Annual Report (2023)

No.35
The NOVARTIS Foundation (Japan)
for the Promotion of Science
2023年度

財団年報 第35号

Annual Report (2023)
No.35
The NOVARTIS Foundation (Japan)
for the Promotion of Science
2023年度

財団年報 第35号

The Novartis Foundation(Japan)
for the Promotion of Science

Ⅰ．Introduction はじめに ………………………………………………………………

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Kuniaki Takata, Ph.D.
Chairman of the Board of Trustees

Ⅱ．Reports from the Recipients of the 35th Novartis Research Grants
第35回研究奨励報告 ………………………………………………………………… 11
1. Molecular mechanism of lysosomal damage repair by TFEB ……………… 12
Shuhei Nakamura
Graduate School of Frontier Biosciences, Osaka University

2. Investigation of cellular dynamics of CD73-expressing mesenchymal stem
cells in the bone marrow microenvironment …………………………………… 15
Kenichi KimuraLife Science Center for Survival Dynamics,
Tsukuba Advanced Research Alliance, University of Tsukuba

3. A new challenge to HFpEF focusing on the cardioprotective effects of anovel lncRNA ……………………………………………………………………… 18
Michio Sato
Institute of Resource Development and Analysis, Kumamoto University

4. Revealing the mechanism to inhibit early senescence initiation in plants byswitching transcription start sites. ……………………………………………… 21
Asuka Higo
Center for Gene Research, Nagoya University

5. Analysis of cellular self-organizing mechanics by novel opto-thermogenetic
approach …………………………………………………………………………… 24
Fumio Motegi
Institute for Genetic Medicine, Div. of Developmental Physiology,
Hokkaido University

6. Structural and functional analysis of primary cation transporters andelucidation of the substrate specificity by gain-of-function mutant ………… 27
Kazuhiro Abe
Graduate school of Pharmaceutical Sciences, Nagoya University

7. Hematopoietic stem cell aging and mitochondria aging ……………………… 29
Takayoshi Matsumura
Division of Inflammation Research, Center for Molecular Medicine.
Division of Cardiovascular Medicine., Jichi Medical University

8. Significance and spatial profiling of phospholipid metabolism in NASH …… 32
Hayato Nakagawa
Department of Gastroenterology and Hepatology, Mie University

9. Investigating the mechanism of vascular aging using iPSC-derived
3D vascular model ………………………………………………………………… 35
Takafumi Toyohara
Division of Medical Science, Tohoku University

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10. Mechanisms of differentiation of highly infiltrating CD8 T cells in the tumor 38
Shiki Takamura
Department of Immunology, Kindai University Faculty of Medicine

11. Dissection of epigenetic reprogramming underlying tumorigenesis
using acute lymphoblastic leukemia inducing model ………………………… 41
Tomokatsu Ikawa
Research Institute for Biomedical Sciences, Tokyo University of Science

12. Exploratory study for the discovery of zinc homeostasis modulators
utilizing a highly sensitive fluorescence imaging probe ……………………… 43
Kensuke Okuda
Laboratory of Bioorganic & Natural Products Chemistry,
Kobe Pharmaceutical University

13. Cryo-electron microscopy studies for disease pathogenesis of
Xeroderma Pigmentosum ……………………………………………………… 46
Syota Matsumoto
Institute for Quantitative Biosciences, The university of Tokyo

14. Analysis of brain aging caused by the disruption of"nuclear-axon crosstalk" machinery …………………………………………… 49
Kenichiro Kuwako
School of Medicine, Department of Neural and Muscular Physiology,
Shimane University

15. Establishment of precision medicine for subsequent solid tumors
after hematopoietic cell transplantation based on comprehensive
genomic analyses ……………………………………………………………… 52
Satoshi Inoue
Division of Immune Response, Aichi Cancer Center Research Institute

16. Development of asymmetric synthesis of sulfoximine analogs asbioisosteres ………………………………………………………………………… 55
Taichi Kano
Graduate School of Engineering, Tokyo University of Agriculture and Technology

17. Achieve a comprehensive understanding of the pathogenesis of type 1
diabetes mellitus, leading to development of the prevention measures …… 58
Keiichiro Mine
Division of Metabolism and Endocrinology,
Department of Internal Medicine, Faculty of Medicine, Saga University

18. Synthesis of amino acid derivatives and its application for the
inhibitors against SARS-CoV2 protease ……………………………………… 61
Hiroyuki Konno
Graduate School of Science and Engineering, Yamagata University

19. Potential implications of enzymatic activity of the multifunctional protease
NRDC as a new therapeutic target for cardiovascular disease …………… 64
Mikiko Ohno
Department of Pharmacology, Shiga University of Medical Science

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20. Elucidating the mechanism of skin stem cell aging by targeting anti-aging
matrix Fibulin-7 …………………………………………………………………… 67
Aiko Sada
International Research Center for Medical Sciences (IRCMS), Kumamoto
University

21. Studies of genetic basis of early onset cardiac conduction systemdisease and disease mechanism-based personalized medicine …………… 70
Kenshi Hayashi
School of Health Sciences, College of Medical,
Pharmaceutical and Health Sciences, Kanazawa University

22. Analysis of the mechanism underlying extracellular release of tau fortreatment of Alzheimer’s disease ……………………………………………… 73
Shigeki Arawaka
Department of Internal Medicine IV, Division of Neurology,
Faculty of Medicine, Osaka Medical and Pharmaceutical University

23. Study of functional deficits of microglia and accelerated onset ofneurodegenerative disorders …………………………………………………… 76
Keiro Shirotani
The Laboratory of Genome-based Drug Discovery, Nagasaki University

24. Modulation of itch by neuro-immune interaction ……………………………… 79
Hiromitsu Hara
Graduate School of Medical and Dental Sciences, Department of Immunology,
Kagoshima University

25. Analysis of ncRNA transcription mediated chromatin modulation mechanism 81
Kouji Hirota
Department of Chemistry, Graduate School of Science, Metropolitan University

26. Communication between tumor cells and neurons regulating brain tumor

84

Naofumi Uesaka
Graduate School of Medical and Dental Sciences,
Tokyo Medical and Dental University

27. Establishment of tissue maturaiton induction method to obtain adult tissues
from pluripotent stem cells ……………………………………………………… 87
Mio Nakanishi
Graduate School of Medicine, Chiba University

28. Intrinsic barrier dysfunction in multiple sclerosis patient …………………… 89
Hideaki Nishihara
Department of Neurotherapeutics, Yamaguchi University

29. Identification of PP cells as an origin of pancreatic ductal adenocarcinoma 92
Yoshio Fujitani
Laboratory for Developmental Biology and Metabolism, Institute for Molecular
and Cellular Regulation, Gunma University

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30. Development of an innovative diagnostic and therapeutic method thatcombines radionuclide therapy and novel boron neutron capture therapy

95

Kazuma Ogawa
Institute for Frontier Science Initiative, Kanazawa University

31. Investigation on adjuvant effect of peptide-stabilized emulsions …………… 98
Rie Wakabayashi
Goto-Kamiya laboratory, Department of Applied Chemistry, Faculty ofEngineering, Kyushu University

32. Development of novel therapy for sarcopenia based on the regulation of
O-linked N-acetylglcosamine motif (O-GlcNacylation) …………………… 101
Shintaro Kinugawa
Department of Cardiovascular Medicine, Faculty of Medical Sciences,
Kyushu University

33. Molecular mechanism of neuroprotection after ischemic stroke ………… 104
Hiroo Takahashi
Department of Molecular Neurobiology, Kagawa University

34. Involvement of fusion proteins on morphogenesis of neuromuscular junction 107
Bisei Ohkawara
Division of Neurogenetics, Nagoya University Graduate School of Medicine

35. Inhibition of cancer metastasis by selective regulation of AM-RAMP2 andAM-RAMP3 systems ……………………………………………………………110
Megumu Tanaka
Department of Cardiovascular Research, Shinshu University School of Medicine

36. Molecular mechanism for the mutual relationship between macrophage
chemotaxis and activation ………………………………………………………113
Etsuko Toda
Department of Analytic Human Pathology, Nippon Medical School

37. Metabolic signaling regulation by RNA modification …………………………116
Akiko Ogawa
Institute of Development, Aging, and Cancer. Dept of Modomics medicine,
Tohoku University

38. Catalytic Asymmetric Indole Halocyclization for Construction of
Bioactive Structures………………………………………………………………118
Keisuke Asano
Molecular Catalyst Research Division, Institute for Catalysis, Hokkaido University,

39. Analysis of structures and functions of plant nuclear actin microfilaments 120
Noriko Inada
Graduate School of Life and Environmental Sciences, Osaka Prefecture University

40. Establishment of embryonic diapause induction technique for invitro culture of animal embryos.……………………………………………… 123
Katsuyoshi TAKAOKA
Laboratory for Embryology, Institute of Advanced Medical Sciences,
Tokushima university

Publication - FY2021 Grant Recipients ………………………………………… 126
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Ⅲ．Reports from the Recipients of Grants for International Meetings
研究集会報告 ……………………………………………………………………… 135
1 20th International Symposium on Spontaneously Hypertensive Rats …… 136
Norihiro Kato
Department of Gene Diagnostics and Therapeutics, Research Institute,
National Center for Global Health and Medicine

2 10th Asian Biological Inorganic Chemistry Conference …………………… 138
Shinobu Itoh
Graduate School of Engineering, Division of Applied Chemistry, Department of
Molecular Chemistry, Biofunctional Chemistry Lab., Osaka University

3 Symposium on Molecular and Cellular Cognition …………………………… 139
Satoshi Kida
Department of Applied Biological Chemistry, Graduate School of Agriculture andLife Sciences, The University of Tokyo

4 11th International Tunicate Meeting ………………………………………… 142
Takehiro Kusakabe
Department of Biology, Konan University

5 JSICR/MMCB 2022 Joint Symposium ……………………………………… 144
Shigeru Kakuta
Laboratory of Biomedical Science, Graduate School of Agricultural andLife Sciences, The University of Tokyo

Ⅳ．The 36th (fiscal year 2022) Promotion Report

第36期 （2022年度） 助成事業報告 ……………………………………………… 146

Ⅴ．The 36th (fiscal year 2022) Financial Report

第36期 （2022年度） 財務報告 …………………………………………………… 158

Ⅵ．List of the Trustees, Auditors, Councilors and Grant Selection Committee
Members
理事 ・ 監事 ・ 評議員 ・ 選考委員名簿 …………………………………………… 160

Ⅶ．Information from the Secretariat
事務局便り …………………………………………………………………………………… 164

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Introduction

Kuniaki Takata, Ph.D.
Chairman of the Board of Trustees
This annual report includes research reports from the 35th Novartis Research Grant
recipients (research from April 2022 to March 2023: 40 research grants and 5 researchmeetings).
The coronavirus (COVID-19) infection that began in 2019 raged around the world. In
2023 the situation has stabilized and society has regained its composure. During the
pandemic period, while human traffic was greatly restricted, remote conferencing andinformation exchange using digital technology became commonplace, causing a major
tectonic shift in the traffic of information. Through these experiences, we have learned
that in many cases, things can be done efficiently and effectively through Internetbased communication. On the other hand, we have also recognized the importanceof direct person-to-person contact. Furthermore, it was a great surprise to see thepractical application of generative AI technology such as ChatGPT, which allows
people to easily communicate with AI through natural languages. Witnessing the rapid
development of AI and other information science technologies, we are reminded once
again what unique creativity of human beings can bring about.
The Novartis Foundation (Japan) for the Promotion of Science was establishedin 1987 with a donation of 1 billion yen from Ciba-Geigy AG (now Novartis AG)
of Switzerland. The purpose of the Foundation is clearly stated in its Articles ofIncorporation as "to promote science by encouraging creative research in the naturalsciences, thereby contributing to the improvement of the health and welfare of thepeople”. In addition, a document titled "Prospectus for Establishment" written in theyear of the Foundation's establishment states that the Foundation "seeks to promoteand support creative research in the natural sciences, which will be the axis of sciencein the coming 21st century, thereby contributing to the welfare of mankind" and that
the Foundation "provides financial support for research and offers opportunities forcross-border exchange”. The policy of the Foundation is to "promote creative researchin the natural sciences and thereby contribute to the welfare of humanity”. Basedon this policy, the Foundation has provided a total of 1,943 grants over the past 36
years, amounting to approximately 2.22 billion yen. In these times of rapid change,
the Foundation is determined to return to this starting point and support the excellent
research that will open the way to the next era.
This annual report summarizes the results of the excellent research supported by
the Foundation. These are admirable accomplishments achieved in the limited time of
one year. The list of past recipients of the Foundation's grants includes many leading
researchers including two Nobel laureates, Dr. Hideki Shirakawa (Chemistry), and Dr.
Tasuku Honjo (Physiology or Medicine). We hope that the recipients of this grant will
use the results of their research to make even greater strides in their fields. I would
like to express my deepest gratitude again to the selection committee members who
selected these outstanding research projects, and to Novartis Pharma K.K., the donor,
and all those involved in supporting the activities of the Foundation.

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はじめに

代表理事

高田

邦昭

本年報には、第 35 回ノバルティス科学振興財団の研究助成金を受けられた方々の研究報告（2022 年４月～ 2023 年３月の研究：研究奨励金 40 件、研究集会 5 件）を収録しました。
2019 年に始まった新型コロナウイルス（COVID-19）感染症は世界中で猛威をふるいましたが、2023 年になり状況が安定するとともに、社会も落ち着きを取り戻してきました。この間、人の往き来が大きく制限された中で、デジタル技術を活用したリモー
トでの会議や情報発信が一般化し、情報流通に大きな地殻変動が起きました。この様な経験を通じて、私達はインターネットによるコミュニケーションで効率良く効果的に事を運ぶことができる場合が多いことを知る一方で、人と人とが直接向き合うことの重要性も認識しました。さらには、ChatGPT に代表される生成 AI 技術が実用化され、自然言語により人が AI と簡単にコミュニケーションできるようになったのは大いなる驚きでした。この様な AI を始めとする情報科学技術の急速な発展を目の当たりにするにつけ、改めて人にしかできない創造性とは何だろうかと考えさせられたりもします。
本財団は 1987 年に、スイス、チバガイギー社（現ノバルティスファーマ社）からの 10 億円のご寄附をもとに設立されたものです。財団の目的は、定款に「自然科学における創造的な研究の奨励等を行うことにより、学術の振興を図り、もって国民の健康と福祉の向上に寄与する」と明記されています。また、財団設立の年に記された「財団設立の趣意」と題する文書には、財団が「来るべき 21 世紀の科学の軸となる自然科学の創造的研究の振興助成をはかり、以って人類の福祉に寄与できれば」とあり、「研究のための資金的な助成、並びに国境を越えた交流の場の提供」がうたわれています。
このような方針のもと、36 年間で総計 1,943 件、金額にして約 22.2 億円の助成を行ってきました。日々目まぐるしい変化の起こる現在においてこそ、財団は改めてこの原点に立ち返り、独自のアイデアを膨らませている方、新たなプロジェクトを考えている方などの、次の時代を拓く優れた研究を支援して行きたいと考えています。
この年報には本財団が支援した優れた研究の成果をまとめています。一年間という限られた時間の中で達成した立派な業績です。過去に当財団の助成を受けた方々のリストには、ノーベル化学賞を受賞した白川英樹博士やノーベル医学・生理学賞を受賞
した本庶佑博士をはじめ各分野をリードする研究者の名前が多数見られます。今回助成を受けた方々が、この研究成果を契機としてさらに大きく飛躍されることを祈念いたします。これらの優れた研究を選考していただいた選考委員の皆様や、出捐者であるノバルティスファーマ社をはじめとして財団の活動を支えて下さっている関係者の皆様に改めて深く感謝いたします。

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Reports from the Recipients ofNovartis Research Grants

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Molecular mechanism of lysosomal damage repair by TFEB
Shuhei Nakamura
shuhei.nakamura@fbs.osaka-u.ac.jp
Osaka University

Summary Abstract
Damaged lysosomes can be detrimental for cell, but their repair mechanism is largely unclear. We have previously found that TFEB, a master transcription factor for autophagy andlysosome biogenesis, is activated upon lysosomal damage and is essential to repair of damaged lysosomes (Nakamura et al., Nat Cell Biol, 2020). In the current study, to further reveal
the regulatory mechanism of TFEB, we identified key molecules which work both upstream
and downstream of TFEB. On-going functional analysis of these key molecules will contributeto the understanding of molecular mechanism to repair damaged lysosomes.
Key Words ： TFEB, lysosome, autophagy

Introduction
Lysosomes are acidic organelles that contain a variety of digestive enzymes and are responsible for the degradation of intracellular and extracellular components. Various irritantparticles such as silica, calcium oxalate crystals, and toxins are delivered to lysosomes by endocytosis pathway, and these are known to often damage lysosomes. Damaged lysosomes
are extremely harmful to cells because they induce inflammation, oxidative stress, and apoptosis by releasing their acidic contents into the cytoplasm, leading to several prominent disease including neurodegenerative disease and kidney disease. In this study, we aim to reveal
how cells repair damaged lysosomes by focusing on TFEB function.

Results
We have recently found that TFEB, a master regulator of autophagy and lysosomal biogenesis, is activated upon lysosomal damage and plays an essential role in the repair of damaged lysosomes (Nakamura et al., Nat Cell Biol, 2020). Autophagy is known as an intracellular bulk degradation system and is strongly induced by starvation and various stresses. When
autophagy is induced, a double-membrane structure called an autophagosome is formed,
which envelopes various unwanted intracellular materials and then fuses with lysosomes for
degradation. Interestingly, this activation of TFEB requires non-autophagic functions of LC3
protein, which normally plays an essential role in autophagy. We newly found that when lysosomes are damaged, LC3 localizes not only to autophagosomes but also to lysosomes andactivates TFEB through the function of calcium channel TRPML1. However, fundamental andimportant questions remain unanswered, such as (1) how LC3 localizes to lysosomes, (2)why LC3 ultimately leads to TFEB activation, and (3) how TFEB repairs damaged lysosomes.
In order to answer these questions, we carried out the research with the following three aims.

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Aim 1: Molecular mechanism of lysosomal localization of LC3 during lysosomal damage
It has been shown that treatment of cells with LLOMe, a lysosome-specific damaging
agent, causes internal calcium leakage when lysosomes are damaged. Therefore, we investigated the possibility that this calcium may be involved in the lysosomal localization of
LC3. We found that LC3 localizes to lysosomes when calcium leakage from lysosomes is
artificially induced by administration of ML-SA1, an agonist of TRPML1, a calcium channel inlysosomes. On the other hand, LLOMe-treated damaged lysosomal localization of LC3 wasinhibited by administration of BAPTA-AM, a calcium chelator. These results indicate that local
calcium efflux from lysosomes is necessary and sufficient for lysosomal localization of LC3.
In addition, analysis of various mutations in ATG16L1, which is responsible for lipid modification and membrane localization of LC3, revealed that a specific region is essential for thelysosomal localization of LC3. We are currently searching for factors that interact with LC3
and ATG16L1 during lysosomal damage and will use this information to elucidate the detailed
localization mechanism.
Aim 2: Elucidation of the mechanism of TFEB activation by lysosomal LC3
The activity of TFEB is known to be regulated by its phosphorylation status. During lysosomal damage, lysosomal-localized LC3 interacts with TRPML1, which promotes calcium efflux, resulting in dephosphorylation, nuclear translocation and activation of TFEB. These findings suggest that there is a phosphatase that is activated by LC3 and calcium, but the entityof this enzyme is unknown. Therefore, we screened 298 phosphatases fusing a RNAi libraryand identified a candidate phosphatase responsible for nuclear translocation and activation of
TFEB upon lysosomal damage. Importantly, this phosphatase played a role in lysosome damage-specific manner but not in other TFEB activation conditions such as starvation. Interestingly, this phosphatase was normally located in the nucleus, but only upon lysosomal damage
it localized on damaged lysosomes. Furthermore, we found that this localization is dependent
on the lipidation of LC3. We plan to further characterize the role of this phosphatase to understand how LC3 activates TFEB in our future study.
Aim 3: Elucidation of the mechanism to repair damaged lysosomes by TFEB
Targets of TFEB during lysosomal damage and their function in lysosomal repair are notwell understood. To reveal how TFEB repairs damaged lysosomes, we performed transcriptome analysis using RNAseq to search for novel targets of TFEB during lysosomal damage.
The results revealed that there are many TFEB targets that are completely different fromthose induced by starvation. Screening of these factors for their ability to repair damaged
lysosomes, followed by ChIP-qPCR analysis, led to the identification of an enzyme working
in one metabolic pathway as a novel direct TFEB target. Interestingly, this enzyme is mainly
localized to mitochondria and plays an essential role both in mitochondrial and lysosomal
homeostasis independently of its enzymatic activity. We are currently identifying factors thatinteract with this metabolic enzyme, and have discovered a new regulatory mechanism of mitochondrial and lysosomal homeostasis through these factors. Although both lysosomes andmitochondria are known to become dysfunctional in aging and various important diseases,
the mechanism by which homeostasis of these two organelles is simultaneously regulated
is not well understood. We expect that the function of this metabolic enzyme downstream of
TFEB identified in this study will be the key to uncover this mystery.

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Discussion & Conclusion
Damaged lysosomes have been found in many diseases such as neurodegenerative diseases, crystalline nephropathy, silicosis, and infectious diseases, as well as in individual aging
and cellular aging processes, suggesting that the failure of damaged lysosome repair mechanisms leads to the onset and development of these diseases and aging. We believe that further functional analysis of the obtained key factors and TFEB using pathological models andaging models lead to the establishment of new therapeutic methods for lysosomal damageassociated diseases and inhibitors of aging itself.

References
1) Nakamura S, Shigeyama S, Minami S, Shima T, Akayama S, Matsuda T, Esposito A, Napolitano G, Kuma A, Namba-Hamano T, Nakamura J, Yamamoto K, Sasai M, Tokumura A,
Miyamoto M, Oe Y, Fujita T, Terawaki S, Takahashi A, Hamasaki M, Yamamoto M, Okada Y,
Komatsu M, Nagai T, Takabatake Y, Xu H, Isaka Y, Ballabio A, Yoshimori T. LC3 lipidation
is essential for TFEB activation during the lysosomal damage response to kidney injury.
Nat Cell Biol. 2020 Oct;22(10):1252-1263. doi: 10.1038/s41556-020-00583-9.
2) Oe Y, Kakuda K, Yoshimura SI, Hara N, Hasegawa J, Terawaki S, Kimura Y, Ikenaka
K, Suetsugu S, Mochizuki H, Yoshimori T, Nakamura S. PACSIN1 is indispensable foramphisome-lysosome fusion during basal autophagy and subsets of selective autophagy.
PLoS Genet. 2022 Jun 30;18(6):e1010264. doi: 10.1371/journal.pgen.1010264.

一般の皆様へ
リソソームは細胞内の分解を担うのみならず様々なシグナル伝達の司令塔として働き、 細胞や個体の恒常性維持に必須の役割を果たす。 リソソーム機能の破綻は神経変性疾患や感染症など多くの疾患に加えて老化の要因になることが示唆されているが、 リソソーム恒常性がどの様に維持されているかはあまりよくわかっていない。 本研究ではリソソーム恒常性維持に必須の働きを持つ TFEB の制御機構について解析を行い、 いくつかこの制御の鍵を握る因子を同定することができた。 今後、 これら因子の病態や老化モデルを用いた機能解析を進めることで、 リソソー
ム機能不全を伴う疾患の新たな治療法や老化そのものの抑制法確立のシーズ創出につながる可能性があると考えている。

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Investigation of cellular dynamics of CD73-expressing
mesenchymal stem cells in the bone marrow microenvironment
Kenichi Kimura
kkimura@tara.tsukuba.ac.jp
University of Tsukuba

Summary Abstract
Mesenchymal stem cells (MSCs) are a multipotent and considered to be of great potential for regenerative medicine. We have currently identified a cell surface enzyme CD73 as anovel marker for MSCs. However, since a part of MSCs lost CD73 expression after differentiation into progenitor cells, their final fate remain unclear. In this study, we generated CD73Cre driver lineage tracing model and tried to analyze their fate during bone development andfracture repair.
Key Words ： Mesenchymal stem cells, bone, cartilage, CD73

Introduction
MSCs have attracted attention as a tool for regenerative medicine that utilizes their multilineage differentiation and tissue regeneration potential, and clinical trials have begun for many
diseases, including bone disease treatment. However, in vivo cellular dynamics and differentiation potential of MSCs remain unclear, and in many cases, clinical application of MSCs has
been preceded by clinical trials with unclear therapeutic mechanism of action. Therefore, it isdesirable to elucidate the properties of MSCs in vivo in order to establish safe and effective
therapeutic approaches. We have currently identified CD73 which labels MSCs efficiently invivo. Therefore, we aim to elucidate the dynamics of CD73-expressing cells in niche formation and remodeling using cell lineage analysis mouse.

Results
MSCs are maintained in vivo by special microenvironments. The bone marrow niche,
known as one of these niches, is formed by sinusoidal endothelial cells, hematopoietic cells,
and pericytes which tightly regulated by various humoral factors, cytokines, and intercellular adhesion. To clarify the localization and properties of MSCs, we performed screening ofgene expression profiles and identified CD73 (5'-Nucleotidase Ecto: Nt5e) as a promising
molecule. We generated CD73-EGFP reporter mouse, in which EGFP is expressed undercontrol of a CD73 bacterial artificial chromosome (BAC) promoter including distal regulatory
elements (Breitbach*, Kimura*, Cell Stem Cell, 2018). CD73-EGFP reporter mouse labeled
a subgroup of MSCs as well as sinusoidal endothelial cells in the bone marrow. CD73-EGFP
positive MSCs displayed in vitro high proliferation potential and differentiated into osteoblastand chondrocyte compared to EGFP negative MSCs. To investigate whether EGFP positive
MSCs contribute to bone repairing, we generated femoral fracture model. At day 4 post surgery, endogenous CD73-EGFP positive MSCs migrated to bone repair sites and differentiatedinto bone and chondrocytes. Furthermore, some CD73-positive MSCs contributed to neovascularization, suggesting that they are involved in niche remodeling.

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To chase their lineage in detail, we generated tamoxifen
inducible Cre knock-in mouse, CD73-CreERT2 mouse,
by CRISPR/Cas9 system. At first, we confirmed whether
CD73-CreERT2 mouse could label MSCs and sinusoidal
endothelial cells in the bone marrow, we administrated 2 mg
tamoxifen for three consecutive days in the CD73-CreERT2crossed with tdTomato reporter mouse at 8 weeks of age.
tdTomato positive cells were appeared at the periosteum,endosteum, articular cartilage and metaphysis, but sinusoidal endothelial cells in the bone marrow were not labeled.
Next, we administrated tamoxifen to pregnant mother toinvestigate when CD73 positive MSCs emerge in the bone.
At postnatal day 14 after tamoxifen administrated at embryonic day 14.5, tdTomato positive cells appeared at themetaphysis and a part of articular cartilage, and the number of tdTomato positive cells rather increased in the cancellous bone, articular cartilage andsynovium at embryonic day 16.5 tamoxifen administration (Figure 1). The tdTomato positive
cells in articular cartilage expressed Sox9, suggesting that they are chondrogenic progenitors.
The tdTomato positive cells localize perivascular region in the metaphysis, where osteoblastic
progenitors are abundant and undergo active bone differentiation and maturation. These tdTomato positive cells expressed osteo progenitor marker Osterix. The tdTomato positive cells
were also observed in the endosteum in the diaphysis, and they differentiated into osteocyteswith aged.
Bone repair is accompanied by disruption of the bone and vascular network, resulting inmassive remodeling of the niche. We examined how MSCs differentiate and migrate during
this process. After labeling CD73 positive cells with tamoxifen administration, we created femur bone fracture model. At day 14 post surgery, tdTomato positive cells formed cluster within
the callus. These tdTomato positive cells positive for chondrocyte marker, indicating that theydifferentiated into chondrocytes and contributed to cartilage formation. tdTomato positive cells
were also found differentiated osteoblast within the hard callus. Thus, these data revealed
that CD73 is suitable marker to enrich for MSCs that give rise to chondrocyte and osteocytes
during fracture repair.

Discussion & Conclusion
These results indicate that CD73 positive MSCs are a population with high proliferativeand osteo-chondrogenic differentiation potential. We found that CD73 positive cells contributeto the regenerative process of fracture repair by promoting callus formation, suggesting that
CD73 marks osteo-chondroprogenitor cells. Since CD73 serve AMP to adenosine in which
adenosine signal regulate osteo and chondrogenic differentiation, CD73 may play role in not
only as a marker for MSCs and also involving in homeostasis in bone and cartilage. Their final
fate upon bone development and fracture repair and the involved mechanisms of intercellularsignaling need to be addressed in future studies.

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References
1. Kenichi K, Breitbach M, Schildberg FA, Hesse M, Fleischmann BK. Bone marrow CD73+
mesenchymal stem cells display increased stemness in vitro and promote fracture healingin vivo. Bone Reports, 15:101133, 2021
2. Breitbach M*, Kimura K*, Luis TC, Fuegemann CJ, Woll PS, Hesse M, Facchini R, Rieck S,
Jobin K, Reinhardt J, Ohneda O, Wenzel D, Geisen C, Kurts C, Kastenmüller W, Hölzel M,
Jacobsen SEW, Fleischmann BK. In vivo labeling by CD73 marks multipotent stromal cells
and highlights endothelial heterogeneity in the bone marrow niche. Cell Stem Cell, 22(2):262-76, 2018.

一般の皆様へ
間葉系幹細胞 (MSCs) は、 再生医療のツールとして骨疾患治療への実用化に向けた研究が進んでいます。 しかし、 治療の有効性や作用機序についてはまだ明らかとなっていないことが多く残されています。 MSCs がどのような性質を持っているのかが分かれば、 MSCs を利用した新
たな治療法の開発に繋がります。 この研究では、 MSCs の局在とその性質を制御する微小環境について明らかにすることを目指しています。 これによって、 MSCs の生体内の機能解明につながり、 安全で効果的な治療法確立へ貢献できると考えています。

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A new challenge to HFpEF focusing on the cardioprotective
effects of a novel lncRNA
Michio Sato, M.D., PhD,.
m_sato@kumamoto-u.ac.jp
Kumamoto University

Summary Abstract
Recently, using a comprehensive Gene Trap approach, the applicants identified a novel
long non-coding RNA Caren (lncRNA cardiomyocyte-enriched non-coding transcript), which
is highly expressed specifically in cardiomyocytes and localized to the cytoplasm. The applicants have shown that Caren acts on multiple targets, attenuating the "DNA damage response" and exerting cardioprotective effects by "enhancing mitochondrial biosynthesis andfunction. While ncRNAs are generally not conserved among species, we succeeded in identifying a human CAREN candidate and confirmed that its Tg mice exhibit cardioprotective effects.
Key Words ： Heart Failure (HF), Mitochondria, Aging, non-coding RNA

Introduction
The number of patients with heart failure (HF) is increasing in world-wide, especially in HF
preserved Ejection Fraction (HFpEF), which accounts for about half of all HF and is associated with a mortality rate no less than that of HF Ejection Fraction (HFrEF). HFpEF accounts
for about half of all HF, and its mortality rate is no less than that of HFrEF. However, HFpEF,
including its molecular basis, is still largely unknown, and effective treatment has not yet been
established. Moreover, patients with HFpEF are predominantly the elderly. We have reported
that identified lncRNA Caren (cardiomyocyte-enriched non-coding transcript) has cardioprotective effects against pressure-load-induced HF, and we investigated whether this Caren
could ameliorate age-related decline in cardiac function including diastolic dysfunction.

Results
Analysis of a human genomic DNA database revealed that the intergenic region between
human HMGB2 and SAP30 gives rise to 6 lincRNA transcripts (Figure1).
Figure1

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This region is the same where mouse Caren exists. Two of them were expressed in humaninducible pluripotent stem cell-derived cardiomyocytes (Figure2).
Next, the expression of lincRNA transcript 1 was inversely
Figure2
correlated with that of NPPB, a marker of left ventricular
dysfunction (&rho; = &ndash;0.77, p = 0.016), although weobserved no correlation between the expression of lincRNAtranscript 3 and NPPB (Figure3).
Figure3

Moreover, we generated transcript1 or 3 Tg mice andevaluated these mice by TAC (transverse aortic constriction)model.
As a result, transcript1 Tg mice showed cardiac protective effects like mice Caren Tg mice. Based on these results, we determined that the identified transcript 1 is Carenhuman homologue. Finally, we expect that the elucidation of
the mechanism of human CAREN lead to the developmentof novel HF therapies in the future.

Discussion & Conclusion
The majority of lncRNAs reported so far are localized in the nucleus, target a single adjacent gene, and exert their biological effects via some form of regulation under "Cis-manner".
Furthermore, among lncRNAs, lncRNAs that have been shown to exert physiological or
pathophysiological effects in vivo. In addition, only a few lncRNAs have been confirmed tohave physiological or pathophysiological5 effects in vivo. On the other hand, the newly identified "Caren" is characterized by the fact that (1) it is localized in the cytoplasm and (2) multiple
actions have been confirmed in vivo. Therefore, the detailed elucidation of the mechanism ofaction of Caren in this study is expected to lead to the development of a new RNA world byunderstanding the regulatory mechanism of "multiple" target molecules by lncRNAs localized
in the cytoplasm, which has not been elucidated. Moreover, the mechanism of cardioprotective action of Caren differs from existing cardiac failure drugs in that it (1) reduces the DNA
damage response and (2) maintains and enhances the energy supply to the heart by maintaining and improving mitochondrial function.
Our research is expected to elucidate the molecular basis of HF (especially HFpEF) andlead to the development of innovative novel therapies.

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References
1. Sato, M., Kadomatsu, T., Miyata, K., Warren, J.S., Tian, Z., Zhu, S., Horiguchi, H., Makaju,
A., Bakhtina, A., Morinaga, J., et al. (2021). The lncRNA Caren antagonizes heart failure byinactivating DNA damage response and activating mitochondrial biogenesis. Nat Commun
12, 2529. 10.1038/s41467-021-22735-7

一般の皆様へ
日本を含めた先進各国において、心不全患者は増加の一途を辿っています。 心不全は、様々な要因 ・ 疾患により、 心臓の機能が低下した状態です。 心不全の死亡率は、 主要な悪性腫瘍に劣りません。 しかし、 心不全の根本的な治療法は確立していません。 そのような状況の中で、私はミトコンドリアに着目した研究を行っています。 加齢や心負荷により、 ミトコンドリア機能が低下すると、 心不全の進展に繋がります。 私は、 タンパクを作らない RNA (non-coding RNA)を用いて、 心臓ミトコンドリアを増やすことにより、 心機能を改善できるのではないかと期待して、研究を進めています。

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Revealing the mechanism to inhibit early senescence initiationin plants by switching transcription start sites.
Asuka Higohigo.asuka@gene.nagoya-u.ac.jp
Center for Gene Research, Nagoya University

Summary Abstract
For plants, controlling the timing and extent of senescence may be important to properly respond to stress, but such molecular mechanisms are unknown. The genes encoding a novel
family of receptor kinases that inhibit the onset of leaf senescence have multiple transcription
start sites and are interestingly characterized by their ability to switch ligand-accepting extracellular domains (ECDs) by changing the transcription start sites. In this study, comprehensive analysis of transcription start sites by CAGE-seq revealed that transcription is initiatedupstream of all ECDs and that the active transcription start sites vary depending on the leaf
age. Detailed expression analysis showed that the spatio-temporal expression patterns variedamong ECDs. Furthermore, this receptor-kinase family is involved in senescence induced bynitrogen deprivation stress in addition to age-related senescence.
Key Words ： Arabidopsis thaliana, senescence, receptor-like-kinase

Introduction
For land plants, it is necessary to sense and respond to changes in the surrounding environment and their own growth stage, and to adjust the growth of each organ and the entire organism. Plants store nutrients obtained through photosynthesis in their leaves and distributethem to all organs of the body as needed to regulate the growth of the entire organism. Whenplants sense stress, they choose whether to initiate senescence in only some organs or in theentire individual as a survival strategy. On the other hand, early senescence would result in alack of leaves for photosynthesis, making it impossible to maintain the individual. Thus, controlling the timing and extent of senescence initiation may be important to properly respond to
stress, but such molecular mechanisms are unknown.

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Results
The genes encoding a novel receptor-kinases family that repress the onset of leaf senescencehave multiple transcription start sites and have the interesting feature of switching the Extracellulardomain (ECD), the ligand-receptive part, by changing the transcription start sites. The purpose ofthis study is to find clues to elucidate the molecular mechanism that suppresses early senescence
initiation by analyzing the usage of each transcript in detail by exploring the growth conditions andstress stimuli that change the transcription start sites of thisfamily. To comprehensively identify thetranscription start sites of each of the four genes encoding the novel receptor-kinase family andto detect changes in transcription start sites, CAGE-seq analysis was performed on young leaves
in the middle of wild-type maturation and old leaves that have started senescence. The results
showed that transcription is initiated upstream of all ECD-encoding exons. Furthermore, we foundthat the actively transcribed ECDs were altered between young leaves and those that had initiatedsenescence. Next, we compared the changes in transcription of each ECD along developmental
stages under normal growth conditions. Using qRT-PCR with total RNA extracted from the thirdand fourth leaves of various day-old plants, we analyzed changes in transcript levels over time inthe entire leaf. The results showed that each ECD showed a different pattern of expression levelchanges along the developmental stages. Some ECDs showed a pattern of continually increasing expression levels along their age, while others showed a pattern of increasing up to a certain
days of age and then decreasing. The degree of up-regulation also differed among the ECDs, withsome ECDs showing only a 1.5-fold up-regulation, while others showed up to a 28-fold up-regulation. Furthermore, a reporter line expressing the GUS (β-glucuronidase) gene under the controlof the promoter region of each ECD was created, and a detailed analysis of the spatial expression
pattern of each ECD sequence revealed that the tissues expressing each ECD were different. All
ECD promoters showed strong activity in vascular tissue, but some showed activity throughout
the vascular bundle, while others showed activity only around the xylem. Some promoters werealso active in leaf fleshy tissue. To examine the differences in function of each ECD in senescenceinduced in response to stress in addition to aging under normal growing conditions, we first examined the stresses in which the receptor kinases we were focusing on were involved. Using a
medium deficient in essential nutrients for growth such as sugar, nitrogen, phosphorus, and potassium, we compared the time of senescence induction in the wild type and mutants of the receptor
kinase. The results showed that the focused receptor kinase was involved in senescence inducedby nitrogen in particular.

Discussion&. Conclusion
This study showed that the spatio-temporal expression pattern of ECDs differs among
ECDs during aging under normal growth conditions. We would like to further verify this interms of changes in the ratio of ECDs along the growth stages by performing absolute quantification of ECDs in the future. We are currently preparing mutants in which only a single ECD
is non-functional by introducing mutations in each ECD with CRISPR-Cas9, and are observing the aging phase. Preliminary experiments suggest that in aging-induced senescence, different ECDs lacking function may have different onset times of senescence, i.e., the contribution of each ECD in suppressing senescence may be different. In addition, we would like toexamine the use of different ECDs in stress-induced senescence by observing the different
expression patterns of each ECD and the onset time of senescence of mutants of each ECD
in the induction system of senescence by nitrogen deprivation established this time.

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一般の皆様へ
動くことのできない植物は、 周囲の環境変化や自身の成長段階を感知して応答し、 それぞれの器官および個体全体の成長を調整する必要があります。 そのような応答の一つに、 老化開始
時期の制御がありますが、 植物が、 様々な情報を統合して老化の開始時期を調節するための分子機構はわかっていません。 本研究で機能解析を進めた受容体キナーゼは、 その遺伝子が欠失すると老化開始時期が早まることから、 老化開始の調節に関わっていることが示唆されます。
本研究での成果を足がかりに、 今後さらに機能解明を進めることで、 植物が老化開始時期を調節する仕組みを明らかにし、 将来的には、 人工的に操作する技術の開発にもつながることが期待されます。

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Analysis of cellular self-organizing mechanics
by novel opto-thermo-genetic approach
Fumio Motegi
motegi@igm.hokudai.ac.jp
Institute for Genetic Medicine, Hokkaido University

Summary Abstract
An embryo undergoes the generation of cells with specific fates, forms, and functions
through collective interactions of biological components, referred to as self-organization. This
project aims to develop new paradigms for morphogenesis by developing novel techniques
for the measurement and manipulation of mechanical forces in the multi-cellular organism, C.
elegans. We employed tools and techniques needed to interrogate the mechanical processes
to analyze how embryonic patterning and oogenesis are regulated through the coupling ofchemical reactions and mechanical forces.
Key Words ： Mechanics, Embryogenesis, Force, Cell polarity, C. elegans

Introduction
The establishment of global order within an organism generally emerges from local interactions among molecules and cells. These collective interactions, commonly referred to as selforganization, give rise to the emergent properties encompassing the fate, form, and functionof cells, ultimately leading to morphogenesis of tissues and organs. Mechanical forces play a
crucial role in driving these transformative changes, and recent investigations have shed lighton their potential to influence cellular fate and function. These findings suggest the existenceof a complex feedback loop between forces and cellular physiology. To elucidate the underlying design principles governing such mechano-chemical feedbacks, it is imperative to develop
advanced technologies capable of quantifying and manipulating forces within biological systems.

Results
This study aimed to develop analytical techniques to investigate the physiological significance of mechanical forces in multi-cellular systems. We attempted to establish a "thermogenetics" method by which the functions of target proteins involved in force generation can bequickly inhibited in multi-cellular stage animal, C. elegans. Using a combination of microfluidic
device and infrared light device, temperature in early embryos and oocytes were globally
maintained at 15ºC and locally increased it to 20-25ºC. This local increase in cellular temperature was sensed by genetically modified cytoskeletons, which could be promptly inactivatedwhen exposed to a restrictive (high) temperature. By utilizing this technology, we were able toelucidate the effects of force generation attributed to microtubules and the actin cytoskeleton
on the patterning of cells and tissues. Our investigation specifically focused on asymmetriccell division during the early-stage embryo development and oogenesis in adult hermaphrodite animals.

