Laboratory for Molecular Dynamics of Mental Disorders, Brain Science Institute, Saitama, Japan

Correspondence to: T Kato, MD, PhD, Laboratory for Molecular Dynamics of Mental Disorders, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama 351-0198, Japan. Tel: +81 48 467 6949; Fax: +81 48 467 6947; E-mail: kato@brain.riken.go.jp

Serotonin 2C receptor (5-HT_2CR) transcripts undergo RNA editing, generating pharmacologically different isoforms. To test whether the RNA editing of 5-HT_2CR is regulated by serotonergic activity, effects of cocaine or reserpine administration in the rat cerebral cortex were examined. Although these drugs have been known to alter serotonin metabolism, no alterations in the RNA editing were found by the sequencing analysis. Towards high throughput analysis, we developed a non-RI method that allows accurate and rapid estimation of RNA editing by combining the primer extension with denaturing high-performance liquid chromatography (DHPLC). By using this, RNA editing efficiencies of 5-HT_2CR in the midbrain and hippocampus as well as the cerebral cortex were examined, and no alterations were found among these regions. Our method using DHPLC is applicable to examine association of RNA editing with various diseases.

The Pharmacogenomics Journal (2002) 2, 335-340 doi:10.1038/sj.tpj.6500130

DHPLC; serotonin 2C receptor; schizophrenia; suicide; depression

INTRODUCTION

Several neurotransmitter receptors undergo A-to-I RNA-editing, by which specific adenosine residues in transcripts are converted into inosine by adenosine deaminases.^1,2,3 Since inosine pair with cytosine and are read as guanosine during translation, this modification can lead to amino acid substitution with altered function. In the case of serotonin (5-HT; 5-hydroxytryptamine) 2C receptor (5-HT_2CR), five adenosine residues (termed site A-to-E) in the second intracellular loop are converted into inosine, resulting in the production of several isoforms.^4,5,6 Importantly, some of them have been shown to possess pharmacological differences, and composition of each 5-HT_2CR isoform is varied among the brain regions.⁴ These suggest that RNA editing plays an important role in the regulation of functional properties of 5-HT_2CR. However, it is not known to what extent RNA editing of 5-HT_2CR physiologically fluctuates.

Several lines of evidence suggest association of 5-HT_2CR with mental disorders. Pharmacological studies revealed the association of 5-HT_2CR with depression⁷ and anxiety.⁸ Cys23Ser polymorphism of 5-HT_2CR is associated with bipolar disorder,⁹ tardive dyskinesia in schizophrenia,¹⁰ and psychotic symptoms in late onset Altzheimer's disease.¹¹ Recently, aberration of RNA editing of 5-HT_2CR has been reported to be associated with suicide and schizophrenia. Elevation of RNA editing in site A was found in the frontal cortex of patients with schizophrenia or depression who have committed suicide,¹² whereas reduction of RNA editing of 5-HT_2CR was found in the frontal cortex of patients with schizophrenia.¹³ Although more studies will be needed to clarify these contradictory findings and evaluate the association of RNA editing of 5-HT_2CR with mental disorders,² assessment of RNA editing efficiency requires exhausitive cloning of RT-PCR products and sequencing, which hampers a replication study in a large number of patients.

Here we tested whether enhancement (cocaine) or reduction (reserpine) of serotonergic neurotransmission affects RNA editing of rat 5-HT_2CR. Cocaine strongly binds to 5-HT uptake sites^14,15 and inhibits the reuptake of 5-HT,¹⁶ and behavioral sensitization to cocaine shares some characteristics with psychoses. Reserpine is a drug that depletes monoamines and has ever been used for one of the classical animal models of depression.^17,18

As well as the extensive analysis of RNA editing of 5-HT_2CR by cloning of RT-PCR products and sequencing, we developed a non-radioactive isotope method (non-RI method) that allows accurate and rapid evaluation of RNA editing, which enables the high throughput estimation. This method uses the primer extension method combined with the denaturing high-performance liquid chromatography (DHPLC).¹⁹ After the RT-PCR of the second intracellular loop region containing the editing sites, primer extension was performed with appropriate dNTPs and ddNTPs. Reaction mixtures were then separated and quantified by DHPLC. We confirmed the reliability of this method by comparing the results obtained by DHPLC with those obtained by the sequencing method, and applied this method to estimate the RNA editing efficiencies in the various brain regions of drug-administrated rats.

