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2O2-1 Effects of amyloid-β and lipopolysaccharide on insulin expression in cultured astrocytes Katsura Takano，Keisuke Koarashi，Mitsuaki Moriyama，Yoichi Nakamura Lab. Integ. Physiol., Vet. Sci., Osaka Pref. Univ. Insulin resistance in brain has been reported in Alzheimer’s diseases (AD). Insulin signaling is important for homeostasis in brain function and reported to be disturbed in neurons leading to tau phosphorylation and neurofibrillary tangles. Many investigations of insulin in neurons have been reported; however, it has not been reported whether astrocytes also produce insulin and insulin released from astrocytes could affect neurons. In the present study, we assessed the expression of insulin and the effects of amyloid β_1-42 (Aβ) and lipopolysaccharide (LPS) in cultured rat brain astrocytes. We found that astrocytes expressed preproinsulin mRNA and insulin protein, and released insulin in the medium measured by using an ELISA kit. Aβ and LPS decreased these preproinsulin mRNA and insulin protein expressions but did not significantly affect insulin release in the medium. Aβ and LPS increased reactive oxygen species (ROS) in astrocytes assessed by using 2’,7’-dichlorodihydrofluorescein diacetate. Antioxidants, glutathione and N-acetylcysteine, restored the decrease in insulin expression by Aβ and LPS. In neuroblastoma SH-SY5Y, conditioned medium of astrocytes significantly increased phosphorylation of Akt, which is known to be a downstream of neuronal insulin signaling. These results suggest that astrocytes might express and secrete insulin, Aβ and LPS decreased the insulin expression, and that ROS elevation might be involved in decreased insulin expression by Aβ and LPS. Aβ could decrease insulin expression in astrocytes as well as neurons and the decreased insulin could be a novel disturbing mechanism for brain insulin signaling in AD.		2O2-2 Microglia-induced astrocytic P2Y₁ receptor down-regulation causes astrocyte scar formation Youichi Shinozaki¹，Keisuke Shibata¹，Eiji Shigetomi¹，Keitaro Yoshida²，Kazuhiro Ikenaka³，Kenji F Tanaka²，Schuichi Koizumi¹ ¹Dept. Neuropharmacol., Interdiscip. Grad. Sch. Med. Univ. Yamanashi，²Dept. Neuropsychiat., Keio Univ. Sch. Med.，³Dev. Neurobiol. Bioinfo., Natl. Inst. Physiol. Sci. Microglia and astrocytes become reactive in response to various brain injuries including traumatic brain injury (TBI). However, the coordination of this reactivity and its relation to pathophysiology are unclear. Here, we report that the reactive microglia enhance reactive astrogliosis and neuroprotective astrocyte scar formation in which down-regulation of P2Y₁ receptor in astrocytes was essential. We found that TBI initially caused microglial activation in the injury core, followed by reactive astrogliosis in the peri-injured region and formation of neuroprotective astrocyte scar. In vitro experiments clarified that TBI-induced reactive microglia produce pro-inflammatory cytokines and down-regulate P2Y₁ receptor expression in astrocytes thereby enhancing reactive astrogliosis. Pharmacological inhibition of P2Y₁ receptors or deletion of P2ry1 gene significantly enhanced astrocytic responses after in vitro TBI. In mice, knocking out of P2ry1 gene accelerated reactive astrogliosis and neuroprotective astrocyte scar formation after TBI. To further confirm the role of astrocytic P2Y₁ receptors, we generated two double transgenic mice: astrocyte-specific P2Y₁ overexpression (Astro-P2Y₁OE) or knockdown (Astro-P2Y₁KD). Astro-P2Y₁OE mice counteracted reactive astrogliosis and astrocyte scar formation, while astro-P2Y₁KD mice showed accelerated scar formation and reduced neuronal damage, respectively. Taken together, our data show that gliotransmitter-mediated interaction between microglia and astrocytes is essential for the formation of neuroprotective astrocytes after brain injury.