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We first screened temperature-sensitive mutations in various cytoskeleton genes and cell
polarity genes that exhibited a rapid response to a temperature shift from 15ºC to 25ºC. We
confirmed that missense mutations in the type II myosin heavy chain NMY-2 (ne3409), theactin nucleator formin CYK-1 (or596), and tubulin TBA-1 (or594) led to the inhibition of cytoskeletal structures within 10 seconds upon the rapid temperature shift. Similarly, temperaturesensitive mutations of the centriole component SPD-2 (or293), the midbody protein CYK-4(or749), the PAR complex proteins PAR-2 (or539) and PKC-3 (ne4246) caused inhibition of
their respective structures within approximately 30 seconds following the temperature shift.
Based on these observations, we successfully demonstrated that local temperature changes
can effectively manipulate mechanical forces within a confined region of multicellular organisms.
We next employed the thermos-genetics technique to investigate the impact of force generation derived from the actin skeleton on the patterning of fertilized zygotes. Shortly after fertilization, the zygote initiates polarization along the antero-posterior axis and segregates the
dedicated polarity regulators, PAR proteins, on the cell cortex during mitosis. Previous studies
have established the significance of the actin cytoskeleton in the initiation and establishment
of PAR polarity. By using the thermo-genetics approach, we aimed to examine whether theactin cytoskeleton also contributes to the maintenance of the established pattern of PAR proteins during late mitosis. We found that inactivation of NMY-2 and CYK-1 during anaphase,but not from prometaphase to metaphase, led to the mis-segregation of PARs during cytokinesis. This finding confirms the efficacy of the thermo-genetics technique in exerting temporaland spatial control over actin cytoskeleton-mediated forces. The thermo-genetics techniqueoffers valuable tool to explore the temporal and spatial control of the actin cytoskeleton-mediated forces.
We are employing this technique to analyze the impact of both microtubule- and actin
skeleton-derived forces on the spatial pattern of multicellular-stage embryos. Previous studies
have highlighted distinct mechanisms in the initiation and pattern formation of PAR proteins
between zygotes and two-cell stage embryos. In the formation of germ cell precursors during
the two-cell-stage, polarity formation relies not only on the actin cytoskeleton but also on microtubules. To investigate this phenomenon further, we compromised the architecture of microtubules by inhibiting the function of tubulin and centrosomes. As a result, we observed that
although PAR proteins initially established an asymmetric pattern, they eventually depolarized
during mitosis. This finding a crosstalk between microtubule- and actin-dependent forces in
the maintenance of the polarity pattern during early embryonic development. By combining
our thermo-genetics technique with these mutant strains, we aim to gain a deeper understanding of the intricate interplay between mechanics and developmental processes.

Discussion & Conclusion
Through the utilization of a hybrid approach involving a microfluidic device and an infrared
light device, we successfully demonstrated that local manipulation of temperature can beused for acute inactivation of cellular forces exerted by the cytoskeletons within multi-cellular
stage embryos. By employing this method, we can delve deeper into understanding the intricate interplay between mechanics and tissue patterning during animal development.

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一般の皆様へ生物は細胞集団の空間パターンを組織 ・ 器官スケールで秩序化することで、 生体の形と機能を創生する。 この細胞集団の秩序化では、 細胞が機械的力の発生を感知 ・ 応答することで化
学シグナル伝達を調節する 「力学化学カップリング」 を必要とすることが示された。 しかしながら、力作用は直接可視化することができないので、 この仕組みと役割には未だに不明な点が多い。
本研究では、力発生の役割を解明するための技術開発を目的とし、細胞内温度変化を利用して、力発生に関わる標的因子の機能を素早く阻害する 「温度遺伝学法」 を開発し、 線虫多細胞期
胚における非対称分裂を司るメカニクスの機能を解析した。 この技術を活用して、 細胞自律的な自己組織化に対して、 細胞非自律的な力学的作用が及ぼす影響を調べることで、 マクロスケー
ルの生体秩序化を司る基盤原理が解明できる。

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Structural and functional analysis of primary cation transporters
and elucidation of the substrate specificity
by gain-of-function mutant
Kazuhiro Abe
kabe@cespi.nagoya-u.ac.jp
Nagoya University

Summary Abstract
We study primary-active transporter via P-type ATPases using functional and structural
analyses to demonstrate that four mutations transform the non-gastric proton pump, a strict
H+-selective electroneutral ATPase, into a bona fide Na+-dependent electrogenic sodium
pump, providing insight on how Na+ binding drives a concerted mechanism leading to Na+/K+
pump phosphorylation.
Key Words ： P-type ATPase, proton pump, sodium pump, cryo-EM, membrane proteins

Introduction
Despite high sequence conservation between proton and sodium pump (more than65%), their transport substrate (H+ or Na+) and their stoichiometry (1H+/1K+ for proton pump,
3Na+/2K+ for sodium pump) is strictly discriminated. In order to gain the molecular insight into
their substrate specificity, we attempted to identify Na+-transporting gain-of-function mutant ofproton pump, and determine its structure by cryo-EM.

Results
1. We determined a crystal structure of non-gastric proton pump (ngHKA), and structurally
defines its transport stoichiometry of 1H+/1K+ per ATPhydrolysis.
2. Staring from WT ngHKA, we design tailored mutants basedon the structural data, previous results and sequence comparison. The substrate specificity of these mutants is evaluated utilizing kinetics, electrophysiology, and cryo-EM analyses.
3. Finally, we success to generate quadruple mutant of ngHKA(SPWC mutant, see figure) that mediate Na+/K+-specific,
electrogenic transport like genuine sodium pump. Cryo-EM
analysis determined three Na+ and two K+ are bound to the
TM cation site in E1 and E2 state, respectively. We further
identified sequential conformational change induced by the
Na+-binding leading autophosphorylation.

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Discussion & Conclusion
We presented the first structure of the ngHKA, defined its transport stoichiometry and proton extrusion mechanism, and revealed the requirements to transform this strict electroneutral
1H+/1K+ pump into a strict electrogenic 3Na+/2K+ pump. This transformation reveals how evolution exploited the same mechanism to build electrochemical gradient of different magnitude
for dissimilar ions.

References
Young V., Nakanishi H., Meyer D.J., Nishizawa T., Oshima A., *Artigas P. & *Abe K. Structure and function of H+/K+ pump mutants reveal Na+/K+ pump mechanism. Nat. Commun.,13, 5270, 2022.
(*correspondence)

一般の皆様へ
細胞膜を隔てたイオンの不均衡分布は、 例えば神経の情報伝達や栄養素の細胞への取り込
み等、 生命現象と深くかかわっています。 これは ATP のエネルギーを利用してイオンを輸送す
る非常によく似たタンパク質、 能動輸送体によって形成されています。 しかしながら、 輸送される
イオンの種類やその個数は厳密に決まっていて、 決して間違えたりはしません。 我々はプロトンポンプをナトリウムポンプに変換することに挑戦し、 たった４つのアミノ酸を置換するだけで、 Na+を輸送するポンプを創る事に成功しました。 構造解析によって、 ３つの Na+ と２つの K+ を結合していることが確認できました。 この研究によって、 プロトンポンプとナトリウムポンプがどのように輸送するイオンの種類を認識しているかが詳しく理解できました。

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Hematopoietic stem cell aging and mitochondria aging
Takayoshi Matsumura
matsut@jichi.ac.jp
Division of Inflammation Research, Center for Molecular Medicine
Division of Cardiovascular Medicine, Jichi Medical University

Summary Abstract
In this study, given the diversity and heterogeneity of aging hematopoietic stem cells(HSC), comprehensive gene expression analysis and epigenomic analysis at the single celllevel (single-cell RNA sequencing and single-cell ATAC sequencing) were performed. Among
HSCs obtained from aged mice, we found some cell populations with gene expression andepigenomic changes similar to those of HSCs obtained from young mice. In the future, wewould like to establish a method to select such HSC populations from elderly individuals anduse them in the treatment of various age-related diseases.
Key Words ： Hematopoietic stem cells, aging, mitochondria

Introduction
Hematopoietic stem cells (HSCs) reside at the apex of the hierarchy of hematopoietic cells,
uniquely capable of dividing without differentiating (self-renewal) and also differentiating to all
the different cellular components within the blood. Thus the precise regulation of HSC self-renewal and differentiation without malignant transformation is critical. However, HSC functions
alter with age: aged HSCs exhibit lower repopulation potential, loss of homing potential, and ahigher risk of malignant transformation. In this project, we have extended our current researchon HSC aging by using single cell RNA-sequencing (scRNA-seq) and single cell assay fortransposase-accessible chromatin using sequencing (scATAC-seq).

Results
Dendra2 mice (Gt(ROSA)26Sortm1.1(CAG-COX8A/Dendra2)Dcc/J mice), whose intracellular mitochondria emit green fluorescence, were used in this study. Slam HSCs
(CD150+CD48-Lin-Sca1+Kit+) were sorted from bone marrow of young (2-4 months old) andold (18 months old) Dendra2 mice using FACS, and further sub-divided into Dendra2-high
HSCs (HSCs with high mitochondrial mass) and Dendra2-low HSCs (HSCs with low mitochondrial mass). Comprehensive gene expression analysis (scRNA-seq) and epigenomic
analysis (scATAC-seq to identify open chromatin regions in the whole genome) were performed for each single cell from 4 groups (young mitochondria-low, young mitochondria-high,old mitochondria-low, and old mitochondria-high HSCs).
Researchers from all over the world have proposed various methods to analyze scRNAseq, and it is not yet clear which one is superior. Furthermore, simultaneous analysis of
scRNA-seq and scATAC-seq is still relatively new, and no established methods are publishedor publicly available on how to integrate multiple scRNA-seq and scATAC-seq data considering their batch effects. Hence we tried and compared several bioinformatic analyses, and

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decided to: 1) merge scRNA-seq data from each sample after individual quality check, 2)
merge scATAC-seq data from each sample after individual quality check, 3) further merge
two pre-merged data using the weighted neighborhood (WNN) analysis and a feature dimension reduction method called Uniform Manifold Approximation Projection (UMAP), 4) conduct
clustering using information both from scRNA-seq and scATAC-seq, 5) perform scRNA-seq
analysis both on each cluster and on the whole dataset, and 6) perform scATAC-seq analysis
both on each cluster and on the whole dataset. The know-how established during this project
was applicable also to scRNA-seq data on normal hematopoietic differentiation (reference 1).
HSCs were divided into four groups in order of cell numbers: 1) average HSCs, 2) HSCs
with a high protein synthesis capacity, 3) HSCs with cell cycling, and 4) HSCs with increased
inflammatory signals. The most striking feature of HSCs fromaged mice was that cells withcell cycling reduced, especially in aged HSCs with high mitochondrial mass. This may reflect
cell cycle arrest or senescence in these cells. Secondly, we noticed that a specific subsetof HSCs increased in HSCs from aged mice, suggesting that this subset represents a cell
population with particularly reduced HSC function. Third, another cell population located on the
opposite side in the UMAP showed a higher protein synthesis capacity than other populations,and was considered to be a population of cells in which stem cell function was maintained.
This population was well preserved even in aged mice, demonstrating gene expression andepigenomic changes very similar to HSCs from young mice. This cell population can be apotential candidate for 'young-like' HSCs in old mice. We are currently investigating whichsurface antigens (cell surface proteins that can be used for FACS sorting) are suitable for
sorting this cell population. In addition, by analyzing the above-mentioned HSCs with particularly
reduced function in detail, we identified a surface protein X was specifically expressed on more
aged HSCs among aged mice. We successfully established the method to sort these cellsby FACS using antibodies against protein X. To sort protein X-negative HSCs can be anotherstrategy to enrich ‘young-like’ HSCs.

Discussion & Conclusion
We have found that a cell population among HSCs obtained from aged mice shows gene
expression and epigenomic changes very similar to HSCs obtained from young mice and that
protein synthesis is enhanced in this cell population compared to other cell populations. In
the future, we would like to establish a method to select such HSC populations from elderly
individuals. When a method to identify and enrich 'young-like' HSCs is established in the near
future, it will be possible to raise the age of bone marrow transplantation donors. In the longer
term, knowledge gained in the process of elucidating HSC aging can be applied to researchon stem cell aging in various tissues other than bone marrow. It could also be applied to personalized medicine to identify individuals likely to develop abnormalities associated with aging, not only in blood diseases but also in diseases of various organs.

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References
1. Matsumura T (corresponding author), Totani H, Gunji Y, Fukuda M, Yokomori R, Deng
J, Rethnam M, Yang C, Tan TK, Karasawa T, Kario K, Takahashi M, Osato M, Sanda T,
Suda T. A Myb enhancer-guided analysis of basophil and mast cell differentiation. Nat
Commun. 2022 13(1):7064.

一般の皆様へ
造血幹細胞は全ての血液細胞になる （分化する） ことが可能な特別な細胞です。 悪性腫瘍化することなく生涯にわたる血液の産生を維持するために、 造血幹細胞の機能は厳密に制御されなければいけません。 しかし、 残念ながら、 造血幹細胞の機能は老化とともに低下し、 悪性腫瘍化の危険性は上昇します。 今回の研究で、 年老いたネズミの造血幹細胞を詳細に解析したところ、 年老いたネズミの中にも比較的その機能が保たれた、 いわば、 “まだ若い” 造血幹細胞がいる可能性があります。 今後は、 このような “若い” 細胞をヒトからも精製する方法を確立したいと考えております。

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Significance and spatial profiling of phospholipid metabolism
in NASH
Hayato Nakagawa
nakagawah@med.mie-u.ac.jp
Department of Gastroenterology and Hepatology, Mie University

Summary Abstract
Enhanced de novo lipogenesis mediated by SREBP is thought to be involved in nonalcoholic steatohepatitis (NASH) pathogenesis. In this study, we assessed the impact of SREBP
inhibition on NASH and liver cancer development in murine models. Unexpectedly, SREBP
inhibition via deletion of SCAP in the liver exacerbated liver injury, fibrosis, and carcinogenesis, despite markedly reduced hepatic steatosis. SCAP–SREBP pathway inhibition altered
the fatty acid (FA) composition of phosphatidylcholines due to both impaired FA synthesis anddisorganized FA incorporation into phosphatidylcholine via LPCAT3 downregulation, which led
to endoplasmic reticulum stress and hepatocyte injury. Thus, excessively strong and broad lipogenesis inhibition was counterproductive for NASH therapy, which will have important clinical implications in NASH treatment.
Key Words ： Nonalcoholic fatty liver disease, Lysophosphatidylcholine acyltransferase,
hepatocellular carcinoma, autophagy, lipidomics

Introduction
Enhanced de novo lipogenesis mediated by SREBP is thought to be involved in NASH
pathogenesis. At present, several drugs targeting enzymes involved in hepatic de novo lipogenesis such as ACC, FASN, and SCD1, which are all regulated by SREBP, are underdevelopment (1). However, to the best of our knowledge, there is no conclusive experimental
evidence demonstrating that SREBP inhibition can prevent NASH or NASH-related hepatocellular carcinoma (HCC) development. Thus, we assessed the impact of SREBP inhibition
on NASH and HCC development in murine models.

Results
We used a well-known NASH-HCC mouse model: a liver-specific phosphatase and tensin homolog (PTEN) knockout mouse (PTENΔL) generated by crossing a Ptenflox/flox
mouse and an albumin-Cre mouse (Alb-Cre). PTENΔL mice show constitutive upregulationof SREBP due to PI3K–Akt pathway activation, which leads to spontaneous fatty liver andsubsequent HCC development. To examine the effects of SCAP–SREBP pathway inhibition
in PTENΔL mice, we generated liver-specific PTEN/SCAP double-knockout mice by crossing PTENΔL mice with Scapflox/flox mice (PTEN/SCAPΔL). Unexpectedly, SREBP inhibition
via deletion of SCAP in the liver exacerbated liver injury, fibrosis, and carcinogenesis, despite markedly reduced hepatic steatosis. These phenotypes were ameliorated by restoring
SREBP function through the transgenic or AAV-mediated approach. Moreover, enhanced
hepatocarcinogenesis through SCAP deletion was observed in another NASH model, thecholine-deficient, L-amino acid-defined, high-fat-diet model.

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Transcriptome and lipidome analyses revealed that SCAP–SREBP pathway inhibition altered the fatty acid (FA) composition of phosphatidylcholines due to both impaired FA synthesis and disorganized FA incorporation into phosphatidylcholine via lysophosphatidylcholine
acyltransferase 3 (LPCAT3) downregulation, which led to endoplasmic reticulum (ER) stressand hepatocyte injury. Supplementation of phosphatidylcholines significantly improved liver
injury and ER stress induced by SCAP deletion. Spatial profiling of phospholipids revealed
that zonal distribution of phosphatidylcholines was observed in wild type mice, while it wasmarkedly disorganized in PTEN/SCAPΔL mice.
Hepatic steatosis is a hallmark of NAFLD. However, hepatic fat deposition is often significantly decreased in advanced NASH, referred to as “burned-out NASH.” We performed
RNA-seq analysis using liver biopsies obtained from NAFLD patients and found that the
SREBP-mediated lipogenesis pathway was significantly downregulated in burned-out NASH.
Furthermore, the activity of SCAP-SREBP-LPCAT3 axis was found inversely associated withliver fibrosis severity in human NASH, suggesting that the downregulation of this axis may be
involved in the pathogenesis of advanced burned-out NASH.
Although phospholipid composition was also disturbed in SCAP single knockout mice
(SCAPΔL), SCAPΔL mice did not exhibit spontaneous liver injury, suggesting that an additional factor induced by PTEN deletion is required for liver injury. We identified that SREBP
inhibition cooperated with impaired autophagy to trigger liver injury. In fact, additional deletionof the autophagy-essential gene ATG5 in SCAPΔL mice significantly aggravated liver injury
and HCC development accompanied by enhanced ER stress .

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Discussion & Conclusion
We found that the strong inhibition of SCAP/SREBP-mediated lipogenesis unexpectedly
exacerbated liver injury, fibrosis, and carcinogenesis in murine NASH via the disturbance ofphospholipid metabolism. These findings are critical to developing NASH treatment strategies
because they indicate that excessively broad and strong inhibition of the lipogenic pathway
may be counterproductive in NASH therapy. Importantly, the SREBP-mediated lipogenesis
pathway was significantly downregulated in patients with advanced burned-out NASH, which
was consistent with a previous report (2). Although the significance of downregulation of
SREBP pathway in advanced NASH has been unknown, our present study arises the possibility that downregulation of SREBP pathway may be a disease-promoting factor in advanced
stage NASH. In fact, less steatosis in patients with NASH-related cirrhosis is associated withhigher risk for HCC development and mortality, even among patients with the same Child–
Turcotte–Pugh score (3). Thus, both excessive and insufficient SREBP activation may resultin NASH disease progression, and appropriate SREBP activity may be essential for maintaining liver homeostasis. Furthermore, phospholipid supplementation or correction of abnormal
FA incorporation into phospholipids may be treatment options for advanced stage NASH (4).

References
1. Nakagawa H, et al. Lipid Metabolic Reprogramming in Hepatocellular Carcinoma. Cancers.
2018;10(11).
2. Nagaya T, et al. Down-regulation of SREBP-1c is associated with the development ofburned-out NASH. J Hepatol. 2010;53(4):724-31.
3. Vilar-Gomez E, et al. Fibrosis Severity as a Determinant of Cause-Specific Mortality inPatients With Advanced Nonalcoholic Fatty Liver Disease: A Multi-National Cohort Study.
Gastroenterology. 2018;155(2):443-57.e17.
4. Kawamura S, et al. Inhibiting SCAP/SREBP exacerbates liver injury and carcinogenesis inmurine nonalcoholic steatoheopatitis. J Clin Invest. 2022;132(11):e151895.

一般の皆様へ
非アルコール性脂肪肝炎 （NASH） は、 肝臓への脂肪沈着 （脂肪肝） をきっかけに、 炎症 ・線維化がおこり、 肝硬変や肝癌に至る進行性の疾患です。 今回の研究から、 進行した NASH
では肝細胞内の脂質、 特にリン脂質のバランスが崩れ、 細胞が障害されやすくなっていることが明らかとなりました。 リン脂質の補充やリン脂質バランスの改善が、 進行 NASH の治療法の一つとなると期待されます。

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Investigating the mechanism of vascular aging
using iPSC-derived 3D vascular model
Takafumi Toyohara
toyohara@med.tohoku.ac.jp
Tohoku University Hospital

Summary Abstract
Lifestyle diseases such as hypertension, diabetes, and dyslipidemia are ultimately 'vasculardiseases' which causes vascular aging. However, no effective way has been developed to accurately predict and treat vascular aging. In this research, 3D blood vessel was created frompatient-derived iPS cells. We are investigating the physical effects of blood flow on vascular
aging using the 3D vasculature.
Key Words ： Vascular aging, iPS cells, Vascular smooth muscle cells, Endothelial cells

Introduction
As average life expectancy is increasing and the population aged 100 and over is expected
to reach 300,000 by 2040 in Japan, it is necessary that elderly people can stay healthy andplay an active role in society. Lifestyle diseases such as hypertension, diabetes, and dyslipidemia are ultimately 'vascular diseases' which cause vascular aging. Despite significant progress in the field, curative treatments have yet to be developed directly targeting the vascular
aging.

Results
Since models using mouse vascular tissue is unable to fully recapitulate human pathophysiology observed in the clinic, due to the differences in gene expression from that of humans,
human iPSC-derived tissue-engineered blood vessels (TEBV) will serve as a more robustmodel to elucidate the mechanisms leading to vascular aging. Although some TEBVs with
vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) have already been established in the field, these models have caveats in their ability to mimic the physiology of thehuman artery. For example, the contraction of these vessels remains weak due to the lack of
circumferential alignment of VSMCs and the lack of flexibility in the scaffolds. These two characteristics are important to address because it has been well established that contraction anddilation play a crucial role in vascular diseases.
In this research, we established a novel rolling-up molding method to form a physiologically
deformable TEBV from human iPSCs. First, a VSMC fiber was formed with a 3D-printed microchannel mold and metallic anchors. The iPSCs-derived VSMCs suspended in a collagenhydrogel spontaneously contracted about 95% in volume and aligned axially in 24 hours. This
fiber is rolled up by a 3D-printed inclined mold to spontaneously form a spiral shape. Finally,rolled-up VSMCs were embedded into collagen hydrogel with perfusion connectors, and ECs
were seeded inside to establish the TEBV. As a proof of concept, our TEBV was also challenged to undergo other physiologically-relevant conditions including, maintenance under

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The Novartis Foundation(Japan)
for the Promotion of Sciencepulsatile perfusion, treatment with U46619, a thromboxane A2 agonist, and normalized with
contraction by KCl. The relative contraction ratio was reported to be 78.0 ± 5.1%, similar topreviously reported ex vivo experiments.
We prepared TEBV using iPS cells derived from patients with Werner syndrome, a type ofprogeria, to create a model of vascular aging, and confirm differences in vascular contractility and compliance between Werner syndrome and healthy control. The TEBV derived froma patient with Werner syndrome tended to show increased contraction, similar to the result
observed in previous studies. We are currently performing computational fluid dynamics using patient clinical data and the TEBV to clarify what types of blood flow affect vascular aging.
Furthermore, RNA-seq, metabolomics, and lipidomics are planned to elucidate the mechanism of vascular aging.

Discussion & Conclusion
Our TEBV can mimic the phenotype of vascular aging, which can be utilized to examine
the effect of blood flow on vascular aging. Once we clarify the type of flow affecting vascular
aging, prediction of the vascular site where aging is likely to occur will be possible. It will also
have the potential to create a method for monitoring vascular aging. This research also has
the potential developing new therapies that can mitigate vascular aging. Taken together, ourmodel will serve as a more physiologically-relevant model to investigate the mechanisms behind vascular aging and thus will lead to therapeutic discoveries.

Figure 1.
A novel rolling-up molding method to form a physiologically deformable TEBV from human iPSCs. The
TEBV is contracted with vasoconstrictors such as thromboxane A2 agonist.

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References
1. Itai S., Tajima H., Onoe H. Double-layer perfusable collagen microtube device for heterogeneous cell culture.
Biofabrication 11: 0150110, 2019.
2. Toyohara T., Roudnicky F., Florido M.H.C., Nakano T., Yu H., Katsuki S., Lee M.J., Torsten
M., Friesen M., Davidow L.S., Ptaszek L., Abe T., Rubin L.L., Pereira A.C., Aikawa M.,
Cowan C.A. Patient hiPSCs Identify Vascular Smooth Muscle Arylacetamide Deacetylase
as Protective against Atherosclerosis.
Cell Stem Cell 27: 147-157, 2020

一般の皆様へ
血管は年齢とともに老化し動脈硬化のリスクとなりますが、 その原因は解明されておらず、 治
療法もありません。 我々は iPS 細胞を用いて3D 血管を作製し、 血流が血管老化に及ぼす影響を解析しています。 本研究によって血管老化を起こす要因を明らかにすることで、 血管の老化を予防、 治療する方法を開発していきたいと思っております。

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Mechanisms of differentiation of highly infiltrating
CD8 T cells in the tumor
Shiki Takamuratakamura@med.kindai.ac.jp
Department of Immunology, Kindai University Faculty of Medicine

Summary Abstract
CD8+ T cells with tissue-resident memory (TRM-like TIL) characteristics are found in the tumor, and their presence is critically associated with preferable clinical outcomes. However, themechanisms by which TRM-like TIL are generated in the tumor are poorly understood. Throughthe analysis of tissue section of human cancer patients, we identified the actual sites where
TRM-like TIL are generated in the tumor, and local antigen signaling are likely to be a factor
that promotes the differentiation of TRM-like TIL. In the future, we will investigate the possibility
that macrophages located in the differentiation sites provide the necessary signaling for thedifferentiation of TRM-like TIL.
Key Words ： Tumor, CD8 T cell, TRM-like TIL,

Introduction
Tissue-resident memory T cells (TRM) that permanently reside in epithelial tissues play a
predominant role in protective immunity1. It has recently been shown in human patients thatsome tumors harbor CD8+ T cells with TRM characteristics (TRM-like TIL) and high frequenciesof these are associated with an improved response to tumor immunotherapy2. However, themechanisms by which TRM-like TIL are generated in the tumor are poorly understood. In thisstudy, by using human tissue samples and the mouse model, we attempt to understand how
TRM-like TIL are generated in the tumor.

Results
Our histological analysis of tumor specimens from patients with head and neck cancer(squamous cell carcinoma) revealed that the generation of TRM-like TIL (double-positive forCD103 and CD8) is unique to some patients and that TRM-like TIL, but not CD103− CD8 T
cells predominantly infiltrate the tumor, suggesting that CD8 T cells acquire TRM characteristics before infiltrating the tumor (in the stroma). We classified patients into three groups basedon the infiltration of CD8 T cells and their differentiation to TRM -like TIL: 1) differentiation of
TRM-like TILs in the stroma and infiltration of these cells in the tumor, 2) infiltration of CD8+
TILs (CD103−) in the stroma without TRM differentiation, and 3) poor infiltration of CD8+ TILs inthe stroma. In group 1, TRM -like TIL have a superior ability to infiltrate deep into the tumor, reflecting their reputational advantage for antitumor immunity. Although TRM-like TIL in the tumor
highly express checkpoint inhibitory molecules PD-1 and Tim-3, most cells are still positive
for IFNγ and perforin, indicating that these cells are not fully exhausted. It is well known that
TCF+ progenitor exhausted CD8 T cells, which is capable of effectively responding to checkpoint blockade immunotherapy, are preferentially generated in the tertiary lymphoid structures

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The Novartis Foundation(Japan)
for the Promotion of Science(TLS)3. On the other hand, TRM -like TIL are rarely found in TLS, indicating that TCF+ progenitor exhausted CD8 T cells and TRM -like TIL are generated by distinct mechanisms. We
identified at least two distinct sites where TRM-like TIL are generated in the stroma: one is at
the tumor border where anti-tumor immunity is actively induced (differentiation site: DS), andthe other is surrounding the areas where tumor cells undergo necrosis (remodeling site: RS).
Given the morphological differences of these sites, we hypothesize that TRM-like TIL in DS and
RS are distinct subsets, effector, and memory, respectively, and that signals necessary for
their differentiation likely differ. In support of this, we found that DS mainly consisted of fibroblasts, whereas RS consisted of αSMA expressing myofibroblasts, a cellular hallmark of tissue regeneration. Utilizing a spatial transcriptomic approach (Nanostring, GeoMx), we foundthat TRM-like TIL in both DS and RS highly express genes related to activation as compared
to CD8+ CD103− TILs in the stroma, suggesting that local antigen signaling plays a role in the
differentiation of TRM-like TIL. However, we did not see large differences between TRM-like TIL
in DS and RS. In this analysis, we realized that CD8 T cells isolated from DS and RS containsignificant amounts of genes related to plasma cells, indicating that the neighbor cells’ genes
are also involved in. Thus, another spatial transcriptome approach with single-cell resolution,such as CosMx, would be preferable.
The lack of a mouse model is a major obstacle to the study of TRM-like TIL as conventional
tumor-cell injection models result in low, if any, generation of TRM -like TIL. To address thisproblem, we have analyzed a genetically engineered mouse model (Cre-mediated tissue-specific genetic alteration) that develops ovarian cancer with a typical clinical course, including
progressive accumulation of hemorrhagic ascites and development of peritoneal metastasis4.
We have found that TRM-like TIL are effectively generated in tumor tissues that have malignantly developed around the peritoneal surfaces. In order to investigate the gene signaturesof TRM-like TIL and their ontogeny in the circulation, we isolate CD8+ T cells from the peritoneal metastasis (tumor sites) and spleen (circulation) and performed single-cell TCR/RNA
sequence. We just received the data and analysis is ongoing.

Discussion & Conclusion
Through the analysis of tissue section of human cancer patients, we identified the actual
sites where TRM-like TIL are generated in the tumor, and local antigen signaling are likely to bea factor that promote differentiation of TRM-like TIL. Our preliminary data showed that, moreover, a large number of macrophages are found in both DS and RS while dendritic cells are
rarely found in those areas, suggesting that macrophages are the provider of antigen signaling that promote differentiation of TRM-like TIL. It is well known that TGFβ is required for upregulation of CD103, a key molecule for differentiation of TRM-like TIL5. Since TGFβ signaling
suppresses effector functions of CD8+ T cells, we expect that TRM-like TIL receive additionalsignaling that overcome TGFβ-mediated suppression. Macrophages in DS and RS could also
be a provider of such signaling. We will investigate the role of these macrophages in the differentiation of TRM-like TIL.

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References
1 Takamura Front Immunol. 2018, 9:1214.
2 Takamura Int Immunol. 2020, 32:571-581.
3 Im et al. Nature 2016, 537:417-421.
4 Chandler et al. Nat Commun. 2015, 6:6118.
5 Mackay et al. Nat Immunol. 2013, 14:1294-1301.

一般の皆様へ
組織滞在型メモリー CD8T 細胞 （TRM） は粘膜組織に定着し、 感染防御免疫の第一線を担う。 これに極めて類似した特徴もつ CD8T 細胞 （TRM-like TIL） は強力な抗腫瘍活性を持つことが知られているが、 この細胞が腫瘍内にてどのように分化するのかは不明である。 我々はヒト腫瘍組織にてその分化部位を特定すると共に、 TRM-like TIL 局所抗原刺激が重要であること、また、 分化部位に存在するマクロファージが抗原刺激の供給源となっている可能性を示唆する結果を得た。 今後はこのマクロファージが TRM-like TIL 分化誘導に果たす役割を追及する。

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Dissection of epigenetic reprogramming underlying
tumorigenesis using acute lymphoblastic leukemia inducing model
Tomokatsu Ikawa
ikawa@rs.tus.ac.jp
Research Institute for Biomedical Sciences, Tokyo University of Science

Summary Abstract
B-precursor acute lymphoblastic leukemia (B-ALL) is the most common childhood tumor
and the leading cause of cancer-related death in children. TCF-PBX1 fusion protein which is
produced from the t(1;19)(q23;p13) translocation is present in about 6% of childhood B-ALL
cases. Although previous studies have shown the pathogenesis of the patient, the mechanisms of TCF3-PBX1 B-ALL disease onset still remain unclear. We have recently establisheda mouse model of TCF3-PBX1 B-ALL. Using this, we dissected the molecular mechanisms
underlying the B-ALL development.
Key Words ： B-ALL, leukemia, hematopoietic stem cell, differentiation, B cell

Introduction
TCF3-PBX1 B-ALL has a poor prognosis, especially in children. Although various mousemodels have been developed to elucidate the pathogenesis of B-ALL, the efficient models
were not established yet. We have previously developed a method that allows unlimited
expansion of mouse and human hematopoietic stem / progenitor cells by inhibiting the function of E2A, a transcription factor essential for B cell differentiation. These cells were named
induced Leukocyte Stem (iLS) cells because they have the ability to differentiate into T, B
lymphocytes and myeloid cells. By transducing the TCF3-PBX1 to the iLS cells, we have established TCF3-PBX1-positive iLS cells.

Results
The TCF3-PBX1-positive iLS cells can be maintained in a same culture system and have
similar morphology and proliferation potential to normal iLS cells.
To determine the differentiation potential of the TCF3-PBX1-positve iLS cells to B lineage
cells, the cells were cultured on OP9 stromal cells in the presence of IL-7 and FLT3-ligand. After 6 days of the culture, the cells were harvested and analyzed by flow cytometer. The number of B cells were considerably fewer than control, suggesting that the TCF3-PBX1 inhibitedthe B cell differentiation. To examine the potential for B-ALL development, the TCF3-PBX1positve iLS cells were transferred to sublethally-irradiated mice. All the mice died at around3-4months of injection. Pro-B-ALL like cells were expanded and infiltrated in bone marrow,spleen and lymph nodes. RT-qPCR analysis demonstrated the downregulation of B-lineage
genes, Pax5, Vpreb1 and Ikzf1, while the expression of metabolism-related genes, Glut1 andInsr was upregulated. RNA-seq analysis also demonstrated that the global expression patternof the pro-B ALL derived from TCF3-PBX1 iLS cells was quite similar to the one of patient
samples reported before. Moreover, the genomic mutation in the Pax5, Ikzf1, Nras, Kras and

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Cdkn2a loci, which is also frequently observed in the TCF3-PBX1 B-ALL patients was foundin the TCF3-PBX1 pro-B ALL cells, indicating that the mouse model faithfully reflected thepathogenesis of the ALL patient.
Next, in order to elucidate the onset mechanism, we analyzed the gene expression patternusing preleukemic cells (TCF3-PBX1-expressing pro-B cells), and found that the expressionof Hoxa9, Lmo2, and Igf1 was markedly elevated. All of these genes have been implicated inassociation with hematopoietic malignancies such as acute myeloid leukemia and T-cell ALL(T-ALL). Interestingly, Hoxa9 is a direct target gene of the transcription factor PBX1 (Bijl et al.
2005), and the expression of Lmo2 and Igf1 is regulated by HOXA9 (Calero-Nieto et al. 2013;
Steger et al. 2015), suggesting that the cascade of TCF3-PBX1 → HOXA9 → LMO2, IGF1 isimportant in the early stage of B-ALL onset.
To determine the role of HOXA9 in early stages of B cell differentiation, HOXA9 inhibitor(DB818) was added to the culture of the TCF3-PBX1-positive iLS cells differentiating into B
cells. The inhibitor did not affect the generation of B cells, suggesting that the HOXA9 is notessential for B cell differentiation of the TCF3-PBX1-positive iLS cells.
To determine if the HOXA9 is involved in the proliferation of multipotent progenitors,
the Hoxa9 of TCF3-PBX1-positive / negative iLS cells was deleted using CRISPR-Cas9
system. Interestingly, the proliferation potential of both iLS cells was affected, while thegrowth of Hoxa9-/-TCF3-PBX1-positive iLS cells was more severely suppressed than that of
Hoxa9+/+TCF3-PBX1-positive iLS cells, indicating that growth of TCF3-PBX1-positive iLS cells
was dependent on the HOXA9 activity.

Discussion & Conclusion
In this study, we succeeded in establishing a mouse model of TCF3-PBX1 B-ALL using iLS
cells. The model faithfully reflected the patient pathogenesis, based on the gene expression
profile or single nucleotide polymorphism (SNP). Using this model, we found that the expression of Hoxa9, Lmo2, and Igf1 was markedly elevated in TCF3-PBX1-positive pro-B cells.
The knockdown of Hoxa9 significantly suppressed the growth of TCF3-PBX1 iLS cells. These
results indicated that the HOXA9 is involved in the early stages of B-ALL development. The
mouse model will be useful for examining the molecular mechanisms of the B-ALL development and the screening of novel drugs. This method, which uses B-ALL-inducing system ofiLS cells to study the mechanism of leukemia onset and to develop a new treatment method,
can be applied to the research of not only TCF3-PBX1 but also leukemias caused by otherfusion proteins and genetic mutation. We hope the study will lead to create a new trend in hematopoietic malignancy research.

一般の皆様へ近年、 急性リンパ性白血病 （ALL） の治療成績は飛躍的に向上したものの、 未だに予後不良な転座型も存在します。 我々は、 難治性 ALL の一つである1;19転座型 （TCF3-PBX 型 ） B
細胞性 ALL （B-ALL） に着目して研究を行いました。 独自に開発した動物モデルを用いて、発症初期の分子メカニズムの一端を解明しつつあります。 今後は本研究で見出した分子機構を
B-ALL の新規治療法の開発に結びつけたいと考えています。

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Exploratory study for the discovery of zinc homeostasis
modulators utilizing a highly sensitive fluorescenceimaging probe
Kensuke Okuda
okuda@kobepharma-u.ac.jp
Kobe Pharmaceutical University

Summary Abstract
As part of our ongoing research to improve a highly sensitive intracellular zinc fluorescentprobe Dpa-LBC, we have successfully developed a MRI probe candidate Dpa-SoxLdiF. Contrary to fluorescence imaging modality with poor permeability, MRI modality is suitable for invivo imaging based on its high permeability. We expect that Dpa-SoxLdiF is a lead compoundto image zinc dynamics in vivo.
Key Words ： zinc, imaging, probe, fluorescence, MRIIntroduction
Lately, mobile zinc has been proposed to function as a mediator in signal transduction pathways. It has also become clear that disruption of zinc homeostasis is associated with various diseases such as Alzheimer's disease, diabetes mellitus, and cancer.
Therefore, targeting zinc metabolism is now attracting attention as a new therapeutic strategy for intractable diseases related to zinc homeostasis disruption. In this context, an assay
system that can efficiently visualize the intracellular status of zinc is needed to accelerate thediscovery of therapeutic agents that can be used to maintain or intervene in the zinc homeostasis of the organism.

Results
Recently, we have successfully developed a small molecule fluorescent probe, Dpa-LBC,
which greatly exceeds the sensitivity of existing fluorescent probes by incorporating a signal
enhancement system into the probe design with respect to fluorescent imaging of intracellularzinc.1) Therefore, we expected that Dpa-LBC could be applicable to a high-content screening system (HCS) to explore for compounds that would regulate mobile zinc in living cells.
However, unlike the high-sensitivity system used in the development of Dpa-LBC, which uses
a confocal fluorescence microscope with an objective lens collecting more light and a glassbottom dish, the usual HCS system uses a widefield fluorescence microscope with a conventional objective lens and a plastic-bottom well plate from the aspect of cost. Preliminary
results using the HCS system indicate that it is difficult to capture zinc perturbations by DpaLBC. The objectives of this study were 1) to modify this novel highly sensitive zinc fluorescentprobe to be applicable to a HCS system to detect mobile zinc in living cells, and 2) to establish a high-throughput screening (HTS) system to explore intracellular zinc modulators.

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To improve the sensitivity of Dpa-LBC, accelerating the zinc-catalyzed reaction of the probe
and increasing the catalytic turnover rate are necessary. Dpa-LBC consists of three substructures: a zinc-recognizing ligand moiety (Dpa: dipicolylamine), a zinc-responsive linker, and aquenched fluorophore. Of these three elements, the ligand moiety and the fluorophore canbe freely converted. Therefore, we aimed to improve the reactivity of the probe by optimizing
the ligand moiety for zinc responsiveness. In parallel, we also attempted to convert the fluorophore for fluorescence imaging to magnetic resonance responsive moiety for magnetic resonance imaging (MRI).
First, in terms of optimizing the Dpa moiety, it is possible to increase the affinity between
the probe ligand and zinc with lower affinity of the decomposed zinc complex after release
reaction of the fluorophore to quickly dissociate the zinc and increase the catalytic turnover.
In this study, we designed and synthesized Dpaderivatives with 4- and/or 6-substituted
pyridine(s) aiming to accelerate the release of fluorophore by adjusting the affinity to zinc andimproving the nucleophilicity of the coordinating water on the zinc complex to trigger the fluorescent turn on reaction. Then we designed a simplified compounds with a straightforward
linker between the modified Dpa ligands and the fluorophore to achieve a prompt evaluationof the zinc-catalyzed reaction, but the synthesis was unexpectedly difficult. Further studies
are currently underway to establish an evaluation system.
On the other hand, some plausible results were obtained on efforts to expand the fluorescent probe to MRI probe. The details are as follows. As a first-generation zinc MRI probe,
Dpa-SoxLdiF was designed and synthesized by replacing the quenched fluorophore, a substructure of Dpa-LBC described above, with 2,6-difluorophenol as a 19F-MRI signal-responsive moiety and converting the sulfide to a sulfoxide and eliminating p-acetoxybenzyl moiety
for synthetic convenience. Dpa-SoxLdiF was expected to release 2,6-difluorophenol by zinccatalyzed reaction like Dpa-LBC. First, 19F-NMR characterization was performed on DpaSoxLdiF. Difference in chemical shifts of 2,6-difluorophenol, which is expected to be releasedby the reaction of zinc with Dpa-SoxLdiF, and Dpa-SoxLdiF itself measured by 19F-NMR, was
6.8 ppm. This difference was sufficiently distinguishable by 19F-MRI. The reaction with catalytic amount of zinc with Dpa-SoxLdiF was subsequently examined by 19F-NMR, which revealed
that Dpa-SoxLdiF reacted completely and was quantitatively converted to 2,6-difluorophenol,
indicating that zinc amplified MR signal by catalyzing the release reaction of 2,6-difluorophenol..

Discussion & Conclusion
The results which I have mentioned above indicate that Dpa-SoxLdiF is a promising 19F-MRI
probe molecule for amplifying zinc signal in buffer solution. Glutathione, which exists in high concentration under intracellular environment, coordinates with zinc and competes with the zinc ligand
moiety of the probe. Therefore, it may inhibit the probe response to zinc. We then evaluated the
response of Dpa-SoxLdiF in the presence of glutathione (10 mM), which mimicked the intracellular environment, and found that the response of Dpa-SoxLdiF to zinc was maintained, althougha slight inhibition was observed. Nevertheless, zinc catalyzed the probe reaction to enhance MR
signal even in the presence of high level glutathione. In conclusion, Dpa-SoxLdiF is useful as alead compound for 19F-MRI probes that can detect intracellular zinc. Evaluating the sensitivity toother divalent metals is currently underway to assess zinc selectivity.

