神経化学教育口演セッション1
Cerebellum
O3-8-3-1
AAVベクター血管内投与による霊長類の広範な中枢神経領域への遺伝子導入
Widespread transduction of neurons in the primate brain using intrathecal injection of AAV vectors

○飯田麻子1, 滝野直美1, 宮内ひとみ1, 柴田宏昭2, 小野文子3, 村松慎一1
○Asako Iida1, Naomi Takino1, Hitomi Miyauchi1, Hiroaki Shibata2, Fumiko Ono3, Shin-ichi Muramatsu1
自治医科大学大学院 医学研究科 神経内科学1, 医薬基盤研究所 つくば霊長類医科学研究センター2, 予防衛生協会 研究支援企画部3
Dept Neurology, Jichi Medical Univ, Tochigi, Japan1, Primate Research Center, National Institute of Biomedical Innovation, Ibaraki, Japan2, The Corporation for Production and Research of Laboratory Primates, Ibaraki, Japan3

Recombinant adeno-associated virus (AAV) vectors are a powerful vehicle with applications in both basic neuroscience and gene therapy for neurological diseases. Infusion of the recombinant AAV vectors via stereotaxic surgery into target brain areas provides continuous, long-term expression of transgenes. However, for diseases that affect large areas of the central nervous system (CNS), local injection of these vectors yields less than optimal results. Recently AAV9-derived vectors have gained much attention because these vectors can cross the blood-brain barrier. Following intravenous injection of AAV9 vectors, transduction of spinal motor neurons has been achieved in fetal and neonate mice as well as in adult cat and pigs; however, in adult mice and nonhuman primate, most of transduced cells are astrocytes. Thus, gene delivery extensively to the neurons in primate CNS remains a challenge. Here, we present an efficient method for selective neuronal transduction in broad areas of primate CNS. Intrathecal administration of pseudotype AAV9/3 vectors with some modifications of the capsid protein resulted in extensive and widespread expression of the transgene in the brain and spinal cord of adult cynomolgus monkeys. Furthermore neuron-specific promoters were used for selective transduction of neurons without significant microglial activation. These AAV vectors provide a new tool for creating animal models and developing gene therapies for neurological disorders.
O3-8-3-2
マウス神経系前駆細胞において膜輸送体OCTN1を介したエルゴチオネインの取り込みは神経細胞への分化能を促進する
Solute carrier OCTN1-mediated ergothioneine uptake promotes neuronal differentiation in mouse neural progenitor cells

○石本尚大1, 中道範隆1, 細谷拓史1, 杉浦智子1, 加藤将夫1
○Takahiro Ishimoto1, Noritaka Nakamichi1, Hiroshi Hosotani1, Tomoko Sugiura1, Yukio Kato1
金沢大学大学院 医学系研究科 創薬科学専攻1
Faculty of Pharmacy, Kanazawa University, Kanazawa1

We have recently demonstrated that polyspecific transporter OCTN1/SLC22A4 is functionally expressed in neural progenitor cells (NPCs). OCTN1 accepts a naturally occurring antioxidant ergothioneine (ERGO) as a typical substrate, although its physiological function has not yet been clarified. Elucidation of physiological function of this polyspecific transporter in NPCs may lead to find a novel target for promotion of neurogenesis with an aim to treat neurodegenerative disorders. The aim of the present study was to clarify it using mouse primary cultured NPCs and embryonic carcinoma P19 cells differentiated into neural progenitor-like cells. In cultured NPCs, on real-time PCR analysis, expression of OCTN1 was much higher than its homologues OCTN2 and OCTN3, whereas that of OCT1-3, MATE1 and PMAT could not be detected. These cells exhibited time-dependent uptake of [3H]ERGO. To examine the role of OCTN1 in differentiation into neuronal and glial cells, NPCs and P19 cells were dispersed after the culture under floating conditions, followed by further culture under adhesion conditions. Exposure to ERGO induced a marked increase in the number of cells immunoreactive for the neuronal marker βIII-tubulin with a concomitant decrease in that for the astroglial marker GFAP in cultured NPCs, whereas knockdown of OCTN1 increased the number of cells immunoreactive for GFAP with a decrease in those immunoreactive for βIII-tubulin in P19 cells. Next, to examine molecular mechanisms underlying promotion of neuronal differentiation by ERGO, we determined expression level of various basic helix-loop-helix (bHLH) transcription factors by real-time PCR. In cultured NPCs, exposure to ERGO induced up-regulation of Math1 gene, a positive regulator of neuronal differentiation, and down-regulation of Hes1 gene, a negative one. These results suggest that OCTN1-mediated ERGO uptake may promote differentiation into neurons possibly via the regulation of bHLH transcription factors in mouse NPCs.
O3-8-3-3
新規化合物Denosominはvimentinを分泌するアストロサイトを介して、脊髄損傷後の軸索伸展を促進する
A novel compound, Denosomin, promotes axonal growth via astrocytes secreting vimentin after spinal cord injury

