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deletion polymorphism in the dopamine D2 receptor gene on the dopamine system in the striatum in patients with schizophrenia
Psychiatry Research 264 (2018) 116–118
Contents lists available at ScienceDirect
Psychiatry Research journal homepage: www.elsevier.com/locate/psychres
Short communication
Effects of the −141C insertion/deletion polymorphism in the dopamine D2 receptor gene on the dopamine system in the striatum in patients with schizophrenia
T
⁎
Junya Matsumotoa, ,1, Atsuko Nagaokaa,1, Yasuto Kuniia,b, Itaru Miuraa, Mizuki Hinoa, Shin-ichi Niwab, Hiroyuki Nawac, Hitoshi Takahashid, Akiyoshi Kakitad, Hirooki Yabea a
Department of Neuropsychiatry, Fukushima Medical University School of Medicine, 1 Hikarigaoka, Fukushima City, Fukushima 960-1295, Japan Department of Psychiatry, Aizu Medical Center, Fukushima Medical University, 21-2 Maeda, Tanisawa, Kawahigashimachi, Aizuwakamatsu City, Fukushima 969-3492, Japan c Department of Molecular Neurobiology, Brain Research Institute, Niigata University, 1-757 Asahimachi-dori, Niigata, Niigata 951-8585, Japan d Department of Pathology, Brain Research Institute, Niigata University, 1-757 Asahimachi-dori, Niigata, Niigata 951-8585, Japan b
A B S T R A C T
The relationships between −141C insertion/deletion (Ins/Del) polymorphisms in the dopamine D2 receptor gene and the two dopamine system integrators, i.e., dopamine- and cAMP-regulated phosphoprotein of molecular weight 32 kDa (DARPP-32) and calcineurin (CaN), are still unclear. In this study, we assessed the effect of this polymorphism on DARPP-32 and CaN protein expression in the postmortem striatum of patients with schizophrenia and control individuals. The expression levels of truncated DARPP and CaN were lower in Del allele carriers. These findings provide important insights into the mechanism by which this genotype could result in a poor response to antipsychotic drugs.
1. Introduction Attenuation of the dopamine system is important for the treatment of schizophrenia, since all clinically used antipsychotics are dopamine D2 receptor (DRD2) antagonists. The −141C insertion/deletion (Ins/ Del) polymorphism of DRD2 (rs1799732) is a deletion and insertion of cytosine at position −141, which is located in the 5′ promoter region of the DRD2 gene and is associated with schizophrenia (Arinami et al., 1997). Del allele carriers are associated with poor antipsychotic drug response in schizophrenia (Zhang et al., 2010). Patients who are carriers of the Del allele and noncarriers of the A1 allele in Taq1A showed treatment resistance (Kondo et al., 2003). The glutamate system is also related to the pathophysiology of schizophrenia. Since dopamine- and cAMP-regulated phosphoprotein of molecular weight 32 kDa (DARPP-32) and calcineurin (CaN) are the primary regulatory molecules for these two systems, they are thought to play central roles in the etiology of schizophrenia. DARPP-32 is expressed either as full-length (FL-DARPP) or as truncated (t-DARPP); tDARPP lacks the N-terminal region containing a phosphorylation site at Thr34 that is responsible for inhibitory regulation of protein phosphatase 1. Thus, t-DARPP cannot regulate the physiological activities of ⁎
1
several key proteins by inhibiting protein phosphatase 1. In this study, we analyzed the association of DARPP-32 and CaN expression with DRD2 gene polymorphism (−141C Ins/Del) in postmortem striatum samples from patients with schizophrenia. 2. Methods Postmortem brain samples from 12 patients with schizophrenia were obtained from the Fukushima Brain Bank, Fukushima Medical University, and those from 12 non-psychiatric control individuals were obtained from the Brain Research Institute, Niigata University. Each patient fulfilled the diagnostic criteria of the Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV). All 24 participants were Japanese. The average age was 58 years among control subjects (6 men and 6 women), and 68 years in the patient group (8 men and 4 women). Genotyping of −141C Ins/Del in DRD2 gene was performed using the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method, as previously described (Miura et al., 2015). FL-DARPP, t-DARPP, and CaN expression levels in the striatum (putamen and caudate) of postmortem tissue samples had been quantified
Corresponding author. E-mail address: [email protected] (J. Matsumoto). These authors contributed equally to this work.
https://doi.org/10.1016/j.psychres.2018.03.029 Received 7 February 2017; Received in revised form 31 January 2018; Accepted 12 March 2018 Available online 29 March 2018 0165-1781/ © 2018 Elsevier B.V. All rights reserved.
