e ポスター

e ポスター 11. 発生、分化、再生 e Poster 11. Development, Differentiation, Regeneration

2020/9/12　13:40～14:40　オンデマンドB-1 P3-01 ヒトiPS細胞由来ミクログリアの特徴 Characterization of human iPS cell-derived microglia 初山麻子¹、永田亮佑¹、西江敏和¹、出野美津子¹、榎竜嗣¹、峰野純一¹ 1. タカラバイオ株式会社 Asako Hatsuyama¹, Ryosuke Nagata¹, Toshikazu Nishie¹, Mitsuko Ideno¹, Tatsuji Enoki¹, Junichi Mineno¹ 1. TAKARA BIO INC. Microglia play important roles in the regulation of CNS homeostasis. Microglial dysfunction is known to cause disorders of the CNS such as Alzheimer's disease. Because primary human microglia are extremely difficult to utilize for various experiments, human iPS (Induced Pluripotent Stem) cell-derived microglia are considered to be very useful tool for basic research. The purpose of this study is to characterize human iPS cell-derived microglia produced by novel established differentiation method. At first, several kinds of human iPS-cell lines were tested for the ability to differentiate into microglia. Among these, we selected ChiPSC12 iPS cell line for further analysis. Morphology of the microglia from ChiPSC12 (ChiPSC12 microglia) was similar to that of primary microglia. Typical microglial markers, IbaI and P2Y12, were expressed in almost all of the ChiPSC12 microglia. Phagocytosis assay using fluorescent-labelled amyroid-β (1-42) showed that more than 10% of the ChiPSC12 microglia phagocytosed amyloid-β and the phagocytosis was suppressed by cytochalasinD which is an inhibitor of actin polymerization for phagocytosis. ChiPSC12 microglia also showed ATP-dependent chemotaxis function involved in accumulation of the lesion site. Furthermore, ChiPSC12 microglia secreted proinflammatory cytokines, such as IL-6, TNF-α and IL-1β, by the LPS (or LPS and BzATP) stimulation, resulting in initiation of inflammation. Our data demonstrated that ChiPSC12 microglia have similar morphology and functions compared to human primary microglia, suggesting that it is useful tool for basic research of microglia.		2020/9/12　13:40～14:40　オンデマンドB-1 P3-02 Gタンパク質Gαi1（GNAI1）の大脳皮質神経細胞の増殖、移動、分化における役割 Role of a heterotrimeric G-protein, Gi1, in the corticogenesis 浜田奈々子¹、永田浩一¹ 1. 愛知県医療療育総合センター Nanako Hamada¹, Koh-ichi Nagata¹ 1. Aichi Developmental Disability Center Based on recent reports indicating critical roles of the α-subunits of Gi/o family heterotrimeric G-proteins, Gαi2 and Gαo1, in brain development, we examined the role of another α-subunit of Gi/o member, Gαi1, during corticogenesis. In situ hybridization, immunohistochemistry and western blotting analyses revealed Gαi1 expression in mouse brain in a developmental stage-dependent manner. Notably Gαi1 was enriched at membrane area in early mitotic cells in the ventricular zone (VZ) in the embryonic stage. Acute knockdown of Gαi1 with in utero electroporation gave rise to abnormally elongated leading process and hampered nucleokinesis, leading to delayed radial migration of excitatory neurons during corticogenesis. This phenotype was at least partially rescued by an RNAi-resistant version of Gαi1. Silencing of Gαi1 also impaired dendritic arbor development and cell cycle of stem/progenitor cells at the VZ. Collectively, these results strongly suggest a crucial role of Gαi1 in cortical development, and disturbance of its function may cause deficits in physiological synaptic network formation, leading to neurodevelopmental disorders.

