GluR2 Gene Research Articles

Absence of the GluR2 receptor sensitizes mouse sympathetic neurons to nerve growth factor deprivation

Over-activation of glutamate receptors is implicated in neurodegeneration. Using mice with a deletion in the GluR2 gene, we studied the sensitivity of sympathetic neurons to reduced levels of nerve growth factor (NGF), which can cause neuronal cell death. Under standard culture conditions of 50 ng/ml NGF, neurons from the superior cervical ganglion survived and grew equally well compared with wild type controls. However, the subsequent reduction of NGF levels caused significantly poorer survival among mutant neurons by 48 h, at 44±13% of control at 10 ng/ml NGF, and dropping further to 14±6% at 0.05 ng/ml NGF. These results suggest that the absence of GluR2 impairs the ability of these NGF-sensitive neurons to survive under limiting amounts of this neurotrophic factor.

Transcriptional Regulation of the GluR2 Gene: Neural-Specific Expression, Multiple Promoters, and Regulatory Elements

To understand how neurons control the expression of the AMPA receptor subunit GluR2, we cloned the 5' proximal region of the rat gene and investigated GluR2 promoter activity by transient transfection. RNase protection and primer extension of rat brain mRNA revealed multiple transcription initiation sites from -340 to -481 bases upstream of the GluR2 AUG codon. The relative use of 5' start sites was different in cortex and cerebellum, indicating complexity of GluR2 transcript expression among different sets of neurons. When GluR2 promoter activity was investigated by plasmid transfection into cultured cortical neurons, cortical glia, and C6 glioma cells, the promoter construct with the strongest activity, per transfected cell, was 29.4-fold (+/- 3.7) more active in neurons than in non-neural cells. Immunostaining of cortical cultures showed that >97% of the luciferase-positive cells also expressed the neuronal marker MAP-2. Evaluation of internal deletion and substitution mutations identified a functional repressor element I RE1-like silencer and functional Sp1 and nuclear respiratory factor-1 (NRF-1) elements within a GC-rich proximal GluR2 promoter region. The GluR2 silencer reduced promoter activity in glia and non-neuronal cell lines by two- to threefold, was without effect in cortical neurons, and could bind the RE1-silencing transcription factor (REST) because cotransfection of REST into neurons reduced GluR2 promoter activity in a silencer-dependent manner. Substitution of the GluR2 silencer by the homologous NaII RE1 silencer further reduced GluR2 promoter activity in non-neuronal cells by 30-47%. Maximal positive GluR2 promoter activity required both Sp1 and NRF-1 cis elements and an interelement nucleotide bridge sequence. These results indicate that GluR2 transcription initiates from multiple sites, is highly neuronal selective, and is regulated by three regulatory elements in the 5' proximal promoter region.

Multiple behavioral anomalies in GluR2 mutant mice exhibiting enhanced LTP

We have previously disrupted the ionotropic glutamate receptor type 2 gene ( GluR2) using gene targeting in embryonic stem cells and generated mice which lacked the GluR2 gene product. Neurophysiological analyses of these mice showed a markedly enhanced long-term potentiation (LTP) and a 9-fold increase in kainate induced Ca 2+ permeability in the hippocampus. Here, we analyze the behavioral and neuroanatomical consequences of GluR2 deficiency in homozygous null mutant and age-matched littermate control mice. We show that despite unaltered gross brain morphology, several aspects of behavior were abnormal in the mutants. Object exploration, rearing, grooming and locomotion were altered in the novel arena. Eye-closure reflex, motor performance on the rotating rod and spatial and non-spatial learning performance in the water maze were also abnormal in the mutants. These abnormalities together with the widespread expression pattern of GluR2 in most excitatory CNS pathways suggest that the absence of GluR2 leads to neurological phenotypes associated with not only the hippocampus but several other brain regions potentially including the cortex and cerebellum. We speculate that GluR2 mutant mice suffer from an overall non-specifically increased excitability that may alter cognitive functions ranging from stimulus processing to motivation and learning.

Transcriptional Regulation of the GluR2 Gene: Neural-Specific Expression, Multiple Promoters, and Regulatory Elements

Transcriptional Regulation of the GluR2 Gene: Neural-Specific Expression, Multiple Promoters, and Regulatory Elements

The GluR2 (GluR-B) hypothesis: Ca 2+-permeable AMPA receptors in neurological disorders

The abnormal influx of Ca 2+ through glutamate receptor channels is thought to contribute to the loss of neurons associated with a number of brain disorders. Until recently, the NMDA receptor was the only glutamate receptor known to be Ca 2+-permeable. It is now well established that AMPA receptors exist not only in Ca 2+-impermeable but also in Ca 2+-permeable forms. AMPA receptors are encoded by four genes designated gluR1 ( gluR-A) through gluR4 ( gluR-D). The presence of the gluR2 subunit renders heteromeric AMPA receptor assemblies Ca 2+-impermeable. Recent studies involving animal models of transient forebrain ischemia and epilepsy show that gluR2 mRNA is downregulated in vulnerable neurons. These observations suggest that downregulation of gluR2 gene expression may serve as a `molecular switch' leading to the formation of Ca 2+-permeable AMPA receptors and enhanced toxicity of endogenous glutamate following a neurological insult.

