若手道場

若手道場7

2021/9/30　18:00～19:00　ZOOM 若手道場 WD7-1 侵害刺激は下行性ノルアドレナリン神経シグナルを介して脊髄後角アストロサイトを活性化する Noxious stimuli activate spinal dorsal horn astrocytes via descending noradrenergic signaling ^○川邉陸, Kohei Yoshihara, Makoto Tsuda 九州大学　大学院薬学府　薬理学分野 ^○Riku Kawanabe, Kohei Yoshihara, Makoto Tsuda Department of Molecular and System Pharmacology, Graduate School of Pharmaceutical Sciences, Kyushu University Astrocytes, which are abundant glial cells in the central nervous system, have become increasingly recognized as critical elements regulating neuronal function including somatosensory information processing in the spinal dorsal horn (SDH) and brain. We have previously shown that astrocytes in the SDH increase intracellular Ca²⁺ levels following intraplantar injection of the noxious irritant, formalin. However, the underlying mechanisms remain unknown. We investigated these mechanisms by focusing on the role of descending noradrenergic (NAergic) signaling because our recent study revealed the essential role of the astrocytic Ca²⁺ responses evoked by intraplantar capsaicin. Using in vivo SDH imaging, we found that the Ca²⁺ levels are increased in SDH astrocytes by intraplantar formalin injection and that the increase was suppressed by ablation of SDH-projecting locus coeruleus (LC)-NAergic neurons. Furthermore, the formalin-induced Ca²⁺ responses were decreased by the loss ofα_1A-adrenaline receptors (ARs) in astrocytes located in the superficial laminae of the SDH. Moreover, similar inhibition was observed in mice pretreated intrathecally with anα_1A-AR-specific antagonist. Therefore, activation ofα_1A-ARs via descending LC-NAergic signals may be a common mechanism underlying astrocytic Ca²⁺ responses in the SDH evoked by noxious stimuli, including chemical irritants.		2021/9/30　18:00～19:00　ZOOM 若手道場 WD7-2 NG2プロモーターを用いたオリゴデンドロサイト前駆細胞と血管壁細胞の光操作 Separate optogenetic manipulation of oligodendrocyte precursor cells and mural cells using the NG2 promoter ^○大石光洋, Kenji F Tanaka 慶應義塾大学　医学部 ^○Mitsuhiro Oishi, Kenji F Tanaka Department of Neuropsychiatry, Keio University School of Medicine Nerve/glial antigen 2 (NG2) is a protein marker of oligodendrocyte precursor cells (OPC) and mural cells, and NG2 promoter activity is utilized to target these cells. As a matter of course, the NG2 promoter cannot target OPC and mural cells separately. However, here, we developed transgenic mice that did target either cell type using the NG2 promoter. We selected a tetracycline-controllable gene induction system for cell type–specific transgene expression, and generated NG2-tTA (tetracycline transactivator) transgenic lines. We crossed tTA lines with the tetO-ChR2-EYFP line to characterize tTA-dependent transgene induction. We isolated two unique NG2-tTA lines: one that induced ChR2-EYFP only in mural cells, likely due to the chromosomal position effect of NG2-tTA insertion, and the other that induced it in both cell types. We then applied a Cre-mediated set-subtraction strategy to the latter case and eliminated ChR2-EYFP from mural cells, resulting in OPC-specific transgene induction. We further demonstrated that tTA-dependent ChR2 expression could manipulate cell function. ChR2-mediated depolarization was observed in OPCs in acute slices, and this was consistent with physiological depolarization levels, indicating the feasibility of this new tool to manipulate OPC function in vivo. Optogenetic mural cell activation decreased cerebral blood flow, as previously reported, indicating that the tTA-mediated ChR2 expression was sufficient to impact cellular function. Since the tTA-tetO combination is expandable, the mural cell–specific NG2-tTA line and the set-subtraction–assisted, OPC-specific NG2-tTA line will permit us to conduct observational and manipulation studies to examine in vivo function of these cells separately.

2021/9/30　18:00～19:00　ZOOM 若手道場 WD7-3 iPS細胞由来ミクログリアの誘導法と疾患モデリング Single Transcription Factor Efficiently Leads Human iPS Cells to Functional Microglia and Disease Modeling ^○孫怡姫, 渡部博貴, 森本悟, 岡野栄之 Department of Physiology, Graduate School of Medicine, Keio University ^○Iki Sonn, 博貴渡部, 森本悟, 栄之岡野 Department of Physiology, Graduate School of Medicine, Keio University Microglia are representative immune cells, also the only residental immune cells in the central nervous system parenchyma. They play vital physiological roles, not only in adult but also during development. Microglia are further in the spotlight, because multiple risk genes of neurodegenerative diseases expressed in microglia have been reported recently, which leading to the abnormal activities of microglia under traumatic or disease condition. In this study, to investigate the multifaced functions of human microglia, we established a novel protocol to generate microglia from human pluripotent stem cells (iPSCs). By forcing the expression of SPI1 during the hemogenic endothelium differentiation, we can harvest a greater deal of microglia within 3 weeks comparing to a widely used previous method. Over 90% of hiMGLs expressed microglia specific markers, such as CX3CR1 and IBA-1. Whole-transcriptome analysis revealed that these hiMGLs resembled characteristics to human primary microglia, different with monocytes/macrophage. Furthermore, specific physiological functions of microglia were confirmed through indices of lipopolysaccharide (LPS) responsiveness, phagocytotic ability and inflammasome formation. By co-culturing hiMGLs with mouse primary neurons, we demonstrated that hiMGLs could regulate the activity and maturation of neurons. Summarily, we established a simple method for generating hiMGLs from iPSCs rapidly and high efficiently, which may shed lights on the future investigation of microglia in both physiological and diseases condition, as well as drug discovery.