軸索再生、組織修復 Axonal Regeneration and Tissue Repair
P2-2-65 ゼブラフィッシュ視神経損傷後のleukemia inhibitory factor (LIF)とSox2の発現上昇について Upregulation of leukemia inhibitory factor (LIF) and Sox2 after optic nerve injury in adult zebrafish ○大貝和裕1, 杉谷加代1, 郡山恵樹2, 中島廣志1, 加藤聖2 ○Kazuhiro Ogai1, Kayo Sugitani1, Yoshiki Koriyama2, Hiroshi Nakashima1, Satoru Kato2 金沢大院・医・保1, 金沢大院・医・脳情報分子2 Div Health Sci, Grad Sch Med Sci, Kanazawa Univ, Ishikawa1, Dept Mol Neurobiol, Grad Sch Med Sci, Kanazawa Univ, Ishikawa2 Fish retinal ganglion cells (RGCs) can survive and regrow their axons following optic nerve injury (ONI) whereas mammalian RGCs cannot survive. Since the number of surviving RGCs following ONI in zebrafish does not change and there is no sign of cell death or proliferation, it is supposed that the state of RGCs might be transit from fully differentiated state to axon regeneration-ready state in which RGCs can regrow their new axons. To date, however, there is no insight into the molecules that are thought to transit the state of injured RGCs in zebrafish. Thus we focused on the expression of Sox2 in zebrafish RGCs after ONI, as Sox2 is one of the "reprogramming factors" that enable somatic cells to transit into pluripotent stem cells (PSCs). Also we focused on the expression of leukemia inhibitory factor (LIF) which activates Sox2 expression in PSCs. RT-PCR analysis revealed that lif mRNA was upregulated at 3 days post-injury (dpi) followed by the sequential upregulation of sox2 mRNA in 5-10 dpi. Interestingly, the expression of lif mRNA might be regulated by alternative splicing. The upregulation of both lif and sox2 mRNA was limited in RGCs, revealed by in situ hybridization. Immunohistochemical staining showed that the upregulation of LIF and Sox2 protein was also limited in RGCs. To understand the function of Sox2 on optic nerve regeneration, Sox2 knockdown experiment by applying sox2-targeted morpholino and detailed analysis of LIF-Jak-Stat3-Sox2 pathway are under way.	P2-2-66 ラット顔面神経核における抑制性ニューロン/シナプスに関する運動ニューロン傷害の影響 Influence of motoneuron lesion on inhibitory neurons/synapses in rat facial nucleus ○菊池麗香1, 高坂新一2, 中嶋一行1,2 ○Reika Kikuchi1, Shinichi Kohsaka2, Kazuyuki Nakajima1,2 創価大学　工学部　生命情報工学科1, 国立精神・神経センター神経研究所2 Dept. of Bioinformatics, Faculty of Engineering, Soka University, Tokyo1, Dept. of Neurochemistry, National Institute of Neuroscience, Tokyo2 Transection of facial nerves in adult rats leads to a retrograde injury in the motoneuron cell bodies. The functioning of the injured motoneurons declines along with the down-regulated levels of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT). These results are a reflection of the depressed state of motoneurons. Accompanying these phenomena, we clarified that glutamic acid decarboxylase (GAD) for synthesizing γ-aminobutyric acid (GABA), vesicular GABA transporter (VGAT) for GABA uptake into vesicle and GABA transporter-1 (GAT-1) for GABA reuptake into pre-synapse decreased during 3-14 days after transection in the ipsilateral nucleus. Likewise, the levels of GABAA receptor (GABAAR) (ionotropic receptor) were demonstrated to reduce in injured nucleus during 3-14 days after insult. The cells expressing GABAAR in facial nucleus were identified as motoneurons, because GABAAR was immunohistochemically co-localized with motoneuron marker N-metyl D-aspartate receptor subtype 3B (NR3B). Although these GAD, GAT-1 and GABAAR were found to be low at 14 days after injury, it was possible that the amounts recover to control levels after 14 days. However, the analysis in the later stage (after 14 days) remains to be elucidated. Therefore, in this study we investigated the influence of motoneuron insult on the levels of GAT-1 and GABAAR at 14-35 days following insult. Immunoblotting and immunohistochemistry revealed that the low levels of GAT-1 and GABAAR were sustained for 35 days. However, Nissl staining signified that facial motoneurons in the transected nucleus were almost all alive. Thus, these results demonstrate that transected adult rat facial motoneurons are functionally down-regulated with low levels of GAT-1 and GABAAR during 35 days following insult.
