Dysbindin Regulates the Transcriptional Level of Myristoylated Alanine-Rich Protein Kinase C Substrate via the Interaction with NF-YB in Mice Brain

et al. (2010) Dysbindin Regulates the Transcriptional Level of Myristoylated Alanine-Rich Protein Kinase C Substrate via the Interaction with NF-YB in Mice Brain. PLoS ONE 5(1): e8773. doi:10.1371/journal.pone.0008773 Dysbindin Regulates the Transcriptional Level of Myristoylated Alanine-Rich Protein Kinase C Substrate via the Interaction with NF-YB in Mice Brain Hiroaki Okuda 0 Ryusuke Kuwahara 0 Shinsuke Matsuzaki 0 Shingo Miyata 0 Natsuko 0 Kumamoto 0 Tsuyoshi Hattori 0 Shoko Shimizu 0 Kohei Yamada 0 Keisuke Kawamoto 0 Ryota Hashimoto 0 Masatoshi Takeda 0 Taiichi Katayama 0 Masaya Tohyama 0 Kenji Hashimoto, Chiba University Center for Forensic Mental Health, Japan 0 1 Department of Anatomy and Neuroscience, Graduate School of Medicine, Osaka University , Osaka , Japan , 2 Department of Second Anatomy, Faculty of Medicine, Nara Medical University , Nara , Japan , 3 The Osaka-Hamamatsu Joint Research Center for Child Mental Development, Graduate School of Medicine, Osaka University , Osaka , Japan , 4 Department of Child Development and Molecular Brain Science, United Graduate School of Child Development, Osaka University, Kanazawa University and Hamamatsu University School of Medicine, Osaka, Japan, 5 Department of Psychiatry, Osaka University Graduate School of Medicine , Osaka , Japan Background: An accumulating body of evidence suggests that Dtnbp1 (Dysbindin) is a key susceptibility gene for schizophrenia. Using the yeast-two-hybrid screening system, we examined the candidate proteins interacting with Dysbindin and revealed one of these candidates to be the transcription factor NF-YB. Methods: We employed an immunoprecipitation (IP) assay to demonstrate the Dysbindin-NF-YB interaction. DNA chips were used to screen for altered expression of genes in cells in which Dysbindin or NF-YB was down regulated, while Chromatin IP and Reporter assays were used to confirm the involvement of these genes in transcription of Myristoylated alanine-rich protein kinase C substrate (MARCKS). The sdy mutant mice with a deletion in Dysbindin, which exhibit behavioral abnormalities, and wild-type DBA2J mice were used to investigate MARCKS expression. Results: We revealed an interaction between Dysbindin and NF-YB. DNA chips showed that MARCKS expression was increased in both Dysbindin knockdown cells and NF-YB knockdown cells, and Chromatin IP revealed interaction of these proteins at the MARCKS promoter region. Reporter assay results suggested functional involvement of the interaction between Dysbindin and NF-YB in MARCKS transcription levels, via the CCAAT motif which is a NF-YB binding sequence. MARCKS expression was increased in sdy mutant mice when compared to wild-type mice. Conclusions: These findings suggest that abnormal expression of MARCKS via dysfunction of Dysbindin might cause impairment of neural transmission and abnormal synaptogenesis. Our results should provide new insights into the mechanisms of neuronal development and the pathogenesis of schizophrenia. - Funding: This work was supported by grants from the Osaka Medical Research Foundation For Incurable Diseases (Shinsuke Matsuzaki) and the Twenty-First Century Center of Excellence (COE) program and the Global COE program (Hiroaki Okuda, Ryusuke Kuwahara, Shinsuke Matsuzaki, Shingo Miyata, Natsuko Kumamoto, Tsuyoshi Hattori, Shoko Shimizu, Kohei Yamada, Taiichi Katayama and Masaya Tohyama). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Schizophrenia is a common and devastating psychiatric disorder. Lack of patient compliance, due to undesirable side effects and efficacy restricted to positive symptoms, highlights the need to develop novel therapeutics. The etiology of the disease remains unknown, but in recent years a convergence of genetic, pharmacological, and neuroanatomical findings suggest that neural transmission [14] and synapse formation [511] are involved in schizophrenia. Recent studies suggest that disturbances of Dysbindin (dystrobrevin-binding protein 1; DTNBP1) are involved in this abnormal neural transmission. The cause of schizophrenia is thought to involve the combined effects of multiple gene components. Genetic linkage and association studies have identified potential susceptibility genes such as Dysbindin [12,13], Neuregulin [14,15], Catechol-O-methyltransferase [1618] and RG4 [1922]. In particular, it has been reported that chromosome 6p is one of the highest susceptibility regions in linkage studies of schizophrenia [23,24]. Among them, genetic variants in a gene 6p22.3 expressing Dysbindin, which is identified as a protein interacting with dystrobevins [25], have been shown to be strongly associated with schizophrenia [12]. In studies on postmortem brain tissue, decreased levels of Dysbindin protein [26] and mRNA [27] have been shown in patients with schizophrenia compared with controls. Chronic treatment of mice with antipsychotics did not affect the expression levels of Dysbindin protein and mRNA in their brains [26,28], suggesting that evidence of lower levels of Dysbindin protein and mRNA in the postmortem brains of schizophrenics is not likely to be a simple artifact of antemortem drug treatment. In addition, previous reports have shown that diverse high-risk single nucleotide polymorphisms (SNPs) and haplotypes could influence Dysbindin mRNA expression [27,29]. These data indicate that the Dysbindin gene may confer susceptibility to schizophrenia through reduced Dysbindin expression. Several lines of evidence suggest that Dysbindin may be associated with brain function. SNPs in Dysbindin have been associated with intermediate cognitive phenotypes related to schizophrenia such as IQ and working and episodic memory, and a Dysbindin haplotype has been associated with higher educational attainment [30,31]. In addition, several reports suggest the involvement of Dysbindin in cognitive functions [3234]. These findings strongly suggest the importance of Dysbindin in brain function. At the cellular level, Dysbindin is located at both pre- and post-synaptic terminals [26,35], and is thought to be involved in postsynaptic density (PSD) function and the trafficking of receptors (NMDA, GABAergic, and nicotinic). Over-expression of Dysbindin increases glutamate release from pyramidal neurons in cell culture, possibly because of its role in vesicular trafficking [36]. Decreases in Dysbindin mRNA and protein levels have been reported in regions previously implicated in schizophrenia: the prefrontal cortex, midbrain, and hippocampus [26,27]. However, the molecular mechanisms of how decreases in Dysbindin expression may contribute to vulnerability to schizophrenia remain unknown. Thus, we examined the interacting partners of Dysbindin using yeast two-hybrid analysis in order to he (...truncated)