RNA editing of serotonin 2C receptor in human postmortem brains of major mental disorders

Neuroscience Letters 346 (2003) 169–172 www.elsevier.com/locate/neulet

RNA editing of serotonin 2C receptor in human postmortem brains of major mental disorders Kazuya Iwamoto, Tadafumi Kato* Laboratory for Molecular Dynamics of Mental Disorders, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-City, Saitama, 351-0198, Japan Received 27 February 2003; received in revised form 28 April 2003; accepted 6 May 2003

Abstract The importance of serotonin 2C receptor (HTR2C) in mental disorders has been implicated by studies of HTR2C-deficient mice and linkage and association studies. Recent studies have revealed that RNA editing of HTR2C is involved in mental disorders. Here we examined RNA editing efficiencies of site A and D of HTR2C in the prefrontal cortex samples of patients with bipolar disorder, schizophrenia, and major depression as well as control subjects by using primer extension combined with denaturing high performance liquid chromatography. Postmortem samples were donated by the Stanley Foundation Brain Collection. We could not find significant alterations of RNA editing efficiencies of these sites in patients. However, we found trends for increased RNA editing efficiencies of site D in depressive patients (P ¼ 0:08) and site A in suicide victims (P ¼ 0:07). These findings are in accordance with the previous findings, and suggest that altered RNA editing of HTR2C may have some significance in major depression and suicide. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Serotonin 2C receptor; Denaturing high performance liquid chromatography; Bipolar disorder; Major depression; Schizophrenia; Prefrontal cortex

Pharmacological studies revealed the involvement of serotonin 2C receptor (HTR2C) in fundamental brain functions including regulation of locomotion, appetite, sexual behavior, and anxiety [1]. HTR2C-deficient mice also exhibited abnormal control of feeding behavior and enhanced seizure susceptibility [15], indicating its regulatory role in complex behavior and development of the central nervous system. Accumulating evidence suggests that HTR2C is involved in various aspects of mental disorders. Its chromosomal locus, Xq24, has been reported to be one of the susceptibility loci of bipolar disorder by linkage studies [2,12]. Allelic variation of Cys23Ser of HTR2C was reported to be associated with depression and bipolar disorder [9], tardive dyskinesia in schizophrenia [13], and psychotic symptoms in late onset Alzheimer’s disease [6]. However, it has been reported that the Cys23Ser allelic variation does not alter the function of HTR2C [8]. In addition to variations of genomic sequence, posttranscriptional modification may also have some pathophysiological significance. HTR2C is functionally diversified by RNA editing, by which specific adenosine residues in * Corresponding author. Tel.: þ 81-48-467-6949; fax: þ81-48-467-6947. E-mail address: [email protected] (T. Kato).

transcripts are converted into inosine by adenosine deaminases that act on RNA (ADARs) [3]. Since inosine pairs with cytosine and is read as guanosine during translation, this modification can lead to amino acid substitution and altered function. To date, five adenosine residues (termed as sites A – E) in the second intracellular loop of HTR2C have been found to be edited into inosine, leading to the production of several isoforms [3,4,10]. Importantly, it was found that different isoforms exhibit different pharmacological responses, and the composition of each isoform varies among brain regions [3]. Altered RNA editing of HTR2C has been reported to be associated with mental disorders. Increased RNA editing in site A was found in patients with schizophrenia or depression who committed suicide [11], whereas decreased RNA editing was found in patients with schizophrenia [14]. Another study reported altered RNA editing in depressive patients who committed suicide [5], and showed that administration of an antidepressant, fluoxetine, to normal mice changed RNA editing efficiency of HTR2C [5]. These studies suggested that RNA editing may be involved in the pathophysiology of mental disorders and the mechanism of action of antidepressants. We previously developed a rapid and accurate method

0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00608-6

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K. Iwamoto, T. Kato / Neuroscience Letters 346 (2003) 169–172

