RNA Editing of the Glutamate Receptor Subunits GluR2 and GluR6 in Human Brain Tissue

Abstract: Editing of mRNA in the coding region of the second transmembrane domain of glutamate receptor subunits GluR2, GluR5, and GluR6 involves a change of the base A in genomic DNA to the base G in mRNA as described in rat brain. To determine whether this reaction occurs in humans as well as rats, we studied RNA editing of GluR2 and GluR6 in human brain. We compared the extent of editing in controls and cases with Huntington's disease. To assay the extent of editing in brain RNA, first strand cDNA was amplified using the polymerase chain reaction yielding a product across the region of the second transmembrane spanning segment in which editing takes place in rats. The PCR product was incubated with the restriction enzyme BbvI, which recognizes the sequence GCAGC present in the nonedited sequence of the mRNA in subunits GluR2 and GluR6. Thus, BbvI cuts the nonedited version but leaves the edited version intact. As in the rat, the GluR2 subunit mRNA was completely edited in human brain. The GluR6 subunit was nearly completely edited in all gray matter structures investigated including cortex, striatum, thalamus, hippocampus, amygdala, and cerebellum with extent of editing ranging from 89% in the cerebellum to 95% in the cortex and striatum. No significant differences in the extent of RNA editing were apparent in control versus Huntington's disease brains. To compare the extent of editing in neurons and glia in the brain, editing in cerebral cortex (predominantly gray matter and thus neurons) was compared with editing in corpus callosum (white matter and thus nearly completely glial cells). In white matter, GluR2 was completely edited, whereas GluR6 was only ～10% edited compared with ～90% edited in gray matter. Thus, these studies indicate that RNA editing is seen in human brain as well as rat brain and that the extent of editing is similar in Huntington's disease compared with controls. The differences in editing in white matter for GluR6, but not for GluR2, suggest that different templates could be subject to different editing activities that undergo tissue-specific regulation.     

Regional Distribution of the GABAA/Benzodiazepine Receptor (α Subunit) mRNA in Rat Brain

A human cDNA clone containing the 5' coding region of the GABAA/benzodiazepine receptor alpha subunit was used to quantify and visualize receptor mRNA in various regions of the rat brain. Using a [32P]CTP-labelled antisense RNA probe (860 bases) prepared from the alpha subunit cDNA, multiple mRNA species were detected in Northern blots using total and poly A rat brain RNA. In all brain regions, mRNAs of 4.4 and 4.8 kb were observed, and an additional mRNA of 3.0 kb was detected in the cerebellum and hippocampus. The level of GABAA/benzodiazepine receptor mRNA was highest in the cerebellum followed by the thalamus = frontal cortex = hippocampus = parietal cortex = hypothalamus much greater than pons = striatum = medulla. In situ hybridization revealed high levels of alpha subunit mRNA in cerebellar gray matter, olfactory bulb, thalamus, hippocampus/dentate gyrus, and the arcuate nucleus of the hypothalamus. These data suggest the presence of multiple GABAA/benzodiazepine receptor alpha subunit mRNAs in rat brain and demonstrate the feasibility of studying the expression of genes encoding the GABAA/benzodiazepine receptor after pharmacological and/or environmental manipulation.     

Editing of AMPA and Serotonin 2C Receptors in Individual Central Neurons,Controlling Wakefulness

(1) Pre-mRNA editing of serotonin 2C (5-HT_2C) and glutamate (Glu) receptors (R) influences higher brain functions and pathological states such as epilepsy, amyotrophic lateral sclerosis, and depression. Adenosine deaminases acting on RNA (ADAR1–3) convert adenosine to inosine on synthetic RNAs, analogous to 5-HT_2cR and GluR. The order of editing as well as mechanisms controlling editing in native neurons is unknown. (2) With single-cell RT-PCR we investigated the co-expression of ADAR genes with GluR and 5-HT_2CR and determined the editing status at known sites in the hypothalamic tuberomamillary nucleus, a major center for wakefulness and arousal. (3) The most frequently expressed enzymes were ADAR1, followed by ADAR2. The Q/R site of GluR2 was always fully edited. Editing at the R/G site in the GluR2 (but not GluR4) subunit was co-ordinated with ADAR expression: maximal editing was found in neurons expressing both ADAR2 splice variants of the deaminase domain and lacking ADAR3. (4) Editing of the 5-HT_2CR did not correlate with ADAR expression. The 5-HT_2CR mRNA was always edited at A, in the majority of cells at B sites and variably edited at E, C and D sites. A negative correlation was found between editing of C and D sites. The GluR4 R/G site editing was homogeneous within individuals: it was fully edited in all neurons obtained from 12 rats and under-edited in six neurons obtained from three rats. (5) We conclude that GluR2 R/G editing is controlled at the level of ADAR2 and therefore this enzyme may be a target for pharmacotherapy. On the other hand, further factors/enzymes besides ADAR must control or influence 5-HT_2CR and GluR pre-mRNA editing in native neurons; our data indicate that these factors vary between individuals and could be predictors of psychiatric disease.     

