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Analysis of neural circuit on the basis of neuroblast lineage in Drosophila
S90
Abstracts
cells, but the total length of the dendrite per Purkinje cell was slightly, but significantly shorter in infected than in non-infected cells. Dendrite branching analyzed by Sholl test was variably impaired in infected cells. In this presentation, based on the data obtained, we will assess the influence of lentiviral vector infection on developing Purkinje neurons and discuss the usefulness and limitation of lentiviral vectors for their application to basic research as well as gene therapy. doi:10.1016/j.neures.2009.09.366
P1-d08 Altered gene expression of histone deacetylases in mood disorder patients Teruyuki Hobara, Shusaku Uchida, Koji Otsuki, Toshio Matsubara, Hiromasa Funato, Koji Matsuo, Masatomo Suetsugi, Yoshifumi Watanabe Div. of Neuropsy, Department Neurosci., Yamaguchi University Grad. Sch. Med., Japan Histone acetylation has been suggested to play an important role in the pathophysiology of mood disorders. In this study, we investigated whether the expression of histone deacetylase (HDAC) genes are altered in mood disorders. We measured mRNA levels of 11 HDACs in leukocytes of major depressive disorder (MDD), bipolar disorder (BPD) patients. We also studied the effect of medications on mRNA levels of 11 HDACs using mice. In MDD, the expression of HDAC2 and -5 mRNA was increased in a depressive state, but not in a remissive state. In BPD, the expression of HDAC4 mRNA was increased only in a depressive state, while the expression of HDAC6 and -8 mRNA was decreased in both depressive and remissive states. In the animal study, the expression of these HDAC mRNAs was not affected by medications. Our data suggest that aberrant transcriptional regulation caused by the altered expression of HDACs is associated with the pathophysiology of mood disorders. doi:10.1016/j.neures.2009.09.367
P1-d09 A role of neuronal RNA binding protein Hu and NF45/NF90 in the nervous system Mana Igarashi, Masato Yano, James Hirotaka Okano, Hideyuki Okano Department Physiol. Keio University Sch. Med., Tokyo, Japan Neuronal RNA binding protein family Hu is homolog of Drosophila ELAV protein, which is essential for differentiation and maintenance of the nervous system. In mammals, Hu proteins are expressed in both early postmitotic and mature neurons and are shown to induce neuronal differentiation by binding to the UTR sequences of specific target mRNAs. To understand the molecular mechanism of the function of Hu, we purified HuB associated complexes. NF90, a double strand RNA binding protein which is one of Hu associated proteins, forms heterodimer with NF45 and interacts with UTRs of p21, p27 and tau mRNA which are also known as Hu targets. Since NF90 is a double strand RNA binding protein, it may recognize and bind to not only a specific RNA sequence but also a secondary structure of target RNAs. Here we investigate the NF90 binding sites in p21 mRNA 3 UTR by using a new in silico methodology to predict a secondary structure of RNA. doi:10.1016/j.neures.2009.09.368
P1-d10 PTBP1 regulates the alternative splicing of dopamine receptor D2 (DRD2) Toshikazu Sasabe, Eugene Futai, Shoichi Ishiura Dept. Life Sci., Univ. of Tokyo, Tokyo, Japan Dopamine receptor D2 (DRD2) has two splicing isoforms, the long (D2L) and the short (D2S), which have distinct functions in the dopaminergic system. However, the regulatory mechanism of the alternative splicing of DRD2 is still unknown. So, in this study, we examined which factor regulates the ratio of D2 receptor isoforms by overexpression of some splicing regulatory proteins in HEK293 cells. We identified PTBP1 as a protein regulating the alternative splicing of DRD2. doi:10.1016/j.neures.2009.09.369
P1-d11 Involvement of integrin-associated protein form 2 in neurona differentiation of neural stem/progenitor cells Kazuhiko Fujimura, Yoriko Shinozuka, Takeshi Kihara, Hachiro Sugimoto, Tetsuhiro Niidome Dept. of Neurosci. Drug Discov., Grad. Sch. Pharmaceutical Sci., Kyoto Univ., Japan
