突起伸展、回路形成 Axonal/Dendritic Growth and Circuit Formation
P2-2-80 皮質脊髄路回路形成における軸索側枝形成因子の探索と解析 Search and analysis of the candidate molecules involved in axon collateralization in the corticospinal tract ○猪口徳一1, 尾身実1,3, 岡雄一郎1,2,3, 佐藤真1,2,3 ○Tokuichi Iguchi1, Minoru Omi1,3, Yuichiro Oka1,2,3, Makoto Sato1,2,3 福井大学 医学部 形態機能医科学講座 組織細胞形態学 神経科学領域1, 福井大学　子どものこころの発達研究センター2, 福井大学　生命科学複合研究教育センター3 Dept Morphol and Physiol Sci, Facul Med Sci, Univ of Fukui, Fukui, Japan1, Research Center for Child Mental Development, Univ of Fukui, Fukui, Japan2, Research and Education Program for Life Sci, Univ of Fukui, Fukui, Japan3 Corticospinal tract is a major subcortical output from the neocortex. It consists of axons from layer V neurons of the cerebral cortex and projects to the spinal cord. During the development of corticospinal tract, axon collaterals protrude form their main shaft toward the nuclei such as superior colliculi, pontine nuclei, and inferior olivary nuclei. Axon collaterals are thought to have an important role in the coordination of neural activities required for the higher brain function. However, molecular mechanisms by which axon collaterals are induced and projected to the specific region of the brain are mostly unknown. Previous reports have suggested that some kind of tropic factor is secreted from the target of the collateral branch extension. To obtain the molecules responsible for the induction of axon collaterals, we performed microarray analyses on the target of axon collaterals (pontine nuclei and superior colliculus). Then, we narrowed down the candidates utilizing in situ hybridization and RT-PCR. We got 6 candidates from the 28868 genes. These candidates were expressed specifically in the target of the collateral branch extension from the corticospinal projection. To analyze the candidate molecules, we developed the analysis method enabling us the manipulation of the gene expression in the pons and the DiI tracing of the corticospinal tract. We also obtained the candidates of the receptor that responds to the signals from the target of axon collaterals searching from our own array data combined with the gene expression profiles of corticospinal neurons.	P2-2-81 難読症関連遺伝子KIAA0319による神経細胞形態制御の分子機構 The molecular mechanism by which KIAA0319 modulates neuronal morphology ○服部光治1, 田頭大志1, 深見瑛1, 梅田健太郎1, 鈴木友美子1, 高嶋悠1, 杉江真梨子1, 松田幸江1, 石井萌1, 馬場敦1, 河野孝夫1 ○Mitsuharu Hattori1, Masashi Tagashira1, Akira Fukami1, Kentaro Umeda1, Yumiko Suzuki1, Yu Takashima1, Mariko Sugie1, Yukie Matsuda1, Moe Ishii1, Atsushi Baba1, Takao Kohno1 名古屋市立大学 大学院薬学研究科 病態生化学分野1 Dept Biomed Sci, Grad Sch Pharmaceut Sci, Nagoya City University1 Developmental dyslexia (DD) is a congenital disorder in which reading and writing is specifically impaired without overall intellectual disability or visual malfunctioning. DD affects as much as 10% of whole population and clarifying its underlying molecular mechanism is critically important. In the last decade, genetic studies identified a few genes that may be involved in the onset of DD, including KIAA0319. KIAA0319 gene encodes a transmembrane protein with its N-terminus and C-terminus in the cytoplasm and the extracellular space, respectively. KIAA0319 has been reported to play a role in neuronal migration in the cerebral cortex, but its underlying mechanism remains unknown.In this study, we investigated expression and subcellular localization of KIAA0319 protein in postnatal brain by taking advantage of newly established monoclonal antibodies. KIAA0319 protein was abundantly expressed in the neurons that were extending dendrites, indicating that it us involved in the regulation of dendrite extension and/or branching. We then found that overexpression of KIAA0319 protein greatly affects neuronal cell morphology. We also identified molecules that bind to the extracellular and intracellular regions of KIAA0319 protein, resepectively. We propose the mechanism how KIAA0319 modulates neuronal morphology, which may lead to understanding, diagnosis, or treatment of DD.
