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Kinetic study on DNA methylation of autism susceptibility gene, SHANK3, in the developing mouse brain
e204
Abstracts / Neuroscience Research 68S (2010) e109–e222
P1-p19 Predicting the effect of the methylphenidate hydrochloride in children with ADHD using multi-channel NIRS Ayaka Takahashi , Yuki Kawakubo, Yukika Nishimura, Hitoshi Kuwabara, Ryu Takizawa, Kiyoto Kasai Dept Psych, Univ of Tokyo,Tokyo Introduction: Temporal trends in the use of stimulant drugs for attention deficit hyperactivity disorder (ADHD) have raised questions about possible over treatment. It is necessary to develop biologically based criteria for treatment. Near-infrared spectroscopy (NIRS) is a noninvasive neuroimaging method that can measure changes in the concentration of oxygenated hemoglobin ([Oxy-Hb]) in cortical tissue in natural settings. The stop signal task is suitable for detecting the dysfunction of response inhibition in ADHD. Objective: The aims of our study were to examine the bilateral prefrontal activation associated with response inhibition by using multi-channel NIRS in children with ADHD, and to develop a neurophysiological index which predicts the effect of continuous administration of methylphenidate hydrochloride (MPH) on the cognitive function evaluated by Das-Naglieri Cognitive Assssment System (DN-CAS) Methods: Participants were 27 children with ADHD and 25 typically developing children. We defined the children with ADHD whose total score or subscale score of DN-CAS was improved after administration of MPH as responder (ADHD-R) and the children with ADHD whose score was not improved as non-responder (ADHD-NR). Result: ADHD-NR showed the lower [Oxy-Hb] increase than ADHD-R and typically developing children during the activation task in right ventrolateral prefrontal cortex (VLPFC) (p < 0.01). Conclusion: The activation of [Oxy-Hb] in right VLPFC represented the difference of the frontal lobe function between responder and non-responder before administration of MPH. This preliminary result showed that NIRS might be potentially useful as the neurophysiological index which predicts the effect of MPH in children with ADHD. doi:10.1016/j.neures.2010.07.2473
P1-p20 Interaction screening of the CDKL5, a causative gene for the intractable epilepsy and developmental disorder syndromes Kohsuke Okuda , Aya Watanabe, Masashi Mizuguchi, Teruyuki Tanaka Dept Developmental Medical Sciences, Univ of Tokyo, Tokyo West syndrome is one of the most intractable epilepsy syndromes in infancy, and characterized by infantile spasms, abnormal EEG, profound mental and motor development delay. Rett syndrome is a progressive neurodevelopmental disorder in female infants, characterized by progressive loss of intellectual functioning, fine and gross motor skills and communicative abilities, and the development of stereotypic hand movements. Approximately 80% of the patients have mutations in X-linked methyl-CpG-binding protein 2 (MECP2) gene. Intriguingly, mutations in the cycline-dependent kinase-like 5 (CDKL5) gene on the X chromosome have been identified in the patients with West syndrome or atypical Rett syndrome without mutations in the MeCP2 gene. CDKL5 is a member of the serine/threonine kinase family, with its N-terminal kinase domain sharing homology to the mitogen-activated protein (MAP) kinase and cycline-dependent kinase (CDK) families. The protein localizes in the nucleus and the cytoplasm, which is considered to be mediated by the Cterminal region. However, it remains unknown how CDKL5 functions in the nervous system. In order to identify the funtional interactions of CDKL5, we performed the yeast two-hybrid screening assay. CDKL5 has the N-terminal kinase domain and the C-terminal region. Each domain was used as the bait. As the results, 21 candidates were obtained, of which 19 were in the kinase domain and 2 in the other domain. To confirm the CDKL5 interaction with each candidate, we have performed co-immunoprecipitation, GST pull-down, and in vitro kinase assays. We will discuss the possible interactions of CDKL5 at the meeting. doi:10.1016/j.neures.2010.07.2474
