若手道場

若手道場5

2021/9/30　15:00～16:00　ZOOM 若手道場 WD5-1 プラダー・ウィリー症候群患者iPS細胞由来視床下部ニューロンを用いた疾患モデルの作製 Modeling Prader-Willi syndrome using hypothalamic neurons from patient-derived induced pluripotent stem cells ^○根本晶沙, Hironobu Okuno, Hideyuki Okano Department of Physiology, Keio University School of Medicine ^○Akisa Nemoto, Hironobu Okuno, Hideyuki Okano Department of Physiology, Keio University School of Medicine Prader-Willi Syndrome (PWS) is a genomic imprinting disorder caused by loss of function of paternally expressed genes (PEGs) in chromosome 15q11-13 region. Hyperphagia leading to obesity, hypogonadism and hypogenitalism, short stature and small hands and feet due to growth hormone deficiency are major clinical manifestations of PWS. These phenotypes have been thought to be closely related to hypothalamic dysfunction, but little is known about the cellular and molecular pathophysiology in PWS. Here, we established models of PWS using hypothalamic neurons from iPSCs by two differentiation methods: (1) 2-dimensional hypothalamic neuronal induction (Merkle et al., 2016; Wang et al., 2016) and (2) 3-dimensional hypothalamic organoid induction (Kasai et al., 2020). iPS cell lines were generated from 5 PWS patients harboring different genetic backgrounds (deletion, maternal uniparental disomy, and translocation). We have succeeded in generating RAX(+), NKX2.1(+), OTP(+) hypothalamic progenitor cells from those PWS-iPSCs (day ~30) as well as several hypothalamic neurons including POMC, AGRP, NPY positive cells which are crucial for the regulation of feeding. Moreover, we found some PEGs, NDN, MAGEL2, and MKRN3 in chromosome 15q11-13 were highly expressed in control hypothalamic neurons, but not in PWS hypothalamic neurons. Notably, these PEGs are known to be highly expressed in hypothalamus and related to PWS clinical symptoms. This indicates that these PWS cell lines genetically recapitulate disease-specific features. Using these in vitro models, we are going to reveal in vitro phenotypes and pathophysiology of PWS.		2021/9/30　15:00～16:00　ZOOM 若手道場 WD5-2 ミクログリアはフォスファチジルセリン依存的に成体新生ニューロンのシナプスを貪食する Phosphatidylserine-dependent synaptic pruning by microglia in adult-born neurons ^○榑松千紘¹, Masato Sawada^1,2, Masaki Ohmuraya³, Motoki Tanaka⁴, Kazuya Kuboyama¹, Takashi Ogino¹, Mami Matsumoto^1,2, Hisashi Oishi¹, Hiroyuki Inada², Shinichi Kohsaka⁵, Nobuhiko Ohno^2,6, Maki K. Yamada⁷, Masato Asai⁴, Masahiro Sokabe⁸, Junichi Nabekura², Kenichi Asano⁹, Masato Tanaka⁹, Kazunobu Sawamoto^1,2 1.名古屋市立大学大学院医学研究科　脳神経科学研究所　神経発達・再生医学分野, 2.National Institute for Physiological Sciences, 3.Hyogo College of Medicine, 4.Aichi Developmental Disability Center, 5.National Center of Neurology and Psychiatry, 6.Jichi Medical University, 7.Tokushima Bunri University, 8.Nagoya University, 9.Tokyo University of Pharmacy and Life Sciences ^○Chihiro Kurematsu¹, Masato Sawada^1,2, Masaki Ohmuraya³, Motoki Tanaka⁴, Kazuya Kuboyama¹, Takashi Ogino¹, Mami Matsumoto^1,2, Hisashi Oishi¹, Hiroyuki Inada², Shinichi Kohsaka⁵, Nobuhiko Ohno^2,6, Maki K. Yamada⁷, Masato Asai⁴, Masahiro Sokabe⁸, Junichi Nabekura², Kenichi Asano⁹, Masato Tanaka⁹, Kazunobu Sawamoto^1,2 1.Nagoya City University, 2.National Institute for Physiological Sciences, 3.Hyogo College of Medicine, 4.Aichi Developmental Disability Center, 5.National Center of Neurology and Psychiatry, 6.Jichi Medical University, 7.Tokushima Bunri University, 8.Nagoya University, 9.Tokyo University of Pharmacy and Life Sciences Adult mammalian brains have a remarkable capacity to generate new functional neurons. Much recent research has focused on the mechanisms that control synapse formation of adult-born neurons. However, how synaptic pruning, another critical step for functional integration of adult-born neurons into mature circuits, is controlled is not fully understood. Phosphatidylserine (PS) is exposed on the outer plasma membrane of the apoptotic cells and serves as an eat-me signal for phagocytes. Recent studies suggest that synapses locally present PS and are phagocytosed by microglia during postnatal brain development, raising the possibility that PS also acts as a synaptic eat-me signal for microglia. However, the function of PS in synaptic pruning by microglia in the adult brain has not been demonstrated. In this study, we show that spine pruning of adult-born neurons by microglia is dependent on PS, whose exposure on dendritic spines is inversely correlated with their input activity. To study the role of PS in spine pruning by microglia in vivo, we developed an inducible transgenic mouse line, in which the exposed PS is masked by a dominant-negative form of milk fat globule-EGF-factor 8 (MFG-E8), MFG-E8D89E. In this transgenic mouse, the spine pruning of adult-born neurons by microglia is impaired in the OB and hippocampus. Furthermore, the electrophysiological properties of these adult-born neurons are altered in MFG-E8D89E mice. These data suggest that PS is involved in the microglial spine pruning and functional maturation of adult-born neurons.

2021/9/30　15:00～16:00　ZOOM 若手道場 WD5-3 Akhirinはミクログリアの活性化を介して神経幹細胞の増殖を制御する Akhirin regulates neural stem cell proliferation through microglial activation ^○工藤三希子, Kunimasa Ohta 基幹教育院 ^○Mikiko Kudo, Kunimasa Ohta Faculty of Arts and science, Kyushu University Previously, we identified Akhirin (AKH) as a novel secretory molecule expressed in the chick embryo’s lens epithelium. AKH contains one LCCL domain and two vWF domains , exhibits heterophilic cell adhesion property (Ahsan et al., 2005). AKH is expressed explicitly in the neural stem cell (NSC) niche region (microenvironment where NSCs are present) of the central nervous system (eye, spinal cord, and brain) and plays a role in their development. (Ahsan et al., 2005, Athary et al., 2015, Anam et al., 2020). The timing of NSC proliferation and differentiation is controlled by the interaction of NSCs, neural progenitor cells, neural ependymal cells, blood vessels, and glial cells with each other in the NSC niche. In the mouse brain, there is a NSC niche in the subventricular zone facing lateral ventricles (LV). AKH is expressed in the ependymal cells at postnatal developing brain. Compared to wild-type mice (AKH+/+), AKH knock out mice (AKH-/-) have aberrant LV expansion and suppressed NSC proliferation. Our findings suggest that AKH plays a role in sustaining NSC proliferation and LV size in the NSC niche. However, the detailed molecular function of AKH in brain development is still unclear. Here, using an anti-AKH monoclonal antibody, we show that AKH is expressed in the blood vessels, neuronal ependymal cells, and choroid plexus of the embryonic mice brain. Further, we revealed the abnormal blood vessels formation and the increase of activated microglia in the AKH-/- brain. We propose that AKH regulates the NSC proliferation and vasculogenesis in the brain through modulating microglial activation. Currently, we are planning to analyze the factors that induce microglial activation under AKH deficiency and unveil its mechanism.