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.     

Regulation of glutamate receptor B pre-mRNA splicing by RNA editing

RNA-editing enzymes of the ADAR family convert adenosines to inosines in double-stranded RNA substrates. Frequently, editing sites are defined by base-pairing of the editing site with a complementary intronic region. The glutamate receptor subunit B (GluR-B) pre-mRNA harbors two such exonic editing sites termed Q/R and R/G. Data from ADAR knockout mice and in vitro editing assays suggest an intimate connection between editing and splicing of GluR-B pre-mRNA.

By comparing the events at the Q/R and R/G sites, we can show that editing can both stimulate and repress splicing efficiency. The edited nucleotide, but not ADAR binding itself, is sufficient to exert this effect. The presence of an edited nucleotide at the R/G site reduces splicing efficiency of the adjacent intron facilitating alternative splicing events occurring downstream of the R/G site.

Lack of editing inhibits splicing at the Q/R site. Editing of both the Q/R nucleotide and an intronic editing hotspot are required to allow efficient splicing. Inefficient intron removal may ensure that only properly edited mRNAs become spliced and exported to the cytoplasm.

     

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.     

ADAR2 A-->I editing: site selectivity and editing efficiency are separate events

ADAR enzymes, adenosine deaminases that act on RNA, form a family of RNA editing enzymes that convert adenosine to inosine within RNA that is completely or largely double-stranded. Site-selective A→I editing has been detected at specific sites within a few structured pre-mRNAs of metazoans. We have analyzed the editing selectivity of ADAR enzymes and have chosen to study the naturally edited R/G site in the pre-mRNA of the glutamate receptor subunit B (GluR-B). A comparison of editing by ADAR1 and ADAR2 revealed differences in the specificity of editing. Our results show that ADAR2 selectively edits the R/G site, while ADAR1 edits more promiscuously at several other adenosines in the double-stranded stem. To further understand the mechanism of selective ADAR2 editing we have investigated the importance of internal loops in the RNA substrate. We have found that the immediate structure surrounding the editing site is important. A purine opposite to the editing site has a negative effect on both selectivity and efficiency of editing. More distant internal loops in the substrate were found to have minor effects on site selectivity, while efficiency of editing was found to be influenced. Finally, changes in the RNA structure that affected editing did not alter the binding abilities of ADAR2. Overall these findings suggest that binding and catalysis are independent events.     

Pin1 and WWP2 regulate GluR2 Q/R site RNA editing by ADAR2 with opposing effects

ADAR2 catalyses the deamination of adenosine to inosine at the GluR2 Q/R site in the pre-mRNA encoding the critical subunit of AMPA receptors. Among ADAR2 substrates this is the vital one as editing at this position is indispensable for normal brain function. However, the regulation of ADAR2 post-translationally remains to be elucidated. We demonstrate that the phosphorylation-dependent prolyl-isomerase Pin1 interacts with ADAR2 and is a positive regulator required for the nuclear localization and stability of ADAR2. Pin1(-/-) mouse embryonic fibroblasts show mislocalization of ADAR2 in the cytoplasm and reduced editing at the GluR2 Q/R and R/G sites. The E3 ubiquitin ligase WWP2 plays a negative role by binding to ADAR2 and catalysing its ubiquitination and subsequent degradation. Therefore, ADAR2 protein levels and catalytic activity are coordinately regulated in a positive manner by Pin1 and negatively by WWP2 and this may have downstream effects on the function of GluR2. Pin1 and WWP2 also regulate the large subunit of RNA Pol II, so these proteins may also coordinately regulate other key cellular proteins.     

Cell Type-Specific Gene Expression and Editing Responses to Chronic Fluoxetine Treatment in the In Vivo Mouse Brain and Their Relevance for Stress-Induced Anhedonia

Recently developed methods for fluorescence-activated cell sorting (FACS) of freshly-isolated brain cells from transgenic mice combining fluorescent signals with cell type-specific markers allow cell-type separation. Based upon previous observations in primary cultures of mouse astrocytes we treated transgenic mice tagged with a neuron-specific or an astrocyte-specific marker with fluoxetine, either acute (10?mg/kg for 2?h) or chronic (10?mg/kg daily for 2?weeks). Acute treatment upregulated cfos and fosB mRNA expression in astrocytes and neurons. Chronic effects on astrocytes replicated those demonstrated in cultures, i.e., upregulation of mRNA and/or protein expression of 5-HT_2B receptors (5-HT_2BR), and GluK2 receptors, and of cPLA_2a and ADAR2, together with increased GluK2 and 5-HT_2BR editing. Neurons showed increased GluK4 and 5-HT_2C receptor expression. To further correlate these findings with major depression we compared the changes in gene expression with those in a mouse model of anhedonia. Three out of 4 genes up-regulated in astrocytes by fluoxetine were down-regulated, whereas the neuronally upregulated 5-HT_2C receptor gene showed no change. References are made to recent review papers discussing potential relations between observed fluoxetine effects and clinical effects of SSRIs, emphasizing that all 5 clinically used SSRIs have identical and virtually equipotent effects on cultured astrocytes.     

