Poster

Poster 8 Development 2 ポスター 8 発生・分化2

P8-1 Role of LOTUS, a neural circuit formation factor in memory function 神経回路形成因子LOTUSの記憶・認知機能における役割 Nishida Ryohei（西田遼平），杉山礼奈，栗原裕司，竹居光太郎 Molecular Medical Bioscience Laboratory, Yokohama City University Graduate School of Medical Life Science, Yokohama, Japan Overcoming the higher brain dysfunction that exhibits memory disorders, such as dementia, is an important and urgent issue for mankind and many researches for the issue are currently conducted all over the world. Nogo binds to Nogo receptor 1 (NgR1) and the binding inhibits axon growth and synapse formation, thereby affects nerve regeneration and memory function. We previously identified lateral olfactory guide substance (LOTUS) as an endogenous NgR1 antagonist. Although LOTUS is abundantly expressed in the adult brain, it was reported that the expression level of LOTUS in the hippocampus decreased in accordance with aging in rodents and the memory function decreased associated with aging. However, the role of LOTUS in memory function has remained to be elucidated. Therefore, we examined whether LOTUS is involved in memory function. We first evaluated roles of LOTUS in memory function using social recognition test in wild type (WT) mice, LOTUS-gene knocking-out (LOTUS- KO) mice and LOTUS gene overexpressing transgenic (LOTUS-Tg) mice. As a result, we found that LOTUS-KO mice impaired social recognition when compared with WT mice. In contrast, LOTUS-Tg mice enhanced social recognition. Next, we examined roles of LOTUS in synapse formation in cultured hippocampal neurons, and found that decrease of synaptic density in LOTUS-KO mice whereas increase of the density in LOTUS-Tg mice. Furthermore, we also found that decrease of neurogenesis in the adult hippocampus of LOTUS-KO mice, while increase in LOTUS-Tg mice. These findings suggest that LOTUS may involve in memory formation accompanied with synapse formation and neurogenesis in hippocampus.		P8-2 Blockade by LOTUS, a neural circuit formation factor, of axonal growth inhibition induced by Nogo-PirB interaction Nogo-PirB相互作用により誘起される軸索伸長阻害に対する神経回路形成因子LOTUSの抑制効果 Kurihara Yuji（栗原裕司），竹居光太郎 Mol. Med. Biosci. Lab., Grad. Sch. of Med. Life Sci., Yokohama City Univ. The neurons re-extend their axons very poorly following damage in the adult mammalian central nervous system (CNS). This limited ability is caused by axonal growth inhibitors such as Nogo, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp), which commonly bind to both receptors of Nogo receptor-1 (NgR1) and paired immunoglobulin-like receptor B (PirB). We previously found that lateral olfactory tract usher substance (LOTUS) interacts with NgR1 and blocks the binding of Nogo, MAG and OMgp to NgR1, resulting in suppression of axonal growth inhibition induced by these inhibitors. However, the role of LOTUS in regulation of PirB function remains to be elucidated. In this study, we identified PirB as a LOTUS binding protein. The Nogo-binding to PirB was abolished in COS7 cells overexpressing both of LOTUS and PirB. Treatment with the soluble form of LOTUS suppressed Nogo-induced growth cone collapse and neurite outgrowth inhibition in cultured dorsal root ganglion neurons from Ngr1-deficient mice, where PirB is expressed. Our data demonstrate that LOTUS functions as an antagonist for PirB as well as NgR1, suggesting that LOTUS may enable the damaged CNS neurons to overcome PirB- and NgR1-mediated axonal growth inhibition and thereby to regenerate their axons.

