binding proteins. They also demonstrate that one fifth of the GluR1-IR neurons in the mouse visual cortex are GABAergic interneurons.     

Sensory deafferentation transsynaptically alters neuronal GluR1 expression in the external plexiform layer of the adult mouse main olfactory bulb

Altered distribution of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit GluR1 has been linked to stimulation-dependent changes in synaptic efficacy, including long-term potentiation and depression. The main olfactory bulb (OB) remains plastic throughout life; how GluR1 may be involved in this plasticity is unknown. We have previously shown that neonatal naris occlusion reduces numbers of interneuron cell bodies that are immunoreactive for GluR1 in the external plexiform layer (EPL) of the adult mouse OB. Here, we show that immunoreactivity of mouse EPL interneurons for GluR1 is also dramatically reduced following olfactory deafferentation in adulthood. We further show that expression of glutamic acid decarboxylase (GAD) 65, 1 of 2 GAD isoforms expressed by adult gamma-aminobutyric acidergic interneurons, is reduced, but to a much smaller extent, and that in double-labeled cells, immunoreactivity for the Ca(2+)-binding protein parvalbumin (PV) is also reduced. In addition, GluR1 expression is reduced in presumptive tufted cells and interneurons that are negative for GAD65 and PV. Consistent with previous reports, sensory deafferentation resulted in little neuronal degeneration in the adult EPL, indicating that these differences were not likely due to death of EPL neurons. Together, these results suggest that olfactory input regulates expression of the GluR1 AMPA receptor subunit by tufted cells that may in turn regulate GluR1 expression by interneurons within the OB EPL.     

Interactions between NEEP21, GRIP1 and GluR2 regulate sorting and recycling of the glutamate receptor subunit GluR2

Trafficking of AMPA-type glutamate receptors (AMPAR) between endosomes and the postsynaptic plasma membrane of neurons plays a central role in the control of synaptic strength associated with learning and memory. The molecular mechanisms of its regulation remain poorly understood, however. Here we show by biochemical and atomic force microscopy analyses that NEEP21, a neuronal endosomal protein necessary for receptor recycling including AMPAR, is associated with the scaffolding protein GRIP1 and the AMPAR subunit GluR2. Moreover, the interaction between NEEP21 and GRIP1 is regulated by neuronal activity. Expression of a NEEP21 fragment containing the GRIP1-binding site decreases surface GluR2 levels and delays recycling of internalized GluR2, which accumulates in early endosomes and lysosomes. Infusion of this fragment into pyramidal neurons of hippocampal slices induces inward rectification of AMPAR-mediated synaptic responses, suggesting decreased GluR2 expression at synapses. These results indicate that NEEP21-GRIP1 binding is crucial for GluR2-AMPAR sorting through endosomes and their recruitment to the plasma membrane, providing a first molecular mechanism to differentially regulate AMPAR subunit cycling in internal compartments.