2020/9/12　13:40～14:40　オンデマンドB-1 P3-03 VLDLRは大脳皮質発生において神経細胞の凝集の促進ではなく辺縁帯内への神経細胞の進入を阻止することによって神経細胞の配置を制御する VLDLR controls the positioning of pyramidal neurons by suppressing neuronal invasion into the marginal zone, rather than by promoting neuronal aggregation, during neocortical development 廣田ゆき¹、仲嶋一範¹ 1. 慶應義塾大学医学部解剖学 Yuki Hirota¹, Kazunori Nakajima¹ 1. Dept. Anatomy, Keio Univ. Sch. Med. In the developing neocortex, newborn excitatory neurons radially migrate toward the brain surface. Upon reaching beneath the marginal zone (MZ), they stop migration and form layers. Reelin plays essential roles in these processes via its receptors, apolipoprotein E receptor 2 (ApoER2) and very low density lipoprotein receptor (VLDLR). We recently reported that Reelin causes neuronal aggregation via ApoER2, which is thought to be important for the subsequent layer formation. As for the significance of VLDLR, it was previously shown that neurons were ectopically distributed in layer I on postnatal day 7 in Vldlr-deficient neocortex. However, it remains unknown whether Vldlr-deficient neurons invade the MZ during the migration stages, and what effect Reelin exerts via the VLDLR. Herein, we found that ectopic Reelin overexpression in the Vldlr-mutant cortex also causes neuronal aggregation, but without a MZ-like cell-sparse central region that is formed when Reelin is overexpressed in the normal cortex. We also found that both the early-born and late-born Vldlr-deficient neurons invade the MZ and exhibit significantly impaired dendrite outgrowth from before birth. Rescue experiments indicate that VLDLR has a cell-autonomous function with respect to suppression of neuronal invasion into the MZ, possibly mediated by Rap1, integrin, and Akt. These results suggest that VLDLR is not a prerequisite for Reelin-induced neuronal aggregation and that the major role of VLDLR is to suppress neuronal invasion into the MZ during neocortical development.		2020/9/12　13:40～14:40　オンデマンドB-1 P3-04 Glial cells missing 1は損傷脳においてグリア細胞分化と血管新生を促進する Glial cells missing 1 promote glial cell differentiation and angiogenesis in the injured brain 林義剛¹、福家聡¹、郷康広²、中林一彦³、Adbullah Asmaa¹、Mohd Zakiyyah¹、守村直子¹、Daun Kenny¹、小山なつ¹、等誠司¹ 1. 滋賀医科大学、2. 生理学研究所、3. 成育医療研究センター Yoshitaka Hayashi¹, Satoshi Fuke¹, Yashuhiro Go², Kazuhiko Nakabayashi³, Asmaa Abdullah¹, Zakiyyah Mohd¹, Naoko Morimura¹, Kenny Daun¹, Natsu Koyama¹, Seiji Hitoshi¹ 1. Shiga University of Medical Science, 2. National Institute for Physiological Science, 3. National Center for Child Health and Development Glial cell missing (gcm) plays a critical role in glial cell development in Drosophila. Overexpression of Gcm1 in the mammalian embryo brain was shown to promote the differentiation of neural precursor cells into astrocytes. However, the function of Gcm1 in the postnatal brain remains to be investigated because Gcm1-deficient mice are embryonic lethal. On the other hand, when the brain was injured, a lot of astrocytes proliferate and play a key role to repair. We revealed that the Gcm1 was upregulated expression in the brain of 3 days after injury. To determine the function of Gcm1, we performed in utero electroporation studies to overexpress Gcm1 together with GFP in neural precursor cells at E14.5 and analyzed at E17.5. The Gcm1 significantly promoted the emergence of GFAP(+) and S100β(+) astrocytes, which is consistent with the precedent studies. Next, we investigated the differentiation into oligodendrocyte lineage cells by immunostaining the Gcm1 electroporated brains. The number of Olig2(+) cells, an oligodendrocyte lineage marker, were increased both in GFP(+) and in GFP(-) populations. Furthermore, we also noticed that Gcm1 overexpression resulted in more angiogenesis. Interestingly, differentiation and angiogenesis were regulated by LIF, VEGFA, and VEGFC secretion. Thus, considering that brain injury requires gliogenesis and angiogenesis to repair the injury, our results suggest that Gcm1 plays an important role in the injured brain and have a possible association with injury treatment.

