一般演題（口述）

一般演題（口述） Glia・Myelin

2O3-01 Entry of circulating molecules is restricted by alternative barrier in sensory circumventricular organs of adult mouse brain Morita-Takemura Shoko¹，Furube Eriko²，Mannari Tetsuya²，Okuda Hiroaki¹，Tatsumi Kouko¹，Miyata Seiji²，Wanaka Akio¹ ¹Department of Anatomy and Neuroscience, Nara Medical University，²Department of Applied Biology, Kyoto Institute of Technology Fenestrated capillaries of the sensory circumventricular organs（CVOs）, including the organum vasculosum of the lamina terminalis, the subfornical organ, and the area postrema,lack completeness of the blood-brain barrier（BBB）to sense a variety of blood-derivedmolecules and convey the information into other brain regions. In the present study, we investigated the restriction of parenchymal entry of circulating molecules in the sensory CVOs. Previous and present tracer assays revealed that molecules more than or equal to molecular weight（MW）10,000 stayed in the perivascular space between endothelial and parenchymal basement membranes. On the other hand, low MW tracers such as fluorescein isothiocyanate（MW：389）entered into parenchyma but did not pass beyond the area of dense astrocytic network in the lateral part of the sensory CVOs. Immunoreactivity of tight junction proteins of claudin-1, occludin, and zonula occludens-1 appeared at the parenchyma of the sensory CVOs, suggesting that parenchymal cells, probably astrocytes express tight junction proteins and have barrier functions, especially for low MW molecules. The present study demonstrates that high MW tracer is trapped in perivascular space and diffusion of low MW tracer into parenchyma is limited, indicating that there are alternative barrier that protect neurons or adjacent brain area from toxic molecules in the sensory CVOs.		2O3-02 GlcNAc6ST-1 regulates sulfation of N-glycans and myelination in the peripheral nervous system. Yoshimura Takeshi¹，Hayashi Akiko²，Uchimura Kenji³，Kadomatsu Kenji³，Yamaguchi Yoshihide²，Baba Hiroko²，Ikenaka Kazuhiro¹ ¹Division of Neurobiology and Bioinformatics, NIPS, NINS，²Dept of Molecular Neurobiology, Tokyo Univ of Pharmacy and Life Sci，³Dept of Biochemistry, Graduate School of Med, Nagoya Univ Myelin is a multilamellar, tightly compacted membrane that surrounds axons in the peripheral nervous system（PNS）and central nervous system（CNS）. Because glycoproteins are prominent components of plasma membranes, a growing number of glycoproteins have been identified and characterized in myelin. In this study, we found that PNS myelin had many anionic N-glycans, especially sulfated N-glycans, harbored on glycoproteins in pigs and mice at a much higher rate than CNS myelin. Major sulfated N-glycans in porcine and mouse PNS myelin were identified. The sulfation at the 6-O-GlcNAc position on glycoproteins was highly conserved in PNS myelin between these species. P0 protein, the most abundant glycoprotein involved in PNS myelin compaction, had 6-O-sulfated N-glycans abundantly. Mice deficient in N-Acetylglucosamine 6-O-Sulfotransferase-1（GlcNAc6ST-1）were impaired in the elaboration of 6-O-sulfated N-glycans in PNS myelin. Further, GlcNAc6ST-1 deficiency in mice caused hypomyelination and axonal degeneration. Taken together, these results indicate that GlcNAc6ST-1 plays critical roles in PNS myelination through the elaboration of 6-O-sulfated N-glycans.

