栄養因子、サイトカイン Trophic Factors and Cytokines
P2-1-80 微小ペプチドタグを用いたBDNF分泌のライブイメージング Live imaging of BDNF secretion using a small peptide-based fluorescent tag ○松田尚人1, 小川糸音1, 浜田駿1, 真鍋俊也1 ○Naoto Matsuda1, Itone Ogawa1, Shun Hamada1, Toshiya Manabe1 東京大学医科学研究所神経ネットワーク分野1 Division of Neuronal Network, Institute of Medical Sciences, University of Tokyo1 Brain-derived neurotrophic factor (BDNF) is a secreted protein that is essential for brain development, survival of neurons and synaptic plasticity. Since mutations that impair the secretion of BDNF cause depression or anxiety disorders, it is important to know how the secretion of BDNF is regulated. Fusion of a protein with green fluorescent protein (GFP) or its variant has been used for real-time imaging of synthesis, localization and secretion of the protein. In the case of BDNF, however, the molecular mass of GFP is approximately twice as large as that of mature BDNF protein, and the GFP tag might affect the secretion and diffusion of the tagged BDNF. In this study, we aimed to develop a novel BDNF fluorescent probe with minimal additional molecular mass. The peptide tag appended at the carboxy-terminus of BDNF contained a tetracysteine motif (BDNF-TC), providing binding sites to two arsenic groups of a fluorescein derivative (FlAsH). The BDNF-TC protein expressed in HEK293T cells was successfully labeled with exogenously added FlAsH. The resultant fluorescent probe (hereafter named BDNF-Lumio) was found to localize to the Golgi complex as expected, and was secreted from the cells. Western blotting revealed that BDNF-Lumio was functional in terms of the ability to phosphorylate the trkB receptor and its downstream signal ERK. Next, we performed live imaging of BDNF-Lumio in transfected cultured hippocampal neurons. Trafficking of secretory vesicles containing BDNF-Lumio was observed similarly to that of the GFP-tagged BDNF. Electrical stimulation of the neurons expressing BDNF-Lumio induced exocytosis of the vesicles. Thus, we propose that the BDNF-Lumio probe is a promising tool that mimics the behavior of endogenous BDNF. Advantages and disadvantages of the TC tag in comparison with the GFP tag will be further discussed in the presentation.	P2-1-81 セロトニン・ドーパミン神経細胞に対する選択的栄養シグナルの探索 Differential neurotrophic signals for serotonergic and dopaminergic development ○岩倉百合子1, 那波宏之1 ○Yuriko Iwakura1, Hiroyuki Nawa1 新潟大学　脳研究所　分子神経生物学分野1 Dept Mol Neurobiol, Brain Res Inst, Niigata Univ, Niigata1 Monoamine such as serotonin (5HT) and dopamine (DA) is a key modulator of brain development and functions. Recent studies also indicate the implication in the etiology or neuropathology of various brain diseases such as schizophrenia and autism. In early developmental stage, progenitors of both 5HT and DA neurons are located in the mid-hindbrain boundary. They split into two groups and move to the midbrain and hindbrain during development and differentially begin to express tryptophan hydroxylase (TPH; a rate-limiting enzyme in 5HT synthesis) and/or tyrosine hydroxylase (TH; a rate-limiting enzyme in DA synthesis). Of note, neurotrophic factors such as BDNF, GDNF and FGF show distinct trophic activities on 5HT and DA neuronal populations. However, the selectivity and the strength of each neurotrophic factor remains to be characterized for 5HT and DA neurons. Here we examined trophic actions of these factors in rat rhombencephalon cultures. Cultured neurons were treated with several neurotrophic factors and immunostained with the anti-TPH and anti-TH antibodies. In our culture condition, there is the cell population expressing TPH and TH. Among the neurotrophic factors examined, bFGF exerted the highest neurotrophic effects on TPH-positive cells than BDNF and GDNF, whereas the effect on TH-positive cells were most prominent with BDNF and less with GDNF. BDNF and GDNF increased the number of the neurons positive for both TH and TPH. Currently we are analyzing the neurotrphic contribution to the phenotypic differentiation to 5HT and DA neurons.