○執行美智子1, 勅使川原匡1, 久保山友晴1, 長田愛子1, 杉本健二2, 松谷裕二2, 東田千尋1
○Michiko Shigyo1, Kiyoshi Teshigawara1, Tomoharu Kuboyama1, Aiko Nagata1, Kenji Sugimoto2, Yuji Matsuya2, Chihiro Tohda1
富山大学 和漢医薬学総合研究所 神経機能学分野1, 富山大学 薬学部 薬品製造学研究室2
Div of Neuromedical Science, Inst of Natural Med, Univ of Toyama1, Lab of Organochem Design and Synthesis, Fac of Pharmaceutical Sci, Univ of Toyama2

In the spinal cord injury (SCI), axonal growth is inhibited by the glial scar, where reactive astrocytes secrete condroitin sulfate proteoglycan (CSPG). We previously found that a novel compound Denosomin improved hindlimb dysfunction, promoted axonal growth and increased astrocyte density in the injured spinal cord of mice. In addition, Denosomin enhanced astrocyte proliferation and secretion of the intermediate filament protein vimentin in cultured astrocytes. In this study, we aimed to examine the role of vimentin in axonal growth and in SCI mice. We examined the effect of vimentin on axonal growth in cortical neurons (ddY mouse, E14). By culture on inhibitory CSPG substrate, axonal growth was inhibited, whereas vimentin (10 ng/ml) enhanced axonal growth even on CSPG substrate. Next, the role of vimentin in SCI mice was examined. Denosomin (20 μmol/kg/day) or vehicle was orally administrated to SCI mice for 7 days from 1h after the injury, and immunohistochemical analyses were performed. Denosomin administration significantly increased the frequency of 5-HT-positive axons associating with vimentin in SCI mice. Denosomin significantly increased the frequency of vimentin-positive astrocytes, but not CSPG-positive astrocytes. The distribution of vimentin primarily overlapped with that of laminin, an extracellular matrix protein, and surrounded β-actin distribution, which is cytoplasmic protein in the scar of Denosomin-treated mice. Then, vimentin (0.4 μg) or vehicle was directly injected into the injured region of SCI mice 5 min after the injury. Vimentin improved motor dysfunction during 10 days after injury in SCI mice. These results indicate that Densomin increases astrocytes secreting vimentin inside glial scars of SCI mice, and 5-HT-positive axonal growth occurrs in a vimentin-associated manner. These findings are the first to demonstrate a novel role of vimentin in SCI mice and that Denosomin converts the role of astrocyte to stimulating axonal growth.
O3-8-3-4
NecdinとBmi1の拮抗的相互作用による神経幹細胞の増殖制御
Regulation of neural stem cell proliferation via antagonistic interplaybetween necdin and Bmi1

○南出良平1, 長谷川孝一1, 藤原一志郎1, 吉川和明1
○Ryohei Minamide1, Koichi Hasegawa1, Kazushiro Fujiwara1, Kazuaki Yoshikawa1
大阪大学大学院 蛋白質研究所 神経発生制御研究室1
Laboratory of Regulation of Neuronal Development, Institute for Protein Research, Osaka University1

During embryonic development, neural stem cells (NSCs), multipotent cells that differentiated into neurons and glial cells, exhibit a high proliferation activity under undifferentiated conditions. However, the molecular mechanism that controls the proliferation rate of NSCs remains elusive. Necdin is predominantly expressed in postmitotic neurons. Necdin interacts with many regulatory proteins, such as E2F1 or p53, and suppresses cell proliferation and apoptosis. It has recently been reported that necdin is expressed in several stem cells in non-neural tissues and regulates their proliferation and quiescence. In this study, we examined whether necdin regulates the proliferation of NSCs during embryonic development. Immunohistochemical analysis revealed that Ki67-positive cells and pH3-positive cells were increased in the cortex of necdin-deficient mice at embryonic day (E) 14.5, whereas expression of the cyclin-dependent kinase inhibitor p16Ink4A was decreased. Because Bmi1 downregulates the expression of p16Ink4A to promote NSC proliferation, we examined the physical and functional interactions between necdin and Bmi1. By immunoprecipitation assay, we detected the endogenous interaction between necdin and Bmi1 in E14.5 mouse forebrain. Necdin bound to helix-turn-helix domain of Bmi1 by co-immunoprecipitation and in vitro binding assays. Furthermore, luciferase reporter assay revealed that necdin canceled Bmi1-induced p16Ink4A promoter suppression, whereas Bmi1 canceled necdin-induced E2F1-promoter suppression. We also demonstrated using BrdU incorporation assay that lentivirus-mediated overexpression of Bmi1 led to a significant increase in the S-phase population of wild-type NSCs, but this effect was diminished in necdin-deficient NSCs. These results suggest that necdin and Bmi1 control the proliferation of embryonic cortical NSCs by their antagonistic interactions.
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