Editing modifies the GABA(A) receptor subunit alpha3

Adenosine to inosine (A-to-I) pre-mRNA editing by the ADAR enzyme family has the potential to increase the variety of the proteome. This editing by adenosine deamination is essential in mammals for a functional brain. To detect novel substrates for A-to-I editing we have used an experimental method to find selectively edited sites and combined it with bioinformatic techniques that find stem-loop structures suitable for editing. We present here the first verified editing candidate detected by this screening procedure. We show that Gabra-3, which codes for the alpha3 subunit of the GABA(A) receptor, is a substrate for editing by both ADAR1 and ADAR2. Editing of the Gabra-3 mRNA recodes an isoleucine to a methionine. The extent of editing is low at birth but increases with age, reaching close to 100% in the adult brain. We therefore propose that editing of the Gabra-3 mRNA is important for normal brain development.     

5-HT2C Receptors in the Basolateral Amygdala and Dorsal Striatum Are a Novel Target for the Anxiolytic and Antidepressant Effects of Exercise

Physical activity reduces the incidence and severity of psychiatric disorders such as anxiety and depression. Similarly, voluntary wheel running produces anxiolytic- and antidepressant-like effects in rodent models. The specific neurobiological mechanisms underlying the beneficial properties of exercise, however, remain unclear. One relevant pharmacological target in the treatment of psychiatric disorders is the 5-HT_2C receptor (5-HT_2CR). Consistent with data demonstrating the anxiogenic consequences of 5-HT_2CR activation in humans and rodents, we have previously reported that site-specific administration of the selective 5-HT_2CR agonist CP-809101 in the lateral/basolateral amygdala (BLA) increases shock-elicited fear while administration of CP-809101 in the dorsal striatum (DS) interferes with shuttle box escape learning. These findings suggest that activation of 5-HT_2CR in discrete brain regions contributes to specific anxiety- and depression-like behaviors and may indicate potential brain sites involved in the anxiolytic and antidepressant effects of exercise. The current studies tested the hypothesis that voluntary wheel running reduces the behavioral consequences of 5-HT_2CR activation in the BLA and DS, specifically enhanced shock-elicited fear and interference with shuttle box escape learning. After 6 weeks of voluntary wheel running or sedentary conditions, the selective 5-HT_2CR agonist CP-809101 was microinjected into either the BLA or the DS of adult Fischer 344 rats, and shock-elicited fear and shuttle box escape learning was assessed. Additionally, in-situ hybridization was used to determine if 6 weeks of voluntary exercise changed levels of 5-HT_2CR mRNA. We found that voluntary wheel running reduced the behavioral effects of CP-809101 and reduced levels of 5-HT_2CR mRNA in both the BLA and the DS. The current data indicate that expression of 5-HT_2CR mRNA in discrete brain sites is sensitive to physical activity status of the organism, and implicates the 5-HT_2CR as a target for the beneficial effects of physical activity on mental health.     

Dimerization of ADAR2 is mediated by the double-stranded RNA binding domain

Members of the family of adenosine deaminases acting on RNA (ADARs) can catalyze the hydrolytic deamination of adenosine to inosine and thereby change the sequence of specific mRNAs with highly double-stranded structures. The ADARs all contain one or more repeats of the double-stranded RNA binding motif (DRBM). By both in vitro and in vivo assays, we show that the DRBMs of rat ADAR2 are necessary and sufficient for dimerization of the enzyme. Bioluminescence resonance energy transfer (BRET) demonstrates that ADAR2 also exists as dimers in living mammalian cells and that mutation of DRBM1 lowers the dimerization affinity while mutation of DRBM2 does not. Nonetheless, the editing efficiency of the GluR2 Q/R site depends on a functional DRBM2. The ADAR2 DRBMs thus serve differential roles in RNA dimerization and GluR2 Q/R editing, and we propose a model for RNA editing that incorporates the new findings.     

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.