2O2-3 Multiple origins of perilesional nestin-expressing reactive astrocytes following closed-head injury Mitsuhiro Morita，Natsuki Okazaki，Hinata Matsuda，Erika Tsuji Dept Biol, Grad Sch Sci, Kobe Univ The expression of a neural stem cell marker, nestin is a common hallmark of perilesional reactive astrocytes, and the multipotency of reactive astrocytes for producing neurons was suggested by culturing cells from brain lesion. These neural stem cell-like aspects of reactive astrocytes are assumed to reflect the injury-induced reprogramming of astrocytes or the migration of SVZ or RMS cells to lesion. In order to address this issue, the origin and fate of nestin-expressing reactive astrocytes were determined by creating closed head injuries in Nestin-CreERT2/CAG-CAT^f/f-GFP mice. The GFP-labelling of pre-existing neural stem cells by a tamoxifen treatment before injury located GFP+ cells perilesionally, as well as along the white matter between lesion and SVZ after injury, indicating the migration of neural stem cells to lesion. Perilesional majority of these cells expressed astrocyte markers, which were lost in Nestin-CreERT2/STAT3^f/f/CAG-CAT^f/f-GFP mice, suggesting the STAT3-dependent astrocyte differentiation of the migrating cells. A tamoxifen treatment after injury labelled, unexpectedly cells lacking astrocyte markers within lesion core at four days after injury. These GFP+ cells gradually expressed astrocyte markers and became indistinguishable from other perilesional reactive astrocytes in 14 days after injury. These GFP+ non-astrocytic cells were NG2+ and partly laminin+, suggesting the conversion of NG2+ glia or pericyte to reactive astrocytes. The neuronal differentiation of GFP+ cells was not found following any tamoxifen treatment, but the majority of these cells were eliminated in two months after injury. The present study has excluded the multipotency of reactive astrocytes and revealed novel multiple processes of gliosis.		2O2-4 Siglec-H is a specific marker for microglia in mice Hiroyuki Konishi¹，Masaaki Kobayashi¹，Katsuaki Sato²，Hiroshi Kiyama¹ ¹Dept Funct Anat & Neurosci, Nagoya Univ Grad Sch Med，²Div Immunol, Dept Infect Dis, Fac Med, Univ Miyazaki The CNS contains several types of myeloid cells. Although microglia are a well-known population, CNS-associated macrophages (meningeal macrophages in the meninges, perivascular macrophages in the perivascular space, and choroid plexus macrophages in the choroid plexus) are also present at the boundary of the CNS. In addition, circulating monocytes infiltrate the CNS parenchyma following breakdown of blood–brain barrier under injured conditions. Because gene expression is largely conserved among these myeloid cells, microglia-specific markers have not been identified until recently. In addition, microglia-specific gene targeting in mice is frustrated by the lack of gene locus that is specifically expressed in microglia. Here we show that a transmembrane lectin, sialic acid-binding immunoglobulin-like lectin H (Siglec-H), is an authentic marker for microglia in mice. Immunohistochemistry using Siglec-H-specific antibody demonstrated that Siglec-H was expressed by microglia, but not by CNS-associated macrophages and CNS-infiltrating monocytes, with a minor exception. We also show that Siglech gene locus is useful for microglia-specific gene manipulation in the nervous system of mice. Furthermore we addressed Siglec-H function in microglia, and found that a Siglec-H-mediated signal suppressed pro-inflammatory responses of activated microglia. Taken together, Siglec-H is concluded as a microglia-specific anti-inflammatory molecule in mice.

2O2-5 Astrocytic protein NDRG2 exacerbates demyelination in mouse experimental autoimmune encephalomyelitis Mika Takarada-Iemata，Thoung Manh Le，Nahoko Okitani，Jureepon Roboon，Tsuyoshi Hattori，Hiroshi Ishii，Osamu Hori Dept Neuroanat. Grad Sch Med Sci, Kanazawa Univ N-myc downstream-regulated gene 2 (NDRG2) is a member of differentiation-related genes, and up-regulated in response to various stresses. We previously reported that NDRG2 is involved in the regulation of astroglial activation after brain injury. In this study, we investigated the relevance of NDRG2 in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. The expression of NDRG2 was enhanced in the acute and chronic phase after induction of EAE. The expression pattern was dominantly seen in gray matter astrocytes, and also in white matter astrocytes to a lesser extent. The severity of EAE is reduced in Ndrg2-deficient mice in association with milder demyelination after EAE induction. In addition, induction of ATF3 expression, a marker of neuronal damage, and reduction of glutamate transporters were attenuated in Ndrg2-deficient mice after EAE. Further analysis using cultured astrocytes revealed that knockdown of NDRG2 increase the expression of glutamate transporters. These results suggest that NDRG2 expressed in astrocytes may play important roles in the regulation of glutamate transporters and contribute to motor neuronal damage and demyelination after CNS inflammation.