ADAR2-dependent RNA editing of AMPA receptor subunit GluR2 determines vulnerability of neurons in forebrain ischemia

ADAR2 is a nuclear enzyme essential for GluR2 pre-mRNA editing at Q/R site-607, which gates Ca2+ entry through AMPA receptor channels. Here, we show that forebrain ischemia in adult rats selectively reduces expression of ADAR2 enzyme and, hence, disrupts RNA Q/R site editing of GluR2 subunit in vulnerable neurons. Recovery of GluR2 Q/R site editing by expression of exogenous ADAR2b gene or a constitutively active CREB, VP16-CREB, which induces expression of endogenous ADAR2, protects vulnerable neurons in the rat hippocampus from forebrain ischemic insult. Generation of a stable ADAR2 gene silencing by delivering small interfering RNA (siRNA) inhibits GluR2 Q/R site editing, leading to degeneration of ischemia-insensitive neurons. Direct introduction of the Q/R site edited GluR2 gene, GluR2(R607), rescues ADAR2 degeneration. Thus, ADAR2-dependent GluR2 Q/R site editing determines vulnerability of neurons in the rat hippocampus to forebrain ischemia.     

Dramatic Increase of the RNA Editing for Glutamate Receptor Subunits During Terminal Differentiation of Clonal Human Neurons

Abstract: RNA editing plays an important role in determining physiological characteristics of certain glutamate-gated receptor (GluR) channels such as Ca²⁺ permeability and desensitization kinetics. In one case, the editing changes a gene-encoded glutamine (Q) to an arginine (R) codon located in the channel-forming domain of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit GluR-B and also the kainate receptor subunits GluR5 and GluR6. Another case of RNA editing alters an arginine (R) to a glycine (G) codon at a position termed the "R/G" site of AMPA subunits GluR-B, C, and D. Double-stranded RNA-specific adenosine deaminases (DRADA) have been implicated as agents involved in the editing. By using a human teratocarcinoma cell line, NT2, we investigated the change of the RNA editing of GluR subunits in conjunction with the expression of two DRADA members, DRADA1 and DRADA2 genes, during neuronal differentiation. Whereas Q/R and R/G site RNA editing both become progressively activated in differentiating NT2 cells, the expression of the two DRADA genes can already be detected even in the undifferentiated NT2 cells. Development of the editing machinery appears to require, in addition to DRADA enzymes, a currently unidentified mechanism(s) that may become activated during neuronal differentiation.     

听原性惊厥易感大鼠下丘GluR2的表达及QR位点编辑水平

听原性惊厥易感大鼠是强直 -阵挛惊厥大发作的一种模型 .一般认为 ,下丘是听原性惊厥发作神经元网络的启动部位 .采用RT PCR、Western印迹、免疫组织化学等方法观察了听原性惊厥易感大鼠 (P77PMC)一次惊厥发作与惊厥点燃状态下AMPA受体亚基GluR2在下丘内表达的改变 ,并采用限制性酶切方法分析了GluR2Q R位点mRNA编辑水平的改变 .研究结果显示 ,一次惊厥发作后下丘内GluR2表达无明显改变 ,惊厥点燃后下丘内GluR2表达降低 ,一次惊厥发作及惊厥点燃状态下GluR2Q R位点处于编辑成熟状态 .提示 ,GluR2表达降低参与了点燃状态下的惊厥发作 ,在听原性惊厥易感大鼠惊厥发作机制中不涉及下丘内GluR2Q R位点编辑水平改变 .     