P2-2-82 マウス大脳皮質長連合ニューロンの選択的標識 Selective labeling of long association neurons in the mouse cerebral cortex ○岡雄一郎1,2,3,4, 猪口徳一1,2,3, 佐藤真1,2,3,4 ○Yuichiro Oka1,2,3,4, Tokuichi Iguchi1,2,3, Makoto Sato1,2,3,4 福井大・子どものこころの発達研究セ1, 福井大医　組織細胞形態学・神経科学2, 福井大　生命科学複合研究教育セ3, 大阪大院　連合小児発達　福井校4 Res Center for Child Mental Dev, Univ of Fukui, Fukui, Japan1, Div Cell Biol Neurosci, Dept Morphol Physiol Sci, Univ of Fukui, Fukui, Japan2, Res Edu Prog for Life Sci, Univ of Fukui, Fukui, Japan3, Dept Child Dev, United Grad Sch of Child Dev, Osaka Univ, Kanazawa Univ, Hamamtsu Univ Sch of Med, Chiba Univ, Univ of Fukui4 Long association fibers (LAFs) are the neuronal connections between distant areas located in different cortical lobes within a cerebral hemisphere. Because cortical areas are distinct in functions, neural connections via LAFs are critical for higher cortical functions such as integration of sensory inputs of different modalities and regulation of voluntary motions. Recent studies found the correlation between deficits in LAFs and human mental/developmental diseases like schizophrenia and autism spectrum disorders, suggesting the importance of LAFs in cognitive functions. However, how the neuronal circuits of LAFs are organized at cellular level and how the circuits form during cortical development are still largely unknown. To study the development of LAFs, we explored the genes preferentially expressed in long association neurons (LANs) that project axons through LAFs. We first labeled the LANs and the callosal neurons (CNs, connecting bilateral cerebral hemispheres) in the mouse primary somatosensory cortex (S1) with a fluorescent retrograde tracer injected in the primary motor cortex and the contralateral S1, respectively. The labeled LANs were found in layers 2/3, 5, and 6b, whereas the labeled CNs were in layers 2/3 and 5, confirming intermingling distribution of LANs and CNs in layers 2/3 and 5 in the S1. We then collected the labeled LANs and CNs in each layer separately using laser microdissection. Comparison of their gene expression profiles by microarray analysis identified candidate genes with higher expression in LANs. Double staining with in situ hybridization and retrograde tracing confirmed that several among the candidate genes were indeed expressed in LANs. Using the promoters of these LAN-specific genes we are establishing the system with which we can visualize and manipulate LANs and LAFs to study their development and functions.	P2-2-83 膜トラフィッキングの不均衡による成長円錐ガイダンスの制御機構 Localized imbalance of membrane trafficking for bidirectional growth cone guidance ○糸総るり香1, 戸島拓郎1,2, 上口裕之1 ○Rurika Itofusa1, Takuro Tojima1,2, Hiroyuki Kamiguchi1 理化学研究所　脳科学総合研究センター　神経成長機構研究チーム1, 科学技術振興機構さきがけ2 Lab. for Neuronal Growth Mechanisms, RIKEN BSI, Saitama, Japan1, PRESTO, Japan Science and Technology Agency, Saitama, Japan2 The axonal growth cone migrates along the correct path to form precise neuronal networks. Asymmetric elevation of cytosolic Ca2+ concentration across the growth cone, which is triggered upon the reception of extracellular guidance cues, can mediate both attractive and repulsive growth cone guidance. We previously showed that the directional polarity of growth cone guidance is determined by the source of Ca2+ signals: Ca2+ influx through plasma membrane Ca2+ channels alone triggers repulsion, whereas the Ca2+ influx together with Ca2+-induced Ca2+ release (CICR) through ryanodine receptors on the ER membrane triggers attraction (Ooashi et al., J. Cell Biol., 170, 1159-1167. 2005; Tojima et al., J. Neurosci., 29, 7886-7897. 2009). We also showed that asymmetric clathrin-mediated endocytosis and VAMP2-mediated exocytosis drive growth cone repulsion and attraction, respectively, downstream of Ca2+ signals (Tojima et al., Nat. Neurosci., 10, 58-66. 2007; Tojima et al., Neuron 66, 370-377. 2010; Tojima et al., Nat. Rev. Neurosci., 12, 191-203. 2011). In the present study, we examine the signaling pathways that link Ca2+ signals with clathrin-mediated endocytosis. The Ca2+/calmodulin-dependent protein phosphatase calcineurin acts downstream of Ca2+ influx to facilitate endocytosis for repulsion. On the other hand, Ca2+/calmodulin-dependent protein kinase II and cyclin-dependent protein kinase 5 act downstream of CICR to inhibit endocytosis for attraction. We also identify an endocytic regulatory protein that operates in Ca2+-mediated growth cone guidance. Our results reveal signaling mechanisms underlying localized imbalance of membrane trafficking that drives bidirectional growth cone guidance.