Psychiatry Research 264 (2018) 116–118
J. Matsumoto et al.
had decreased expression of t-DARPP while CaN expression tended to decrease in the striatum. Recent studies have indicated an association between −141C Ins/ Del polymorphism and antipsychotic drug response; Del allele carriers show poor response to antipsychotic drugs (Zhang et al., 2010), although mechanism remains unclear. Noncarriers of the A1 allele and carriers of the Del allele were found to show poor responses to antipsychotic drugs (Kondo et al., 2003); therefore, A1 allele carriers may show good treatment responses. In our previous study, we reported that the expression of t-DARRP was elevated in A1 allele carriers (Kunii et al., 2014). Thus, these findings suggested that increased expression of t-DARPP might lead to increased response to antipsychotic drugs, whereas decreased expression of tDARRP might reduce the response to antipsychotic drugs. There are several limitations to the present study. First, there is no data regarding the DRD2 density in the striatum. In future studies, we need to clarify the relationship between DARPP and CaN by analyzing the DRD2 density simultaneously. Second, we could not consider these postmortem samples’ drug responses to antipsychotic drug treatment of the patients directly, because we did not have enough information about drug response data. Third, the sample size was small. Public databases such as the braincloud or the ExSNP cannot solve this problem, because they did not contain the 141C Ins/Del (rs1799732) data though this polymorphism is quite important in the treatment by antipsychotics. Further study with large sample size is needed to validate our data. Finally, we should consider the possible effect of antipsychotics on levels of protein expression (Rushlow et al., 2005). Further study with postmortem brain sample from patients with schizophrenia who are drug naïve is needed. In spite of these limitations, we believe that this report is important, because this report is the first to focus on this clinically significant polymorphism, with expression data on DARPP and CaN. In this study, we showed that t-DARPP expression was decreased and the expression of CaN tended to decrease in the striatum of patients with schizophrenia harboring the Del allele. Although this study was relatively small and provided only a preliminary analysis, these results may reflect the potential molecular mechanisms of non-response to antipsychotics in patients with schizophrenia who are Del allele carriers. Further studies with larger cohorts are needed to confirm these findings.
by immunoblotting in our previous study (Kunii et al., 2014). Micro BCA kits (Pierce Chemical, Rockland, IL) were used to determine the protein content in each supernatant. For each tissue sample, 20 µg of protein was subjected to 10%-polyacrylamide gel electrophoresis in sodium dodecyl sulfate and then transferred to nitrocellulose membranes (Schleicher and Schull, Dassel, Germany). Protein expression levels in patients with schizophrenia did not correlate with the daily dosage of antipsychotics. The average daily dosage of antipsychotics, prescribed during the 3 months immediately preceding death, was 323 mg. Additionally, none of the individuals had any record of substance abuse, except for some who smoked tobacco. Correlation between the protein expression levels and −141C Ins/ Del genotype in the DRD2 gene was analyzed by analysis of covariance (ANCOVA) using Statistica Ver. 10.0 (Statsoft Inc., Tulsa, OK), and differences with p < 0.05 were considered significant. The genotype was set as an independent variable, and sex, age at death, and postmortem interval (PMI) were set as covariates. Pearson's chi-squared test was used to examine deviations from the Hardy–Weinberg equilibrium. However, post hoc correction was not carried out. This study was approved by the Ethics Committees of Fukushima Medical University and Niigata University and was performed in accordance with the Declaration of Helsinki. 3. Results We determined the −141C Ins/Del genotype (Ins/Ins, Ins/Del, or Del/Del) in all participants. Among the 24 participants, 18 (75%) were homozygous for the Ins allele, five (21%) were heterozygous for the Ins/Del alleles, and one (4%) was homozygous for the Del allele. The allele distribution was in Hardy–Weinberg equilibrium (χ2 = 0.64, df = 1, P > 0.05). The expression of t-DARPP in the putamen of postmortem brains with schizophrenia correlated with the −141C Ins/Del genotype; specifically, the Del allele was associated with significantly reduced t-DARPP expression (ANCOVA: F [1, 7] = 6.1889, P = 0.042; Fig. 1a). CaN expression in the caudate of postmortem brains with schizophrenia also correlated with −141C Ins/Del; the Del allele tended to reduce CaN expression (ANCOVA: F [1, 7] = 5.5660, P = 0.050; Fig. 1b). 4. Discussion In this study, we found that Del allele carriers with schizophrenia
Fig. 1. Effect of −141C genotypes on protein expression. Mean expression of t-DARPP in the putamen (a) or CaN in the caudate (b) of patients with schizophrenia. tDARPP, truncated DARPP-32; CaN, calcineurin. 117