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References.
1) I. Takashima, Y. Inoue, N. Matsumoto, A. Takagi, and K. Okuda, Chem. Commun. 56(87),13327−13330 (2020).

一般の皆様へ亜鉛ホメオスタシスの崩壊とアルツハイマー病、 糖尿病、 がんなどの多様な疾患との関連性が明らかとなっており、 亜鉛ホメオスタシスを維持あるいは介入するために使用可能な治療薬の発見を加速するためには、 亜鉛の細胞内の状態を効率的に可視化できるアッセイ系が必要である。
本研究では私たちが開発した高感度細胞内亜鉛蛍光プローブの改良を行って本アッセイ系の構築を試みるとともに、 個体レベルでの生体イメージングに適した MRI プローブへの展開を図った
ところ、 第一世代の亜鉛 MRI プローブの開発に成功した。

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Cryo-electron microscopy studies for disease pathogenesis of
Xeroderma Pigmentosum
Syota Matsumotomatssyot@iqb.u-tokyo.ac.jp
Institute for Quantitative Biosciences, The University of Tokyo

Summary Abstract
Xeroderma pigmentosum (XP) is a genetic disease caused by the defect of nucleotide excision repair (NER). UV-DDB is a specific recognition protein for NER, and efficiently binds to
UV lesions in chromatin, in which DNA is tightly packed. However, it is unclear how UV-DDB
recognize DNA lesions in chromatin. Our result indicated by cryo-electron microscopy how
UV-DDB binds to DNA lesions in chromatin. This achievement may contribute to the development of treatments for XP patients.
Key Words ： DNA repair, NER, chromatin, cryo-EM, nucleosome

Introduction
Xeroderma Pigmentosum (XP) is a representative disease caused by the defect of nucleotide excision repair (NER) [Ref. 1]. NER is one of the important pathway to eliminate DNA lesions generated by ultraviolet (UV) from genome. In NER, UV-damaged DNA binding protein(UV-DDB) recognizes DNA lesions on genome, and then, recruits other NER factors to damaged sites, which is important to proceed NER [Ref. 2]. On the other hand, DNA is wrappedaround histone proteins in cells, and forms high order structures, called chromatin. It is still
unclear how NER efficiently works in chromatin, which could be a barrier for UV-DDB to recognize lesions on DNA.

Results
To understand the detail of NER mechanism in chromatin, we tried to directly observe UVDDB bound to DNA lesion in chromatin by cryo-electron microscopy (cryo-EM). We focusedon in vitro reconstitution of UV-DDB bound to nucleosome, a minimum unit of chromatin,
containing UV lesion [Figure]. Nucleosomes consist of four types of histone protein (each two
molecules of H2A, H2B, H3, H4) and about 145 bp DNA. To reconstitute damaged nucleosomes in vitro, we purified all histones from E. coli. We chemically synthesized DNA lesion on
145 bp DNA. After mixing purified histones with damaged DNA, we reconstituted damaged
nucleosomes in vitro. We expressed UV-DDB complexes using baculo virus in insect cells,
and purified UV-DDB complex. To assess the binding activity of UV-DDB to damaged nucleosomes, we performed gelshift and fluorescent polarization (FP) assay. These results suggested that UV-DDB has the capability to bind to DNA lesion even in nucleosomes. As a next,
to reveal how UV-DDB binds to DNA lesion in nucleosomes, we reconstituted UV-DDB-NCP
complex, and applied for cryo-EM analysis. By cryo-EM analysis, we successfully revealed
the structure of UV-DDB-NCP complex at high resolution. This structure indicated that UV-

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DDB directly binds to UV lesion in nucleosomesand UV-DDB doesn’t affect the core structure ofNCP by its binding.
In addition to UV-DDB, we also investigatedthe downstream factors, Xeroderma pigmentosum complementation group C (XPC) and group
A (XPA). We purified XPC and XPA in the same
method as we described above. Gelshift assay
with damaged nucleosomes and these factors
indicated that XPC and XPA potentially binds todamaged nucleosomes.
Now we are working on revealing UV-DDBXPC-XPA assembled on damaged nucleosomes.
We hope further research could investigate how
XPC and XPA are recruited in damaged nucleosomes.
Recently, the structure of XPC, XPA and its
downstream factor, transcription factor IIH (TFIIH),
bound to damaged DNA was solved by cryo-EM[Ref. 3]. Following these structure, XPC binds to
the DNA lesion from the opposite direction of UVDDB. Our result raises the question that how XPC
recognizes DNA lesion after UV-DDB binding inchromatin because the binding structure of XPC
seems to crash into the histone core in nucleosome. This is also interesting mechanism and further studies are needed to figure them out.

Discussion & Conclusion
Our result indicated how UV-DDB recognizes UV lesions in nucleosomes, and it may help
us to understand the basic mechanism of the damage detection in chromatin in NER. Our
cryo-EM structure of UV-DDB-NCP might be also important for screening seeds of potential
drugs affecting the DNA repair mechanism including NER. The defect of DNA repair pathway
is related to various diseases, and XP is one of them, which derived from the defect of NER.
Therefore, in the near future, our achievement of this research could contribute to develop thetreatments for XP patients. Not only these patients, our achievements also might propose thedevelopments of the methods of UV protection from sunlight, which risks are increasing because of the global warming and ozone layer depletion in the world.

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References.
1. Lehmann AR. DNA repair-deficient diseases, xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Biochimie 85, 1101–1111 (2003).
2. Sugasawa K, Okuda Y, Saijo M, Nishi R, Matsuda N, Chu G, Mori T, Iwai S, Tanaka K, Tanaka K, Hanaoka F. UV-induced ubiquitylation of XPC protein mediated by UV-DDB-ubiquitin
ligase complex. Cell 121, 387–400 (2005).
3. Kim J, Li CL, Chen X, Cui Y, Golebiowski FM, Wang H, Hanaoka F, Sugasawa K, Yang W.
Lesion recognition by XPC, TFIIH and XPA in DNA excision repair. Nature 617, 170–175(2023)

一般の皆様へ
私たちの遺伝情報はゲノムと呼ばれる DNA 配列に保存されています。 遺伝子の設計図とも言われる DNA は、 ほぼ全ての細胞に等しく保存されており、 それは安定に子孫へと引き継がれていきます。 ところが紫外線など外から強力なエネルギーを受けると、 DNA が損傷を受け、 遺伝
子が変異を起してがんが発生するリスクが高まります。 この損傷を修復するメカニズムの一つが、ヌクレオチド除去修復であり、 絶えず私たちに生じた損傷を取り除き続けています。
ヌクレオチド除去修復に異常が起こると、 紫外線による損傷を修復できなくなり、 さまざまな疾
患が引き起こされます。 色素性乾皮症もその一つであり、 特定難病に指定された治療法がない
遺伝病です。 私たちはクライオ電子顕微鏡という最先端の顕微鏡により、 この修復メカニズムを可視化して理解することを目指しています。 この研究が進めば、 紫外線によるがんの発生を抑制できると期待され、 将来的に色素性乾皮症の治療法の確立へと繋がることも夢ではありません。

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Analysis of brain aging caused by the disruption of“nuclear-axon crosstalk” machinery
Ken-ichiro Kuwako
kuwako@med.shimane-u.ac.jp
Department of Neural and Muscular Physiology, Shimane University School of Medicine

Summary Abstract
The linker of nucleoskeleton and cytoskeleton (LINC) complex, a nuclear membrane protein complex, plays an important role in the regulation of cytoskeletal network. In this study,
we show that the LINC complex is essential for the regulation of neural activity via axon initial
segment (AIS) which is required for the generation of action potential. Furthermore, we foundthat the expression levels of the LINC complex components on the nuclear envelope weremarkedly reduced with aging, leading to structural abnormality in the AIS of aged neurons.
These findings indicate that the LINC complex-mediated AIS regulation may be involved inphysiological brain aging.
Key Words ： Brain aging, Neural activity, Axon initial segment, LINC complex

Introduction
As our country enters the era of super-aging, extending the healthy life span of the brain
has become an extremely important issue. Physiological brain aging is thought to be caused
by a gradual decline in the activity and plasticity of individual neurons, leading to a decreasein neurological function (Burke and Barnes, Nat Rev Neurosci 2006). However, the mechanism for physiological brain aging is still largely unknown, and effective strategies to prevent it
based on solid molecular mechanisms have yet to be established.

Results
In this study, we analyzed the mechanism of brain aging by focusing on the nucleus-mediated regulation of neural activity. The LINC complex that penetrates the nuclear envelope is a
nuclear membrane complex composed of Sun proteins on the inner membrane and Nesprin
proteins on the outer membrane. By binding of Nesprin to the cytoskeleton, including microtubules and actin fibers, the LINC complex functions as the center of the cytoskeletal network
and is involved in various cellular events such as cell polarity formation and migration (Horn,
Curr Top Dev Biol, 2014). AIS is located at the proximal site of axon in neurons and is a physical and physiological border between the somatodendritic and axonal domains. AIS contains
specific cytoskeletal structures and voltage-gated sodium and potassium channels, and playsa pivotal role in regulating neuronal excitability via action potential generation (Leterrier, J
Neurosci 2018). Recent studies demonstrate that AIS alters its length, position and molecularcomposition in response to changes in neural activity level, indicating that AIS is one of theimportant factors subserving neuronal plasticity.

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To clarify the importance of the LINC complex in AIS regulation, we first inhibited LINC
complex function by forced expression of the dominant negative Nesprin1 mutant (Nesprin1
DN) in mouse primary neurons. We found that the AIS length was significantly shortenedin Nesprin1 DN-expressing neurons including cortical neurons, hippocampal neurons andcerebellar Purkinje cells, compared to GFP-expressing control neurons. The expression of
Nesprin1 DN in cortical pyramidal neurons in vivo by in utero electroporation also significantly
shortened their AIS length. We next examined the structural plasticity of AIS

Figure 1. LINC complex-mediated AIS regulation and neuronal aging

Discussion & Conclusion
In this study, we show for the first time that the LINC complex-mediated AIS regulation isimportant for the regulation of neuronal excitability. We also show that the disruption of thissystem affects brain functions such as locomotion and emotion in mice. Furthermore, weshow that aging causes a reduction in the expression of the LINC complex and the structuralabnormality of AIS, which may lead to the decline of brain functions.
In the future, it will be necessary to elucidate the detailed molecular mechanisms of AIS
regulation via the LINC complex in order to fully understand the system of the nucleus-mediated neural activity control. Furthermore, it is very important to verify whether preventing the
reduction of the LINC complex expression with aging can deter the decline in brain function in
old age. The success of these future studies is expected to lead to the implementation of innovative preventive medicine aimed at extending the healthy life span of the brain.

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References.
Burke SN and Barnes CA. Neural plasticity in the ageing brain. Nat Rev Neurosci 7, 30-40,(2006)
Horn HF. LINC complex proteins in development and disease. Curr Top Dev Biol 109, 287321, (2014)
Leterrier C. The Axon Initial Segment: An Updated Viewpoint. J Neurosci 38, 2135-2145,(2018)

一般の皆様へ
私たちの脳は、 痴呆症などの神経疾患に罹らなくても、 加齢に伴って徐々にその機能が衰えていきます。 しかし、 どのようにしてその生理的変化が起こるのかはよくわかっておらず、 有効な予防法もありません。 今回の研究では、 これまで知られていなかった神経細胞の活動レベルを制御する新たなしくみを発見し、 さらに老化によってその機構が破綻することで脳機能の衰退が起こる可能性を見出しました。 今後は、 今回発見した機構を老齢期でも正常に維持することで加齢による脳機能の低下を抑止できるかどうかを検証していきます。 そして、 将来的には、 これらの知見を脳老化の予防法の開発につなげたいと思います。

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Establishment of precision medicine for subsequent solid
tumors after hematopoietic cell transplantation based oncomprehensive genomic analyses
Satoshi Inoues.inoue@aichi-cc.jp
Aichi Cancer Center Research Institute

Summary Abstract
Long-term survivors of hematopoietic cell transplantation represent significant risk of diagnosis of subsequent malignant neoplasms (SMNs). The genomic features of SMNs are
unknown. The genomic features of SMNs are unknown. Here, we used next-generation sequencing of SMNs, to detect distinct mutational signatures (BRCAness and MSI) compared
to primary malignant neoplasms without transplantation, raising a possibility that distinct molecular pathogenesis caused by a series of therapy of hematopoietic diseases such as chemotherapy, radiation and graft versus host disease.
Key Words ： Hematopoietic cell transplantation, Subsequent malignant neoplasms, Genomic features, Graft versus host disease

Introduction
The population of long-term survivors of hematopoietic cell transplantation (HCT) is growing because of improvement of HCT-based therapy. Long-term survivors of HCT have an increased risk of diagnosis of subsequent malignant neoplasms (SMNs). Although HCT therapy
is effective to cure primary hematopoietic cancer, it also produces harmful genetic damages
within normal cells. Several factors are thought to be contributed to the development ofSMNs, including irradiation, genotoxic agents, immunosuppressive therapy, and chronic graftversus-host disease (GHVD). The genomic features of SMNs are unknown.

Results
To perform comprehensive genomic analyses of SMN, we collected tumor samples from86 individuals with a mean age of 59 years. In 20 individuals, chronological multi-sampling oftumor from SMNs were collected. We also collected multi-regional sampling of tumor and adjacent normal tissues from 12 SMN individuals.
High coverage whole exome sequencing (WES) was performed on these FFPE or frozen
samples with a mean coverage of 100-150x in tumor tissues and adjacent normal tissues;and ~50x in both donor- and recipient-derived germline control samples. Both somatic synonymous and nonsynonymous mutations were called using three publicly available mutation
callers. We developed effective pipelines to eliminate mutations were artificially caused by
FFPE, so called FFPE artefact.
We successfully detected reliable somatic mutations from 86 SMNs and 77 primary malignant neoplasms (PMNs) without transplantation (non-SSC). No obvious differences of

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recurrent driver mutations were observed between PMNs and SMNs (eg: APC and TP53 incolorectal cancer; TP53 in esophagus cancer; TP53 and CDKN2A in oral cancer. However,tumor mutation burden (TMB) of SMNs is higher than PMNs in oral and colorectal cancers.
Indeed, we detected genomic alterations of TMB-H-related genes and mutational signaturesin some TMB-H tumors. As TMB-H has been proposed as a potential biomarker for immunotherapy such as anti-PD1 Ab, raising a possibility that immunotherapy is more efficacious
than chemotherapy in SMNs of oral and colorectal cancers.
Chronological multi-sample sequencing revealed tumors with same individual are primarily
clonally heterogenous in 75% (15/20) of SMNs, suggesting these tumors developed independently rather than relapsed from primary tumors (Figure 1).

Sequencing of non-tumor regions in individuals with SMNs show that marked genomic
alterations are observed compared to individuals with PMNs. Sequencing of multi-regional
sampling of semi-microdissected regions unveiled extremely heterogenous, multiple/widespread expanded clones with pathogenic mutations such as TP53 are developed all overthe normal tissues (oral and esophagus) (Figure 2), suggesting extensive heterogeneity anddistinct mutagenesis across clones in normal epithelial tissues a potential explanation of different clinical and pathological features of SMNs caused by exposure of a series of mutagens
and/or GVHD during the therapy. In contrast, there is no obvious “field cancerization” wereobserved in colorectal cancer of SMNs, explaining that lack of recurrent colorectal cancers
developed in SMNs. Taken together, these insights into the biology of these distinct conditions
may enable future efforts to optimize the treatment and surveillance of individuals with moreof this diagnosis.

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Discussion & Conclusion
Our novel NGS-based study provides fresh insights into the pathogenesis of SMNs. We
have shown that: i) TMB of SMN is higher than that of PMN; ii) SMNs tumors developed independently rather than relapsed from primary tumors (Figures 1); and iii) extensive heterogeneity and distinct mutagenesis across clones in normal epithelial tissues (Figure 2). Obviously, further investigations are required to establish the validity of this approach, our findings
urge requirement of genetically-guided therapy and/or relapse risk assessment for SMNs.

References
1. Ikegami M, Kohsaka S, Hirose T, Ueno T, Inoue S, Konomata N, Yamauchi H, Mori T, Sekine S, Inamoto Y, Yatabe Y, Kobayashi H, Tanaka S, Mano H. MicroSEC; Sequence error
filtering pipeline for formalin-fixed and paraffin-embedded samples. Commun Biol 2021 4:1396.

一般の皆様へ
同種造血細胞移植は、 白血病などの難治性血液疾患の根治を可能とする治療法です。 移植
技術の向上に伴い、 長期生存者数が増加しています。 しかしながら移植治療による副作用として正常細胞がダメージを受けてしまい、 口腔や食道などの2次固形がんの発症に至ってしまいます。 しかしその原因や病態は不明であるため、 1次がんに準じた治療法が選択せざる得ないのが現状です。 そこで大規模2次がん検体バンクと空間 ・ 経時的マルチサンプリングゲノム解析を組み合わせることで、 移植症例に対するゲノム医療による個別化至適治療の実現に向けた分子基盤を構築することを目的としました。 その結果、 2次がんに特徴的なゲノム異常が認められ、免疫チェックポイント阻害剤などの個別化至適治療法の適用対象になり得る可能性が示唆されました。

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Development of asymmetric synthesis ofsulfoximine analogs as bioisosteres
Taichi Kano
kano@go.tuat.ac.jp
Tokyo University of Agriculture and Technology

Summary Abstract
Key Words ： Sulfoximine, Sulfinamidine

Introduction
Sulfoximines with a chiral center on the sulfur atom have attracted attention in recent years
as new substructures for pharmaceuticals and agrochemicals. Asymmetric synthesis methods
for such sulfoximines have been established to date.1,2 On the other hand, asymmetric synthesis of nitrogen analogues of sulfoximines, such as sulfonediimines and sulfonimidoamides,
is quite difficult, although they are expected to be new transition state analogs. In this study,
we established a simple asymmetric synthesis method for sulfinamidines which are the precursors of these compounds.

Results
First, we began our investigation on an O-selective alkylation of N-pivaloyl-protected sulfinamide 1a (Fig. 1). The reaction using methyl iodide as an alkylating agent and Cs2CO3 as abase in DMF at 50 °C gave a low yield of the desired product, sulfinimidate ester 2 (R = Me)along with a substantial amount of S-alkylation product 3 and N-alkylation product 4. Use ofethylating agent led to suppression of the N-alkylation while providing a significant amount of
the undesired S-alkylation product 3 (R = Et). The bulky tert-butyl group of the pivaloyl group
may shield the nitrogen atom and prevent the approach of the ethylating agent. Use of sterically more hindered isopropyl iodide dramatically improved the oxygen-selectivity, giving 69%
yield of 2a (R = i-Pr). We then investigated the influence of base and solvent on the productratio in the reaction of 1a with isopropyl iodide at 70 °C. The choice of base also significantly
affected the reaction, and the less expensive K2CO3 outperformed the other bases tested.
Among the solvents screened, DMPU (N,N'-dimethylpropyleneurea) afforded optimal yield.
With the optimized conditions in hand, we examined the substrate scope in the O-selective
alkylation of sulfinamides, and various aryl and alkyl sulfinamides were found to be applicableto the present method. Chiral HPLC analysis confirmed that most of the sulfinimidate esters
were obtained without racemization.
Fig. 1

Cs2CO3(1.2 equiv)RO
O NPiv
Piv
S
S
R
N
p-tol
NHPiv
NPiv p-tol
p-tol
p-tol
DMF (0.1 M)
2
3
4 R(S)-1a(2.0 equiv)
Piv = COt-Bu
R = Me18%
36%
47%
R = Et38%
46%<5%
R = i-Pr69%
12%
not detected
R = i-Pr86%
7%
not detected
and DMPU instead of Cs
and DMF.)(Use of K2CO3
2CO3
O
S

+ R I

O
S

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Next, we tested chiral sulfinamidine synthesis by the reaction of sulfinimidate ester 2a witha magnesium amide (Fig. 2). The reaction of sulfinimidate ester 2a with magnesium amidethat was generated from pyrrolidine (1.5 equiv) and ethylmagnesium bromide (1.2 equiv) gave
sulfinamidine 5a in good yield without racemization. In this reaction, the reduction product 6
was not observed, while a substantial amount of 6 was formed as byproduct in the reaction
using excess amounts of ethylmagnesium bromide (2 equiv).
Fig. 2
p-tol

O
S
2a

i-Pr
NPiv

pyrrolidine EtMgBr(1.5 equiv) (1.2 equiv)

Piv
NS
p-tol * N
5a
91%

p-tol

HN
S

Piv

6
not observed

We then investigated the substrate scope of magnesium amides in the sulfinamidine synthesis. The reactions of sulfinimidate ester 2a with various magnesium amides were performed under the optimized reaction conditions. In the case of the magnesium N,N-diethylamide that was generated from ethylmagnesium bromine and diethylamine, the corresponding
sulfinamidine was obtained in good yield (86%) without racemization (99% ee). These results
suggested that both cyclic and acyclic aliphatic amides can be introduced stereospecifically.
On the other hand, when N-ethylaniline was used as an amide source, the reaction gave
the corresponding sulfinamidine in moderate yield (54%) with decreased optical purity (60%ee). In this reaction, a less nucleophilic magnesium amide was generated from the aromatic
amine, which may decrease the reaction rate of the desired reaction and increase the undesired racemization. Additionally, the reaction using a primary amine (e.g. cyclopropylmethylamine) afforded the desired sulfinamidine in good yield (70%). However, the optical purity ofthe obtained sulfinamidine was found to be drastically decreased (3% ee). We are currently
investigating the appropriate conditions to suppress optical purity degradation of sulfinamidines when using aromatic amines and primary amines. In addition, since the stereochemistryof the obtained sulfinamidine is not clear, we are investigating a method for determining thestereochemistry.

Discussion & Conclusion
In summary, we have developed an efficient method for the synthesis of chiral sulfinamidines from easily available chiral sulfinamides. The key step of this method is stereospecificO-selective alkylation of chiral sulfinamides, which is accomplished by using the appropriatebase, solvent, and sterically hindered isopropyl iodide as the alkylating agent. Various chiralsulfinamidines are obtained in high yield without racemization by the nucleophilic addition of
the magnesium amides derived from secondary amines to chiral sulfinimidate esters. However, the reaction of magnesium amides derived from aromatic amines and primary amines wasfound to give the racemized sulfinamidines, and it is necessary to find the optimum conditions
to suppress the racemization. The further transformation of the obtained chiral sulfinamidines
to other compounds containing an S=N bond is underway in our laboratory.

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References
1. Aota, Y.; Kano, T.; Maruoka, K. Asymmetric Synthesis of Chiral Sulfoximines via the SArylation of Sulfinamides. J. Am. Chem. Soc. 2019, 141, 19263-19268.
2. Aota, Y.; Kano, T.; Maruoka, K. Asymmetric Synthesis of Chiral Sulfoximines through the SAlkylation of Sulfinamides. Angew. Chem. Int. Ed. 2019, 58, 17661-17665.

一般の皆様へ
医薬品や農薬などの生物活性物質には、 硫黄原子を含むスルホキシイミンと呼ばれるものがあります。 スルホキシイミンにはその鏡像となるものが存在するため、 どちらか一方だけを合成す
る不斉合成法の開発が必要となります。 本研究では、 スルホキシイミンの酸素原子を窒素原子で置換したスルフィンアミジンの不斉合成法を開発し、 新しい生物活性物質の合成が可能となりました。 今後は医薬品開発への応用が期待されます。

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Achieve a comprehensive understanding of the pathogenesis oftype 1 diabetes mellitus, leading to development of
the prevention measures
Keiichiro Mine
sv7899@cc.saga-u.ac.jp
Division of Metabolism and Endocrinology, Department of Internal Medicine,
Faculty of Medicine, Saga University.

Summary Abstract
Type 1 diabetes (T1D) is caused by insulin-producing pancreatic β-cell destruction, usuallyleading to absolute insulin deficiency. Genetic and environmental factors have been widelyreported as risk or protection factors for T1D. We have reported that Tyrosine kinase 2 (Tyk2)
is a susceptibility gene for virus-induced T1D in mice and humans. Tyk2, a member of Janus
kinase (Jak) family, is involved in cytokine signaling. Since the association between Tyk2 andautoimmune T1D has not been reported, we examined the role of Tyk2 in autoimmune T1D
to understand the pathogenesis of T1D from various viewpoints. In this study, we found thatTyk2 plays an important role in the development of autoimmune T1D.
Key Words ： Type 1 diabetes, NOD mouse, Autoimmunity

Introduction
Type 1 diabetes (T1D) is caused by destruction of insulin producing pancreatic β-cells. The
pathogenesis of T1D is related to genetic and environmental factors. Viruses are one of theimportant environmental factors in T1D (1). We have reported that Tyrosine kinase 2 (Tyk2) isa susceptibility gene for virus-induced diabetes in mice and humans (2, 3). Tyk2, a member ofJanus kinase (Jak) family (Jak1, Jak2, Jak3, and Tyk2), is involved in cytokine signalings including type 1 interferons (IFNs), interleukin (IL)-6, and IL-10. After binding of the cytokine(s)to the cell surface receptor(s), Tyk2 gets activated, phosphorylate intracellular tail of thereceptor(s), and recruits Signal transducers and activators of transcriptions (Stats). Recruited
Stats become phosphorylated themselves, dimerize, and translocate to the nucleus to regulate gene expression by direct binding to DNA regulatory elements (4). The Tyk2 mediatedsignaling plays a role in anti-microorganism responses; however, aberrant activation of thesignaling has been implicated in the pathogenesis of inflammatory diseases such as psoriasis(5, 6). Since the association between Tyk2 and autoimmune T1D has not been reported, we
examined the role of Tyk2 in autoimmune T1D to understand the pathogenesis of T1D fromvarious viewpoints and develop the safety prevention measures for T1D.

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Results
Tyk2 knockout NOD mice showed reduced
incidence of autoimmune T1D compared withTyk2 sufficient NOD mice (Figure 1, p<0.001).
The transfer experiments of naïve NY8.3
CD8 T cells into NOD mice suggested that Tyk2
expression in antigen-presenting cells (APCs)
is important for the proliferation of CD8 T cells
in pancreatic lymph node. We found that MHC
class 1 expression in CD8-positive tissue-resident dendritic cells (CD8+ rDC) was reduced by
Tyk2 deficiency. To test the T cell priming capacity of CD8+ rDC, sorted CD8+ rDC was cultured with naïve NY8.3 CD8 T cells and IGRP peptide. The proliferation of NY8.3 CD8 T cell was reduced when it cultured with Tyk2 KO CD8+
rDC compared with Tyk2 WT CD8+ rDC, indicating that Tyk2 KO CD8+ rDC has a reduced
ability to proliferate of islet autoreactive CD8 T cells.
RNA-sequencing analysis revealed that Tyk2 KO CD8 T cells in the pancreas showed decreased gene expression of interferon (IFN)- γ and granzyme.
These results suggest that Tyk2 is a potential target for the prevention of T1D onset. Fourweeks of Tyk2 inhibitor treatment starting at 6 weeks of age prevented the onset of diabetes;
this inhibition was not observed when treatment was started at 12 weeks of age.

Discussion & Conclusion
Our results indicate that Tyk2 plays an important role in the development of autoimmune
T1D. Reduced expression of Tyk2 in CD8+ rDC and CD8 T cells suppresses its function in
the acquired immune response. Although the risk of infection must be considered, Tyk2 inhibition is a potential methods to prevent the onset of autoimmune T1D.

References
1. Op de Beeck, A., Eizirik, D. Viral infections in type 1 diabetes mellitus — why the β cells? Nature Revew of Endocrinology 12, 263–273 (2016).
2. Izumi, K., Mine, K., et al. Reduced Tyk2 gene expression in β-cells due to natural mutation
determines susceptibility to virus-induced diabetes. Nature Communications 6, 6748 (2015).
3. Nagafuchi, S., et al. EBioMedicine. TYK2 Promoter Variant and Diabetes Mellitus in theJapanese. EBioMedicine 2, 744-749 (2015).
4. O'Shea, John J., et al. The JAK-STAT Pathway: Impact on Human Disease and Therapeutic Intervention. Annual Review of Medicine 66. 311-328 (2015).
5. Papp, K., et al. Phase 2 Trial of Selective Tyrosine Kinase 2 Inhibition in Psoriasis. New
England Journal of Medicine 379. 1313-1321 (2018).
6. Nakamura, R., et al. Tyk2-signaling plays an important role in host defense against Escherichia coli through IL-23-induced IL-17 production by gammadelta T cells. Journal of Immunology 181, 2071-2075 (2008).

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一般の皆様へ
本研究は血糖値を下げるホルモンであるインスリンを作る細胞が破壊され糖尿病 （1型糖尿病）になる機序を解析することで、 有効で安全な1型糖尿病の発症予防法を確立することを目的としています。 1型糖尿病は生涯にわたってインスリンを補充する以外に治療法がなく、 現在のところ不治の病です。 我々の研究によって1型糖尿病の発症を防ぐ方法を開発し、 患者数の減少と人々のQOL の向上へ貢献したいと考えています。
ある遺伝子とその遺伝子から作られるタンパク質の働きを阻害することで、 1型糖尿病の発症が抑制されることが明らかになりました。 その発症抑制機序を詳細に解析し、 標的として最適だと考えられる細胞の種類を特定することができました。
この研究成果は、 副作用を限りなく抑えた1型糖尿病発症予防法の開発へ繋がると考えられます。

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Synthesis of amino acid derivatives and its application for the
inhibitors against SARS-CoV2 protease
Hiroyuki Konno
konno@yz.yamagata-u.ac.jp
Graduate School of Science and Engineering, Yamagata University

Summary Abstract
Synthesis of amino acid derivatives using chiral Ni(II) Schiff base are described. For the
first time, dynamic kinetic resolution of unprotected amino acids with active side chains was
attempted to afford chiral Ni(II) Schiff base derivatives in good yield and diastereoselectivity.
After the conversion of free side chain, protected amino acids were isolated by removal of chiral ligand.
Key Words ： Chiral Ni(II) Schiff base, dynamic kinetic resolution, tailor-made amino acids,
SARS-CoV2 protease inhibitor

Introduction
Tailor-made amino acids are important structural units in the development of modern pharmaceuticals, biomaterials, and so on. Synthesis of tailor-made amino acids via Ni(II) complex
intermediates is one of the effective preparation method. We have reported that synthesis ofunusual amino acids for the total synthesis of peptidyl natural products using chiral Ni(II) glycine Schiff base [1].
On the other hand, SARS-CoV2 protease is responsible for the processing of two virus
precursors polypeptides produced during virus replication. Therefore, SARS-CoV2 proteaseis essential for life cycle and reasonable target protein for the drug discovery [2][3].

Results
In the present study, we attempted dynamic kinetic resolutions or (S)/(R) interconversionsof unprotected amino acids with active side chains. The amino acids derivatives we synthesized are used for the creation of SARS-CoV2 protease inhibitors.
All dynamic kinetic resolutions of amino acids were conducted under the following conditions as a synthetic protocol. The (R)-chiral ligands (0.1 mmol) with (rac or L)-amino acids (0.11mmol) and Ni(OAc)2.4H2O (0.11 mmol) in the presence of K2CO3 (0.6 mmol) in MeOH (1.6mL) were treated at 50 oC in N2 atmosphere. Each reaction was monitored by TLC, NMR and/or HPLC. After the completion of the reactions, the mixtures were quenched by 10% AcOH (3.2mL) and concentration in vacuo. Crude compounds were protected with active side chainsand purified by column chromatography over silica gel (CHCl3-MeOH) to afford the Ni(II) complexs (R,R)-form. Each diastereomeric excess of the Ni(II) complexs was determined by 1HNMR analysis. Using above a synthetic protocol, dynamic kinetic resolution of various (rac orL)-amino acids with active functional groups on the side chains was performed using (R)-chiralligand and Ni(OAc)2. Resultantly, dynamic kinetic resolutions of amino acids with active side
chains (Gln, Orn, Lys, Glu, Tyr) without protecting groups even for linear (Nle, Met), branched

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chain (Val, Ile, Leu) proceeded to give (R,R)-forms in 68-99% yields and high diastereoselectivity (>93%de). Surprisingly, Orn and Lys with two free amino groups and Glu with two freecarboxylic acids constructed with chiral ligand to afford selectively the Ni(II) complexs (R,R)forms in excellent yield. However, the dynamic kinetic resolution of Asp and Cys did not workand those of Asn showed low stereoselectivity (~10%de). The use of Ser and Thr gave undesired diastereoisomer (R,S)-forms in high yield compared with the desired isomers (R,R)forms. On the other hand, homoCys, homoSer, Asp(OtBu), His(Trt), Ser(tBu), and Thr(tBu)
were successful to obtain the corresponding products in good yields. It is noted that aminoacids with hetero atoms on β-carbon were unsuccessful. It guess that the formation of aminoacids and Ni(II) directly proceeded and functionalities of side chains of amino acids react toNi(II) complex (R,R)-forms. Cheap (rac or L)-amino acids are converted easily to expensive(D)-amino acids by the methodology. Additionally, cheap (rac)-amino acids can be convertedto (L)-amino acids. Moreover, the synthesized Ni(II) complex Lys type (R,R)-form was attempted to modify the side chain by Cbz, Ac, Boc, Fmoc, Alloc, phthalimide, and urea derivatives in satisfactory chemical yields. The homoSer derivative was converted to TBS and Ac
forms in good yields. By these reactions, the side chains were protected and the corresponding compounds can be used as protected amino acids for the peptide synthesis. The synthesized Ni(II) complexs could be disassembled with HCl to give amino acids and chiral ligand inmoderate yields [4][5][6].
Next, we attempted the preparation of α,α-disubstituted amino acids using the methodology. As use of Ni(II) complex Phe type (R,R)-form, α-substituted reaction was smoothly
proceeded to give Ni(II) complex Phe type (R,R)-form with methyl group in the α position inmoderate yield. However, benzyl group was not introduced in the α position to give dibenzyl
derivatives. DOPA and its derivatives did not react with chiral ligand with Ni(OAc)2.4H2O. To
produce SARS-CoV2 3CL protease inhibitors from the docking simulation, preparation of several amino acid derivatives was also hard work.

Discussion & Conclusion
By the use of Ni(II) chiral Schiff base, dynamic kinetic resolution and (S)/(R) interconversions of amino acids and alkylation with Gly Schiff base can be performed to give tailor madeamino acids. In this study, we found the reaction using unprotected amino acids with activeside chains proceeds to afford desired Ni(II) complexs. These results mean that this chemistry has great potential for the amino acids synthesis and creates new chemical space for the
drug discovery and material science. There are several tasks to be solved, molecular design,
optimized conditions. For the production of new SARS-CoV2 3CL protease inhibitors, we willattempt new amino acids synthesis and evaluate the inhibitory activities.

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References[1] Nagaoka, K.; Nakano, A.; Han, J.; Sakamoto, T.; Konno, H.; Moriwaki, H.; Abe, H.; Izawa, K.;
Soloshonok, V. A. Chirality 2021, 33, 685-702.
[2] Akaji, K.; Konno, H. Molecules 2020, 25, 3920.
[3] Konno, H.; Akaji, K. Org. Syn. Chem. Jpn 2021, 79, 2-10.
[4] Nakano, A.; Nagaoka, K.; Sakamoto, T.; Moriwaki, H.; Abe, H.; Konno, K. Peptide Science
2022, 2023, 45-46.
[5] Nakano, A.; Nagaoka, K.; Han, J.; Sakamoto, T.; Moriwaki, H.; Abe, H.; Izawa, K.; Soloshonok, V. A.; Konno, K. Peptide Science 2021, 2022, 43-44.
[6] Nakano, A.; Nagaoka, K.; Moriwaki, H.; Konno, K. manuscript in preparation.

一般の皆様へアミノ酸は最も重要な部分構造の一つであり、 医薬品、 農薬、 食品添加物などに利用されている。 アミノ酸は多彩な側鎖構造を持つことから需要が高く、 その効率的な製造技術の開発は重要課題となっている。 それらの製造プロセスにおいて、 一般性があり、 環境にやさしく、 なお
かつ安全、 安価な方法論の確立が求められていた。 筆者は光学活性なニッケルリガンドを用いて安価な L 体を高価な D 体に変換しつつ、保護基を効率的に導入しうる方法の開発に成功した。
この結果は従来法と比較して様々な利点があり、 社会実装への展開が期待される。

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Potential implications of enzymatic activity of the multifunctional
protease NRDC as a new therapeutic target for
cardiovascular disease
Mikiko Ohno
moono@belle.shiga-med.ac.jp
Shiga University of Medical Science

Summary Abstract
Nardilysin, a multifunctional protease abbreviated as NRDC, is a potential therapeutic target for cardiovascular diseases. We analyzed mutant NRDC mice lacking enzyme activity toclarify the pathophysiological significance of enzyme activity.
Key Words ： metalloprotease, enzymatic activity

Introduction
We reported that nardilysin (NRDC), an M16 family metalloprotease, is a multifunctional
protein dependent on the cellular localization. NRDC acts as an enhancer of ectodomain
shedding making a complex with ADAM17 and enhances ectodomain shedding of membraneanchored proteins on the cell surface (ref.1-4), and acts as a transcriptional co-regulator making complexes with other transcriptional factors such as PGC1α, Islt1, p53, and HDACs in the
nucleus (ref. 5-9, 14-15). Systemic NRDC knockout mice (Nrdc-/-) show the cardiovascular
phenotypes such as bradycardia and hypotension despite increased sympathetic nerve activity (ref. 10). These findings suggest that NRDC may be a key player of the regulation of vascular tone. We also reported that serum NRDC concentration was elevated in ACS patientscompared with healthy volunteer and that NRDC is a novel biomarker for the early detection of ACS (ref.11-13). However, the pathophysiological significance of enzymatic activity of
NRDC in cardiovascular disease is still unclear. In this study, we generated Nrdc E>A knockin mice (Nrdc E>A KI/KI) lacking enzymatic activity of NRDC by changing the glutamic acid in
the center of enzyme activity to alanine and analyzed.

Results
This research explored the following major themes.
1) Cardiac phenotypic analysis of adult Nrdc E>A KI/KI.
To clarify the effect of enzymatic activity of NRDC regulating heart rate, Nrdc E>A KI/KI
were analyzed using an electrocardiographic telemetry system. The average heart rate
after the stimulation of atropine and metoprolol to remove the autonomic nerve effect wassignificantly lower in Nrdc E>A KI/KI compared with control mice littermates indicatingthat heart rate is regulated by NRDC enzymatic activity.
Furthermore, we confirmed decreased mRNA expression of ion channels essential forsinus node automaticity formation in Nrdc E>A KI/KI hearts using RT-PCR. In addition,
we performed RNA-seq using Nrdc E>A KI/KI hearts and found that several novel mRNA
expressions essential for the cardiac conduction system were altered compared tocontrol mice.

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The Novartis Foundation(Japan)
for the Promotion of Science2) Significance of NRDC in a model of cardiac disease (takotsubo cardiomyopathy model)
We have previously shown that serum NRDC levels were significantly elevated in humanacute coronary syndrome (ACS) and takotsubo cardiomyopathy (TSS) compared tohealthy controls and patients with stable angina pectoris (ref. 8 and unpublished). We
generated a model of takotsubo cardiomyopathy (isoproterenol overdose injection) inwild-type and cardiovascular-specific Nrdc-deficient mice (Sm22α-Nrdc-KO) as well as
in Nrdc E>A KI/KI, which is a model of stress-induced cardiomyopathy believed to becaused by overexcitation of the sympathetic nervous system. We focused on how the
enzymatic activity of NRDC is involved in catecholamine-induced myocardial damage,focusing on changes in downstream signaling of β-receptors and reactive oxygen
species.
3) Screening of compounds which reduce the enzymatic activity of NRDC
MEF cells derived from wild-type, Nrdc E>A KI/KI, and Nrdc-KO mice were treated withsmall molecule compounds from a library that have been previously shown to inhibit
the enzymatic activity of NRDC, and we examined how the expression of the target
molecules is altered in these cells.

Discussion & Conclusion
Cardiac phenotyping of mutant NRDC knock-in mice lacking enzyme activity revealed thatthese mice, like Nrdc-KO mice, exhibit bradycardia and reduced expression of ion channels
responsible for sinus node automaticity. Previously, we reported that NRDC forms complexes
with other transcription factors, suggesting that the enzymatic activity of NRDC may regulate
ion channel expression in coordination with other transcription factors. However, the detailed
mechanism by which transcription factors NRDC forms complexes in the heart and how
NRDC modulates these transcription factors enzymatically remain to be elucidated and require further investigation.