P2-2-84 神経突起伸長におけるNB-2/contactin-5とAPLP1の相互作用の関与 Involvement of the interaction between NB-2/contactin-5 and APLP1 in neurite outgrowth ○霜田靖1, 長田恭平1, 中野悟司1, 平田晴菜1, 渡邉和忠1,2 ○Yasushi Shimoda1, Kyohei Osada1, Satoshi Nakano1, Haruna Hirata1, Kazutada Watanabe1,2 長岡技科大・生物1, 長岡高専2 Dept Bioeng, Nagaoka Univ Tech, Niigata, Japan1, Nagaoka Natl Coll Tech, Niigata, Japan2 NB-2, also referred to as contactin-5, is a GPI-anchored cell adhesion molecule in the immunoglobulin superfamily. NB-2 forms cis-complex with amyloid precursor-like protein 1 (APLP1) belonging to amyloid precursor protein (APP) family. While APP is well know as the precursor to amyloid peptide implicated in Alzheimer's disease, it has been suggested that APP has such biological functions as cell adhesion, neurite outgrowth and synaptogenesis. We have demonstrated that NB-2 exhibits neurite outgrowth-promoting activity toward cortical neurons by activating Fyn. It has been reported that APP trafficking and processing could be regulated by Fyn-dependent phosphorylation. Then we hypothesize that the interaction between NB-2 and APLP1 may be involved in neurite outgrowth mediate by Fyn. Here we examined the interaction between NB-2 and APLP1 in the mouse cerebral cortex and the ectopically expressing cells. First, using newly generated anti-APLP1 and anti-NB-2 antibodies, we carried out immunostaining of the mouse brain, showing that NB-2 and APLP1 proteins were co-localized in the cerebral cortex. Next, we studied the effect of co-expression of NB-2 and APLP1 on the properties of these molecules. To test whether NB-2 affects the phosphorylation or dimerization of APLP1, we transfected HEK293 cells with APLP1 and NB-2 or mock, and subsequently performed immunoprecipitation and western blotting of lysates of the transfected cells. The anti-phosphotyrosine antibody detected no band in the immunoprecipitates with anti-APLP1 either in the presence or absence of NB-2, suggesting that NB-2 may have no effect on APLP1 phosphorylation. Using cells co-transfected with APLP1-HA and -myc, APLP1-myc was detected in the precipitates with anti-HA in the absence of NB-2, but not in the presence of NB-2, suggesting that APLP1 dimerization might be inhibited by interacting with NB-2. There is a possibility that NB-2 might promote neurite outgrowth by inhibiting dimerization of APLP1.	P2-2-85 SPIG1はBDNFのプロセッシングを負に制御する SPIG1 negatively regulates BDNF maturation ○鈴木亮子1, 加藤彰1, 米原圭祐1, 藤川顕寛1, 松本匡史1, 新谷隆史1,2, 作田拓1,2, 野田昌晴1,2 ○Ryoko Suzuki1, Akira Kato1, Keisuke Yonehara1, Akihiro Fujikawa1, Masahito Matsumoto1, Takafumi Shintani1,2, Hiraki Sakuta1,2, Masaharu Noda1,2 基礎生物学研究所　統合神経生物学研究部門1, 総合研究大学院大学2 National Institute for Basic Biology, Okazaki, Japan1, The Graduate University for Advanced Studies, Okazaki, Japan2 In the formation of topographic neural projections, region-specific branch formation along the axon shaft is the first step to establish functional circuits. However, molecular mechanisms underlying the axonal branch formation at a specific position are not fully elucidated. In our large-scale screen for asymmetrically expressing molecules in the embryonic chick retina, we previously isolated SPARC-related protein containing immunoglobulin domains 1 (SPIG1, also known as Follistatin-like protein 4) as one of the dorsal-retina specific molecules expressed in retinal ganglion cells (RGCs). Here, we found that knockdown of SPIG1 in the developing chick retina induces ectopic branching in dorsal retinal axons and fails to form a tight termination zone at the proper position on the tectum. Knockdown of SPIG1 in primary cultured RGCs also enhanced axon branching. Interestingly, it was canceled out by adding a neutralizing antibody against brain-derived neurotrophic factor (BDNF) to the culture medium. SPIG1 showed interaction with pro-form of BDNF, but not with mature BDNF in in vitro experiments. When SPIG1 was coexpressed with BDNF in HEK293T cells, mature BDNF protein released to the culture medium was significantly decreased. We also found that the expression of mature BDNF is increased in the hippocampus of the SPIG1 knockout mouse, compared with the wild-type control. Taken altogether, our findings suggest that SPIG1 regulates the axonal branching through the control of BDNF maturation.