for estimating the RNA editing efficiency by combining primer extension with denaturing high performance liquid chromatography (PE-DHPLC) [7]. This high throughput analysis enables examination of a large number of subjects. Here we investigated RNA editing efficiencies of site A and site D of HTR2C in postmortem brains of patients with major mental disorders. Postmortem prefrontal cortex (Brodmann’s Area 10) samples were donated by the Stanley Foundation Brain Collection. These were derived from patients with bipolar disorder, depression and schizophrenia as well as control subjects. Each group consisted of 15 subjects. Diagnoses had been made according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) criteria. Although they were well matched for age, gender, race, postmortem interval (PMI), pH, side of brain, and quality of mRNA, RT-PCR product of HTR2C could not obtained from some of these samples. A summary of demographic information of subjects used in this study is shown in Table 1. Detailed information of the original set of subjects was described elsewhere [16]. Total RNA was extracted from frozen tissue using TRIzol (Invitrogen, Groningen, The Netherlands) and it was purified with an RNAeasy column (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. One microgram of total RNA was used for cDNA synthesis by SuperScript II reverse transcriptase (Invitrogen) and oligo(dT) primers (Invitrogen). Amplification of the HTR2C was performed using primers 50 -TCTGGATTTCTTTAGATGTTTTA-30 (P1, the 50 end at nucleotide (nt) 386; the A in the initiation codon of ATG was designated as nucleotide þ 1) and 50 -GTCCCTCAGTCCAATCACAG-30 (P2, the 50 end at nt 588). Using 1% of the reaction mixture of PCR product as template, second round PCR was performed with primers P1 and 50 -TAGAAATTGCCCAAACAATA-30 (P3, the 50 end at nt 547). This additional PCR could eliminate undesirable amplification of alternative splicing forms of HTR2C. Primer extension reactions and DHPLC analysis were performed as reported previously [7] with minor modifications. In brief, primer extension reactions contained 50 ng of purified RT-PCR product, 50 mM of the appropriate Table 1 Summary of demographic information of subjects used in this study

C BP MD SZ

n

Age (years)

PMI (h)

Gendera Medicationb Suicidec

15 12 11 13

48.1 ^ 10.7 42.3 ^ 12.4 45.8 ^ 10.0 43.5 ^ 13.6

23.7 ^ 10.0 29.0 ^ 13.3 26.8 ^ 11.9 33.0 ^ 14.9

6F:9M 4F:8M 4F:7M 5F:8M

0M:15MF 10M:2MF 8M:3MF 11M:2MF

0S:15NS 7S:5NS 5S:6NS 4S:9NS

C, control; BP, bipolar disorder; MD, major depression; SZ, schizophrenia; PMI, postmortem interval. aFemale and male ratio. F, female; M, male. bMedication status. One, three, and none of the subjects were treated with fluoxetine in bipolar disorder, major depression, and schizophrenia, respectively. M, medicated subjects; MF, medication-free subjects. cCause of death: S, suicide; NS, non-suicide.

dNTPs and ddNTPs, 1 mM of primer, 1.25 units of ThermoSequenase (Amersham Bioscience, Uppsala, Sweden), and the reaction buffer provided by the manufacturer. For the site A analysis, dATP, ddTTP and ddGTP were included in the reaction buffer and 50 -CGCTGGATCGGTATGTAGC-30 (the 50 end at nt 446) was used as the extension primer. For the site D analysis, ddTTP and ddCTP were included and 50 -AATTGAACCGGCTATGCTCAA-30 (the 50 end at nt 499) was used for an extension primer. Reaction parameters were 94 8C for 2 min followed by 50 cycles (94 8C, 30 s; 55 8C, 30 s; 60 8C, 30 s). The denaturing HPLC was performed using a WAVE DNA fragment analysis system with the DNASep column (Transgenomic, Omaha, NE). The gradient was prepared by mixing 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. The column temperature was set at 80 8C. The eluted products were monitored at 260 nm by the UV detector. The RNA editing efficiency was calculated by comparing the area of peaks corresponding to edited and non-edited extension products. The RNA editing efficiency of each sample was determined from the results of two independent PE-DHPLC analyses. The general reproducibility and reliability of PEDHPLC were discussed in the previous report [7]. In summary, the RNA editing efficiency of HTR2C obtained by PE-DHPLC was reproducible (r ¼ 0:958), and highly correlated with that measured by the cloning and sequencing method (r ¼ 0:948). A Mann –Whitney U-test and Spearman’s correlation analysis were used for statistical analysis. P , 0:05 was defined as significant, whereas 0:05 , P , 0:10 was defined as being a trend. The RNA editing efficiencies of site A and site D in postmortem prefrontal cortex samples measured by the PEDHPLC method are shown in Fig. 1. There were no significant correlations between RNA editing efficiencies and age (site A, r ¼ 20:20, P ¼ 0:893; site D, r ¼ 20:050, P ¼ 0:739) or PMI (site A, r ¼ 0:155, P ¼ 0:294; site D, r ¼ 20:043, P ¼ 0:776) of samples. There were no significant alterations in RNA editing efficiencies of site A (bipolar disorder (BP), 74.8 ^ 14.9, n ¼ 11, P ¼ 0:885; major depression (MD), 71.4 ^ 10.9, n ¼ 11, P ¼ 0:140; schizophrenia (SZ), 72.1 ^ 13.3, n ¼ 13, P ¼ 0:479) and site D (BP, 58.8 ^ 4.5, n ¼ 10, P ¼ 0:648; MD, 65.6 ^ 10.5, n ¼ 10, P ¼ 0:085; SZ, 62.3 ^ 9.1, n ¼ 13, P ¼ 0:264) compared with control subjects (site A, 77.3 ^ 11.7, n ¼ 13; site D, 56.6 ^ 13.8, n ¼ 13). However, we found a trend for increased RNA editing efficiency of site D in depressive patients. Since fluoxetine administration in normal mice showed decreased RNA editing at site E and increased RNA editing at site D [5], RNA editing in major depressives may be influenced by the effects of medication. To examine the possible effect of fluoxetine, depressive patients were