References
1）

2）

3）

4）

5）

6）

7)

Nishi E, Hiraoka Y, Yoshida K, Okawa K, Kita T. Nardilysin enhances ectodomain sheddingof heparin-binding epidermal growth factor-like growth factor through activation of tumor
necrosis factor-alpha-converting enzyme. J Biol Chem. 2006 Oct 13;281(41):31164-72.
Hiraoka Y, Ohno M, Yoshida K, Okawa K, Tomimoto H, Kita T, Nishi E. Enhancementof alpha-secretase cleavage of amyloid precursor protein by a metalloendopeptidase
nardilysin. J Neurochem. 2007 Sep;102(5):1595-1605.
Hiraoka Y, Yoshida K, Ohno M, Matsuoka T, Kita T, Nishi E. Ectodomain shedding of TNFalpha is enhanced by nardilysin via activation of ADAM proteases. Biochem Biophys Res
Commun. 2008 May 23;370(1):154-8.
Ohno M, Hiraoka Y, Matsuoka T, Tomimoto H, Takao K, Miyakawa T, Oshima N, Kiyonari
H, Kimura T, Kita T, Nishi E. Nardilysin regulates axonal maturation and myelination in thecentral and peripheral nervous system. Nat Neurosci. 2009 Dec;12(12):1506-13.
Hiraoka Y, Matsuoka T, Ohno M, Nakamura K, Saijo S, Matsumura S, Nishi K, Sakamoto
J, Chen PM, Inoue K, Fushiki T, Kita T, Kimura T, Nishi E. Critical roles of nardilysin in themaintenance of body temperature homoeostasis. Nat Commun. 2014;5:3224.
Nishi K, Sato Y, Ohno M, Hiraoka Y, Saijo S, Sakamoto J, Chen PM, Morita Y, Matsuda
S, Iwasaki K, Sugizaki K, Harada N, Mukumoto Y, Kiyonari H, Furuyama K, Kawaguchi Y,
Uemoto S, Kita T, Inagaki N, Kimura T and *Nishi E. Nardilysin is Required for maintaining
Pancreatic β-Cell Function. Diabetes 65: 3015-27, 2016
Kanda K, Sakamoto J, Matsumoto Y, Ikuta K, Goto N, Morita Y, Ohno M, Nishi K, Eto
K, Kimura Y, Nakanishi Y, Ikegami K, Yoshikawa T, Fukuda A, Kawada K, Sakai Y, Ito
A, Yoshida M, Kimura T, Chiba T, Nishi E and Seno H. Nardilysin controls intestinal
tumorigenesis through HDAC1/p53–dependent transcriptional regulation. JCI Insight 2018;
3(8): e91316

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8)

9)

10)

11)

12)
13)

14)

15)

Morita Y, Ohno M, Nishi K, Hiraoka Y, Saijo S, Matsuda S, Kita T, Kimura T and Nishi E.
Genome-wide profiling of nardilysin target genes reveals its role in epigenetic regulationand cell cycle progression. Sci Rep. 7: 14801, 2017
Kasai Y, Toriguchi K, Hatano E, Nishi K, Ohno M, Yoh T, Fukuyama K, Nishio T, Okuno M,
Iwaisako K, Seo S, Taura K, Kurokawa M, Kunichika M, Uemoto S, *Nishi E. Nardilysin
promotes hepatocellular carcinoma through activation of signal transducer and activator oftranscription 3. Cancer Sci. 108: 910-917, 2017
Ohno M, Nishi K, Hiraoka Y, Niizuma S, Matsuda S, Iwasaki H, Kimura T and Nishi E.
Nardilysin controls cardiac sympathetic innervation patterning through regulation of p75
neurotrophin receptor. FASEB J. 2020; 34 (9): 11624-11640
Chen PM, Ohno M, Hiwasa T, Nishi K, Saijo S, Sakamoto J, Morita Y, Matsuda S,
Watanabe S, Kuwabara Y, Ono K, Imai M, Inoue K, Murai T, Inada T, Tanaka M, Kita T,
Kimura T and Nishi E. Nardilysin is a promising biomarker for the early diagnosis of AcuteCoronary Syndrome. Int J Cardiol. 243: 1-8, 2017
Chen PM, Ohno M, Kimura T, Nishi E. Reply: Nardilysin is a promising biomarker for the
early diagnosis of acute coronary syndrome. Int J Cardiol. Vol.265,236,2018
Response to Letter of Stephenson et al.: Nardilysin: A potential biomarker for the early
diagnosis of acute coronary syndrome. Ohno M, Chen PM, Kimura T, Nishi E. Int J Cardiol.
277 249 2019
Saijo S, Ohno M, Iwasaki H, Matsuda S, Nishi K, Hiraoka Y, Ide N, Kimura T, Nishi E.
Nardilysin in adipocytes regulates UCP1 expression and body temperature homeostasis.
Sci Rep. 2022
Yasuda D, Hiraoka Y, Ohno M, Nishi K, Iwasaki H, Kita T, Nishi E, Kume N. Deficiency
of Nardilysin in the Liver Reduces Serum Cholesterol Levels. Biol Pharm Bull.
2021;44(3):363-371.

一般の皆様へ
我々は、 プロテアーゼの一つであるナルディライジンについて、 生体内でどのような役割を持
つのか長年研究しています。 遺伝子改変マウスの解析系やヒト血清ナルディライジン値を測定できるシステムを立ち上げ、様々な病態での役割を明らかにしてきました。 今回の研究ではナルディ
ライジンによる心臓の拍動 （心拍数） 調節のしくみを明らかにしました。 また、 その酵素活性による意義を明らかにするために、 ナルディライジンの酵素活性を持たない変異型ナルディライジ
ンを持つマウスや、 ナルディライジンの酵素活性を抑制する小分子化合物を解析し、 心筋梗塞
やタコツボ型心筋症などの心臓病において、 ナルディライジンが新しい治療薬開発の標的となり得るかどうかを検討しました。 今後、 ナルディライジン酵素活性を抑制することによって、 実際に心臓病の改善が見込めるかどうか、 また、 さらに詳細なメカニズムを解析し、 将来の治療薬候補を探索していきます。

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Elucidating the mechanism of skin stem cell aging
by targeting anti-aging matrix Fibulin-7
Aiko Sada
aisada@kumamoto-u.ac.jp
International Research Center for Medical Sciences (IRCMS), Kumamoto University

Summary Abstract
Tissue stem cells divide infrequently as a protective mechanism against internal and external stresses associated with aging. Here, we demonstrated that mice lacking fibulin-7, an
extracellular matrix (ECM), show early impairments resembling epidermal stem cell aging,such as the loss of fast-cycling clones, delayed wound healing, and increased expression of
inflammation- and differentiation-related genes. Fibulin-7 interacts with ECM proteins, and theoverexpression of fibulin-7 in primary keratinocytes results in slower proliferation. Our results
suggest that fibulin-7 maintains epidermal stem cell heterogeneity, thereby protecting skinfrom the detrimental effects of aging and maintaining long-term tissue resilience.
Key Words ： skin aging, epidermal stem cells, extracellular matrix, microenvironment, Fibulin-7

Introduction
The extracellular matrix (ECM) provides an essential microenvironment for the tissue stem
cells, and its alterations can induce aging-associated skin dysfunction during aging. Amongthe genes upregulated in epidermal stem cells in aged skin with the unknown function wasfibulin-7, a secreted glycoprotein belonging to the short fibulin family of ECM proteins1. Fibulin-7 regulates cell differentiation and migration through interaction with other ECM proteins,heparin, and cell surface receptors such as β1 integrins to mediate binding to odontoblasts,monocytes, and endothelial cells2,3,4. However, the role of fibulin-7 in the skin remains largely
unknown.

Results
1. Loss of fibulin-7 leads to age-dependent depletion of fast-cycling stem cell clones
and increased inflammatory response
The fibulin-7 protein was localized to the basement membrane, and the expression was
significantly lower in the aged skin. Mice lacking fibulin-7 showed an aging skin-like phenotype, with an accelerated decrease in the number of fast-cycling epidermal stem cell clones
and a delay in wound healing. To understand how fibulin-7 maintains epidermal stem cells
at the molecular level, we performed RNA sequencing using epidermal stem cells isolated
from the skin of fibulin-7-deficient mice. The results showed that the expression of a groupof inflammation-related genes, including antigen presentation and cytokine production, waselevated in fibulin-7-deficient mice. In a previous report, abnormal expression of a group ofgenes that define the properties of epidermal stem cells was reported as part of the inflammatory response of the skin. Analysis of these marker genes revealed that the characteristic

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molecular changes seen in aged mice, such as enhanced epidermal differentiation markers,
were already present in fibulin-7-deficient mice at the age of 1 year. This suggests that the
aging phenotype of epidermal stem cells may be accelerated in fibulin-7 deficient mice.
2. Fibulin-7 binds to extracellular matrix proteins and regulates the microenvironmentof epidermal stem cells
To address the biochemical function of Fibulin-7, proteins that bind to fibulin-7 were identified by affinity chromatography and mass spectrometry, and included basement membrane
structural proteins, growth regulators, matricellular proteins, and matrix proteases. Fibulin-7
interacted with basement membrane structural proteins such as Collagen IV, and fibulin-7-deficient mice showed irregular basement membrane thickening patterns and abnormal expression. This aligns with a previous report describing the upregulation of col IV in thickened BM,
which led to niche stiffness in aged skin5. Moreover, collagen XVII proteolysis promotes skinaging6, and we confirmed that collagen XVII staining was significantly decreased in the aged
WT mice as well as in the 1-y-old fibulin-7 KO mice. These results indicate that fibulin-7 maintains the microenvironment of epidermal stem cells through physical interaction with extracellular matrix proteins.
3. Fibulin-7 regulates the undifferentiated state and proliferative response to growth
factors in epidermal stem cells
Finally, we investigated the possibility that overexpression of fibulin-7 could exert a protective effect on epidermal stem cells using cultured epidermal stem cells. Epidermal stem cells
overexpressing fibulin-7 are maintained in an undifferentiated and slow-cycling state. The
regulation of proliferation and differentiation was shown to be dependent on different domainsof the fibulin-7 protein.
Fibulin-7 overexpression was able to inhibit differentiation markers not only in the basal
state but also during calcium induction in the presence or absence of col IV coating, albeit amore significant suppression was observed with col IV coating. Binding with col IV may enhance fibulin 7 function through their protein–protein interactions.
To mimic the microenvironment of aging skin, we examined the effects of low serum or
inflammation-like stress conditions and found that fibulin-7 maintains low proliferation of epidermal stem cells under both conditions. These results suggest that fibulin-7 may act as an"anti-aging matrix" that maintains epidermal stem cell capacity over the long-term.

Discussion & Conclusion
Young skin is supported by heterogeneous stem cell populations having the high capacity
for injury recovery and resilience to stress. We demonstrated that the deletion of fibulin-7
accelerated the age-dependent loss of fast-cycling stem cell clones, accompanied by
defective skin regeneration after tissue damage7. Fibulin-7 maintains epidermal stem cells inpart by regulating inflammatory stress responses and the fate balance of stem cells throughdirect binding to ECM proteins. Our results suggest that regulation of the extracellularenvironment by fibulin-7 maintains heterogeneous epidermal stem cell populations over the
long-term, which may be an essential mechanism in controlling skin aging.

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The slower cycling rate induced by fibulin-7 overexpression was only partially enhanced
by IL-6, a positive regulator of keratinocyte proliferation8. This indicates that fibulin-7 may
dampen keratinocyte response to excess inflammatory cytokine signaling. Fibulin-7 could
represent a potential molecule for protecting epidermal stem cells against the detrimental
effects of aging-associated inflammation, lineage misspecifications, and a premature
differentiation-like state. Taken together, fibulin-7 could be a valuable candidate for theintervention of chronic wounds observed more frequently in older patients.

References
1. de Vega S et al (2007) J Biol Chem 282: 30878–30888
2. Ikeuchi T et al (2018) Sci Rep 8: 17654
3. Sarangi PP et al (2018) FASEB J 32: 4889–4898
4. Tsunezumi J et al (2018) Matrix Biol 74: 5–20
5. Koester J et al (2021) Nat Cell Biol 23: 771–781
6. Liu N et al (2019) Nature 568: 344–350
7. Raja et al (2022) EMBO Rep. 23(12): e55478
8. Grossman RM et al (1989) Proc Natl Acad Sci USA 86: 6367–6371
9. Dusadeemeelap C, Rojasawasthien T, Matsubara T, Kokabu S, Addison WN. Inhibition of
TET-mediated DNA demethylation suppresses osteoblast differentiation. FASEB J. 2022
Feb;36(2):e22153.

一般の皆様へ皮膚は、 外的 ・ 内的ストレスを常に受けながらも、 柔軟に応答し、 組織を回復する力を持つ
レジリエンスの高い臓器の一つですが、 加齢とともに徐々にその能力を喪失していきます。 本研
究では、 細胞外ではたらくタンパク質である fibulin-7が、 表皮幹細胞周囲の微小環境を構築することで、 皮膚の老化を防ぐ鍵になる因子の一つであることを解明しました。 本研究成果は、 老化した表皮幹細胞や環境因子を標的とした皮膚の老化予防 ・ 制御法の創出へとつながることが期待されます。

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Studies of genetic basis of early onset cardiac
conduction system disease and disease mechanism-based
personalized medicine
Kenshi Hayashi
kenshi@med.kanazawa-u.ac.jp
Kanazawa University Graduate School of Medical Sciences

Summary Abstract
High-throughput sequencing targeting 180 arrhythmia and cardiomyopathy-related genes
with functional studies identified 21 pathogenic or likely pathogenic variants in 20 of 51 earlyonset (<65 years) cardiac conduction system disease (CCSD) probands (39%), who showed
an AV block and/or a sick sinus syndrome (SSS) with pacemaker implantation (PMI) or afamily history of PMI. Functional analyses of a cellular electrophysiological study and in vivo
zebrafish cardiac assay might be useful for determining the pathogenicity of rare variants inpatients with CCSD. LMNA and SCN10A may be the major development factors in CCSD.
Key Words ： Cardiac conduction system disease, 2015 ACMG standards and guidelines,
CRISPR/Cas9-mediated gene knock-out in zebrafish, Cellular electrophysiological study

Introduction
The genetic cause of cardiac conduction system disease (CCSD) has not been fully elucidated. One of the main challenges facing genetic testing is that interpretation of the testingis complicated by the high prevalence variants of uncertain significance (VUS), which cannot
be applied in clinical practice. We aimed to identify pathogenic or likely pathogenic variants
in CCSD patients by using the targeted next-generation sequencing of candidate genes and2015 American College of Medical Genetics and Genomics (ACMG) standards and guidelines as well as evaluating the usefulness of functional studies for determining them.

Results
We performed the targeted next-generation sequencing of 180 candidate genes linked toarrhythmogenic diseases or cardiomyopathies of 51 probands diagnosed with early-onset(<65 years) CCSD, who showed an AV block and/or a sick sinus syndrome (SSS) withpacemaker implantation (PMI) or a family history of PMI.
The mean age of the study participants was 45±19 years at
the diagnosis of CCSD. Of 51 subjects, 27 (53%) were women, 28 (55%) had SSS, 30(59%) had AV block, and 39 (76%) underwent PMI. Seventeen (33%) had a first-degree family history of PMI. Echocardiographic data revealed a normal mean ejection fraction. Atrial
fibrillation (AF) and muscular disease were complicated in 17 (33%) and 2 (4%) subjects, respectively.
Gene analysis showed that 15 variants with pathogenic or likely pathogenic were identified in LMNA (n=5), EMD (n=2), MYH7 (n=2), SCN5A (n=1), SCN10A (n=1), KCNA5 (n=1),

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TNNT2 (n=1), OBSCN (n=1) and LAMP2 (n=1) in 15 probands (29%). In addition to these 15
variants, 25 variants with VUS were identified in 21 genes in 15 probands (29%).
To evaluate the functional changes brought about by these variants, we generated a knockout zebrafish with CRISPR-mediated insertions or deletions of the EMD or LMNA homologs
in zebrafish. At 48 hpf after the microinjection of sgRNA and Cas9 protein, the mean heart
rate and conduction velocities in the CRISPR/Cas9-injected embryos were significantly decreased. We also evaluated F2 zebrafish embryos. The mean heart rate and conduction
velocities of lmnadel/del embryos were significantly decreased. We next conducted functional
study of one MYH6 variant using zebrafish. We performed targeted myh6 knockdown with
ATG-blocking morpholino antisense oligonucleotide (ATG-MO). Myh6 morphants showeda significantly slower HR and conduction velocities of ventricle compared with non-injected
embryos. Co-injection of MYH6 WT cRNA partially rescued the reduced mean conduction
velocities, while co-injection of MYH6 variant cRNA did not.
We also conducted cellular electrophysiological study to evaluate 2 variants in KCNH2and SCN5A, 3 variants in SCN10A. Electrophysiological study demonstrated that 4 variants
in KCNH2, SCN5A, SCN10A showed loss-of-function effect, and one variant in SCN10A
showed gain-of-function effect.
Six variants in MYH6, KCNH2, SCN5A and SCN10A changed their clinical significancefrom ‘uncertain significance’ to ‘likely pathogenic’ when added the functional study results.

Discussion & Conclusion
High-throughput sequencing targeting 180 arrhythmia and cardiomyopathy-related genes
with functional studies identified 21 pathogenic or likely pathogenic variants in 20 of 51 CCSD
probands (39%). Functional analyses of a cellular electrophysiological study and in vivo zebrafish cardiac assay might be useful for determining the pathogenicity of rare variants in patients with CCSD. LMNA and SCN10A may be the major development factors in CCSD.

References
1. Usuda K, Hayashi K, et al. Mechanisms of fever-induced QT prolongation and torsades
de pointes in patients with KCNH2 mutation. EP Europace. 2023 in press.
2. Cui S, Hayashi K, Kobayashi I, Hosomichi K, Nomura A, Teramoto R, Usuda K, Okada H,
Deng Y, Kobayashi-Sun J, Nishikawa T, Furusho H, Saito T, Hirase H, Ohta K, Fujimoto
M, Horita Y, Kusayama T, Tsuda T, Tada H, Kato T, Usui S, Sakata K, Fujino N, Tajima A,
Yamagishi M, Takamura M. The utility of zebrafish cardiac arrhythmia model to predict thepathogenicity of KCNQ1 variants J Mol Cell Cardiol. 2023 Apr;177:50-61. doi: 10.1016/
j.yjmcc.2023.03.001.
3. Hayashi K（Corresponding author）, Teramoto R, Nomura A, Asano Y, Beerens M, Kurata Y,
Kobayashi I, Fujino N, Furusho H, Sakata K, Onoue K, Chiang DY, Kiviniemi TO, Buys E,
Sips P, Burch ML, Zhao Y, Kelly AE, Namura M, Kita Y, Tsuchiya T, Kaku B, Oe K, Takeda Y,
Konno T, Inoue M, Fujita T, Kato T, Funada A, Tada H, Hodatsu A, Nakanishi C, Sakamoto Y,
Tsuda T, Nagata Y, Tanaka Y, Okada H, Usuda K, Cui S, Saito Y, MacRae CA, Takashima
S, Yamagishi M, Kawashiri MA, Takamura M. Impact of functional studies on exome sequence variant interpretation in early-onset cardiac conduction system diseases. Cardiovasc Res. 2020 Nov 1;116(13):2116-2130. doi: 10.1093/cvr/cvaa010.

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一般の皆様へ
若年発症徐脈性不整脈の原因遺伝子の詳細は明らかにされていない。 また、 遺伝子解析によって見いだされるバリアントの中にはその病的意義が不明なものが数多く含まれる。 我々は次世代シーケンサーを用いて65才未満で発症した徐脈性不整脈51症例に対して拡大候補遺伝子解析を行った。 遺伝子解析の結果、 15症例に15個の病的バリアントを見出し、 15症例に25個の病的意義不明のバリアントを見出した。 病的意義不明のバリアントに対してゼブラフィッシュあ
るいは動物培養細胞を用いて機能解析を行ったところ、 6個のバリアントの機能異常を確認した。
最終的に若年発症徐脈性不整脈51症例のうち、 20症例に21個の病的バリアントを見出し、 陽性率は39% であった。 その中には LMNA 遺伝子と SCN10A 遺伝子のバリアントが多く含まれ、 若年発症徐脈性不整脈の主要原因遺伝子であることが示唆された。

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Analysis of the mechanism underlying extracellular release of
tau for treatment of Alzheimer’s disease
Shigeki Arawaka
shigeki.arawaka@ompu.ac.jp
Department of Internal Medicine IV, Division of Neurology, Osaka Medical andPharmaceutical University Faculty of Medicine

Summary Abstract
Cell-to-cell propagation of tau pathology is thought to cause spread of neuronal degeneration in Alzheimer’s disease. Extracellular release of tau proteins is a key step for this propagation. However, the mechanism underlying extracellular release of tau is not fully understood.
In this study, we found that treatment with autophagy inducer rapamycin facilitated physiological release of tau in mouse primary cortical neurons. Additionally, treatment with lysosomal
inhibitor cocktail facilitated release of tau. These findings suggest that tau was physiologicallyreleased via an autophagy-based mechanism, and this release is facilitated during inhibitionof lysosome function as a compensatory phenomenon.
Key Words ： Alzheimer’s disease, tau, secretion, autophagy

Introduction
Cell-to-cell propagation of tau pathology is thought to cause spread of neuronal degeneration in Alzheimer’s disease. Extracellular release of tau proteins is a key step for this propagation. However, the mechanism underlying extracellular release of tau is not fully understood.
To elucidate the pathway that regulates release of tau, we tested whether tau was physiologically released via an autophagy-based mechanism in mouse primary cortical neurons.

Results
In this study, to obtain cell lysates, mouse primary cortical neurons were suspended in ice
cold lysis buffer, sonicated at 30W for 3 sec 5 times, and kept on ice for 30 min. Samples
were centrifugated at 12,000×g for 30 min at 4°C. The resultant supernatants were collected
as cell lysates and stored at −80°C until use. Protein concentration was measured by BCA
Protein Assay Kit. To assess extracellular tau, collected conditioned media (CM) were centrifuged at 6,000×g for 5 min at 4°C to remove cells and cell debris, and then 1/4 volume of
100% trichloroacetic acid was added to CM. CM were kept on ice for 30 min and centrifuged
at 20,000×g for 30 min at 4°C. The resultant pellets were washed 3 times with 1 ml of coldacetone, air dried, and dissolved in 100 µl of Laemmli’s sample buffer containing 2.5% 2-mercaptoethanol with brief sonication. For assessment of intracellular levels and extracellularrelease of target proteins, we performed SDS-PAGE and western blotting. For quantitative
analysis, the signals of b-actin were used as a loading control. To obtain detergent-insolubletau, neurons were sonicated on ice in buffer containing 1% Triton X-100 and centrifuged at
100,000 × g for 30 min at 4°C. The supernatant was collected as 1% Triton X-100 soluble
fraction. After washing pellets with buffer, resultant pellets were dissolved in the equal aliquot

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The Novartis Foundation(Japan)
for the Promotion of Scienceof the solution containing 8 M urea and 2% SDS and centrifuged at 100,000 × g for 30 min.
The supernatant was collected as 1% Triton X-100 insoluble fraction.
To explore a mechanism underlying extracellular release of tau, we investigated the effects
of autophagy on release of tau in mouse primary cortical neurons. Treatment with autophagy
inducer rapamycin facilitated release of tau and decreased intracellular levels of soluble tau.
Treatment with rapamycin increased intracellular generation of LC3-II with reducing intracellular levels of p62, indicating that rapamycin stimulated autophagy flux under the present condition. Also, treatment with rapamycin did not increase LDH release, showing that the elevationof tau release was not caused by drug-induced cell death. These findings suggested that tau
was physiologically released via an autophagy-based mechanism.
Then, we investigated whether degradative function of autophagy affected release of tau.
To address this issue, we treated primary neurons with cocktail of protease inhibitors, E-64
and leupeptin, which inhibit lysosomal degradation. Degradative function of autophagy isexerted by fusion of lysosomes with autophagic vacuoles. Treatment with these inhibitors
facilitated extracellular release of tau without altering LDH release, whereas it increased intracellular levels of soluble and detergent-insoluble tau. Protease inhibitor cocktail increased
intracellular levels of LC3-II and p62, showing that it blocked autophagy flux. These findings
suggested that autophagic release of tau was facilitated in a fashion compensatory to dysfunction of autophagic degradation.
To see the relationship between extracellular release and degradation in autophagy, we investigated the effects of rapamycin on tau metabolism under inhibition of lysosomal degradation. When we co-treated primary neurons with rapamycin and cocktail of proteasome inhibitors E-64 and leupeptin, extracellular release of tau was increased more significantly than thatin treatment with either rapamycin or cocktail of proteasome inhibitors. However, proteasome
inhibitor-induced accumulation of soluble and insoluble tau was not reduced by addition of rapamycin. Increased levels of LC3-II and p62 were also sustained. These findings suggested
that rapamycin further facilitated extracellular release of tau under inhibition of autophagic
degradation, but this effect did not lead to reduction in intracellular accumulation of soluble
and insoluble tau.

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Discussion & Conclusion
Autophagy plays a pivotal role in maintenance of proteostasis by remove unnecessary organelles and misfolded proteins under basal and stress conditions. In addition to this degradative function, accumulating lines of evidence have indicated the role of extracellular releaseof “leaderless proteins”, which lack an endoplasmic reticulum-translating sequence. We foundthat treatment with autophagy inducer rapamycin facilitated physiological release of tau inmouse primary cortical neurons. This finding suggests that tau was physiologically released
via an autophagy-based mechanism as one of leaderless proteins. Additionally, treatment
with lysosome inhibitor cocktail facilitated release of tau. Co-treatment with rapamycin andlysosome inhibitor cocktail further increased extracellular release of tau, whereas rapamycin
failed to reduce lysosome inhibitor-induced accumulation of soluble and insoluble tau. Thesefindings suggest that autophagic release of tau is facilitated during inhibition of autophagic
degradation as a compensatory phenomenon, but this effect was insufficient to normalize
intracellular accumulation of soluble and insoluble tau. Proteostasis of tau protein may be
maintained by cooperation of degradative and releasing functions of autophagy. Dysregulation of autophagic functions may lead to cause the formation of tau aggregates and cell-to-cell
propagation of tau pathology in Alzheimer’s disease.

References
In preparation for submission.

一般の皆様へアルツハイマー病の発症には、 タウタンパク質の細胞間伝播が深く関与している。 本研究では、
細胞間伝播に必須のステップであるタウタンパク質の細胞外放出機構に焦点をあてて解析を行った。
その結果、 マウス大脳皮質初代神経細胞において、 タウタンパク質は生理的にオートファジーを介して細胞外に放出されること、 この放出はオートファジーの分解機能を阻害すると代償的に亢進することを見出した。 これらの所見は、オートファジーの機能異常は、タウのタンパク質恒常性維持を乱し、アルツハイマー病の病勢進展に関与する可能性を示唆していた。

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Study of functional deficits of microglia and accelerated onset ofneurodegenerative disorders
Keiro Shirotani
keiroshiro@nagasaki-u.ac.jp
Graduate School of Biomedical Sciences, Nagasaki University

Summary Abstract
Knock-in mice whose exon 2 was replaced with human exon 2 with Alzheimer’s diseaserisk allele R47H (hE2R47H), were generated and its mRNA levels were determined to be atsimilar levels as knock-in mice with wild-type human exon 2 (hE2WT) or wild-type mouse,suggesting the successful generation without unwanted splicing as described before. The
Trem2hE2WT/hE2WT and Trem2hE2R47H/hE2R47H homo knock-in mice were crossed with AppNLF/NLF Alzheimer’s disease model mice and are being aged and analyzed. In phagocytosis assay using primary macrophages, the phagocytic activities were not different between wild-type andTrem2 knock-out mice. However, phagocytic activities of wild-type macrophages stimulated
by lipopolysaccharide (LPS) were higher than those of Trem2 knock-out. Finally, phagocytic
activities of Trem2hE2WT/hE2WT and Trem2hE2R47H/hE2R47H homo knock-in mice were compared but
no difference was observed even when stimulated by LPS.
Key Words ： Alzheimer’s disease, microglia, TREM2Introduction
Genetic studies identified triggering receptor expressed on myeloid cells 2 (TREM2) as an
Alzheimer’s disease (AD)-associated risk factor and so far researchers have tried to establish knocked-in mouse models with TREM2 risk allele (R47H). However, the knock-in mice
showed unwanted splicing and did not express Trem2 mRNA or proteins. Our research group
have succeeded in generation of the Trem2 knock-in mice model by exchanging mouseexon2 with human exon2 including R47H mutation. In this study we characterize the expression of Trem2 mRNA in the mouse brain and investigated the function of Trem2 R47H allele
hE2R47H compared with Trem2 wild type hE2WT.

Results
Trem2 mRNA levels of the generated homozygous knock-in mice whose exon2 were replaced with human exon2 wild-type (hE2WT) or human exon2 R47H risk allele (hE2R47H)
were quantified by qPCR in adult mouse cerebral cortex. I have found that Trem2 mRNAlevels of both knocked-in mice were expressed at similar levels as wild-type mice, suggesting
the successful generation of the knock-in mice without unwanted splicing.
The Trem2hE2WT/hE2WT and Trem2hE2R47H/hE2R47H homo knock-in mice were crossed with AppNLF/NLF Alzheimer’s disease model mice and about thirty mice of Trem2hE2WT/hE2WT/AppNLF/NLF
and Trem2hE2R47H/hE2R47H/AppNLF/NLF male or female were obtained. Amyloid accumulation wasinvestigated in cerebral cortex and hippocampus of AppNLF/NLF mice and I have found that few
amyloid plaques were observed in 12 months old mice. Therefore chronology of amyloid ac-

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The Novartis Foundation(Japan)
for the Promotion of Sciencecumulation study was determined as 12, 18, 24 months. Currently Trem2hE2WT/hE2WT/AppNLF/NLF
and Trem2hE2R47H/hE2R47H/AppNLF/NLF mice are being aged, and their brains are removed and analyzed at these time points.
Phagocytosis assay, which measures the microglial important function for onset and progression of Alzheimer’s disease, was established. In brief, pHrodo-labelled E.coli, which is
non-fluorescent outside the cell at neutral pH but fluorescent in intracellular acidic pH environments, were incubated with primary microglia prepared from wild-type mice. However,
because microglial cell numbers obtained from these mice were not enough, primary bonemarrow-derived macrophages (BMDM) which are naïve immune cells as microglia and haveoverlapped functions, were used. First, todemonstrate that Trem2 is involved in thephagocytosis, phagocytic activities of wildtype and Trem2 knock-out (KO) BMDM
were compared. Unexpectedly, phagocytic
activities were not different between those
BMDM. However, phagocytic activities ofwild-type BMDM stimulated by lipopolysaccharide (LPS) were higher than thoseof Trem2 knock-out BMDM (Figure). Finally, phagocytic activities of hE2WT andhE2R47H BMDM homo knock-in mice
(Trem2 hE2WT/hE2WT and Trem2 hE2R47H/hE2R47H)
were compared but no difference was observed.

Discussion & Conclusion
So far Trem2 R47H knock-in mice displayed unexpected splicing and lost Trem2 expression. In this study the Trem2 hE2R47H knock-in mice generated by our group expressed
Trem2 at similar levels as that in Trem2 hE2WT control mice and wild-type mice. These newmouse models should help understanding how TREM2 increases the incidence of Alzheimer’s disease. At the moment aged model mice are under investigation how Trem2 affects
amyloid accumulation, microglial activation, and tau phosphorylation.
We established phagocytosis assay which are related to Trem2 function because wild-type
macrophages stimulated by LPS enhanced phagocytic activity much more than Trem2 knockout macrophages. In this system, however, Trem2hE2R47H homozygous macrophages showedsimilar phagocytic activity as Trem2hE2WT homozygous macrophages even when stimulated by
LPS. This suggests that Trem2 activates phagocytic activity induced by LPS but this function
may not be related to pathology of Alzheimer’s disease. That may suggest that functional deficits of Trem2hE2R47H should be analyzed in vivo. We are currently investigating the pathologicalaspects in aged mice.

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References
1. The role of TREM2 N-glycans in trafficking to the cell surface and signal transductionof TREM2.
Shirotani K, Hatta D, Wakita N, Watanabe K, Iwata N. J. Biochem. 2022; 172(6):347-353
2. Alterations of ATG4A and LC3B in neurons derived from Alzheimer's disease patients.
Shirotani K, Watanabe K, Hatta D, Kutoku Y, Ohsawa Y, Sunada Y, Kondo T, Inoue H, Iwata
N. Genes Cells. 2023; 28(4):319-325.
3. Enhancement of Neprilysin Activity by Natural Polyphenolic Compounds and Their Derivatives in Cultured Neuroglioma Cells.
Hori Y, Watanabe K, Yassen ASA, Shirotani K, Tanaka T, Iwata N. Biol Pharm Bull. 2023;
46(3):446-454
4. アミロイド病理における神経炎症とミクログリア , Trem2 の役割
城谷 圭朗、岩田 修永 Dementia Japan 36:232-243, 2022

一般の皆様へアルツハイマー病の根本治療薬はアミロイドの抗体医療薬のみが米国で上市されている。 しかし認知機能改善効果は弱いので他の作用点の治療薬開発が必要である。 脳の免疫細胞ミクログリアが発症や進行に関わっているという最近の知見から本研究ではミクログリアが持つ TREM2というタンパク質を標的とした創薬を行っている。 これまでの研究上の問題点はアルツハイマー病の発症頻度を高める TREM2R47H 変異を導入したモデルマウスでは TREM2自身が発現しないことであった。 本研究ではこの問題点を克服しモデルマウスの作製に成功し、 実際に TREM2が発現していることを確認した。 また初代培養細胞の実験では TREM2の変異があっても貪食機能に大きな影響を与えないことがわかった。 これよりモデルマウスでの機能解析研究が非常に重要であることが示唆され、 今後は本研究で作製したマウスでアルツハイマー病の発症機序あるいは治療薬開発研究を進めていく。

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Modulation of itch by neuro-immune interaction
Hiromitsu Hara
harah@m2.kufm.kagoshima-u.ac.jp
Kagoshima University Graduate School of Medical and Dental Sciences

Summary Abstract
The type 2 cytokines IL-4, IL-13 and IL-31 act as itch mediators associated with allergic
dermatitis directly through their receptors expressed on pruritic neurons in dorsal root ganglia.
IL-27 is an immunoregulatory cytokine that signals via its heterodimeric receptor composed of
WSX-1 and gp130. Although IL-27 is known to suppress Th2 and ILC2 responses, the effectof IL-27 on the neural system is unknown. Here we show that IL-27 acts directly on pruritic
sensory neurons and suppresses itch.
Key Words ： itch, cytokine, IL-27

Introduction
Physiological itch is essential for host defense as scratching behavior leads to the removalof potentially harmful organisms from the skin. However, in chronic diseases such as atopic
dermatitis, itch is associated with unpleasant effects. Recent reports have shown that the type2 cytokines IL-4, IL-13 and IL-31 control itch in allergic dermatitis by directly activating and/or
sensitizing pruriceptors in the dorsal root ganglion (Ref.1). IL-27 is an immunoregulatory cytokine that signals via its heterodimeric receptor composed of WSX-1 and gp130. Although IL27 suppresses Th2 and ILC2 responses, the role of IL-27 in the neural system is unknown.

Results
To investigate whether IL-27 is involved in itch sensation, we intradermally injected several
pruritogens into WSX-1-deficient (WSX-1-/-) and wild-type control mice and monitored itch
behavior. We found that WSX-1-/- mice showed significantly enhanced scratching behavior
to several pruritogens, suggesting that IL-27 controls itch sensation in the physiological state.
Conversely, we found that intradermal administration of recombinant IL-27 could suppress
pruritogen-induced scratching behavior in wild-type mice. Since IL-27 receptor engages
Jak1/2-STAT1/3 pathway as a downstream signaling, we next investigated a contribution of
JAKs in the suppressive effect of IL-27 on itch by using a JAK(1/2) inhibitor, ruxolitinib. Although administration of recombinant IL-27 suppressed histamine-induced scratching behavior, treatment with the JAK inhibitor partially but significantly reduced the suppressive effectof rIL-27, suggesting that the effect of IL-27 on itch sensation is dependent on JAK activation.
Next, to investigate whether IL-27 acts directly or indirectly on pruriceptive neurons, we generated conditional knockout mice, Nav1.8-Cre/WSX-1flox/flox, which lack WSX-1 expression
specifically in nociceptive neurons including pruriceptors. We found that the suppressive effect of IL-27 on itch sensation was abolished in Nav1.8-Cre/WSX-1flox/flox mice, suggestingthat IL-27 acts directly on sensory neurons to suppress itch sensation. To identify a possible
source of IL-27 in normal skin, we used immunohistochemistry of skin from p28-Venus report-

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Results

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for the Promotion of Scienceer mice, which express the fluorescent protein Venus in synchrony with the expression of p28,
a component of IL-27. We found that IL-27 was produced mainly by CD11c+ dermal dendritic
cells (DCs). Flow cytometric analysis of skin DC subsets revealed that CD207-CD11b+ dermal DCs most abundantly produced IL-27.

Discussion & Conclusion
Our data suggest that dermal DCs constitutively produce IL-27 in the skin under steadystate conditions and suppress excessive itch sensation by directly acting on the pruriceptive DRG neuron. The significance of IL-27 regulation of itch in pathogenic conditions suchas allergic dermatitis is still unknown and requires further investigation. This study provides
important new insights into the regulation of peripheral sensation by immune and nervous interactions. Further studies would open up the IL-27-Jak pathway as a potential target for thetreatment of pruritus.

References

Results(1) Sensory Neurons Co-opt Classical Immune Signaling Pathways to Mediate Chronic Itch.
Oetjen LK, Mack MR, Feng J, Whelan TM, Niu H, Guo CJ, Chen S, Trier AM, Xu AZ, Tripathi SV, Luo J, Gao X, Yang L, Hamilton SL, Wang PL, Brestoff JR, Council ML, Brasington R,
Schaffer A, Brombacher F, Hsieh CS, Gereau RW 4th, Miller MJ, Chen ZF, Hu H, Davidson
S, Liu Q, Kim BS. Cell. 2017 Sep 21;171(1):217-228.e13. doi: 10.1016/j.cell.2017.08.006.
Fig. 1 ncRNA initiations induce new meiotic DSBs in fbp1 upstream region(left) Schematic drawing of fbp1 locus. Arrows represent expressed ncRNA andmRNA of fbp1. Gray boxes 一般の皆様へ
represent cis element required for fbp1 expression.
White circle represents mlonBox required for the initiation of mlonRNA-c. (right)
アトピー性皮膚炎に代表される慢性の痒みは患者のQOL を著しく低下させる。
今回の我々
DSBs introduced in fbp1 upstream region are induced
by glucose starvationstress.
の研究により、 これまでアトピー性皮膚炎などの炎症を抑制するサイトカインとして知られていた
IL-27が、 痒みを伝達する感覚神経にも直接作用して痒みを抑制することが明らかとなった。 これは、 免疫細胞が産生するサイトカインが痒み感覚の抑制に働くことを示した初めて発見であり、この研究をさらに進めることで、 新しい掻痒治療薬の開発につながるものと期待される。

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Analysis of ncRNA transcription mediated chromatin
modulation mechanism
Kouji Hirota
khirota@tmu.ac.jp
Tokyo Metropolitan University

Summary Abstract
In eukaryotic cells, transcription from genomic regions that do not contain protein-coding
information has been found, and such transcripts are called non-coding RNAs. Among them,long non-coding RNAs that are expressed at promoters and involved in the regulation ofgene expression are called promoter non-coding RNAs, but such RNA’s functions remain unknown. The non-promoter-coding "mlonRNA" originally discovered by the applicant, which isexpressed on the fission yeast fbp1 promoter, is expressed during glucose starvation stressand contributes to transcriptional activation by promoting the binding of transcription factors
by opening the chromatin structure of the passed region of transcription polymerase. In thisstudy, we elucidate whether the pioneer polymerase transcribing mlonRNAs and opening
chromatin, is involved in DNA associated reaction other than transcription such as recombination in fission yeast genome.
Key Words ： Chromatin, non-coding RNA, transcription, fbp1, fission yeast

Introduction
Many forms of life, including humans, engage in sexual reproduction, which involves theexchange of the genome between two individuals, a male and a female. This process produces offspring with a divergent genotype that differs from that of the parent, which is advantageous in responding to crises such as environmental change. In sexual reproduction, thisgenetic diversity is brought about by an exchange reaction between the father and motherthrough homologous recombination of the genome between the father and mother, which isaccompanied by the halving of the genome through meiosis. Meiotic homologous recombination is initiated by the introduction of a DNA double-strand break (DSB) in the genome. DSBs
are not randomly distributed; some regions of the genome frequently introduce DNA dsDNA
breaks while others do not, but the regulatory mechanism of DSB distribution remains unresolved. In addition, since many of the meiotic recombination sites in fission yeast localize similarly to non-coding RNA transcription sites, the possibility of DSB regulation by non-coding
RNA has been suggested, but its molecular mechanism remains completely unknown.

Results
To investigate the possible roles of ncRNA expression in the determination of meiotic DSB
sites, we compared the frequency and distribution of meiotic DSBs in fbp1 upstream region in
the presence and absence of glucose. In the presence of glucose, we found faint DSB signal
around UAS1 in the fbp1 upstream region at 4 and 6 hours after induction of meiosis (Fig. 1).

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By contrast, we found at least two prominent meiotic DSB signals around UAS1 and UAS2
in fbp1 upstream region (Fig. 1). These results indicate redistributions and upregulations of
meiotic DSB sites are induced in fbp1 upstream region in response to glucose starvation. To
address the involvement of ncRNA transcriptions in the redistribution of meiotic DSBs, we examined the expression profile of mlonRNAs and mRNA upon glucose starvation stress in meiosis. In contrast to previous study (1) , leaky expressions of mlonRNAs and mRNA was observed before glucose starvation in meiosis. This might be caused by the difference of culture
conditions. To synchronously induce meiosis, we cultured cells in the medium lacking nitrogensource to arrest cell cycle in G1 phase once and released in meiotic condition at time 0. Thus,
the observed leaky expressions may be caused by nitrogen starvation condition. Nevertheless, this slight difference of fbp1 induction upon glucose starvation in mitosis and meiosis,
each mlonRNA (a, b, and c) and mRNA of fbp1 were induced in stepwise manner during glucose starvation in meiosis, while such inductions were not seen in glucose-added condition.
We analyzed chromatin configuration in fbp1 upstream region by investigating the distributionof micrococcal nuclease (MNase) sensitive sites. The distribution patterns of MNase sensitive sites in mitotic cells, G1-arested cells and meiotic cells in glucose-added condition werecomparable, whereas meiotic cells in glucose starvation condition exhibited drastic changesof the distribution patterns; A couple of MNase sensitive sites appeared at UAS1 and intense
MNase sensitive bands appeared around UAS2–TATA box region at 60–180 min after glucose starvation, which were coincident with the robust fbp1 mRNA transcription. Collectively,
these results indicate that transcriptions of mlonRNA/mRNA of fbp1 in response to glucose
starvation stress are associated with chromatin conversions and this transcription-associatedchromatin changing induces redistribution and upregulation of meiotic DSB sites in fbp1 upstream region.
We next wish to study which transcription, mlonRNA or mRNA, is involved in the introduction of meiotic DSBs in fbp1 upstream region. To this end, we mutated several critical cis elements required for these transcriptions. We analyzed mutant cells carrying a point mutationin UAS1 or UAS2 (33), the critical binding sites for transcription factor Atf1-Pcr1 or Rst2, respectively. Both elements are required for fbp1-mRNA expression, while UAS1 but not UAS2
is required for the inductions of mlonRNA. In addition to these two cis elements, we analyzed
mlonRNA initiation element (mlon-IE), which is involved in the mlonRNA-c transcription. Moreover, we also analyzed fbp1-TATA-box, which is required for fbp1-mRNA expression but not

Fig. 1 ncRNA initiations induce new meiotic DSBs in fbp1 upstream region(left) Schematic drawing of fbp1 locus. Arrows represent expressed ncRNA andmRNA of fbp1. Gray boxes represent cis element required for fbp1 expression. White
circle represents mlonBox required for the initiation of mlonRNA-c. (right) DSBs
introduced in fbp1 upstream region are induced by glucose starvation stress.