P2-2-86 樹状突起形成時におけるGABAシグナル伝達経路とユビキチンプロテアソームシステムの関係 The ubiquitin proteasome system acts downstream of GABA signaling and regulates dendrite development ○渡辺康仁1 ○Yasuhito Watanabe1, Konstantin Khodosevich1, Hannah Monyer1 Department of Clinical Neurobiology, The German Cancer Research Center1 Department of Clinical Neurobiology, The German Cancer Research Center, Heidelberg, Germany1 The stimulating effect of GABA (gamma-aminobutyric acid) signaling on dendrite growth has been indirectly linked with downstream suppression of DISC1 (disrupted in schizophrenia 1) and its interacting molecule FEZ1 (fasciculation and elongation protein zeta 1). How DISC1 and FEZ1 are downregulated is currently unknown. We speculated whether and how these players interact with the ubiquitin proteasome system (UPS), the latter is also known to be involved in dendrite development. Our experiments provide evidence that FEZ1 interacts with Cdc20 (cell division cycle 20) /APC (adenomatosis polyposis coli) complex, and is degraded by UPS. We will discuss our findings in the context of dendrite development in postnatal dentate granule neurons, and will provide new data explaining the underlying mechanism.	P2-2-87 コンドロイチン硫酸はOtx2取り込みを促し臨界期を持続的に制御する Persistent contribution of chondroitin sulfate (CS) to Otx2 uptake in cortical plasticity ○侯旭濱1, 武内恒成2, 宮田真路3, 吉岡慧史1, 北川裕之3, 五十嵐道弘2, 杉山清佳1 ○Xubin Hou1, Kosei Takeuchi2, Shinji Miyata3, Satoshi Yoshioka1, Hiroshi Kitagawa3, Michihiro Igarashi2, Sayaka Sugiyama1 新潟大院・医歯・神経発達研1, 新潟大院・医歯・分子細胞機能2, 神戸薬大・生化3 Lab of Neuronal Development, Grad Sch Medical and Dental Sci, Univ of Niigata, Niigata, Japan1, Division of Molecular and Cellular Biology, Grad Sch Medical and Dental Sci, Univ of Niigata, Niigata, Japan2, Dept Biochem, Kobe Pharma Univ, Kobe, Japan3 Binocular vision is established in the primary visual cortex (V1) through activity-dependent competition during early postnatal life. Neuronal circuit rewiring reflects a well-orchestrated balance of excitation and inhibition. A previous report showed that transfer of Otx2 homeoprotein into parvalbumin (PV)-cells regulates the cortical plasticity. Otx2 uptake accelerates maturation of PV-cell circuits which are enwrapped by perineuronal net (PNN) structures enriched with chondroitin sulfate (CS) proteoglycans. Here, we show that CS enrichment conversely promotes Otx2 uptake into PV-cells to regulate the plasticity throughout life. CS N-acetylgalctosaminyltransferase-1 is one of the enzymes synthesizing CS, which catalyzes the key step reaction in the CS synthesis pathway. In the null mice, total amount of CS was reduced by half in V1, similar to the reduction of an interaction of Wisteria floribunda agglutinin (WFA)-lectin. Visual evoked potentials (VEPs) recorded from the binocular zone revealed that visual acuity was normally developed in those mice, however the onset of the plasticity was impaired. Importantly, both Otx2 accumulation and PV-cell maturation were disrupted. Infusion of Otx2 protein into V1 rescued the onset of the critical period, increasing WFA-positive cells. Extracellular recording showed that prolonged discharge which reflects immature inhibition was increased. Then, the plasticity was recovered by enhancing GABA function with diazepam treatment (> 4 days). Once triggered by diazepam (P24-28), the plastic state was strikingly sustained even one month later (< P60). This persistent plasticity was degraded by another treatment of diazepam (P60-64), indicating that diazepam triggered both the onset and offset of the plasticity. Taken together, the enrichment of CS sugar chains may serve as a positive feedback loop to promote Otx2 uptake and further CS maturation, ultimately regulating the cortical plasticity over a lifetime.
P2-2-88 RhoファミリーGタンパク質による細胞形態形成の定量数理モデル Quantitative modeling of cell morphogenesis by Rho family small GTPases ○作村諭一1,2, 田中大河3, 池田和司3, 中村岳史4 ○Yuichi Sakumura1,2, Taiga Tanaka3, Kazushi Ikeda3, Takeshi Nakamura4 愛知県大・情報1, 奈良先端大・バイオ2, 奈良先端大・情報3, 東京理大・生命医4 Sch Info Sci and Tech, Aichi Pref Univ1, Grad Sch Bio Sci, NAIST2, Grad Sch Info Sci, NAIST3, Res Inst Bio Sci, Tokyo Univ of Sci4 Rho family GTPases (Cdc42/Rac1/RhoA) are known as key molecules for cell morphogenesis including neuron; Cdc42 and Rac1 accelerate actin polymerization for extending a cell edge and RhoA activates Myosin II to make the edge contraction. However, this biochemical framework is not consistent with the analyses by the recent studies that suggest that the edge protrusion activates Cdc42 and Rac1 and is induced by RhoA. In most studies, a time-shift cross-correlation analysis is used for extracting causality between molecular activities and cell edge movement. The inconsistency between biochemical and morphological viewpoints and the corresponding molecular mechanism for cell morphogenesis are still open questions. In this study, first we computed what the time-shift cross-correlation analysis extracts from the time series of Rho GTPase activities and cell edge velocity. Our result suggests that the time-shift cross-correlation analysis does not necessarily show causality between two time series. Secondly, using experimental data, we developed the quantitative mathematical model which describes cell edge velocity regulated by GTPase activities and mechanical forces which change cell edge location. Using the model, we show that the complicated relationship between cell edge velocity and GTPase activities can be quantitatively explained. The model raises the possibility that a nonlinear process related to mechanical forces exists between the velocity and the GTPase activities	P2-2-89 マウス脊髄灰白質内皮質脊髄軸索投射の生後発達の定量解析 Quantitative analysis of postnatal development