Annual Report (2017)

No.29
The NOVARTIS Foundation (Japan)
for the Promotion of Science
平成29年度

財団年報 第29号

The Novartis Foundation(Japan)
for the Promotion of Science

Contents
Ⅰ．Introduction はじめに ……………………………………………………………………… 5
Akimichi Kaneko, MD, PhD
Chairman of the Board of Trustees

Ⅱ．Reports from the Recipients of Novartis Research Grants

研究報告 ………………………………………………………………………………………… 7
1. LY6E; a Conductor of malignant tumor growth through modulation of the PTEN/PI3K/
Akt/HIF-1 axis ……………………………………………………………………………… 8
Hiroshi HARADA
Radiation Biology Center, Kyoto University

2. Control of host immune responses by virulent cell-wall lipids of Mycobacterium
tuberculosis through innate lipid pattern-recognition receptors. ……………………… 11
Hiromitsu Hara
Department of Immunology, Graduate School of Medical and
Dental Sciences, Kagoshima University

3. Research of glycosylation that dynamically regulates innate immunity ………………… 13
Satoshi Goto
Rikkyo University

4. Development of therapy for multiple sclerosis by regulation of neural circuit formation
factor LOTUS expression …………………………………………………………………… 15
Kohtaro Takei
Yokohama City University Graduate School of Medical Life Science

5. Molecular mechanism of epigenetic dysregulation in myelofibrosis-initiating cells …… 18
Goro Sashida
Kumamoto University, International Research Center for Medical Sciences

6. Leukemogenesis caused by a defect of the ribosomal RNA processing ………………… 21
Hirotaka Matsui
Department of Molecular Laboratory Medicine, Faculty of Life Sciences, Kumamoto
University

7. Induction of antigen-specific regulatory T cells by dendritic cells in human …………… 24
Sayuri Yamazaki
Nagoya City University, Department of Immunology

8. Study on the neuronal mechanisms for depression and anxiety induced by chronic stressand pain ……………………………………………………………………………………… 27
Masabumi Minami
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Hokkaido University

9. Pathogenesis of cardiomyopathy by RBM20 mutations modeled in genetically modified
human iPS cells ……………………………………………………………………………… 30
Yuichiro Miyaoka
Tokyo Metropolitan Institute of Medical Science

The Novartis Foundation(Japan)
for the Promotion of Science

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

Contents
Ⅰ．Introduction はじめに ……………………………………………………………………… 5
Akimichi Kaneko, MD, PhD
Chairman of the Board of Trustees

Ⅱ．Reports from the Recipients of Novartis Research Grants

研究報告 ………………………………………………………………………………………… 7
1. LY6E; a Conductor of malignant tumor growth through modulation of the PTEN/PI3K/
Akt/HIF-1 axis ……………………………………………………………………………… 8
Hiroshi Harada
Radiation Biology Center, Kyoto University

2. Control of host immune responses by virulent cell-wall lipids of Mycobacterium
tuberculosis through innate lipid pattern-recognition receptors. ……………………… 11
Hiromitsu Hara
Department of Immunology, Graduate School of Medical and
Dental Sciences, Kagoshima University

3. Research of glycosylation that dynamically regulates innate immunity ………………… 13
Satoshi Goto
Rikkyo University

4. Development of therapy for multiple sclerosis by regulation of neural circuit formation
factor LOTUS expression …………………………………………………………………… 15
Kohtaro Takei
Yokohama City University Graduate School of Medical Life Science

5. Molecular mechanism of epigenetic dysregulation in myelofibrosis-initiating cells …… 18
Goro Sashida
Kumamoto University, International Research Center for Medical Sciences

6. Leukemogenesis caused by a defect of the ribosomal RNA processing ………………… 21
Hirotaka Matsui
Department of Molecular Laboratory Medicine, Faculty of Life Sciences, Kumamoto
University

7. Induction of antigen-specific regulatory T cells by dendritic cells in human …………… 24
Sayuri Yamazaki
Nagoya City University, Department of Immunology

8. Study on the neuronal mechanisms for depression and anxiety induced by chronic stressand pain ……………………………………………………………………………………… 27
Masabumi Minami
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Hokkaido University

9. Pathogenesis of cardiomyopathy by RBM20 mutations modeled in genetically modified
human iPS cells ……………………………………………………………………………… 30
Yuichiro Miyaoka
Tokyo Metropolitan Institute of Medical Science

The Novartis Foundation(Japan)
for the Promotion of Science

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

10. Chemical investigation of the food-poisoning epidemics caused by the mushroom
Pleurocybella porrigens. ……………………………………………………………………… 33
Tomohiro Suzuki
Center for Bioscience Research and Education, Utsunomiya University

11. Developing analysis of genome replication in single cell to elucidate molecular
dynamics of DNA polymerases …………………………………………………………… 36
Yasukazu Daigaku
Frontier Research Institute for Interdisciplinary Sciences, Tohoku University

12. A novel lincRNA, Linc-Heart, regulates cell cycle and proliferation of adultcardiomyocytes ……………………………………………………………………………… 39
Masaharu Kataoka
Department of Cardiology, Keio University School of Medicine

13. Thymic positive selection impacts T cell immune responses in vivo …………………… 43
Kensuke Takada
Institute of Genome Research, Univetsity of Tokushima(Present affiliation: Faculty of Veterinary Medicine, Hokkaido University)

14. Role of junctophilin for the proper function and intracellular distribution of L-type
calcium channels …………………………………………………………………………… 46
Tsutomu Nakada
Shinshu University School of Medicine

15. Epigenetic regulation in cell reprogramming and chimera formation. ………………… 49
Daiji Okamura
Department of Advanced Bioscience, Graduate School of Agriculture, Kindai University

16. Analysis of T2DM pathogenic mechanisms caused by susceptibility genes using
human iPS cells ……………………………………………………………………………… 51
Shun-ichiro Asahara
Kobe University Graduate School of Medicine

17. Molecular mechanism of plant cell growth via NIMA-related kinases ………………… 53
Hiroyasu Motose
Okayama University

18. Molecular mechanism of overeating regulated by cellular stress response …………… 56
Takao Iwawaki
Kanazawa Medical University

19. Analysis of a signal through ER required for the induction of axon regeneration …… 59
Naoki Hisamoto
Graduate school of Science, Nagoya University

20. Development of molecular delivery system using cell membrane penetrating DNA
nanostructures ………………………………………………………………………………… 62
Masayuki Endo
Institute for Integrated Cell-Material Sciences, Kyoto University

21. Studies toward total syntheses of biologically active, bridged polycyclic
meroterpenoids ……………………………………………………………………………… 65
Seiichi Nakamura
Nagoya City University

The Novartis Foundation(Japan)
for the Promotion of Science

2

The Novartis Foundation(Japan)
for the Promotion of Science22. Functional significance of chandelier cell in cortical microcircuit ……………………… 68
Yoshiyuki Kubota
National Institute for Physiological Sciences

23. Characterization of T-follicular regulatory cells in human blood and tumor tissues … 71
WING, James
Immunology Frontier Research Center (IFReC), Osaka University

24. Conformational plasticity of JRAB regulates transition between mesenchymal andamoeboid invasiveness in cancer cells. …………………………………………………… 74
Ayuko Sakane
Tokushima University Graduate School of Medical Sciences, Department of Biochemistry

25. Development of apoptosis inhibitor targeting mitochondria …………………………… 77
Kenji Matsumoto
Tokushima Bunri University, Faculty of pharmaceutical sciences

26. Cortical synaptic plasticity for coexistence of chronic pain and emotions …………… 80
Kohei Koga
Department of Neurophysiology, Hyogo College of Medicine

27. Functions and expression of synaptotagmin 13 driving peritoneal metastasis of gastriccancer cells …………………………………………………………………………………… 83
Mitsuro Kanda
Department of Gastroenterological Surgery (Surgery II),
Nagoya University Graduate School of Medicine

28. Epigenetic and biological analysis of human cancer-associated fibroblasts …………… 85
Masayuki Shimoda
Keio University School of Medicine

29. Study on the RNA splicing checkpoint mechanism to prevent the accumulation ofpre-mRNA …………………………………………………………………………………… 88
Daisuke Kaida
University of Toyama

30. Sensing ECM stiffness may trigger the transition from Solid-like to Liquid-likebehaviour in collective cell migration of cranial neural crest cells …………………… 90
Sei Kuriyama
Akita University

31. Functional analysis of a novel silencing mechanism in higher plants ………………… 93
Kei-ichiro Mishiba
Graduate School of Life and Environmental Science, Osaka Prefecture University

32. The origin of animal multicellular system – an approach from genome editing of
unicellular holozoans ………………………………………………………………………… 96
Hiroshi Suga
Department of Environmental Life Sciences, Prefectural University of Hiroshima

33. Regulating microtubule arrays for neuronal dendrite growth and for neuronal
regeneration …………………………………………………………………………………… 99
Adrian Walton Moore
RIKEN Brain Science Institute

The Novartis Foundation(Japan)
for the Promotion of Science

3

The Novartis Foundation(Japan)
for the Promotion of Science

34. A novel network between mature adipocytes and endothelial cells that regulates
adipose tissue angiogenesis and homeostasis ………………………………………… 102
Koji Ikeda
Kobe Pharmaceutical University

35. Investigation of the mechanism of cell fate determination independent fromtranscriptional factor ……………………………………………………………………… 104
Ai Kotani
Department of Hematology and Oncology, Tokai University School of Medicine
Department of Hematological Malignancy, Tokai University The Institute of Medical
Sciences

Ⅲ．Reports from the Recipients of Grants for International Meetings
研究集会報告………………………………………………………………………………… 106
1. KTCC 2017:
7th International Workshop of Kyoto T Cell Conference ……………………………… 107
Hiroshi Kawamoto
Kyoto University/Professor

2. CeNeuro2016 & Nagoya BNC Joint Meeting …………………………………………… 109
Ikue Mori
Professor, Graduate School of Science, Nagoya University

3. 20th International Symposium on Homogeneous Catalysis …………………………… 111
Kyoko Nozaki

4. The 4th International Symposium on Salivary Glands in Honor of Niels Stensen …… 112
Masataka Murakami

5. 5th International Conference for Food and Environmental Virology (ISFEV2016) … 113
Hiroyuki Katayama
University of Tokyo (ISFEV2016 Chair)

Ⅳ．The 30th (fiscal year 2016) Grant Report

第30期（2016年度）助成事業報告 ……………………………………………………… 115

Ⅴ．The 30th (fiscal year 2016) Financial Report

第30期（2016年度）財務報告 …………………………………………………………… 121

Ⅵ．List of the Board Members

役員名簿 ……………………………………………………………………………………… 123

Ⅶ．Information from the Secretariat

事務局便り …………………………………………………………………………………… 127

The Novartis Foundation(Japan)
for the Promotion of Science

4

The Novartis Foundation(Japan)
for the Promotion of Science

Introduction

Akimichi Kaneko, MD, PhD
Chairman of the Board of Trustees
We have compiled here the research accomplishments of the grantees of the NOVARTISFoundation (Japan) for the Promotion of Science in FY2015. The Foundation was originallyestablished on September 4, 1987 with the generous donation of JPY 1 billion from CibaGeigy AG, Switzerland, for the purpose of “contributing to academic development and thus
improving public health and welfare of the people by subsidizing to promote creative researchand international exchange in the fields of biological and life sciences and related chemistry”.
Since then, the Foundation has granted nearly JPY 2 billion to approximately 1,700 researchcases in 29 years. Research funds provided by the Foundation may be only a part of the cost of
the entire research, however, we have continued to subsidize in belief that we could contributeto the development of excellent research. Joy of grantees could be felt in the acknowledgmentof the papers received by the Secretariat, and we are pleased with our humble contribution.
The Foundation supports creative research in the fields of biological and life sciences, related
chemistry and information science. Numerous entries submitted are examined by specialized
researchers in a strict, fair and impartial manner. While we value originality as selection criteria,
considerations are given to those start-up laboratories in urgent needs of research funds, aswell as age and women researchers. In each case, we believe that excellent research is selected.
In fact, we are informed that the quality of the research funded by the Foundation is highly
esteemed by researchers in Japan, proving our purposes have not been wrong. We hope thatthese researches will lead the development of life sciences, and eventually to the developmentof new drugs and therapies.
This Annual Report summarizes the excellent research outcomes. It is an outstandingachievement accomplished within a limited period of one year. We are deeply grateful toeveryone who supported the activities of the Foundation, including the selection committee
members who have selected these excellent researches.

The Novartis Foundation(Japan)
for the Promotion of Science

5

The Novartis Foundation(Japan)
for the Promotion of Science

はじめに

代表理事

金子

章道

ここに2015年度にノバルティス科学振興財団研究助成金を受けられた方々の研究報告を収録いたしました。当財団は1987年９月４日、スイス、チバガイギー社からの10億円のご寄附をもとに、「生物・生命科学および関連する化学の領域において、創造的な研究ならびに国際交流への助成を行うことにより、学術の振興を図り国民の健康と福祉の向上に寄与する」ことを目的に設立されました。爾来29年間に1,700件
近く、金額にしておよそ20億円の助成を行ってまいりました。当財団が提供する研究
費は、研究全体に要する費用の一部かも知れませんが、優れた研究の発展に少しでも寄与できればと考え、助成して参りました。事務局に寄せられた論文等の謝辞からも受賞者の喜びが伝わり、多少なりとも貢献できているものと嬉しく思っております。
当財団は、生物・生命科学、関連する化学および情報科学の領域における創造的な研究を助成しております。数多く寄せられた応募を専門の研究者により厳正に公正
かつ公平に審査しております。選考基準と致しまして独創性を重視しておりますが、研究室を新たに立ち上げ研究資金に逼迫しているケースや年齢、女性研究者にも配慮しており、いずれも優れた研究が選択されたものと存じます。事実、当財団で助成を受けた研究の質が高いと国内の研究者の間で評価されていることを聞くにつけ、我々の目指すところは間違っていないことを確信する次第であります。このような研究が生命科学の発展、ひいては新しい治療薬の開発や治療法の開発に発展することを期待しております。
この年報にはこのような優れた研究の成果をまとめております。１年間と限られた時間の中で達成した立派な業績であります。なお、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をはじめ、財団の活動を支えて下さっている関係者の皆様に深く感謝いたします。

The Novartis Foundation(Japan)
for the Promotion of Science

6

The Novartis Foundation(Japan)
for the Promotion of ScienceII.
Reports from the Recipients ofNovartis Research Grants

The Novartis Foundation(Japan)
for the Promotion of Science

7

The Novartis Foundation(Japan)
for the Promotion of Science

LY6E; a Conductor of malignant tumor growth through modulation of
the PTEN/PI3K/Akt/HIF-1 axis
Hiroshi Harada
Harada.hiroshi.5e@kyoto-u.ac.jp
Radiation Biology Center, Kyoto University