P8-3 The N-terminal region of soluble LOTUS forms a stable structure for secretion and functions in inhibition of Nogo receptor -mediated signaling 神経回路形成因子LOTUSの可溶型タンパク質のN末端側29-532aaはNgR1シグナル阻害機能をもつ構成的分泌ドメインである Tezuka Haruna（手塚はるな），川上裕，高宅博和，栗原裕司，竹居光太郎 Molecular Medical Bioscience Laboratory, Yokohama City University Graduate School of Medical Life Science Lateral olfactory tract usher substance (LOTUS) is a potent antagonist for Nogo receptor-1 (NgR1), and is expected to promote axonal regeneration after injuries in the central nervous system. We previously reported that the soluble form of LOTUS (s-LOTUS: 29-606 amino acid (aa) in number) inhibits NgR1-mediated signaling, resulting in inhibition of growth cone collapse and neurite outgrowth inhibition induced by all NgR1 ligands. The current protein domain databases shows that LOTUS consists of three independent domains: FG-GAP (29-445aa), UA (446-540aa) and EC (541-606aa). Molecular evolution analysis suggests it is speculated that the N-terminal region of s-LOTUS without EC domain contains functional domain. Here, we examined the molecular characteristics of the N-terminal region of s-LOTUS. The FG-GAP domain alone was not secreted from the transfected cells, while each of UA or EC domain alone was secreted. We also found that (29-532aa) was secreted, whereas (29-531aa) was little secreted. These findings are well consistent with the estimate by disorder prediction analysis. These results implicate that (29-532aa) is a minimal region to form a stable structure for secretion. Next, we examined the binding capacity, and found that (29-532aa) bound to both of NgR1 and its co-receptor p75NTR. Furthermore, the functional analysis revealed that (29-532aa) had the potential to inhibit NgR1-mediated signaling in olfactory blub neurons from lotus-deficient mice. The data implicate that the N-terminal region of s-LOTUS (29-532aa), including FG-GAP domain and the large part of UA domain, forms a stable structure, which is presumably consisted of beta-propeller structure, and have antagonistic function of s-LOTUS to inhibit NgR1-mediated signaling.		P8-4 Expression pattern of LOTUS, a neural circuit formation factor, in the central nervous system 神経回路形成因子LOTUSの中枢神経系における発現様式 Norisue Yui（則末優衣），西田遼平，栗原裕司，竹居光太郎 Mol. Med. Biosci. Lab., Yokohama City Univ. Grad. Sch. of Med. Life. Sci., Yokohama Japan Lateral olfactory tract usher substance (LOTUS), a neural circuit formation factor, plays an important role in lateral olfactory tract formation through its antagonistic action to Nogo receptor-1 (NgR1) function which induces axon growth inhibition in the central nervous system (CNS). Therefore, LOTUS is an endogenous antagonist of NgR1. However, expression pattern of LOTUS protein remains to be elucidated. Here, we examined LOTUS expression in the CNS by immunohistochemistry. First, we confirmed specificity of monoclonal antibody used in this study in LOTUS-knocking out (LOTUS-KO) mice and found no detectable immunodeposit of this antibody in LOTUS-KO mice, indicating that the antibody recognizes LOTUS protein specifically. LOTUS was expressed in many neural projection pathways in the CNS. Interestingly, we found LOTUS expression in axonal projections of Papez circuit that has been well known to control emotion and memory. LOTUS expression in Papez circuit implies possible association of LOTUS physiological function with memory and emotion. Next, we examined LOTUS expression in cultured hippocampal neurons. We found that LOTUS was expressed in axon and dendrite of the hippocampal neurons and also post-synaptic region of the dendrite. Since it has been reported that Nogo and NgR1 are expressed in synaptic region, LOTUS expressed in synapse may function in the regulation of synaptic activities through antagonism for NgR1 function.