Neurogenesis from neural stem/progenitor cells (NSPCs) is determined by an interplay between intrinsic genetic mechanisms and extrinsic cues. Recent studies show that NSPCs temporally change their differentiation potential; however, its molecular mechanism is largely unknown. In this study, we first demonstrated that the percentage of TUJ1-positive cells from 13 DIV NSPCs was higher than that from 20 DIV NSPCs under differentiation conditions. Using differential display techniques, we next demonstrated that the expression levels of integrin-associated protein form 2 (IAP2) mRNA in 13 DIV NSPCs was higher than those in 20 DIV NSPCs. Interestingly NSPCs expressed only IAP2 mRNA although IAP has been reported to express four splicing variants (IAP1-4). Retroviral overexpression of IAP2 promoted the neuronal differentiation of 13 DIV NSPCs, whereas it had no effect on the neuronal differentiation of 20 DIV NSPCs. These results suggest that IAP2 plays a critical role in the neuronal differentiation of stage-specific NSPCs. doi:10.1016/j.neures.2009.09.370
P1-d12 Docosahexaenoic acid controls the expression of Hes1 and p27 on the neural stem cell differentiation Masanori Katakura, Michio Hashimoto, Hossain M. Shahdat, Shyuji Gamoh, Toshiyuki Okui, Osamu Shido Shimane University, Japan We previously demonstrated that docosahexaenoic acid (DHA) promotes neurogenesis from neural stem/progenitor cells (NPCs). However the underlying mechanism remains largely unknown. In this study, we investigated the effects of DHA on the expression of bHLH transcriptional factors and cell cycle of cultured NPCs. NPCs from E14.5 rats were cultured in N2 medium containing bFGF. Treatment with DHA without bFGF increased Tuj-1 and MAP2 positive cells and their protein levels. DHA significantly decreased the Hes1 mRNA and protein level; on the other hand, DHA increased NeuroD mRNA level. DHA decreased the percentage of S-phase cells, which correlated with prolonged expression of cyclin-dependent kinase inhibitor p27kip1 , suggesting that DHA enhances neuronal differentiation of NSCs, in part, by controlling the bHLH transcription factors and promoting cell cycle exit. doi:10.1016/j.neures.2009.09.371
P1-d13 Analysis of neural circuit on the basis of neuroblast lineage in Drosophila Masayoshi Ito 1,2 , Keita Endo 1 , Kei Ito 1,2 1
IMCB, The University of Tokyo, Tokyo, Japan; 2 Dept Life Sci., Grad. Sch. of Arts and Sci., The University of Tokyo, Tokyo, Japan
In the larval brain of Drosophila, there are about 100 neuroblasts per hemisphere in the cerebrum. During larval neurogenesis, neuroblasts repeat asymmetric cell divisions to generate clonally related progeny, which we call clonal unit. A clonal unit consists of a cluster of cell bodies and a bundle of neurites that innervate distinct areas of the brain. We analyzed the structure of each clonal unit by visualizing neuroblasts and their progeny with the MARCM technique. We have so far identified 56 candidates for clonal units in the cerebrum. On the basis of detailed brain region map, we made a database of cell body sites, projection patterns and distribution of the pre-synaptic sites. Analysis of this database revealed that a particular brain region received multiple inputs from several brain regions not only from nearby regions but also from a distance. We were able to classify clonal units into several groups by their characteristic distribution of the pre-synaptic sites, e.g. those who have presynapses only in the distal parts of their neurites or across the entire arborization areas. doi:10.1016/j.neures.2009.09.372
P1-d14 Analysis of cell surface glycoproteins on neural stem cells Makoto Hamanoue 1 , Hirotaka Okano 2 , Hideyuki Okano 2 , Ken Takamatsu 1 1
Dept. Phsiol., Toho University, Tokyo, Japan; sity, Tokyo, Japan
2
Dept. Physiol., Keio Univer-
Complex type Asparagine-linked oligosaccharides (N-glycans) are one of the cell surface oligosaccharides on neural stem cells (NSCs), and we have been reported that NSCs can be isolated by using cell attachment between cell surface complex type N-glycans and the plant lectin, phaseolus vulgaris erythroagglutinating lectin (E-PHA), which bind to biantennary complex-type N-glycans.In this study, the cell surface glycoproteins prepared from primary NSCs were analyzed by lectin blot analysis using E-PHA. Lectin blot analysis revealed that E-PHA bound to membrane fraction of the cultured NSCs lysate, and this binding was diminished by glycosidase treatment, indicating the N-glycan dependency. Among these molecules, we demonstrated that CD133 and EGF-Receptor (EGF-R), which have been reported