Summary Abstract
Lymphocyte antigen 6 complex, locus E (LY6E) has been implicated in the malignant progressionof various types of cancers; however, the underlying mechanism remains unclear. Here, we identified
LY6E as an activator of HIF-1 and revealed their mechanistic and functional links in malignant
tumor growth(1). The aberrant overexpression of LY6E increased HIF-1α gene expression principallyat the transcription level. This, in turn, led to the expression of the pro-angiogenic factors, VEGFA
and PDGFB, through decreases in the expression levels of PTEN mRNA and subsequent activationof the PI3K/Akt pathway. The LY6E-HIF-1 axis functioned to increase tumor blood vessel densityand promoted tumor growth in immunodeficient mice. LY6E expression levels were significantly
higher in human breast cancers than in normal breast tissues, and were strongly associated with thepoor prognoses of various cancer patients. Our results characterized LY6E as a novel conductorof tumor growth through its modulation of the PTEN/PI3K/Akt/HIF-1 axis and demonstrated thevalidity of targeting this pathway for cancer therapy.
Key Words：tumor hypoxia; hypoxia-inducible factor 1 (HIF-1); Lymphocyte antigen 6 complex,
locus E (LY6E); PTEN/PI3K/Akt/HIF-1 Axis; angiogenesis

Introduction
Accumulating evidence has shown that HIF-1 plays critical roles in radioresistance of cancers andtumor recurrence post-radiotherapy(2) and eventually causes death of cancer patients. Clinical studies
have demonstrated consistent data that HIF-1 could be used as an adverse prognostic factor for not
only local tumor recurrence but also distant tumor metastasis in cancer patients. These findings
justify targeting HIF-1 for cancer therapies(2).
HIF-1, a transcription factor composed of an α-subunit (HIF-1α) and a β-subunit (HIF-1α), its
activity is known to depend on the expression levels of HIF-1α protein(2). Under normoxic conditions,
HIF-1α protein is actively degraded through the hydroxylation- and subsequent ubiquitinationmediated proteolysis reactions. On the contrary, HIF-1α becomes active under hypoxic conditions
because of the inactivation of the hydroxylases, and then, interacts with its binding partner, HIF1β. Resultant heterodimer, HIF-1, binds to its cognate enhancer sequence, the hypoxia-responsive
element (HRE), and induces transcriptions of various genes related to the escape from hypoxia(invasion and metastasis of cancer cells) as well as the improvement of oxy-gen-availability(angiogenesis) and adaptation of cellular metabolism to hypoxia (metabolic reprogramming)(2).

Results
In order to explore novel genes which are responsible for the HIF-1-mediated tumor malignant
progression, we recently established a new genetic screening method (1,3,4) and found that

The Novartis Foundation(Japan)
for the Promotion of Science

8

The Novartis Foundation(Japan)
for the Promotion of Science

overexpression of lymphocyte antigen 6 complex, Fig. 1
locus E (LY6E) functions in the activation of
HIF-1(1). In the present study, we analyzed boththe molecular mechanisms underlying the LY6Emediated activation of HIF-1 and the involvementof the LY6E-HIF-1 axis in malignant progressionof cancers.
Forced expression of LY6E using a plasmidbased expression vector for LY6E increased HIF1α gene expression principally at the transcription
level (Figure 1).
Fig. 2
This, in turn, led to the expression of the proangiogenic factors, VEGFA and PDGFB, throughdecreases in the expression levels of PTEN mRNAand subsequent activation of the PI3K/Akt pathway.
The LY6E-HIF-1 axis functioned to increase tumor
blood vessel density and promoted tumor growth inimmune-deficient mice (Figure 2).
LY6E expression levels were significantly higher in human breast cancers than in normal breast
tissues, and were strongly associated with the poor prognoses of various cancer patients (Figure 3).
Fig. 3

Discussion & Conclusion
Our results characterized LY6E as a novel conductor of tumor growth and tumor radioresistancethrough its modulation of the PTEN/PI3K/Akt/HIF-1 axis and demonstrated the validity of targeting
this pathway for cancer therapy.

References(1) Yeom CJ. et al. Oncotarget. 11670. 2016.
(2) *Harada H. J Radiat Res. 57:99-105. 2016.
(3) Zeng L. et al. Oncogene. 34:4758-4766. 2015.
(4) Goto Y. et al. Nature Communications. 6: 6153.2015.

The Novartis Foundation(Japan)
for the Promotion of Science

9

The Novartis Foundation(Japan)
for the Promotion of Science

一般の皆様へ
がんには個性があり、治療が効きやすいタイプや効きにくいタイプ、転移をし易いタイプやしにくいタイプなど、患者さんによって患っているがんのタイプは様々です。私達は、がんの悪性度と治療抵抗性を左右する原因遺伝子の一つとして、LY6E を発見しました。そして LY6E
蛋白質を豊富に含むがんは増殖が極めて速く、治療がなかなか効きにくいこと、そして結果的に患者さんの生存期間が短くなってしまうことを確認しました。この研究結果を基に、LY6Eを標的とする新たな治療法の開発に向け、研究を続けています。

The Novartis Foundation(Japan)
for the Promotion of Science

10

The Novartis Foundation(Japan)
for the Promotion of Science

Control of host immune responses by virulent cell-wall lipids of
Mycobacterium tuberculosis through innate lipid
pattern-recognition receptors.
Hiromitsu Hara
harah@m2.kufm.kagoshima-u.ac.jp
Department of Immunology, Graduate School of Medical and
Dental Sciences, Kagoshima University

Summary Abstract
Mycolic acid (MA) and Phenolic glycolipid (PGL) are lipids identified as virulent lipids of
Mycobacterium tuberculosis that suppress host immunity. We identified ITAM-coupled innate
immune receptors, MAR and PGLR, that recognize MA and PGL, respectively. Macrophage
response to MAR or PGLR was markedly impaired in MAR or PGLR-deficient cells, respectively.
In addition, MAR-deficient cells as well as mice showed enhanced Mincle-mediated innate immune
response essential for host protection to M. tuberculosis. Thus, MAR acts as a negative regulator of
Mincle and may contribute to immune evasion of M. tuberculosis through the recognition of MA.
Key Words：Tuberculosis, innate immunity

Introduction
M. tuberculosis contains various unique lipids in the cell walls that act on the activation or
suppression of immunity. Although structure and function of such lipids have been extensively
studied, the host receptor for them was not well understood. Mycolic acid (MA) and Phenolic
glycolipid (PGL) are lipids identified as virulent factors of M. tuberculosis that suppress hostimmunity. We have identified novel ITAM-coupled receptors expressed on innate immune cells,
MAR and PGLR, that recognize MA and PGL, respectively. In this study, we aim to clarify the roleof these receptors in the pathogenesis of tuberculosis.

Results
We found that macrophage stimulation with the glycosylated MAs, TDM and GMM, produced
the macrophage chemoattractant MCP-1 as well as the inflammatory cytokine TNF and inducible
nitric oxide synthase (iNOS), both of which are known to be essential for tuberculosis control inmacrophages, while macrophage stimulation with the non-glycosylated MAs, free MA and GroMM,
induced MCP-1 but only few or no production of TNF and iNOS. The non-glycosylated MA-induced
chemokine production required MAR but not Mincle, while the responses to the glycosylated MAs
required Mincle but not MAR. Interestingly, MAR could bind to the glycosylated MAs albeit itis weaker than Mincle, but its binding appeared to inhibit Mincle-mediated response as MAR
deficiency enhanced TDM-induced MCP-1, TNF and iNOS productions. Consistent with theseresults, MAR-deficient mice showed accelerated elimination of Mycobacterium bovis BCG in vivo.
Similar to MA, we found that PGL induced MCP-1 but not TNF and iNOS by macrophages invitro, and the MCP-1 production is completely dependent on PGLR.
Together, these results suggest that the mycobacterium virulent lipids, non-glycosylated MAs
and PGL, may show its immnosuppressive activity through their host receptors, MAR and PGL,
respectively.
The Novartis Foundation(Japan)
for the Promotion of Science

11

The Novartis Foundation(Japan)
for the Promotion of Science

Discussion & Conclusion
Therapy with anti-TNF antibodies for rheumatoid arthritis and inflammatory bowel diseasepromotes relapse of tuberculosis, which suggests that suppression of host TNF production iseffective means of avoiding immunity and reactivation. We identified MAR as a pattern-recognition
receptor for non-glycosylated MAs, known as virulent lipids with immunosuppressive activity.
Interestingly, MAR is DAP12-associated receptor that induce Mφ chemotactic factor MCP-1 upon
stimulation, while not inducing TNF and NO production. Given enhanced TNF production in
response to mycobacterium lipids in MAR-deficient Mφ, a therapeutic strategy can be expected toenhance the anti-tuberculosis immune response by inhibiting the binding of the lipid ligand to MAR
or its signaling, thereby promoting elimination and dormancy of M. tuberculosis. On the contrary,by stimulating this inhibition pathway, development of anti-inflammatory/immunosuppressive drugs
with a new concept can also be expected.

一般の皆様へ
結核は世界人口の1/3が罹患する世界最大の細菌感染症であり、年間200万人を超える人が命を失い続けています。しかし、結核を薬で完治させることは難しく、効果的なワクチンも未だに存在しません。結核を克服するためには、宿主免疫による結核菌の認識と応答の仕組みと、結核菌が免疫を回避する仕組みの両方を分子レベルで理解する必要があリます。我々の研究に
よって結核菌の病原性脂質による免疫抑制の分子機構が明らかになれば、新しい概念での結核の予防薬や治療薬の開発に繋がるものと期待しています。

The Novartis Foundation(Japan)
for the Promotion of Science

12

The Novartis Foundation(Japan)
for the Promotion of Science

Research of glycosylation that dynamically regulates innate immunity
Satoshi Goto
stgoto@rikkyo.ac.jp
Rikkyo University

Summary Abstract
Innate immunity is the first line of defense to combat invaded pathogens. In this study, we foundthat glucosaminoglycan (GAG) regulates immune activity. GAG suppresses hyper-activation of
the Toll pathway in the steady state. Interestingly, pathogen infection reduces the expression of a
GAG synthesizing enzyme to enhance immune responses. These results indicate that GAG plays animportant role in both suppression of runaway inflammation and enhancement of immune responses.
Key Words：Glycosylation, Glucosaminoglycan, Innate immunity

Introduction
Innate immunity is the first line of defense to combat invaded pathogens. Delayed and/or
inadequate innate immune responses can result in failure to combat pathogens, whereas excessive
and/or inappropriate responses cause runaway inflammation. Therefore, immune responses aretightly regulated from initiation to resolution and are repressed during the steady state.
Glycosylation is well known to control activities of target proteins. Innate immunity is also
regulated by glycosylation (1). However, only a few glycans have been reported to be involved ininnate immunity so far. Glycosylation dependent regulation of innate immunity remains largely
unrevealed.

Results
First, we screened the Drosophila RNAi library to identify what glycosylenzymes are involved
in innate immune responses and found that the immune responses are hyper-activated in mutantsof tout-velu (ttv) and brother of tout-velu (botv) genes required for synthesis of glucosaminoglycan(GAG). GAG is a long sugar chain with a tetra-saccharides at the reducing end and disaccharide
repeats extended to the non-reducing end. Since Ttv and Botv are enzymes to generate disaccharide
repeats, most part of GAG are deleted in ttv and/or botv mutants. Thus, our results suggest that GAG
suppresses activation of innate immune responses in the steady state.
We next investigated what tissue requires GAG to regulate innate immune responses. To approach
this question, we knocked down ttv gene in several immune tissues including the lymph glands,
lymphoid cells and fat bodies. The lymph glands contain most immune precursor cells and a partof mature lymphoid cells. The mature lymphoid cells are released into body fluid. The lymphoidcells play various roles in immune responses such as release of humoral immune factors and antimicrobial peptides, phagocytosis and encapsulation. When invaded pathogens are recognized byhumoral immune factors, humoral signals activate expression of anti-microbial peptides in the fatbodies. Our result showed that immune responses in the fat bodies were hyper-activated when ttv
was knocked down in the lymph glands. This result raised two possibilities. One is that a large
number of immune precursor cells may differentiate into mature cells in ttv knockdown, causing

The Novartis Foundation(Japan)
for the Promotion of Science

13

The Novartis Foundation(Japan)
for the Promotion of Science

hyper-activation of innate immune responses. The other is that the lymph glands in ttv knockdown
may send excess signal to fat bodies. To distinguish these two possibilities, we examined whether
precursor cells differentiate into mature cells in the lymph glands in which ttv was specificallyknocked down. Mature cells were hardly found in the lymph glands. This result precludes the former
possibility. However, the possible signal from the lymph glands to the fat bodies has not been
identified. This indicates that there is a new signal sent from the lymph glands to the fat bodies.
Taken together, GAG in the lymph glands suppresses activation of immune responses in the fatbodies during the steady state. This raises a next question. When pathogens invade into bodies,
GAG still continues suppression of immune responses or not. We measured transcriptional changeof seven genes encoding GAG synthesizing enzymes with pathogen invasion. Among the genes,
mRNA level of botv by pathogen challenge was reduced to ~60% compared to the steady state. To
examine whether 60% reduction is significant level for immune activation, we measured immune
response level in heteroallelic mutant of botv (botv+/-) in which botv mRNA is expected to be
reduced to ~50%. In the mutant, immune response level was significantly enhanced, suggestingthat 60% reduction of botv mRNA is enough to activate immune responses. These results suggest
an interesting regulatory machinery that GAG suppresses hyper-activation of immune responses inthe steady state and once pathogens invade into bodies, expression of Botv, the GAG synthesizing
enzyme is reduced to enhance immune responses.

Discussion & Conclusion
Immune responses are tightly regulated from initiation to resolution and are repressed during thesteady state. The current study demonstrates that GAG in the lymph glands suppresses activationof immune responses during the steady state and once pathogens invade into bodies, expressionof the GAG synthesizing enzyme is reduced to enhance immune responses for prompt eliminationof pathogens. We have previously reported that galactose-containing glycans suppresses hyperactivation of innate immune responses in the steady state as well (1). Taken together with the report,
the current study strongly suggests that more than one glycosylation machineries play an importantrole in regulation of innate immunity.

