Elsevier

Gene

Volume 391, Issues 1–2, 15 April 2007, Pages 186-197
Gene

Editing of the serotonin 2C receptor pre-mRNA: Effects of the Morris Water Maze

https://doi.org/10.1016/j.gene.2006.12.023Get rights and content

Abstract

The pre-mRNA encoding the serotonin 2C receptor, HTR2C (official mouse gene symbol, Htr2c), is subject to adenosine deamination that produces inosine at five sites within the coding region. Combinations of this site-specific A-to-I editing can produce 32 different mRNA sequences encoding 24 different protein isoforms with differing biochemical and pharmacological properties. Studies in humans have reported abnormalities in patterns of HTR2C editing in psychiatric disorders, and studies in rodents show altered patterns of editing in response to drug treatments and stressful situations. To further explore the biological significance of editing of the Htr2c mRNA and its regulation, we have examined patterns of Htr2c editing in C57BL/6J mice after exposure to the hidden platform version of the Morris Water Maze, a test of spatial learning that, in mice, is also associated with stress. In brains of both swimming controls and mice trained to find the platform, subtle time dependent changes in editing patterns are seen as soon as 1 h after a probe trial and typically last less than 24 h. Changes in whole brain with cerebellum removed differ from those seen in isolated hippocampus and cortex. Unexpectedly, in hippocampi from subsets of mice, abnormally low levels of editing were seen that were not correlated with behavior or with editing levels in cortex. These data implicate responses to spatial learning and stress, in addition to stochastic processes, in the generation of subtle changes in editing patterns of Htr2c.

Introduction

The serotonin 2C receptor, Htr2c, is unique among the serotonin receptors in that the pre-mRNA is subject to deamination of adenosine to inosine (Burns et al., 1997). This editing occurs at five sites within a 15 nucleotide segment encoding the second intracellular loop (reviewed in Schmauss, 2003, Sanders-Bush et al., 2003). Because inosine is interpreted by the ribosome as guanosine, editing alters individual codons, most often also altering the encoded amino acid. Fig. 1a illustrates the repertoire of codon combinations that can result in a total of 24 different protein sequences. Within the protein sequence, the edited region starts two amino acids downstream from the conserved DRY motif that is believed to be involved in G-protein coupling (Ballesteros et al., 1998, Moro et al., 1993). Both mutation experiments and computational approaches predict that the sequence diversity created by editing will modulate protein function (Ballesteros et al., 1998, Moro et al., 1993, Visiers et al., 2001), as originally suggested by Burns et al. (1997). Indeed, in vitro studies have shown differences among several isoforms in biochemical and pharmacological properties that include affinity for serotonin, G-protein coupling, and responses to atypical antipsychotics (Niswender et al., 1999, Niswender et al., 2001, Herrick-Davis et al., 1999, Fitzgerald et al., 1999, Wang et al., 2000, Price and Sanders-Bush, 2000, Berg et al., 2001, Price et al., 2001, McGrew et al., 2004, Marion et al., 2004).

In vivo studies have shown that most of the 32 possible mRNA variants are produced in human and rodent brain (Burns et al., 1997, Niswender et al., 1999). In a recent work, we have detected 28 of 32 mRNA variants in mouse brain, encoding 20 of 24 possible protein isoforms (Du et al., 2006). However, there are both species-specific and brain region-specific differences in the proportions of the different variants, and only five or six variants typically are found at high frequency, with the remainder together accounting for only a few percent. Abnormalities in patterns of HTR2C editing have been reported in subsets of patients with schizophrenia and depression (Gurevich et al., 2002a, Niswender et al., 2001, Dracheva et al., 2003), and baseline differences have also been reported among different inbred mouse strains (Englander et al., 2005, Hackler et al., 2006, Du et al., 2006). Changes in editing patterns have been induced, in rodents, by exposure to tests of anxiety, including the forced swim test and learned helplessness, and by drugs, such as fluoxetine (Gurevich et al., 2002b, Iwamoto et al., 2005, Englander et al., 2005, Yang et al., 2004). Most of the changes in editing patterns induced by disease, genetic background, stress and pharmacological treatment are subtle and the corresponding diversity at the protein level has not yet been studied. Nevertheless, the cumulative data suggest that HTR2C pre-mRNA editing is biologically relevant.

Cavallaro et al. (2002) classed the Htr2c as one of a number of “learning and memory genes” because transcript levels, as measured by microarray analysis, changed with time in rats exposed to the learning phase of the Morris Water Maze compared with those exposed to swimming alone. This observation prompts the question: Do editing patterns change when transcript levels are dynamic and, if they do, how dynamic are the changes and are they relevant to learning?

Editing of HTR2C is effected by two adenosine deaminases that act on RNA, ADAR1 and ADAR2 (reviewed in Maas et al., 2003). The ADAR2 gene (official gene name ADARB1) maps to human chromosome 21 and therefore is a candidate gene for the cognitive and behavioral features of Down syndrome (Villard et al., 1997). If expression of ADAR2 is increased in Down syndrome, as predicted from gene dosage (trisomy of chromosome 21), it may alter patterns of HTR2C mRNA editing.

Based on these observations, we generated transgenic mice that carry an extra genomic copy of the human ADAR2 gene and examined editing of Htr2c at timed intervals after exposure to the Morris Water Maze. While results do not inform the role of ADAR2 in Down syndrome, they illustrate dynamic patterns of editing that differ between brain regions, and that are associated with the stress of swimming and spatial learning. Results also suggest that stochastic processes contribute to editing patterns.

Section snippets

Mouse brain tissue

Transgenic mice, lines Tg(Adarb1)14Dn (Tg14Dn) and Tg(Adarb1)15Dn (Tg15Dn), carry the human BAC RP11-581A12 (BacPac Resources) which contains the entire ADAR2 gene. Both lines are in a C57BL/6J background and were produced by the transgenic facility at The Jackson Laboratory (Bar Harbor, ME). For the first set of experiments, using whole brain minus cerebellum, transgenic mice and wild type littermates were used. For the second set of experiments, using hippocampus and cortex, wild type

Editing patterns in whole brain minus cerebellum

The first set of experiments was designed to assay the molecular and behavioral phenotypes of transgenic mice carrying a genomic copy of the human ADAR2 gene. For the molecular phenotype, two lines, Tg14Dn and Tg15Dn, were verified to express the human ADAR2 mRNA in addition to normal levels of mouse Adar2 (Supplementary Fig. 1). However, while levels and proportions of alternatively spliced variants of the mouse transcripts were normal, those for the human transcripts showed some

Discussion

The MWM was chosen as a behavioral task because previous work by Cavallaro et al. (2002) showed that it causes changes, that differ between the SC and WM mice, in the level of Htr2c mRNA during the 24 h after the last exposure to swimming. Those experiments measured Htr2c mRNA levels in rat hippocampus by microarrays, and while not dramatic (the greatest was a four-fold decrease), changes were significant. Such changes were not replicated here in mice by quantitative RT-PCR, but there are

Acknowledgements

This work was supported by grants from the Cullpepper Foundation and the National Institutes of Health (MH62638) to KG and by a contract from the National Institutes of Health (HD73265) to MTD. The authors thank Cecilia Schmidt for technical assistance.

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    Present address: Division of Clinical Pharmacology and Toxicology and Department of Psychiatry, University of Colorado at Denver and the Health Sciences Center, United States.

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