Developmentally regulated, combinatorial RNA processing modulates AMPA receptor biogenesis

The subunit composition determines AMPA receptor (AMPA-R) function and trafficking. Mechanisms underlying channel assembly are thus central to the efficacy and plasticity of glutamatergic synapses. We previously showed that RNA editing at the Q/R site of the GluR2 subunit contributes to the assembly of AMPA-R heteromers by attenuating formation of GluR2 homotetramers. Here we report that this function of the Q/R site depends on subunit contacts between adjacent ligand binding domains (LBDs). Changes of LBD interface contacts alter GluR2 assembly properties, forward traffic, and expression at synapses. Interestingly, developmentally regulated RNA editing within the LBD (at the R/G site) produces analogous effects. Our data reveal that editing to glycine reduces the self-assembly competence of this critical subunit and slows GluR2 maturation in the endoplasmic reticulum (ER). Therefore, RNA editing sites, located at strategic subunit interfaces, shape AMPA-R assembly and trafficking in a developmentally regulated manner.     

Cis-acting elements stimulating kinetoplastid guide RNA-directed editing

The coding sequence of several mitochondrial mRNAs of the kinetoplastid protozoa is created through the insertion and deletion of specific uridylates. The editing reactions are required to be highly specific in order to ensure that functional open reading frames are created in edited mRNAs and that potentially deleterious modification of normally nonedited sequence does not occur. Selection-amplification and mutagenesis were previously used to identify the optimal sequence requirements for in vitro editing. There is, however, a minority of natural editing sites with suboptimal sequence. Several cis-acting elements, obtained from an in vitro selection, are described here that are able to compensate for a suboptimal editing site. An A + U sequence element within the 5'-untranslated region of cytochrome b mRNA from Leishmania tarentolae is also demonstrated to function as a cis-acting guide RNA and is postulated to compensate for a suboptimal editing site in vivo. Two proteins within an enriched editing extract are UV-cross-linked to two different in vitro selected editing substrates more efficiently than poorly edited RNAs. The results suggest that these proteins contribute to the specificity of the editing reaction.     

Partially edited mRNAs for cytochrome b and subunit III of cytochrome oxidase from Leishmania tarentolae mitochondria: RNA editing intermediates

Partially edited mRNAs were selected by the polymerase chain reaction and sequenced. In the case of cytochrome b, 102 out of 106 clones displayed patterns of editing that were consistent with a strictly progressive 3' to 5' editing process, as predicted by the guide RNA model of RNA editing. In the case of cytochrome oxidase subunit III (COIII), 177 out of 304 clones displayed strictly progressive 3' to 5' patterns of editing. However, the remaining 127 COIII clones displayed unexpected patterns in which upstream editing preceded downstream editing, uridines were inserted at sites not normally edited, and purine residues were deleted. We suggest that many of these RNAs are produced by normal 3' to 5' editing of the COIII mRNA with incorrect guide RNA molecules.     

AMPA receptor tetramerization is mediated by Q/R editing

AMPA-type glutamate receptors (AMPARs) play a major role in excitatory synaptic transmission and plasticity. Channel properties are largely dictated by their composition of the four subunits, GluR1-4 (or A-D). Here we show that AMPAR assembly and subunit stoichiometry are determined by RNA editing in the pore loop. We demonstrate that editing at the GluR2 Q/R site regulates AMPAR assembly at the step of tetramerization. Specifically, edited R subunits are largely unassembled and ER retained, whereas unedited Q subunits readily tetramerize and traffic to synapses. This assembly mechanism restricts the number of the functionally critical R subunits in AMPAR tetramers. Therefore, a single amino acid residue affects channel composition and, in turn, controls ion conduction through the majority of AMPARs in the brain.     

Evidence for low GluR2 AMPA receptor subunit expression at synapses in the rat basolateral amygdala

Fast excitatory synaptic responses in basolateral amygdala (BLA) neurons are mainly mediated by ionotropic glutamate receptors of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) subtype. AMPA receptors containing an edited GluR2 subunit are calcium impermeable, whereas those that lack this subunit are calcium permeable and also inwardly rectifying. Here, we sought to determine the extent to which synapses in the rat BLA have AMPA receptors with GluR2 subunits. We assessed GluR2 protein expression in the BLA by immunocytochemistry with a GluR2 subunit-specific antiserum at the light and electron microscopic level; for comparison, a parallel examination was carried out in the hippocampus. We also recorded from amygdala brain slices to examine the voltage-dependent properties of AMPA receptor- mediated evoked synaptic currents in BLA principal neurons. At the light microscopic level, GluR2 immunoreactivity was localized to the perikarya and proximal dendrites of BLA neurons; dense labeling was also present over the pyramidal cell layer of hippocampal subfields CA1 and CA3. In electron micrographs from the BLA, most of the synapses were asymmetrical with pronounced postsynaptic densities (PSD). They contained clear, spherical vesicles apposed to the PSD and were predominantly onto spines (86%), indicating that they are mainly with BLA principal neurons. Only 11% of morphological synapses in the BLA were onto postsynaptic elements that showed GluR2 immunoreactivity, in contrast to hippocampal subfields CA1 and CA3 in which 76% and 71% of postsynaptic elements were labeled (p < 0.001). Synaptic staining in the BLA and hippocampus, when it occurred, was exclusively postsynaptic, and particularly heavy over the PSD. In whole-cell voltage clamp recordings, 72% of BLA principal neurons exhibited AMPA receptor-mediated synaptic currents evoked by external capsule stimulation that were inwardly rectifying. Although BLA principal neurons express perikaryal and proximal dendritic GluR2 immunoreactivity, few synapses onto these neurons express GluR2, and a preponderance of principal neurons have inwardly rectifying AMPA-mediated synaptic currents, suggesting that targeting of GluR2 to synapses is restricted. Many BLA synaptic AMPA receptors are likely to be calcium permeable and could play roles in synaptic plasticity, epileptogenesis and excitoxicity.     