References
1. Dynamic regulation of innate immune responses in Drosophila by Senju-mediated glycosylation.
Yamamoto-Hino, M., Muraoka, M., Kondo, S., Ueda, R., Okano, H. and Goto, S., Proc. Natl.
Acad. Sci. USA, 112 (18), 5809-5814 (2015) DOI: 10.1073/pnas.1424514112

一般の皆様へ
免疫は私達の身体を守るのに重要な役割を果たしています。その免疫は、感染していないときには働かないように抑えられていなければなりません。一方、いったん病原体などが感染したときには免疫は十分に活性化されなければなりません。本研究では、その免疫の動的な制御に糖鎖が重要な役割を果たしていることを見出しました。非感染時には糖鎖が免疫の活性化を抑え、感染時には糖鎖が減少することで免疫を十分に活性化させていました。将来的には、糖鎖を使った免疫の活性化を制御する技術が期待されます。

The Novartis Foundation(Japan)
for the Promotion of Science

14

The Novartis Foundation(Japan)
for the Promotion of Science

Development of therapy for multiple sclerosis by regulation ofneural circuit formation factor LOTUS expression
Kohtaro Takei
kohtaro@yokohama-cu.ac.jp
Yokohama City University Graduate School of Medical Life Science

Summary Abstract
Multiple sclerosis (MS) is recognized as an autoimmune demyelinating disorder of the central
nervous system with axonal degeneration induced by Nogo receptor-1 (NgR1)-mediated signaling.
We identified lateral olfactory tract usher substance (LOTUS) as an endogenous antagonist of
NgR1. We therefore examined the relationship between LOTUS and experimental autoimmune
encephalomyelitis (EAE), an animal model for MS. Disease severity in EAE was improved in
LOTUS-deficient mice, whereas that was increased in LOTUS-overexpressing mice. Furthermore,
the binding of LOTUS to the CD4-positive T-cells from EAE mice increased IL-17 and IFN-γ
secretions with cell proliferation of T-cell. These findings suggest that LOTUS may be involved in
EAE induction through inflammation response.
Key Words：LOTUS, Multiple sclerosis, EAE, Therapy, Inflammation

Introduction
Multiple sclerosis (MS) is generally recognized as an autoimmune demyelinating disorder of thecentral nervous system (CNS), and it has been recently considered that axonal degeneration inducedby Nogo receptor-1 (NgR1)-mediated signaling is an important pathological feature in the first andthe secondary progressive phases of MS (Petratos et al., 2012). We previously identified lateralolfactory tract usher substance (LOTUS) as an endogenous antagonist of NgR1 (Sato et al., 2011;
Kurihara et al., 2014). We also found that the LOTUS concentration in cerebrospinal fluid (CSF) was
significantly decreased in patients with MS when compared with normal controls (Takahashi et al.,2015). Therefore, we examined the relationship between LOTUS expression levels in the CNS anddisease activities in experimental autoimmune encephalomyelitis (EAE), an animal model for MS.

Results
Since variations in LOTUS levels in CSF were well correlated with disease activity in MS, it could
be considered that low LOTUS concentrations in the CNS may be possibly involved in enhancementof NgR1 signaling, thereby loss of LOTUS may induce axonal degeneration in MS. Therefore, we
predicted pathological condition of MS may grow seriously in accordance with decrease of LOTUS
expression level. To address this issue, we analyzed disease activity of EAE in LOTUS-knocking
out (LOTUS-KO) and LOTUS-overexpressing transgenic (LOTUS-Tg) mice. In contrast to our
expectation, disease severity in onset of EAE was improved in LOTUS-KO mice, whereas that wasslightly increased in LOTUS-Tg mice when compared with wild type mice. These surprising findings
suggest that LOTUS may be involved in EAE induction with inflammatory response.
We next examined whether LOTUS binds to lymphocytes and induces cytokines secretion related
to inflammation response. We found that LOTUS bound to lymphocytes from EAE mice and the

The Novartis Foundation(Japan)
for the Promotion of Science

15

The Novartis Foundation(Japan)
for the Promotion of Science

LOTUS binding to cultured lymphocytes from EAE mice significantly increased IL-17 and IFN-γ
secretions, although the LOTUS binding did not change in the level of IL-2 secretion. Furthermore,
MTT assay revealed that IL-17 secretion induced by LOTUS binding increased cell proliferationof lymphocyte. These findings suggest that binding of LOTUS to lymphocyte may be involved ininflammatory response in acute phase of EAE. We also confirmed these findings in CD4-positive
T-cells from EAE mice. Namely, LOTUS bound CD4-positve T-cells and the binding of LOTUS to
the T-cells increased IL-17 and IFN-γ secretions with cell proliferation.
Next, We examined whether LOTUS binds to lymphocyte from NgR1-knocking out (NgR1-KO)
mice, since LOTUS binds to NgR1 and NgR1 is also expressed in lymphocytes. LOTUS bound tolymphocytes from EAE-induced NgR1-KO mice, indicating that LOTUS may interact with unknown
binding partner in lymphocytes except for NgR1. This is also confirmed in CD4-positive T-cells from
NgR1-KO mice. Thus, binding of LOTUS to unidentified molecule expressed in lymphocyte andCD4-positive T-cells may be involved in the induction of EAE and the association of LOTUS withthe unidentified molecule may be a possible therapeutic target in acute phase of MS.

Discussion & Conclusion
It was found that the LOTUS concentration in CSF was significantly decreased in patients with MS(Takahashi et al., 2015). The reason why LOTUS expression is decreased in accordance with thedisease activity of MS remains completely unknown. LOTUS expressed in the CNS may decreasein compensation for the damage LOTUS has given. As a step in elucidating molecular mechanismof the regulation of LOTUS expression, we are making an attempt to search for some factors thatinduce decrease of LOTUS expression. It is also necessary to examine whether EAE is induced bytransplantation of lymphocytes sensitized to LOTUS in wild type mice in order to validate the EAE
induction by LOTUS. In addition, identification of the binding partner of LOTUS in lymphocyte
is required for development of future therapy for MS, because the association of LOTUS with theunidentified molecule may be a possible therapeutic target in acute phase of MS. In conclusion,
binding of LOTUS protein in blood to lymphocytes may induce EAE induction and the associationof LOTUS with unidentified molecule on lymphocytes may be a novel therapeutic target in MS.

References
・ Kurihara, Y., Iketani, M., Ito, H., et al., LOTUS suppresses axon growth inhibition by blocking
interaction between Nogo receptor-1 and all four types of its ligand. Molecular Cellular
Neuroscience, 61: 211−218 (2014).
・ Petratos, S., Ozturk, E., Azari, M.F. et al., Limiting multiple sclerosis related axonopathy byblocking Nogo receptor and CRMP-2 phosphorylation. Brain, 135: 1794-1818 (2012) .
・ Sato, Y., Iketani, M., Kurihara, Y., et al., Cartilage acidic protein-1B (LOTUS), an endogenous
Nogo receptor antagonist for axon tract formation. Science, 333: 769-773 (2011).
・ Takahashi, K., Kurihara, Y., Suzuki, Y. et al., Association of cerebrospinal fluid levels of lateralolfactory usher substance protein with disease activity in multiple sclerosis. JAMA Neurology,
72(2): 176-179 (2015).

The Novartis Foundation(Japan)
for the Promotion of Science

16

The Novartis Foundation(Japan)
for the Promotion of Science

一般の皆様へ
国の難病に指定されている多発性硬化症に見られる神経変性は Nogo 受容体という分子が関与していると報告されているため、私達が発見した Nogo 受容体の働きを阻止する LOTUS とい

う分子とこの病気との関連について病態モデルマウスを用いて調べたところ、予想に反して、LOTUS は血中のリンパ球と結合して炎症を引き起こすサイトカインの一種をリンパ球から分泌させて炎症を引き起こす可能性が示されました。即ち、LOTUS はこの病気の発症に関連することが示唆されました。LOTUS が引き起こす炎症を止めることでこの病気の発症や進行を抑制することができるのではないかと考え、その方策を明らかにするための研究を行っています。

The Novartis Foundation(Japan)
for the Promotion of Science

17

The Novartis Foundation(Japan)
for the Promotion of Science

Molecular mechanism of epigenetic dysregulation
in myelofibrosis-initiating cells
Goro Sashidasashidag@kumamoto-u.ac.jp
Kumamoto University, International Research Center for Medical Sciences

Summary Abstract
Myeloproliferative neoplasms (MPNs) are clonal hematopoietic malignancies that originate
from HSCs and are characterized by the excess production of mature myeloid cells and active
extramedullary hematopoiesis. HMGA2, one of the polycomb target genes, has been implicated in thepathogenesis of MPNs including myelofibrosis. In this study, in order to understand the oncogenic
function of Hmga2, we generated a novel conditional Hmga2 knock-in mouse.
Key Words：Myeloproliferative neoplasms (MPNs), Myelofibrosis (MF), Hematopoietic stem cells(HSCs), HMGA2, Epigenetics

Introduction
MPNs are clonal hematopoietic malignancies that originate from HSCs and are characterized
by the excess production of mature myeloid cells and active extramedullary hematopoiesis. The
identification of a somatic activating mutation in JAK2 (JAK2V617F) in patients with MPN, including
those with polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis(PMF), underlined the importance of the constitutive activation of the JAK-STAT signaling pathway
in MPN pathogenesis (Levine at al. 2007). Recent genome-wide sequencing studies revealed
frequent co-occurring mutations in epigenetic regulators such as TET2, ASXL1, and EZH2 (Shih etal. 2012). Loss-of-function mutations in EZH2 have been identified in approximately 10% of patientswith PMF (Guglielmelli et al. 2011). We previously reported that Ezh2-deficient mice showed MPNlike phenotypes, such as increased platelet counts with EMH and myeloid-biased hematopoiesis at
the expense of lymphopoiesis (Mochizuki-Kashio et al. 2011; Muto et al. 2013). We then attempted
to determine how the loss of Ezh2 impacted on oncogene expression associated with the propagationof MF, and found that Hmga2, a potent oncogene, was higly rexpressed in MF stem/progenitor cells,
relavant to enhanced expression of HMGA2 in CD34+ stem/progenitor cells of patients with PMF.
Here, we exmeined how Hmga2 enhances the funciton of MF-initiating cells and leads to promoting
the development of MF via generating a novel conditional Hmga2 knock-in mice.

Results
We observed that the expression of Hmga2 was significantly elevated upon the loss of Ezh2 in
LSKs and MEPs regardless of the presence of JAK2V617F, and its activation was more evident in
MEPs, in which Hmag2 physiologically undergoes transcriptional repression (data not shown). As
the transduction of Hmga2 in wild-type HSCs has been shown to promote megakaryopoiesis (Oguro
et al. 2012), we examined the effects of the ectopic expression of Hmga2 in combination with
JAK2V617F on the production of megakaryocytes. Indeed, the transduction of Hmga2 in WT HSCs
promoted the cell growth and increased the frequencies of megakaryocytes in culture in the presence

The Novartis Foundation(Japan)
for the Promotion of Science

18

The Novartis Foundation(Japan)
for the Promotion of Scienceof SCF and TPO.
In contrast, overexpression of Hmga2 in
JAK2V617F HSCs, which showed limited
proliferation in culture, did not enhancecell growth. Instead, it markedly promoted
the megakaryocytic differentiation of
JAK2V617F HSCs, and induced dysplastic
megakaryocytes characterized by loose
lobulation and various sizes in vitro (datanot shown). These findings implicate thederepressed Hmga2 in the expansion ofabnormal megakaryocytes in aggressive
MF mice. This result also appeared tobe relevant to the activation of HMGA2
in CD34 + cells in patients with PMF(Guglielmelli et al. 2011).
Given that Hmga2 appeared to promote the development of MF in vivo, we successfully generated
a novel conditional Hmga2 knock-in mouse. To do this, we utilized a vector of Rosa26 loxP-STOPloxP HA-tag-Hmga2 IRES-GFP to inject it into mouse ES cells and selected properly knockedin clone mice. To understand how Hmga2 promotes the function of normal as well as cancer stem
cells, we crossed this mouse with a transgenic mouse, which has a hematopoietic stem cells-specific
Cre-ERT (in a collaboration with Prof Motomi Osato at NUS Singapore)(See a figure in the firstpage). We saw successful transduction of Hmga2 preferentially in myeloid cells, but not T-cells,
which suggested the HSCs-specific induction of Hmga2 in vivo. We are now studying the molecularmechanisms of Hmga2 in the pathogenesis of MF.

Discussion & Conclusion
Hmga2, one of the polycomb-repressive complex 2 target genes de-repressed in the absence ofEzh2, markedly enhanced the megakaryocytic differentiation of HSCs in concert with JAK2V617F
in cultures. This result is consistent with previous findings in which the overexpression of Hmga2
induced MPN in mice lacking Hmga2 3’UTR or Bmi1/Cdkn2a double knockout mice (Oguro et al2012; Ikeda et al 2011), thereby strongly supporting the importance of HMGA2 in the pathogenesisof PMF in patients harboring EZH2 mutations. However, it remains unknown how Hmga2 promotes
the initiation of MPN-stem cells, because of the lacking of mouse model, which can induce theexpression of Hmga2 in a HSCs-specific manner. Once, we successfully got the Hmga2 knock-in
mice, we will be able to understand how Hmga2 regulate the expression of target genes in stem cells
via modulating chromatin formation.

References
1. Levine, R.L., A. Pardanani, A. Tefferi, et al. Role of JAK2 in the pathogenesis and therapy of
myeloproliferative disorders. Nat. Rev. Cancer. 2007 7:673–683.
2. Shih, A.H., O. Abdel-Wahab, J.P. Patel, at al. The role of mutations in epigenetic regulators inmyeloid malignancies. Nat. Rev. Cancer. 2012 12:599–612.

The Novartis Foundation(Japan)
for the Promotion of Science

19

The Novartis Foundation(Japan)
for the Promotion of Science

3. Mochizuki-Kashio, M., Y. Mishima, S. Miyagi, et al. Dependency on the polycomb gene Ezh2
distinguishes fetal from adult hematopoietic stem cells. Blood. 2011 118:6553–6561.
4. Muto, T., G. Sashida, M. Oshima, et al. Concurrent loss of Ezh2 and Tet2 cooperates in thepathogenesis of myelodysplastic disorders. J. Exp. Med. 2013 210:2627–39.
5. Oguro, H., J. Yuan, S. Tanaka, et al. Lethal myelofibrosis induced by Bmi1-deficient hematopoietic
cells unveils a tumor suppressor function of the polycomb group genes. J. Exp. Med. 2012
209:445–454.
6. Guglielmelli, P., R. Zini, C. Bogani, at al. Molecular profiling of CD34+ cells in idiopathic
myelofibrosis identifies a set of disease-associated genes and reveals the clinical significance ofWilms’ tumor gene 1 (WT1). Stem Cells. 2007 25:165–173.
7. Ikeda, K., P.J. Mason, and M. Bessler. 3’UTR-truncated Hmga2 cDNAcauses MPN-likehematopoiesis by conferring a clonal growth advantage at the level of HSC in mice. Blood. 2011
117:5860–5869.