P8-5 The soluble form of LOTUS suppresses BLyS-and CSPG-induced Nogo receptor signaling 神経回路形成因子LOTUSの可溶型タンパク質はBLyS, CSPGsに誘起されるNgR1シグナルを抑制する Kawakami Yutaka（川上裕），齋藤優，中川良太，栗原裕司，竹居光太郎 Department of Medical Life Science, Yokohama City University Graduate School, Yokohama, Japan Lateral olfactory tract usher substance (LOTUS) is a potent antagonist for Nogo receptor type 1 (NgR1). LOTUS antagonizes NgR1 functions induced by all five ligands: myelin-associated inhibitors (Nogo, myelin-associated glycoprotein, oligodendrocyte myelin glycoprotein), B lymphocyte stimulator (BLyS) and chondroitin sulfate proteoglycans (CSPGs). We have recently reported that the soluble form of LOTUS (s-LOTUS) inhibits NgR1 signaling induced by myelin-associated inhibitors, leading to suppression of growth cone collapse and neurite outgrowth inhibition induced by these inhibitors. In this study, we examined whether s-LOTUS inhibited NgR1 signaling induced by other ligands such as BLyS and CSPGs. In line with our previous report that has shown the binding of LOTUS with BLyS, s-LOTUS bound with BLyS and blocked the binding of BLyS to membrane-bound LOTUS expressed in COS7 cells. However, treatment or pretreatment with s-LOTUS did not inhibit the binding of BLyS to NgR1. In functional analysis, s-LOTUS inhibited growth cone collapse and neurite outgrowth inhibition induced by BLyS and CSPGs in cultured chick dorsal root ganglion neurons. Furthermore, exogenously administered s-LOTUS compensated for the lack of the suppressive function of endogenous LOTUS in NgR1-mediated signaling in olfactory bulb neurons of lotus-knockout mice. These findings indicate that s-LOTUS inhibits NgR1-mediated signaling induced by all five ligands including BLyS and CSPGs, and thereby s-LOTUS has the potential as a therapeutic agent for neuronal regeneration after CNS injuries.		P8-6 The role of AUTS2 gene in the corticogenesis 自閉症感受性遺伝子AUTS2の大脳皮質構築における役割 Shimaoka Kazumi（嶋岡可純）¹，堀啓¹，坂本亜沙美¹，阿部学²，崎村建司²，星野幹雄¹ ¹Dept. of Biochem. & Cell. Biol., NCNP, Tokyo ²Dept. of Cell. Neurobiol.,BRI, Umiv. of Niigata Autism susceptibility candidate 2 (AUTS2) has been implicated as the gene associated with various psychiatric disorders such as autism spectrum disorders (ASDs) and intellectual disabilities (ID). In mouse developing CNS, AUTS2 is highly expressed at several brain regions responsible for the higher brain functions such as cerebral cortex and hippocampus. We have previously demonstrated that cytoplasmic AUTS2 regulated the neuronal migration and neurite formation in the developing cerebral cortex. Furthermore, nuclear AUTS2 has been reported to be involved in the transcriptional regulation of multiple genes for neural development by interacting with the Polycomb group protein complex 1 (PRC1). However, there remain many questions about the physiological roles for AUTS2 in the brain development. In this study, we analyzed the function of AUTS2 in the cerebral corticogenesis. We found that loss of Auts2 in mice caused the reduction in the thickness of cortical plate. Pulse-labeling assay revealed that the number of EdU-positive cortical neurons was significantly decreased in the Auts2 mutant mice, and most notably in the upper layer neurons. On the other hand, the number of neural progenitor cells at the ventricular zone was not different between wild-type (WT) and Auts2 mutant mice whereas the number of neural progenitor cells at M-phase or S-phase was dramatically decreased in the Auts2 mutant mice compared to WT mice, implying that defects of AUTS2 induced the cell cycle delay of neural progenitor cells and resulted in a decrease in the production of cortical neurons in Auts2 mutant mice. Taken together, these results suggest that AUTS2 plays a key role for the control of neural progenitor to postmitotic cell transition during later cerebral corticogenesis.

