一般口演（若手道場）

一般口演（若手道場）若手道場　疾患モデリング Wakate Dojo: Disorder Modeling 座長：澤本和延（名古屋市立大学）・野田万理子（愛知県医療療育総合センター）

2022年7月1日　10:00～10:15　沖縄コンベンションセンター会議場A2　第7会場 2WD07m2-01 神経系・免疫系における炎症マーカーとしてのネオプテリン生合成機構の解析 Analysis of biosynthetic mechanisms of neopterin as an inflammatory marker in the nervous and immune systems 木村真尋(1)、阪本吉彬(1)、原怜(1)、一瀬宏(1) 1. 東京工業大学 Mahiro Kimura(1), yoshiaki sakamoto(1), satoshi hara(1), hiroshi ichinose(1) 1. Tokyo Institute of Technology Keyword: neopterin, inflammation, biopterin [背景] テトラヒドロビオプテリン(BH₄)はモノアミン神経伝達物質を生合成する芳香属アミノ酸水酸化酵素や一酸化窒素合成酵素の補酵素として、脳内で重要な生理機能を営んでいる。 BH₄は、グアノシン三リン酸から3段階の酵素反応を経て生合成される。その第1段階を担うGTPシクロヒドロラーゼI(GCH)は、GTPをジヒドロネオプテリン三リン酸(NPH₂TP)に変換する。 NPH₂TPはBH₄への生合成系に使われる一方で、NPH₂TPのもう1つの代謝物であるネオプテリン(NP)への生合成系にも使われる。 NPは細胞性免疫活性化の優れた指標となることがわかっている。私達の研究室でも、脳内の炎症反応の亢進が示唆されている家族性パーキンソン病Park8の患者において、脳脊髄液中のネオプテリンが有意に増加していること(Ichinose et al. J Neural Transm. 2017)、SARS-CoV-2ウィルス感染症であるCOVID-19患者血漿中でネオプテリンが著明に増加すること(Hara et al. 投稿中)を見出している。しかし、通常はBH₄の生合成中間体であるNPH₂TPから、なぜNPが作られるようになるのか、どのような酵素によって、どこでNPH₂TP の3つのリン酸基の脱リン酸化が行われてNPになるのかについても明らかとされていない。本研究の目的は、NPH₂TPの脱リン酸化機序の詳細を解析して、NPの生合成機構やその生理的意義を明らかとすることである。 [方法] NPH₂TPからNPに変換される途中の中間代謝産物を定量するために、まず酸化型NP類である、ネオプテリン三リン酸(NPTP)、ネオプテリン二リン酸(NPDP)、ネオプテリン一リン酸(NPMP)、ネオプテリン(NP)を分離するHPLC条件を検討した。その後、神経系・免疫系に由来する種々の培養細胞を用いて、NPH₂TPの代謝がどのように進行していくか解析した。 [結果と考察] 種々のカラムを検討してNPのリン酸化体を分離できる条件を検討した。その結果、サイズ排除クロマトグラフィと逆相モードの複合的相互作用により分離するマルチモードカラムを使用することにより、NPTP, NPDP, NPMP,NPを分離することができた。細胞内に存在する中間代謝産物を調べると、ヒト単球性白血病由来のTHP-1細胞やマウスマクロファージ由来RAW264.7細胞では、NPTPのみしか検出できなかった。しかし、T細胞性白血病由来のjurkat細胞では、細胞内にNPTPに加えてNPTPの脱リン酸化代謝産物NPDP,NPMP,NPが存在していた。このことから、細胞種によって、NPTPからNPへの代謝は異なるということが推測できた。また、jurkat細胞内では、NPへの脱リン酸化が起こっている可能性も推測できた。		2022年7月1日　10:15～10:30　沖縄コンベンションセンター会議場A2　第7会場 2WD07m2-02 Deciphering the role of Tob in the brain: An insight into stress coping machinery Mohieldin M Youssef(1), Hiro Hamada(2), Esther Lai(3), Yuji Kiyama(4), Mohamed Eltabbal(5), Bernd Kuhn(5), Tadashi Yamamoto(1) 1. Cell Signal Unit, Okinawa Institute of Science and Technology OIST, 2. Neural Computation Unit, Okinawa Institute of Science and Technology OIST, 3. Neural Circuit Unit, Okinawa Institute of Science and Technology OIST, 4. Laboratory of Biochemistry and Molecular Biology, Graduate school of medical and dental sciences, Kagoshima University, Japan, 5. Optical Neuroimaging Unit, Okinawa Institute of Science and Technology OIST Keyword:* Stress, Fear and extinction, Depression, Tob We face stressful events during our daily life, to which our body generates responses and memories of these events are stored in order to cope with their future occurrence. The increased stress/anxiety-related disorders in modern society prompt an understanding of the mechanisms underlying stress responses and identifying of potential therapeutic targets.Transducer of ErbB2 (Tob), a member of the Tob/BTG anti-proliferative family proteins, has been implicated in cell proliferation, differentiation and the DNA damage response. In the brain, there are reports suggesting that Tob regulates complex behavioral processes such as learning and memory, and Tob downregulation is correlated with increased occurrence of major depressive disorder (MDD). Since stress is one of the common predisposing factors for depression, we investigated the role of Tob in response to stress.For the first time, we show increased expression of Tob in the brain after exposure to different acute stress models. Interestingly, Tob knockout mice exhibit abnormal stress-related behavioral phenotypes such as increased