一般の皆様へ
原発性骨髄線維症とは骨髄の中で血小板を作る巨核球と顆粒球系細胞が腫瘍性に増殖して骨髄の線維化と造血障害に加えて、脾臓などで異所性造血を生じる予後不良ながんである。病気の原因として、遺伝子変異があり、約半数の患者で JAK2キナーゼの活性化変異があり、その他に共存する遺伝子変異としてエピゲノム制御因子である EZH2や TET2が知られ、悪性度との関連が言われている。骨髄線維症マウスの造血幹細胞を採取して、患者の腫瘍細胞でも発現上昇が知られる Hmga2を骨髄線維症の病態進展に責任のあるがん遺伝子として同定した。

今後、新規に作製した Hmga2発現を造血幹細胞で特異的に誘導できるマウスを用いて、詳細な骨髄線維症の発症メカニズムを解明するとともに、標的遺伝子の検索を通した新規治療戦略の進展が期待できる。

The Novartis Foundation(Japan)
for the Promotion of Science

20

The Novartis Foundation(Japan)
for the Promotion of Science

Leukemogenesis caused by a defect of the ribosomal RNA processing
Hirotaka Matsuihmatsui@kumamoto-u.ac.jp
Department of Molecular Laboratory Medicine, Faculty of Life Sciences, Kumamoto University

Summary Abstract
In this study, we screened for the DDX41 mutation of CD34-positive tumor cells and identified thep.R525H mutation in three cases among 23 AML/MDS patients. These patients commonly exhibited
AML with peripheral blood cytopenias and low blast counts, suggesting that the mutation inhibits
growth and differentiation of hematopoietic cells. Data from cord blood cells and leukemia celllines suggest a role for DDX41 in the pre-ribosomal RNA processing, in which the expression of themutant causes a certain ribosomopathy phenotype by suppressing MDM2-mediated RB degradation,
thus triggering the inhibition of E2F activity. This study uncovered a pathogenic role of p.R525H
DDX41 in the slow growth rate of tumor cells.
Key Words：DDX41, pre-rRNA processing, Acute Myeloid Leukemia

Introduction
Current comprehensive sequencing approaches led to the identification of rare but reproducible
somatic gene mutations in myeloid malignancies. Among them, there is a somatic mutation in
the DDX41 gene encoding a DEAD-box type ATP-dependent RNA helicase. However, thus far,the detailed physiological function of DDX41, as well as the perturbation of the function by the
mutant DDX41, has not been clarified. In this study, we propose a role for DDX41 as a pre-rRNA
processing factor, in which the p.R525H mutation affects ribosome biogenesis. We found thatribosome biogenesis was widely affected when cells were transfected with DDX41 p.R525H, thus
compromising cell cycle progression through impaired E2F function.

Results(1) Identification of DDX41 p.R525H mutation in three AML patients.
We identified heterozygous somatic DDX41 c.G1574A (p.R525H) mutation in three cases among
23 patients, which was confirmed by sanger sequencing. Intriguingly, these patients exhibited AML
with peripheral and bone marrow cytopenias and low blast counts. According to our transcriptome
data on patients’ CD34-positive cells, almost half of the sequenced tags at c.G1574 were consideredmutated in each patient. Therefore, almost all CD34-positive cells in these patients may harborheterozygous DDX41 p.R525H mutation. On the other hand, CD34-negative cells carrying themutation were fewer compared with CD34-positive cells, suggesting a block in the differentiation of
CD34-positive DDX41 mutant cells into negative cells.
(2) Nuclear localization of DDX41 protein
We next clarified the localization of DDX41 protein. Ectopically expressed as well as endogenous
DDX41 was mostly nuclear regardless of the p.R525H mutation and hardly co-localized with FLAGSTING, a protein shown to localize in ER. While previous reports described DDX41 as a cytosolic

The Novartis Foundation(Japan)
for the Promotion of Science

21

The Novartis Foundation(Japan)
for the Promotion of Science

DNA sensor that recognizes nucleic acids of pathogens, our study did not suggest this function basedon the protein localization.
(3) Growth inhibition of p.R525H DDX41 expressing cells due to the impaired pre-rRNA processing
To investigate the molecular functions of DDX41 in hematopoietic cells, cord blood-derived humanCD34-positive cells were transduced with wild-type (WT) or p.R525H DDX41. After a 30-day
culture, p.R525H cells showed decreased proliferation compared with WT cells, accompanied by the
suppression of mRNAs encoding ribosomal proteins. A GSEA analysis showed that gene expression
pattern of p.R525H cells negatively correlated with ribosomal gene sets. These data suggest that acertain ribosomopathy may occur in the mutant DDX41 expressing cells.
These findings and a highly-conserved DEAD-box type RNA helicase domain of DDX41 led us
to speculate that DDX41 protein might be involved in pre-rRNA processing. As expected from theposition of the somatic mutation (p.R525), which is within the helicase core where the ATP binds andis hydrolyzed, the mutant helicase domain displayed a lower ATPase activity. In addition, Northernblot analysis probing ITS1 and ITS2 of pre-rRNA showed increased signals of 47S and 41S prerRNAs in THP-1 cells expressing p.R525H DDX41. Although the precise phase at which DDX41
takes part in the pre-rRNA processing has not been elucidated, this series of experiments suggests arole for DDX41 in the trimming of 5’ ETS and/or ITS 2.
(4) Activation of RB pathway occurring in p.R525H DDX41 expressing cells
Recent studies on ribosomopathies revealed an activation of the MDM2-p53 pathway in thepathogenesis of the diseases. It is now widely recognized that RPL5 and RPL11, not incorporated
into the 60S ribosome, preferentially bind to MDM2; therefore, inhibition of p53 by MDM2 iscompromised, thus causing a stimulation of the p53 pathway. We initially assumed that the same
defect might be involved in the growth impairment of p.R525H DDX41 cells. Nevertheless, GSEA
did not indicate p53 activation in the cells.
On the other hand, GSEA instead revealed a negative enrichment of cell cycle-promoting genes
regulated by the RB-E2F axis in p.R525H DDX41 cord blood cells. Cell cycle inhibition throughthe suppression of E2F activity was also detected in patient-derived samples. In our study, we foundincreased RPL5 and RPL11 bound to MDM2 in p.R525H DDX41 cells. Since the total amount ofthese ribosomal proteins was not apparently altered by the enforced expression of p.R525H DDX41,
the mutant DDX41 might increase free ribosomal proteins that are not incorporated into the 60S
ribosome; these proteins may eventually form a complex with MDM2.

Discussion & Conclusion
Here, we propose a mechanism of growth defect in hematopoietic cells triggered by p.R525H
DDX41 occurring in the following order: (i) p.R525H mutant inhibits pre-rRNA processing; (ii)compromised ribosomal biogenesis as a result of impaired rRNA synthesis causes a release ofribosomal proteins that bind to MDM2; (iii) MDM2-mediated RB degradation is suppressed, thus
eventually activating the RB pathway and resulting in the inhibition of E2F activity. Although thisstudy uncovered a pathogenic role of p.R525H DDX41 in the slow growth rate of tumor cells, how
the mutation induces AML development and inhibits cell differentiation is still not understood.
Considering late occurrence of AML in patients harboring the mutation, it might require agedependent epigenetic alterations or other somatic changes for this mutation to fully transformhematopoietic cells.

The Novartis Foundation(Japan)
for the Promotion of Science

22

The Novartis Foundation(Japan)
for the Promotion of Science

References
1. Kadono M, Kanai A, Nagamachi A, Shinriki S, Kawata J, Iwato K, Kyo T, Oshima K, Yokoyama
A, Kawamura T, Nagase R, Inoue D, Kitamura T, Inaba T, Ichinohe T, Matsui H. Biologicalimplication of somatic DDX41 p.R525H mutation in acute myeloid leukemia. Exp. Hematol.
44(8): 745-754, 2016.
2. Polprasert C, Schulze I, Sekeres MA, et al. Inherited and Somatic Defects in DDX41 in Myeloid
Neoplasms. Cancer Cell. 2015;27(5):658-670.
3. Lewinsohn M, Brown AL, Weinel LM, et al. Novel germ line DDX41 mutations define families
with a lower age of MDS/AML onset and lymphoid malignancies. Blood. 2016;127(8):10171023.
4. Parvatiyar K, Zhang Z, Teles RM, et al. The helicase DDX41 recognizes the bacterial secondary
messengers cyclic di-GMP and cyclic di-AMP to activate a type I interferon immune response.
Nat Immunol. 2012;13(12):1155-1161.
5. Tafforeau L, Zorbas C, Langhendries JL, et al. The complexity of human ribosome biogenesis
revealed by systematic nucleolar screening of Pre-rRNA processing factors. Mol Cell.
2013;51(4):539-551.
6. Linder P, Jankowsky E. From unwinding to clamping - the DEAD box RNA helicase family. Nat
Rev Mol Cell Biol. 2011;12(8):505-516.
7. Dutt S, Narla A, Lin K, et al. Haploinsufficiency for ribosomal protein genes causes selective
activation of p53 in human erythroid progenitor cells. Blood. 2011;117(9):2567-2576.
8. Sun XX, Wang YG, Xirodimas DP, Dai MS. Perturbation of 60 S ribosomal biogenesis resultsin ribosomal protein L5- and L11-dependent p53 activation. J Biol Chem. 2010;285(33):2581225821.
9. Abdul-Ghani M, Hartman KL, Ngsee JK. Abstrakt interacts with and regulates the expression ofsorting nexin-2. J Cell Physiol. 2005;204(1):210-218.
10. Donati G, Brighenti E, Vici M, et al. Selective inhibition of rRNA transcription downregulates
E2F-1: a new p53-independent mechanism linking cell growth to cell proliferation. J Cell Sci.
2011;124(Pt 17):3017-3028.
11. Uchida C, Miwa S, Kitagawa K, et al. Enhanced Mdm2 activity inhibits pRB function via
ubiquitin-dependent degradation. EMBO J. 2005;24(1):160-169.

一般の皆様へ
近年の遺伝子解析研究の飛躍的な進歩により、白血病や骨髄異形成症候群といった血液腫瘍の原因となる遺伝子異常は、ほぼ全て明らかにされたといえます。一方で、こうした遺伝子異常が実際にどのように血液細胞を腫瘍細胞へと変貌させるのか、という根源的な疑問は、遺伝子解析だけで解くことができないものであり、地道な分子生物学的研究を必要とします。こうしたなか私たちは、最近血液腫瘍の原因となることがわかった DDX41 という遺伝子の変異に着目し、この変異により作られる異常なDDX41 が、リボソームと呼ばれる細胞内のタンパク質精製工場の機能を障害することを、多面的な解析手法により示してきました。今後さらに詳細なメカニズムを解明すべく、研究を続けていきたいと考えています。

The Novartis Foundation(Japan)
for the Promotion of Science

23

The Novartis Foundation(Japan)
for the Promotion of Science

Induction of antigen-specific regulatory T cells by dendritic cells inhuman
Sayuri Yamazakiyamazas@med.nagoya-cu.ac.jp
Nagoya City University, Department of Immunology

Summary Abstract
To control autoimmune disease, allergy and graft rejection in a specific way, we have been working
on inducting antigen specific regulatory T cells in mice and found that dendritic cells are criticalantigen presenting cells for regulatory T cells to proliferate in an antigen-specific manner. In thisstudy, we tried to translate our findings in mice into human.
Key Words：Regulatory T cells, Dendritic cells, antigen-specificity

Introduction
Regulatory T cells suppress important immune responses such as autoimmunity, anti-tumor
immunity, graft rejection, allergy and responses to infection. Regulatory T cell-based therapy is
promising, however, polyclonal regulatory T cells could lead to cancer and serious infection becauseof the global immune suppression. To control autoimmune disease, allergy and graft rejection in a
specific way, we tried to induce antigen specific regulatory T cells using human cells.

Results
Regulatory T cells suppress important immune responses such as autoimmunity, anti-tumor
immunity, graft rejection, allergy and responses to infection. Regulatory T cell-based therapy is
promising, however, polyclonal regulatory T cells could lead to cancer and serious infection becauseof the global immune suppression. To control autoimmune disease, allergy and graft rejection ina specific way, we have been working on inducting antigen specific regulatory T cells in mice andfound that dendritic cells are critical antigen presenting cells for regulatory T cells to proliferate in an
antigen-specific manner. Thus, in this study, we tried to translate our findings in mice into human.
For the dendritic cell preparation, we first purified dendritic cell subsets from peripheral blood byflow cytometer. Human peripheral dendritic cell subsets were divieded into CD141+(BDCA3+)
dendritic cells, CD1c+(BDCA1+) and BDCA2+ plasmacytoid dendritic cells. We were able to sortthese dendritic cell subsets with >90% purity by flow cytometer. However, the cell numbers from
20ml blood were very low and were not enough for the titration or time course experiments.
Therefore, next, CD14+monycytes were purified from peripheral blood and monocytederived dendritic cells were generated in the culture with GM-CSF and IL-4. We determined theconcentration of GM-CSF and IL-4, and were able to generate enough numbers of monocyte-derived
dendritic cells. Monocyte-derived dendritic cells were treated with or without inflammatory cytokine
cocktails, i.e., TNF-a, IL-1b, IL-6 and PGE2. Monocyte-derived dendritic cells were analyzed byflow cytometer and the phenotype was checked. We confirmed the cytokine cocktailes matured them

The Novartis Foundation(Japan)
for the Promotion of Science

24

The Novartis Foundation(Japan)
for the Promotion of Science

into hith CD86 and HLADR.
When monocyte-derived dendritic cells were cultered with CFSE-labeled allogeneic naïve CD4+T
cells from peripheral blood, we confirmed that the dendritic cells could induce allogeneic T cellproliferation. Foxp3 is the transcription factor for differentiation of regulatory T cells. In addition toFoxp3, functional human regulatory T cells can be distinguished by the expression of CD45RA or
CD127. Using these markers, we did time course analysis at several time points and determined thebest condition for regulatory T cell induction.
We also found that effector T cells and regulatory T cells proliferated in the presence of FCS andcould not detect the difference between allogeneic and syngeneic responses. Therefore, we chose acertain type of serum free medium and were able to reduce the non-specific proliferation to FCS.
Thus, using allogeneic systems, we were able to determine what type of dendritic cells could be
used, how long cells should be cultured, and how many cells should be in the culture.
To determine the function of induced regulatory T cells, it is important to perform suppression
assay in vitro. To set up the suppression assay in human cells, we first used naïve CD25highCD4+T
cells and added into responder allogenic CFSE-labeled CD4+T cells. We determined the condition
which the proliferation of responder T cells were suppressed by CD25highCD4+T cells. Using thesame condition, we will be able to see the antigen specific suppression of induced regulatory T cellsby titrating induced regulatory T cell numbers.
Collectively, in this study, we were able to determine the best condition to induce Foxp3+regulatory
T cells using dendritic cells in human cells. We will use this condition and try to expand functional
antigen specific regulatory T cells for the future therapy.

Discussion & Conclusion
It is very important to use antigen specific regulatory T cells to prevent global immune suppression.
We were able to determine the best condition to induce Foxp3+regulatory T cells using dendritic
cells in human cells by phenotype analysis. It seems that human dendritic cells derived from
CD14+monocytes are able to induce antigen-specific regulatory T cells, although we still need toinvestigate the in vitro suppressive activity. We used allogeneic mixed leukocyte reaction, but othertypes of antigens are also needed to be investigated. We will continue this study and hope to translate
into therapy in the future.