Psychiatry Research 264 (2018) 116–118
J. Matsumoto et al.
Acknowledgments
dopamine D2 receptor gene polymorphisms as a possible predictor of treatment-resistance to dopamine antagonists in schizophrenic patients. Prog. Neuropsychopharmacol. Biol. Psychiatry 27, 921–926. Kunii, Y., Miura, I., Matsumoto, J., Hino, M., Wada, A., Niwa, S.I., Nawa, H., Sakai, M., Someya, T., Takahashi, H., Kakita, A., Yabe, H., 2014. Elevated postmortem striatal tDARPP expression in schizophrenia and associations with DRD2/ANKK1 polymorphism. Prog. Neuropsychopharmacol. Biol. Psychiatry 53C, 123–128. Miura, I., Kanno-Nozaki, K., Hino, M., Horikoshi, S., Ota, T., Mashiko, H., Niwa, S., Yabe, H., 2015. Influence of −141C Ins/Del polymorphism in DRD2 gene on clinical symptoms and plasma homovanillic acid levels in the treatment of schizophrenia with aripiprazole. J. Clin. Psychopharmacol. 35, 333–334. Rushlow, W.J., Seah, Y.H., Belliveau, D.J., Rajakumar, N., 2005. Changes in calcineurin expression induced in the rat brain by the administration of antipsychotics. J. Neurochem. 94, 587–596. Zhang, J.P., Lencz, T., Malhotra, A.K., 2010. D2 receptor genetic variation and clinical response to antipsychotic drug treatment: a meta-analysis. Am. J. Psychiatry 167, 763–772.
This work was supported by JSPS KAKENHIGrant Number JP15K19739, JP16H06277, the Collaborative Research Project (2016–2809) of the Brain Research Institute, Niigata University, the Strategic Research Program for Brain Science from Japan Agency for Medical Research and Development, AMED. References Arinami, T., Gao, M., Hamaguchi, H., Toru, M., 1997. A functional polymorphism in the promoter region of the dopamine D2 receptor gene is associated with schizophrenia. Hum. Mol. Gen. 6, 577–582. Kondo, T., Mihara, K., Suzuki, A., Yasui-Furukori, N., Kaneko, S., 2003. Combination of
118
Contents lists available at ScienceDirect
Psychiatry Research journal homepage: www.elsevier.com/locate/psychres
Short communication
Effects of the −141C insertion/deletion polymorphism in the dopamine D2 receptor gene on the dopamine system in the striatum in patients with schizophrenia
T
⁎
Junya Matsumotoa, ,1, Atsuko Nagaokaa,1, Yasuto Kuniia,b, Itaru Miuraa, Mizuki Hinoa, Shin-ichi Niwab, Hiroyuki Nawac, Hitoshi Takahashid, Akiyoshi Kakitad, Hirooki Yabea a
Department of Neuropsychiatry, Fukushima Medical University School of Medicine, 1 Hikarigaoka, Fukushima City, Fukushima 960-1295, Japan Department of Psychiatry, Aizu Medical Center, Fukushima Medical University, 21-2 Maeda, Tanisawa, Kawahigashimachi, Aizuwakamatsu City, Fukushima 969-3492, Japan c Department of Molecular Neurobiology, Brain Research Institute, Niigata University, 1-757 Asahimachi-dori, Niigata, Niigata 951-8585, Japan d Department of Pathology, Brain Research Institute, Niigata University, 1-757 Asahimachi-dori, Niigata, Niigata 951-8585, Japan b
A B S T R A C T
The relationships between −141C insertion/deletion (Ins/Del) polymorphisms in the dopamine D2 receptor gene and the two dopamine system integrators, i.e., dopamine- and cAMP-regulated phosphoprotein of molecular weight 32 kDa (DARPP-32) and calcineurin (CaN), are still unclear. In this study, we assessed the effect of this polymorphism on DARPP-32 and CaN protein expression in the postmortem striatum of patients with schizophrenia and control individuals. The expression levels of truncated DARPP and CaN were lower in Del allele carriers. These findings provide important insights into the mechanism by which this genotype could result in a poor response to antipsychotic drugs.