Global Ischemia Induces Downregulation of Glur2 mRNA and Increases AMPA Receptor-Mediated Ca2+Influx in Hippocampal CA1 Neurons of Gerbil

Transient, severe forebrain or global ischemia leads to delayed cell death of pyramidal neurons in the hippocampal CA1. The precise molecular mechanisms underlying neuronal cell death after global ischemia are as yet unknown. Glutamate receptor-mediated Ca2+ influx is thought to play a critical role in this cell death. In situ hybridization revealed that the expression of mRNA encoding GluR2 (the subunit that limits Ca2+ permeability of AMPA-type glutamate receptors) was markedly and specifically reduced in gerbil CA1 pyramidal neurons after global ischemia but before the onset of neurodegeneration. To determine whether the change in GluR2 expression is functionally significant, we examined the AMPA receptor-mediated rise in cytoplasmic free Ca2+ level ([Ca2+]i) in individual CA1 pyramidal neurons by optical imaging with the Ca2+ indicator dye fura-2 and by intracellular recording. Seventy-two hours after ischemia, CA1 neurons that retained the ability to fire action potentials exhibited a greatly enhanced AMPA-elicited rise in [Ca2+]i. Basal [Ca2+]i in these neurons was unchanged. These findings provide evidence for Ca2+ entry directly through AMPA receptors in pyramidal neurons destined to die. Downregulation of GluR2 gene expression and an increase in Ca2+ influx through AMPA receptors in response to endogenous glutamate are likely to contribute to the delayed neuronal death after global ischemia.

Seizure-related changes in the glutamate R2 and R5 receptor genes expression in the rat hippocampal formation.

The expression of mRNA coding for AMPA selective glutamate (Glu) R2 receptor and kainate selective GluR5 receptor was studied in the rat hippocampal formation in two animal models of limbic seizures evoked by systemic administration of pilocarpine (400 mg/kg i.p.) or kainate (15 mg/kg i.p.). As shown by an in situ hybridization study, pilocarpine decreased the GluR2 flip mRNA level in CA1 and CA3 areas of the hippocampus after 3h and kainate after 24h, e.g. at the time preceding neuronal degeneration. No changes in the GluR2 flop or GluR5 mRNA level were found in those regions. In the dentate gyrus, resistant to neurodegeneration, pilocarpine and kainate differentially affected the expression of GluR2 and GluR5 mRNAs. After 72h pilocarpine, but not kainate, increased the GluR2 flop mRNA level and decreased the flip one, which suggests attenuation of the GluR2 sensitivity. On the other hand, kainate, elevated the GluR2 flip and GluR5 mRNA level in the dentate gyrus after 72h. All in all the above data suggest that changes in the GluR2 gene expression may play some role in the neuronal damage to vulnerable areas (CA1, CA3). However, differences in the kainate-and pilocarpine-induced changes in the dentate gyrus at the late time points indicate that alterations in the stoichiometry of GluR2 forms of GluR5 gene expression in this brain region are not a common causal factor responsible for delayed neuronal hyperexcitability.

Editing for an AMPA receptor subunit RNA in prefrontal cortex and striatum in Alzheimer's disease, Huntington's disease and schizophrenia

Animal studies and cell culture experiments demonstrated that posttranscriptional editing of the transcript of the GluR-2 gene, resulting in substitution of an arginine for glutamine in the second transmembrane region (TM II) of the expressed protein, is associated with a reduction in Ca 2+ permeability of the receptor channel. Thus, disturbances in GluR-2 RNA editing with alteration of intracellular Ca 2+ homeostasis could lead to neuronal dysfunction and even neuronal degeneration. The present study determined the proportions of edited and unedited GluR-2 RNA in the prefrontal cortex of brains from patients with Alzheimer's disease, in the striatum of brains from patients with Huntington's disease, and in the same areas of brains from age-matched schizophrenics and controls, by using reverse transcriptase-polymerase chain reaction, restriction endonuclease digestion, gel electrophoresis and scintillation radiometry. In the prefrontal cortex of controls, < 0.1% of all GluR-2 RNA molecules were unedited and *> 99.9% were edited; in the prefrontal cortex both of schizophrenics and of Alzheimer's patients ∼ 1.0% of all GluR-2 RNA molecules were unedited and 99% were edited. In the striatum of controls and of schizophrenics, ∼ 0.5% of GluR-2 RNA molecules were unedited and 99.5% were edited; in the striatum of Huntington's patients nearly 5.0% of GluR-2 RNA was unedited. In the prefrontal white matter of controls, ∼ 7.0% of GluR-2 RNA was unedited. In the normal human prefrontal cortex and striatum, the large majority of GluR-2 RNA molecules contains a CGG codon for arginine in the TMII coding region; this implies that the corresponding AMPA receptors have a low Ca 2+ permeability, as previously demonstrated for the rat brain. The process of GluR-2 RNA editing is compromised in a region-specific manner in schizophrenia, in Alzheimer's disease and Huntington's Chorea although in each of these disorders there is still a large excess of edited GluR-2 RNA molecules. Disturbances of GluR-2 RNA editing leading to excessive Ca 2+ permeability, may contribute to neuronal dysfunction in schizophrenia and to neuronal death in Alzheimer's disease and Huntington's disease.

