Oral Session

Oral Session 2 一般口演2

O2-1 LRP2 expression and effects of lipopolysaccharide on the expression in cultured astrocytes and microglia グリア細胞におけるLRP2発現とリポポリサッカライドによる変化 Takano Katsura（高野桂），的場教起，森山光章，中村洋一 Laboratory of Integrative Physiology in Veterinary Sciences, Osaka Prefecture University, Osaka, Japan Abnormal accumulation and aggregation of Aβ in brain is a characteristic pathology in Alzheimer’s diseases (AD), and it is reported in sporadic AD that Aβ production rate is not different from healthy brain while Aβ clearance is impaired. Dynamic changes in Aβ, production and clearance, have been investigated. To elucidate the mechanism of Aβ uptake in astrocytes and microglia would be important for inhibition of Aβ accumulation. Insulin resistance in brain also has been reported in AD. Insulin signaling is important for homeostasis in brain function and reported to be disturbed in neurons leading to tau phosphorylation and neurofibrillary tangles. Low density lipoprotein (LDL) receptor-related protein (LRP) is a transmembrane glycoprotein belonging to LDL receptor family. LRP1 expressing in endothelial cells in BBB was reported to be involved in Aβ clearance. LRP2 in proximal tubule was reported to associate with insulin uptake; however, the expression of LRP2 and its changes in glial cells in brain has not been reported. In the present study, we assessed the expression of LRP2 and the effects of lipopolysaccharide (LPS) in cultured rat brain astrocytes and mouse microglial cell line BV-2. We found that both astrocytes and BV-2 cells expressed LRP2 mRNA. LPS decreased LRP2 mRNA in astrocytes and increased in BV-2 cells. In astrocytes, LPS decreased Aβ uptake assessed by immunostaining and FITC-conjugated insulin incorporation. On the other hand, in BV-2 cells, LPS increased incorporations of Aβ and FITC-conjugated insulin. These results suggest that LRP2 expressing in glial cells might be involved in uptake of Aβ and insulin.		O2-2 Clearance of microglial debris by reactive astrocytes under absence of functional microglia ミクログリア除去モデルマウスにおけるミクログリア残骸のアストロサイトによる貪食 Konishi Hiroyuki（小西博之）¹，岡本峻幸¹，佐藤克明²，木山博資¹ ¹Dept. Funct. Anat. & Neurosci., Nagoya Univ. Grad. Sch. Med. ²Div. Immunol., Dept. Infect. Dis., Fac. Med., Univ. Miyazaki Microglia are the main phagocytes in the CNS. We recently established microglial ablation model without affecting other CNS-related mononuclear cells, using microglia-specific diphtheria toxin (DT) receptor-knockin (Siglech ^dtr) mice. In this model, DT treatment specifically kills microglia, and intriguingly the microglial debris are somehow rapidly removed even under absence of functional microglia, raising a question how the debris were cleared. The microglial ablation did not cause infiltration of mononuclear phagocytes, such as perivascular macrophages and circulating monocytes, in CNS parenchyma, suggesting that the other cell populations were involved in the clearance. We found that astrocytes became activated with GFAP upregulation upon microglial ablation, and extended their processes to microglial debris. Confocal and electron microscopy showed that activated astrocytes contained a significant number of microglial debris in their cell bodies as well as processes. Furthermore the microglial debris were observed in the lysosome of cultured astrocytes, when astrocytes were co-cultured with apoptotic microglia. Gene knockdown experiment revealed that some of phagocytic receptor molecules expressed by astrocytes were involved in the clearance. These results suggest that astrocytes have a potential to compensate for the dysfunction of microglial phagocytic activity in the CNS.

O2-3 The Roles of Notch and MAPKs on Temporal Changes of Differentiation Potential of Neural Stem Cells 神経幹細胞/前駆細胞の時間経過に伴う分可能の変化の解析 Okano Yuji（岡野雄士），加瀬義高，岡野栄之 Department of Physiology, Keio University School of Medicine Neural stem/progenitor cells (NSPCs) varies their characters regulated by spatial and temporal factors during the development. Previous studies showed that NSPCs produce neurons first and subsequently glial cells during the development of the central nervous system. However, the mechanism and the regulator of this phenomenon is still not fully understood. In this study, we analyzed how Notch and MAPKs work, and also what kind of relations they have with the developmental plasticity of NSPCs, using the ES cells-derived neurosphere. First, we confirmed of culture period-dependent change of differentiation competency of neurospheres from neurogenic to gliogenic in vitro and the effect of DAPT (γ-secretase inhibitor), which inhibits Notch signaling. Here, surprisingly, DAPT-treatment increased the ratio of glial cells among the cells differentiated from neurospheres in spite of some positive actions of Notch in glial development that were previously reported. We investigated the action of DAPT focusing its roles in the regulations of MAPKs. Since Notch signaling induces expression of DUSP, a phosphatase which de-phosphorylates MAPKs, we investigated the temporal change of expression of various MAPKs and effect of DAPT-treatment on relative phosphorylation level of MAPK families during passages of neurospheres. We found that only p38’s expression level changed in a temporarily regulated fashion among them and phosphorylation form (active form) of p38 was increased by DAPT-treatment. It still needs to clarify how p38 activation is involved in glial cell differentiation. Collectively, in this study, we found that inhibition of Notch signals by DAPT-treatment promotes phosphorylation of p38. We hypothesize that this could lead to preferred differentiation to glia.