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for the inductions of mlonRNA. The cells carrying a mutation in UAS2 and cells lacking fbp1TATA-box showed very similar DSB distribution compared to wild-type cells in glucose starvation condition, indicating that expression of fbp1-mRNA is not important for the creation of
DSB sites in fbp1 upstream region upon glucose starvation stress. The cells with a mutationin UAS1 showed critical reduction of DSBs around UAS 1 and UAS2 compared to wild-type
cells. Moreover, mutant cells in which mlon-IE is replaced with a part of act1 sequence also
showed reduction of DSB around UAS2 compared to wild-type cells. These results indicatethat inductions of mlonRNAs rather than fbp1-mRNA are required for meiotic DSB formationin fbp1 in response to glucose starvation stress.

Discussion & Conclusion
Evolution is a continuous process of genetic variation and phenotypic selection (2). At amolecular level, mutations in genes make diversity of the functionality of genes, and it is believed that genes possessing advantages to fit environmental condition are selected fromsuch diverse genes. Meanwhile, a study using bacteria model showed that whole genomeshuffling via homologous recombination between homologous DNA sequences has great
advantage over the simple mutation-mediated improvement of gene function. In the current
study, we showed that transcription events induced by environmental changes trigger meiotic
recombination events. These events might be induced around gene-promoter region, sincetranscription factor binding elements are usually located in gene promoter region, and thus
DNA sequences not only within gene body but also in promoter region should be efficiently
diversified. This view is consistent with the recent study showing a pivotal role of mutations
in non-coding regulatory DNA sequences such as promoter sequences. Moreover, frequent
recombination-mediated genome-shuffling between homologous DNAs at the actively usedgenes in response to various stresses might be reasonable for the rapid molecular evolution
upon the environmental clue.

References(1) Hirota, K., T. Miyoshi, K. Kugou, C. S. Hoffman, T. Shibata, and K. Ohta. 2008.
Stepwise chromatin remodelling by a cascade of transcription initiation of non-coding
RNAs. Nature 456:130-134.
(2)Darwin, C. 1859. The origin of species by means of natural selection : or, The preservationof favored races in the struggle for life. John Murray, London.

一般の皆様へ
本研究では、 子孫に遺伝子を繋ぐ機構、 減数分裂における、 配偶子の多様性の創出機構を新規に解明しました。 子孫の多様性は、 環境変動への生物種としての対応や進化の原動力として重要であることは皆さんご存知のことと思います。 例えば、接木で全国にクローン増殖した 「桜」の木は、 一度病原菌や虫におかされると全滅してしまいますね。 本研究をさらに発展させ、 減
数分裂期の 「組換え」 による子孫の多様性創出の秘密をさらに解明し、 効率的な品種改良技術など応用研究にも繋げたいと思います。

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Communication between tumor cells and neurons
regulating brain tumor
Naofumi Uesaka
uesaka.cnb@tmd.ac.jp
Tokyo Medical and Dental University

Summary Abstract
Key Words ： Brain tumor, synapse, Neural activity, glia, cell-cell communication

Introduction
It is now known that not only the characteristics of the tumor cells, but also the surrounding microenvironment is important in the malignant transformation of brain tumors. However,it remains unclear how the interaction between brain tumor cells and the microenvironment
causes brain tumors to become malignant or benign. In this study, we aim to test the hypothesis that "tumor cells form networks with normal cells in the microenvironment and exploit bioactive substances from normal cells to promote tumor cell growth.

Results
We first investigated the onset of tumor cell growth and glial cell changes in the brains ofglioma model mice. It is known that tumors (gliomas) are generated in the mouse brain byknocking out or knocking down three tumor suppressor genes, Trp53, Pten, and Nf1, whichhave been found mutated in patients with human brain tumors, by the CRISPR/Cas9 system.
Using this model mouse, we analyzed the growing tumor cells and glial cells. The results
showed that glioma increased cell division from around 4 weeks of age, and the number ofneurons and oligodendrocytes decreased markedly from 8 weeks of age due to tumor cell
progression. In contrast, microglia increased within the tumor from 6 weeks of age, and astrocytes increased in the peritumoral area. These results indicate that tumor cell-neuron andtumor cell-glial cell interactions can be analyzed using this model system.
Next, to analyze the mode of interaction of tumor cells with normal cells and tumor progression, we aimed to develop an in vivo experimental system to detect activity dynamics andtransitions in the neuron-tumor cell network. Calcium imaging has been used in neurosciencefield to measure cell activity and analyze intercellular networks, and the applicant applied
this method to tumor cells. The applicant used adeno-associated virus and electroporation toexpress calcium sensor genes (genetically encoded calcium indicator) in neurons and tumor
cells. One week later or late, the craniums of tumor model mice were opened and the tumor
cells and neurons expressing the calcium sensor gene were observed under an objective
lens. We found that brain tumor cells exhibit calcium activity, albeit at low frequency (approximately once every 3 minutes), and that specific cell populations are synchronously active. In
addition, neurons surrounding tumor cells showed calcium transients. We also screened forthe best calcium-directed genes for detecting calcium activity in tumor cells and found that jGCamP8m best detected calcium activity in tumor cells.

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We used the calcium imaging method to analyze the transition of tumor cell activity during
tumor progression, and found that only a few tumor cells showed activity at 4-5 weeks of age,soon after tumor initiation. However, at 7-8 weeks of age, when tumor progression is more
pronounced, more than half of the tumor cells showed calcium activity. These results suggestthat the number of tumor cells receiving transmission and sensory input from neurons increases with tumor progression, supporting the hypothesis of this study that tumor cells form
networks with neurons and glial cells during tumor progression.
To investigate whether neuronal activity and tumor cell activity affect tumor progression, weused adeno-associated viruses to express artificial synthetic receptors (DREADD) on neurons and tumor cells, which, when expressed in cells, respond to their ligand CNO and induce
calcium activity in these cells. We analyzed tumor model mice expressing DREADD in neurons and intraperitoneally injected with CNO to increase neuronal activity. Compared to tumor
model mice without elevated neuronal activity, tumor model mice with elevated neuronal activity had an increased number of Ki67-positive cells, a marker of mitotic cells, in brain tumor
tissue. Elevated neural activity also increased the number of microglia and oligodendrocytes
in the tumors. Moreover, elevated tumor cell activity resulted in the increased number of Ki67positive cells. These results indicate that neuronal activity promotes tumor progression andrecruits peritumoral brain endogenous cells into the tumor.

Discussion & Conclusion
This research has significance in establishing “brain tumor physiology” by combining cutting-edge technologies in neuroscience and basic medical research, ultimately leading to thecure of brain tumors. By elucidating the contribution of neurotransmission and gliotransmission to tumor progression, this research will provide a foothold for developing a new therapeutic approach that "targets factors that support tumor progression," rather than the conventional
approach of targeting the tumor cells themselves. If we can identify specific neurotransmission and gliotransmission, we will be able to predict which neuronal network will become
abnormal at an early stage of development and establish a new diagnostic method for brain
tumors to detect the disease in the preliminary stage when the tumor grows and the patient
recognizes the disease through pain. This could lead to new diagnostic methods for detecting
brain tumors before the tumor has grown and the patient is aware of the disease in pain. The
strategies in this study can be applied to other types of brain tumors and tumors arising in organs where nerves are present (e.g., the intestine where the vagus nerve is located).

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References
Broekman, M.L., Maas, S.L.N., Abels, E.R., Mempel, T.R., Krichevsky, A.M., and Breakefield, X.O.
(2018). Multidimensional communication in the microenvirons of glioblastoma. Nat Rev Neurol 14,
482-495.
Chia, K., Keatinge, M., Mazzolini, J., and Sieger, D. (2019). Brain tumours repurpose endogenous
neuron to microglia signalling mechanisms to promote their own proliferation. Elife 8.
Johung, T., and Monje, M. (2017). Neuronal activity in the glioma microenvironment. Curr Opin
Neurobiol 47, 156-161.
Magnon, C., Hall, S.J., Lin, J., Xue, X., Gerber, L., Freedland, S.J., and Frenette, P.S. (2013). Autonomic nerve development contributes to prostate cancer progression. Science 341, 1236361.
Monje, M., Borniger, J.C., D'Silva, N.J., Deneen, B., Dirks, P.B., Fattahi, F., Frenette, P.S., Garzia, L.,
Gutmann, D.H., Hanahan, D., et al. (2020). Roadmap for the Emerging Field of Cancer Neuroscience. Cell 181, 219-222.
Osswald, M., Jung, E., Sahm, F., Solecki, G., Venkataramani, V., Blaes, J., Weil, S., Horstmann, H.,
Wiestler, B., Syed, M., et al. (2015). Brain tumour cells interconnect to a functional and resistantnetwork. Nature 528, 93-98.
Peterson, S.C., Eberl, M., Vagnozzi, A.N., Belkadi, A., Veniaminova, N.A., Verhaegen, M.E., Bichakjian, C.K., Ward, N.L., Dlugosz, A.A., and Wong, S.Y. (2015). Basal cell carcinoma preferentially arises from stem cells within hair follicle and mechanosensory niches. Cell Stem Cell 16,
400-412.
Stosiek, C., Garaschuk, O., Holthoff, K., and Konnerth, A. (2003). In vivo two-photon calcium imaging of neuronal networks. Proc Natl Acad Sci U S A 100, 7319-7324.
Venkataramani, V., Tanev, D.I., Strahle, C., Studier-Fischer, A., Fankhauser, L., Kessler, T., Korber, C., Kardorff, M., Ratliff, M., Xie, R., et al. (2019). Glutamatergic synaptic input to glioma cells
drives brain tumour progression. Nature 573, 532-538.
Venkatesh, H.S., Morishita, W., Geraghty, A.C., Silverbush, D., Gillespie, S.M., Arzt, M., Tam, L.T.,
Espenel, C., Ponnuswami, A., Ni, L., et al. (2019). Electrical and synaptic integration of glioma into
neural circuits. Nature 573, 539-545.
Zuckermann, M., Hovestadt, V., Knobbe-Thomsen, C.B., Zapatka, M., Northcott, P.A., Schramm,K., Belic, J., Jones, D.T., Tschida, B., Moriarity, B., et al. (2015). Somatic CRISPR/Cas9-mediatedtumour suppressor disruption enables versatile brain tumour modelling. Nat Commun 6, 7391.

一般の皆様へ
脳が正常に働くには脳内の環境が安定していることが必要不可欠です。 脳疾患の原因や病態として、 脳内の局所環境が乱れていることが考えられています。 脳腫瘍は脳内の局所環境が乱れている疾患の１つです。 脳腫瘍は、 腫瘍細胞自体が持つ性質だけでなく、 周りに存在する正常細胞が進展や悪化に影響することが分かっています。 しかし、 腫瘍細胞と周囲の脳内環境にある正常細胞とがどのように相互作用して脳腫瘍を進行させるのかはほとんど分かっていません。
この研究では、 正常な神経細胞やグリア細胞と腫瘍細胞との相互作用に注目しています。 腫瘍細胞は、 正常な神経細胞やグリア細胞と相互作用して、 腫瘍細胞にとって必要な脳内環境を形成していると考えられています。 この研究では、 腫瘍細胞が神経細胞やグリア細胞からの物質を悪用して腫瘍を進行させる可能性を調べました。 今後、 それらの物質を標的とする脳腫瘍の新規治療法の開発につなげることを目指しています。

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Establishment of tissue maturation induction method to obtain
adult tissues from pluripotent stem cells
Mio NAKANISHI
mnakanishi@chiba-u.jp
Chiba University

Summary Abstract
In this research, we aimed to develop a new method to induce cellular maturation frompluripotent stem cells to functional hematopoietic stem cells. We identified fetal-liver derived
niche factors via in silico and in vitro screening using human induced pluripotent stem cells(hiPSCs) derived pre-hematopoietic stem cells (pre-HSCs). Using these newly identified
niche factors, we successfully established a method to induce maturation of the hiPSCs-derived pre-HSCs into HSCs expressing CD201, an essential marker of transplantable fetal-liver
HSCs.
Key Words ： pluripotent stem cells, hematopoietic stem and progenitor cells, fetal liver, maturation,

Introduction
Recently, enormous number of studies have been performed to induce directed differentiation of human pluripotent stem cells (hPSCs) into various cells and tissues, attempting toapply them to future regenerative medicine and drug development. However, these hPSCderived cells and tissues are still immature in terms of gene-expression, epigenetic states,
structures, and functions and are most similar to the counterparts at fetal stages. This 'roadblock to maturation' in hPSC differentiation is thought to be a most serious obstacle to hPSC
application to regenerative medicine and drug development. In this research project, I aim to(1) understand a common mechanism underlying functional maturation, and (2) remove the'roadblock to maturation' by manipulating the maturation-machinery and obtain adult-like cells/
tissues from hPSCs in a shorter time.

Results
First step of human hematopoietic stem cell (HSC) development takes place in a specificregion of mesoderm (AGM region). Approximately 1 month after fertilization, epithelial cells
at the AGM region differentiate into primitive blood cells called pre-HSCs Two months after
fertilization, the pre-HSCs translocate to fetal liver and undergo proliferation and maturation
into fetal-liver HSCs. As early as 3 months after fertilization, CD201(+) fetal-liver HSCs obtaintransplantability and bone-marrow reconstitution ability. Among such multistep paths of maturation into functional HSCs, the maturity of human pluripotent stem cell (hPSC) derived hematopoietic cells remained at the primitive CD45(-) pre-HSCs (Nat Biotechnol 34 1168).
By using recently-develped monolayer-culture method, we first optimized conditions to
induce differentiation of hPSCs into pre-HSCs and successfully obtained culture containing>75% CD34(+) CD43(+) CD45(+) pre-HSCs, that have not been obtained with >5% efficiency

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for the Promotion of Sciencethrough the previous methods. Flow cytometric and transcriptome analyses revealed thatthese triple-positive cells were most similar to pre-HSCs when translocating to fetal-liver 2
months after fertilization in the human development.
To induce further maturation of the pre-HSCs to fetal-liver harboring transplantibility andbone-marrow reconstitution ability, we sought a way to find unknown fetal-liver-derived nichefactors that presumably enhance maturation of the pre-HSCs into HSCs. To this end, we performed in silico analysis of single-cell transcriptome data for fetal-liver cells containing both
hematopoietic and non-hematopoietic niche cells (Nature 574 365). Based on the transcriptome data, we predicted fetal-liver niche factors that were supposed to be secreted by fetal
hepatocytes, fibroblasts, epithelial cells, or macrophages and responded by pre-HSCs. Then
we tested >20 predicted niche factors by in-vitro screening system using the hPSC-derived
pre-HSCs. As a result, we could successfully identified several potential niche factors thatenhanced maturation of pre-HSCs into feta-liver-HSC-like cells expressing CD201. Of note,
CD201 was recently identified as a specific marker for transplantable fetal-liver HSCs (NatComms 13 1103).
Our ongoing analyses on the CD201(+) HSC-like cells may allow us to confirm their bonemarrow reconstitution ability and transcriptional- and epigenetic similarities between the hPSC-derived HSCs and bona fide fetal-liver HSCs.

Discussion & Conclusion
Overall, despite a slight change from the approach described in the proposal, we successfully established a novel method to induce fetal HSC-like cells from pluripotent stem cells.
Further experiments are ongoing to develop additional method to induce further maturationfrom the fetal-liver HSCs to neonatal- (e.g. cord blood) and adult HSCs that takes longer timein human development. Strategy of our research should be applicable to other studies aiming
to identify unknown maturation factors for other cells and tissues.

References
Nat Biotechnol 34 1168
Nature 574 365
Nature Communications 13 1103

一般の皆様へ
本研究ではヒト多能性幹細胞由来の未熟な細胞を試験管内で短期間に成人の細胞へと成熟させることを目的に、 造血幹細胞をモデルとして成熟化誘導法の開発をおこなった。 従来の方法 で ヒ ト 多 能 性 幹 細 胞 か ら 得 ら れ る の は、 CD45陰 性 の primitive な pre-HSC に 留 ま っ て い たが、 研究代表者は造血細胞の更なる成熟の場である胎児肝臓内のニッチ因子同定をおこない、同定された胎児肝臓ニッチ因子を使って造血再構築能をもった細胞に特異的な CD201陽性の
胎児肝臓造血幹細胞様の細胞を得た。 現在、 これらの細胞の機能を詳細に解析中であるほか、
胎児型幹細胞から成人幹細胞へと成熟化技術をより進化させるため、さらなる研究を進めている。

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Intrinsic barrier dysfunction in multiple sclerosis patients
Hideaki Nishihara
hidehara@yamaguchi-u.ac.jp
Department of Neurotherapeutics, Yamaguchi University, Japan

Summary Abstract
Recently, disease susceptibility genes for multiple sclerosis (MS) have been localized toendothelial cells1, making blood-brain barrier (BBB)-forming endothelial cells as an interesting candidate for novel therapeutic targets. However, research on the BBB has been limited
due to difficulties in accessing patient-derived BBB samples. We generated BBB models from
MS patients-derived induced pluripotent stem cells (iPSCs) to overcome this issue. We havedemonstrated that our in vitro BBB model can replicate BBB abnormalities observed in postmortem brains of MS patients. We identified over 400 differentially expressed genes in MS
compare to healthy individual. We further demonstrated that inhibition of candidate gene X in
BBB-forming endothelial cells derived from healthy individuals led to downregulation of claudin-5 and increased permeability of small molecules, thus replicating BBB dysfunction similar
to MS patients.
Key Words ： Multiple Sclerosis (MS), induced pluripotent stem cells (iPSCs), blood-brain
barrier (BBB)

Introduction
The detailed pathogenesis of MS is not yet fully understood, and there is currently nocure for the disease. It is widely accepted that the infiltration of immune cells into the central
nervous system and the breakdown of the blood-brain barrier (BBB) are early pathologicalhallmarks in MS lesions2. However, due to the limited access to brain samples, how BBB
changes in MS contribute disease development and progress is poorly understood. Here, we
employed induced pluripotent stem cells (iPSCs) to establish BBB model from MS-patient andexplored active contribution of BBB-forming endothelial cells for MS pathogenesis.

Results
1. Transcriptome analysis MS patients-derived BBB-forming endothelial cells containmore than 400 genes with altered expression compared to healthy controls.
We confirmed that our method (extended endothelial cell culture method-derived brain
microvascular endothelial cell-like cell: EECM-BMEC-like cell) recapitulates tight junction disruption and enhanced adhesion molecule expression observed in autopsy brain samples of
MS patients as previously published3. To identify candidate genes involved in BBB disruption
in MS patients, transcriptome analysis was conducted on EECM-BMEC-like cells. There areover 400 differential expression genes (DEGs) in EECM-BMEC-like cells derived from MS
patients compared to healthy individuals. Reactome pathway analysis based on DEGs identified several pathways. Among these pathways, two pathways have been previously reported
to be associated with BBB development and maintenance. Furthermore, we identified that a

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for the Promotion of Sciencecandidate X, which has been reported as a susceptibility gene for MS (literature omitted), issignificantly decreased in MS patients.
2. Candidate X induced tight junction disruption in healthy individual-derived EECMBMEC-like cells as observed in MS patients.
To analyze the function of candidate X in BBB-forming endothelial cells, multiple small molecules (compound A-D) known to inhibit the function of candidate X were applied to healthy
individual-derived EECM-BMEC-like. None of these compounds altered the expression of VEcadherin, which is an adherence junction protein, and cell-cell junctions were maintained (Figure 1). Interestingly, compound A and B disrupted claudin-5, which is a tight junction protein
in EECM-BMEC-like cells, while Compound C and D, known to inhibit Candidate X, did notcause claudin-5 disruption (Figure 1).

Figure 1. BBB disruption caused by candidate X in healthy individual-derived EECM-BMEClike cells
The healthy individual-derived EECM-BMEC-like cells were cultured on chamber slides andtreated with multiple small molecules (Compound A-D) known to inhibit candidate X, as well
as a control (DMSO). Immunostaining was performed for claudin-5, a tight junction protein,and VE-cadherin, an adherence junction protein, shown in red. Nuclei were stained withDAPI (blue). (Scale bar: 50 μm).

Next, we examined whether compound A, which was found to disrupt tight junctions, affected the permeability of small molecules, an important function of BBB. EECM-BMEC-likecells were cultured on cell culture inserts with 0.4 μm pores in the presence of compound A orDMSO as a control. After 6 days of culture to establish a confluent monolayer, the permeability of a small molecule, sodium fluorescein (NaFl; molecular weight 376 Da), was evaluated.
In healthy individual-derived EECM-BMEC-like cells treated with compound A, the permeability of the small molecule was significantly increased (Figure 2).
Figure 2. The effect of compound A on small
molecule permeability
Healthy individual-derived EECM-BMEC-likecells were cultured on cell culture insertswith 0.4 μm pores and treated with small
molecules (compound A) known to inhibit
candidate X, as well as a control (DMSO).
After 6 days of culture, permeability tosodium fluorescein (NaFl) was measured aspreviously described4, 5. Statistical analysis
using t-test showed a significant change inpermeability (P=<0.0001).

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Discussion & Conclusion
These results indicate that BBB disruption observed in MS patients can be reproduced in
artificially manipulated healthy individual-derived EECM-BMEC-like cells. It is speculated thatcandidate X has multiple functions and the mode of inhibition differs for each small molecule,
resulting in differential downstream signaling and varying degrees of claudin-5 disruption.
Further investigation of differences among the compounds will help to identify the detailed
molecular mechanism and downstream signaling of candidate X in BBB function. This leads
to the establishment of 1) strategies to strengthen BBB integrity and 2) potential applications
in drug delivery to the central nervous system by transiently disrupting BBB..

References
1. Lake BB, Chen S, Sos BC, et al. Integrative single-cell analysis of transcriptional and epigenetic states in the human adult brain. Nature biotechnology 2018;36:70-80.
2. Nishihara H, Engelhardt B. Brain Barriers and Multiple Sclerosis: Novel Treatment Approaches from a Brain Barriers Perspective. Handbook of experimental pharmacology
2020.
3. Nishihara H, Perriot S, Gastfriend BD, et al. Intrinsic blood-brain barrier dysfunction contributes to multiple sclerosis pathogenesis. Brain : a journal of neurology 2022.
4. Nishihara H, Gastfriend BD, Soldati S, et al. Advancing human induced pluripotent stem
cell-derived blood-brain barrier models for studying immune cell interactions. FASEB journal : official publication of the Federation of American Societies for Experimental Biology
2020;34:16693-16715.
5. Nishihara H, Gastfriend BD, Kasap P, Palecek SP, Shusta EV, Engelhardt B. Differentiationof human pluripotent stem cells to brain microvascular endothelial cell-like cells suitable tostudy immune cell interactions. STAR Protocols 2021;2.

一般の皆様へ
血液脳関門という脳を守るバリアシステムに着目して多発性硬化症の病態解明、 創薬研究を行いました。 本研究では患者さん由来の血液脳関門モデルを iPS 細胞の技術を用いて実験室に再現しました。 また、 健常人由来の血液脳関門モデルを人為的に改変することで多発性硬化症患者にみられる変化を再現することができました。 これらの発見は、 今後血液脳関門を標的とした、 血液脳関門を強固にする治療法、 もしくは脳内に薬物を届ける薬物輸送研究に発展できると期待しています。

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Identification of PP cells as an origin of pancreatic ductal
adenocarcinoma
Yoshio Fujitani, MD, PhD
fujitani@gunma-u.ac.jp
Institute for Molecular and Cellular Regulation, Gunma University

Summary Abstract
Unlike previously known pancreatic adenocarcinomas arising from pancreatic acinar or
ductal cells, we have revealed the existence of a new type of pancreatic adenocarcinoma development mechanism that arises from PP cells (Ppy-expressing cells). This study is expected to provide new insights into the diagnosis and treatment of pancreatic cancer in the future.
Key Words ： PP cells, Ppy gene, Pancreatic islet, Pancreatic cancer, PDAC,

Introduction
The applicants focused on PP cells, one of the endocrine cells of the pancreas whosefunction has not been well analyzed, and reported the details of their gene profile by single
cell analysis (1). To expand this work, we attempted to generate a cell line of PP cells byconstructing a mouse model in which PP cells express the oncogene SV40 Large T. Initially,
the mice were expected to develop PPoma, an endocrine tumor, but surprisingly, tumors thatappeared to be pancreatic ductal adenocarcinoma (PDAC) developed very early, at 3 to 4
weeks of age, and all died by 7 weeks of age.

Results
By crossing Ppy-Cre mice with Rosa26-Lox-Stop-Lox-Large T (Rosa26LargeT) mice, we generated a mouse model Ppy-Cre;Rosa26LargeT that expresses an oncogene by targeting Ppyexpressing cells. The mice were expected to develop PPoma, an endocrine tumor, in the pancreas, but contrary to expectations, pancreatic adenocarcinoma (PDAC) development wasobserved in all cases as early as 3 to 4 weeks of age.
The average survival time of Ppy-Cre;Rosa26LargeT mice was 4.7 weeks, and none of theanalyzed mice survived longer than 7 weeks of age. Body weight and blood glucose levels
were significantly lower than those of control mice, and at 4 weeks, most of the pancreas
was occupied by abnormal duct structures, tumors and stromal tissue, and the amount of differentiated islets and acinar cells was markedly reduced. The most characteristic feature isthat the abnormal duct-like structures appear to extend directly from the islets from about 2
weeks of age. It is considered that SV40 Large T antigen is expressed in the periphery of theislets where PP cells are originally located, and these Large T-expressing cells protrude andextend from the islets while proliferating, forming abnormal pancreatic duct structures. It wassuggested that Ppy-Cre;Rosa26LargeT/tdTomato mice develop pancreatic adenocarcinoma (PDAC)
within 3 weeks after birth. As one of the methods to verify that the lesions are cancerous,transplantation experiments into immunodeficient mice were conducted. Islets were isolated
at 7 days of age and transplanted subcutaneously into athymic mice, and tumors developed

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within one month. Ppy-Cre;Rosa26LargeT/tdTomato mouse islets were shown to develop typical CK19 positive PDAC with glandular duct structures. The PDAC was also considered to be derived from Ppy-expressing cells since they were Tomato-positive.
To elucidate what kind of changes in gene expression profiles were induced by the activation of Large T in Ppy-expressing cells, we sorted tomato-positive cells from PpyCre;Rosa26tdTomato mice and Ppy-Cre;Rosa26LargeT/tdTomato mice, respectively, using FACS. Then,
cDNA libraries were prepared from the RNA, and RNA-sequence was performed using anext-generation sequencer to identify the variable genes. As a result, 1362 genes were observed as variable expression genes. Among them, we observed decreased expression of
pancreatic endocrine cell marker genes such as Ppy, Gcg, Pyy, etc., increased expressionof duct cell markers such as Krt19 (CK-19), increased expression of DNMT1, which is known
to induce hypermethylation of tumor suppressor gene promoters (2), increased expression ofmycL1, a known reprogramming gene. In addition, increased expression of mycL1 and sox2,
which are known as reprogramming genes, were observed. In addition, a number of genesthat have been reported to be associated with PDAC in the literature were listed. Pathway
analysis revealed that pathways related to secretion such as peptide secretion and peptide
transport were down-regulated, while those related to cell proliferation such as DNA replication and DNA repair were enriched as up-regulated pathways. These results strongly suggestthat the differentiation of pancreatic endocrine cells into cancer cells (PDAC) is induced fromthe viewpoint of gene signature.

Discussion & Conclusion(1) It has been believed that pancreatic acinar cells and duct cells are the cells of origin ofpancreatic adenocarcinoma. One of the reasons for this is that lesions diagnosed as pancreatic cancer morphologically show an adenoductal structure. In addition, animal models
have been developed in which pancreatic carcinogenesis occurs when oncogenes such
as KRAS are activated and tumor suppressor genes are inactivated in pancreatic acinar
cells or duct cells, but no animal model has been reported in which carcinogenesis occurs
from endocrine cells (3).
(2) RNA sequencing analysis in this study revealed a number of genes with altered expression levels associated with the development of pancreatic cancer from Ppy-expressing
cells. By comparing the results of RNA sequencing analysis with those of mouse modelsof pancreatic cancer (Ptf1a-Cre;LSL-KRASG12D;Trp53f/+) and human pancreatic cancer
that have been deposited so far, we plan identify several unique marker gene groups forpancreatic cancer arising from Ppy-expressing endocrine cells created in this study. We
will also compare the results with those of RNA sequencing of human pancreatic cancers.
Using these marker genes, we plan to test whether similar pathways exist in human pancreatic cancer.
This study reveals a previously undescribed pathway for the development of pancreatic adenocarcinoma from Ppy-expressing cells, which are pancreatic endocrine cells.

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References(1) Fukaishi T et al. Characterisation of Ppy-lineage cells clarifies the functional heterogeneityof pancreatic beta cells in mice. Diabetologia. 2021 Dec;64(12):2803-2816.
(2) Wong KK et al. DNMT1 as a therapeutic target in pancreatic cancer: mechanisms andclinical implications. Cell Oncol (Dordr) 2020 Oct;43(5):779-792.
(3) Mallya K et al. Modeling pancreatic cancer in mice for experimental therapeutics.
Biochim Biophys Acta Rev Cancer. 2021 Aug;1876(1):188554

一般の皆様へ膵がんは早期発見が困難な癌種であり , 本邦における5年生存率は7% と他の癌に比べて極端に低い . 従って、 膵がんの発生進展様式を理解することは喫緊の課題である .
膵臓は腺房細胞 , 膵管細胞そして膵ランゲルハンス島 （内分泌細胞） という3つのパートから成るが , 膵がんは腺房細胞あるいは導管細胞を源として発生すると現在は考えられている . 我々は ,膵ランゲルハンス島に存在する内分泌細胞の一つである PP 細胞を起源として再現性よく膵がんを発生するユニークな動物モデルを開発した . このモデル動物を詳細に分子レベルで調べることによって , これまでには報告されていない , ユニークな膵癌の発症様式が存在することが明らかになった . 我々の発見は , あらたな膵がんの診断や治療法の開発につながる可能性がある .

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Development of an innovative diagnostic
and therapeutic method that combines radionuclide therapy
and novel boron neutron capture therapy
Kazuma Ogawa
kogawa@p.kanazawa-u.ac.jp
Institute for Frontier Science Initiative, Kanazawa University

Summary Abstract
This study aimed to synthesize and evaluate a probe closododecaborate-(Ga-DOTA)c(RGDfK) (1) containing closo-dodecaborate ([B12H12]2−) as a boron cluster, a [67Ga]Ga-DOTA
derivative for nuclear medicine imaging, and an RGD peptide for tumor targeting. Moreover,
we prepared a radioiodinated probe [125I]2 in which I-125 is introduced into a closo-dodecaborate moiety of 1. [67Ga]1 and [125I]2 showed high stability and high uptake in cancer cells in vitro. Biodistribution experiments in tumor-bearing mice revealed similar biodistribution patterns
between [67Ga]1 and [125I]2. Meanwhile, [125I]2 exhibited higher accumulation in most tissues,
including the tumor, than [67Ga]1, probably because of higher albumin binding.
Key Words ： BNCT, αvβ3 integrin, RGD peptide

Introduction
A boron neutron capture therapy (BNCT) system, using boron-10-introduced agents coupled with companion diagnostics, is anticipated as a promising cancer theranostic. To develop
a novel BNCT system using drugs with the same chemical structure between a compoundcontaining 10B for BNCT and a PET probe for companion diagnostics, I designed, synthesized, and evaluated a probe closo-dodecaborate-(Ga-DOTA)-c(RGDfK) 1 containing closododecaborate ([B12H12]2–) as a boron cluster to introduce multiple 10B molecules into a probe,a [68Ga]Ga-DOTA derivative as a stable 68Ga complex for PET imaging, and an arginine–glycine–aspartic acid (RGD) peptide for tumor-targeting.

Results67

Ga was used as an alternative radionuclide instead of 68Ga because of its relatively long
half-life 1. [67Ga]1 was prepared via conjugation of a [67Ga]Ga-DOTA-c(RGDfK) derivative witha dodecaborate derivative by alkyne-azide cycloaddition. Radioiodinated compound [125I]2
was prepared from 1 using chloramine T as an oxidant. [67Ga]1 and [125I]2 were synthesizedwith a radiochemical purity of over 95%.
The affinity of 1 for αvβ3 integrin was determined via a competitive binding assay using
U-87 MG human glioblastoma cells with high αvβ3 integrin expression. Figure The half maximal inhibitory concentration (IC50) value (nM) for 1 was 10.9 ± 1.4, which was not significantly
different from that of c(RGDfK) (10.9 ± 4.2 nM) in our previous study 2. Therefore, introducing
a relatively large molecule, a Ga-DOTA complex conjugated dodecaborate derivative, intoc(RGDfK) via the ε-amino group of the lysine residue did not significantly impede the affinity of c(RGDfK) for αvβ3 integrin. Cellular uptake experiments with a human glioma cell line,

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U-87 MG, and biodistribution experiments of [67Ga]1 and [125I]2 were performed. The uptakesof [67Ga]1 and [125I]2 in U-87 MG cells were similar and increased in a time-dependent manner. These results indicate that the introduction of iodine into a dodecaborate structure hardly
alters their affinity for αvβ3 integrin. The biodistribution experiments of [67Ga]1 and [125I]2 intumor-bearing mice were similar, such as high tumor uptake and relatively low uptake in other
non-target tissues, except the kidney, liver, and intestine. Therefore, it indicates that [67Ga]1
and [125I]2 could be distributed to these tissues in intact forms. The result of urine analysis reconfirmed their high metabolic stabilities of [67Ga]1 and [125I]2 since most radioactivity, 89.3%
for [67Ga]1 and 84.5% for [125I]2, was observed as intact forms in urine at 1 h postinjection.
Meanwhile, unexpectedly, the radioactivity of [125I]2 in most tissues, including the tumor, washigher than that of [67Ga]1. We supposed that the higher [125I]2 radioactivity should be derived
from delayed blood clearance due to its higher protein binding in plasma. Thus, to confirm
the difference in blood clearance and protein binding between [67Ga]1 and [125I]2 in detail, we
performed the biodistribution experiments of them in many time points of normal mice andthe in vitro protein binding experiment. As the results the halflives of the distribution phase (αphase) of [67Ga]1 and [125I]2 from the blood were calculated as 7.7 and 5.7 min, respectively.
And then, we revealed the percentages of the protein bindings of [67Ga]1 and [125I]2 in murine
plasma as 56.2 ± 4.3 and 80.2 ± 1.9%, respectively (p < 0.001), and the binding percentages
in the purified human serum albumin (HSA) as 82.1 ± 0.8 and 94.9 ± 0.3%, respectively (p <0.001). The results indicate that introducing an iodine atom into closo-dodecaborate significantly increased the protein binding ratio in the plasma, supporting the difference in the blood
clearance and biodistribution between [67Ga]1 and [125I]2.

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Discussion & Conclusion
We synthesized a 67Ga-labeled compound ([67Ga]1) and a corresponding nonradioactive
compound containing RGD peptide, Ga-DOTA, and closo-dodecaborate for BNCT and its
companion diagnostics. Moreover, we synthesized an I-125-introduced compound ([125I]2) into
a closo-dodecaborate of 1. Biodistribution experiments in tumor-bearing mice revealed that
[67Ga]1 and [125I]2 showed high tumor uptake and similar biodistribution patterns. Meanwhile,[125I]2 showed higher accumulation in most tissues, including the tumor, derived from higher
albumin binding than [67Ga]1. The higher accumulation in the tumor is desirable for BNCT,
and the iodo-closo-dodecaborate site may work as an albumin binder. These results provide
useful information for developing novel drugs for BNCT and its companion diagnostic imaging. However, determining the boron element in the biodistribution experiments and the therapeutic study by neutron irradiation is necessary for the future because this report is a preliminary study.

References
Satpati, D. Recent Breakthrough in 68Ga-Radiopharmaceuticals Cold Kits for Convenient
PET Radiopharmacy. Bioconjugate Chem. 2021, 32, 430−447.
2
Ogawa, K.; Echigo, H.; Mishiro, K.; Hirata, S.; Washiyama, K.; Kitamura, Y.; Takahashi, K.;
Shiba, K.; Kinuya, S. 68Ga- and 211AtLabeled RGD Peptides for Radiotheranostics with Multiradionuclides. Mol. Pharm. 2021, 18, 3553−3562.
1

一般の皆様へBNCT は、2020年に世界に先駆けて日本で承認された最先端のがん治療法です。ホウ素（ 10 B）
をがんに集積させて中性子線を照射し、 ホウ素の分裂により放出される放射線によりがんを殺傷する治療法であるため、 ホウ素をがんに運ぶ薬剤が非常に重要です。 本研究コンセプトのような同一構造化合物による PET 診断情報を利用した精密な治療計画を行うことにより、 それぞれの患者により最適な BNCT 治療の供給へつながると考えられます。 現在、 構造を最適化した更なる薬剤開発など、 実用化を目指した研究を進めています。

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Investigation on adjuvant effect of peptide-stabilized emulsions
Rie Wakabayashi

wakabayashi.rie.122@m.kyushu-u.ac.jp
Kyushu University

Summary Abstract
This study aimed to develop adjuvants that exhibit immunostimulant effects against protein
antigens. We developed oil-in-water emulsions with interface stabilized with self-assembling
peptides that can directly carry protein antigens by enzymatic reaction. The enhanced antigen
delivery to immune cells was confirmed depending on the design of the self-assembling peptides.
Key Words ： vaccine, adjuvant, oil-in-water emulsion, self-assembly peptide

Introduction
Vaccines are effective tools to prevent various infectious diseases or to prevent the severityof symptoms. The use of adjuvants, which are immunostimulants, is important to induce effective immune responses by vaccines. Therefore, the development of effective adjuvants withidentified mechanisms of action is one of the most important issues in vaccine development.
In this study, we focused on an oil-in-water (O/W) emulsion as the adjuvant formulation andaimed to develop self-assembled peptide-based O/W emulsions that show adjuvant effects
for vaccines carrying protein antigens.

Results
We have prepared self-assembled peptide-based O/W emulsions by using peptide amphiphiles (PAs) (Fig. 1A). PAs spontaneously self-assemble in aqueous solutions by multiple intermolecular interactions, such as hydrophobic interaction and hydrogen bond formation. We
have previously designed PAs with microbial transglutaminase (MTG) enzyme recognition sequences and demonstrated that proteins can be integrated on their self-assembled structures
via the MTG reaction.1,2 In this study, we synthesized several PAs with aromatic Pyrenyl andFmoc groups in the hydrophobic part and LLQG sequence as an MTG recognition sequence.
Additional amino acid was introduced between the aromatic groups and the LLQG sequence
to investigate the influence of PA design on self-assembly, MTG reactivity, emulsion formation, and interaction with immune system. The obtained PAs were subjected to self-assemble
in several aqueous solutions (water, citrate and phosphate buffers). Spectroscopic analyzes(fluorescence and Fourier transform infrared spectra) revealed that these PAs self-assembledby π-π stacking interactions between aromatic groups and hydrogen bond formation between
peptides. The stacking interactions were influenced by the PA design or solution conditions,while hydrogen bond formation remained similar among PAs used in this study. The formationof fibrous structures was confirmed for all PAs by transmission electron microscopy and confocal laser scanning microscopy (CLSM) images.
A green fluorescent protein containing an MTG-reactive Lys-containing peptide sequence98

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for the Promotion of Science(Ktag-EGFP) was used as a model antigen protein. Ktag-EGFP was reacted with pre-formed
PA fibrous assemblies by the addition of MTG at 37 °C for 2 h. More than 80% of the KtagEGFP proteins reacted with PAs, which was confirmed by high performance liquid chromatography. The accumulation of Ktag-EGFP on the PA fibers was directly observed by CLSM.
The antigen protein-loaded fibers were used to make O/W emulsions with squalene as the oilphase. Emulsion particles with diameters ranging from several hundred nanometers to several micrometers were produced, depending on the type of PA used. CLSM showed that mostof the antigenic proteins and PA fibers located at the interface between the oil and aqueous
phases, suggesting that these fibrous structures could stabilize the O/W emulsions (Fig. 1B).
Murine dendritic cells, DC2.4 cells, were used as immune cells for in vitro antigenic protein
delivery study. The cells were treated with the O/W emulsions prepared above. As controls,
fibers loaded with Ktag-EGFP protein, and Ktag-EGFP protein alone were also applied. The
delivery rate of Ktag-EGFP into the DC2.4 cells was quantified by flow cytometry. The amountof proteins delivered to the cells was significantly higher when emulsions or fibers with direct
conjugation of Ktag-EGFP were applied than when Ktag-EGFP alone was applied. The protein delivery efficiency showed the dependance on the design of the PAs, and tended to behigher when PAs containing basic or hydrophobic amino acids in the peptide sequence wereused. The observation by CLSM indicated that the PA assemblies with higher protein delivery
nature have tendency to accumulate on/near the cell membranes. The higher protein delivery
efficiency by O/W emulsions or PA fibers was not observed when proteins were not directly
bound to PAs by MTG reaction but were physically mixed with emulsions or fibers. Moreover,the difference in the cellular internalization pathway was suggested for emulsions and fibrousstates.

Discussion & Conclusion
The loading of antigenic proteins directly onto the fiber-like structures formed by PAs using
enzymatic reactions was shown to improve the protein delivery ratio to immune cells in a PA
design-dependent manner. The accumulation of a large number of proteins on PA structures
to form larger objects and the moderate hydrophobicity of the PA aggregates may have enhanced their affinity to cells. Using this PA fiber, a new form of O/W emulsion-type vaccinewith antigenic proteins integrated at the O/W emulsion interface was successfully created. Although the immunizing effect could not be verified using experimental animals, the newly developed formulation in this study is expected to show high vaccine efficacy, since the delivery
ability to immune cells was improved in vitro.

Fig. 1 (A) Schematic representation and (B) confocal microscopic image of O/W emulsion adjuvant
developed in this study. Bar: 10 µm.

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References
1. Rie Wakabayashi, Ayumi Suehiro, Masahiro Goto, Noriho Kamiya, Designer aromatic peptide amphiphiles for self-assembly and enzymatic display of proteins with morphology control,
Chem. Commun., 2019, 55, 640–643.
2. Rie Wakabayashi, Ayato Higuchi, Hiroki Obayashi, Masahiro Goto, Noriho Kamiya, pHresponsive self-assembly of designer aromatic peptide amphiphiles and enzymatic postmodification of assembled structures, Int. J. Mol. Sci., 2021, 22, 3459.