of corticospinal axons projecting to the spinal gray matter (C7) in the mouse ○村部直之1, 桜井正樹1 ○Naoyuki Murabe1, Masaki Sakurai1 帝京大・医・生理1 Dept. Physiol. Teikyo Univ. Sch. Med.1 Rodent corticospinal (CS) axons postnatally show dynamic changes of axon and synapse distributions in the spinal cord. Previously, we showed that the CS neurons distribute densely in a large area of infant cortex followed by their decrease in density by the adult. This study addresses postnatal development of CS axons in the spinal gray matter at the cervical enlargement segment C7, which is involved in control of forelimb movement. To visualize every CS axon, we used transgenic mice, referred to as CST-YFP mice, in which the CS axons express yellow fluorescent protein (YFP). These animals express YFP under a neuron-specific promoter thy-1 only when forebrain specific Cre recombinase excises a sequence that prevents expression of YFP. Distribution of the CS axons in the gray matter at C7 and its change during postnatal development were investigated with a laser scanning confocal microscope. GFP-immunoreactive axons ran through the ventralmost of the dorsal column where the CS tract is located in rodents and enter the gray matter. At postnatal day 7 (P7), fine CS axons extended radially and reached the margin of the gray matter. Small fusiform/granular varicosities were evenly spaced along the CS axons. These had small dots that were vesicular glutamate transporter I (VGluT1) immunoreactive. As development proceeds, distribution of CS axons becomes segregated to four regions. The gray matter received dense projection of the CS axons dorsomedially and laterally, and moderate projections ventromedially, and light projections ventrolaterally. CS axons had large varicosities with VGluT1 immunoreactive dots. Quantification of CS axons running ventrolaterally showed an increase in number from P7 to P14. Densities of CS axons peaked at P14, and then declined thereafter. These results, together with previous results, suggest that a population of the CS neurons eliminate synapses after innervating infant gray matter.
P2-2-90 マウスプルキンエ細胞樹状突起形態形成および維持におけるRORαの時期特異的役割 Stage-specific roles of RORα in formation and maintenance of Purkinje cell dendrites in mice ○竹尾ゆかり1, 三浦会里子1, 掛川渉1, 柚崎通介1 ○Yukari Takeo1, Eriko Miura1, Wataru Kakegawa1, Michisuke Yuzaki1 慶應義塾大学　医学部　生理学1 School of Medicine, Keio University1 Morphology of dendrites determines how information is received and integrated in neurons. The shape of dendrites is not only determined genetically but also modified by neuronal activities during development. Nevertheless, little is known about molecular mechanisms underlying such dynamic dendritogenesis. Cerebellar Purkinje cells (PCs) show well-characterized dynamic morphological changes in dendrites during development. At birth, PCs exhibit a fusiform shape with long primitive dendrites. In a few days, the primitive dendrites regress and PCs turn into the stellate-cell stage with multiple short dendrites. Around P8 in mice, thicker stem dendrites emerge and in the next 2 weeks highly branched mature dendrites are formed. Retinoid-related orphan receptor alpha (RORα) is the responsible gene of staggerer mutant mice which display severe cerebellar ataxia. Previous studies suggest that RORα is responsible for dendritic regression at the fusiform stage. However, specific roles of RORα have not been directly analyzed in vivo. Moreover, although the expression of RORα persists throughout life, its significance in mature PCs has been unclear. Here we addressed these questions using in utero electroporation of PCs and tamoxifen (4-OHT)-induced shRNA-based knockdown of RORα. When knockdown vector was directly overexpressed in PCs from the embryonic stage, the dendrites of PCs exhibited fusiform-like abnormality. In addition, when RORα was knocked down by injecting 4-OHT at P4, PCs showed striking defects in dendritic formation, indicating that RORα are necessary also for normal dendritogenesis after the stellate-cell stage. Furthermore, we injected 4-OHT at P21 and tested whether RORα also played a role in the maintenance of mature PC dendrites. These results indicate that RORα not only regulates the exit from the fusiform stage, but also plays new stage-specific roles during development of PCs in vivo.	P2-2-91 大脳皮質神経回路形成におけるT－カドヘリンの役割 Involvement of T-cadherin in axonal pathfinding of neocortical neurons ○早野祐紀1, , 山本亘彦1 ○Yuki Hayano1, Hong Zhao1, Nobuhiko Yamamoto1 大阪大学大学院　生命機能研究科　細胞分子神経生物学1 Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan1 During development, axons having the same destination form bundles and pathways in the CNS. Here, we studied the molecular mechanism for axonal pathway formation of cortical efferents. We previously demonstrated that T-cadherin (T-cad), one of the classical cadherins, was expressed in deep layer cell axons projecting to subcortical structures, but not in upper layer callosal axons projecting to the contralateral cortex. To understand the functional role of T-cad during development, we performed ectopic expression and knock-down of T-cad using an in vivo electroporation technique. Over expression in upper layer cells, which normally do not express T-cad, resulted in abnormal projection towards the internal capsule and the cerebral peduncle where endogenous T-cad is expressed. When a shRNA plasmid was introduced into deep layer cells, knocked-down axons oriented to the contralateral cortex along the pathway in which T-cad is not expressed. These findings indicate that T-cad is involved in pathway formation by being expressed in subsets of cortical neurons and by regulating fasciculation or interactions with the surrounding cells.