P1-p21 Kinetic study on DNA methylation of autism susceptibility gene, SHANK3, in the developing mouse brain Chikako Waga 1 , Hirotsugu Asano 1 , Reiko Kato 1 , Masayuki Itoh 2 , Yu-ichi Goto 2 , Shigeo Uchino 1 , Shinichi Kohsaka 1 1
Neurochemistry, National Institute of Neuroscience tokyo Japan Department of Mental Retardation and Birth Defect Research, National Institute of Neuroscience, Tokyo, Japan 2
Shank3, a multidomain protein containing SH3 and PDZ domains, is localized in the postsynaptic density, and interacts with various synaptic molecules including PSD-95 and glutamate receptors. Furthermore, SHANK3 gene is reported as a major causative gene of 22q13.3 deletion syndrome, showing severe language and speech delay, mental retardation, hypotonia and autistic feature. Therefore, it has been thought that Shank3 plays important roles in the formation and function of synapse in the developing brain and is involved in higher brain function. SHANK3 gene has five CpG islands (CpG island-P, CpG island-2, −3, −4, −5) whose methylation is involved in tissue specific expression of SHANK3 gene. However, DNA methylation of CpG islands in the developing brain remains unknown. In this study, we examined DNA methylation of five CpG islands in the developing mouse brains (E17-12w) by using Hpa II-McrBC-PCR method, and confirmed that CpG island-P was unmethylated and CpG island-3 was methylated in all developing stages. In contrast, the rate of methylation in CpG island-2, −4, and −5 significantly increased after postnatal day 7. To elucidate the related molecules involved in DNA methylation of SHANK3 gene, we here focused on Methyl CpG binding protein 2 (MeCP2), which was identified as a responsible gene for Rett syndrome and has been thought to regulate gene transcription, mRNA splicing, and chromatin structure. We examined whether the MeCP2 binds to methylated CpG islands of SHANK3 gene using a chromatin immunoprecipitation (ChIP) assay, and found that MeCP2 bound to the CpG islands-2, −3 and −4 at postnatal day 14. We recently found mutations within a CpG island of SHANK3 gene in autistic patients with mental retardation. To clarify the effect of methylation of SHANK3 gene in the developing brain and the involvement in development disorders including autism and mental retardation, further study is now on going. doi:10.1016/j.neures.2010.07.2475
P1-p22 Behavioral profiles of HATANO rats Toshiyuki Himi 1 , Maiko Kawaguchi 1 , Kaori Morohoshi 2 , Gen Watanabe 3,4 , Kazuyoshi Taya 3,4 , Masatoshi Morita 2,5 , Yasuhiko Kondo 6 , Hideki Imai 7 , Nobumasa Kato 8 1
Faculty of Pharmacy, Musashino University 2 National Inst. Environ. Stud. Tsukuba, Japan 3 Inst. Symbiotic Sci. and Tec., Tokyo Univ. Agri., Tokyo, Japan 4 Dept. Basic Vet. Sci., Uni. Grad. Sch. Vet. Sci., Gifu Univ., Gifu, Japan 5 Dept. Biores., Fac. Agri., Ehime Univ., Japan 6 Dept. Physiol., Nippon Med. Sch., Tokyo, Japan 7 Dept. Soc. Med., Fac. Med., Univ. Miyazaki, Japan 8 Showa University, Tokyo, Japan HATANO rats were consisted of the 2 strains selected from Sprague-Dawley rats by the learning scores in the active avoidance tests in shuttle box: Hatano high-avoidance (HAA) and low-avoidance (LAA). The 2 strains has been reported to show the different patterns in the hormonal secression during suckling and on stress. Here we studied the effects of estradiol on social, emotional and learning behaviors, using ovariectomized (OVX) female HATANO rats. LAA OVX rats showed lower social activity, and this decreased social activity was turned to the normal level in the presence of estradiol. LAA rats showed lower passive avoidance, and estradiol did not affect this performance. Althought no difference was observed in elevated plus maze, LAA showed higher scores in marble burrying test than HAA, which was turned to the same level in the presence of estradiol, suggesting LAA showed higher anziety or impulsive behaviors. Both in the presence or absence of estradiol, LAA showed lower passive avoidance performance. doi:10.1016/j.neures.2010.07.2476
P1-p23 Scn1a mice exhibit hyperactivity, autism-like behavioral deficits and learning impairments Ikuo Ogiwara 1 Yamakawa 1 1
, Susumu Ito 1 , Kazuyuki Yamada 2 , Kazuhiro
Lab Neurogenetics, RIKEN-BSI 2 Supp Unit Animal Experiments, RIKEN-BSI