一般の皆様へワクチンは、 感染症の感染予防あるいは感染による症状の重篤化を抑えるために有効です。
ワクチン効果を高めるために、 アジュバントと呼ばれる添加剤が使われます。 本研究は、 水の中に油滴が分散したエマルションという形状が示すアジュバント効果に着目しました。 特に、 水中で繊維状の構造体を形成する自己組織化ペプチドを用いて、 水と油の界面に抗原となるタンパ
ク質を集めることが可能な、 新しいワクチンアジュバントを開発し、 免疫細胞へ効率的に抗原タ
ンパク質を運ぶことに成功しました。

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Development of novel therapy for sarcopenia based on theregulation of O-linked N-acetylglcosamine motif
Shintaro Kinugawa
kinugawa.shintaro.786@m.kyushu-u.ac.jp
Kyushu University

Summary Abstract
In this study, we clarified whether O-GlcNAcylation of skeletal muscle proteins is involved in
the progression of age-related skeletal muscle atrophy. Skeletal muscle in mice atrophied withage. This age-related skeletal muscle atrophy is achieved by an imbalance between protein
synthesis and degradation. At the same time, O-GlcNAcylation of skeletal muscle proteins
increased and GFAT1 protein expression increased. It was suggested that O-GlcNAcylation
might be involved in the progression of skeletal muscle atrophy.
Key Words ： Sarcopenia, skeletal muscle atrophy, O-GlcNAcylation

Introduction
The development of preventive and therapeutic methods for age-related sarcopenia is anextremely important research topic. Skeletal muscle atrophy is caused by an imbalance between protein synthesis and degradation [1]. O-GlcNAcylation is one of the post-translational
modifications of proteins, which alters the functions of proteins in response to cellular stresses[2]. However, the role of O-GlcNAcylation in skeletal muscle atrophy has not been elucidated
so far. The purpose of this study was to clarify whether O-GlcNAcylation of various proteins
involved in protein synthesis and degradation plays an important role in the progression ofage-related skeletal muscle atrophy.

Results
2-month-old, 5-month-old, and 11-month-old C57BL/6J mice were purchased (Japan SLC
Co., Ltd.). Each mouse was housed under a 12-hour light/dark cycle for one month in anenvironment with free access to standard diet and water. All animal experiments, breeding
and storage of mice used in experiments, and experimental plans were approved by the Kyushu University Animal Care and Use Committee, and were carried out in accordance with
the Kyushu University Animal Experiment Practice Manual. At 3 months, 6 months, and 12
months of age, lower extremity skeletal muscle and epididymal fat were excised from eachmouse under deep anesthesia (intraperitoneal administration of 200 mg/kg of pentobarbital).
After weighing, skeletal muscle tissue was used for histological evaluation (hematoxylin-eosin
staining), and Western blotting. Protein extraction, electrophoresis, and immunoblotting fromskeletal muscle tissue samples were performed by standard methods. As primary antibodies,Akt, phospho-Akt, mTOR, phospho-mTOR, atrogin-1, O-GlcNAc, MuRF1, glutamine-fructose6-phosphate amidotransferase (GFAT) 1, GFAT2, O-GlcNAc transferase (OGT), An antibody
against O-GlcNAcase (OGA) was used.

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The body weight of mice was increased significantly with age (3 months old: 28±1 g, 6months old: 28±2 g, 12 months old: 32±3 g, p<0.05). Consistent with the increase in bodyweight, the fat weight-to-body weight ratio of 12-month-old mice was increased by 93% compared to 3-month-old mice. On the other hand, the lower extremity skeletal muscle weightbody weight ratio was decreased with aging (3 months old: 14.3±0.3 mg/g, 6 months old:13.4±0.6 mg/g, 12 months old: 12.3±0.5 mg/g, p <0.05). Histological evaluation of skeletal
muscle showed a 10% reduction in cell cross-sectional area in skeletal muscle of 12-monthold mice compared with 3-month-old mice. Skeletal muscle atrophy was found to occur withaging. Therefore, we evaluated molecules related to protein synthesis and degradation systems. Akt and mTOR protein expression did not differ at each age. Akt phosphorylation levels were reduced by 22% in skeletal muscle of 12-month-old mice compared to 3-month-old
mice, and mTOR phosphorylation levels were reduced by 19%. Furthermore, atrogin-1 andMuRF1 protein expression were increased by 43% and 71% in skeletal muscle of 12-monthold mice compared with 3-month-old mice. Therefore, the balance of the protein synthesis/
degradation system was broken. O-GlcNAcylation modification of skeletal muscle proteins
was significantly increased in skeletal muscle of 12-month-old mice compared with 3-monthold mice (p<0.05). Among molecular proteins regulating O-GlcNAcylation, GFAT1 was significantly increased. On the other hand, GFAT2, OGT and OGA did not change significantly.

Discussion & Conclusion
In this study, skeletal muscle atrophy in aged (12 months old) mice was observed. This wasachieved by decreasing the protein synthesis and increasing the protein degradation. At thistime, it became clear that the proteins subjected to O-GlcNAc modification were increased. Increased O-GLcNAcylation was thought to be regulated by an increase in GFAT1. O-GlcNAcylation modification of skeletal muscle extract protein increased with age-related skeletal
muscle atrophy. In this study, we could not clarify the causal relationship between increased
O-GlcNAcylation and skeletal muscle atrophy. Treatment of cultured C2C12 skeletal muscle
cells with dexamethasone has been shown to cause cell atrophy [3]. In this experiment, OGlcNAcylation was increased, decreased Akt phosphorylation and increased Atrogin-1,
MuRF1 expression [3]. This cultured cell result supports our current findings. In addition, OGlcNAcylation in the myocardium was increased in a pressure-overload hypertrophic heartmodel, which was closely related to the activation of Akt phosphorylation, a key molecule for
cardiac hypertrophy signaling [4]. Since the sites of O-GlcNAcylation and phosphorylation occur at or near the same site, it is thought to play an important role in regulating protein phosphorylation [5]. Therefore, it is suggested that age-related skeletal muscle atrophy, decreased
skeletal muscle Akt phosphorylation, and O-GlcNAcylation modification are closely related. It
is necessary to examine the causal relationship of this point in detail in the future.

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References[1] Kinugawa S, Takada S, Matsushima S, Okita K, Tsutsui H. Skeletal muscle abnormalities inheart failure. Int Heart J. 2015;56(5):475-84. PMID: 26346520 DOI: 10.1536/ihj.15-108[2] Hart GW, Housley MP, Slawson C. Cycling of O-linked beta-N-acetylglucosamine on nucleocytoplasmic proteins. Nature.2007;446(7139):1017-22. PMID: 17460662 DOI: 10.1038/nature05815[3] Massaccesi L, Goi G, Tringali C, Barassi A, Venerando B, Papini N. Dexamethasone-induced skeletal muscle atrophy increases O-GlcNAcylation in C2C12 cells. J Cell Biochem.
2016;117(8):1833-42. PMID: 26728070 DOI: 10.1002/jcb.25483[4] Ishikita A, Matsushima S, Ikeda S, Okabe K, Nishimura R, Tadokoro T, Enzan N, Yamamoto T, Sada M, Tsutsui Y, Miyake R, Ikeda M, Ide T, Kinugawa S, Tsutsui H. iScience.
2021;24(12):103517. PMID: 34934932 DOI: 10.1016/j.isci.2021.103517[5] Butkinaree C, Park K, Hart GW. O-linked beta-N-acetylglucosamine (O-GlcNAc): Extensive
crosstalk with phosphorylation to regulate signaling and transcription in response to nutrientsand stress. Biochim Biophys Acta. 2010;1800(2):96-106. PMID: 19647786 DOI:10.1016/
j.bbagen.2009.07.018

一般の皆様へ
本研究において、 加齢に伴う骨格筋萎縮の進展に骨格筋タンパクの O-GlcNAc 化が関係していることを明らかにした。 骨格筋萎縮の新たな分子機序を明らかにした点が極めて意義が高く、今後 O-GlcNAc 化を制御する薬剤の開発により、 現代の社会的な問題である老化によるサルコ
ペニアの克服に貢献できると考える。

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Molecular mechanism of neuroprotection after ischemic stroke
Hiroo Takahashi
takahashi.hiroo@kagawa-u.ac.jp
Department of Molecular Neurobiology, Faculty of Medicine, Kagawa University

Summary Abstract
Ischemic stroke is the frequent cause of death and disability in adults worldwide. We investigated endogenous neuroprotective mechanisms for ischemic stroke, by using animal models. Our findings reveal that the condition of the brain just before stroke has a great impact on
ischemic cell death. Transcription factor Npas4-dependent and Npas4-independent neuroprotective mechanisms function to promote cell survival in ischemic neurons.
Key Words ： Ischemic stroke, neuroprotection, Npas4

Introduction
Ischemic stroke is the second leading cause of death and the most frequent cause of disability in adults worldwide. Stroke initiates complex pathological events in the brain, resulting
in cell damage and loss of neurological function. It is important to know the factors that influence neuronal survival or death after stroke. In this study, we focused on endogenous neuroprotective mechanisms for ischemic stroke.

ResultsT
When the brain is pre-exposed to a brief period of ischemia, it can withstand subsequent
lethal ischemia. This phenomenon is called ischemic tolerance. Intriguingly, non-noxious
stimuli could also cause ischemic tolerance. For example, exercise for several weeks helps
develop ischemic tolerance. However, it was unknown whether short-term exposure to nonnoxious stimuli can induce ischemic tolerance. Recently, we found that mice pre-exposed toan enriched cage (provided with tubes, hiding place, glove etc) for a short period (40 min) acquired resistance to ischemic cell death (1). Activation of cortical neurons using AAV vectors
carrying the DREADD (Designer Receptors Exclusively Activated by Designer Drugs) systemshowed that transient neuronal activity is sufficient to activate endogenous neuroprotective
mechanisms. We named this phenomenon as “activity-dependent ischemic tolerance” (2).
The activity-dependent transcription factor Npas4 (neuron PAS domain protein) is required forthis activity-dependent ischemia tolerance (1, 2). The condition of the brain just before stroke
has a great impact on ischemic cell death.
Previous study has reported that stroke onset time influences the effect of drugs on ischemic stroke (3). It suggests that brain conditions altered by circadian rhythms may affectischemic resistance of the brain. Therefore, we investigated whether stroke onset time has asignificant effect on cell death by stroke. We performed permanent MCAO (middle cerebral
artery occlusion) surgery for wild-type mice (male, 6 weeks) during the day (zeitgeber time (ZT)
6) and nighttime (ZT6). Twenty-four hours after the surgery, live cells were stained with TTC(2,3,5-triphenyl tetrazolium chloride) and the volume of the dead cell area was measured.

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Interestingly, infract volume in the cerebral cortex was remarkably reduced, when a stroke
occurred at ZT15 compared to when a stroke occurred at ZT 6. It suggests that endogenous
neuroprotective mechanisms function prominently at night. Since mice are nocturnal animals,
their activity increases significantly at night. We speculated that the expression of the activitydependent neuroprotective gene Npas4 may remarkably increase in mice brain at night andfacilitate cell survival after stroke. To reveal this point, we analyzed Npas4 expression patternin the cerebral cortex due to circadian rhythm. As expected, Npas4 was strongly expressedin the cerebral cortex during the night compared to the day. Next, we studied the contributionof increased Npas4 expression to neuroprotective mechanisms activated at night, by using
Npas4-KO mice (male, 6 weeks).
Unexpectedly, Npas4-KO mice also showed the reduced cell death after stroke, when astroke occurred at ZT 15 compared with at ZT 6. This result suggests that this time-dependent
reduction in cell death is Npas4-independent. To reveal the molecular mechanisms underlying this Npas4-independent neuroprotection, we searched for genes that were remarkably
increased (or decreased) in the cerebral cortex at night, and affected the cell survival after
stroke. We found that some clock genes are more strongly expressed in cortical neurons atnight than during the day. To reveal the neuroprotective function of these genes, we prepareddissociated cultured neurons from cerebral cortex at embryonic day 15. After the electroporation of candidate genes, cultured neurons were treated oxygen-glucose deprivation conditions(1% oxygen, no glucose). By using in vitro ischemic culture conditions, we are currently analyzing the neuroprotective functions of these candidate genes.

Discussion & Conclusion
Our findings suggest that several neuroprotective mechanisms function in ischemic neurons. One is activated by neuronal activity. This mechanism is induced by Npas4 expression.
We found that Npas4 induces the expression of the small Ras-like GTPase Gem, and Gem
prevents the abnormal activation of L-type voltage gated calcium channels and promotes cell
survival in ischemic neurons (1, 2). On the other hand, Npas4-independent neuroprotective
mechanism is strongly activated in mice brain during nighttime. We found several candidate
molecules that are strongly induced in the cerebral cortex at night. We are currently analyzing
the neuroprotective functions of these candidate genes.

References(1) Ras-like Gem GTPase induced by Npas4 promotes activity-dependent neuronal tolerance
for ischemic stroke. Takahashi H, Asahina R, Fujioka M, Matsui T, Kato S, Mori E, Hioki H,
Yamamoto T, Kobayashi K, Tsuboi A.
Proc. Natl. Acad. Sci. USA. (2021) 118,e2018850118.
(2) Molecular mechanisms underlying activity-dependent ischemic tolerance in the brain.
Takahashi H, Yamamoto T, Tsuboi A.Neuroscience Research (2023) 186, 3-9.
(3) Potential circadian effects on translational failure for neuroprotection. Esposito E, Li W, T
Mandeville E, Park JH, Şencan I, Guo S, Shi J, Lan J, Lee J, Hayakawa K, Sakadžić S, Ji X,
Lo EH.
Nature (2020) 582, 395-398.

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一般の皆様へ
脳梗塞により、 脳では大規模な神経細胞死が起こります。 一度失われた神経細胞は再生しな
いため、 神経細胞を守ることが予後の改善に極めて重要です。 私達は、 脳梗塞の直後に、 脳が自身を守るためのメカニズムを活性化することを見出しました。 これらのメカニズムを人為的に強く活性化することができれば、 新たな脳梗塞の治療法に繋がると考え、 その基盤となる基礎研究を進めています。

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Involvement of fusion proteins on morphogenesis ofneuromuscular junction
Bisei Ohkawara
biseiohkawara@med.nagoya-u.ac.jp
Nagoya University Graduate School of Medicine Neurogenetics

Summary Abstract
Skeletal muscle fiber is a large syncytium with multiple and evenly distributed nuclei.
We performed single-nucleus RNA sequencing (snRNA-seq) analysis of embryonic mouse
diaphragms, and found that some mRNA for cell fusion were specifically detected in neuromuscular junction (NMJ) nuclei in the transcriptome analysis. With further experiments, werevealed that proteins, which are encoded by the mRNA for cell fusion, were not specifically
localized in NMJ of the skeletal muscles and acetylcholine receptors-accumulated regions ofin vitro myotubes. We here revealed that some scaffold proteins for NMJ formation was involved in NMJ formation.
Key Words ： Neuromuscular junction, snRNA-seq

Introduction
The neuromuscular junctions exploit a coordinated interaction between a presynaptic region comprised of the spinal motor neurons (SMNs) and a postsynaptic region comprised ofmultinucleated myofibers. The formation of postsynaptic compartmentalization in myofibers
involves multiple mechanisms to create a sharp delineation between the synaptic and extrasynaptic domains of the myofibers. This has been most extensively studied by analyzing theexpression patterns of acetylcholine receptor (AChR) subunits and signaling molecules tomake AChR clusters. We already know that these proteins are specifically expressed in thesubsynaptic myonuclei.

Results
We performed single-nucleus RNA sequencing (snRNA-seq) analysis of diaphragms inmouse embryos at embryonic day (E)12.5, 15.5, 18.5, and postnatal day (P)1.5. Furthermore, we investigated the transcriptional profiles of myonuclei in the embryonic myofibers by
Seurat software. To our knowledge, this is the first report of snRNA-seq of embryonic skeletal
muscles at different developmental stages. However, our analysis had a following limitation;
snRNA-seq analysis at four developmental stages of E12.5, E15.5, E18.5 and P1.5 was likely
to be insufficient to clearly delineate the lineage of nuclei of our interest.
Anyway, we found that each myonucleus had a distinct transcriptome pattern during the
NMJ formation in embryogenesis (unpublished data). By the analyses, we found some mRNAfor cell fusion including Cav3, which were specifically detected in neuromuscular junction(NMJ) myonuclei. Using transfection of fusion proteins, Cav3-GFP and clathrin heavy chainGFP in myofibers, we revealed that Cav3 proteins, which are encoded by the Cav3, were
not specifically localized in the subsynaptic NMJ region of the skeletal muscles, stained with

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AChR clustering (Figure 1). Surprisingly, we also found that protein for clathrin heavy chain were
specifically colocalized in theregions with AChR clustering. It
suggested that membrane characters for subsynaptic region for
NMJ were different to the otherregions.
We thus focused on the genes,
which encode proteins for clathrin-dependent structures withcertain scaffold proteins and are expressed together with the genes for AChR subunits andsignaling molecules, Dok7. We found that some genes for the scaffold protein complex beneath the plasma membrane were also expressed specifically, significantly, and correlatedly
in the subsynaptic myonuclei together with genes for NMJ formation by the transcriptomic
analysis. Using quantitative real-time PCR (qRT-PCR), we also confirmed that the genes for
scaffold proteins were significantly expressed (1) in the center of E18.5 mouse diaphragms,
and (2) in the in vitro C2C12 myotubes when the cells were differentiated and fused for 2days. Using small hairpin RNA (shRNA) against some genes for the scaffold proteins, we revealed that the scaffold proteins were only involved in size and number of AChR clustering in
C2C12 myotube, but did not affect gene expressions for the signaling pathway X in the myotubes.
By analyzing snRNA-seq, we counted number of myonuclei for total, NMJ, and MTJ as wellas number of nuclei for the other cells such as; tendon cells, Schwann cells, perichondrium,
proximal mesenchymal cells, endothelium cells, and chondrocytes in diaphragms during embryogenesis. These data newly revealed the characters of myogenic cells and other cells inembryonic diaphragms. In addition to subsynaptic myonuclei for NMJ, we also found expression profiles of myonuclei, which were likely localized closely to the myotendinous junction(MTJ) in the embryonic myofibers. We revealed that MTJ myonuclei expressed genes not
only for myofibers but also for extracellular matrix. Interestingly, we found that both NMJ and
MTJ myonuclei had gene profiles which showed activated signaling pathway of “X”. Previous
reports suggested SMNs and cells in cartilage tissues expresses ligands which activate thesignaling pathway X during embryogenesis to develop skeletal myofibers. In total, our further
analysis resulted in novel coordinated gene expressions in embryonic NMJ and MTJ myonuclei.

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Discussion & Conclusion
Taken together with all results, we have identified gene expression profiles of embryonicsubsynaptic nuclei at the NMJ and MTJ in myofibers of the skeletal muscles. Genes which
are associated with signaling pathway X and other NMJ-specific genes are attractive candidates to dissect the formation of the embryonic NMJ at the molecular level. Similarly, MTJ
myonuclei has activated signaling pathway X and is likely to be stimulated by the ligands
released from perichondrium and cartilage. Dissection of genes for the compartments of skeletal myofibers will be able to unveil finely tuned communications among myofibers and othercells in the skeletal muscle tissues during embryogenesis.

References
Currently, a manuscript with the data of snRNA-seq is submitted to J. Neurochemistry.

一般の皆様へ
本研究の目的は、 身体の中で比較的大きな細胞で、 多核である筋肉細胞が胎生期にどのように発達し、 領域を決定しているのかを、 最新の技術であるシングル核 RNA-seq という手法で明らかにしたものです。 本研究の結果は各発生段階の筋細胞の特徴を明らかにしただけでなく、今後筋肉の病態研究を行う際の基盤となる知識を含んでいます。

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Inhibition of cancer metastasis by selective regulation of
AM-RAMP2 and AM-RAMP3 systems
Megumu Tanaka
organregen@shinshu-u.ac.jp
Department of Cardiovascular Research, Shinshu University School of Medicinel

Summary Abstract
Adrenomedullin (AM) is a multifunctional peptide regulated by receptor activity-modifying
proteins (RAMPs). We investigated the actions of the AM-RAMP2 and 3 systems in the tumor microenvironment and their impact on metastasis. PAN02 pancreatic cancer cells were
injected into the spleens of mice, leading to spontaneous liver metastasis. Tumor metastasis
was enhanced in vascular endothelial cell-specific RAMP2 knockout mice (DI-E-RAMP2-/-).
By contrast, metastasis was suppressed in RAMP3-/- mice, where the number of podoplanin(PDPN)-positive cancer-associated fibroblasts (CAFs) was reduced. Because PDPN-positive
CAFs are a hallmark of tumor malignancy, we assessed the regulation of PDPN and foundthat Src/Cas/PDPN signaling is mediated by RAMP3. Activation of RAMP2 in RAMP3-/- mice
suppressed both tumor growth and metastasis.
We suggest that upregulation of PDPN in DI-E-RAMP2-/- mice increases malignancy, while
downregulation of PDPN in RAMP3-/- mice reduces it.
Key Words ： Metastasis, Adrenomedullin, RAMP

Introduction
Adrenomedullin (AM) is a peptide that is widely produced throughout the body and has
a wide variety of biological activities. AM activity is mainly regulated by the receptor activity modifying proteins RAMP2 and RAMP3. We have previously shown that AM-/- mice and
RAMP2-/- mice are embryonic lethal due to abnormal vascular development. We have established mice that can induce the loss of RAMP2 in vascular endothelial cells (DI-E-RAMP2-/-)in adults and performed subcutaneous transplantation of melanoma cells. We found thatmetastasis to the lungs was enhanced and reported that vascular homeostasis by the AMRAMP2 system suppressed cancer metastasis.
On the other hand, an important problem is that pancreatic cancer has a poor prognosis
and is prone to postoperative liver metastasis. It has also been reported that RAMP3 expression is elevated in human pancreatic cancer samples.
In this study, we investigated the significance of the AM-RAMP2 and 3 system in organ-toorgan metastasis using DI-E-RAMP2-/- and RAMP3-/- mice.

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Results
PAN02 pancreatic cancer cells were transplanted into the spleen and examined for metastasis to the liver. In DI-E-RAMP2-/-, primary tumor size was reduced, whereas metastasis to the liver was enhanced, confirming the reproducibility of the previous experimental
results. Observing liver metastases in DI-E-RAMP2-/-, we found increased fibrosis aroundthe cancer and an increase in αSMA-positive cancer-associated fibroblasts (CAFs), a marker of myofibroblasts, and these CAFs were podoplanin (PDPN)-positive. Clinically, cancers
with PDPN-positive CAFs have been reported to have a poor prognosis. In contrast, DI-ERAMP2-/- had a compensatory increase in RAMP3 expression with a decrease in RAMP2
expression. Therefore, we next investigated a similar organ-to-organ transition model in
RAMP3-/- mice.
Cancer metastasis experiments using RAMP3-/- showed that, contrary to DI-E-RAMP2-/-,
metastasis to the liver was suppressed. Furthermore, contrary to DI-E-RAMP2-/-, PDPNpositive CAFs around the tumor were reduced. Therefore, we proceeded to investigate therelationship between RAMP3 and PDPN in fibroblasts. Knockdown of RAMP3 in fetal-derived fibroblasts (MEFs) also reduced PDPN expression. Furthermore, in RAMP3-/- MEFs
stimulated with PAN02 cell supernatants, the activities of Src and Cas, which are involved
in regulating PDPN expression, were also reduced, indicating the existence of a Src-CasPDPN system downstream of RAMP3.
Next, we proceeded to examine primary cultures of CAFs from around metastases, andfound reduced expression of αSMA in RAMP3-/- derived CAFs. In RAMP3-/- CAFs, intracytoplasmic stress fiber formation was suppressed, while actin formation just below theplasma membrane was enhanced, suggesting that mesenchymal epithelial transition (MET)
occurred. Furthermore, RAMP3-/- CAFs suppressed cell migration and proliferation in a coculture system with PAN02 cells. In array analysis, RAMP3-/- CAFs showed decreased expression of cancer-promoting factors and increased expression of inhibitory factors. Therefore, when cell supernatants of RAMP3-/- CAFs were added to PAN02 cells, the expressionof the mesenchymal marker Vimentin was reduced and the proliferation and migration of
PAN02 cells were also inhibited. In fact, when RAMP3-/- CAFs were mixed with PAN02
cells and transplanted into mice, tumour size was suppressed and liver metastasis was reduced, suggesting that RAMP3-/- CAFs are so to speak good CAFs that suppress cancer
growth and metastasis.
Finally, when AM was continuously administered to RAMP3-/- to selectively activate the
AM-RAMP2 system while the AM-RAMP3 system was blocked, cancer metastasis was further suppressed than in RAMP3-/-.

Discussion & Conclusion
These results suggest that in DI-E-RAMP2-/-, compensatory enhancement of the AMRAMP3 system resulted in increased PDPN expression and enhanced cancer malignancy. In
contrast, in RAMP3-/-, the reduction of malignant PDPN-positive CAFs was considered to bea consequence of reduced cancer metastasis. Selective RAMP2 activation and RAMP3 inhibition are expected to be a new therapeutic approach to suppress cancer metastasis.

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References
Tanaka M, Koyama T, Sakurai T, Kamiyoshi A, Ichikawa-Shindo Y, Kawate H, Liu T, Xian X,
Imai A, Zhai L, Hirabayashi K, Owa S, Yamauchi A, Igarashi K, Taniguchi S, Shindo T.
The endothelial adrenomedullin-RAMP2 system regulates vascular integrity and suppresses tumor metastasis.
Cardiovasc Res. 111(4):398-409. 2016

一般の皆様へ
この研究から、 AM-RAMP2系が血管の恒常性維持作用を介して癌の転移を抑制するのに対して、 AM-RAMP3系が癌関連線維芽細胞の悪性度を高め、 癌転移を促進させることが分かりました。 選択的な AM-RAMP 系の制御が、癌転移を抑制する治療法に繋がることが期待されます。

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Molecular mechanism for the mutual relationship between
macrophage chemotaxis and activation
Etsuko Todat-etsuko@nms.ac.jp
Department of Analytic Human Pathology, Nippon Medical School

Summary Abstract
The activation state of macrophages and their precursor cells, monocytes, is closely linked tothe pathogenesis of inflammatory diseases and presents a potential therapeutic target. In thisstudy, we explored the role of FROUNT, a molecule regulating the migration of monocytes/macrophages, in their activation. We discovered that inhibiting FROUNT suppressed not only the accumulation of monocytes and macrophages at inflammatory sites but also the expression of inflammatory cytokines, resulting in the amelioration of nephritis. These findings suggest that FROUNT
may be a promising therapeutic target capable of regulating both the excessive accumulation andactivation of monocytes/macrophages in inflammatory diseases.
Key Words ： chemokine, macrophage, FROUNT, chemotaxis

Introduction
Activated monocytes/macrophages play a crucial role in promoting immune and inflammatory
responses in various inflammatory diseases. Chemokines are known to mediate the migration
and activation of immune cells at sites of inflammation (1). However, the molecular mechanismsof chemokine-mediated cellular activation are not yet fully understood. We previously reported thatFROUNT mediates monocyte/macrophage chemotaxis by promoting the signaling of chemokine
receptors CCR2 and CCR5 (2, 3). More recently, we demonstrated that disulfiram (DSF), an established drug used for treating alcoholism, potently inhibits FROUNT function (4). In the current
study, we investigate the molecular mechanism of FROUNT-mediated monocyte-macrophageactivation, as well as the inhibitory effects of DSF as a potential immune response modulator.

Results
To clarify the involvement of the chemokine signaling regulator FROUNT in the regulation ofmacrophage activation, morphological and gene expression analyses of inflammatory tissue froma nephritis model and bone marrow-derived macrophages were performed as follows:
1. Involvement of FROUNT in macrophage activation in response to various stimuli.
To assess the involvement of FROUNT in macrophage activation, cultured macrophages
prepared from the bone marrow of FROUNT-deficient mice and macrophages treated with the
FROUNT inhibitor disulfiram (DSF) were subjected to various stimuli, including LPS and Fc receptor ligation. Total RNA prepared from the stimulated macrophages were examined for cytokine
expression by reverse transcription-quantitative PCR (RT-qPCR). Results show that FROUNT
deficiency or DSF treatment reduces the expression of inflammatory cytokines and chemokines instimulated macrophages. To investigate the signaling pathways affected in the FROUNT-deficientstate, Western blot analysis was performed. The results showed that some kinases showed re-

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duced phosphorylation, while others showed up-regulated phosphorylation in FROUNT-deficientmacrophages. Morphological observations revealed that control macrophages showed lamellipodia-like pseudopodia upon stimulation. In contrast, FROUNT-deficient macrophages showedfilopodia-rich pseudopodia before stimulation and showed little stimulation-dependent morphological changes.
2. Evaluation of FROUNT-targeting therapy in a model of macrophage-associated inflammation
The efficacy of targeting FROUNT on monocyte/macrophage activation and migration wasinvestigated in a WKY rat model of anti-glomerular basement membrane (GBM) glomerulonephritis. In the control group, urinary albumin excretion, which reflects glomerular damage, gradually
increased from day5, and a large amount of albuminuria was observed on day7. In contrast, no
increase in albuminuria was observed in the DSF-treated group during the observation period. In
accordance with them, crescentic formation and necrotizing glomeruli, the pathological feature ofthis disease, is significantly suppressed in DSF-treated group. DSF markedly reduced monocyte/macrophage numbers in glomeruli on day3, before glomerular damage occurred. In
controls, monocytes accumulating in the glomeruli developed pseudopodia in the glomerular
capillaries, whereas pseudopodia formation was inhibited in the DSF-treated group. This isconsistent with in vitro observations that DSF blocks chemokine-dependent pseudopodia
formation and chemotaxis of bone marrow-derived monocytes/macrophages. As for CD163positive M2-like macrophage, they appear on day 7 in the control group, but are hardly found in
DSF group. The RT-qPCR analysis showed that glomerular mRNA of inflammatory cytokines andchemokines such as TNFα, CCL2, and CXCL9 were significantly downregulated in the DSF-treated group compared with the control group. In addition, podocyte loss, which is reported to be
induced by inflammatory cytokines such as TNFα, was reduced in the DSF group, suggestingthat DSF inhibits the activation of macrophages accumulated in glomeruli and prevents glomerular damage with further monocyte recruitment and macrophage activation.
3. Crosstalk analysis of migration and activation signals.
To investigate whether there is crosstalk between chemokine receptor signaling and activation signaling, and to clarify the interconnection between cell motility and activation signaling,
we tested the cytokine response of activated macrophages in CCR2-deficient condition or in
the presence of the actin polymerization inhibitors, which alter cell motility and morphology.
Macrophages from each condition were examined for morphological changes and cytokine
expression. Although the results were not entirely consistent with FROUNT deficiency, they
did provide new insights into the relationship between changes in cell movement and morphology and cytokine expression.

Discussion & Conclusion
These results suggest that both FROUNT deficiency and its inhibition via DSF treatment suppress the chemotactic response of monocytes/macrophages and their subsequent activation,
leading to a reduction in the production of cytokines and chemokines. This results in a significant
decrease in monocyte/macrophage numbers at inflammation sites. In cases of anti-GBM nephritis, this dampened inflammatory cytokine response leads to an attenuation of podocyte loss.
Therefore, targeting FROUNT with disulfiram could serve as a highly effective and safe strategy
in the treatment of glomerulonephritis, blocking both the monocyte/macrophage chemotactic
response and their activation within the glomerulus (5). This study revealed the dual regulation

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for the Promotion of Scienceof monocytes/macrophage migration and activation via FROUNT, offering a potential new way
to control harmful and excessive immune and inflammatory responses with fewer side effects.
In conclusion, the strategy of targeting FROUNT appears promising in controlling macrophagerelated responses. We plan to investigate further the underlying molecular mechanism in futurestudies.

References
1. F. Bachelerie, A. Ben-Baruch, A. M. Burkhardt, C. Combadiere, J. M. Farber, G. J. Graham, R.
Horuk, A. H. Sparre-Ulrich, M. Locati, A. D. Luster, A. Mantovani, K. Matsushima, P. M. Murphy,
R. Nibbs, H. Nomiyama, C. A. Power, A. E. Proudfoot, M. M. Rosenkilde, A. Rot, S. Sozzani, M.
Thelen, O. Yoshie, A. Zlotnik, International Union of Basic and Clinical Pharmacology. [corrected].
LXXXIX. Update on the extended family of chemokine receptors and introducing a new nomenclature for atypical chemokine receptors. Pharmacol Rev 66, 1-79 (2014).
2. Y. Terashima, N. Onai, M. Murai, M. Enomoto, V. Poonpiriya, T. Hamada, K. Motomura, M.
Suwa, T. Ezaki, T. Haga, S. Kanegasaki, K. Matsushima, Pivotal function for cytoplasmic protein
FROUNT in CCR2-mediated monocyte chemotaxis. Nature immunology 6, 827-835 (2005).
3. E. Toda, Y. Terashima, T. Sato, K. Hirose, S. Kanegasaki, K. Matsushima, FROUNT is a common regulator of CCR2 and CCR5 signaling to control directional migration. J Immunol 183,6387-6394 (2009).
4. Y. Terashima, E. Toda, M. Itakura, M. Otsuji, S. Yoshinaga, K. Okumura, F. H. W. Shand, Y.
Komohara, M. Takeda, K. Kokubo, M. C. Chen, S. Yokoi, H. Rokutan, Y. Kofuku, K. Ohnishi,
M. Ohira, T. Iizasa, H. Nakano, T. Okabe, H. Kojima, A. Shimizu, S. Kanegasaki, M. R. Zhang,
I. Shimada, H. Nagase, H. Terasawa, K. Matsushima, Targeting FROUNT with disulfiram suppresses macrophage accumulation and its tumor-promoting properties. Nat Commun 11, 609(2020).
5. E. Toda, A. Sawada, K. Takeuchi, K. Wakamatsu, A. Ishikawa, N. Kuwahara, Y. Sawa, S. Hatanaka, K. Kokubo, K. Makino, H. Takahashi, Y. Endo, S. Kunugi, M. Terasaki, Y. Terasaki, K.
Matsushima, Y. Terashima, A. Shimizu, Inhibition of the chemokine signal regulator FROUNT
by disulfiram ameliorates crescentic glomerulonephritis. Kidney Int, (2022).

一般の皆様へ
私たちはこれまで、 炎症 ・ 免疫反応において重要な働きを担う単球 ・ マクロファージの遊走を促進する分子、 FROUNT の機能について解析してきました。 新たに、 FROUNT が活性化に伴う炎症性サイトカインの発現にも関与することを明らかにしました。 ラットの腎炎モデルにおいては、
FROUNT の阻害活性をもつ既存の嫌酒薬ジスルフィラムが腎炎を強力に抑制することを見出しました。 単球 ・ マクロファージの遊走と活性化は相互に関連しており、 これらが過剰に働きすぎると組織傷害や病的な線維化の原因ともなることから、 FROUNT はこれら双方を制御可能な標的分子として期待されます。

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Metabolic signaling regulation by RNA modification
Akiko Ogawa
akiko.ogawa.e5@tohoku.ac.jp
Tohoku University, Institute of Development, Aging and Cancer

Summary Abstract
Key Words ： RNA, RNA modification, metabolism, epitranscriptome, GPCR

Introduction
Among the soluble factors in vivo, factors of nucleic acid-type is limited to adenosine and ATP,and their diversity is extremely poor compared to other soluble factors. Recently, it has been discovered that RNA has as many as 150 different chemical modifications, which are responsible
for post-transcriptional regulation of gene expression, and that abnormal RNA modifications cancause disease onset. In this study, we aimed to clarify the metabolism and physiological significance of soluble factors (modified nucleosides) derived from RNA modifications.

Results
1. Establishment of a comprehensive detection method for RNA modification metabolism
Biological samples were stored at -80°C after collection, thawed on the day of measurement,
deproteinized by adding organic solvent, and then water was added to separate the liquid layer
into two layers. The aqueous layer was collected after ultrafiltration. The collected aqueous layer
was centrifugally concentrated and redissolved in ultrapure water, and the RNA-modified metabolites were detected by mass spectrometry.
2. Examination of bioactivity of modified RNA-derived catabolites
Using the above detection method, the distribution of RNA catabolites in extracellular fluid ofvarious species including human was determined. Since these metabolites are modified nucleosides, we screened them for their activities as GPCR ligands. As a result, we identified N6-methyladenosine (m6A) as having specific activity against the adenosine A3 receptor. The activity was
more than 10-fold stronger than that of unmodified adenosine, and m6A had activity against the
adenosine A3 receptor even at only 1 nM.
3. Elucidation of the mechanism of activity of RNA-modified metabolites and identificationof their physiological actions
To elucidate the mechanism of the potent
receptor activity of m6A, we investigated thepredicted structure by homology modeling andfurther supported by mutant assays to identifyhydrophobic amino acid residues specific for
m6A binding.

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Next, we examined the regulatory mechanism of m6A by applying various external stimuli, andfound that extracellular m6A increases by lysosomal degradation during cytotoxic stimulation suchas cell injury, as demonstrated by the generation of a knockout cell line of the modifying enzyme.
Furthermore, downstream signaling of the m6A-adenosine A3 receptor increases the productionof type I allergy or local inflammatory cytokines in vivo(fig a & b).

Discussion & Conclusion
Receptor screening of modified nucleosides present in the human body using the shedding
method revealed that m6A (N6-methyladenosine) binds specifically to the adenosine A3 receptor.
The binding potency was about 10 times stronger than that of unmodified adenosine. Structural
analysis identified amino acid residues involved in the specificity of m6A. m6A caused phosphorylation of ERK and elevation of intracellular calcium concentration, and these signaling pathways
were confirmed to be adenosine A3 receptor dependent by inhibitor administration. In both themast cell degranulation test and the passive cutaneous anaphylaxis (PCA) model in mice, m6A
elicited allergic reactions. Furthermore, m6A administration to mice increased inflammatory cytokines and chemokines such as IL6 and MCP-1.
Recently, it has become clear that not only DNA and proteins but also RNA undergoes chemical modifications such as methylation and acetylation, and RNA modification is becoming a newresearch field next to epigenetics. In this study, we focused on RNA modification metabolism,
optimized its comprehensive detection, and analyzed various samples using this method. Furthermore, a new metabolic pathway of m6A is currently being analyzed and is in preparation for
submission (unpublished data not shown in this report). We were also able to clarify the RNA
modification kinetics of the global pandemic COVID-19 vaccine (Ref2) or the receptor activity of
the adverse effects of nucleic acid therapeutics (Ref1).

References
1) Akiko Ogawa, Seiya Ohira, Yuri Kato, Tatsuya Ikuta, Shota Yanagida, Xinya Mi, Yukina Ishii,
Yasunari Kanda, Motohiro Nishida, Asuka Inoue, and Fan-Yan Wei.
Activation of the urotensin-II receptor by remdesivir induces cardiomyocyte dysfunction.
Communications Biology, 2023, in press.
2) 小川 亜希子、 松尾 紀孝、 齋藤 一創、 魏 范研 .
COVID-19 ワクチン接種後の RNA 修飾代謝物排泄の変動 . 痛風と尿酸 ・ 核酸 ( 日本痛風 ・ 尿酸核酸学会誌 )2022 年 46 巻 2 号 p. 105-113* 直接の成果となっている論文は投稿前です。

一般の皆様へ近年、 DNA やタンパク質だけでなく、 RNA もメチル化やアセチル化といった化学修飾を受けることが明らかになり、 RNA 修飾がエピジェネティクスに次ぐ新たな研究分野として定着しつつある。
本研究では RNA 修飾代謝に着目し、 その網羅的検出の最適化を行い、 更にこの手法を用いて様々な検体を解析することにより成果を得た。 更に m6A の新たな代謝経路についても現在解析を進め、 投稿準備中である。 また、 世界的パンデミック COVID-19ワクチンの RNA 修飾動態あるいは核酸治療薬の副作用の受容体活性についても明らかにすることができた。

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Catalytic Asymmetric Indole Halocyclization for Construction of
Bioactive Structures
Keisuke Asano
asano@cat.hokudai.ac.jp
Institute for Catalysis, Hokkaido University

Summary Abstract
Cooperative catalysis which consists of a trans-cyclooctene derivative, which retards background reactions, and an asymmetric catalyst, which provides enantioselectivity, enabledcatalytic asymmetric induction in rapid halogenation reactions which have been difficult to
conduct due to their background reactions. The concept of the catalyst system was also applied to catalytic asymmetric indole halocyclization.
Key Words ： trans-cyclooctene, asymmetric catalyst, background reaction, bromination, indole

Introduction
Bromination is indispensable synthetic reactions because bromo-organic compounds areuseful as functional molecules and synthetic intermediates. However, fast non-catalyzed
background reactions disturb the catalytic control of enantioselectivity even when using Nbromoamide reagents, the reactivity of which is milder than that of bromine (Br2). Bromocyclization of Indole derivatives also suffered from the problem. In this study, we developed catalyst systems to solve the problem and enable catalytic asymmetric indole bromocyclization.

Results
We developed a trans-cyclooctene (TCO) derivative to retard background reaction pathways in several bromocyclization reactions. In addition, mechanistic studies including NMR
and Raman analyses of the reaction mixtures and control experiments revealed that a small
amount of Br2 generated in situ from N-bromosuccinimide (NBS) causes the backgroundreactions. This is the first study to clarify the mechanism of the rapid background reactions
which take place even when NBS is employed as a mild brominating reagent. Moreover, wefound that the TCO reacts with Br2 to deactivate it, thereby retarding the background reactions. It is notable that the use of less than stoichiometric amount of the TCO was sufficient
to shut out the non-catalyzed reactions based on the Br2-involved chain mechanism. Furthermore, the key species which was formed through the reaction of the TCO and Br2 was isolated by gel permeation chromatography (GPC) purification, which allowed for investigationsof its reactivity and characterization of its structure.
Next, based on these results, we developed cooperative catalysis which consists of the
TCO and a chiral bromination catalyst. First, proof of concept was obtained in asymmetricbromocyclization of allylic amides: the cooperative catalysis improved the enantioselectivity
and alleviated the reaction rate. Catalytic asymmetric bromocyclization of indole derivatives
was also investigated, and some chiral catalysts provided enantioselectivity. However, this118

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reaction was halted in the presence of the TCO even with the chiral bromination catalyst. It
indicates that Br2 was also involved in the reaction mechanism of the chiral bromination catalyst cycle, and this mechanism efficiently inhibited the background reaction pathway. Thus, inthis reaction, the chiral bromination catalyst proved to play two roles as the catalyst decelerating the background reaction and as the chiral bromination catalyst. These results provided
another methodology controlling the background reactions involving Br2.In addition, throughthe above-mentioned research, we focused on the ability of the TCO to inhibit not only noncatalytic background reaction pathways but also catalytic reaction pathways involving Br2.
Thus, we consider that the TCO serves as an indicator to investigate whether Br2 is involvedin the mechanism of unknown bromination reactions. Thus, we added the TCO to a catalytic
asymmetric reaction which was reported to involve Br2 in the catalytic reaction pathway,1 andthe TCO completely inhibited the catalytic reaction. Thus, we developed a function of the
TCO as an indicator useful for mechanistic studies on bromination reactions like 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), which has been often used in mechanistic studies on radical reactions.