2O3-03 The role of myelin sheaths in the regulation of axonal homeostasis Ishibashi Tomoko¹，Hinohara Kiyoshiro¹，Mizuno Juri¹，Takahashi Saki¹，Mikoshiba Katsuhiko²，Baba Hiroko¹ ¹Tokyo Univ. of Pharmacy and Life Sciences，²RIKEN BSI In vertebrates, most axons are insulated with myelin sheaths, and the action potentials that enable the rapid salutatory propagation of the nerve impulses are regenerated at the nodes of Ranvier. Myelin sheaths are not only important for the salutatory propagation, but are also involved in many aspects of neural functioning. However, little is known about the contribution of myelin sheaths to axonal homeostasis such as the regulation of Ca²⁺. The current study examines the distribution of type 1 inositol 1,4,5-trisphosohate receptor（IP₃R1）in Purkinje axons in developmental mice and cerebroside sulfotransferase（CST；a sulfatide synthetic enzyme）deficient mice, which partially lack paranodal axo-glial junctions（PNJs）in both the CNS and PNS. IP₃R1 is a Ca²⁺ channel on the endoplasmic reticulum（ER）and is a predominant isoform in the brain among the three types of IP₃Rs. At 8 days of age before myelination, IP₃R1 was stained throughout Purkinje axons. After formation of complete PNJs, by 21 days of age IP3R1-positive areas had gradually concentrated into myelinated internodes. In CST-deficient mice, IP₃R1 formed focal small swellings in Purkinje internodal axons at 12 days of age, and the number and the size of the swellings increased with age. Although CST-deficient mice do not display any neurological symptoms until 4 to 6 weeks of age, the alteration of the distribution of IP₃R1 was already observed when compact myelin formed. Contrary to in the cerebellum, in the sciatic nerves, the distribution of vesicular ER at nodes of Ranvier differed between CST mutant and wild control mice. These results suggest that the state of myelin sheaths plays a role in the distribution of IP₃R1 and ER in axons and in the regulation of axonal homeostasis.		2O3-04 Upregulation of TN-C and GFAP in reactive astrocytes in injured brain and in primary culture is dependent on aquaporin-4 expression IKESHIMA-KATAOKA Hiroko^1,2，FURUKAWA Motoko²，INUI Sayaka²，IMAMURA Manae²，YASUI Masato² ¹Faculty of Science and Engineering, Waseda University，²Department of Pharmacology ＆ Neuroscience, Keio University School of Medicine We have previously reported that one of the main water channel family, aquaporin 4（AQP4）is exclusively expressed in the endofoot of astrocytes in the brain, and its expression is upregulated after the stab wound to mouse brains or injection of MeHg in common marmosets. Moreover, glial activation was induced by the stab wound injury enhanced by a neuroimmunological function of AQP4 involving osteopontin, which is an inflammatory cytokine inducer. It is already reported that expression of glial fibrillary acidic protein（GFAP）and tenascin-C（TN-C）is prominently upregulated in reactive astrocytes around injury site of the brain by us and other researchers, however the functional roles of these molecules are poorly known. Since AQP4 is highly expressed not only at the membrane of endofoot of astrocyte but also in the cytoplasm of activated astrocytes, we analyzed the functional correlation among GFAP, TN-C and AQP4 using wild type（WT）and AQP4-deficient mice（AQP4/KO）. By the immunohistochemistry and Western blot analysis, high levels of GFAP and TN-C expression were observed in activated astrocytes in WT mice brain；however, insignificant in AQP4/KO mice. Furthermore, lipopolysaccharide（LPS）stimulation activated the primary culture of astrocytes and upregulated GFAP and TN-C expression in the cells from WT mice, while it was slightly upregulated in the cells from AQP4/KO mice. Moreover, mRNA expression level of inflammatory cytokines was examined in primary culture of astrocytes or microglial cells treated with or without LPS, and found that inflammatory cytokines were upregulated in the cells from WT mice, while modest increases were observed in the cells from AQP4/KO mice. Here, we propose that upregulation of GFAP and TN-C in reactive astrocytes induced by stab wound in mouse brain and LPS-stimulated primary culture of astrocytes is dependent on upregulation of AQP4 expression.

2O3-05 Mechanism of process tip localization of astrocytic glutamate transporters Hayashi Mariko，Yasui Masato Dept.Pharmacol. Keio Univ. School of Medicine Following synaptic activation, excitatory neurotransmitter glutamate released to synapses must be removed to terminate the signal and to protect neurons from excitotoxicity. Astrocytes are mostly responsible for the clearance of glutamates. They extend thousands of thin cellular processes among the networks of neurons to approach synapses for this purpose.In this study, we found that the trimeric transmembrane transporter domain of glutamate transporters has a property to localize to the tips of filopodia, while their N- and C-terminal cytoplasmic tails are not required. A transporter domain fragment of a neutral amino acid transporter ASCT1, another trimeric transporter family member, similarly localized to the filopodia tips. Neither transporter activity nor astrocyte specific protein was required for this filopodia tip localization. We also found that the transporter core within filopodia tips strengthens the attachment of filopodia to external substrates, thereby stabilizing the filopodia. The process tip localization of glutamate transporter is hyaluronan dependent. However, CD44, a representative hyaluronan receptor, was not required. Instead, hyaluronan synthase showed hyaluronan dependent interaction with glutamate transporters.		2O3-06 Calcium imaging method for the visualization of subtle and local activity of astrocytes in intact brain Kanemaru Kazunori¹，Sekiya Hiroshi¹，Kitajima Nami¹，Takagi Miki¹，Tanaka Kenji²，Iino Masamitsu¹ ¹Dept. Pharmacol., Grad. Sch. Med., Univ. Tokyo，²Dept. Neurospychiat., Sch. Med., Keio Univ. Astrocytes generate dynamic changes in the intracellular Ca²⁺ level（Ca²⁺ signals）that are thought to regulate their function. In vivo analysis of Ca²⁺ signals with high spatiotemporal resolution may be instrumental in unveiling enigmatic functions of astrocytes. Here we report a method for in vivo astrocytic Ca²⁺ imaging using transgenic mice expressing the ultrasensitive ratiometric Ca²⁺ indicator YC-Nano50 in astrocytes. Using the method, we succeeded in detecting a previously unidentified pattern of spontaneous Ca²⁺ signals（Ca²⁺ twinkles）, which occur predominantly in the fine processes but not the cell body. Upon sensory stimulation, astrocytes initially responded with Ca²⁺ signals at the fine processes, and the Ca²⁺ signal subsequently propagated to the cell body. Ca²⁺ twinkles and evoked Ca²⁺ signals were partially and fully dependent on the Ca²⁺ release via the type 2 IP₃ receptor, respectively. These results suggest that astrocytic fine processes function as a high-sensitivity detector of neuronal activities, and indicate the importance of intracellular Ca²⁺ stores in the regulation of astrocytic functions. Thus, the current method provides a useful tool to uncover the functions of astrocytes in the intact brain.