P2-2-92 ドパミンニューロンによる線条体ニューロンの神経支配おけるにインテグリンα5β1の関与 Involvement of integrin α5β1 in dopaminergic innervation of striatal neruons ○泉安彦1, 脇田誓子1, 神原知里1, 中井利恵1, 久米利明1, 赤池昭紀1,2 ○Yasuhiko Izumi1, Seiko Wakita1, Chisato Kanbara1, Toshie Nakai1, Toshiaki Kume1, Akinori Akaike1,2 京都大院・薬・薬品作用解析1, 名大院・創薬2 Dept Pharmacol, Grad Sch Pharm Sci, Kyoto Univ, Kyoto, Japan1, Grad. Sch. Pharm. Sci., Nagoya Univ. Nagoya, Japan2 The main pathological feature of Parkinson disease is a selective degeneration of the nigrostriatal pathway, and regeneration of the neuroprojection is one of the promising therapeutic approaches. However, the precise mechanisms by which dopaminergic axons innervate striatal neurons are still unclear. Integrins, heterodimers of α and β subunits that function as cell adhesion molecules, play an important role in embryonic development of central nervous system. In this study, we investigated the involvement of integrins in dopaminergic innervation of striatal neurons. We established a novel method to evaluate dopaminergic innervation of striatal neurons by using primary dissociated cells. Mecencephalic and striatal cells were dissected from rat embryos, then a mecencephalic cell region was formed adjacent to a striatal cell region on a plastic coverslip. After paired-culturing for 11 days, dopaminergic neurons in the mesencephalic cell region extended their axons to the striatal cell region. Integrin β1 subunit plays a central role in the function. Neutralizing integrin β1 antibody suppressed dopaminergic innervation of the striatal neurons. To identify the integrin α subunit, we examined the effect of selective integrin-blocking peptides and neutralizing integrin α antibody. As a result, an integrin α5β1-blocking peptide A5-1 (VILVIF) and neutralizing integrin α5 antibody attenuated the dopaminergic innervation of striatal neurons. Integrin α5 and β1 subunits were expressed at the axonal growth cones and somas of dopaminergic neurons. In addition, dopaminergic axonal outgrowth was enhanced by coating with fibronectin, a ligand of integrin α5β1. These results suggest that dopaminergic axons innervate striatal neurons through integrin α5β1.	P2-2-93 マウスバレル皮質のシナプス結合特異性はDnmt3bのエピジェネティックな制御を受ける Dnmt3b epigenetically regulates specificity of synaptic connections in mouse barrel cortex ○足澤悦子1,3,6, 豊田峻輔2,3, 小林俊寛4,5, 三宝誠6, 平林真澄3,6, 八木健2,3, 吉村由美子1 ○Etsuko Tarusawa1,3,6, Syunsuke Toyoda2,3, Toshihiro Kobayashi4,5, Makoto Sanbo6, Masumi Hirabayashi3,6, Takeshi Yagi2,3, Yumiko Yoshimura1 生理研・神経分化1, 阪大大学院・生命機能・心生物学研究室2, JST-CREST3, 東大医科研・幹細胞治療研究センター4, JST-ERATO5, 生理研・遺伝子改変動物作製室6 Div Dev of neurophysiol, NIPS, Okazaki1, KOKORO-biology, Lab. for Integrated Biology, Grad. Sch. of Front. Biosci., Osaka Univ. Osaka2, JST, CREST, Osaka univ. Osaka3, Cent. for Stem Cell Biol. and Regenerative Med., Inst of Med.l Sci., Univ of Tokyo, Tokyo4, JST, ERATO, Tokyo5, Sec. Mammalian transgenesis , NIPS, Okazaki6 Clustered protocadherin (cPcdh), one of the cell adhesion molecules, consists of 58 isoforms. Some of these isoforms are stochastically expressed in individual neurons. We have recently demonstrated that this stochastic expression is epigenetically regulated by the DNA methyltransferase Dnmt3b during early development. In cells lacking Dnmt3b, many types of cPcdh isoforms were commonly expressed and the diversity of cPcdh expression was decreased. This diversity might be implicated in cell recognition leading to the generation of specific synaptic connections, because cPcdh isoforms interact homophilically and contribute to spine formation. To test this hypothesis, we established iPS cells from embryonic lethal Dnmt3b KO mice and produced chimera mice by injection of the iPS cells into wild-type (WT) mouse blastocysts. Dnmt3b KO cells were kept in the chimera mice until synaptic connections were established. We conducted whole-cell recordings from layer 4 cells in barrel cortical slices prepared from 2 week-old mice, and analyzed EPSCs evoked in WT and KO cells by firing of other cortical cells in layer 2-6, resulting from focal glutamate uncaging using laser scanning photostimulation. WT cells received strong excitatory inputs, mostly from cells in layer 4, but KO cells received substantial inputs from all layers. Photostimulation of layer 4 evoked larger EPSCs in amplitude in WT than KO cells. The effect of Dnmt3b KO was further characterized by dual whole-cell recordings from layer 4 cells. There was no difference in the detection probability of excitatory connections and EPSC amplitude in connected pairs between WT and KO cell pairs, but reciprocal connections were found less frequently in KO than WT cell pairs. These results suggest that cPcdh expression with diverse combinations of isoforms, regulated by Dnmt3-mediated DNA methylation, contributes to the establishment of specific synaptic connections in barrel cortex.