Discussion & Conclusion
In summary, we revealed that in situ generated Br2 causes the non-catalyzed reaction pathways in several bromocyclization, and the TCO derivative was developed to efficiently retard
the background reactions. Notably, the use of less than stoichiometric amount of the TCO
was sufficient to shut out the non-catalyzed reactions based on the Br2-involved chain mechanism. Furthermore, the TCO was useful as an additive for improving the enantioselectivity inthe catalytic asymmetric reaction. The cooperative catalytic system using the TCO with chiralcatalysts offers alternative methods for catalytic asymmetric induction in bromination that suffers from non-selective background reactions. In addition, the extended methodology using
the asymmetric catalysts playing the two roles was useful for catalytic asymmetric indole bromocyclization. Moreover, the TCO also served as the indicator of Br2 in the catalytic reaction
pathway, and thus it was demonstrated to be useful for mechanistic studies to know whether
Br2 is involved in bromination reactions of interest.

References(1) Yamashita, K.; Hirokawa, R.; Ichikawa, M.; Hisanaga, T.; Nagao, Y.; Takita, R.; Watanabe,K.; Kawato, Y.; Hamashima, Y. J. Am. Chem. Soc. 2022, 144, 3913.

一般の皆様へ
医薬品などの機能性分子を効率よく合成することは、 その安価 ・ 安定供給や新物質創成につながる基盤技術になり、 人々の生活を豊かにします。 不斉触媒はそれを実現する武器になりま
すが、 速すぎる反応にはこれが利用できませんでした。 この問題を解決するために、 反応をあえて遅くする触媒を開発し、 ハロゲン化反応による生物活性分子の合成を短工程化する反応原
理を創り出しました。 これをきっかけに、 不斉触媒による医薬品合成がこれまで以上に発展すると考えています。

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Analysis of structures and functions of plant nuclear
actin microfilaments
Noriko Inada
norikoinada@omu.ac.jp
Osaka Metropolitan University

Summary Abstract
Recent studies using animal cells showed that actin microfilaments (AFs), one of cytoskeletons that functions in intracellular transports, are transiently formed in the nucleus and function in regulation of gene expression. However, the presence of nuclear AFs in plants was
unknown. This study strongly suggested that nuclear AFs do exist and function in regulationof various plant physiologies.
Key Words ： Actin microfilaments, Arabidopsis thaliana, gene expression, nucleus

Introduction
We previously found that Arabidopsis thaliana mutants of ACTIN DEPOLYMERIZING FACTOR (ADF), actin binding protein that regulates organization and dynamics of actin microfilaments (AFs), functions in the nucleus to regulate responses against pathogen. In addition,
our preliminary data showed that adf mutants showed altered nuclear morphology and gene
expression. Together with the recent animal research regarding nuclear AFs that transiently
formed to regulate gene expression, we hypothesized that A. thaliana ADF regulates gene
expression through regulation of nuclear AFs.

Results
A. thaliana genome possesses 11 ADF genes, which are divided into 4 subclasses. Subclass I ADF, containing ADF1, -2, -3 and -4, express throughout the plant (Inada, 2017). We
previously showed that adf4 knockout mutant and ADF1-4 knockdown plants (ADF1-4Ri)
showed an increased resistance against powdery mildew fungus (Inada et al., 2016). In
addition, both adf4 and ADF1-4Ri exhibited an increase of plant size and an enhancementof endoreplication (Inada et al., 2021). Both pathogen resistance and increased plant size
phenotypes were more significantly observed in ADF1-4Ri compared to adf4. Furthermore,
we showed that nuclear localization of ADF4 is important for response against powdery mildew fungus (Inada et al., 2016).
While the main function of plant AFs is in regulation of intracellular transport at the cell
periphery, recent research using animal cells showed that transiently formed nuclear AFs
have various functions including regulation of gene expression. Gene expression is also
affected by nuclear morphology (Pontvianne&Grob 2020). Based on those results, we hypothesized that A. thaliana ADFs regulate expression of genes for pathogen responses andplant growth through regulation of organization and dynamics of nuclear AFs.

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To test this hypothesis, we first analyzed nuclear morphology in epidermal cells of fixedmature leaves of wild type (WT) Col-0, adf4 and ADF1-4Ri. The nuclei were stained with
4ʹ,6-diamidino-2-phenylindole (DAPI) and observed with confocal laser scanning microscope (CLSM). Obtained fluorescent images were quantitatively analyzed using ImageJ.
We found that adf4 and ADF1-4Ri showed a significant decrease in size of heterochromatin, which is a nuclear domain in which chromatins are highly condensed and gene expression is suppressed. Neither heterochromatin number per nucleus nor nuclear size werealtered in adf4 and ADF1-4Ri (Matsumoto et al, 2023).
If alteration in heterochromatin size in adf4 and ADF1-4Ri was caused through ADF’s
function in regulation of AFs, loss of actin could cause similar alteration in heterochromatin. A. thaliana genome encode 8 ACTIN genes, of which ACT2, ACT7, and ACT8 are expressed in vegetative tissues, where subclass I ADFs express (Kandasamy et al.,2010).
While act2/8 double mutant did not show alteration in heterochromatin size, we found thatthe size of heterochromatin in act7 increased compared to WT (Matsumoto et al, 2023).
Then we performed microarray analysis using Col-0, adf4 and ADF1-4Ri to see if loss of
ADFs causes alteration in gene expression. We found that expression of more than 1800
genes was altered in adf4 and ADF1-4Ri. Particularly, genes related to pathogen responses
showed altered expression. Those results clearly showed that ADF functions in regulationof nuclear organization and gene expression (Matsumoto et al., 2023).
To examine the possibility that regulation of nuclear AFs by ADF functions in regulationof nuclear organization and gene expression, we established A. thaliana lines for nuclear
AF visualization. Actin-binding peptide Lifeact conjugated with GFP and nuclear localization
signal (NLS) was expressed in Col-0, adf4 and ADF1-4Ri. We found filamentous structuresin nuclei in roots of Col-0, adf4, and ADF1-4Ri. The frequency that nuclei with filamentous
structures was higher in ADF1-4Ri compared to Col-0 and adf4. The length of the filamentous structures was longer in ADF1-4Ri compared to Col-0 and adf4. Those filamentous
structures labeled with Lifeact-GFP-NLS were more often observed in root tips, where cells
were undifferentiated.

Discussion & Conclusion
By a microscopic observation of the nuclei, we showed that A. thaliana ADFs and ACTs
function in regulation of nuclear organization. Furthermore, large-scale expression analysis
revealed that a number of genes showed altered expression in adf mutants, indicating thatADFs function in regulation of gene expression. We hypothesized that ADFs regulate organization and dynamics of nuclear AFs, which regulate gene expression. We established plantsthat express actin filament-binding peptide conjugated with fluorescent protein and nuclear
localization signal, and found that filamentous structures were observed in root cells. The frequency that filamentous structures were observed was higher, and length of nuclear filamentous structures was longer in adf mutants. Those results strongly suggest that ADF function in
regulation of nuclear AFs, which are involved in regulation of gene expression.

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References
Inada, N., Higaki, T., and Hasezawa, S. (2016) Nuclear function of subclass I Actin Depolymerizing Factor contributes to susceptibility in Arabidopsis to an adapted powdery mildew
fungus. Plant Physiology 170, 1420-1434.
Inada, N. (2017) Plant Actin Depolymerizing Factor – Actin microfilament disassembly andmore. Journal of Plant Research 130, 227-238.
Inada, N., Takahashi, N., and Umeda, M. (2021) Arabidopsis thaliana subclass I ACTINDEPOLYMERIZING FACTORs and vegetative ACTIN2/8 are novel regulators of endoreplication. Journal of Plant Research 134, 1291-1300.
Kandasamy, M.K., McKinney, E.C., and Meagher, R.B. (2010) Differential sublocalization ofactin variants within the nucleus. Cytoskeleton 67, 729-743.
Matsumoto, T., Higaki, T., Takatsuka, H., Kutsuna, N., Ogata, Y., Hasezawa, S., Umeda, M., and Inada, N. (2023) Arabidopsis thaliana subclass I ACTIN DEPOLYMERIZING FACTORs regulate nuclear organization and gene expression. Biorxiv doi: https://doi.
org/10.1101/2023.04.19.537409
Pontvianne, F., and Grob, S. (2020) Three-dimensional nuclear organization in Arabidopsis
thaliana. Journal of Plant Research 133, 479-488.

一般の皆様へ
細胞骨格の一種であるアクチン繊維は、 人や植物などの生物を構成する細胞内において、 主に物質の輸送を制御する器官である。 最近の動物における研究で、 アクチン繊維が細胞核の中でも遺伝子発現制御に働くことが明らかになっている。 私たちは本研究で、 これまで一部の動物細胞でしか解析されていなかった細胞核内アクチン繊維が、 植物細胞でも存在し、 遺伝子発現制御を通して植物の病害応答や成長に寄与していることを強く示唆した。 今後は、 植物生理機能の制御における植物細胞核内アクチン繊維の役割を更に明らかにしていきたい。

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Establishment of embryonic diapause induction techniquefor in vitro culture of animal embryos.
TAKAOKA, Katsuyoshi
takaoka.katsuyoshi@tokushima-u.ac.jp
Institute of Advanced Medical Sciences, Tokushima University,

Summary Abstract
Many conventional studies on developmental regeneration have predominantly focusedon "activity" and have achieved a certain degree of success. However, human and mouseembryos implanted in vitro or reconstructed from undifferentiated cultured cells bypass the"developmental quiescence" observed in vivo and proceed to the implantation process at adevelopmental stage unsuitable for successful implantation, giving rise to various functionaland efficiency issues. From this study, we found that apparently developmentally diapause
embryos are a mixture of embryos that can and cannot reactivate.
Key Words ： Embryonic diapause, Implantation

Introduction
Embryonic diapause is a phenomenon exhibited by mammalian embryos, wherein thematernal intrauterine environment undergoes alterations in response to environmental cues,
resulting in the temporary arrest of the cell cycle and cell differentiation in preimplantation embryos. The induction of developmental pauses acts as a regulatory mechanism for embryonicself-organization and facilitates a stable pregnancy by temporarily halting the embryo at adevelopmental stage conducive to implantation. Therefore, embryonic diapause represents a
critically significant embryonic event; however, the molecular mechanisms underlying this process remain largely unknown.

Results
While developmental processes encompass both "active" and "quiescent" aspects, conventional research has predominantly focused on the former and achieved a certain levelof success. Presently, worldwide efforts are underway to develop organoid technologies forregenerative medicine, aiming to generate diverse tissues and organs from embryonic stem(ES) or induced pluripotent stem (iPS) cells. However, significant challenges persist regarding
functionality, size, and quality of these organoids. One contributing factor is the prevailing bias
towards techniques that promote the release and activation of the quiescent state during embryonic development. Notably, human and mouse embryos implanted in vitro or reconstructedusing undifferentiated cultured cells bypass the "Embryonic diapause" that would normallyoccur in vivo. Consequently, they proceed to the implantation stage at a developmental phase
unsuitable for successful implantation, resulting in numerous functional and efficiency-related
issues.

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The objective of this study is to establish the groundwork for the realization of complete in vitro
culture of embryos from embryonic animals through fundamental research on developmental arrest. By investigating the mechanisms underlying developmental pauses, this research endeavors
to lay the foundation for achieving comprehensive in vitro embryonic culture.
In conventional studies on embryonic diapause, there has been a general assumption that
all mouse embryos possess the ability to undergo embryonic diapause due to the observation of a significant number of reactivating mouse embryos during this developmental stage.
However, through this study conducted by the principal investigator, which specifically aimed
to explore individual variations, it was discovered that only approximately 70% of mouse embryos are capable of successfully reemerging, even when they exhibit apparent dormancy. In
essence, the population of diapausing mouse embryos consists of a combination of genuinely
hibernating embryos and those falsely categorized as diapause.
The transcriptome analysis using RNA-seq on individual embryos revealed the presence of two
distinct groups. Furthermore, the identification of 35 differentially expressed genes (DEGs) associated with principal components (PC) provided insight into their potential involvement in determining the regenerative capacity of the embryos. Notably, some of these candidate factors underwent
phenotypic screening, wherein knockout (KO) embryos for four of them exhibited disrupted developmental pauses. Based on this evidence, we posit a strong likelihood of an association between
the PC component and the presence or absence of regenerative potential.
Moving forward, our future investigations will aim to ascertain whether a correlation exists
between the individual variations observed in dormant embryos, as mentioned above, and theoccurrence of regenerative processes. And we aim for in vitro culture of future embryonic embryos of embryonic animals.
The academic results obtained will lead to a breakthrough in the essential understanding of
the phenomenon of developmental arrest, which will lead to the discovery of developmental
arrest in primates, including human embryos, and its applied research. For example, they willlead to the development of technologies that have been considered an unattainable dream,such as artificial birth timing control and complete in vitro culture technology for embryos ofembryonic animals, and their impact will be felt in many fields, including animal husbandry
and human clinical practice. In this way, this research has the potential to produce innovationsof great significance in both the academic and technological fields.

Discussion & Conclusion
Reflecting upon the trajectory of scientific progress, the domains of apoptosis, a form of cell
death that garnered the Nobel Prize in Physiology or Medicine, and autophagy, an intracellular degradation mechanism, have evolved as academic disciplines through the identification and systematic understanding of fundamental principles underlying diverse biologicalphenomena. By elucidating a segment of the foundational model concerning inactivation
phenomena through the proposed research, it becomes feasible to detect and analyze suchinactivation phenomena in various contexts, including latent cancer. Moreover, through thediscovery of fundamental principles governing inactivity across a broad spectrum of biologicalevents and the elucidation of their biological significance, we will establish a novel researchparadigm of programmed inactivity, akin to apoptosis (programmed cell death) and autophagy(programmed self-degradation).

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一般の皆様へ
本研究では、 発生休止というこれまで分子学的に研究されてこなかった現象の分子メカニズムを明らかにします。 得られた知見は、 将来の胎生動物胚の完全試験管培養といった夢のような技術の開発につながります。

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Publication - FY2021 Grant Recipients

126

Title of the research
project

Molecular mechanism of lysosomal damage repair by TFEB

Recipient (Institution)

Shuhei Nakamura (Graduate School of Frontier Biosciences, Osaka University)

Journal article / othermaterial

PLOS Genetics doi.org/10.1371/journal.pgen.1010264

Title of the paper

PACSIN1 is indispensable for amphisomelysosome fusion during basal
autophagy and subsets of selective autophagy

Title of the research
project

Structural and functional analysis of primary cation transporters and elucidationof the substrate specificity by gain-of-function mutant

Recipient (Institution)

Kazuhiro Abe (Graduate school of Pharmaceutical Sciences, Nagoya
University)

Journal article / othermaterial

Nature communications 09 Sept. 2022
doi.org/10.1038/s41467-022-32793-0

Title of the paper

Structure and function of H+/K+ pump mutants reveal Na+/K+ pumpmechanisms

Title of the research
project

Hematopoietic stem cell aging and mitochondria aging

Recipient (Institution)

Takayoshi Matsumura (Division of Inflammation Research, Center for Molecular
Medicine. Division of Cardiovascular Medicine., Jichi Medical University)

Journal article / othermaterial

Nature Communications | ( 2022) 13:7064
doi.org/10.1038/s41467-022-34906-1

Title of the paper

A Myb enhancer-guided analysis of basophil and mast cell differentiation

Title of the research
project

Significance and spatial profiling of phospholipid metabolism in NASH

Recipient (Institution)

Hayato Nakagawa (Department of Gastroenterology and Hepatology, Mie
University)

Journal article / othermaterial

J Clin Invest. 2022;132(11):e151895. doi.org/10.1172/JCI151895.

Title of the paper

Inhibiting SCAP/SREBP exacerbates liver injury and carcinogenesis in murine
nonalcoholic steatohepatitis

The Novartis Foundation(Japan)
for the Promotion of Science

The Novartis Foundation(Japan)
for the Promotion of Science

Title of the research
project

Cryo-electron microscopy studies for disease pathogenesis of Xeroderma
Pigmentosum

Recipient (Institution)

Syota Matsumoto (Institute for Quantitative Biosciences, The university ofTokyo)

Journal article / othermaterial

Cancer Science.23-May-2023, doi.org/10.1111/cas.15850

Title of the paper

Chromatin structure related to oncogenesis

Title of the research
project

Analysis of brain aging caused by the disruption of "nuclear-axon crosstalk"machinery

Recipient (Institution)

Kenichiro Kuwako (School of Medicine, Department of Neural and Muscular
Physiology , Shimane University)

Journal article / othermaterial

Development (2022) 149, dev201214.
doi:10.1242/dev.201214

Title of the paper

N-WASP-Arp2/3 signaling controls multiple steps of dendrite maturation in
Purkinje cells in vivo

Title of the research
project

Elucidating the mechanism of skin stem cell aging by targeting anti-aging
matrix Fibulin-7

Recipient (Institution)

Aiko Sada (International Research Center for Medical Sciences (IRCMS),
Kumamoto University)

Journal article / othermaterial

EMBO Rep . 2022 Dec 6;23(12):e55478.
doi: 10.15252/embr.202255478. Epub 2022 Oct 24.

Title of the paper

The extracellular matrix fibulin 7 maintains epidermal stem cell heterogeneity
during skin aging

Title of the research
project

Studies of genetic basis of early onset cardiac conduction system disease anddisease mechanism-based personalized medicine

Recipient (Institution)

Kenshi Hayashi (School of Health Sciences, College of Medical,
Pharmaceutical and Health Sciences, Kanazawa University)

Journal article / othermaterial

Journal of Molecular and Cellular Cardiology doi.org/10.1016/
j.yjmcc.2023.03.001

Title of the paper

The utility of zebrafish cardiac arrhythmia model to predict the pathogenicity of
KCNQ1 variants

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for the Promotion of Science

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Title of the research
project

Study of functional deficits of microglia and accelerated onset ofneurodegenerative disorders

Recipient (Institution)

Keiro Shirotani (The Laboratory of Genome-based Drug Discovery, Nagasaki
University)

Journal article / othermaterial

J. Biochem 2022;00(00):1–7 doi.org/10.1093/jb/mvac073

Title of the paper

The role of TREM2 N-glycans in trafficking to the cell surface and signaltransduction of TREM2

Title of the research
project

Intrinsic barrier dysfunction in multiple sclerosis patient

Recipient (Institution)

Hideaki Nishihara (Department of Neurotherapeutics, Yamaguchi University)

Journal article / othermaterialAdv. Healthcare Mater. 2022, 11, 2200804
doi: 10.1002/adhm.202200804

Title of the paper

The Modular μSiM: A Mass Produced, Rapidly Assembled,and Reconfigurable
Platform for the Study of Barrier Tissue Models In Vitro

Title of the research
project

Molecular mechanism of neuroprotection after ischemic stroke

Recipient (Institution)

Hiroo Takahashi (Department of Molecular Neurobiology, Kagawa University)

Journal article / othermaterial

Neuroscience Research 186 (2023) 3.9
doi: 10.1016/j.neures.2022.10.005. Epub 2022

Title of the paper

Molecular mechanisms underlying activity-dependent ischemic tolerance in thebrain

Title of the research
project

Molecular mechanism for the mutual relationship between macrophage
chemotaxis and activation

Recipient (Institution)

Etsuko Toda (Department of Analytic Human Pathology, Nippon Medical
School)Journal article / othermaterial

Kidney Int.2022 Dec. doi.org/10.1016/j.kint.2022.07.031

Title of the paper

nhibition of the chemokine signal regulator FROUNT by disulfiram ameliorates
crescentic glomerulonephritis

The Novartis Foundation(Japan)
for the Promotion of Science

The Novartis Foundation(Japan)
for the Promotion of Science

Title of the research
project

Catalytic Asymmetric Indole Halocyclization for Construction of Bioactive
Structures

Recipient (Institution)

Keisuke Asano (Institute for Catalysis, Hokkaido University)

Journal article / othermaterial

Chem. Rec. 2022, e202200200 doi.org/10.1002/tcr.202200200
tcr.wiley-vch.de

Title of the paper

Organocatalytic Access to Tetrasubstituted Chiral Carbons Integrating
Functional Groups

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for the Promotion of Science＜論文掲載＞ 2021 年度受賞者

130

助成タイトルTFEB による損傷リソソーム修復機構の解明

受賞者

中村 修平 ( 大阪大学 大学院生命機能研究科 )論文掲載誌 ・ 書誌事項

PLOS Genetics doi.org/10.1371/journal.pgen.1010264

論文タイトルPACSIN1 is indispensable for amphisomelysosome fusion during basal autophagy andsubsets of selective autophagy

助成タイトル

カチオン能動輸送体の構造機能解析と機能獲得変異体による
基質特異性の解明

受賞者

阿部 一啓 ( 名古屋大学 創薬科学研究科 )論文掲載誌 ・ 書誌事項

Nature communications 09 Sept. 2022
doi.org/10.1038/s41467-022-32793-0

論文タイトルStructure and function of H+/K+ pump mutants reveal Na+/K+ pump mechanisms

助成タイトル

ミトコンドリアに着目した老化造血幹細胞多様性の解析

受賞者

松村 貴由 ( 自治医科大学 分子病態治療研究センター 炎症 ・ 免疫研究部
兼任 循環器内科 )論文掲載誌 ・ 書誌事項

Nature Communications | ( 2022) 13:7064
doi.org/10.1038/s41467-022-34906-1

論文タイトルA Myb enhancer-guided analysis of basophil and mast cell differentiation

助成タイトル非アルコール性脂肪肝炎におけるリン脂質代謝異常の意義とその空間的理解

受賞者

中川 勇人 ( 三重大学 大学院医学系研究科消化器内科学 )論文掲載誌 ・ 書誌事項

J Clin Invest. 2022;132(11):e151895. doi.org/10.1172/JCI151895.

論文タイトルInhibiting SCAP/SREBP exacerbates liver injury and carcinogenesis in murine
nonalcoholic steatohepatitis

The Novartis Foundation(Japan)
for the Promotion of Science

The Novartis Foundation(Japan)
for the Promotion of Science

助成タイトル

クライオ電子顕微鏡を用いた色素性乾皮症発症メカニズムの解明

受賞者

松本 翔太 ( 東京大学 定量生命科学研究所 胡桃坂研究室 )論文掲載誌 ・ 書誌事項

Cancer Science.23-May-2023, doi.org/10.1111/cas.15850

論文タイトルChromatin structure related to oncogenesis

助成タイトル“核 - 軸索クロストーク制御システム” の破綻による脳機能老化

受賞者

桑子 賢一郎 ( 島根大学 医学部 神経 ・ 筋肉生理学 )論文掲載誌 ・ 書誌事項

Development (2022) 149, dev201214.
doi:10.1242/dev.201214

論文タイトルN-WASP-Arp2/3 signaling controls multiple steps of dendrite maturation in
Purkinje cells in vivo

助成タイトル

抗老化マトリクス Fibulin-7 に着眼した皮膚幹細胞老化メカニズムの解明

受賞者

佐田 亜衣子 ( 熊本大学 国際先端医学研究機構 )論文掲載誌 ・ 書誌事項

EMBO Rep . 2022 Dec 6;23(12):e55478.
doi: 10.15252/embr.202255478. Epub 2022 Oct 24.

論文タイトルThe extracellular matrix fibulin 7 maintains epidermal stem cell heterogeneity during
skin aging

助成タイトル

若年発症心臓刺激伝導障害の遺伝子基盤解明とそれに基づく個別化医療の実践

受賞者

林 研至 ( 金沢大学 医薬保健研究域保健学系 )論文掲載誌 ・ 書誌事項

Journal of Molecular and Cellular Cardiology doi.org/10.1016/j.yjmcc.2023.03.001

論文タイトルThe utility of zebrafish cardiac arrhythmia model to predict the pathogenicity of
KCNQ1 variants

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for the Promotion of Science

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for the Promotion of Science

132

助成タイトル

ミクログリアの機能障害と神経変性疾患の発症機序の研究

受賞者

城谷 圭朗 ( 長崎大学 ゲノム創薬学研究室 )論文掲載誌 ・ 書誌事項

J. Biochem 2022;00(00):1–7 doi.org/10.1093/jb/mvac073

論文タイトルThe role of TREM2 N-glycans in trafficking to the cell surface and signaltransduction of TREM2

助成タイトル

多発性硬化症患者に存在する血液脳関門破綻の遺伝的素因の解明

受賞者

西原 秀昭 ( 山口大学 神経 ・ 筋難病治療学講座 )論文掲載誌 ・ 書誌事項

Adv. Healthcare Mater. 2022, 11, 2200804
doi: 10.1002/adhm.202200804

論文タイトルThe Modular μ SiM: A Mass Produced, Rapidly Assembled,and Reconfigurable
Platform for the Study of Barrier Tissue Models In Vitro

助成タイトル

脳梗塞から神経細胞を守る分子メカニズムの解析

受賞者

高橋 弘雄 ( 香川大学 分子神経生物学 )論文掲載誌 ・ 書誌事項

Neuroscience Research 186 (2023) 3.9
doi: 10.1016/j.neures.2022.10.005. Epub 2022

論文タイトル

Molecular mechanisms underlying activity-dependent ischemic tolerance in the brain

助成タイトル

マクロファージの 「動き」 と 「活性化」 の相互連関メカニズム

受賞者

遠田 悦子 ( 日本医科大学 解析人体病理学 )論文掲載誌 ・ 書誌事項

Kidney Int.2022 Dec. doi.org/10.1016/j.kint.2022.07.031

論文タイトルnhibition of the chemokine signal regulator FROUNT by disulfiram ameliorates
crescentic glomerulonephritis

The Novartis Foundation(Japan)
for the Promotion of Science

The Novartis Foundation(Japan)
for the Promotion of Science

助成タイトルインドール類の触媒的不斉ハロ環化反応による生物活性骨格構築

受賞者

浅野 圭佑 ( 北海道大学 触媒科学研究所 )論文掲載誌 ・ 書誌事項

Chem. Rec. 2022, e202200200 doi.org/10.1002/tcr.202200200
tcr.wiley-vch.de

論文タイトル

Organocatalytic Access to Tetrasubstituted Chiral Carbons Integrating Functional
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Ⅲ.
Reports from the Recipients of Grants
for International Meetings

The Novartis Foundation(Japan)
for the Promotion of Science

Report on Research Meeting
1. Name of Research Meeting / Conference
The 20th International Symposium on Spontaneously Hypertensive Rats
2. Representative
Norihiro Kato (chair of the meeting)
3. Opening period and Place
October 16-17, 2022
Shiraikaikan Annex, 11-1 Yoshida-Konoe-cho, Sakyo-ku, Kyoto, JAPAN4. Number of participants / Number of participating countries and areas
53 participants / 9 countries in Asia, Europe, North America and Oceania
5. Total cost
JPY 1,419,618
6. Main use of subsidy
Equipment operating expenses
7. Result and Impression
The International SHR (Spontaneously Hypertensive Rats) Symposium was first
held in Kyoto in 1971 with the aim of contributing to the development of preventive andtherapeutic methods for hypertension and stroke by bringing together researchers fromaround the world who are engaged in various research projects related to hypertension
using disease models such as SHR to present and discuss their research results and toraise awareness of research on hypertension and stroke and other organ complications.
Since then, the symposium has continued to be held about once every two years,
and a total of nine countries have hosted the symposium so far. Since the International
Society of Hypertension (ISH) held in Fukuoka in 2006, the symposium has beenrecognized as a satellite symposium of the ISH.
In 2022, the ISH would be held in Japan (Kyoto), and this meeting was planned as asatellite symposium to discuss (1) achievements and prospects of disease models and(2) prevention strategies based on precision medicine.
The meeting was initially planned to be held in a hybrid format, with a significant
number of local (i.e., on-site) participants and viewers expected. However, due to the
COVID-19 pandemic, we decided to hold the two-day conference in a semi-hybrid format,
which reduced the number of on-site participants as much as possible and increased theweight of online participation.
On the afternoon of the first day (October 16), there were oral presentations at two
sessions and poster presentations. On the morning of the second day (October 17), oralpresentations were given at two more sessions. This conference (all English session)featured presentations and Q&A sessions by a total of 21 leading researchers from Japanand abroad. The titles of the sessions and symposiums are as follows

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for the Promotion of Science

Session 1: System-oriented study on hypertension and cardiovascular complications.
① Symposium 1 (Yamori et al., 4 speakers): Hypertension model research in thepast, present and future.
② Symposium 2 (presented by Kato et al.): Mechanistic understanding of
hypertension.
Session 2: Precision medicine and prevention of hypertension-related disease③ Symposium 3 (presented by Charchar et al.): Recent progress in thepathophysiology of hypertension④ Symposium 4 (presented by Munroe et al.): Bridging the gap between bench andbedsideThus, the participants were able to discuss and exchange new knowledge with researchers
from Japan and abroad regarding research using disease models for lifestyle-relateddiseases.
8. Additional description
Due to the time difference between Japan and the local area, the order of thepresentations was slightly changed. However, many researchers participated in real timefrom overseas despite the late night and early morning, and we had a lively exchange of
opinions.
Lastly, we would like to express our sincere gratitude to the NOVARTIS Foundation(Japan) for the Promotion of Science for their support.

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Report on Research Meeting
2022/12/19
1. Name of Research Meeting / Conference
10th Asian Biological Inorganic Chemistry Conference (AsBIC10)
2. Representative
Shinobu Itoh, Professor
Osaka University, Graduate School of Engineering, Division of Applied Chemistry
2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
3. Opening period and Place
November 28 – December 3, 2022
Kobe International Conference Center, Ariston Hotel Kobe, Kobe Portopia Hotel
4. Number of participants / Number of participating countries and areas
354 participants / 26 countries / Japan, Korea, India, Hong Kong, China, Australia,
Singapore, Taiwan, New Zealand, Philippine, Israel, Malaysia, Cyprus, USA, UK, France,
Germany, Switzerland, Poland, Canada, Belgium, Spain, Italy, Iceland, Hungary, South
Africa
5. Total cost
¥33,950,000
6. Main use of subsidy
Support for the accommodations of plenary and award lectures
7. Result and Impression
AsBIC10 will bring together researchers, educators, postdoctoral fellows, andstudents working on ‘bioinorganic chemistry’ to share the latest information and promote
the development of this field and international mutual understanding as well as to foster
young researchers. To this end, 10 plenary lectures, 5 AsBIC award lectures, 32 keynote
lectures, 99 invited talks, 25 oral presentations, and 127 poster presentations were
presented related on the following themes: (1) Metal Homeostasis, (2) Metallodrugs,(3) Metal-based Mechanism in Diseases, (4) Metal Sensing, (5) Natural and Artificial
Photosynthesis, (6) Engineering Metalloproteins, (7) Metalloproteins and Functional
Models, (8) Models and Mechanism, (9) Bioinspired Catalysis, (10) Nano/Tech Material,(11) Integrated Biometal Sciences, (12) Molecular Movies in Structural Metallo-biology.
8. Additional description
This is an unprecedented large-scale, full face-to-face international conference
carried out a difficult condition due to the Corona Disaster. We hope that the success of
this conference will serve as a foundation for future international conferences.

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ReportonResearchMeeting
1. Name of Research Meeting / Conference
Symposium on Molecular and Cellular Cognition
2. Representative
Satoshi Kida; President of MCCS-Asia, Professor, the University of Tokyo
3. Opening period and Place
Date: June 27-28, 2022
AM9-PM7, June 27
AM9-AM5:30, June 28
Venue: Ito Hall, The Ito International Research Center, The University of Tokyo 7-3-1,
Hongo, Bunkyou-ku, Tokyo ZIP 113-0033
https://www.u-tokyo.ac.jp/adm/iirc/en/hall.html

4. Number of participants / Number of participating countries and areas
The number of participants; about 200 (Approximately 70 people participated in theevent locally)
Number of participating countries and areas;9 countries including Japan (Australia,
Canada, China, France, Israel, South Korea, UK, USA)
5. Total cost
400,000yen
6. Main use of subsidy
Travelexpensesforspeakers
7. Result and Impression
MCCS-Asia is the Asian branch of MCCS (headquartered in the United States), whichholds an international symposium in Asian countries every two years. The symposium
was originally scheduled to be held in 2021, but due to the difficulty of holding the
symposium because of the COVID-19 pandemic, it was postponed for one year and heldon June 27-28, 2022, inviting top-class researchers from Japan and abroad. The themeof the symposium focused on research to elucidate the mechanisms of brain function
and psychiatric disorders. Since the impact of the COVID-19 pandemic is still strong
and we have travel restrictions to Japan, we decided to have the meeting in a hybridmanner. Researchers who were unable to come to Japan were asked to make online
presentations. Registration was required for those who wished to participate, but there
was no charge for participation. We also took thorough measures to prevent infection at
COVID-19, such as installing acrylic partitions on the podium and requiring the wearing ofmasks.
On both June 27 and 28, from 9:00 a.m. to 5:00 p.m., a total of 30 speakers gave
20-minute talks followed by a 5-minute Q&A. Alicia Che (Yale University, USA), Timothy

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W. Bready (University of Queensland, Australia), Anatol Kreitzer (University of California,
San Francisco, USA), Ted Abel (University of Iowa, USA), Mazen Kheirbek (Universityof California, San Francisco, USA), and Juan Song (University of North Carolina, USA),
Matthew Girgenti (Yale University, USA), Paul Frankland (University of Toronto, Canada),
Takashi Kitamura (University of Texas, USA), Gisella Vetere (ESPCI Paris, France), andYong-Seok Lee (Seoul National University, Korea) visited Japan and gave lectures there.
On the other hand, Jelena Radulovic (Albert Einstein College of Medicine, USA), Gustavo
Turecki (McGill University, Canada), Weidong Li (Shanghai Jiao Tong University, China),
Zhihua Gao (Zhejiang University, China), Hyungju Park (Korea Brain Res Inst, Korea),
Kobi Rosenblum (University of Haifa, Israel), Gemma Modinos (King's College London,
UK), and Justin Lee (Inst Basic Sci, Korea) gave their lectures online. 11 researchers
from Japan gave them in the Ito hall: Yasunori Hayashi (Kyoto University), Satoshi
Kida (University of Tokyo), Tadafumi Kato (Juntendo University), Kanzo Suzuki (Tokyo
University of Science), Takanobu Nakazawa (Tokyo University of Agriculture), Akiko
Hayashi (RIKEN), Takeshi Imai (Kyushu University), Shusaku Uchida (Kyoto University),
Tomoyuki Furuyashiki (Kobe University), Atsushi Kasai (Osaka University), and Takuya
Sasaki (Tohoku University). The presentations were at the cutting edge of research
results. The Q&A session was so active that many questions were asked both onsite andonline, so much so that the break time was shortened.
In addition, 19 poster presentations were given on June 27 from 5:00 p.m. to 7:00
p.m. The speakers who attended the conference room scored the poster presentations,and four of them received the Poster Best Presentation Award.
Although there were significant restrictions due to the COVID-19 pandemic, lecturesin the Ito Hall and online performances from overseas were also conducted without
delay. In addition, online delivery and question-and-answer sessions online as well as
locally were conducted without problems. In addition, poster presentations were also
successful, although only locally. As described above, the international symposium wasconducted the same way as before the COVID-19 pandemic, and it was more convenient
to conduct the symposium because it allowed online presentations and participation.
I was grateful that researchers from overseas also understood and cooperated withthe mask-wearing requirement in Japan, although they are not required to wear masks
in their own countries. I would also like to thank almost all the participants from mylaboratory who took part in this symposium and played a behind-the- scenes role. I haveno information that any participants were infected with the COVID-19 after participating inthe symposium.
8. Additional description
I would like to express my sincere gratitude to the NOVARTIS Foundation (Japan)
for the Promotion of Science for their cooperation in organizing such an excellent
symposium.

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The Novartis Foundation(Japan)
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The pictures of the conference.

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Report on Research Meeting
Date of Report: September 13, 2022
1. Name of Research Meeting / Conference
11th International Tunicate Meeting (11th ITM)
2. Representative
TAKEHIRO KUSAKABE
Professor
Graduate School of Natural Science, Konan University
3. Opening period and Place
July 11, 2022 - July 15, 2022 (5 days)
Konan University - Okamoto Campus 8-9-1 Okamoto, Higashinada-ku Kobe 658-8501,
Japan
4. Number of participants / Number of participating countries and areas
151 participants / 18 countries (Japan and 17 others)
5. Total cost
2,091,500yen
6. Main use of subsidy
Labor costs: Graduate students and undergraduate students of Department of
Biology and Department of Nanobiochemistry of Konan University were hired as meeting
staffs.
Travel fellowships: Travel fellowships (called ITM-NOVARTIS Travel Fellowship)
were awarded to four graduate students (three from Japanese Universities and one fromabroad) who presented a high quality paper at the meeting and whose travel expenses
were not fully supported by their own institutes and other fellowships or grants.
7. Result and Impression
Under the Covid-19 pandemic situation, we decided to organize the meeting in a
hybrid style, combination of on-site and on-line presentations/participation. Every paper
was given by sharing the presentation slides in zoom meeting, irrespective of whether
speakers are on site or not. When talks were given in Zoom by online speakers, thetalks were also projected on the screen in the meeting room. Zoom movies of talks wererecorded in real-time during the meeting days and were uploaded for on-demand viewing
shortly in the same day. Preparation and running of the hybrid system was very hard due
to both technical reasons and time differences among countries and areas, but the selfsacrificing works of the meeting staffs, most of them were graduate students hired by the
grant from the NOVARTIS Foundation for the Promotion of Science, made it possible.
As we reported above, we had 151 participants, including 8 oversea on-site attendees(from USA, Belgium, France, Italy, and Israel). Among them, 67 are residents in Japan

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The Novartis Foundation(Japan)
for the Promotion of Scienceand 84 are from other countries. Ratio of principal investigators/senior researchers :post-docs/students are 81:70. Thus, nearly half of the participants were post-docs andstudents and we could support their attendance by the travel fellowship from the grantfrom the NOVARTIS Foundation for the Promotion of Science. We had 74 regular talks aswell as a technical session and round table discussion. The round table discussion wasscheduled at 24:00-25:00 on 14th of Japanese time, because this would be a time slot at
which most of people on the globe was able to attend the session.
The following are some of the messages from the meeting participants that we
received after the meeting:“Dear organizers, What a wonderful conference! You all did such a terrific job inorganizing and putting everything together in a marvelous way. Please also deliver
my deepest thanks to the students that worked so hard behind the scenes.”“Thanks to the organizers and participants for a very successful 11th International
Tunicate Meeting in Kobe (and around the world!). Despite all the challenges
presented by the uncertainties of the past few years, and the hybrid format of themeeting, the organizers were able to create a fully functional international meetingand meet a critical need for the tunicate research community. The science was
as strong as at any prior meeting, showing the great resilience of the community
during the coronavirus pandemic. Congratulations to all who contributed to theorganization, and to all who presented their work. All very much appreciated.”
As we can tell from the above messages from participants, the meeting was frankand enthusiastic atmosphere and we had very active discussion throughout the meeting.
As a whole, the meeting was really successful. On behalf of the Organizing Committee,
I would like to express our sincere gratitude to the NOVARTIS Foundation for the
Promotion of Science.

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Report on Research Meeting
1. DateNov. 29, 2022
2. Name of Research Meeting / Conference
JSICR/MMCB 2022 Joint Symposium
3. Representative
Shigeru Kakuta, PhD., DVM.
Chair, The 86th Annual Meeting of the Japanese Society of Interferon & Cytokine
Research (JSICR2022)
Laboratory of Biomedical Science, Graduate School of Agricultural and Life Sciences,
The University of Tokyo
4. Opening period and Place June 9-10, 2022
Yayoi Auditorium, The University of Tokyo
5. Number of participants / Number of participating countries and areas
Number of participants: 185
Number of participating countries: 5
6. Total cost
JPY 9,252,0007. Main use of subsidy
Labor cost for stuffs (¥150,000)
Others (website creation and maintenance ¥50,000; Video device and streming ¥200,000)
8. Result and Impression
The main theme of JSICR/MMCB 2022 Joint Symposium was “New horizons in
Immune- mediated homeostasis and diseases.” 4 symposiums by each 4-5 invited
speakers and 2 special talks by Dr. Matsushima and Dr. Yoshimura were held by onsite (Yayoi Auditorium)/on-line (Zoom) hybrid style. Among invited speakers from foreigncountries, Glen Barber (Miami University), Helen Goodridge (Cedars-Sinai Medical
Center), Simon Yona (Hebrew University) and Dmitry Gabrilovich (AstraZeneca)came to Japan and talked about their research on-site, and the others joined online.
We could directly discuss with speakers using excellent online tools. In addition, 56
regular presentations were held by on-demand video presentation style, but interactive
Q&A was possible using ONLINE CONF system. Among these presentations, JSICR/
MMCB 2022 Young Investigator Award was given to 6 young researchers, Kohei Soga,
Ryosuke Hiranuma (The University of Tokyo), Tanakorn Srirat (Keio University), Haruka
Shimizu (Tokyo University of Science), Ka Man Tse (Kyoto University) and Takashi Kato
(Wakayama Medical University). Although an official party could not be held on during
this symposium, participants enjoyed and got the latest scientific information.

144

The Novartis Foundation(Japan)
for the Promotion of Science

The Novartis Foundation(Japan)
for the Promotion of Science

As special plans, Symposium 1 (June 9) “Immune Regulation against viral infection
and type 2 immunity” was cosponsored by AMED iD3 catalyst unit and held as “The7th Translational and Regulatory Sciences Symposium”. 120 non-members attended tothis symposium by Zoom via AMED iD3 catalyst unit. Symposium 1 (June 10) “Immune
Regulation by Innate Immune Sensor Molecules” was held as “The 2nd ARS seminar,2022” in Department of Animal Resource Sciences, Graduate School of Agricultural andLife Sciences, The University of Tokyo. 49 graduate students of The University of Tokyoattended to this symposium by Zoom to earn a credit. As a result, not only our society
members, but also many participants form various community joined to this symposium.