Dravet syndrome, or severe myoclonic epilepsy in infancy, is an intractable epileptic disorder that begins in children under age one year. Besides
Abstracts / Neuroscience Research 68S (2010) e109–e222
P1-p19 Predicting the effect of the methylphenidate hydrochloride in children with ADHD using multi-channel NIRS Ayaka Takahashi , Yuki Kawakubo, Yukika Nishimura, Hitoshi Kuwabara, Ryu Takizawa, Kiyoto Kasai Dept Psych, Univ of Tokyo,Tokyo Introduction: Temporal trends in the use of stimulant drugs for attention deficit hyperactivity disorder (ADHD) have raised questions about possible over treatment. It is necessary to develop biologically based criteria for treatment. Near-infrared spectroscopy (NIRS) is a noninvasive neuroimaging method that can measure changes in the concentration of oxygenated hemoglobin ([Oxy-Hb]) in cortical tissue in natural settings. The stop signal task is suitable for detecting the dysfunction of response inhibition in ADHD. Objective: The aims of our study were to examine the bilateral prefrontal activation associated with response inhibition by using multi-channel NIRS in children with ADHD, and to develop a neurophysiological index which predicts the effect of continuous administration of methylphenidate hydrochloride (MPH) on the cognitive function evaluated by Das-Naglieri Cognitive Assssment System (DN-CAS) Methods: Participants were 27 children with ADHD and 25 typically developing children. We defined the children with ADHD whose total score or subscale score of DN-CAS was improved after administration of MPH as responder (ADHD-R) and the children with ADHD whose score was not improved as non-responder (ADHD-NR). Result: ADHD-NR showed the lower [Oxy-Hb] increase than ADHD-R and typically developing children during the activation task in right ventrolateral prefrontal cortex (VLPFC) (p < 0.01). Conclusion: The activation of [Oxy-Hb] in right VLPFC represented the difference of the frontal lobe function between responder and non-responder before administration of MPH. This preliminary result showed that NIRS might be potentially useful as the neurophysiological index which predicts the effect of MPH in children with ADHD. doi:10.1016/j.neures.2010.07.2473
P1-p20 Interaction screening of the CDKL5, a causative gene for the intractable epilepsy and developmental disorder syndromes Kohsuke Okuda , Aya Watanabe, Masashi Mizuguchi, Teruyuki Tanaka Dept Developmental Medical Sciences, Univ of Tokyo, Tokyo West syndrome is one of the most intractable epilepsy syndromes in infancy, and characterized by infantile spasms, abnormal EEG, profound mental and motor development delay. Rett syndrome is a progressive neurodevelopmental disorder in female infants, characterized by progressive loss of intellectual functioning, fine and gross motor skills and communicative abilities, and the development of stereotypic hand movements. Approximately 80% of the patients have mutations in X-linked methyl-CpG-binding protein 2 (MECP2) gene. Intriguingly, mutations in the cycline-dependent kinase-like 5 (CDKL5) gene on the X chromosome have been identified in the patients with West syndrome or atypical Rett syndrome without mutations in the MeCP2 gene. CDKL5 is a member of the serine/threonine kinase family, with its N-terminal kinase domain sharing homology to the mitogen-activated protein (MAP) kinase and cycline-dependent kinase (CDK) families. The protein localizes in the nucleus and the cytoplasm, which is considered to be mediated by the Cterminal region. However, it remains unknown how CDKL5 functions in the nervous system. In order to identify the funtional interactions of CDKL5, we performed the yeast two-hybrid screening assay. CDKL5 has the N-terminal kinase domain and the C-terminal region. Each domain was used as the bait. As the results, 21 candidates were obtained, of which 19 were in the kinase domain and 2 in the other domain. To confirm the CDKL5 interaction with each candidate, we have performed co-immunoprecipitation, GST pull-down, and in vitro kinase assays. We will discuss the possible interactions of CDKL5 at the meeting. doi:10.1016/j.neures.2010.07.2474
P1-p21 Kinetic study on DNA methylation of autism susceptibility gene, SHANK3, in the developing mouse brain Chikako Waga 1 , Hirotsugu Asano 1 , Reiko Kato 1 , Masayuki Itoh 2 , Yu-ichi Goto 2 , Shigeo Uchino 1 , Shinichi Kohsaka 1 1