RESULTS

After the decapitation of drug-administrated rats, cerebral cortex, midbrain and hippocampus were dissected. Total RNA was isolated from each brain region. RT-PCR was performed to amplify the second intracellular loop region containing the five editing sites. RT-PCR products amplified from cerebral cortex cDNA were subjected to cloning and sequencing analysis. Of the 24 possible isoforms (summarized in Figure 1), 15 isoforms could be identified in the cerebral cortex through the extensive sequencing analysis (Figure 2a). All of them were previously identified isoforms. We could not find marked alterations in the composition of each isoform at the amino acid level (Figure 2a) or at the cDNA level (data not shown). Next, we compared RNA editing efficiencies of the five editing sites (Figure 2b) and the total number of the edited sites (Figure 2c). RNA editing efficiencies of control rats were consistent with those previously reported.⁴ Although statistical analysis could not be applied because of the small number of rats in each group, we could not find a striking effect of cocaine or reserpine administration on RNA editing in the rat cerebral cortex.

Although estimation of RNA editing efficiencies by cloning and sequencing method provides complete data including editing efficiencies of all sites and composition of isoforms, this exhaustive method is clearly unable to process large number of samples. Alternative approach often used is the primer extension method with radiolabeled ddNTPs. This method is also time-consuming and inadequate for rapid estimation. Towards high throughput estimation, we developed a non-RI method to estimate the RNA editing efficiency by primer extension method combined with DHPLC (PE-DHPLC). Principle of this method was summarized in Figure 3. Extension of primer was terminated by the incorporation of ddNTPs (Figure 3a) and this reaction mixture was separated and quantified by DHPLC. An example of typical chromatogram of site A analysis was shown in Figure 3b. Since retention time of the oligonucleotide is dependent on the base composition as well as the length of oligonucleotide, both of which collectively determines total hydrophobicity of the oligonucleotide, same length of oligonucleotides differing at 3' end can be separated. In our case, we successfully separated and quantified the same length of products generated by the site D analysis by DHPLC (Figure 4). Results obtained by PE-DHPLC were highly reproducible (Pearson's correlation coefficient between two measurements; r=0.958, P<0.001, n=54), and editing efficiency estimated by PE-DHPLC was significantly correlated with that measured by cloning and sequencing method (r=0.948, P<0.001, n=16; Figure 5).

We applied this method to estimate RNA editing efficiencies of site A and site D in the midbrain and hippocampus as well as the cerebral cortex (Figure 6). Through the studies of the mice deficient in the RNA editing enzymes, it has been suggested that site A is edited mainly by adenosine deaminase that act on RNA 1 (ADAR1)²⁰ and site D is edited exclusively by ADAR2.²¹ Thus, no significant alterations in editing efficiencies of sites A and D may suggest maintained activities of these two ADARs.

DISCUSSION

Since both cocaine and reserpine alter the 5-HT metabolism, we assumed that administration of these drugs might have some effects on RNA editiNg of 5-HT_2CR if it is physiologically regulated by serotonergic activity. In addition, some inconsistency, that is enhanced sensitivity to 5-HT₂R agonists^22,23 without the changes of 5-HT₂R densities,^24,25 has been reported in the rats chronically administrated with cocaine. This inconsistency has been attributed to the changes of the functional properties of 5-HT₂R, and thus alterations in RNA editing efficiencies or pattern of 5-HT_2CR might be involved. However, the present study demonstrated that administration of these drugs did not influence the RNA editing of rat 5-HT_2CR. Comparison of composition of isoforms, RNA editing efficiencies at each site, and the total number of the edited sites revealed that there were no marked aberrations in RNA editing of rat 5-HT_2CR in the cerebral cortex (Figure 2). In the midbrain and hippocampus as well as the cortex, we examined RNA editing efficiencies of sites A and D by PE-DHPLC (Figure 6). There were also no aberrations in the editing efficiencies in these sites. Our results indicate that the regulatory system of RNA editing is not perturbed by these drugs and relatively stable against alteration of serotonergic activity. Thus, inconsistency found in the rats chronically administrated with cocaine could be explained by other mechanisms. Recently, alternative splicing variants of 5-HT_2CR were reported.^26,27,28 Since this results in the generation of non-functional truncated forms of 5-HT_2CR, other posttranscriptional alterations such as changes in the composition of the variants might be involved.