Intron sequence directs RNA editing of the glutamate receptor subunit GluR2 coding sequence.

The Ca2+ permeability and the rectifying properties of the glutamate receptors assembled from the subunits GluR1-GluR4 depend upon a critical Arg in the GluR2 subunit located in a domain that has been proposed to span the membrane. The GluR2 subunit gene encodes a Gln (CAG) at this position, whereas the mRNA is edited so that it encodes an Arg (CGG) at this position [Sommer, B., Kohler, M., Sprengel, R. & Seeburg, P. H. (1991) Cell 67, 11-20]. The editing process is specific since only the GluR2 subunit RNA is edited even though the GluR1, GluR3, and GluR4 RNAs have a similar sequence. We show that this selective RNA editing depends upon a critical intron sequence in the GluR2 gene. This critical intron sequence is sufficient to cause editing of the GluR3 subunit exon in a chimera minigene constructed so that the GluR3 exon is placed upstream to the GluR2 intron sequence. Transfections of a neuronal cell line, N2a, with minigene constructs encoding different fragments of the GluR2 gene demonstrate that the 5' part of the 3' intron is essential for editing. Part of the exon and this critical intron sequence contains an inverted repeat that can fold into a structure consisting of three helical elements. Similar conclusions were reached by Higuchi, M., Single, F. n., Köhler, M., Sommer, B., Sprengel, R. & Seeburg, P. H. [(1993) Cell 75, 1361-1370]. These experiments demonstrate that the low Ca2+ permeability of the ionotropic non-N-methyl-D-aspartate glutamate receptors depends upon RNA editing, which requires a sequence in an intron 3' to the exon.

Switch in glutamate receptor subunit gene expression in CA1 subfield of hippocampus following global ischemia in rats.

Severe, transient global ischemia of the brain induces delayed damage to specific neuronal populations. Sustained Ca2+ influx through glutamate receptor channels is thought to play a critical role in postischemic cell death. Although most kainate-type glutamate receptors are Ca(2+)-impermeable, Ca(2+)-permeable kainate receptors have been reported in specific kinds of neurons and glia. Recombinant receptors assembled from GluR1 and/or GluR3 subunits in exogenous expression systems are permeable to Ca2+; heteromeric channels containing GluR2 subunits are Ca(2+)-impermeable. Thus, altered expression of GluR2 in development or following a neurological insult or injury to the brain can act as a switch to modify Ca2+ permeability. To investigate the molecular mechanism underlying delayed postischemic cell death, GluR1, GluR2, and GluR3 gene expression was examined by in situ hybridization in postischemic rats. Following severe, transient forebrain ischemia GluR2 gene expression was preferentially reduced in CA1 hippocampal neurons at a time point that preceded their degeneration. The switch in expression of kainate/AMPA receptor subunits coincided with the previously reported increase in Ca2+ influx into CA1 cells. Timing of the switch indicates that it may play a causal role in postischemic cell death.

Are Ca 2+-permeable kainate/AMPA receptors more abundant in immature brain?

The permeability properties of kainate/AMPA receptors are determined by subunit composition. The GluR1 and GluR3 subunits form Ca 2+-permeable channels and exhibit inward rectification; heteromeric receptors containing the GluR2 subunit are Ca 2+-impermeable and electrically linear. These observations raise the possibily of a developmental ‘switch’ in which turning on or off of GluR2 expression regulates the level of Ca 2+ permeable kainate/AMPA receptors. We examined the ratio of GluR1 and GluR3 to GluR2 gene expression in developing and adult rat brain by in situ hybridization. A larger value of this ratio is likely to be associated with greater Ca 2+ permeability. Our data suggest that in neocortex, striatum and cerebellum the number of Ca 2+-permeable kainate/AMPA receptors is high at P4 and declines monotonically with age. In hippocampus, the number increases from P7 to P21, after which it declines. These findings provide evidence for a developmental ‘switch’ in which Ca 2+ permeable glutamate receptors are turned off following early developmental events.