1. Introduction Attenuation of the dopamine system is important for the treatment of schizophrenia, since all clinically used antipsychotics are dopamine D2 receptor (DRD2) antagonists. The −141C insertion/deletion (Ins/ Del) polymorphism of DRD2 (rs1799732) is a deletion and insertion of cytosine at position −141, which is located in the 5′ promoter region of the DRD2 gene and is associated with schizophrenia (Arinami et al., 1997). Del allele carriers are associated with poor antipsychotic drug response in schizophrenia (Zhang et al., 2010). Patients who are carriers of the Del allele and noncarriers of the A1 allele in Taq1A showed treatment resistance (Kondo et al., 2003). The glutamate system is also related to the pathophysiology of schizophrenia. Since dopamine- and cAMP-regulated phosphoprotein of molecular weight 32 kDa (DARPP-32) and calcineurin (CaN) are the primary regulatory molecules for these two systems, they are thought to play central roles in the etiology of schizophrenia. DARPP-32 is expressed either as full-length (FL-DARPP) or as truncated (t-DARPP); tDARPP lacks the N-terminal region containing a phosphorylation site at Thr34 that is responsible for inhibitory regulation of protein phosphatase 1. Thus, t-DARPP cannot regulate the physiological activities of ⁎
1
several key proteins by inhibiting protein phosphatase 1. In this study, we analyzed the association of DARPP-32 and CaN expression with DRD2 gene polymorphism (−141C Ins/Del) in postmortem striatum samples from patients with schizophrenia. 2. Methods Postmortem brain samples from 12 patients with schizophrenia were obtained from the Fukushima Brain Bank, Fukushima Medical University, and those from 12 non-psychiatric control individuals were obtained from the Brain Research Institute, Niigata University. Each patient fulfilled the diagnostic criteria of the Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV). All 24 participants were Japanese. The average age was 58 years among control subjects (6 men and 6 women), and 68 years in the patient group (8 men and 4 women). Genotyping of −141C Ins/Del in DRD2 gene was performed using the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method, as previously described (Miura et al., 2015). FL-DARPP, t-DARPP, and CaN expression levels in the striatum (putamen and caudate) of postmortem tissue samples had been quantified
Corresponding author. E-mail address: [email protected] (J. Matsumoto). These authors contributed equally to this work.
https://doi.org/10.1016/j.psychres.2018.03.029 Received 7 February 2017; Received in revised form 31 January 2018; Accepted 12 March 2018 Available online 29 March 2018 0165-1781/ © 2018 Elsevier B.V. All rights reserved.
Psychiatry Research 264 (2018) 116–118
J. Matsumoto et al.
had decreased expression of t-DARPP while CaN expression tended to decrease in the striatum. Recent studies have indicated an association between −141C Ins/ Del polymorphism and antipsychotic drug response; Del allele carriers show poor response to antipsychotic drugs (Zhang et al., 2010), although mechanism remains unclear. Noncarriers of the A1 allele and carriers of the Del allele were found to show poor responses to antipsychotic drugs (Kondo et al., 2003); therefore, A1 allele carriers may show good treatment responses. In our previous study, we reported that the expression of t-DARRP was elevated in A1 allele carriers (Kunii et al., 2014). Thus, these findings suggested that increased expression of t-DARPP might lead to increased response to antipsychotic drugs, whereas decreased expression of tDARRP might reduce the response to antipsychotic drugs. There are several limitations to the present study. First, there is no data regarding the DRD2 density in the striatum. In future studies, we need to clarify the relationship between DARPP and CaN by analyzing the DRD2 density simultaneously. Second, we could not consider these postmortem samples’ drug responses to antipsychotic drug treatment of the patients directly, because we did not have enough information about drug response data. Third, the sample size was small. Public databases such as the braincloud or the ExSNP cannot solve this problem, because they did not contain the 141C Ins/Del (rs1799732) data though this polymorphism is quite important in the treatment by antipsychotics. Further study with large sample size is needed to validate our data. Finally, we should consider the possible effect of antipsychotics on levels of protein expression (Rushlow et al., 2005). Further study with postmortem brain sample from patients with schizophrenia who are drug naïve is needed. In spite of these limitations, we believe that this report is important, because this report is the first to focus on this clinically significant polymorphism, with expression data on DARPP and CaN. In this study, we showed that t-DARPP expression was decreased and the expression of CaN tended to decrease in the striatum of patients with schizophrenia harboring the Del allele. Although this study was relatively small and provided only a preliminary analysis, these results may reflect the potential molecular mechanisms of non-response to antipsychotics in patients with schizophrenia who are Del allele carriers. Further studies with larger cohorts are needed to confirm these findings.
