No abstract

Serotonin 2C receptor (5-HT2CR) mRNA has been reported to receive editing at 5 nucleotide positions (named sites A-E) which are located inconsecutively on the nucleotide sequence encoding the 2nd intracellular loop of the receptor protein. To clarify the physiological role of 5-HT2CR mRNA editing, we investigated developmental changes in editing frequencies at sites A-E in the rat cerebral cortex and primary cultured cortical neurons. The editing at sites A and B increased in parallel with the rat brain development and reached a plateau of 80-100% frequency at postnatal days 1-3. Although editing frequency at site C was low compared to those detected at other sites except site E during a developmental period, it reached the maximal value of 30% during a first 7-d period after birth and then decreased gradually to the negligible level at PN49. Site D exhibited almost constant susceptibility (about 60%) to editing, while no editing at site E was occurred during rat brain development. Similar changes during development in editing frequencies at these sites were observed in primary cultured cortical cells during the cultivation period. These findings indicated that editing sites A-D on 5-HT2CR mRNA have different susceptibility and that the frequencies at these sites are not always constant during development.

The adenosine to inosine RNA editing process mediated by adenosine deaminase acting on RNA (ADARs) has been reported to play critical roles in the functional development of the central nervous system by modulating the functions of neurotransmitter receptors. Serotonin 2C receptor (5-HT2CR) mRNA undergoes editing by these ADARs at 5 nucleotide positions (sites A-E) located in the sequence encoding the second intracellular loop of 5-HT2CR. This editing allows for the generation of 32 mRNA variants and 24 protein isoforms of the receptor differing in G-protein coupling efficiency.1) The involvement of 5-HT2CR mRNA editing in psychiatric disorders has been hypothesized based on etiological and pharmacological studies. Altered editing of 5-HT2CR mRNA was detected in the postmortem brains of patients with major depression, schizophrenia and bipolar disorder.2-4) Yang et al. 5) found that 5-HT2CR mRNA editing frequency in human glioblastoma cell lines was altered by treatment with interferon which causes depression as an adverse effect. These findings also raised the possibility that compounds with the ability to inhibit/stimulate the editing of 5-HT2CR mRNA are useful for clarifying the role of 5-HT2CR mRNA editing in psychiatric disorders.Our previous studies have shown that the editing efficacy of 5-HT2CR mRNA and ADAR-2 pre-mRNA and the level of ADAR-2 pre-mRNA are altered during the development of the brain in rats.6,7) These results suggested that 5-HT2CR mRNA editing and these enzymes play a role in the brain's maturation. To find a pharmacological tool with which to investigate the roles of 5-HT2CR mRNA editing, we examined the effect of erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA), a well-known inhibitor of adenosine deaminase (ADA) which breaks extracellular adenosine, 8) on the editing of 5-HT2CR mRNA at sites A-D in cultured cortical cells. MATERIALS AND METHODSCultured cortical cells prepared from E20 embryos as described previously 6,7) were used for the experiments. The cells were exposed to vehicle (control) or EHNA included in culture media at concentrations of 30 and 60 mM for 6 d.After a 6-d cultivation period, total RNA was extracted, and RT-PCR was carried out as described previously.6,7) The specific primers for target genes to identify the bases at editing sites were: 5-HT2CR mRNA (NM_008312); 5Ј-atg tcc cta gcc att gct gat atg ctg gtg-3Ј (sense), 5Ј-atg cca cga agg acc cga tga gaa cga agt-3Ј (antisense), and Texas Red-labeled: 5Ј-ata ttt gtg ccc cgt cgt ga; ADAR-2 pre-mRNA (NW_047598), 5Ј-atg tgc tgg tga act cac ag-3Ј (sense), 5Ј-gtg gtg ccc aga aag agt gg-3Ј (antisense) and Texas Red-labeled: 5Ј-taa gta gga gag gca ata ggt ccg-3Ј; and GluR2 (NM_017261), 5Ј-tat atg agg agt gca gag cc-3Ј (sense), 5Ј-ata ctc cag caa cgt tgc tc-3Ј (antisense) and Texas Red-labeled: 5Ј-ctg tgt ttg tga gga cta cc-3Ј. Based on pilot studies where we ran specific primers for target genes, the proper cycle number for the amplification of each target gene was chosen (5-HT2CR mRNA including the editing sit...