References
1. Yamazaki S, Iyoda T, Tarbell K, Olson K, Velinzon K, Inaba K, Steinman RM.
Direct expansion of functional CD25+ CD4+ regulatory T cells by antigen processing dendritic
cells.
J Exp Med 198:235-247, 2003.
2. Yamazaki S, Patel M, Harper A, Bonito A, Fukuyama H, Pack M, Tarbell KV, Talmor M, Ravetch
JV, Inaba, K, Steinman RM.
Effective expansion of alloantigen-specific Foxp3+ CD25+ CD4+ regulatory T cells by dendritic

The Novartis Foundation(Japan)
for the Promotion of Science

25

The Novartis Foundation(Japan)
for the Promotion of Sciencecells during the mixed leukocyte reaction.
Proc Natl Acad Sci USA 103: 2758-2763, 2006.
3. Yamazaki S*, Bonito AJ, Spisek R, Dhodapkar M, Inaba K, Steinman RM.
Dendritic cells are specialized accessory cells along with TGF- for the differentiation of
Foxp3+CD4+ regulatory T cells from peripheral Foxp3- precursors.
Blood 110:4293-4302,2007. *Corresponding author
4. Yamazaki S*, Dudziak D, Heidkamp GF, Fiorese C, Bonito AJ, Inaba K, Nussenzweig MC,
Steinman RM.
CD8+ CD205+ splenic dendritic cells are specialized to induce Foxp3+ regulatory T cells.
J Immunol 181: 6923-6933, 2008. *Corresponding author
5. Yamazaki S* et al. Submitted.

一般の皆様へ
様々な免疫反応を抑制する制御性 T 細胞の抗原特異性を樹状細胞という特別な抗原提示細胞でコントロールする研究をこれまでマウスで行ってきた。抗原特異的な制御性 T 細胞を誘導する事ができれば、癌や感染症に対する免疫反応を抑制せずに、自己免疫疾患、アレルギー、移植片拒絶を治療する事が可能となる。本研究では、これをヒトの細胞に応用することを行った。
ヒトの樹状細胞でも抗原特異的に制御性 T 細胞を誘導できる条件決定を行うことができた。

The Novartis Foundation(Japan)
for the Promotion of Science

26

The Novartis Foundation(Japan)
for the Promotion of Science

Study on the neuronal mechanisms for depression and anxiety inducedby chronic stress and pain
Masabumi Minami
mminami@pharm.hokudai.ac.jp
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Hokkaido University

Summary Abstract
The present study demonstrated that the activation of neurons projecting from the BNST to the
CeA increased anxiety-like behaviors, and that these neurons were Ih-positive type II neurons.
Furthermore, the results showed that neurotransmission via CRF1 receptors within the BNST wascontinuously activated under the chronic pain condition, and thereby increased the excitatory inputs
to typeⅡ BNST neurons. These findings suggest that enhanced neuronal excitability in type II BNST
neurons may be responsible for the increased anxiety level in chronic pain. Such a neuroplastic
alteration under the pathological condition may be one of the underlying mechanisms of anxiety/depression disorders.
Key Words：Anxiety, Bed nucleus of the stria terminalis, Depression, Extended amygdala, Pain

Introduction
Mammals including humans are thought to have acquired and evolved neuronal mechanisms
for negative emotion, such as depression and anxiety, as a biological defense system that protects
themselves by suppressing their activities and raising vigilance against the surroundings, when they
are in dangerous condition. Therefore, in order to understand the neuronal mechanisms of depressive
disorders and anxiety disorders, it is necessary to clarify the neuronal mechanisms for negative
emotion from the viewpoint as a biological defense system and to analyze the changes of suchmechanisms in patients and disease model animals.

Results
The bed nucleus of the stria terminalis (BNST) together with the central amygdala (CeA) forms
a forebrain functional unit described as an “extended amygdala”, which have been implicated in
the regulation of negative emotional states such as anxiety, fear, and aversion. Although these twobrain regions bidirectionaly send dense neural projections to each other, it remains to be elucidated
the roles of these neural projections in the regulation of negative emotional states. In this study, we
introduced channelrhodpsin2 into the BNST and activated the neuronal projection from the BNST
to the CeA by applying photostimuation to the CeA. This activation increased anxiety-like behaviors
in an elevated plus maze test. On the other hand, recent study reported that activation of the neuralprojection from the BNST to the lateral hypothalamus (LH) suppressed anxiety-like behaviors. Since
activation of the neural projections from the BNST to these two brain regions (CeA and LH) showed
the opposite effects on anxiety-like behaviors, we compared the electrophysiological propertiesof the BNST neurons projecting to these two brain regions. We injected retrograde tracers into
the CeA or LH, and conducted whole-cell patch clamp recording from the BNST neurons labeledwith retrograde tracers. Majority of the CeA-projecting BNST neurons showed hyperpolarization-

The Novartis Foundation(Japan)
for the Promotion of Science

27

The Novartis Foundation(Japan)
for the Promotion of Science

activated cation current (Ih current) in response to the hyperpolarizing current injection, whilemost of the LH-projecting BNST neurons did not show Ih current. These results suggest that CeAprojecting BNST neurons are Ih-positive type II neurons, and activation of these neurons increases
anxiety-like behaviors, while LH-projecting BNST neurons are Ih-negative type III neurons andactivation of these neurons suppresses anxiety-like behaviors.
The BNST is densely innervated with corticotropin-releasing factor (CRF) containing fibers.
We previously reported that CRF depolarizes typeⅡ neurons and enhances inhibitory synaptictransmission to typeⅢ neurons in the BNST. We also demonstrated the critical role of CRF
neurotransmission within the BNST in aversive responses induced by formalin-evoked pain.
However, the roles of CRF within the BNST in the regulation of negative emotions under the chronic
pain condition remain to be elucidated. In this study, we examined the effects of CRF and a CRF
type 1 receptor antagonist on the synaptic currents in the BNST neurons using the whole-cell patchclamp recordings. Brain slices including the BNST were prepared from chronic pain model rats inwhich neuropathic pain was induced by spinal nerve ligation (SNL). In sham-operated rats, spinal
nerves were exposed without ligation. Bath application of CRF significantly increased the amplitudeof eEPSC in typeⅡ neurons of the sham-operated rats, but not of the SNL model rats. By contrast,
the amplitude of eEPSC was significantly decreased by bath application of NBI27914 (a CRF
type 1 receptor antagonist) in typeⅡ neurons of the SNL model rats, but not of the sham-operated
rats. These data suggest that neurotransmission via CRF type 1 receptors within the BNST arecontinuously activated under the chronic pain condition, thereby increased the amplitude of eEPSC
in typeⅡ BNST neurons.

Discussion & Conclusion
The present study demonstrated that the activation of neuronal projection from the BNST to the
CeA increased anxiety-like behaviors in an elevated plus maze test, and that the neurons projectingfrom the BNST to the CeA are Ih-positive type II neurons. Furthermore, the results showed thatneurotransmission via CRF type 1 receptors within the BNST was continuously activated under thechronic pain condition, and thereby increases the excitatory inputs to typeⅡ BNST neurons. Thesefindings suggest that enhanced neuronal excitability in type II BNST neurons may be responsible
for the increased anxiety level in chronic pain. Such a neuroplastic alteration under the pathologicalcondition may be one of the underlying mechanisms of anxiety/depression disorders.

References
Classification of neurons in the dorsolateral bed nucleus of the stria terminalis by theirelectrophysiological properties and projecting brain regions.
Yamauchi, N., Nagano, Y., Takahashi, D., et al.
Int. J. Neuropsychopharmacol. 19 (2016) 56-57.
Alteration of the effects of corticotropin-releasing factor on synaptic transmission in the dorsolateral
bed nucleus of the stria terminalis of chronic pain model rats.
Takahashi, D., Amano, T., Minami, M.
Int. J. Neuropsychopharmacol. 19 (2016) 277-278.

The Novartis Foundation(Japan)
for the Promotion of Science28

The Novartis Foundation(Japan)
for the Promotion of Science

一般の皆様へ
ヒトを含む哺乳類は、身を守るための生体防御システムとして、抑うつや不安などの負情動生成機構を獲得・進化させてきたと考えられます。したがって、うつ病や不安障害のメカニズ
ムを理解するためには、生体防御システムとしての負情動生成機構を明らかにした上で、患者や病態モデル動物における変化を解析することが必要となります。私たちは、分界条床核と呼ばれる脳部位に着目して研究を進め、分界条床核から扁桃体中心核に投射する神経細胞の活性化が不安情動を引き起こすことを明らかにした。また、慢性痛時に分界条床核内シナプス伝達の可塑的変化が起こることも見いだしています。これらの神経機構がうつ病や不安障害に関与している可能性があります。

The Novartis Foundation(Japan)
for the Promotion of Science

29

The Novartis Foundation(Japan)
for the Promotion of Science

Pathogenesis of cardiomyopathy by RBM20 mutations modeled in
genetically modified human iPS cells
Yuichiro Miyaoka
miyaoka-yi@igakuken.or.jp
Tokyo Metropolitan Institute of Medical Science

Summary Abstract
Point mutations in a splicing factor, RNA binding motif protein 20 (RBM20), are known to be
responsible for cardiomyopathy. However, it remains elusive how these mutations lead to dysfunctionof cardiomyocytes at the molecular level. We generated an RBM20 mutant allelic series in human
iPS cells and differentiate them into cardiomyocytes. These iPS cell-derived cardiomyocytes (iPSCMs) recapitulated the cellular phenotypes of cardiomyocytes in patients. Deep sequencing oftranscripts of these iPS-CMs revealed that these point mutations are not simple loss-of-function
mutations. Our study provides a key insight into pathogenesis of cardiomyopathy by the RBM20
point mutations.
Key Words：Cardiomyopathy, iPS cells, Genome editing, RBM20, Splicing

Introduction
Dilated cardiomyopathy (DCM) is the most common indication for heart transplantation 1-3.
Mutations in a cardiac-specific splicing protein, RBM20, account for up to 3% of observed DCM4-6.
Interestingly, these mutations display a striking pattern of seven tightly clustered DCM-associatedpoint mutations in RBM204,7. As currently available rodent models are knockouts for Rbm20,
they do not reveal the pathogenic mechanism caused by the RBM20 point mutations. Therefore,
we introduced the RBM20 R636S and loss-of-function mutations into human iPS cells, anddifferentiated them into iPS-CMs. By transcriptomic analysis of these cells, we addressed the effectsof the RBM20 mutations on cardiac splicing regulation.

Results
We introduced heterozygous and homozygous RBM20 R636S point mutations in human iPS
cells derived from a healthy male patient, WTC118. For this purpose, we applied a method that wedeveloped recently based on digital-PCR and repeated rounds of limited dilutions without using
any selection marker genes8,9. We also introduced a heterozygous 1-bp insertion mutation in the
open reading frame of RBM20 by using the same method, which should result in loss-of-function
mutations due to frameshifting. These iPS cell lines were then differentiated into cardiomyocytes bysmall molecules that modulate Wnt signaling10. The iPS-CMs were purified from other cell types bylactate treatment11. We performed immunofluorescent staining of Actinin in the iPS-CMs to visualize
the sarcomeric structure that is a functional unit of cardiomyocytes. The mutant iPS-CMs displayed
disarrayed sarcomere compared to WT iPS-CMs demonstrating that the mutant iPS-CMs faithfully
recapitulated the cellular phenotypes of cardiomyocytes in DCM patients. To elucidate molecularpathogenesis behind the observed cellular phenotypes, total RNA purified from the iPS-CMs was
subjected to a paired-end 100 bp deep sequence by illumina HiSeq. The obtained reads were mapped

The Novartis Foundation(Japan)
for the Promotion of Science

30

The Novartis Foundation(Japan)
for the Promotion of Science

onto the reference human genome, and the mapped data was then analyzed by AltAnalyze forgenome-wide splicing profile12. AltAnalyze quantified the frequency of an exon being expressed astranscripts, so that we could analyze the splicing pattern based on how often each exon was included
in mRNAs.
First, we compared the splicing profiles of wild-type (WT), heterozygous R636S mutant (R636S
Het), and homozygous R636S mutant (R636S Homo) iPS-CMs. Because DCM patients areheterozygous for the R636S mutation, we expected to see
splicing defects in R636S Het iPS-CMs. As we expected,
R636S Het showed aberrant splicing compared to WT
in genes associated with functions of cardiomyocytes
including TTN, MYLK, and MEF2A (Figure 1). Somegenes shifted their splicing patterns to more immatureones due to the mutation. Currently, we are analyzing
how these splicing defects lead to the cellular phenotypes
that we see in DCM patients. Interestingly, we observed
distinct splicing phenotypes between R636S Het andFigure 1. Splicing profiles of WT, R636S Het, andR636S Homo iPS-CMs. For example, R636S Homo R636S Homo iPS-CMs
Total RNA of WT (8 samples), R636S Het (8exhibited severer splicing defects than R636S Het in some samples), and R636S Homo (3 samples) wasby RNAseq for splicing patterns. The
genes including COL16A1, RYR2, CDC14B, GPBP1L1, analyzed
frequency for each exon being included in transcripts
and TTN, indicating an additive effect of the R636S is color-coded (yellow and blue for high and low,respectively). Genes with altered exons are listed on
mutation. However, both R636S Het and R636S Homo the right.
iPS-CMs showed splicing defects in some genes such as IMMT,
NEO1, MICAL3, and DMD at a comparable level (Figure 1).
Then, we compared the splicing profiles of WT, R636S Het,
and the heterozygous 1-bp insertion mutant (1-bp Ins Het)
to directly address whether the R636S mutation is a loss-offunction mutation. As a result, R636S Het and 1-bp Ins Het
cells displayed rather different splicing patterns. Some exons
upregulated in R636S Het cells were downregulated in 1-bp
Ins Het, and vice versa. Many of them were genes involved in
cardiomyocyte functions (Figure 2). If the R636S mutation werea simple loss-of-function mutation, R636S Het and 1-bp Ins Het Figure 2. Splicing profiles of R636S Het
and 1-bp Ins Het iPS-CMs
mutations would have similar impacts on splicing regulation. Our Total RNA of R636S Het (8 samples) and1-bp Ins Het (3 samples) was analyzed byresults indicate that the R636S mutation is not a simple loss-of- RNAseq for splicing patterns.
function mutation.

Discussion & Conclusion
If the pathogenic RBM20 point mutations were simple loss-of-function mutations, there would havebeen more frameshift and nonsense mutations distributed throughout the RBM20 gene. However,at least 7 pathogenic point mutations are accumulated in a stretch of only 5 amino acid residues,
indicating that these point mutations may not be simple loss-of-function ones. Our findings based on
RNAseq also support this hypothesis.
This stretch of amino acids is R634 to P638, RSRSP, in RBM20. This stretch is highly characteristic
both in electric charge and structure, and two serines can be phosphorylated. Moreover, this stretch
is located in the Arg-Ser (RS) domain of RBM20, which is suggested to be involved in protein

The Novartis Foundation(Japan)
for the Promotion of Science

31

The Novartis Foundation(Japan)
for the Promotion of Scienceinteractions. We speculate that these RBM20 point mutations have dominant negative effects byinterfering with the protein interactions via the RS domain. We are now analyzing iPS-CMs with ahomozygous RBM20 loss-of-function mutation to further elucidate molecular pathogenesis of DCM.