Abstracts
cells, but the total length of the dendrite per Purkinje cell was slightly, but significantly shorter in infected than in non-infected cells. Dendrite branching analyzed by Sholl test was variably impaired in infected cells. In this presentation, based on the data obtained, we will assess the influence of lentiviral vector infection on developing Purkinje neurons and discuss the usefulness and limitation of lentiviral vectors for their application to basic research as well as gene therapy. doi:10.1016/j.neures.2009.09.366
P1-d08 Altered gene expression of histone deacetylases in mood disorder patients Teruyuki Hobara, Shusaku Uchida, Koji Otsuki, Toshio Matsubara, Hiromasa Funato, Koji Matsuo, Masatomo Suetsugi, Yoshifumi Watanabe Div. of Neuropsy, Department Neurosci., Yamaguchi University Grad. Sch. Med., Japan Histone acetylation has been suggested to play an important role in the pathophysiology of mood disorders. In this study, we investigated whether the expression of histone deacetylase (HDAC) genes are altered in mood disorders. We measured mRNA levels of 11 HDACs in leukocytes of major depressive disorder (MDD), bipolar disorder (BPD) patients. We also studied the effect of medications on mRNA levels of 11 HDACs using mice. In MDD, the expression of HDAC2 and -5 mRNA was increased in a depressive state, but not in a remissive state. In BPD, the expression of HDAC4 mRNA was increased only in a depressive state, while the expression of HDAC6 and -8 mRNA was decreased in both depressive and remissive states. In the animal study, the expression of these HDAC mRNAs was not affected by medications. Our data suggest that aberrant transcriptional regulation caused by the altered expression of HDACs is associated with the pathophysiology of mood disorders. doi:10.1016/j.neures.2009.09.367
P1-d09 A role of neuronal RNA binding protein Hu and NF45/NF90 in the nervous system Mana Igarashi, Masato Yano, James Hirotaka Okano, Hideyuki Okano Department Physiol. Keio University Sch. Med., Tokyo, Japan Neuronal RNA binding protein family Hu is homolog of Drosophila ELAV protein, which is essential for differentiation and maintenance of the nervous system. In mammals, Hu proteins are expressed in both early postmitotic and mature neurons and are shown to induce neuronal differentiation by binding to the UTR sequences of specific target mRNAs. To understand the molecular mechanism of the function of Hu, we purified HuB associated complexes. NF90, a double strand RNA binding protein which is one of Hu associated proteins, forms heterodimer with NF45 and interacts with UTRs of p21, p27 and tau mRNA which are also known as Hu targets. Since NF90 is a double strand RNA binding protein, it may recognize and bind to not only a specific RNA sequence but also a secondary structure of target RNAs. Here we investigate the NF90 binding sites in p21 mRNA 3 UTR by using a new in silico methodology to predict a secondary structure of RNA. doi:10.1016/j.neures.2009.09.368
P1-d10 PTBP1 regulates the alternative splicing of dopamine receptor D2 (DRD2) Toshikazu Sasabe, Eugene Futai, Shoichi Ishiura Dept. Life Sci., Univ. of Tokyo, Tokyo, Japan Dopamine receptor D2 (DRD2) has two splicing isoforms, the long (D2L) and the short (D2S), which have distinct functions in the dopaminergic system. However, the regulatory mechanism of the alternative splicing of DRD2 is still unknown. So, in this study, we examined which factor regulates the ratio of D2 receptor isoforms by overexpression of some splicing regulatory proteins in HEK293 cells. We identified PTBP1 as a protein regulating the alternative splicing of DRD2. doi:10.1016/j.neures.2009.09.369
P1-d11 Involvement of integrin-associated protein form 2 in neurona differentiation of neural stem/progenitor cells Kazuhiko Fujimura, Yoriko Shinozuka, Takeshi Kihara, Hachiro Sugimoto, Tetsuhiro Niidome Dept. of Neurosci. Drug Discov., Grad. Sch. Pharmaceutical Sci., Kyoto Univ., Japan