P8-7 The role of Auts2 in the dentate gyrus development マウス発生期海馬歯状回における自閉症感受性遺伝子AUTS2の役割 Egusa Saki（江草早紀）¹，堀啓¹，坂本亜紗美¹，阿部学²，崎村建司²，郷康広³，星野幹雄¹ ¹Dept. of Biochem. & Cell. Biol., NCNP, Tokyo ²Dept. of Cell. Neurobiol., BRI, Univ. of Niigata ³Dept. of Brain Sci., Center for Novel Sci. Initiative, NIPS, Okazaki, Aichi The Dentate gyrus (DG) is a part of the limbic system crucial for higher brain functions, and malformation of the DG is associated with neuropsychiatric disorders. The molecular mechanisms underlying the pathogenesis are, however, largely unknown. Autism susceptibility candidate 2 (AUTS2) is a gene associated with a broad range of psychiatric illnesses. Auts2 is expressed at multiple brain regions as prefrontal cortex, hippocampus and DG as well as cerebellum. It has been previously reported that the cytoplasmic AUTS2 is involved in the regulation of neuronal migration and neuritogenesis in the developing cerebral cortex whereas the nuclear AUTS2 acts as a transcriptional activator. Although many evidences suggest that AUTS2 plays the crucial roles for the neurocognitive function, the physiological function of AUTS2 in brain development, however, remains to be elucidated. In this study, we investigated the role of AUTS2 in the DG development using the mutant mice that Auts2 gene is conditionally ablated at telencephalon. Immunohistochemistry shows that AUTS2 is expressed in the neural progenitor cells and the granule neurons, but is not detected in neural stem cells . We found that, the size of DG in the Auts2 mutant mice was drastically reduced in both the developing and mature brains. In the mutant mice, the number of granule neurons was decreased, and unexpectedly, the neural stem cells were also significantly reduced in total number and tertiary matrix, but in VZ of DG, the number of the neural stem cells were increased. It is suggested that AUTS2 may be involved in not only in the regulation of neuronal migration, but also the regulation of the granule cell differentiation. Based on these results, we demonstrate the crucial role of Auts2 for the DG development.		P8-8 CD38 is Required for Dendritic Organization in Visual Cortex and Hippocampus 視覚野および海馬の樹状突起形成におけるCD38の役割 Matsuzaki Hideo（松崎秀夫） Research Center for Child Mental Development, University of Fukui Morphological screening of mouse brains with known behavioral deficits can give great insight into the relationship between brain regions and their behavior. Oxytocin- and CD38-deficient mice have previously been shown to have behavioral phenotypes, such as restrictions in social memory, social interactions, and maternal behavior. CD38 is reported as an autism spectrum disorder (ASD) candidate gene and its behavioral phenotypes may be linked to ASD. To address whether these behavioral phenotypes relate to brain pathology and neuronal morphology, here we investigate the morphological changes in the CD38-deficient mice brains, with focus on the pathology and neuronal morphology of the cortex and hippocampus, using Nissl staining, immunohistochemistry, and Golgi staining. No difference was found in terms of cortical layer thickness. However, we found abnormalities in the number of neurons and neuronal morphology in the visual cortex and dentate gyrus (DG). In particular, there were arborisation differences between CD38-/- and CD38+/+ mice in the apical dendrites of the visual cortex and hippocampal CA1 pyramidal neurons. The data suggest that CD38 is implicated in appropriate development of brain regions important for social behavior.

P8-9 SAM68-specific splicing controls proper 3'UTR isoform selection of interleukin 1-receptor accessory protein through ALE usage SAM68はシナプス形成因子IL1RAcPの3'UTRアイソファーム選択を制御する Iijima Takatoshi（飯島崇利）¹，飯島陽子¹，田中正視¹，鈴木暁子¹，Scheiffele Peter² ¹Tokai University Institute of Innovative Science and Technology, Medical Division ²Biozentrum, University of Basel, Basel, Switzerland Neuronal alternative splicing is dynamically regulated in a spatiotemporal fashion. We previously found that STAR family proteins (SAM68, SLM1, SLM2) regulate spatiotemporal alternative splicing in the nervous system. However, the whole aspect of alternative splicing programs governed by STARs remains unclear. We deciphered the alternative splicing programs of SAM68 and SLM1 proteins using transcriptomics. Here, we reveal that SAM68 and SLM1 encode distinct alternative splicing programs; SAM68 preferentially controls alternative last exon (ALE) splicing. Interleukin 1-receptor accessory protein (Il1rap) is a novel target for SAM68. The usage of Il1rap ALEs results in mainly two variants encoding two functionally different isoforms, a membrane-bound (mIL1RAcP) and a soluble (sIL1RAcP) type. The brain exclusively expresses mIL1RAcP. SAM68 knockout results in remarkable conversion into sIL1RAcP in the brain, which significantly disturbs IL1RAcP neuronal function. Thus, we uncovered the critical role of proper neuronal isoform selection through ALE choice by the SAM68-specific splicing program.