References
1 Jefferies, J. L. & Towbin, J. A. Dilated cardiomyopathy. Lancet 375, 752-762, doi:10.1016/
S0140-6736(09)62023-7 (2010).
2 McNally, E. M., Golbus, J. R. & Puckelwartz, M. J. Genetic mutations and mechanisms in dilated
cardiomyopathy. J Clin Invest 123, 19-26, doi:10.1172/JCI62862 (2013).
3 Norton, N. et al. Evaluating pathogenicity of rare variants from dilated cardiomyopathy in theexome era. Circ Cardiovasc Genet 5, 167-174, doi:10.1161/CIRCGENETICS.111.961805 (2012).
4 Li, D. et al. Identification of novel mutations in RBM20 in patients with dilated cardiomyopathy.
Clin Transl Sci 3, 90-97, doi:10.1111/j.1752-8062.2010.00198.x (2010).
5 Maatz, H. et al. RNA-binding protein RBM20 represses splicing to orchestrate cardiac premRNA processing. J Clin Invest 124, 3419-3430, doi:10.1172/JCI74523 (2014).
6 Wells, Q. S. et al. Whole exome sequencing identifies a causal RBM20 mutation in a large
pedigree with familial dilated cardiomyopathy. Circ Cardiovasc Genet 6, 317-326, doi:10.1161/
CIRCGENETICS.113.000011 (2013).
7 Brauch, K. M. et al. Mutations in ribonucleic acid binding protein gene cause familial dilated
cardiomyopathy. J Am Coll Cardiol 54, 930-941, doi:10.1016/j.jacc.2009.05.038 (2009).
8 Miyaoka, Y. et al. Isolation of single-base genome-edited human iPS cells without antibiotic
selection. Nat Methods 11, 291-293, doi:10.1038/nmeth.2840 (2014).
9 Miyaoka, Y. et al. Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease,and cell type on genome-editing. Sci Rep 6, 23549, doi:10.1038/srep23549 (2016).
10 Lian, X. et al. Robust cardiomyocyte differentiation from human pluripotent stem cells via
temporal modulation of canonical Wnt signaling. Proc Natl Acad Sci U S A 109, E1848-1857,
doi:10.1073/pnas.1200250109 (2012).
11 Tohyama, S. et al. Distinct metabolic flow enables large-scale purification of mouse andhuman pluripotent stem cell-derived cardiomyocytes. Cell Stem Cell 12, 127-137, doi:10.1016/
j.stem.2012.09.013 (2013).
12 Emig, D. et al. AltAnalyze and DomainGraph: analyzing and visualizing exon expression data.
Nucleic Acids Res 38, W755-762, doi:10.1093/nar/gkq405 (2010).

一般の皆様へ
拡張型心筋症は、心臓移植の最多の要因である。近年、RBM20という遺伝子の変異が拡張型心筋症の原因であることが明らかとなったが、ラットなどのモデル生物を用いた研究では、心筋症発症機序の理解が十分ではなかった。そこで、私達はヒト iPS 細胞に RBM20の変異を導入し、心筋細胞へと分化させ、詳細に解析することにより、これまでに知られていなかった
RBM20の変異が心筋症発症につながる分子機構を解明した。本研究の成果が、新たな治療法の開発につながることが期待される。

The Novartis Foundation(Japan)
for the Promotion of Science

32

The Novartis Foundation(Japan)
for the Promotion of Science

Chemical investigation of the food-poisoning epidemics caused by themushroom Pleurocybella porrigens.
Tomohiro Suzuki
suzukit@cc.utsunomiya-u.ac.jp
Center for Bioscience Research and Education, Utsunomiya University

Summary Abstract
In autumn, 2004, 55 people got poisoned by eating an edible wild mushroom, Pleurocybella
porrigens and 17 people among them died of acute encephalopathy. We have already reported thepurification and characterization of a lectin from the mushroom (PPL). We have purified the lethal
glycoprotein (B3), and the mixrture (PPL-B3) have showed protease activity and disrupted theblood-brain barrier in mice. However, B3 is insoluble with any organic solvent, and the primarystructure of B3 is still unknown.
We have tried to obtain the gene of the lethal glycoprotein from P. porrigens by a yeast two-hybrid
method. As a result, we selected 2 clones which might be truly positive.
Key Words：Mushroom, Pleurocybella porrigens,

Introduction
In autumn, 2004, 55 people got poisoned by eating an edible wild mushroom, Pleurocybella
porrigens and 17 people among them died of acute encephalopathy. We have already reported thepurification, characterization, and cDNA cloning of a lectin from the mushroom (PPL). We havepurified the lethal glycoprotein (B3) which was extracted with boiling water, and the mixrture(PPL-B3) have showed protease activity and disrupted the blood-brain barrier in mice. Thus, thepurpose of this research is to reveal structure of these proteins, complex formation mechanism andtoxicity mechanism, and to clarify the mechanism of the onset of acute encephalopathy.

Results
A lethal toxic substance, named B3, has been isolated from boiling water-soluble part of P.
porrigens and characterized by us in previous study. A lectin nemed PPL has been also isolated fromwater-soluble part of the mushroom and characterized, and its cDNA was obtained. The mixrtureof PPL and B3 (PPL-B3) showed protease activity and disrupted the blood-brain barrier in mice.
Furthermore, PPL-B3 complex showed novel exo-protease activity to cleave the substrates from bothN- and C-terminus.
However, B3 is insoluble with any organic solvent, and the primary structure of B3 is still
unknown. Thus, we have tried to obtain the gene of B3 from P. porrigens by a yeast two-hybrid
method and have obtained 64 positive clones. Thus, we tried to obtain B3 gene in 64 positive clones
by new screening method, and to express recombinant PPL in basidiomycete Phanerochaete sordida
YK-624 in the present study.
We analyzed these gene sequences inserted in 64 positive clones, and 4 clones were selected bythese information of transcriptome analysis and LC-MS/MS analysis of B3. These selected 4 genes
were expressed using heterologous expression systems of E. coli BL21 and P. sordida YK-624.

The Novartis Foundation(Japan)
for the Promotion of Science

33

The Novartis Foundation(Japan)
for the Promotion of Science

However, all recombinants showed no protease activity although SDS-PAGE analysis of these clones
revealed that each recombinant was successfully expressed. PPL-B3 complex showed novel exoprotease activity to cleave the substrates from both N- and C-terminus as described above. Thus, wedeveloped a new protease assay for the selection of real positive clones by using fluorogenic andchromogenic substrate(s). As a result, we selected 2 clones which might be truly positive.
Occasionally PPL showed the protease activity when it contained a minor peak at m/z 13,000 in
MALDI-TOF-MS. Therefore, we thought that this m/z 13,000 substance was concerned with theprotease activity and named this substance PP-13,000. We isolated PP-13,000 by reversed phaseliquid chromatography, and finally determined its primary structure of PP13,000.
Next, we tried heterologous expression of recombinant PPL by basidiomycete P. sordida YK-624.
The glyceraldehyde 3-phosphate dehydrogenase gene promoter was used to drive the expression of
the PPL gene in P. sordida YK-624. Furthermore, the signal peptide of lignin peroxidase which is an
extracellular protein was used to secrete rPPL into extracellular region. The heterologous expressionof rPPL by P. sordida YK-624 was confirmed by SDS-PAGE, lectin activity by the hemagglutination
assay, indicating that the extracellular secretion of rPPL as active form was successful. Mixture of
PP-13,000 and rPPL expressed in P. sordida YK-624 showed protease activity that hydrolyzed thesubstrates from both N- and C- terminal ends.
We previously analyzed a set of whole genome sequencing and transcriptome sequencing data
in P. porrigens using the Illumina technology and released the compiled data from a web database
A-WING1, 2) Our results suggested that the genome of the mushroom had a unique structure andcontained numerous novel genes1), while the whole picture of the genetic information including
the exact genome size and a full set of genes was not obtained because of the fragmented sequenceassemblies. In the present study, we analyzed a draft genome sequence of P. porrigens to decode theunique genetic information. The improved genome and transcriptome assemblies constructed in thisstudy allowed for characterization of the unique features in paralogous gene clusters and predictionof protein-coding genes.

Discussion & Conclusion
In present study, we obtained 2 positive clones by a yeast two-hybrid method. Furthermore, we
succeeded in purification and primary structure determination of PP-13,000. These proteins might
have caused the poisoning of P. porrigens. Further study is needed to clarify the toxicity mechanismof this mushroom.
rPPL was successfully expressed by using basidiomycete P. sordida YK-624 expression systems.
We are now trying to elucidate the molecular mechanism for this acute encephalopathy using thisrPPL.
The draft genome sequence and annotations obtained in this study were released at the web database
A-WINGS20. Those will give aids to identify not only molecules that act on morphological andphysiological traits in the mushroom, but also useful genes that control rare metabolite biosynthesis
for drug development.

References(†; equal contribution)1) †Suzuki, T., †Igarashi, K., Dohra, H., Someya, T., Takano, T., Harada, K., Omae, S., Hirai, H.,
Yano, K*. and Kawagishi, H*. A new omics data resource of Pleurocybella porrigens for gene

The Novartis Foundation(Japan)
for the Promotion of Science

34

The Novartis Foundation(Japan)
for the Promotion of Science

discovery. Plos One, 8, e69681(2013)
2) Yamamoto, N., †Suzuki, T., †Kobayashi, M., Dohra, H., Sasaki, Y., Hirai, H., Yokoyama, K.,
Kawagishi H*. and Yano, K*. A-WINGS: an integrated genome database for Pleurocybella
porrigens (Angel’s wing oyster mushroom, Sugihiratake) BMC Research Notes, 7, 866(2014)†

一般の皆様へ
スギヒラタケは東北、北陸、中部地方を中心に広く食用とされてきたキノコですが、2004年

以降、スギヒラタケの摂食者が急性脳症を発病しました。本課題を遂行することによって、このキノコの毒性に関与する可能性がある毒性物質数種の構造決定を行う事ができました。この酵素は新たな酵素学として科学的価値だけでなく、様々な応用の可能性を秘めており、新たな酵素の創出も期待できます。さらにスギヒラタケ摂取患者に見られる病変は、既存の病気のものとは異なる未知の発生機序を強く示唆しています。今後も毒性メカニズムの解明のために、研究を継続する予定です。

The Novartis Foundation(Japan)
for the Promotion of Science

35

The Novartis Foundation(Japan)
for the Promotion of Science

Developing analysis of genome replication in single cell to elucidate
molecular dynamics of DNA polymerases
Yasukazu Daigaku
ydaigaku@m.tohoku.ac.jp
Frontier Research Institute for Interdisciplinary Sciences, Tohoku University

Summary Abstract
Synthesis by DNA polymerase is subjected to accidental posing due to many obstacles on genomic
DNA: DNA damage, sequence-specific secondary structure, etc. The heterogenous nature of DNA
replication arise at least from their stochastic behaviour of DNA polymerase. However, no studies
have given insight into any clear specificity in terms of molecular dynamics of DNA polymerase.
This study aim to develop a new experimental system to assess usage of DNA polymerase in asingle cell in order to elucidate how multiple DNA polymerases cooperatively function to complete
duplication of genome.
Key Words：DNA replication, Genome stability

Introduction
Duplication of cells must be faithfully carried out in proliferating cells. DNA replication is thestage in which cells are subjected to various DNA insults, which become truly dangerous andpotentially causes cell death or genomic instability. The replication machinery is therefore endowedwith plasticity that provides means to tolerate various replication stress. Each single cells initiate
replication at different locations and various DNA polymerases are to be functionalised in response toaccidental events in a course of DNA synthesis. In this study, we aimed to establish the experimental
system to characterise DNA polymerase usage globally in a single cell and elucidate stochastic
nature of DNA replication.

Results
To characterise functions of each DNA polymerase globally, we previously devised a new
methodology to identify usage of DNA polymerase at genome wide level (Pu-seq) [1]. In thismethod, first, we established mutants of two major DNA polymerase: Polε and Polδ in which
excessive ribonucleotides (rNMP) are incorporated into genomic DNA. Second, incorporated rNMP
was mapped by high-throughput sequencing based technology. Finally, usage of these polymerases
was calculated from abundancy of incorporated rNMP. Our analysis revealed that the divisionof labour of two polymerases is broadly maintained: Polε is responsible for leading strand DNA
synthesis while Polδ is for lagging strand synthesis. Although this result provided the evidence thatdivision of labour between two major DNA polymerases, their molecular dynamics remain unclear.
The profiles of these polymerases were derived from a mass of cultured cells; however, behaviour of
DNA polymerase is expected to be different in each single cell. This study therefore aims to develop
the method to investigate the genome-wide profile of DNA polymerase in a single cell (single cell
pu-seq). While this method is primarily being developed using fission yeast cells, we plan to apply
the method in C.elegans germline cells to elucidate variation in the molecular mechanism of DNA

The Novartis Foundation(Japan)
for the Promotion of Science

36

The Novartis Foundation(Japan)
for the Promotion of Science

replication during the process of aging.
Regarding the development single cells Pu-seq, we required to examine rNMP incorporation in asingle cell. As alkaline treatment cleaves DNA strands at incorporated rNMP, we are able to map
rNMP by identifying location of 5’ ends of cleaved fragments. Therefore, we aimed to perform
whole genome amplification (WGA) from alkaline treated single cells. Among many WGA methods,
multiple annealing and looping-based amplification: MALBAC method has been reported to cause
relative lesser application bias [2]. After optimisation of this procedure, we successfully amplified
genome DNA from alkaline treated single cells. However, further optimisation is still required toincrease the amount of yielded DNA and probability of successful application before proceeding topreparation of DNA library for high-throughput sequencing.
To construct rNMP-incorporating DNA polymerase mutant in C.elegans, we utilised CRISPR cas9system adapted in this organism [3]. In this system, donor single-stranded DNA bearing a desired
mutation is co-injected with plasmids encoding Cas9 and a gRNA specific for the targeted locus
into the C. elegans germline. An amino acid residue proximal or the steric gate in the catalytic
site of Polε and Polδ was to change to a hydrophobic or small residue. As a result of multiple time
injection, both desired Polε and Polδ mutants were isolated. In the case of the Polε mutant, we only
integrated the mutation into one copy of Polε genes and thus worms carrying bi-allele mutation
was screened after self-fertilisation. To examine whether these mutant polymerases incorporaterNMP into genomic DNA, we examined polymerase mutant worms in a condition where RNaseH2
was inactivated by RNA interference. In the absence of RNaseH2, rNMP incorporated by DNA
polymerase would not be eliminated, resulting in its accumulation in genomic DNA. Consistent
with this prediction, when RNaseH2 was inactivated in Polε or Polδ mutants, hatching rates of next
generation were dramatically decreased. These results indicate that integrated Polε or Polδ mutations
cause accumulation of rNMP during DNA replication in germline cells or early embryo, leading to
failure of development.