Neurogenesis from neural stem/progenitor cells (NSPCs) is determined by an interplay between intrinsic genetic mechanisms and extrinsic cues. Recent studies show that NSPCs temporally change their differentiation potential; however, its molecular mechanism is largely unknown. In this study, we first demonstrated that the percentage of TUJ1-positive cells from 13 DIV NSPCs was higher than that from 20 DIV NSPCs under differentiation conditions. Using differential display techniques, we next demonstrated that the expression levels of integrin-associated protein form 2 (IAP2) mRNA in 13 DIV NSPCs was higher than those in 20 DIV NSPCs. Interestingly NSPCs expressed only IAP2 mRNA although IAP has been reported to express four splicing variants (IAP1-4). Retroviral overexpression of IAP2 promoted the neuronal differentiation of 13 DIV NSPCs, whereas it had no effect on the neuronal differentiation of 20 DIV NSPCs. These results suggest that IAP2 plays a critical role in the neuronal differentiation of stage-specific NSPCs. doi:10.1016/j.neures.2009.09.370
P1-d12 Docosahexaenoic acid controls the expression of Hes1 and p27 on the neural stem cell differentiation Masanori Katakura, Michio Hashimoto, Hossain M. Shahdat, Shyuji Gamoh, Toshiyuki Okui, Osamu Shido Shimane University, Japan We previously demonstrated that docosahexaenoic acid (DHA) promotes neurogenesis from neural stem/progenitor cells (NPCs). However the underlying mechanism remains largely unknown. In this study, we investigated the effects of DHA on the expression of bHLH transcriptional factors and cell cycle of cultured NPCs. NPCs from E14.5 rats were cultured in N2 medium containing bFGF. Treatment with DHA without bFGF increased Tuj-1 and MAP2 positive cells and their protein levels. DHA significantly decreased the Hes1 mRNA and protein level; on the other hand, DHA increased NeuroD mRNA level. DHA decreased the percentage of S-phase cells, which correlated with prolonged expression of cyclin-dependent kinase inhibitor p27kip1 , suggesting that DHA enhances neuronal differentiation of NSCs, in part, by controlling the bHLH transcription factors and promoting cell cycle exit. doi:10.1016/j.neures.2009.09.371
P1-d13 Analysis of neural circuit on the basis of neuroblast lineage in Drosophila Masayoshi Ito 1,2 , Keita Endo 1 , Kei Ito 1,2 1
IMCB, The University of Tokyo, Tokyo, Japan; 2 Dept Life Sci., Grad. Sch. of Arts and Sci., The University of Tokyo, Tokyo, Japan
In the larval brain of Drosophila, there are about 100 neuroblasts per hemisphere in the cerebrum. During larval neurogenesis, neuroblasts repeat asymmetric cell divisions to generate clonally related progeny, which we call clonal unit. A clonal unit consists of a cluster of cell bodies and a bundle of neurites that innervate distinct areas of the brain. We analyzed the structure of each clonal unit by visualizing neuroblasts and their progeny with the MARCM technique. We have so far identified 56 candidates for clonal units in the cerebrum. On the basis of detailed brain region map, we made a database of cell body sites, projection patterns and distribution of the pre-synaptic sites. Analysis of this database revealed that a particular brain region received multiple inputs from several brain regions not only from nearby regions but also from a distance. We were able to classify clonal units into several groups by their characteristic distribution of the pre-synaptic sites, e.g. those who have presynapses only in the distal parts of their neurites or across the entire arborization areas. doi:10.1016/j.neures.2009.09.372
P1-d14 Analysis of cell surface glycoproteins on neural stem cells Makoto Hamanoue 1 , Hirotaka Okano 2 , Hideyuki Okano 2 , Ken Takamatsu 1 1
Dept. Phsiol., Toho University, Tokyo, Japan; sity, Tokyo, Japan
2
Dept. Physiol., Keio Univer-
Complex type Asparagine-linked oligosaccharides (N-glycans) are one of the cell surface oligosaccharides on neural stem cells (NSCs), and we have been reported that NSCs can be isolated by using cell attachment between cell surface complex type N-glycans and the plant lectin, phaseolus vulgaris erythroagglutinating lectin (E-PHA), which bind to biantennary complex-type N-glycans.In this study, the cell surface glycoproteins prepared from primary NSCs were analyzed by lectin blot analysis using E-PHA. Lectin blot analysis revealed that E-PHA bound to membrane fraction of the cultured NSCs lysate, and this binding was diminished by glycosidase treatment, indicating the N-glycan dependency. Among these molecules, we demonstrated that CD133 and EGF-Receptor (EGF-R), which have been reported