若手道場口演（Wakate Dojo）

Wakate Dojo Neural Excitability, Synapse and Glia 4 若手道場口演神経興奮性・シナプス・グリア4

7月26日（金）15:10～15:30　第10会場（万代島ビル　6F　会議室） 2WD10ba1-1 アンモニアによるアストロサイトのグルタミン合成調節機序 Sari Kishikawa（岸川咲吏），Miho Terunuma（照沼美穂）新潟大学大学院　医歯学総合研究科　口腔生化学分野 Astrocytes, one of the major cells in the central nervous system, are the only cells in the brain that remove ammonia using enzyme called glutamine synthetase (GS). Neurotoxic glutamate and ammonia are taken up from astrocytes and converted into glutamine. This glutamate-glutamine cycle regulated by GS is essential for synaptic transmission and brain homeostasis, however the mechanisms through which GS expression and degradation is regulated are still unclear. Here we used cultured astrocytes derived from rat fetus and examined the cellular stability of GS by ammonia stimulation. We found that ammonia significantly decreased the expression of GS in a time and concentration-dependent manner. Interestingly, the reduction of GS was accelerated by L-glutamate suggesting that both ammonia and glutamate are required for GS down-regulation. In addition, glutamine, an end-product of ammonia and glutamate, significantly reduced GS expression. To understand if GS down-regulation was due to reduced GS synthesis, we examined mRNA levels by qPCR. Ammonia significantly reduced GS mRNA similar to what we found in GS proteins. Previous reports suggested that ammonia induces autophagy in liver cells. To determine the mechanism of ammonia-induced GS degradation, we examined autophagy in astrocytes. LC3-II, an autophagy induction marker was significantly increased under ammonia stimuli and GS down-regulation was synchronized with the amount of LC3-II. These results indicated that GS degradation may be due to autophagy activation induced by ammonia. Currently, we are examining the signaling pathways for GS degradation.		7月26日（金）15:30～15:50　第10会場（万代島ビル　6F　会議室） 2WD10ba1-2 ニューロロイキンはアストロサイトからのニューロロイキン分泌を促し、神経細胞の軸索伸展を促す Yoshitaka Tanie（谷江良崇），Norio Tanabe（田辺紀生），Tomoharu Kuboyama（久保山友晴），Chihiro Tohda（東田千尋）富山大学　和漢医薬学総合研究所　病態制御研究部門　神経機能学分野 Under pathological conditions in the central nervous system (CNS), such as traumatic injury, astrocytes are known to show both detrimental effects and beneficial effects against neurons. If it is possible to enhance beneficial effects of astrocytes after injury, dysfunctions of the injured CNS might be improved. However, a way of promotion of beneficial functions in astrocytes has not been elucidated. In this study, we focused on neuroleukin (NLK) that was known to have axonal growth activities in neurons. Although it was reported that NLK was secreted from astrocytes, the function of NLK in astrocytes is rarely understood. Therefore, we aimed to clarify the functional significance of secreted NLK from astrocytes and the mechanism of NLK secretion in astrocytes. Furthermore, we also aimed to clarify the mechanism of NLK-induced axonal growth in neurons. Stimulation with recombinant NLK significantly elevated the secretion of NLK from cultured astrocytes. Furthermore, astrocyte conditioned medium treated with NLK increased the axonal density in cultured cortical neurons. We also confirmed that NLK itself promoted axonal growth. These results indicated that extracellular NLK is an axonal growth factor secreted from astrocytes, and secretion of NLK from astrocytes is stimulated by extracellular NLK. In addition, cell surface 78 kDa glucose regulated protein (GRP78) was identified as a receptor for NLK in astrocytes and neurons. GRP78 was directly involved in both NLK-induced NLK secretion in astrocytes and axonal growth in neurons. When NLK was injected to the lesion site in spinal cord injured mice, NLK expression and axonal density in the injured region was significantly increased and hindlimb motor function was also improved. These results suggest that NLK-GRP78 signaling plays important roles in beneficial effects of astrocytes and axonal growth in neurons. We are now trying to identify downstream signaling of NLK-GRP78.

7月26日（金）15:50～16:10　第10会場（万代島ビル　6F　会議室） 2WD10ba1-3 ミクログリアのアレキサンダー病の病態への関与 Kenji Kobayashi（小林憲司）¹，Eiji Shigetomi（繁富英治）¹，Kozo Saito（齋藤光象）^1,2，Kazuhiro Ikenaka（池中一裕）³，Kenji F Tanaka（田中 F 謙二）⁴，Schuichi Koizumi（小泉修一）¹ ¹山梨大学大学院総合研究部薬理 ²京都府立医科大学大学院医学研究科神経内科学 ³自然科学研究機構生理学研究所分子神経生理部門 ⁴慶應義塾大学医学部精神・神経科学 Astrocytes and microglia play pivotal roles in balancing each other and maintaining brain homeostasis. In many brain disorders including neurodegenerative diseases, they both become activated, and lose their appropriate relationship and balance. However, the interaction of these activated glial cells and its significance in the pathogenesis have remained unclear. Alexander Disease (AxD) is a rare neurodegenerative disorder which is caused by dominant gain of function mutations in GFAP gene. Reactive astrocytes in AxD display pathological hallmarks called Rosenthal Fibers, which is comprised of GFAP accumulations with αB-crystalline. AxD is in fact the primary astrocytic disorder, but microglial activation is also found. Our previous data showed the increase in the number of Iba1-positive microglia in the hippocampus from hemizygotes of 60TM AxD model mice, overexpressing human GFAP with R239H mutation (60TM mice). To investigate the role of activated microglia on AxD pathogenesis in the early developmental stages, we treated 60TM mice with PLX5622 (PLX), a CSF-1 receptor antagonist, between P21 and P42 to deplete microglia from 60TM mice and evaluated its effect on the AxD pathology. PLX eliminated almost all Iba1-positive signals and its mRNA level in the hippocampus. Strikingly, PLX significantly increased reactive astrocyte markers such as GFAP, Vimentin, and CD44, in the hippocampus, suggesting exacerbation of AxD pathology. Immunoreactivity of those markers were significantly higher in stratum lacunosum moleculare of the hippocampus. In addition, the area of both GFAP- and Vimentin-positive signals were also increased. Fluoro-Jade B staining, a marker of Rosenthal fibers, in the hippocampus was stronger in PLX-treated groups, suggesting more Rosenthal fibers appeared by microglial depletion. PLX also augmented mRNA levels of reactive astrocytes markers such as Vim, C3 and Cd44 in the hippocampus. Overall data indicate that elimination of activated microglia by PLX in AxD makes reactive astrocytes a more exacerbate phenotype, suggesting that activated microglia in AxD should have a rather beneficial role on AxD pathogenesis at early developmental stage.