Discussion & Conclusion
This study aims to elucidate DNA replication dynamics which is different among each single
cell. To gain technical advance for this purpose, Using fission yeast cells, WGA prior to highthroughput sequencing has been optimized to achieve mapping of rNMP in single cells. Whilefurther optimisation is required to complete sequencing procedures of this experiment system, we
successfully constructed mutants of Polε or Polδ in C.elegans which incorporate rNMP into genomic
DNA. Thus, we currently prioritise the completion of single Pu-seq system and application of thissystem in c.elegans will follow to elucidate the mechanism of DNA replication, which is predicted to
be variable in response to changes in metabolic status during process of ageing.

References[1] Daigaku et al. Nat. Struct. Mol. Biol. 22 192–198 2015[2] Zong et al. Science 338 1622-1626 2012
[3] Arribere et al. Genetics 198 837–846 2014

The Novartis Foundation(Japan)
for the Promotion of Science

37

The Novartis Foundation(Japan)
for the Promotion of Science

一般の皆様へ
個々の細胞には個性があります．遺伝情報であるゲノムを複製するシステムはすべての細胞において，全く同じパターンで起きるわけではありません．複製が開始される場所，細胞に多種存在するＤＮＡを合成する酵素：ＤＮＡポリメラーゼの使われ方が異なっています．本研究では，１つ１つの細胞で，ゲノムＤＮＡ全体に渡り，個々のＤＮＡポリメラーゼ分子の役割を明らかにする実験系の構築を行いました．

The Novartis Foundation(Japan)
for the Promotion of Science

38

The Novartis Foundation(Japan)
for the Promotion of Science

A novel lincRNA, Linc-Heart, regulates cell cycle and proliferationof adult cardiomyocytes
Masaharu Kataoka
m.kataoka09@keio.jp
Department of Cardiology, Keio University School of Medicine

Summary Abstract“Heart” is a dynamic tissue that maintains pumping by conserved contraction. However, once
myocardial infarction (MI) occurs, the infarcted myocardium loses its capacity, resulting in theformation of scar tissue. The remaining myocardium is forced to compensate, often leading to heart
failure and decreased survival. Meanwhile, long non-coding RNAs (lncRNAs) are transcribed RNA
molecules of >200 nucleotides in length. Thousands of lncRNAs have recently been identified andmany of them are expressed in a cell- and tissue-specific manner. However, the biological functionsof these lncRNAs remain largely unknown. Intriguingly, a few recent studies have demonstrated
some lncRNAs have crucial roles during developmental process. Thus, we hypothesize that there areunrecognized novel lncRNAs that control cell size, proliferation, and contractility of cardiomyocytes.
This study was designed to identify and characterize novel lncRNAs that have crucial roles in
the heart. In particular, we focused on the role of lncRNAs of the regulation of cell biologicalsystems such as cell size and cell cycle of cardiomyocyte after MI. In fact, we demonstrated that theexpression of lncRNAs was dramatically changed after MI, and that a novel lncRNA, that we named“linc-Heart”, can regulate cell cycle of adult cardiomyocytes. The findings in this study would
lead to era with new therapeutic strategy for cardiac diseases by regulation of cell cycle in adultcardiomyocytes in the future.
Key Words：cardiomyocytes, cell cycle, long non-coding RNA, myocardial infarction, proliferation

Introduction
It is now recognized that the majority of the genome is actively transcribed to produce thousands of
non-coding transcripts in many cell types and tissues.1-3 A subset of these non-coding transcripts areclassified as lncRNAs, transcribed RNA molecules greater than 200 nucleotides in length that do not
encode proteins.2-4 The functions of lncRNAs have recently been investigated in diverse biologicalprocesses such as stem cell pluripotency, immune responses, and cell-cycle regulation.5-7 However,
little is known about the expression and functional significance of lncRNAs in the heart.
Recently, a novel lncRNA, Braveheart (Bvht), has been defined as a critical regulator of
cardiovascular commitment from embryonic stem cells.8 Bvht represents the first lncRNA thatdefines cardiac cell fate and lineage specificity, linking the function of lncRNAs to cardiac
development. In their study, the authors showed that the Bvht transcript is expressed in several adultmouse tissues including brain, colon, heart, and muscle with its highest expression in the heart. It
will be important to further characterize the spatiotemporal expression of Bvht in development anddisease. Most importantly, it will be essential to define the in vivo function of Bvht in mice, using
genetic loss-of-function approaches.9 Furthermore, another novel lncRNA, Fendrr, has been recently
identified.10 The lateral mesoderm-specific lncRNA, Fendrr, is essential for proper heart and bodywall development in the mouse. Importantly, any lncRNAs having crucial roles in the maintenance

The Novartis Foundation(Japan)
for the Promotion of Science

39

The Novartis Foundation(Japan)
for the Promotion of Scienceof adult heart functions have yet to be studied. It will be interesting to study whether novel lncRNAs
might also participate in cardiac regeneration or be used to stimulate cellular reprogramming.
Clearly, the discovery of Bvht and Fendrr will significantly impact the cardiovascular research field
and human cardiovascular disease.
Therefore, the purposes of our study are 1) to identify key lncRNAs which regulate essential
biological process in the heart, and 2) to explore the crucial functions of the identified lncRNAs in
the heart.

Resultsa) Myocardial infarction modelFirst of all, our important step is to screen potential candidate novel lncRNAs. To identify key
lncRNAs which regulate essential biological process of adult heart, we used mouse model with
MI. MI surgery was performed in male mice (C57BL/6J, 8 weeks old) by ligating left anterior
descending artery under artificial ventilator and thoracotomy. Heart was dissected and myocardium
from the border-zone area and that from the remote area was collected to obtain RNA at day 3 andday 14 after MI surgery, respectively.
b) LncRNA microarray screening
LncRNA microarray analysis was performed by using a lncRNA microarray kit (Agilent). RNA
samples from myocardium after MI surgery or Sham surgery were used. Among more than 10,500
lncRNAs that the kit can measure, about 7,500 lncRNAs could be detected in our samples. The
results of lncRNA microarray were further analyzed by Gene-Spring software (Agilent). The
analysis of 3D PCA scores, hierarchical clustering, and profile plot of lncRNA expression suggested
that RNAs from myocardium of border-zone area at 3 days after MI surgery (3POD) had the mostdramatically altered lncRNA expression levels.
c) Primary characterization of potential candidate lncRNAs
Our purpose is to discover the potential lncRNAs that have crucial roles to maintain heart functions.
Therefore, we focused on the specific lncRNAs whose expression levels are high in normal adult
hearts compared to other tissues, and whose expression levels dramatically decrease or increase
in myocardium following MI. At first, fold change analysis was performed, and we selected > 5.0
fold-changed lncRNAs in MI myocardium by qPCR. Next, we planned to confirm the expression
levels of above lncRNAs by qPCR. Some of lncRNAs have high-homologous sequences as proteincoding mRNAs. Thus, we selected lncRNAs that don’t have those homologous sequences and can be
expected to be detected by qPCR independently. After measurement of expression levels by qPCR,
the expression patterns of lncRNAs that could be detected by qPCR were compared to the resultsof lncRNA microarray, and lncRNAs whose expression patterns were similar between microarrayand qPCR were selected for further analysis. The expression levels of candidate lncRNAs in several
mouse tissues, such as brain, heart, lung, liver, kidney, and skeletal muscle, were measured byquantitative real-time PCR (qPCR) assays. The expression level of only 1 lncRNA was significantly
greater in the heart compared to other tissues. Therefore, we decided to focus on this one as thepotential candidate lncRNA, which we named “linc-Heart”.
The expression pattern of linc-Heart in staged hearts was determined by qPCR. Intriguingly, we
found that its expression level was low until post-natal day7, but after growing up, the linc-Heartwas highly expressed in the adult heart.

The Novartis Foundation(Japan)
for the Promotion of Science

40

The Novartis Foundation(Japan)
for the Promotion of Science

We also checked the expression level of linc-Heart in several tissues by Northern blotting aswell as qPCR, and estimated the size of linc-Heart transcripts. Northern blotting revealed that theexpression level of linc-Heart was high in the heart compared to other tissues, and that its level wascomparable between in 2 months-age heart and in 6 months-age heart. The predictive size of lincHeart transcripts was 1.9Kb.
d) In vivo function analysis
To investigate the functions of “linc-Heart“, 9 adeno-associated virus (AAV) containing linc-Heart(AAV-linc-Heart) was generated. Hearts injected with AAV-linc-Heart had high expression levelsof linc-Heart. Hypertrophic hearts were created by transverse aortic constriction (TAC) surgery in
8-week old mice. Surprisingly, the size of cardiomyocytes in the hearts injected with AAV-linc-Heartwas maintained within almost normal size, and the total count of cardiomyocytes was increased.
Cell proliferation marker, ph3, was positive in the cardiomyocytes at post-TAC surgery day 3 hearts
injected with AAV-linc-Heart.

Discussion & Conclusion
So far, nothing has been known about the functional significance of lncRNAs in the adult heart.
Importantly, this study suggested that a novel lncRNA, linc-Heart, has a crucial role in the heart,which can regulate the cell cycle of cardiomyocytes in the adult heart. This lncRNA study is novel
and will greatly impact cardiovascular biology and medicine. We will perform in vitro study todetermine the detailed mechanisms mediated by linc-Heart, in near future.

References
1. Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B. Mapping and quantifying
mammalian transcriptomes by RNA-Seq. Nature Methods. 2008;5:621-628.
2. Guttman M, Garber M, Levin JZ, Donaghey J, Robinson J, Adiconis X, Fan L, Koziol MJ,
Gnirke A, Nusbaum C, Rinn JL, Lander ES, Regev A. Ab initio reconstruction of cell typespecific transcriptomes in mouse reveals the conserved multi-exonic structure of lincRNAs.
Nature Biotechnol. 2010;28:503-510.
3. Cabili MN, Trapnell C, Goff L, Koziol M, Tazon-Vega B, Regev A, Rinn JL. Integrative
annotation of human large intergenic noncoding RNAs reveals global properties and specificsubclasses. Genes Dev. 2011;25:1915-1927.
4. Mercer TR, Dinger ME, Sunkin SM, Mehler MF, Mattick JS. Specific expression of longnoncoding RNAs in the mouse brain. Proc Natl Acad Sci U S A. 2008;105:716-721.
5. Guttman M, Amit I, Garber M, et al. Chromatin signature reveals over a thousand highly
conserved large non-coding RNAs in mammals. Nature. 2009;458:223-227.
6. Hung T, Wang Y, Lin MF, et al. Extensive and coordinated transcription of noncoding RNAs
within cell-cycle promoters. Nature Genet. 2011;43:621-629.
7. Ravasi T, Suzuki H, Pang KC, Katayama S, Furuno M, Okunishi R, Fukuda S, Ru K, Frith MC,
Gongora MM, Grimmond SM, Hume DA, Hayashizaki Y, Mattick JS. Experimental validation of
the regulated expression of large numbers of non-coding RNAs from the mouse genome. Genome
Res. 2006;16:11-19.
8. Klattenhoff CA, Scheuermann JC, Surface LE, et al. Braveheart, a long noncoding RNA requiredfor cardiovascular lineage commitment. Cell. 2013;152:570-583.

The Novartis Foundation(Japan)
for the Promotion of Science

41

The Novartis Foundation(Japan)
for the Promotion of Science

9. Kataoka M, Huang ZP, Wang DZ. Build a braveheart: the missing linc (RNA). Circ Res.
2013;112:1532-1534.
10. Grote P, Wittler L, Hendrix D, et al. The tissue-specific lncRNA Fendrr is an essential regulator ofheart and body wall development in the mouse. Dev Cell. 2013;24:206-214.

一般の皆様へ
本研究では成熟心筋細胞の細胞周期を調整する新規の lincRNA を同定し、linc-Heart と命名した。今後、linc-Heart が成熟心筋細胞の細胞周期を調整する詳細なメカニズムを解明し、心筋細胞周期の再活性化による病的心臓治療への応用の可能性を広げたいと考える。

本研究成果を応用することによって、これまでの試みから逸脱した全く新しい概念として、もともと存在している正常な成熟心筋細胞を細胞増殖させることにより治療する、という発想が可能な時代が到来する可能性がある。ヒト心筋梗塞後のリモデリング抑制や心筋症での心機能改善等へ応用できる可能性が広がり、臨床への将来的な貢献が多いに期待できる。

The Novartis Foundation(Japan)
for the Promotion of Science

42

The Novartis Foundation(Japan)
for the Promotion of Science

Thymic positive selection impacts T cell immune responses in vivo
Kensuke Takada
takada@vetmed.hokudai.ac.jp
Institute of Genome Research, Univetsity of Tokushima(Present affiliation: Faculty of Veterinary Medicine, Hokkaido University)

Summary Abstract
Thymic positive selection is generally recognized to shape antigen specificity repertoire of
T cells, but its additional role in functional education of T cells has been recently suggested.
Thymoproteasomes mediate positive selection of CD8 T cells by producing MHC class I-associatedself peptides in thymic cortex. Herein, we report that the loss of thymoproteasomes affects in vivo
T cell responses, including effector T cell differentiation and memory formation. This result suggestthat thymoproteasome-dependent positive selection, an early step of T cell development, determines
the fate of post-activated T cells.
Key Words：Lymphocyte, T cell, thymus, positive selection, proteasome

Introduction
In the thymus, early thymocyte repertoire is shaped through multiple selection steps to generate
immunocompetent T cells. Positive selection is induced by low affinity engagement of antigen
receptors with self-peptide-MHC complexes in thymic cortex and is recognized as a process to form
an usefull antigen recognition repertoire. It is becoming evident that protein degradation machineries
unique to thymic cortex facilitate the production of self peptides inducing positive selection1). Basedon our previous finding that thymoproteasome-dependent positive selection determines the antigen
responsiveness of mature CD8 T cells2), the present study examined the effects of thymoproreasomedependent positive selection on in vivo immune responses.