P2-2-94 大脳皮質ニューロン軸索でみられるネトリン-1依存的な糸状仮足形成ならびに軸索分岐形成の形態学的解析 Morphological analysis of netrin-1-induced filopodial protrusion and axon branching from cortical axon shafts ○松本英子1, 永島雅文1 ○Hideko Matsumoto1, Masabumi Nagashima1 埼玉医大・医・解剖1 Dept Anatomy, Saitama Med Univ, Saitama1 It is well known that a multifunctional axon guidance cue netrin-1 induces axon outgrowth in developing cerebral cortical neurons via one of its receptors DCC (deleted in colorectal cancer). On the other hand, we and others reported that netrin-1 induced axon branching in dissociated cortical neurons prepared from hamster neonates: In this case, it is believed that netrin-1 first induces filopodial protrusions from cortical axon shafts and then a portion of the protrusions develops into axon branches. The aim of this study is to elucidate the regulation of these two functions of netrin-1, that is, induction of axon outgrowth and induction of axon branching, in cortical neurons. We observed that the numbers of axon branches were increased in dissociated cortical cultures prepared from hamster neonates and from embryonic day (E) 16 mouse embryos by stimulating the neurons with netrin-1 for 4 h. The netrin-1-induced axon branching was attenuated by an application of function-blocking antibody against DCC, suggesting a significant contribution of DCC in axon branching. No increase was found in the number of axon branches in cortical neurons prepared from E14 mice, indicating that the role of netrin-1 changes during development of cortical neurons. We also carried out morphological analysis of filopodial protrusions from cortical axon shafts shortly after netrin-1 application, employing a novel type of scanning electron microscope.	P2-2-95 ショウジョウバエ成虫感覚ニューロンにおける受容野形成の制御機構 Adult Drosophila sensory neurons use two distinct mechanisms to specify their dendritic fields ○安永桂一郎1, 榎本和生1 ○Kei-ichiro Yasunaga1, Kazuo Emoto1 （公財）大阪バイオサイエンス研究所・神経細胞生物学1 Dept Cell Biol, OBI, Osaka1 Dendritic arbors achieve appropriate coverage of their receptive fields in the nervous system. For example, in retinal ganglion cells, dendritic arbors of functionally related neurons achieve complete but non-overlapping coverage of a specific layer, a pattern called tiling. We have studied cellular and molecular mechanisms underlying dendritic field specification using Drosophila sensory neurons as a model system. Drosophila larval sensory neurons tile the whole body wall. We previously showed that this tiling arrangement is dominantly mediated by dendro-dendritic repulsive interactions between neighboring cells. Here, we report that adult sensory neurons use two distinct mechanisms to specify their dendritic fields. Our time-lapse imaging experiments revealed that adult sensory neurons tiled dorsal and lateral parts of the body wall along the anterior-posterior axis. Genetic ablation caused neighboring cells to extend their dendrites into the territory of ablated neurons, indicating that sensory neurons tile through dendro-dendritic repulsive interactions between neighboring neurons. Next, we focused on the ventral part, where dendritic arbors ceased their growth before contacting with sternites, epithelial tissues localized in the most ventral area. We performed a genetic screen for genes involved in this growth regulation and found that local dendritic growth around sternites was negatively regulated through Wnt-mediated signaling, raising the possibility that Wnts are secreted from sternites and act as a repulsive signaling molecule. These results provide a novel mechanistic insight into dendritic field specification.