by immunoblotting in our previous study (Kunii et al., 2014). Micro BCA kits (Pierce Chemical, Rockland, IL) were used to determine the protein content in each supernatant. For each tissue sample, 20 µg of protein was subjected to 10%-polyacrylamide gel electrophoresis in sodium dodecyl sulfate and then transferred to nitrocellulose membranes (Schleicher and Schull, Dassel, Germany). Protein expression levels in patients with schizophrenia did not correlate with the daily dosage of antipsychotics. The average daily dosage of antipsychotics, prescribed during the 3 months immediately preceding death, was 323 mg. Additionally, none of the individuals had any record of substance abuse, except for some who smoked tobacco. Correlation between the protein expression levels and −141C Ins/ Del genotype in the DRD2 gene was analyzed by analysis of covariance (ANCOVA) using Statistica Ver. 10.0 (Statsoft Inc., Tulsa, OK), and differences with p < 0.05 were considered significant. The genotype was set as an independent variable, and sex, age at death, and postmortem interval (PMI) were set as covariates. Pearson's chi-squared test was used to examine deviations from the Hardy–Weinberg equilibrium. However, post hoc correction was not carried out. This study was approved by the Ethics Committees of Fukushima Medical University and Niigata University and was performed in accordance with the Declaration of Helsinki. 3. Results We determined the −141C Ins/Del genotype (Ins/Ins, Ins/Del, or Del/Del) in all participants. Among the 24 participants, 18 (75%) were homozygous for the Ins allele, five (21%) were heterozygous for the Ins/Del alleles, and one (4%) was homozygous for the Del allele. The allele distribution was in Hardy–Weinberg equilibrium (χ2 = 0.64, df = 1, P > 0.05). The expression of t-DARPP in the putamen of postmortem brains with schizophrenia correlated with the −141C Ins/Del genotype; specifically, the Del allele was associated with significantly reduced t-DARPP expression (ANCOVA: F [1, 7] = 6.1889, P = 0.042; Fig. 1a). CaN expression in the caudate of postmortem brains with schizophrenia also correlated with −141C Ins/Del; the Del allele tended to reduce CaN expression (ANCOVA: F [1, 7] = 5.5660, P = 0.050; Fig. 1b). 4. Discussion In this study, we found that Del allele carriers with schizophrenia
Fig. 1. Effect of −141C genotypes on protein expression. Mean expression of t-DARPP in the putamen (a) or CaN in the caudate (b) of patients with schizophrenia. tDARPP, truncated DARPP-32; CaN, calcineurin. 117
Psychiatry Research 264 (2018) 116–118
J. Matsumoto et al.
Acknowledgments
dopamine D2 receptor gene polymorphisms as a possible predictor of treatment-resistance to dopamine antagonists in schizophrenic patients. Prog. Neuropsychopharmacol. Biol. Psychiatry 27, 921–926. Kunii, Y., Miura, I., Matsumoto, J., Hino, M., Wada, A., Niwa, S.I., Nawa, H., Sakai, M., Someya, T., Takahashi, H., Kakita, A., Yabe, H., 2014. Elevated postmortem striatal tDARPP expression in schizophrenia and associations with DRD2/ANKK1 polymorphism. Prog. Neuropsychopharmacol. Biol. Psychiatry 53C, 123–128. Miura, I., Kanno-Nozaki, K., Hino, M., Horikoshi, S., Ota, T., Mashiko, H., Niwa, S., Yabe, H., 2015. Influence of −141C Ins/Del polymorphism in DRD2 gene on clinical symptoms and plasma homovanillic acid levels in the treatment of schizophrenia with aripiprazole. J. Clin. Psychopharmacol. 35, 333–334. Rushlow, W.J., Seah, Y.H., Belliveau, D.J., Rajakumar, N., 2005. Changes in calcineurin expression induced in the rat brain by the administration of antipsychotics. J. Neurochem. 94, 587–596. Zhang, J.P., Lencz, T., Malhotra, A.K., 2010. D2 receptor genetic variation and clinical response to antipsychotic drug treatment: a meta-analysis. Am. J. Psychiatry 167, 763–772.
This work was supported by JSPS KAKENHIGrant Number JP15K19739, JP16H06277, the Collaborative Research Project (2016–2809) of the Brain Research Institute, Niigata University, the Strategic Research Program for Brain Science from Japan Agency for Medical Research and Development, AMED. References Arinami, T., Gao, M., Hamaguchi, H., Toru, M., 1997. A functional polymorphism in the promoter region of the dopamine D2 receptor gene is associated with schizophrenia. Hum. Mol. Gen. 6, 577–582. Kondo, T., Mihara, K., Suzuki, A., Yasui-Furukori, N., Kaneko, S., 2003. Combination of
118