The Novartis Foundation(Japan)
for the Promotion of Science

145

The Novartis Foundation(Japan)
for the Promotion of Science

36th Grant Report (FY2022)

The foundation has been conducting public interest activities such as research grant,meeting grant and international exchange programs since its establishment on Sep. 4,
1987 in Japan under authorization of the Ministry of Education, Science, Sports andCulture, followed by a transition to a public interest incorporated foundation on Apr. 1,
2012. The grants conducted in FY 2022 are as follows.
36th Novartis Research Grant: 39 Researchers (JPY 1.0 mil.), Subtotal JPY 39.0mil.
Research Meeting Grant:
5 Meetings (JPY 0.4 mil.),
Subtotal JPY 2.0mil.
Total JPY 41.0mil.

36th Novartis Research Grant(FY2022)

The Grant is to aim supporting creative research in Japan in the field of Bio, life scienceand relevant chemistry
#
1

2

3

4

5

Institution

Title

Research Project

Junior
Associate Y Chromosomal Genes Protective
Professor / against Heart Failure
Lecturer

Soichi Sano

Department of Cardiology,
Graduate Schoolof Medicine, Osaka
Metropolitan University

Susumu Goyama

Division of Molecular
Oncology, Graduate School
Professorof Frontier Sciences, The
Tokyo University

Development of in vivo genetherapy using HSC-targeting lipid
nanoparticles

Mika Nomoto

Center for Gene Research,
Nagoya University

Assistantprofessor

Elucidation of the molecularmechanism of plant immune
response activated bymechanosensory trichomes

Juro Sakai

Division of Metabolic
Medicine, The Universityof Tokyo, Research Center
for Advanced Science andTechnology

Professor

Elucidation of anti-obesity effect bysignal sensing epigenetic modifiermediated mitochondrial biogenesis

Kojiro Mukai

Laboratory of Organelle
Pathophysiology, Graduate
School of Life Sciences,
Tohoku University

Assistant
Professor

The molecular mechanism governing
STING activation

Tomoaki Tanaka

Department of Molecular
Professor
Diagnosis, Chiba University

Elucidation of Novel Regulatory
Mechanism of Glucose Metabolism
in Liver Type 2 Innate Lymphocytes
by Single Cell Analysis and Spatial
Transcriptome Analysis

7

Haruko Takano

Department of MolecularPathophysiology, Institute of Assistant
Advanced Medical Science,, professor
Nippon Medical School

Elucidation of the role of alveolar
capillary in alveolar formation forclinical application

8

Kazuya Kanemoto

Graduate School of
Pharmaceutical Sciences,
Tohoku University

Assistant
Professor

Development of three component
reaction for site-selective peptide
modification

9

Takayuki Nojima

Medical institute of
Bioregulation, Kyushu
University

Associate
Professor

Study about a mechanism of cancerspecific transcription termination that
produces noncoding RNA

6

146

Name

The Novartis Foundation(Japan)
for the Promotion of Science

The Novartis Foundation(Japan)
for the Promotion of Science

#

Name

Institution

Title

Research Project

10 Junki Miyamoto

Institute of Global
Innovation Research, Tokyo Associate
University of Agriculture and Professor
Technology

Diet intervention and chronic
obstructive pulmonary disease

11 Masahito Kawazu

Research Institute, Divisionof Cell Therapy, Chiba
Cancer Center

Division
head

Immunogenomic analysis to elucidate
the mechanism of immune evasionin endometrial cancer with highmicrosatellite instability

12 Hisaaki Hirose

Institute for Chemical
Research, Kyoto University

ProgramSpecific
Associate
Professor

Molecular basis of calcium ionregulated cellular uptake of nutrients
into cancer cells

13 Yoshiaki Abe

Department of Hematology,
Faculty of Medicine,
University of Tsukuba

Postgraduatestudent

Exploring follicular lymphoma-specificimmunity

14 Hiroki Kobayashi

Laboratory of Oncology,
Assistant
School of Life Sciences,
Professor
Tokyo University of
Pharmacy and Life Sciences

Development of novel PFK1 inhibitors
for the therapy of mitochondrial
diseases

15 Koji Kikuchi

Department of Chromosome
Biology, Kumamoto
Lecturer
University

Elucidating the mechanism of Sertoli
cell polarity that plays a crucial role inthe differentiation of male germ celllineage.

16 Osamu Tskamoto

Medical Biochemistry,
Osaka University Graduate
School of Frontier
Biosciences

Associate
Professor

Clarification of the transcriptional
regulatory mechanism of natriuretic
peptide genes by the heart-specificsuper-enhancer CR9 that senses
hemodynamic loads

17 Yoji Kawano

Institute of Plant Scienceand Resources, Okayama
University

Professor

Evolution and activation mechanisms
of NLR immune receptors

18 Kyota Aoyagi

Department of Cellular
Biochemistry, Kyorin
University School of
Medicine

Associate
Professor

Defective mitophagy in pancreaticbeta-cells leads to diabetes

19 Yosuke Tanaka

IRCMS, Laboratory of Stem
Cell Regulation, Kumamoto Lecturer
University

Differential Regulation of Stem
Cell Ability by Calcium Signaling in
Normal and Abnormal Hematopoietic
Stem Cells and Therapeutic
Applications

20 Satoru Okamoto

Department of Agriculture,
Niigata University

Assistantprofessor

A study about a molecularmechanism of regulation of floweringfrom roots

21 Soichiro Yamanaka

Department of Biological
Sciences, Graduate Schoolof Science, The Universityof Tokyo

Associate
Professor

The analysis on the re-establishmentof epigenome in mouse gonocyte

22 Di Wu

Animal Cell Function
Laboratory, Nagoya
University

Assistant
Professor

Research on the vertebrate CMPsialic acid synthetase as a novel
regulatory protein of neurogenesis byinteracting with particular proteins23 Jun Shirakawa

Institute for Molecular
and Cellular Regulation(IMCR),Gunma University

Professor

Interorgan networks of islets withliver, fat, or macrophages to regulate
β-cell mass

24 Takumi Oti

Faculty of Science,
Department of Biological
Science, Kanagawa
University

Assistant
Professor

Copulatory experience and neuralmechanisms controlling male sexualfunction: focusing on the spinal
gastrin-releasing peptide receptor
neurons

25 Hirosuke Shiura

Graduate Faculty ofInterdisciplinary Research,
University of Yamanashi

Assistant
Professor

The mechanism of early placental
development regulated by a virusderived gene

The Novartis Foundation(Japan)
for the Promotion of Science

147

The Novartis Foundation(Japan)
for the Promotion of Science

#

148

Name

Institution

Title

Research Project26 Hiroki Ohara

Department of Functional
Pathology, Shimane
University

LectureDevelopment of novel preventive
and therapeutic approaches for
hypertensive disease based on
elucidation of the immunogenetic
mechanisms

27 Jun Hamazaki

Laboratory of Protein
Metabolism, The Universityof Tokyo

Assistant
Professor

Elucidation of the proteasome
maintenance mechanism and its
application to drug discovery

28 Yasuhiro Horibata

Department of Biochemistry,
Associate
Dokkyo Medical University
Professor
School of Medicine

Elucidation of the diseasedevelopment caused by disruptionof a novel phospholipid transport
factor required for mitochondrial
homeostasis

29 Mineko Kengaku

Institute for Advanced Study,
Institute for Integrated CellProfessor
Material Sciences, Kyoto
University

Molecular mechanism of dendrite
remodeling during the critical periodof brain development

30 Akira Takai

Dept. of Cell Biol., Grad.
AssistantSch. of Med.,The University
Professorof TokyoPhase separation dynamics ofpathogenic RNA granules uncovered
with novel visualization andmanipulation method of endogenousRNAs

31 Shunsuke Kitajima

Cancer Institute, Cell
Biology, JapaneseFoundation for Cancer
Research

Molecular mechanism of intrinsic
resistance to KRAS G12C inhibitor
associated with LKB1/KEAP1
mutation

32 Tsuyoshi Udagawa

Graduate School of
Pharmaceutical Sciences,
Laboratory of Biological
Chemistry, Nagoya City
University

Associate
Professor

33 Minako Hirano

Faculty of Interdisciplinary
Science and Engineering in
Health Systems, Okayama
University

Associate
Professor

Search and creation of photoactivated adenylyl cyclases for
optogenetics tools

34 Ryuichi Ohgaki

Department of Bio-system
Pharmacology, Graduate
School of Medicine, Osaka
University

Associate
Professor

Novel lipid metabolism essential forthe ceramide homeostasis in stratum
corneum

35 Takeshi Fuchigami

Laboratory of ClinicalAnalytical Sciences,
Associate
Graduate School of Medical
Professor
Sciences, Kanazawa
University

Development of multifunctional
molecular probes with high sensitivity
for detecting precancerous lesions inpancreatic cancer

36 Tatsuo Mano

Department of Degenerative
Neurological Diseases,
SectionNational Institute of
Chief
Neuroscience, National
Center of Neurology andPsychiatry

Stratification of Alzheimer's Disease
by Integrative Multi-Omics Analysis

37 Haruki Ochi

Institute for Promotion of
Medical Science Research,
Yamagata University

Research for the assembly of
PML nuclear bodies regulated by
Neddylation and the promotion of
kidney regeneration

38 Kazuhiko Matsuo

Division of Chemotherapy,
Faculty of Pharmacy, Kindai Lecturer
University

Pathological roles of a novel receptor
for chemokine ELC/CCL19 inpsoriasis

39 Tetsuya Tsujihara

Department of Medicinaland Organic Chemistry,
School of Pharmacy,Iwate
Medical University

Development and applicationof stereoselective synthesis of
cyclopropane-fused heteropolycyclic
compounds

The Novartis Foundation(Japan)
for the Promotion of Science

Staff
Scientist

Associate
Professor

Associate
Professor

Optimization of protein amino (N)terminal regions of artificial mRNAs
by an evolutionary engineering
approach

The Novartis Foundation(Japan)
for the Promotion of Science

FY2022 Research Meeting Grant(JPY 400 thousand x5 = 2.0 million)#

Meeting

Place /
Date

Institution

Name

DNA29‐29th International
1 Conference on DNA Computing
and Molecular Programming

Department of Robotics,
Sendai/
Graduate School of2023.9.11-15 Engineering, Tohoku
University/ Professor

Satoshi Murata

2nd Asian Palaeontological2
Congress

Tokyo/
2023.8.3-7

Department of Earthand Planetary Science,
The University of Tokyo/
Professor

Kazuyoshi Endo

“Molecular Movies” International
3 Symposium 2023: MolecularMovies; to be continued

Department of Life
Awaji/
Science, Graduate School
2023.11.30-12.1 of Science, University of
Hyogo/ Professor

4 10th International MDM2 workshop

Tokyo/
2023.10.15-18

5International symposium on skinstem cell dynamics

Laboratory of Fundamental
Oncology, National Cancer
Center Research Institute/
Laboratory Head

Center for Biosystems
Dynamics Research,
Tokyo/
Laboratory for Tissue2023.5.14-15
Microenvironment, RIKEN/Team Leader

Minoru Kubo

Rieko Ohki

Hironobu Fujiwara

The Novartis Foundation(Japan)
for the Promotion of Science

149

The Novartis Foundation(Japan)
for the Promotion of Science

第36期 （2022年度） 助成事業報告

当財団は、 文部大臣の認可を得て1987年9月4日に設立されて以来、 研究助成を中心とした公益事業を行って来ました。 2012年4月1日には、 制度改革に伴い、 公益財団法人へ移行しております。 2022年度は、 下記の総額4,200万円の助成事業を実施しました。
第36回ノバルティス研究奨励金
研究集会助成39件 （1件100万円）
5件 （1件 40万円）

3,900万円
200万円
総額 4,100万円

第36回ノバルティス研究奨励金 （2022年度）
この事業は、 生物 ・ 生命科学および関連する化学の領域において、 我が国で行われる創造的な研究の助成を目的としています。
（受付順、 敬称略、 所属職位は申請時、 贈呈額 ： １件 100万円）
No.

150

氏

名

所

1 佐野

宗一

2 合山

進

3 野元

4 酒井

職位

研 究 課 題大阪公立大学 大学院医学研究科循環器内科学

特任講師

心不全の悪化に関連する Y 染色体遺伝子の特定とそのメカニズムの解明

東京大学 新領域創成科学研究科
先進分子腫瘍学分野教

授

造血幹細胞指向型脂質ナノ粒子を活用した
生体内遺伝子治療法の開発

美佳

名古屋大学 遺伝子実験施設

助教

植物葉面の毛状突起トライコームが活性化する免疫応答の分子機構解明

寿郎

東京大学先端科学技術
研究センター 代謝医学分野

教

授シグナル感知型エピゲノム修飾因子による
ミトコンドリア生合成を標的とした抗肥満効果の解明

5 向井康治朗

東北大学 大学院生命科学研究科
細胞小器官疾患学分野

助教

自然免疫シグナル STING 経路の活性化機
構の解明

6 田中

知明

千葉大学 分子病態解析学講座

教

授

シングルセル解析と空間的トランスクリプ
トーム解析から捉える肝臓 2 型自然リンパ球の新たな糖代謝調節機構の解明

7 高野

晴子

日本医科大学 先端医学研究所
病態解析学部門

助教

肺胞形成機構における血管内皮細胞の役割の解明と肺胞再生治療への応用

8 金本

和也

東北大学大学院 薬学研究科

助教

ペプチドのピンポイント修飾を可能にする
三成分連結反応の開発

9 野島

孝之

九州大学 生体防御医学研究所

准教授

非コード RNA を創出するがん特異的な
転写終結機構の解明

10 宮本

潤基

テニュア
食事介入による慢性閉塞性肺疾患モデル
東京農工大学 大学院
トラック
マウスに対する治療法の確立
グローバルイノベーション研究院
准教授

11 河津

正人

千葉県がんセンター 研究所
細胞治療開発研究部

部

長

マイクロサテライト高度不安定性子宮体
がんの免疫回避のメカニズム解明

12 広瀬

久昭

京都大学 化学研究所

特定准教授

Ca イオンを基軸とした、がん細胞の栄養
取り込み機構の分子基盤

13 安部

佳亮

筑波大学 血液内科研究室

日本学術振興会特 濾胞性リンパ腫特異的免疫環境の探索
別研究員

The Novartis Foundation(Japan)
for the Promotion of Science属

The Novartis Foundation(Japan)
for the Promotion of Science

No.

氏

名

所

属

職位

14 小林

大貴

東京薬科大学 生命科学部
腫瘍医科学研究室

助教

ミトコンドリア病を標的とした新規ホスホ
フルクトキナーゼ（PFK1）阻害剤開発

15 菊池

浩二

熊本大学 染色体制御分野

講師

生殖細胞系列の分化を支えるセルトリ細胞の極性化メカニズムの解明

16 塚本

蔵

大阪大学大学院生命機能研究科
医化学講座

准教授

力学的負荷を感知する心臓特異的スーパーエンハンサー CR9 によるナトリウム利尿ペプチド遺伝子発現制御機構の解明

17 河野

洋治

岡山大学 資源植物科学研究所

教

授

NLR 免疫受容体の進化と活性化機構の解明

18 青柳

共太

杏林大学 医学部 細胞生化学准教授

膵β細胞におけるマイトファジー不全による糖尿病発症機構

19 田中

洋介

正常・異常造血幹細胞におけるカルシウ
熊本大学 国際先端医学研究機構
特任講師 ムシグナルによる幹細胞性制御機構の相幹細胞制御研究室
違点の解明と治療応用

20 岡本

暁

新潟大学 農学部助

21 山中総一郎

東京大学 理学系研究科
生物科学専攻准教授

マウス胎仔期雄性生殖細胞におけるエピ
ゲノム再構築原理の解明

22 呉

迪

名古屋大学 動物細胞機能研究室

助教

CMP- シアル酸合成酵素が神経形成を制御する新奇相互作用分子であることの研究

23 白川

純

群馬大学 生体調節研究所
代謝疾患医科学分野

教

授

膵島と肝臓、脂肪、マクロファージとの相互作用による膵β細胞量調節機構

24 越智

拓海

神奈川大学 理学部生物科学科

交 尾 経 験 に 着 目 し た 脊 髄 GRP 受 容 体
特別助教 ニューロンによる性機能制御メカニズム
の解明

25 志浦

寛相

山梨大学 大学院総合研究部

助教

ウイルス由来遺伝子がもたらした哺乳類
胎盤初期分化メカニズムの解明

26 大原

浩貴

島根大学 医学部病理学講座
病態病理学

講

師

免疫遺伝学的メカニズムの解明に基づく
高血圧性疾患の新たな予防・治療法の開発

27 濱崎

純

東京大学 大学院 薬学系研究科
蛋白質代謝学教室

助教

プロテアソーム維持機構の解明と創薬へ
の応用

28 堀端

康博

獨協医科大学 医学部生化学講座

准教授

ミトコンドリア恒常性に必要な新規リン脂質輸送因子の破綻による疾患発症機構の解明

29 見學美根子

京都大学 高等研究院
物質 - 細胞統合システム拠点教

授

臨界期脳の樹状突起リモデリングを制御する分子シグナルの解明

30 高井

啓

東京大学大学院
医学系研究科 細胞生物学分野

助教

内在性 RNA の可視化・制御法で解き明かす病原性 RNA 顆粒の相分離動態

31 北嶋

俊輔

公益財団法人がん研究会
がん研究所 細胞生物部

研究員

LKB1/KEAP1 遺 伝 子 変 異 に 伴 う KRAS
G12C 阻害剤治療抵抗性の誘導とその分子機序解明

名古屋市立大学 大学院薬学研究科遺伝情報学分野

准教授

進化工学的アプローチによる人工 mRNA のタンパク質アミノ（N）末端領域の最適化

33 平野美奈子

岡山大学 学術研究院
ヘルスシステム統合科学学域

准教授

cAMP の高時間分解能計測による光活性化 cAMP 産生酵素の探索と創製

34 大垣

大阪大学 大学院医学系研究科
生体システム薬理学

准教授

角質層セラミドの恒常性に不可欠な新たな脂質代謝機構の解明

32 宇田川剛隆一

教

研 究 課 題根から花成を制御する分子機構の研究

The Novartis Foundation(Japan)
for the Promotion of Science

151

The Novartis Foundation(Japan)
for the Promotion of Science

No.

152

氏

名

35 淵上

剛志

36 間野

職位

研 究 課 題金沢大学 医薬保健研究域薬学系 臨床分析科学研究室

准教授

膵癌の前癌病変を高感度に検出できる多機能性分子プローブ開発

達雄

国立精神・神経医療研究センター
神経研究所 疾病研究第四部

室長

統合的マルチオミクス解析によるアルツ
ハイマー病の層別化

37 越智

陽城

山形大学 医学部
メディカルサイエンス推進研究
所 生化学解析センター准教授

ネジル化修飾を介した PML ボディの凝集と腎組織再生促進の研究

38 松尾

一彦

近畿大学 薬学部
化学療法学研究室

講師

乾癬病態形成におけるケモカイン ELC/
CCL19 の新規受容体の病理的役割の解明

39 辻原

哲也

岩手医科大学 薬学部 薬科学講
座 創薬有機化学分野

准教授

シクロプロパンが縮環した複素多環式化合物の立体選択的合成法の開発と応用

The Novartis Foundation(Japan)
for the Promotion of Science

所属

The Novartis Foundation(Japan)
for the Promotion of Science

2022年度研究集会助成
この事業は、 生物 ・ 生命科学および関連する化学の領域において、 我が国で開催される国際色豊かな研究集会の助成を目的としています。 2022年度は５件の助成を行いました。
（受付順、 敬称略、 所属 ・ 職位は申請時、 贈呈額 ： １件40万円）
No.

氏

名

所

属

1

村田

智

東北大学 大学院
工学研究科
ロボティクス専攻

2

遠藤

一佳

東京大学 理学系
研究科
地球惑星科学専攻

稔

兵庫県立大学
大学院理学研究科
生命科学専攻

職位

教

教

開催地 /
開催日

研 究 集 会 名授

仙台 /2023.9.11-15

DNA29‐第 29 回 DNA
コンピューティングと分子プログラミングに関する国際会議授

東京 /2023.8.3-7

第 2 回アジア古生物学会議

授

淡路 /2023.11.30-12.1「高速分子動画」
国際シンポジウム 2023:
高分子動画 ; 今後の展開に
繋げて

3

久保

4

大木理恵子

国立がん研究センター
研究所
独立ユニット長
基礎腫瘍学ユニット

東京 /2023.10.15-18

第 10 回 国際 MDM2
ワークショップ

5藤原

理化学研究所 生命機能科学研究センター
細胞外環境研究チーム

東京 /2023.5.14-15

国際シンポジウム「皮膚幹細胞ダイナミクス」

裕展

教

チーム
リーダー

The Novartis Foundation(Japan)
for the Promotion of Science

153

The Novartis Foundation(Japan)
for the Promotion of Science

Promotion Results according to the PROGRAM(Unit : mil yen)
Research Grants
Year

Amount# ofpeopleAmountJapan-Europe
Research Exchange# ofpeopleOversea Reseach Trip
# ofpeopleAmountAmount

1987

18

1,800

8

400

0

0

0

0

1988

39

3,900

8

400

7

1,740

0

0

1989

42

4,200

8

400

9

2,260

0

0

1990

51

7,650

10

500

8

2,440

0

0

1991

55

11,000

11

550

9

2,710

9

250

1992

50

10,000

10

500

10

3,315

8

265

1993

50

10,000

10

500

11

3,511

9

300

1994

50

10,000

10

500

8

2,530

6

155

1995

50

6,500

10

500

7

2,020

6

170

1996

45

5,850

10

500

6

1,600

4

120

1997

41

4,920

10

500

6

1,610

2

55

1998

41

4,920

10

500

4

1,070

8

160

1999

41

4,920

10

500

4

710

8

160

2000

41

4,100

8

400

3

660

0

0

2001

41

4,100

7

350

2

440

0

0

2002

40

4,000

8

400

0

0

0

0

2003

40

4,000

4

200

0

0

0

0

2004

45

4,500

5

200

0

0

0

0

2005

45

4,500

5

200

0

0

0

0

2006

46

4,600

6

240

0

0

0

0

2007

50

5,000

6

240

0

0

0

0

2008

45

4,500

7

280

0

0

0

0

2009

30

3,000

6

240

0

0

0

0

2010

38

3,800

5

200

0

0

0

0

2011

41

4,100

6

240

0

0

0

0

2012

40

4,000

6

240

0

0

0

0

2013

42

4,200

5

200

0

0

0

0

2014

42

4,200

6

240

0

0

0

0

2015

35

3,500

6

240

0

0

0

0

2016

35

3,500

5

200

0

0

0

0

2017

41

4,100

5

200

0

0

0

0

2018

37

3,700

5

200

0

0

0

0

2019

37

3,700

5

200

0

0

0

0

2020

39

3,900

9

360

0

0

0

0

2021

40

4,000

5

200

0

0

0

0

2022
Total

154

# of
peopleMeeting Grants

The Novartis Foundation(Japan)
for the Promotion of Science

39

3,900

5

200

0

0

0

0

1,502

178,560

260

11,92094

26,61660

1,635

The Novartis Foundation(Japan)
for the Promotion of Science

Promotion Results according to the PROGRAM(Unit : mil yen)Year

Travel Expense to
Japan
# of
peopleSpecial Grant# ofpeopleAmountTotal # ofpeopleTotal
AmountAmount

1987

0

0

0

0

26

2,200

1988

0

0

0

0

54

6,040

1989

0

0

0

0

59

6,860

1990

0

0

0

0

69

10,590

1991

0

0

0

0

84

14,510

1992

0

0

0

0

78

14,080

1993

0

0

0

0

80

14,311

1994

0

0

2

110

76

13,295

1995

0

0

1

50

74

9,240

1996

0

0

0

0

65

8,070

1997

0

0

1

30

60

7,115

1998

0

0

0

0

63

6,650

1999

0

0

4

130

67

6,420

2000

0

0

3

142

55

5,302

2001

0

0

3

120

53

5,010

2002

0

0

0

0

48

4,400

2003

0

0

0

0

44

4,200

2004

0

0

0

0

50

4,700

2005

0

0

0

0

50

4,700

2006

0

0

0

0

52

4,840

2007

5

1,000

0

0

61

6,240

2008

5

1,000

0

0

57

5,780

2009

3

600

0

0

39

3,840

2010

0

0

0

0

43

4,000

2011

0

0

0

0

47

4,340

2012

0

0

0

0

46

4,240

2013

0

0

0

0

47

4,400

2014

0

0

0

0

48

4,440

2015

0

0

0

0

41

3,740

2016

0

0

0

0

40

3,700

2017

0

0

0

0

46

4,300

2018

0

0

0

0

42

3,900

2019

0

0

0

0

42

3,900

2020

0

0

0

0

48

4,260

2021

0

0

0

0

45

4,200

2022

0

0

0

0

44

4,100

13

2,600

14

582

1,943

221,913Total

The Novartis Foundation(Japan)
for the Promotion of Science

155

The Novartis Foundation(Japan)
for the Promotion of Science

助成金実績一覧表（単位：万円）
研究奨励金
年号

助成額

人数

日欧研究交流

助成額

人数

海外出張助成

助成金額

人数

助成金額

1987

18

1,800

8

400

0

0

0

0

1988

39

3,900

8

400

7

1,740

0

0

1989

42

4,200

8

400

9

2,260

0

0

1990

51

7,650

10

500

8

2,440

0

0

1991

55

11,000

11

550

9

2,710

9

250

1992

50

10,000

10

500

10

3,315

8

265

1993

50

10,000

10

500

11

3,511

9

300

1994

50

10,000

10

500

8

2,530

6

155

1995

50

6,500

10

500

7

2,020

6

170

1996

45

5,850

10

500

6

1,600

4

120

1997

41

4,920

10

500

6

1,610

2

55

1998

41

4,920

10

500

4

1,070

8

160

1999

41

4,920

10

500

4

710

8

160

2000

41

4,100

8

400

3

660

0

0

2001

41

4,100

7

350

2

440

0

0

2002

40

4,000

8

400

0

0

0

0

2003

40

4,000

4

200

0

0

0

0

2004

45

4,500

5

200

0

0

0

0

2005

45

4,500

5

200

0

0

0

0

2006

46

4,600

6

240

0

0

0

0

2007

50

5,000

6

240

0

0

0

0

2008

45

4,500

7

280

0

0

0

0

2009

30

3,000

6

240

0

0

0

0

2010

38

3,800

5

200

0

0

0

0

2011

41

4,100

6

240

0

0

0

0

2012

40

4,000

6

240

0

0

0

0

2013

42

4,200

5

200

0

0

0

0

2014

42

4,200

6

240

0

0

0

0

2015

35

3,500

6

240

0

0

0

0

2016

35

3,500

5

200

0

0

0

0

2017

41

4,100

5

200

0

0

0

0

2018

37

3,700

5

200

0

0

0

0

2019

37

3,700

5

200

0

0

0

0

2020

39

3,900

9

360

0

0

0

0

2021

40

4,000

5

200

0

0

0

0

2022
Total

156

人数

研究集会

The Novartis Foundation(Japan)
for the Promotion of Science

39

3,900

5

200

0

0

0

0

1,502

178,560

260

11,92094

26,61660

1,635

The Novartis Foundation(Japan)
for the Promotion of Science

助成金実績一覧表（単位：万円）
海外受入
年号

特別助成
人数

助成金額

人数計

金額合計

助成人数

助成金額

1987

0

0

0

0

26

2,200

1988

0

0

0

0

54

6,040

1989

0

0

0

0

59

6,860

1990

0

0

0

0

69

10,590

1991

0

0

0

0

84

14,510

1992

0

0

0

0

78

14,080

1993

0

0

0

0

80

14,311

1994

0

0

2

110

76

13,295

1995

0

0

1

50

74

9,240

1996

0

0

0

0

65

8,070

1997

0

0

1

30

60

7,115

1998

0

0

0

0

63

6,650

1999

0

0

4

130

67

6,420

2000

0

0

3

142

55

5,302

2001

0

0

3

120

53

5,010

2002

0

0

0

0

48

4,400

2003

0

0

0

0

44

4,200

2004

0

0

0

0

50

4,700

2005

0

0

0

0

50

4,700

2006

0

0

0

0

52

4,840

2007

5

1,000

0

0

61

6,240

2008

5

1,000

0

0

57

5,780

2009

3

600

0

0

39

3,840

2010

0

0

0

0

43

4,000

2011

0

0

0

0

47

4,340

2012

0

0

0

0

46

4,240

2013

0

0

0

0

47

4,400

2014

0

0

0

0

48

4,440

2015

0

0

0

0

41

3,740

2016

0

0

0

0

40

3,700

2017

0

0

0

0

46

4,300

2018

0

0

0

0

42

3,900

2019

0

0

0

0

42

3,900

2020

0

0

0

0

48

4,260

2021

0

0

0

0

45

4,200

2022
Total

0

0

0

0

44

4,100

13

2,600

14

582

1,943

221,913

The Novartis Foundation(Japan)
for the Promotion of Science

157

The Novartis Foundation(Japan)
for the Promotion of Science

36th Financial Report

Balance Sheet

Statement of Net Assets

As of March 31, 2023

From April 1 st, 2022 to March 31, 2023
（Unit：JP Yen）

Account

Account

AmountⅠ Assets

Ⅰ General Net Assets Changes

1. Current Assets

1. Ordinary income & Expenditure

Current Assets Total

20,972,711

2. Fixed Assets(1) Basic Fund
Basic Fund Total

1,100,000,000(2) Specific Assets
Specific Assets Total-

Other Long - term Assets Total
Fixed Assets Total
Assets Total

85,037,657

17,442,402

Donation

40,010,594

Other Income

649,205
58,102,201(2) Ordinaty Expenditure
Project Expense

10,396,837

Grant Expense

41,000,000

Novartis Research Grant

39,000,000

1,206,010,368

Research Meeting Grant

2,000,000

Administrative Expense

3,956,142

Ordinary Expenditure Total

55,352,979

41,126,432

Ordinary Balance without Appraisal Profit or Loss

2,749,222

41,126,432

2. Nonrecurring Profit & Loss

1. Current Liabilities

Liabilities Total

Interest from basic fund

1,185,037,657

Ⅱ Liabilities

Current Liabilities Total

Amount(1) Ordinary income

Ordinary Income Total(3) Other Long - term Assets

Ⅲ Equity（Net Assets）

Nonrecurring Balance of Current Period

01. Designated Net Assets

General Net Assets Ending Balance

164,883,936

Designated Net Assets Total（Amount Appropriating to basic Fund）(Amount Appropriating to specific assets)

158（Unit：JP Yen）

1,000,010,000

Ⅱ Designated Net Assets Changes(1,000,000,000)

Designated Net Assets Change(10,594)0

Designated Net Assets Ending Balance

1,000,000,000

2. General Net Assets

164,883,936

Ⅲ Net Assets Balance Ending Balance

1,164,883,936（Amount Appropriating to）(100,000,000)

Equity Total（Net Assets）

1,164,883,936

Liabilities & Equity Total

1,206,010,368

The Novartis Foundation(Japan)
for the Promotion of Science

The Novartis Foundation(Japan)
for the Promotion of Science

第36期 （2022年度） 財務報告

貸借対照表

正味財産増減計算書

2023 年 3 月 31 日現在

2022 年 4 月 1 日から 2023 年 3 月 31 日まで（単位：円）

科

目

金 額

科

Ⅰ資産の部

20,972,711⑴ 基本財産1,100,000,000⑵ 特定資産特定資産合計固定資産合計資産合計-

85,037,657

649,205
58,102,201⑵ 経常費用

支払助成金

10,396,837
41,000,000

研究集会助成金

2,000,000

管理費

3,956,142

経常費用 計

55,352,979

41,126,432

当期経常増減額

2,749,222

41,126,432

２． 経常外増減の部
当期経常外増減額
一般正味財産期末残高

1,000,010,000（うち基本財産への充当額）(1,000,000,000)（うち特定資産への充当額）(0)

負債及び正味財産合計

雑収益

1,206,010,368

指定正味財産合計

正味財産合計40,010,594

39,000,000

１． 指定正味財産（うち基本財産への充当額）

受取寄付金

ノバルティス研究奨励金

Ⅲ正味財産の部

２． 一般正味財産

17,442,402

1,185,037,657

１． 流動負債

負債合計

基本財産運用益

事業費

Ⅱ負債の部

流動負債合計⑴ 経常収益

経常収益 計⑶ その他固定資産
その他固定資産合計

金 額

１． 経常増減の部

２． 固定資産

基本財産合計目

Ⅰ一般正味財産増減の部

１． 流動資産
流動資産合計（単位：円）

164,883,936

0
164,883,936

Ⅱ指定正味財産増減の部
当期指定正味財産増減額
指定正味財産期末残高
Ⅲ正味財産期末残高(10,594)
1,000,000,000
1,164,883,936(100,000,000)
1,164,883,936
1,206,010,368

The Novartis Foundation(Japan)
for the Promotion of Science

159

The Novartis Foundation(Japan)
for the Promotion of Science[Board of Trustees] 5 trustees, 2 auditors

As of July 1, 2023

Post

Name

Chairman

Kuniaki Takata,
Ph.D.

President, Gunma Prefectural Public University Corporation

Sadayoshi Ito,
M.D., Ph.D.

Special Administrator, Katta General Hospital
Professor Emeritus, Tohoku University

Akimichi Kaneko,
M.D., Ph.D.

Professor Emeritus, Keio University

Fujio Murakami,
Ph.D.

Professor Emeritus, Osaka University

Trustee

Leo Lee

Title

President, Novartis Pharma K.K.

Tokuzo Nakajima,
CPA

Representative, Tokuzo Nakajima CPA Firm

Masanori Fuse

Financial Advisor, Novartis Pharma K.K.

Auditor[Board of Councilors] 10 councilors

As of July 1, 2023

Post

Name

Title

ChairmanTakao Shimizu,
M.D., Ph.D.

Director, Institute of Microbial Chemistry, Microbial Chemistry
Research Foundation
Project Leader, National Center for Global Health and Medicine
Professor Emeritus, University of Tokyo

Masamitsu Iino,
M.D., Ph.D.

Special Advisor, International Research Center for
Neurointelligence, University of Tokyo
Professor Emeritus, University of Tokyo

Hiroyuki Kawashima,
Ph.D

Former Professor, Niigata University

Tsuneyoshi Kuroiwa,
Ph.D.

Member of the Japan Academy;
Professor Emeritus, University of Tokyo

Masakatsu Shibasaki, President, Microbial Chemistry Research Foundation
Ph.D.
Professor Emeritus, University of Tokyo
Councilor

160

The Novartis Foundation(Japan)
for the Promotion of Science

Akihiko Nakano,
Ph.D.

Senior Advisor/Deputy Director, RIKEN Center for Advanced
Photonics
Professor Emeritus, University of Tokyo

Yoichi Nabeshima,
M.D., Ph.D.

Special Appointed Professor, Kyoto University Graduate School of
Medicine
Professor Emeritus, Kyoto University

Toyoshi Fujimoto,
M.D., Ph.D.

Research Professor, Juntendo University
Professor Emeritus, Nagoya University

Miwako Mori,
Ph.D.

Professor Emeritus, Hokkaido University

Tohru Hirose,
Ph.D.

Managing Director, Novartis Pharma K.K.
Head, Global Drug Development, Novartis Pharma K.K.

The Novartis Foundation(Japan)
for the Promotion of Science[Grantee Selection Committee] 20 members

As of July 1, 2023

Post

Name

Title

Chairman

Yasuteru Urano,
Ph.D.

Professor, University of Tokyo Graduate School of Pharmaceutical
Sciences

Masaki Ieda,
M.D., Ph.D.

Professor, Keio University School of Medicine

Erina Kuranaga,
Ph.D.

Professor, Tohoku University Graduate School of Life Sciences

Mitinori Saito,
M.D., Ph.D.

Professor, Kyoto University Institute for Advanced Study

Fumitoshi Kakiuchi,
Ph.D.

Professor, Keio University School of Fundamental Science andTechnology

Hiroshi Kawasaki,
M.D., Ph.D.

Professor, Kanazawa University Faculty of Medicine

Shoen Kume,
Ph.D.

Professor, Tokyo Institute of Technology School of Life Science andTechnology

Kiyoshi Takeda,
M.D., Ph.D.

Professor, Osaka University Graduate School of Medicine

Kazuhiro Nakamura,
Ph.D.

Professor, Nagoya University Graduate School of Medicine

Tetsuya Higashiyama,
Professor, University of Tokyo Graduate School of Science
Ph.D.
Member

Sachiko Miyake,
M.D., Ph.D.

Professor, Juntendo University Graduate School of Medicine

Hozumi Motohashi,
M.D., Ph.D.

Professor, Tohoku University Institute of Development, Aging andCancerYoshihiro Sato,
Ph.D

Professor, Hokkaido University Graduate School of Pharmaceutical
Sciences

Yutaka Takahashi
M.D., Ph.D.

Professor, Nara Medical University

Yasushi Sakata,
M.D., Ph.D.

Professor, Osaka University Graduate School of Medicine

Osamu Takeuchi,
M.D., Ph.D.

Professor, Kyoto University Graduate School of Medicine

Osamu Nureki,
Ph.D.

Professor, University of Tokyo Graduate School of Science

Akiko Hayashi-Takagi,
Team leader, RIKEN Center for Brain Science
M.D., Ph.D.
Yu Hayashi,
Ph.D.

Professor, University of Tokyo Graduate School of Science

Yasuyuki Fujita,
M.D., Ph.D

Professor, Kyoto University Graduate School of Medicine

The Novartis Foundation(Japan)
for the Promotion of Science

161

The Novartis Foundation(Japan)
for the Promotion of Science

公益財団法人ノバルティス科学振興財団［理事 ・ 監事］
任期2022年6月15日～ 2024年6月

2023年7月1日現在 ( 敬称略 )

職 名

氏 名

代表理事

高田 邦昭

群馬県公立大学法人 理事長

伊藤 貞嘉

公立刈田綜合病院 特別管理者
東北大学名誉教授

金子 章道

慶應義塾大学名誉教授

村上富士夫

大阪大学名誉教授

レオ リー

ノバルティス ファーマ株式会社 代表取締役社長

理 事

現

職

任期2020年6月17日～ 2024年6月
中嶋 德三

公認会計士 中嶋德三事務所

布施 正則

ノバルティス ファーマ株式会社 企画管理本部ファイナンシャルアドバイザー

監 事

［評議員］
任期2020年6月17日～ 2024年6月
職 名

氏 名

評議員長

清水 孝雄

公益財団法人 微生物化学研究会 微生物化学研究所 所長
国立国際医療研究センター プロジェクト長
東京大学名誉教授

飯野 正光

東京大学ニューロインテリジェンス国際研究機構 機構長特別補佐
東京大学名誉教授

川島 博行

元新潟大学大学院医歯学総合研究科 教授

黒岩 常祥

日本学士院会員
東京大学名誉教授

柴﨑 正勝

公益財団法人 微生物化学研究会 理事長
東京大学名誉教授

中野 明彦

理化学研究所 光量子工学研究センター 特別顧問 ・ 副センター長
東京大学名誉教授

鍋島 陽一

京都大学 大学院医学研究科 特任教授
京都大学名誉教授

藤本 豊士

順天堂大学 大学院医学研究科 特任教授
名古屋大学名誉教授

森 美和子

北海道大学名誉教授

廣瀬

ノバルティス ファーマ株式会社 常務取締役グローバル医薬品開発本部長

評議員

162

2023年7月1日現在 ( 敬称略 )The Novartis Foundation(Japan)
for the Promotion of Science

徹

現

職

The Novartis Foundation(Japan)
for the Promotion of Science［選考委員］
2023年7月1日現在 （敬称略）
職 名

氏 名

選考委員長

浦野 泰照

東京大学 大学院薬学系研究科 教授

家田 真樹

慶應義塾大学 医学部 教授

倉永英里奈

東北大学 大学院生命科学研究科 教授

斎藤 通紀

京都大学 高等研究院 教授

垣内 史敏

慶應義塾大学 理工学部 教授

河崎 洋志

金沢大学 医学系 教授

粂昭苑

東京工業大学 生命理工学院 教授

竹田潔

大阪大学 大学院医学系研究科 教授

選考委員

現

中村 和弘

名古屋大学 大学院医学系研究科 教授

東山 哲也

東京大学 大学院理学系研究科 教授

三宅 幸子

順天堂大学 大学院医学研究科 教授

本橋ほづみ

東北大学 加齢医学研究所 教授佐藤 美洋

北海道大学 大学院薬学研究院 教授

高橋

奈良県立医科大学 教授

裕

坂田 泰史

大阪大学 大学院医学系研究科 教授

竹内理

京都大学 大学院医学研究科 教授

濡木理

東京大学 大学院理学系研究科 教授

職

林 ( 高木 ) 朗子

理化学研究所 脳神経科学研究センター チームリーダー

林悠東京大学 大学院理学系研究科 教授

藤田 恭之

京都大学 大学院医学研究科 教授

The Novartis Foundation(Japan)
for the Promotion of Science163

事 務 局 便 り

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事務局より
2023年度も、 おかげさまで財団年報を発行する運びとなりました。 2020年に世界中に大きな影響を及ぼした新型コロナウイルス感染症はいまだ収束しない状況ですが、 ワクチンやデジタルの発展により、 新しいスタイルのもとで かなりコロナ前の状況に戻った感があります。
当財団の研究奨励金助成や研究集会助成事業については、 今年度も多くのご応募がありました。 基礎研究にむけた研究者の方々の熱意を感じております。
当財団は1987年9月の財団設立以来、 助成件数は総数で1,943件、 総額で約22.2億円となりました。 当財団は、 自然科学の創造的研究への助成によって、日本の学術発展に寄与することを目指しており、 助成を受けられた研究成果がすぐに応用につながらなくとも、 将来、 新分野につながることを夢見て、 この事業を継続して参ります。
今まで助成事業が継続できていることも、 偏に、 助成事業にご理解 ・ ご支援をいただいた方々、 助成を受けられて研究を継続された皆様および財団関係者のお力添えの賜物であると、 心より感謝申し上げます。 引き続きご指導、 ご支援の程よろしくお願い申し上げます。
事務局長 原 健記
2023年10月13日〒105-6333 東京都港区虎ノ門1-23-1
虎ノ門ヒルズ森タワー
TEL：03-6899-2100
E-メール：foundation.japan@novartis.com
ホームページ：https://japanfoundation.novartis.org/