Regional Distribution of Neuropeptide Y and Its Receptor in the Porcine Central Nervous System

The regional distribution of neuropeptide Y (NPY) immunoreactivity and receptor binding was studied in the porcine CNS. The highest amounts of immunoreactive NPY were found in the hypothalamus, septum pellucidum, gyrus cinguli, cortex frontalis, parietalis, and piriformis, corpus amygdaloideum, and bulbus olfactorius (200-1,000 pmol/g wet weight). In the cortex temporalis and occipitalis, striatum, hippocampus, tractus olfactorius, corpus mamillare, thalamus, and globus pallidus, the NPY content was 50-200 pmol/g wet weight, whereas the striatum, colliculi, substantia nigra, cerebellum, pons, medulla oblongata, and medulla spinalis contained less than 50 pmol/g wet weight. The receptor binding of NPY was highest in the hippocampus, corpus fornicis, corpus amygdaloideum, nucleus accumbens, and neurohypophysis, with a range of 1.0-5.87 pmol/mg of protein. Intermediate binding (0.5-1.0 pmol/mg of protein) was found in the septum pellucidum, columna fornicis, corpus mamillare, cortex piriformis, gyrus cinguli, striatum, substantia grisea centralis, substantia nigra, and cerebellum. In the corpus callosum, basal ganglia, corpus pineale, colliculi, corpus geniculatum mediale, nucleus ruber, pons, medulla oblongata, and medulla spinalis, receptor binding of NPY was detectable but less than 0.5 pmol/mg of protein. No binding was observed in the bulbus and tractus olfactorius and adenohypophysis. In conclusion, immunoreactive NPY and its receptors are widespread in the porcine CNS, with predominant location in the limbic system, olfactory system, hypothalamoneurohypophysial tract, corpus striatum, and cerebral cortex.     

The role of RNA editing of kainate receptors in synaptic plasticity and seizures

The ionotropic glutamate receptor subunit GluR6 undergoes developmentally and regionally regulated Q/R site RNA editing that reduces the calcium permeability of GluR6-containing kainate receptors. To investigate the functional significance of this editing in vivo, we engineered mice deficient in GluR6 Q/R site editing. In these mutant mice but not in wild types, NMDA receptor-independent long-term potentiation (LTP) could be induced at the medial perforant path-dentate gyrus synapse. This indicates that kainate receptors with unedited GluR6 subunits can mediate LTP. Behavioral analyses revealed no differences from wild types, but mutant mice were more vulnerable to kainate-induced seizures. Together, these results suggest that GluR6 Q/R site RNA editing may modulate synaptic plasticity and seizure vulnerability.     

RNA editing of the col mRNA throughout the life cycle of Physarum polycephalum

Editing of RNA via the insertion, deletion or substitution of genetic information affects gene expression in a variety of systems. Previous characterization of the Physarum polycephalum cytochrome c oxidase subunit I (col) mRNA revealed that both nucleotide insertions and base substitutions occur during the maturation of this mitochondrial message. Both types of editing are known to be developmentally regulated in other systems, including mammals and trypanosomatids. Here we show that the col mRNA present in Physarum mitochondria is edited via specific nucleotide insertions and C to U conversions at every stage of the life cycle. Primer extension sequencing of the RNA indicates that this editing is both accurate and efficient. Using a sensitive RT-PCR assay to monitor the extent of editing at individual sites of C insertion, we estimate that greater than 98% of the steady-state amount of col mRNA is edited throughout the Physarum developmental cycle.