anxiety, depression and fear memory. Therefore, we hypothesize that Tob protein plays a functional role in moderating stress responses. We analyzed the functional neuronal connectivity between different brain regions in awake Tob knockout mice using functional Magnetic Resonance Imaging (fMRI) and observed altered connectivity between the hippocampus and pre-frontal cortex, which could account for the augmented response to stress in Tob knockout mice. Moreover, AAV-mediated expression of Tob in the hippocampus of Tob knockout mice rescued the behavioral phenotype. At the cellular level, Tob may regulate the activity of neuronal signal transduction pathways involved in the response to stress. This study sets the stage for investigating the potential function of Tob in the brain in response to stress and its involvement in stress-related neurological disorders.

2022年7月1日　10:30～10:45　沖縄コンベンションセンター会議場A2　第7会場 2WD07m2-03 Differential compartmentalization of myeloid cell phenotypes and responses in Alzheimer’s disease. Camila Fernandez Zapata(1,2), Ginevra Giacomello(3), Eike J. Spruth(2), Jinte Middeldorp(4), Gerardina Gallacio(1), Adeline Dehlinger(1), Julia K. H. Leman(8), Stephan Schlickeiser(5), Desiree Kunkel(5), Elly M Hol(6), Friedemann Paul(7), Maria K. Parr(3), Josef Priller(2,9), Chotima Böttcher(1,2) 1. ECRC, Max Delbrueck Center for Molecular Medicine and Charité – Univ, Berlin, Germany, 2. Dept of Neuropsychiatry and Laboratory of Molecular Psychiatry, Charité – Univ, Berlin, Germany, 3. Inst of Pharmacy, Freie Univ Berlin, Berlin, Germany, 4. Dept of Immunobiology, Biomedical Primate Research Center, Rijswijk, The Netherlands, 5. Inst of Medical Immunology and Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité – Univ, Berlin, Germany, 6. Dept of Translational Neuroscience, Univ Medical Center Utrecht Brain Center, Utrecht Univ, The Netherlands, 7. NeuroCure Clinical Research Center, Charité - Univ, Berlin, Germany, 8. Inst for biology, Humboldt Univ, Berlin, Germany, 9. Clinic and Polyclinic for Psychiatry and Psychotherapy, Technic Univ Munich, Munich, Germany. Keyword:* Neurodegeneration, Myeloid cells, Mass Cytometry, Immune profiling The view of the brain as an immune privileged site, only depending on resident microglia cells for maintenance, has been challenged by recent research in mice showing that even under steady-state conditions multiple immune cells can reside within certain niches in the central nervous system (CNS) and that the two systems are coordinated and in constant communication. An influence of the immune system in neurodegeneration is also now widely discussed, as studies in mouse models of Alzheimer’s disease (AD) have shown specific myeloid subpopulations associated with the pathology which have been proposed as key players modifying disease progression and outcome in AD patients. However, little is known about the continuum of phenotypic changes and functional responses of myeloid cells across different body compartments during AD and other neurodegenerative diseases. Therefore, a detailed study of the immune populations along different body compartments (mainly blood, cerebrospinal fluid, and CNS parenchyma) during health and neurodegeneration in humans could provide critical information as to the influence of these cells during disease progression. Here, we have applied multiple state-of-the-art analyses including single-cell mass cytometry (measuring a total of 80 antibodies), 13C-labelled metabolic tracing, Luminex protein array as well as ex vivo