In the frontal cortex of patients with schizophrenia, reduction of RNA editing was found.¹³ This reduction resulted in significant increase of non-edited isoform, 5-HT_2C-INI, and decrease of common isoforms, 5-HT_2C-VSV and 5-HT_2C-VNV. Since the non-edited isoform, 5-HT_2C-INI, has been shown to keep the high affinity state of G-protein coupling,⁶ these changes should result in enhanced activity of 5-HT_2CR in schizophrenia.¹³ Contradictory to this finding, other groups reported that no aberration of RNA editing efficiencies was found in patients with schizophrenia and depression, but elevation of RNA editing efficiency of site A was found in patients who have committed suicide.¹² To date, the cause of discrepancy between these findings is unclear. Although cocaine administration is used as an animal model of schizophrenia, it can represent only the positive symptoms of schizophrenia.^29,30 Therefore, our finding may suggest that positive symptoms of schizophrenia are not caused by aberration of RNA editing of 5-HT_XR in rats. It is interesting to examine the effect of drugs such as phencyclidine that evoke both positive and negative symptoms of schizophrenia.^29,30,31

To date, RNA editing was detected in a small number of transcripts. In mammalian, A-to-I change was detected in 5-HT_2CR, five glutamate receptors (GluR-B, GluR-C, GluR-D, GluR-5, and GluR-6) and ADAR2 itself.^1,2,3 This is clearly underestimation of numbers of the edited transcripts since inosine was reported to be appeared at a frequency of 1 in 17 000 nucleotides in the brain.³² There would be other unidentified transcripts that undergo RNA editing. In addition, C-to-U change was detected in apolipoprotein B^33,34 and neurofibromatosis type1 (Nf1) transcripts.³⁵ A growing body of evidence indicates that the aberration of RNA editing was involved in various diseases. Aberration of RNA editing of GluR-B was reported in patients with Alzheimer disease and Huntington disease as well as patients with schizophrenia,³⁶ and aberration of RNA editing of GluR-B³⁷ and Nf1³⁸ was reported in tumor cells. Considering the importance of testing the association of RNA editing efficiencies with mental disorders in various transcripts and in various types of tissues, methods that allow high throughput estimation of editing efficiency must be needed. PE-DHPLC described here is an appropriate method for such purpose. This non-RI method requires only about 10 min per sample. Results obtained by this method showed high reproducibility and good agreement with those obtained by the standard method (Figure 5). By using this method, association of RNA editing efficiency with mental disorders can be further explored in animal models as well as postmortem brain samples.

In conclusion, our data suggest that RNA editing of 5-HT_2CR is not altered by drug administration in various brain regions, and indicate that PE-DHPLC is an accurate and rapid method for estimating RNA editing efficiency.

MATERIALS AND METHODS

Animals and Drug Administration

Male Sprague-Dawley rats (Japan SLC) weighing 200 g were housed in a constant temperature (23±2°C) and light-controlled conditions (12 h light-dark cycle, lights on 08:00 h) with free access to food and water. Rats were handled for 7 days before the experiments. Rats received cocaine (15 mg/kg, intraperitoneally; i.p., n=4) or saline (0.9% NaCl, 1 ml/kg, i.p., n=3) on seven consecutive days. For the reserpine treatment, rats received reserpine (1 mg/kg, i.p., n=2) on two consecutive days and subsequently saline on five consecutive days. Rats were killed by decapitation 1 day after the last administration. After the removal of the brain, cerebral cortex, midbrain, and hippocampus were dissected. They were submerged in the RNA later (Ambion) solution and stored at -20°C until use.

Total RNA Isolation and RT-PCR

Total RNA was extracted using the TRIzol (GIBCO BRL) reagent as described by the manufacturers. Residual genomic DNA was digested with RNase-free DNase I (TAKARA), and integrity of total RNA was tested by denaturing-agarose gel electrophoresis. One microgram of total RNA was used for cDNA synthesis by SuperScript II reverse transcriptase (Invitrogen) and oligo(dT). For amplification of the second intracellular loop of rat 5-HT_2CR, two rounds of PCR were performed. Primers used in the first round PCR were P1 (5'-TGGATTTCACTAGATGTGCT-3') and P2 (5'-GTCCCT-CAGTCCAATCACAG-3'). These primers were set on two neighboring exons to discriminate the RT-PCR products generated from residual genomic DNA. Those used in the second PCR were P1 and P3(5'-TTGATATTGCCCAAACGATG-3'). First round PCR parameters were 94°C for 10 min, 30 cycles (94°C, 30 s; 59°C, 30 s; 74°C, 30 s), followed by 74°C for 7 min. One percent of the first round PCR product was reamplified in a second round PCR reaction. Second round PCR parameters were 94°C for 10 min, 30 cycles (94°C, 30 s; 61°C, 30 s; 74°C, 30 s), followed by 74°C for 7 min. A portion of RT-PCR products amplified from cerebral cortex cDNA was TA-cloned into the pCR2.1 vector (Invitrogen), and the reaction mixture was used for transformation of E. coli as described by the manufacturers. Sequencing analysis was performed in at least 50 cDNA clones derived from single bacterial colonies per sample.