P2-2-96 発生期のほ乳類大脳皮質でのBtbd3を介した神経活性依存的な樹状突起の形態制御について BTB/POZ domain containing Btbd3 mediates activity-dependent dendritic field patterning in mammalian developing cortex ○松居亜寿香1, , 吉田彩1, 下郡智美1 ○Asuka Matsui1, May Tran1, Aya Yoshida1, Tomomi Shimogori1 理研・BSI・視床発生1 BSI, RIKEN, Wako1 Regulation of dendritic patterning by neuronal activity is one of the most remarkable features to assemble efficient neuronal circuit in the developing brain. For example, in the cat and monkey primary visual cortex, deprivation of visual input affects dendritic field patterning to remodel the circuit. To elucidate the molecular mechanisms of this circuit development, we first labeled ferret primary visual cortical neurons with fluorescent protein using in utero electroporation. In normal condition, neurons have symmetric basal dendrites, however, when neuronal input from contralateral eye is altered, cortical neurons changed dendritic field toward column with input from ipsilateral eye. In contrast, in mouse visual cortex, dendritic field is not affected by monocular deprivation. We have previously shown that Btbd3, BTB/POZ domain containing 3, controls dendritic patterning in mouse barrel field. We tested its expression in the mouse and ferret visual cortex and revealed that Btbd3 is strongly expressed in the ferret but not in the mouse primary visual cortex. This result suggested that expression of Btbd3 in ferret visual cortex allow cortical neurons to change dendritic pattern toward higher input. To test this hypothesis, Btbd3 is ectopically expressed in the binocular region of mouse primary visual cortex and shape of dendritic field is tested after monocular deprivation. As a consequence, mouse visual cortex neurons that received ectopic Btbd3 responded to neuronal input and changed pattern of dendritic morphology. Taken together, we revealed conserved function of Btbd3 in activity-dependent dendrite remodeling in different cortical areas and in different species.	P2-2-97 BDNF pro-peptideは軸索伸長を誘導し、ヒトBDNFの一塩基多型によってその効果が異なる Human BDNF pro-peptide elicits axon growth and the extension activity was modulated by human BDNF polymorphism ○小塚孝司1,2,3, 水井利幸1,2, 熊ノ郷晴子1,2, 清末和之1,2, 小島正己1,2 ○Takashi Kozuka1,2,3, Toshiyuki Mizui1,2, Haruko Kumanogoh1,2, Kazuyuki Kiyosue1,2, Masami Kojima1,2 産総研・健康工学・バイオインターフェース1, 科学技術振興機構　戦略的創造研究推進事業2, 関西大学・化学生命・生命生物3 National Institute of Advanced Industrial Science and Technology (AIST), Ikeda, Japan1, Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Kawaguchi, Japan2, Laboratory of Neurobiology, Department of Life Science and Biotechnology, Materials and Bioengineering, Kansai University, Suita, Japan3 Since its discovery, emerging evidence demonstrates brain-derived neurotrophic factor (BDNF) promotes multiple neuronal events, including neuronal survival, differentiation, development and synaptic modulation. BDNF is initially synthesized as a precursor, proBDNF, and subsequently proteolytic processing leads to the production of BDNF and its pro-peptide (BDNF pro-peptide). We here shows that the BDNF pro-peptide has an activity to promote axon growth and the amino acid substitution in the pro-peptide (human BDNF polymorphism Val66Met) lost the growth activity of the BDNF pro-peptide. To assay the activity of the BDNF pro-peptides to the neurite growth, dissociated hippocampal neurons were prepared from embryonic 17-day mice by Banker Method, and, to visualize the growing neurite, GFP-expressing plasmid was transfected. Cultured neurons were treated with wild-type (Val) or mutant (Met) BDNF pro-peptide (10 ng / ml). Two days later, GFP-positive neurons were imaged and then morphological analysis was done. Interestingly, the wild-type pro-peptide promoted axon growth whereas the Met pro-peptide did not had the activity. However, the biological activity of Val-BDNF pro-peptide was not seen in knockout animals lacking a pan-neurotrophin receptor p75NTR. These data demonstrate that the BDNF pro-peptide elicits axon growth probably through the activation of p75NTR, and that the val66met BDNF polymorphism impairs the pro-peptide activity.
P2-2-98 LOTUSの神経突起伸長作用 Promoting effect of LOTUS on neurite outgrowth ○栗原裕司1, 伊藤拓夢2, 池谷真澄1, 五嶋良郎2, 竹居光太郎1 ○Yuji Kurihara1, Hiromu Ito2, Masumi Iketani1, Yoshio Goshima2, Kohtaro Takei1 横浜市立大学　医学群　生体システム医科学系　生命医科学部門1, 横浜市立大学大学院 医学研究科 分子薬理神経生物学教室2 Div. of Med. Life Sci., Sch. of Med., Yokohama City Univ., Yokohama, Japan1, Dept. of Mol. Pharmacol. & Neurobiol., Grad. Sch. of Med., Yokohama City Univ., Yokohama, Japan2 Axon growth inhibitors such as Nogo proteins, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp) and B lymphocyte stimulator (BLyS) bind to Nogo receptor-1 (NgR1) commonly, which prevents the functional recovery after injury in the adult central nervous system. Lateral olfactory tract usher substance (LOTUS) serves for axonal bundle formation through the antagonism for NgR1 on Nogo. Recently, we have revealed that LOTUS functions as a potent endogenous antagonist for NgR1 on all of the four ligands. However, another function of LOTUS remains absolutely unknown. In this study, we found that neurite outgrowth was promoted in retinal ganglion cell (RGC) and dorsal root ganglion (DRG) neurons on LOTUS substrate. Moreover, RGC neurons in ngr1-deficient mice also showed the similar promoting effect of LOTUS on neurite outgrowth. We are searching for unidentified molecule(s) which mediate the promoting effect of LOTUS on neurite outgrowth with Neuro2A cells derived from mouse neuroblastoma cell line. LOTUS bound to retinoic acid (RA)-treated Neuro2A cells. Furthermore, neurite outgrowth was promoted in RA-treated Neuro2A cells on LOTUS substrate. The data suggest that LOTUS promotes neurite outgrowth in RGC and DRG neurons and this promoting action is mediated by unidentified LOTUS binding molecule(s) which may be also expressed in RA-treated Neuro2A cells.

上部に戻る

前に戻る