P2-2-67 新生血管由来のプロスタサイクリンは成体中枢神経回路の修復を促進する Angiogenesis promotes neuronal rewiring through vessel-derived prostacyclin in the adult central nervous system ○村松里衣子1, 高橋千里1,2, 三宅周蔵1,2, 山下俊英1,2 ○Rieko Muramatsu1, Chisato Takahashi1,2, Shuzo Miyake1,2, Toshihide Yamashita1,2 大阪大学大学院　医学系研究科　分子神経科学1 Dept Mol Neurosci, Grad Sch of Med, Osaka Univ, Osaka1, JST-CREST2 Angiogenesis is a prominent feature of central nervous system (CNS) disease and has roles in both the continued promotion of inflammation and the subsequent repair processes. Here we report that prostacyclin (or prostaglandin I2 (PGI2) derived from new vessels promotes axonal remodeling of injured neuronal networks after CNS inflammation. In a localized model of experimental autoimmune encephalomyelitis (EAE), new vessels formed around the inflammatory lesion, followed by sprouting of adjacent corticospinal tract (CST) fibers. These sprouting fibers formed a compensatory motor circuit, leading to recovery of motor function. Capillary endothelial cell derived prostacyclin bound to its receptor, the type I prostaglandin receptor (IP receptor), on CST neurons, promoting sprouting of CST fibers and contributing to the repair process. Inhibition of prostacyclin receptor signaling impaired motor recovery, whereas the IP receptor agonist iloprost promoted axonal remodeling and motor recovery after the induction of EAE. These findings reveal an important function of angiogenesis in neuronal rewiring and suggest that prostacyclin is a promising molecule for enhancing functional recovery from CNS disease.	P2-2-68 成体マウスの海馬における血管新生 Angiogenesis in the dentate gyrus of adult mouse brains ○澤速人1, 萬成哲也1, 宮田清司1 ○Hayato Sawa1, Tetsuya Mannari1, Seiji Miyata1 京都工芸繊維大院・応用生物学1 Dept of Appl Biol, Kyoto Inst of Technol, Kyoto, Japan1 The major subclasses of proliferating cells have been characterized in the subgranular zone (SGZ)of the hippocampal dentate gyrus even in adult. Continuous neurogenesis in the SGZ is shown to be responsible for learning and memory and deficiency of neurogenesis results in serious impairment of them. Although the proliferation of endothelial cells is demonstrated in the SGZ and the vascular density is increased by environmental enrichment and exercise in adults, little is known about angiogenesis processes in the hippocampus. In the present study, we observe that angiogenic filopodia were present in the hippocampus of adult mouse by using immunostaining of a vascular basement membrane marker laminin. The angiogenic filopodia were fine, usually less than several µm in diameter, and extended from the existing thick microvessels. The density of angiogenic filopodia was higher at the DG than the CA1 and CA3 regions. Surprisingly, angiogenic filopodia were more frequently seen at molecular layer and dentate hilus than granular layer, although proliferating endothelial cells were specifically detected at SGZ of granular cell layer in the dentate gyrus. There was no intimate association between doublecortin-positive neural progenitor cells and angiogenic filopodia. In conclusion, the present study firstly demonstrates the presence of elongating fine filopodia of vasculature in hippocampal dentate gyrus of adult mice.