P2-1-82 GDNFの神経突起伸長促進機構におけるgalectin-1およびgalectin-3の役割 Involvement of galectin-1 and galectin-3 in GDNF-induced neurite outgrowth from adult rat DRG neurons ○三五一憲1, 高久静香1, 柳澤比呂子1, 渡部和彦1, 作見邦彦2, 中別府雄作2堀江秀典4, 門屋利彦5 ○Kazunori Sango1, Shizuka Takaku1, Hiroko Yanagisawa1, Kazuhiko Watabe1, Kunihiko Sakumi2, Yusaku Nakabeppu2, Francoise Poirier3, Hidenori Horie4, Toshihiko Kadoya5 東京都医学研・神経変性病理1, 九大・生医研・脳機能制御2, （株）テクノマスター4, 前橋工科大・生物工学5 ALS/Neuropathy Project, Tokyo Met. Inst. Med. Sci., Tokyo, Japan1, Div. Neurofunc. Genomics, MIB, Kyushu Univ., Fukuoka, Japan2, Institut Jacques Monod, Paris-Diderot Univ., Paris, France3, TechnoMaster Co. Ltd., Yokohama, Japan4, Dept. Biotech., Maebashi Inst. Technol., Maebashi, Japan5 Galectin-1 (GAL-1), a member of a family of β-galactoside binding animal lectins, is predominantly expressed in isolectin B4 (IB4)-binding small non-peptidergic (glial cell line-derived neurotrophic factor (GDNF)-responsive) sensory neurons in the sections of adult rat dorsal root ganglia (DRG). We observed that recombinant GDNF (50 ng/ml) significantly enhanced neurite outgrowth and protein expression of GAL-1 in cultured adult rat DRG neurons. These GDNF effects were attenuated by anti-GDNF family receptor (RET) antibody and phosphatidyl inositol-3'-phosphate-kinase (PI3K) inhibitor LY294002, suggesting that the neurite-outgrowth promoting activity of GDNF is attributable, at least partially, to the upregulation of GAL-1 through RET-PI3K pathway. On the contrary, no significant differences were observed between GAL-1 knockout and wild-type mice in DRG neurite outgrowth in the presence or absence of GDNF. Considerable immunohistochemical colocalization of GAL-3 with GAL-1 in DRG sections and GDNF-induced upregulation of GAL-3 in cultured DRG neurons imply the functional redundancy between these galectins.	P2-1-83 母体免疫活性化により胎児脳脊髄液中の白血病抑制因子が変動する Concentration of leukemia inhibitory factor in fetal cerebrospinal fluid is altered following maternal immune activation ○塚田剛史1, 島村英理子2, 島田ひろき2, 赤井卓也1, 飯塚秀明1, 八田稔久2 ○Tsuyoshi Tsukada1, Eriko Simamura2, Hiroki Shimada2, Takuya Akai1, Hideaki Iizuka1, Toshihisa Hatta2 金沢医科大学脳神経外科1, 金沢医科大学　医学部　解剖学12 Dept Neurosurg, Kanazawa Med Univ, Ishikawa, Japan1, Dept Anat, Kanazawa Med Univ, Ishikawa, Japan2 Epidemiological studies suggest that maternal infection increases the risk of schizophrenia and autism in human offspring. Recent studies suggest that maternal immune activation (MIA) by infection is a cause of schizophrenia and autism in rodent offspring. Interleukin-6 (IL-6) induced by MIA is a possible key mediator of MIA on fetal brain development. However, the effects of maternal IL-6 in fetal neurogenesis remain unclear. In our previous study, we revealed that the maternal-fetal leukemia inhibitory factor (LIF)-adrenocorticotropic hormone (ACTH)-LIF signaling relay pathway (maternal-fetal LIF signal relay) promotes fetal neurogenesis via the placenta in rats. We hypothesized that MIA-induced IL-6 in dams interferes with the maternal-fetal LIF signal relay, resulting in insufficient fetal brain development. In the present study, we examined an alteration in the maternal-fetal LIF signal relay affected by MIA in mice. Polyriboinosinic-polyribocytidylic acid (polyI:C), a synthetic analog of double-stranded RNA, was used to induce MIA. Pregnant dams were intraperitoneally injected with 4 mg/kg or 20 mg/kg polyI:C at 12.5 days post coitum (dpc). The concentrations of ACTH and LIF in fetal serum (FS), and that of LIF in fetal cerebrospinal fluid (CSF) were subsequently measured by ELISA 3 h after the injection. Injection of polyI:C at 4 mg/kg induced an elevation in the mRNA level of Pomc in the placenta and in the concentrations of ACTH and LIF in FS, and that of LIF in fetal CSF. Injection of polyI:C at 20 mg/kg did not affect the mRNA level of Pomc in the placenta and the concentrations of ACTH and LIF in FS, and that of LIF in fetal CSF. The injection of polyI:C at 12.5 dpc altered the mRNA level of Pomc in the placenta and the concentrations of ACTH and LIF in FS, and that of LIF in fetal CSF according to severity of MIA. These results suggest that MIA affects the maternal-fetal LIF signal relay, which causes impairment in neural development of fetuses.