Primer Extension Reactions

RT-PCR products were purified by the ExoSAP-IT kit (Amersham Pharmacia Biotech) as described by the manufacturers. Primer extension reactions contained 50 ng of RT-PCR product, 50 M of the appropriate dNTPs and ddNTPs, 1 M of primer, 1.25 units of ThermoSequenase (Amersham Pharmacia Biotech), and the buffer provided by the manufacturer. For site A analysis, dATP, ddTTP and ddGTP were used and PA (5'-CGCTGGACCGGTATGTAGC-3') was used for extension primer. For site D analysis, ddTTP and ddCTP were used and PD (5'-AATTGAACCGGCTATGCTCAA-3') was used for extension primer. To avoid the possible detection of multiple peaks in DHPLC analysis, primers used for primer extension reaction in this experiment should not contain other editing sites than a target site. Since five editing sites of 5-HT_2CR are closely located as shown in Figure 1, we designed PA in sense, and PD in anti-sense orientation. Reaction parameters were 94°C for 2 min followed by 50 cycles (94°C, 30 s; 50°C, 30 s; 60°C, 30 s). Four cDNA clones derived from bacterial single colonies were used as standards for the calibration in DHPLC experiments. These edited and non-edited cDNA in either site A or site D were identified by sequencing analysis.

DHPLC Analysis

The denaturing HPLC was performed using a WAVE DNA fragment analysis system with the DNASep column (Transgenomic). Gradient was prepared by mixing the buffer A (0.1 M triethylammonium acetate buffer (TEAA), pH 7.0) and buffer B (25% acetonitrile in 0.1 M TEAA). Primer extension products were eluted using a linear gradient from 18%B to 38%B at a flow rate of 0.9 ml/min for 7 min. Column temperature was set at 80°C. The eluted products were monitored at 260 nm by the UV detector. After each elution, column was washed and equilibrated with the gradient of 90%B and 18%B for 1 min, respectively. The peaks were identified using the standard cDNA templates described above. RNA editing efficiency was calculated by comparing the area of peak corresponding to edited and non-edited extension products. RNA editing efficiency of each sample was determined in duplicate.

Statistical Analysis

Pearson's correlation coefficients were performed using SPSS software (SPSS Japan Co. Ltd.).

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Figure 1 Amino acid changes associated with RNA editing of the 5-HT_2CR.

Figure 2 RNA editing efficiencies of 5-HT_2CR in the cerebral cortex of rats administrated with cocaine (n=3), reserpine (n=2), and saline (n=3). 5-HT_2CR cDNA derived from single bacterial colonies were sequenced in at least 50 clones per sample. The sum of the number of sequenced clones was 461: (a) Composition of each isoform (%), (b) RNA editing efficiencies in each site, (c) total number of the edited sites. Values are the mean of the results, and error bars in cocaine and saline administration indicate standard deviation of the means.

Figure 3 Schematic representation of the method for estimating the RNA editing efficiency of site A of 5-HT_2CR by PE-DHPLC. (a) Primer extension of site A analysis. Extension of the primer is terminated by the incorporation of ddGTP or ddTTP, resulting in two products containing either two or three nucleotides extending primer. (b) A typical chromatogram obtained by the site A analysis. The peak identities were predetermined by profiling the control reactions using the cDNA derived from single bacterial colonies as described in the materials and methods section.

Figure 4 Schematic representation of the method for estimating the RNA editing efficiency of site D of 5-HT_2CR by PE-DHPLC. (a) Primer extension of site D analysis. Extension of the primer is terminated by the incorporation of ddTTP or ddCTP. (b) A typical chromatogram obtained by the site D analysis. Primer extension generates two 22-mer products, both of which can be separated.

Figure 5 Comparison of sequencing method and PE-DHPLC. RNA editing efficiencies of site A (closed square) and site D (open square) are plotted.

Figure 6 RNA editing efficiencies of 5-HT_2CR of site A (a) and site D (b) determined by PE-DHPLC in the various rat brain regions. Each sample was determined in duplicate by DHPLC: n=3 (saline), 4 (cocaine), and 2 (reserpine). Values are the mean of the editing efficiencies, and bars in cocaine and saline administration indicate standard deviation of the means.