experiments, to elucidate the phenotypes and roles of the myeloid subpopulations through different body compartments (peripheral blood, CSF, choroid plexus, and cortex) from healthy donors and patients with neurodegenerative diseases (Huntington’s disease, mild cognitive impairment, and Alzheimer’s disease). Among our findings are the discovery of previously unidentified choroid plexus and CSF-specific populations of myeloid cells showing increased expression of inflammatory and metabolism related markers. Interestingly, we detected a population of myeloid cells in the CSF showing expression of the purinergic receptor P2Y₁₂, commonly used as a marker for microglia cells. Some of these alterations were found enhanced in AD as compared to healthy controls, patients with mild cognitive impairment or Huntington´s disease. To our knowledge this is the first study describing multi-compartmental phenotypic analysis of myeloid cells and together this data provides a valuable resource to study the implications of the myeloid populations during neurodegeneration in humans.		2022年7月1日　10:45～11:00　沖縄コンベンションセンター会議場A2　第7会場 2WD07m2-04 C9ORF72遺伝子変異FTD患者iPS細胞由来神経細胞を用いた病態モデリング Pathophysiological model using iPSC-derived cortical neurons from FTD patients with C9ORF72 repeat expansion 佐藤月花(1)、今泉研人(1)、岡野栄之(1) 1. 慶應義塾大学 Tsukika Sato(1), Kent Imaizumi(1), Hideyuki Okano(1) 1. Keio University School of Medicine Keyword: C9ORF72, induced pluripotent stem cell (iPSC), Frontotemporal dementia (FTD) The C9ORF72 hexanucleotide repeat expansion is the most common genetic cause of　familial frontotemporal dementia (FTD), which mainly affects the frontal and temporal lobes of the cerebral cortex. Some studies have recently suggested that one of the major pathomechanisms underlying FTD is the generation of toxic dipeptide repeat (DPR) proteins produced by C9ORF72 expansion. However, DPR-driven pathogenesis of FTD has not been fully verified in human neural cell models. While the development of human induced pluripotent stem cell (iPSC) technology has enabled the generation of patient-derived neural cells in a dish, there are no reports on iPSC-based modeling of C9ORF72-mediated FTD. In this study, we aimed to generate pathophysiological disease models using iPSCs-derived cortical neurons from FTD patients with C9ORF72 repeat expansion. First, we succeeded in generating frontal lobe-specific neurons from patient-derived iPSCs by modulating Wnt and FGF8 signaling pathway. Gene expression patterns of generated neurons were closely similar to those of human embryonic frontal lobes. Next, we found that p62 protein, which associated with autophagy, were accumulated in frontal lobe-specific neurons derived from FTD patient iPSCs, whereas such phenotypes were not detected in neurons with other brain region identities than the frontal lobe, suggesting that the frontal lobe-specific phenotypes of FTD could be recapitulated in our culture system. p62 accumulation was also observed when DPR proteins were overexpressed in neurons from healthy control iPSCs. Furthermore, the number of LAMP1 protein in frontal lobe-specific neurons derived from FTD patient iPSCs, which are major protein components of the lysosomal membrane is decreased, which found that C9ORF72 repeat expansion cause loss of function of lysosomal. These results indicate that DPR protein toxicity would primarily underlie the FTD pathomechanisms. Further studies into the DPR protein toxicity by C9ORF72 repeat expansions should be explored in this iPSC-based FTD models.