P2-2-69 脊髄損傷後の軸索再生におけるCRMPの機能解明 The roles of CRMP in axonal sprouting and recovery after spinal cord injury ○長井淳1, 北村圭輝1, 山下直也2, 五嶋良郎2, 大島登志男1 ○Jun Nagai1, Yoshiteru Kitamura1, Naoya Yamashita2, Yoshio Goshima2, Toshio Ohshima1 早稲田大学大学院　先進理工学研究科　生命医科学専攻1, 横山市立大・院・医・分子薬理神経生物2 Dept. of Life Sci. Med. Biosci., Sch. of Adv. Sci. Eng., Waseda Univ., Tokyo1, Dept. of Mol. Pharmacol. Neurobiol., Grad. Sch. of Med., Yokohama City Univ., Tokyo2 Spinal cord injury (SCI) currently has no promising treatment because severed spinal axons do not become restroed through the adult central nervous system (CNS), whereas axotomized peripheral axons exhibit a robust regenerative response after axotomy. An axon guidance molecule semaphorin3A and myelin-associated inhibitors, including myelin-associated glycoprotein (MAG), potently restrict axonal elongation in CNS and greatly contribute to the failure of CNS axonal regeneration. However, the genetic deletion of a single molecule of these inhibitors does not provide sufficient regeneration after CNS axonal injuries. Thus, manipulating multiple factors controlling axonal growth failure by focusing on common downstream molecules of these signaling pathways may facilitate the development of more effective therapies for CNS trauma. Collapsin response mediator proteins (CRMPs), especially CRMP2 and long-form CRMP4, have been reported to regulate cytoskeletal dynamics in these inhibitory signaling pathways, but the roles in axonal regeneration after CNS injury in vivo remain elucidated. Here, we focus on the role of CRMP4 on neurite and axonal growth in adult neurons via combined in vitro and in vivo approaches using the CRMP4KO mutant mouse model. In cultured dorsal root ganglion (DRG) neurons, loss of CRMP4 prevents MAG-induced inhibition of axonal outgrowth and growth cone collapse. In the control mice, stable microtubules were reduced in injured axons at 2 h after SCI, whereas this reduction was significantly suppressed in CRMP4KO mice. Moreover, axonal sprouting of raphe spinal fibers in CRMP4KO mice correlates with improved locomotion. We are now analyzing axonal sprouting and functional recovery after SCI in CRMP2 knock-in mice in which phosphorylation leading to inactivation of CRMP2 are eliminated. These results will provide us new insights of CRMPs as a possible therapeutic target of SCI treatment.	P2-2-70 マウス脳梗塞モデルにおける徐放化製剤のニューロン新生促進効果 Sustained release of growth factors from gelatin hydrogel microspheres enhances neurogenesis in the adult mouse brain ○中口加奈子1, 神農英雄1,2, 金子奈穂子1, 澤田雅人1, 匹田貴夫1, 斎藤伸治2, 田畑泰彦3, 澤本和延1 ○Kanako Nakaguchi1, Hideo Jinnou1,2, Naoko Kaneko1, Masato Sawada1, Takao Hikita1, Shinji Saitoh2, Yasuhiko Tabata3, Kazunobu Sawamoto1 名古屋市立大学大学院医学研究科再生医学分野1, 名古屋市立大学大学院医学研究科新生児・小児医学2, 京都大学再生医学研究所生体組織工学研究部門3 Dept. of Dev. Regen. Biol., Nagoya City Univ. Grad. Sch. Med. Sci., Nagoya, Japan1, Dept. of Neonatol. Pediatr., Nagoya City Univ. Grad. Sch. Med. Sci., Nagoya, Japan2, Dept. of Biomaterials., Kyoto Univ. Institute for Frontier Medical Sciences., Kyoto, Japan3 New neurons are continuously generated by endogenous neural stem cells in the ventricular-subventricular zone (V-SVZ) of the adult mammalian brain. The new neurons form chain-like aggregates and migrate to the olfactory bulb through the rostral migratory (RMS). Using a middle cerebral artery occlusion (MCAO) model that causes infarction of ipsilateral striatum and adjacent neocortex, we have previously reported that the V-SVZ-derived new neurons migrate radially in the injured striatum along the blood vessels. However, number of these migrating neurons is too small to compensate the lost neurons. We tested whether delivering growth factors via gelatin hydrogel microspheres would support neurogenesis in the V-SVZ. Insulin-like growth factor-1 (IGF-1)-containing microspheres increased the number of new neurons in the V-SVZ. Hepatocyte growth factor (HGF)-containing microspheres increased the number of new neurons migrating from the V-SVZ towards the injured striatum in a stroke model in mouse. These results suggest that the strategy of using gelatin hydrogel microspheres to achieve the sustained release of growth factors holds promise for the clinical regeneration of damaged brain tissues from endogenous neural stem cells in the adult V-SVZ.

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