Neurochemistry, National Institute of Neuroscience tokyo Japan Department of Mental Retardation and Birth Defect Research, National Institute of Neuroscience, Tokyo, Japan 2
Shank3, a multidomain protein containing SH3 and PDZ domains, is localized in the postsynaptic density, and interacts with various synaptic molecules including PSD-95 and glutamate receptors. Furthermore, SHANK3 gene is reported as a major causative gene of 22q13.3 deletion syndrome, showing severe language and speech delay, mental retardation, hypotonia and autistic feature. Therefore, it has been thought that Shank3 plays important roles in the formation and function of synapse in the developing brain and is involved in higher brain function. SHANK3 gene has five CpG islands (CpG island-P, CpG island-2, −3, −4, −5) whose methylation is involved in tissue specific expression of SHANK3 gene. However, DNA methylation of CpG islands in the developing brain remains unknown. In this study, we examined DNA methylation of five CpG islands in the developing mouse brains (E17-12w) by using Hpa II-McrBC-PCR method, and confirmed that CpG island-P was unmethylated and CpG island-3 was methylated in all developing stages. In contrast, the rate of methylation in CpG island-2, −4, and −5 significantly increased after postnatal day 7. To elucidate the related molecules involved in DNA methylation of SHANK3 gene, we here focused on Methyl CpG binding protein 2 (MeCP2), which was identified as a responsible gene for Rett syndrome and has been thought to regulate gene transcription, mRNA splicing, and chromatin structure. We examined whether the MeCP2 binds to methylated CpG islands of SHANK3 gene using a chromatin immunoprecipitation (ChIP) assay, and found that MeCP2 bound to the CpG islands-2, −3 and −4 at postnatal day 14. We recently found mutations within a CpG island of SHANK3 gene in autistic patients with mental retardation. To clarify the effect of methylation of SHANK3 gene in the developing brain and the involvement in development disorders including autism and mental retardation, further study is now on going. doi:10.1016/j.neures.2010.07.2475
P1-p22 Behavioral profiles of HATANO rats Toshiyuki Himi 1 , Maiko Kawaguchi 1 , Kaori Morohoshi 2 , Gen Watanabe 3,4 , Kazuyoshi Taya 3,4 , Masatoshi Morita 2,5 , Yasuhiko Kondo 6 , Hideki Imai 7 , Nobumasa Kato 8 1
Faculty of Pharmacy, Musashino University 2 National Inst. Environ. Stud. Tsukuba, Japan 3 Inst. Symbiotic Sci. and Tec., Tokyo Univ. Agri., Tokyo, Japan 4 Dept. Basic Vet. Sci., Uni. Grad. Sch. Vet. Sci., Gifu Univ., Gifu, Japan 5 Dept. Biores., Fac. Agri., Ehime Univ., Japan 6 Dept. Physiol., Nippon Med. Sch., Tokyo, Japan 7 Dept. Soc. Med., Fac. Med., Univ. Miyazaki, Japan 8 Showa University, Tokyo, Japan HATANO rats were consisted of the 2 strains selected from Sprague-Dawley rats by the learning scores in the active avoidance tests in shuttle box: Hatano high-avoidance (HAA) and low-avoidance (LAA). The 2 strains has been reported to show the different patterns in the hormonal secression during suckling and on stress. Here we studied the effects of estradiol on social, emotional and learning behaviors, using ovariectomized (OVX) female HATANO rats. LAA OVX rats showed lower social activity, and this decreased social activity was turned to the normal level in the presence of estradiol. LAA rats showed lower passive avoidance, and estradiol did not affect this performance. Althought no difference was observed in elevated plus maze, LAA showed higher scores in marble burrying test than HAA, which was turned to the same level in the presence of estradiol, suggesting LAA showed higher anziety or impulsive behaviors. Both in the presence or absence of estradiol, LAA showed lower passive avoidance performance. doi:10.1016/j.neures.2010.07.2476
P1-p23 Scn1a mice exhibit hyperactivity, autism-like behavioral deficits and learning impairments Ikuo Ogiwara 1 Yamakawa 1 1
, Susumu Ito 1 , Kazuyuki Yamada 2 , Kazuhiro
Lab Neurogenetics, RIKEN-BSI 2 Supp Unit Animal Experiments, RIKEN-BSI
Dravet syndrome, or severe myoclonic epilepsy in infancy, is an intractable epileptic disorder that begins in children under age one year. Besides