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

Abstract

(1) Pre-mRNA editing of serotonin 2C (5-HT2c) 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-HT2cR 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-HT2cR 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-HT2cR did not correlate with ADAR expression. The 5-HT2cR 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-HT2cR and GluR pre-mRNA editing in native neurons; our data indicate that these factors vary between individuals and could be predictors of psychiatric disease.

Fluoxetine affects GluK2 editing, glutamate-evoked Ca2+ influx and extracellular signal-regulated kinase phosphorylation in mouse astrocytes

“…The level of expression of ADAR2 does not correlate directly with the enzyme activity alone, but also depends on isoform distribution. 13,14,60 We found that fluoxetine upregulated all 4 isoforms, although it upregulated Ld to a larger extent than the Sd isoform and Sc to a larger extent than the Lc isoform. High levels of expression of both the Sd and Sc isoforms indicate an increase of editing activity, at least of the GluK2 Q/R site in hypothalamic tuberomamillary neurons.…”

“…High levels of expression of both the Sd and Sc isoforms indicate an increase of editing activity, at least of the GluK2 Q/R site in hypothalamic tuberomamillary neurons. 14 Thus, fluoxetine not only upregulates the expression of ADAR2 but also enhances its editing activity. The signalling events linking 5-HT 2B activation with upregulation of ADAR2 are unknown, but we have previously shown that upregulation of cPLA 2 was dependent on metalloproteinase-mediated release of an epidermal growth factor (EGF) receptor agonist and subsequent stimulation of the EGF receptor by this agonist, leading to ERK 1/2 activation and entry of p-ERK 1/2 into the nucleus.…”

“…13 The 4 isoforms have been named ADAR2Sd and ADAR2Ld, which are produced by splicing in the deaminase domain, and ADAR2Sc and ADAR2Lc, which are produced by splicing in the 5′-end of the coding sequence. 14 The different splicing variants and combinations of variants result in the production of proteins with different deaminase activity. [13][14][15] The GluK2 receptor has been demonstrated in astrocytes in the rat hippocampus 16 and spinal cord, 17 and S100β-positive astrocytes obtained by fluorescence-activated cell sorting from dissociated rat brain cortical tissue show GluK2 gene expression that is about half as pronounced as in the corresponding neurons.…”

“…14 The different splicing variants and combinations of variants result in the production of proteins with different deaminase activity. [13][14][15] The GluK2 receptor has been demonstrated in astrocytes in the rat hippocampus 16 and spinal cord, 17 and S100β-positive astrocytes obtained by fluorescence-activated cell sorting from dissociated rat brain cortical tissue show GluK2 gene expression that is about half as pronounced as in the corresponding neurons. 18 It is unknown whether chronic treatment with fluoxetine affects editing of GluK2 in astrocytes, let alone what effect this has on kainate receptor function.…”

Effects of antidepressants on GluR2 Q/R site-RNA editing in modified HeLa cell line

“…In addition, mice chronically treated with fluoxetine also exhibited decreased 5-HT 2c R E site-editing in the brain (Gurevich et al, 2002). Although the expression level of ADAR2 mRNA is one determinant of the efficiency of GluR2 Q/R site-editing, it has been reported that editing extents of the various A-to-I editing sites in 5-HT 2c R mRNA correlated with the mRNA expression level of none of the members of ADAR families in cells from the rat hypothalamic tuberomamillary nucleus (Sergeeva et al, 2007). Taking our data and these 15 reports together, antidepressants might have modulatory effects on A-to-I RNA editing sites in various mRNAs by direct upregulation of ADAR2 mRNA or other mechanisms.…”