2020/9/12　13:40～14:40　オンデマンドB-1 P3-05* マウス小脳顆粒前駆細胞におけるヘパラン硫酸プロテオグリカンシンデカン-3の役割 Role of heparan sulfate proteoglycan syndecan-3 in mouse cerebellar granule progenitor cells 渡邉雛¹、池田夏実¹、福田彩華²、橋本恵³、宮本泰則^1,2,3 1. お茶の水女子大学人間文化創生科学研究科ライフサイエンス専攻生命科学コース、2. お茶の水女子大学理学部生物学科、3. お茶の水女子大学ヒューマンライフイノベーション研究所 Hiina Watanabe¹, Natsumi Ikeda¹, Ayaka Fukuda², Kei Hashimoto³, Yasunori Miyamoto^1,2,3 1. Graduate School of Humanities and Sciences, Ochanomizu Univ., 2. Department of Biology, Ochanomizu Univ., 3. Insitute for Human Life Innovation, Ochanomizu University In the developing mouse cerebellum, cerebellar granule precursor cells (CGCPs) proliferate in the outer granular layer (EGL) on the surface and exit the cell cycle in the inner EGL until two weeks after birth. After that, the GCGPs begin differentiating while radially migrating to the deeper layer and complete the terminal differentiation into cerebellar granule cells (CGCs). Syndecan-3 (Syn3), a heparan sulfate proteoglycan, has been confirmed to be expressed in the developing CGCPs, but how Syn3 is involved in the proliferation and differentiation of CGCP remains largely unclear. In this study, we aimed to clarify the effect of Syn3 on the proliferation and differentiation of CGCPs by the knockdown (KD) and overexpression (over) of Syn3. In the primary culture of CGCPs, the progress of cell proliferation and differentiation from 3 day- to 5 day-culture was analyzed. To trace the CGCP passing from the S-phase to cell cycle exit, BrdU was incorporated into CGCPs in S- phase at 24 h before fixation. Immunofluorescence staining using Ki67, a cell cycling marker, and BrdU showed that Syn3KD decreased the number of both Ki67 and BrdU positive cells and Syn3over increased in the 4-day culture. This result indicated that Syn3 suppresses the cell cycle exit of CGCP. Furthermore, to investigate how Syn3 regulates cell cycle exit, double staining of Ki67 and TAG1, an early differentiation marker, was performed. As a result, the number of Ki67- and TAG1-positive cells decreased in Syn3KD CGCPs and increased in Syn3over CGCPs in the 4 day-culture. This result was suggested that Syn3 shortens the period before the cell cycle exit of CGCP. Taken together, these results demonstrate that Syn3 regulates the timing of cell cycle exit in CGCPs.		2020/9/12　13:40～14:40　オンデマンドB-1 P3-06 プロテアソームによるAISの可塑性制御は損傷運動ニューロン軸索のミトコンドリア輸送に関与する The proteasome-mediated AIS plasticity contributes to the increased transport of mitochondria in damaged motor axons 桐生寿美子¹、松下鈴佳¹、田代善崇²、高橋良輔²、吉村武³、井口洋平¹、勝野雅央¹、木山博資¹ 1. 名古屋大学大学院医学系研究科、2. 京都大学大学院医学研究科、3. 大阪大学連合小児発達学研究科 Sumiko Kiryu-Seo¹, Reika Matsushita¹, Yoshitaka Tashiro², Ryousuke Takahashi², Takeshi Yoshimura³, Yohei Iguchi¹, Masahisa Katsuno¹, Hiroshi Kiyama¹ 1. Nagoya University Graduate School of Medicine, 2. Kyoto University Graduate School of Medicine, 3. Osaka University United Graduate School of Child Development Motor neurons are susceptible to the proteasome-mediated proteolysis whose dysfunction causes neuronal degeneration in motor neuron diseases such as ALS. It is likely that the responses occurring in motor neurons after axonal injury and in those during the disease-onset are somewhat similar. To examine the role of proteasome in damaged motor neurons, we have established a mouse model (Rpt3 CKO) in which a subunit of proteasome is ablated and mitochondria are labeled by GFP in injury-dependent manner. The injury-specific Rpt3 deletion and mitochondrial labeling are accomplished by using the transcriptional regulation of Atf3 gene, a well-known injury marker. Injured motor neurons of Rpt3 CKO resulted in the accumulation of TDP-43 protein and rapid degeneration. Before the time point, multiple proteins were properly down-regulated upon injury in proteasome-deficient motor neurons, probably due to the compensatory autophagy system. Intriguingly, injured motor neurons of wild type mouse (WT) eliminated the axon initial segment (AIS), a specialized compartment in the proximal axon, while those of Rpt3 CKO remained the AIS. Contrary to the absence of the AIS, mitochondria were abundantly found in the region of the disappeared AIS in WT, while not in the remaining AIS of Rpt3 CKO. The in vivo live imaging showed that the number and motility of mitochondria was increased in damaged axons of WT, but not in those in Rpt3 CKO. The remaining AIS was also detected in ATF3-positive motor neurons in ALS model mouse, suggesting the common mechanism with injured motor neurons of Rpt3 CKO. These findings suggest that proteasome-sensitive AIS plasticity is a potential mechanism to supply enough amounts of mitochondria and satisfy energy demand for damaged motor axons to regenerate.