P2-1-84 Withdrawn	P2-1-85 培養細胞を用いたbFGFによるmicroRNA増加のメカニズム解析 Fibroblast growth factor-dependent upregulation of microRNAs in cultured neuronal and glial cells ○山本宜子1, 沼川忠広1,2, 大島淑子1, 岸宗一郎3, 橋戸和夫3, 安達直樹1,2, 功刀浩1,2 ○Noriko Yamamoto1, Tadahiro Numakawa1,2, Yoshiko Ooshima1, Soichiro Kishi3, Kazuo Hashido3, Naoki Adachi1,2, Hiroshi Kunugi1,2 国立精神・神経医療研究センター・神経研・疾病三部1, 科学技術振興機構2, 国立精神・神経医療研究センター・ラジオアイソトープ管理室3 Department of Mental Disorder Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan.1, Core Research for Evolutional Science and Technology Program (CREST), Japan Science and Technology Agency (JST), Saitama, Japan.2, Administrative Section of Radiation Protection, National Institute of Neuroscience, NCNP, Tokyo, Japan.3 MicroRNAs play roles in regulating expression of mRNA and contribute to cell functions. Interestingly, its possibility as a marker for diagnosis of disease has been suggested. Recently, we showed expression of miR-132, one of the brain-specific microRNAs, is elevated in cortical neurons following brain-derived neurotrophic factor (BDNF) stimulation (Numakawa et al 2011.Neurochem Int.). We also examined the effect of BDNF in astroglial cultures although miR-132 levels weren't changed. On the other hand, we examined influence of different growth factor, basic fibroblast growth factor (bFGF), application both neuronal and astroglial cultures and found that significant increased miR-132 levels in both cultures by the bFGF. However, no alteration of miR-9 and 124a, which are other brain-specific microRNAs, in neuronal and glial cultures were observed.
P2-1-86 うつ病患者の脳脊髄液中においてVEGFが増加している Increased cerebrospinal fluid vascular endothelial growth factor levels in patients with major depressive disorder ○若林千里1, 服部功太郎1, 篠山大明1,2, 樋口輝彦3, 功刀浩1 ○Chisato Wakabayashi1, Kotaro Hattori1, Daimei Sasayama1,2, Teruhiko Higuchi3, Hiroshi Kunugi1 （独）国立精神・神経医療研究センター　神経研究所　疾病研究第三部1, 信州大学医学部精神医学講座2, （独）国立精神・神経医療研究センター3 Department of Mental Disorder Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry1, Department of Psychiatry, Shinshu University School of Medicine2, National Center of Neurology and Psychiatry3 Previously, we reported that peripheral levels of vascular endothelial growth factor (VEGF) were increased in patients with major depressive disorder (MDD). However, little information is available for cerebrospinal fluid (CSF) VEGF levels of MDD patients. The present study examined whether CSF VEGF and basic fibroblast growth factor (bFGF) levels are altered in patients with MDD and those with schizophrenia. Race, gender and age-matched healthy control subjects were recruited from local advertisements. Lumbar punctures were performed in 27 patients with schizophrenia, 28 with MDD, and 25 healthy controls. VEGF and bFGF in CSF and plasma levels were determined by BDTM Cytometric Bead Array (CBA) system. VEGF levels in the MDD patients were significantly increased in CSF compared to the controls (P = 0.033). No significant alteration was observed in schizophrenia. There were no alterations for bFGF between healthy control and patients with MDD or those with schizophrenia. Our findings suggest that VEGF of central origin may be involved in the pathophysiology of MDD.

上部に戻る

前に戻る