K. Iwamoto, T. Kato / Neuroscience Letters 346 (2003) 169–172

Fig. 1. RNA editing efficiencies of HTR2C determined by PE-DHPLC in postmortem prefrontal cortex samples. (A) RNA editing efficiency of site A. (B) RNA editing efficiency of site D. Bars indicate mean RNA editing efficiencies. The RNA editing efficiency of each sample was determined in duplicate by the PE-DHPLC method. C, control; BP, bipolar disorder; MD, major depression; SZ, schizophrenia. The asterisk indicates 0:05 , P , 0:10.

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was no significant difference of RNA editing at site D between suicide victims and controls (Fig. 1B). This study revealed (1) increased RNA editing efficiency of site D in depressive patients, (2) no alteration of RNA editing in bipolar disorder, and (3) increased RNA editing efficiency of site A in suicide victims. The first finding of increased RNA editing efficiency of site D in depressive patients seemed not to be attributable to the effect of medication since drug-free patients also showed the increased RNA editing efficiency. This finding seems to be contradictory to the previous report that RNA editing of site D was decreased in postmortem brains in drug-free depressives [5]. These discrepancies may be due to the limited number of subjects without medication, and the difference of characteristics of samples. Secondly, this is the first study of RNA editing in postmortem brains of patients with bipolar disorder. The present study revealed that alteration of RNA editing in HTR2C dose not play an important role in the etiology of bipolar disorder. The third finding replicated the previously reported observation of increased RNA editing at site A in suicide victims [11]. Although the mental state at death, either mania or depression, is difficult to define in postmortem brain studies, suicide is unambiguously associated with a certain mental state before death. Our finding together with the previous report suggests a possible association of altered RNA editing efficiencies of site A and suicide. Altered serotonergic neurotransmission associated with RNA editing status might be associated with suicidal ideation or impulsiveness causing suicide. To confirm a possible relationship among depression, antidepressant treatment, and RNA editing of HTR2C avoiding postmortem changes, a further study using animal models of depression will be needed.

Acknowledgements divided into three groups: drug-free patients, patients treated with fluoxetine, and patients treated with other drugs. There were no significant alterations in RNA editing efficiencies of site A in drug-free patients (64.1 ^ 17.9, n ¼ 3, P ¼ 0:122) or patients treated with other drugs (77.8 ^ 3.7, n ¼ 5, P ¼ 0:961) compared with controls. We found a trend for decreased RNA editing in patients treated with fluoxetine (68.1 ^ 6.8, n ¼ 3, P ¼ 0:065) compared with control subjects. Statistical analysis also revealed a trend for an increased RNA editing efficiency of site D in drug-free patients (69.3 ^ 5.8, n ¼ 3, P ¼ 0:093) and no significant alterations in patients treated with fluoxetine (60.7 ^ 9.9, n ¼ 3, P ¼ 0:113) or other drugs (64.0 ^ 12.0, n ¼ 4, P ¼ 0:840) compared with controls. We next examined whether suicide alters the RNA editing efficiency of HTR2C. The RNA editing efficiency of site A showed an increased trend in suicide victims (74.4 ^ 15.1, n ¼ 16, P ¼ 0:07) compared with nonsuicide patients (71.3 ^ 10.8, n ¼ 19) (Fig. 1A). There

Postmortem brains were donated by the Stanley Foundation Brain Collection courtesy of Drs Michael B. Knable, E. Fuller Torrey, Maree J. Webster, and Robert H. Yolken. The authors thank Dr K. Ikeda for continuous support of our study. This work was supported by a Grant-in-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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