Influence of an Adenosine Deaminase Inhibitor, Erythro-9-(2-hydroxy-3-nonyl) Adenine Hydrochloride, on 5-HT2CR mRNA Editing in Primary Cultured Cortical Cells

Abstract

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...

Structural and functional effects of inosine modification in mRNA

“…To date, no FDA-approved ADAR editing modulatory drugs are available in the market. However, some published reports suggest the use of small molecule nucleoside analogs of adenosine as ADAR editing inhibitors ( Hang et al 2010 ; Zipeto et al 2016 ; Ding et al 2020 ; Ramírez-Moya et al 2020 ). For example, 8-azaadenosine (8-aza-A) has been used as an inhibitory tool compound to evaluate ADAR function in chronic myeloid leukemia and thyroid cancer ( Zipeto et al 2016 ; Ramírez-Moya et al 2020 ).…”

ADAR Family Proteins: A Structural Review

“…Known ADAR inhibitors include O-phenanthroline, a zinc chelator, and N-ethylmaleimide (NEM), an alkylating reagent, both inhibitors of hRED1 [156], ZYS-1, a new inhibitor of ADAR1 that shows promise as a therapeutic in prostate cancer [157], Rebecsinib, an inhibitor of splicing mediated ADAR1 activation [158], and 8-azanebularine which inhibits ADAR2 [159] (Figure 8). Also reported to inhibit ADAR1 is erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA) and compounds alendronate, etidronate, and zoledronate which inhibit the Zα domain of ADAR1p150 (Figure 8) [160,161]. Other studies have suggested new small molecule inhibitors against the CDD IHP binding site of ADAR2 [61], the RNA binding loop of ADAR2 [162], and the Zα domain of ADAR1 [161].…”

RNA modification: mechanisms and therapeutic targets

“…Primary cultured cortical cells treated with the ADAR inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA) significantly reduced the editing efficacy of 5-HT2CR mRNA in a dose-dependent manner (Fig. 7 d) [ 480 ].…”