Results
1. Primary responses of T cells generated in thymoproteasome-deficient thymus
To generate monoclonal CD8 T cells positively selected in the absence of thymoproteasomes, lethally
irradiated mice deficient of a proteolytic subunit of thymoproteasomes, β5t encoded by Psmb11 gene,
were reconstituted with bone marrow cells from Rag1-/-OT-I transgenic mice2-4). Ovalbmin (OVA)specific naïve OT-I T cells selected in the presence or absence of thymoproteasomes were transferred
into Ly5.1 recipient mice. To activate donor OT-I cells in vivo, recipients were subsequently
injected with bone-marrow-derived dendritic cells pulsed with OVA peptide and CpG adjuvant. At
different time points after immunization, donor OT-I cells in recipient spleens were analyzed byflow cytometry. In both groups, donor cells similarly expanded by day 5 post immunization and thendecreased overtime until day 30. This result indicats that CD8 T cells selected in thymoproteasomedeficient thymus normally generate primary antigen responses in terms of cell number.
2. Phenotype of thymoproteasome-independent T cells in effector and memory phases
T cells which are activated in response to antigens differentiate into several subsets of effector

The Novartis Foundation(Japan)
for the Promotion of Science

43

The Novartis Foundation(Japan)
for the Promotion of Science

T cells. Terminally differentiated effector cells (KLRG1+CD127-) are relatively short-lived anddisappear overtime after the clearance of pathogens, while a part of memory precursor effector cells(KLRG1-CD127+) persist longer and ultimately differentiate into memory cells. Accordingly, we
analyzed the expression of KLRG1 and CD127 in donor OT-I cells at different time points after
immunization. OT-I cells positively selected in the absence of thymoproteasomes showed higher
frequency of terminally differentiated effector cells than normally selected OT-I cells did 4 and 30
days after immunization, but not in other time points including day 5, 10 and 15. Together with theformer finding that absolute donor cell number was similar between control and thymoproteasomedeficient groups, this result indicates that CD8 T cells selected in thymoproteasome-deficient thymus
are biased to terminally differentiated effector cells in early expansion and memory phases.
3. Cytokine production by thymoproteasome-independent T cells
To understand the effect of thymoproteasome deficiency on the effector functions after activation in
the periphery, at day 5 post immunization, the peak of response in cell number, production of effector
cytokines was examined. Spleen cells from recipient mice were cultured in the presence of OVA
peptide and brefeldin A, and intracellular cytokines were analyzed by flow cytometry. The frequency
of cells producing IFNγ and TNFα was significantly decreased in thymoproteasome-independent
OT-I cells in comparison with control OT-I cells. Thus, positive selection through thymoproteasomes
affects the effector functions of post-activated mature T cells.
4. Memory responses of thymoproteasome-independent T cells
Functions of memory cells was investigated by testing the proliferative responses of donor
OT-I cells following secondary immunization in vivo. At day 40 post primary immunization, thefrequency and the absolute number of donor OT-I cells in the recipient spleens were similar between
thymoproteasome-deficient and control groups. Four days after given secondary immunization,
significant increase of donor OT-I cells was seen in control but not in thymoproteasome-deficientgroups. In thymoproteasome-deficient group, donor cell numbers before and after secondary
immunization were equivalent, indicating that secondary expansion was almost completely impaired
in memory OT-I cells positively selected in the absence of thymoproteasomes. The data suggestthat positive selection through thymoproteasomes dictates the functional quality of memory T cells
during the early developmental stage of T cells in the thymus.

Discussion & Conclusion
The results of this study indicate that thymoproteasome-dependent positive selection instructs
developing thymocytes to acquire optimal functions after maturation. Especially, impacts ofthymoproteasome deficiency on immunological memory extends the significance of functional
education through positive selection that we previously suggested in in vitro studies 2, 5). Since
its discovery in 1970’s, positive selection has been understood as a process to shape the antigen
recognition specificity repertoire. However, recent advances in our knowledge on thymic
microenvironment and thymus-specific protein degradation machineries, including cathepsin L,
thymus-specific serine protease, and constitutive autophagy 1), may provide opportunities to revisit
the roles of positive selection in the immune system.

The Novartis Foundation(Japan)
for the Promotion of Science

44

The Novartis Foundation(Japan)
for the Promotion of Science

References
1. Klein L, Kyewski B, Allen PM, Hogquist KA. Positive and negative selection of the T cell
repertoire: what thymocytes see (and don't see). Nature Reviews Immunology 14: 377-391 (2014)
2. Takada K, Van Laethem F, Xing Y, Akane K, Suzuki H, Murata S, Tanaka K, Jameson SC, Singer
A, Takahama Y. TCR affinity for thymoproteasome-dependent positively selecting peptides
conditions antigen responsiveness in CD8+ T cells. Nature Immunology 16: 1069-1076 (2015)
3. Murata S, Sasaki K, Kishimoto T, Niwa S, Hayashi H, Takahama Y, Tanaka K. Regulation of
CD8+ T cell development by thymus-specific proteasomes. Science 316: 1349-1353 (2007)
4. Nitta T, Murata S, Sasaki K, Fujii H, Mat Ripen A, Ishimaru N, Koyasu S, Tanaka K, Takahama
Y. Thymoproteasome shapes immunocompetent repertoire of CD8+ T cells. Immunity 32: 29-40(2010)
5. Takada K, Kondo K, Takahama Y. Generation of peptides that promote positive selection in thethymus. Journal of Immunology 198: 2215-2222 (2017)

一般の皆様へ
リンパ球のひとつである T 細胞は、病原体や癌細胞に対する防御に不可欠な役割を果たしています。T 細胞は胸腺という器官で非常に複雑な過程を経て作られます。私たちは、胸腺に存在する特殊な酵素の解析を通し、胸腺での分化の過程が、成熟した後の T 細胞の機能を決定づけることを明らかにしてきました。今回、ワクチンの基本原理としても重要な免疫記憶というT 細胞の機能に、胸腺での分化過程が関わっている可能性を見出しました。機能的に優れた T

細胞が作り出される仕組みを理解することは、免疫の異常が関係する様々な病気の予防や治療、優れたワクチンの開発に役立つと期待されます。

The Novartis Foundation(Japan)
for the Promotion of Science

45

The Novartis Foundation(Japan)
for the Promotion of Science

Role of junctophilin for the proper function and intracellular
distribution of L-type calcium channels
Tsutomu Nakada
tnakada@shinshu-u.ac.jp
Shinshu University School of Medicine

Summary Abstract
Specific localization of the L-type calcium channel (LTCC) to the junctional membrane (JM)of skeletal muscle is critical for the efficient excitation-contraction (EC) coupling; however, thetargeting mechanism is still unclear. This study showed that the physical interaction between
junctophilins (JPs) and the C-terminus of CaV1.1 subunits of LTCC is essential for the proper JMtargeting of LTCC, the physical interaction between LTCC and ryanodine receptors (RyR) andtherefore the efficient EC coupling in the skeletal muscle.
Key Words：L-type calcium channel, junctophilin, junctional membrane, skeletal muscle

Introduction
Skeletal muscle LTCC is localized to JM where the sarcolemma are closely apposed to thesarcoplasmic reticulum membrane. Although this JM targeting of LTCC is critical for the EC
coupling by supporting the physical coupling between LTCC and RyR, its molecular mechanism is
not fully elucidated. JPs are known to stabilize the JM complex by bridging the plasma membraneand sarcoplasmic reticulum. Here, we explored that JPs support the proper JM-targeting of LTCC bybinding to the CaV1.1 subunit of LTCC and the physical interaction between LTCC and RyR, andthereby ensure the efficient EC coupling in the skeletal muscle.

Results
We first examined the effect of siRNAs against JP1 and JP2 on the localization and function of
LTCC in cultured myotubes. Immunocytochemical analysis revealed that the JP1 or JP2 knockdown
significantly inhibited the JM-targeting of LTCC. Calcium imaging analysis using a fluorescentcalcium indicator Fluo-4 showed that the JP1 or JP2 knockdown significantly decreased the numberof myotubes exhibiting a calcium transient in response to electrical stimulation. These results
indicate that JPs contribute to the proper localization and function of LTCC in skeletal myotubes.
Co-immunoprecipitation studies revealed that CaV1.1, a pore subunit of skeletal muscle LTCC,
physically interacted with JP1 and JP2 in the mouse skeletal muscle in vivo. Pull down assay with
GST-fusion proteins bearing the cytosolic regions of CaV1.1 indicated that the proximal C-terminusof CaV1.1 is necessary for the binding of CaV1.1 with JPs. This JP binding domain (JPD) was wellconserved between CaV1.1 and cardiac CaV1.2 which is also targeted to JM when expressed incultured myotubes. However, JPD was not conserved in neuronal CaV2.1, which did not localize to
JM when expressed in cultured myotubes. Alanine substitutions of several residues in JPD reduced
the binding of the GST fusion proteins to JPs. The same alanine substitutions were introduced into
the JPD of CaV1.1, and these mutant CaV1.1 were transiently expressed the channel subunit incultured myotubes. Immunocytochemical analysis revealed that the JM-targeting rate of the mutant

The Novartis Foundation(Japan)
for the Promotion of Science

46

The Novartis Foundation(Japan)
for the Promotion of Science

CaV1.1 was significantly reduced compared with the wild-type. These data show that the physicalinteraction of CaV1.1 with JPs via the JPD is important for the proper JM-targeting of LTCC.
We next examined the effect of transient expression of transmembrane domain deletion mutantof JP1 (JP1ΔTM) in cultured myotubes. This mutant was not localized to JM but diffusely to theentire plasma membranes. Moreover, co-expression of JP1ΔTM inhibited the JM-targeting of LTCC.
Thus, this mutant serves as a dominant negative molecule inhibiting the JM-targeting of LTCC. To
investigate the physiological significance of the JM-targeting of LTCC in vivo, JP1ΔTM was next
expressed in the tibialis anterior (TA) and flexor digitorum brevis (FDB) muscles of C57BL/6 miceby using the adeno-associated virus (AAV) vector system. Ten days after infection, the expressionof JP1ΔTM was observed in >80% of muscle fibers. Interestingly, the contractile force and specificforce of TA muscle were significantly decreased at any stimulation frequency between (1-200 Hz)
with the JP1ΔTM-AAV injection. However, no changes in cross sectional area were observed
between the muscles injected and not injected with JP1ΔTM-AAV. These results indicate that the
JM-targeting of LTCC is essential for the efficient EC coupling of the skeletal muscle in vivo.
Furthermore, the effect of JP1ΔTM-AAV on twitch and tetanus calcium transients of FDB muscle
fibers were analyzed. JP1ΔTM expression significantly decreased the peak amplitude of calcium
transients. Immunocytochamical analysis showed that JP1ΔTM was localized more abundantly tothe sarcolemma than the T-tubule membranes in FDB muscle fibers as was the case for myotubes.
Compared with control FDB fibers, the JP1ΔTM expressed-fibers showed more abundant
immunoreactivity of CaV1.1 in the sarcolemma than T-tubular membranes. Finally, the proximity
ligation assay revealed that JP1ΔTM expression significantly decreased the physical coupling
between LTCC and RyR. These data clearly show that JPs support the physical coupling between
LTCC and RyR, and thereby, ensure the efficient EC-coupling in the skeletal muscle.

Discussion & Conclusion
It has been proposed that the proper JM-targeting of LTCC is critical for the efficient EC couplingof the skeletal muscle. It was also reported that the C-terminus of CaV1.1 and CaV1.2 subunits
was necessary for the proper JM-targeting of LTCC in myotubes (1,2). However, the molecularmechanism underlying the JM-targeting is still not fully elucidated.
Our knockdown experiments showed that it is JPs that support the proper localization andfunction of LTCC. We also showed biochemically the physical interaction of CaV1.1 with JPs via
its C-terminal JPD. Therefore, it is likely that the interaction of JPs and the C-terminus of CaV1.1
subunits ensures the proper intracellular localization and function of LTCC.
JP1ΔTM inhibited the JM-targeting of LTCC. By using this tool, we showed that JP1ΔTM
expression decreased the specific force of mouse skeletal muscles by disrupting the JM targeting of
LTCC and thereby the interaction between LTCC and RyR.
From these results, we conclude that the physical interaction between the C-terminus of CaV1.1
and JPs is essential for the proper JM-targeting of LTCC, the physical interaction between LTCC andRyR and therefore the efficient EC coupling in the skeletal muscle.

References
1. Nakada T, Flucher BE, Kashihara T, Sheng X, Shibazaki T, Horiuchi-Hirose M, Gomi S, Hirose
M, Yamada M., The proximal C-terminus of α1C subunits is necessary for junctional membranetargeting of cardiac L-type calcium channels. Biochem J. 448: 221-231. 2012

The Novartis Foundation(Japan)
for the Promotion of Science

47

The Novartis Foundation(Japan)
for the Promotion of Science

2. Flucher BE, Kasielke N, Grabner M., The triad targeting signal of the skeletal muscle calcium
channel is localized in the COOH terminus of the alpha1S subunit. J Cell Biol. 151: 467-478.
2000

一般の皆様へ
骨格筋が正常に収縮するためには，L 型カルシウムチャネルが結合膜構造と呼ばれる部位に集積することが必要不可欠である。しかし，その集積の詳細な分子メカニズムは明らかになっていない。本研究では，結合膜構造に存在するジャンクトフィリンと呼ばれる分子が，L 型カ

ルシウムチャネルに結合し，正常な結合膜への局在化や機能を調節していることを見いだした。
今回得られた結果は，骨格筋の収縮メカニズムを解明する上で，重要な知見のひとつであると考えられる。

The Novartis Foundation(Japan)
for the Promotion of Science

48

The Novartis Foundation(Japan)
for the Promotion of Science

Epigenetic regulation in cell reprogramming and chimera formation.
Daiji Okamura
dokamura@nara.kindai.ac.jp
Department of Advanced Bioscience, Graduate School of Agriculture, Kindai University

Summary Abstract
Our study will be the first of its kind to demonstrate: 1) a simple epigenetic modifier (VitC) could
help derive a stable cell line from the developing PGCs 2) distinct pluripotent state can arise from thegermline, in addition to “naïve state” in mouse ES/ iPS cells and the “Primed state” in conventional
mouse EpiSCs (Epiblast Stem Cells).
Key Words：Primordial Germ Cells, Reprogramming, Epigenetic, Chimera formation

Introduction
In the mice, primordial germ cells (PGCs), the common precursor of oocytes and spermatozoa,
are specified from competent epiblast cells receiving inductive signals from the extra-embryonicectoderm. Thereafter, PGCs increase in number while migrating through the developing hindgut
and finally colonize the emerging gonads, where they initiate differentiation into either oocytes orspermatozoa. One unique property of progenitors of germ cell lineages is they are susceptible forbeing converted to pluripotent cell types without the need to introduce exogenous reprogramming
factors as normally used for obtaining iPSCs from somatic cell types.

Results
The primordial germ cells could be converted to pluripotent embryonic germ cells (EGCs) with the
combination treatments of LIF, FGF2 and SCF factors. This suggests epigenetic forces driven by the
combination of these growth factors may contribute to reprogramming of PGCs to a pluripotent state.
It is unknown, however, whether direct manipulation with epigenetic modifier could endow PGCs
with pluripotent features. In this study, we examined the effect of Vitamin-C on freshly isolated
PGCs. Vitamin-C, also know as L-ascorbic acid, has recently gained considerable attention largely
due to its role in facilitating the efficiency as well as quality of iPSC reprogramming (Esteban et al,
Cell Stem Cell, 2010; Stadtfeld, Nature Genetics, 2012). In addition, Blaschke et al (Nature, 2013)
demonstrated that Vitamin-C promotes ICM-like DNA methylation state in mouse ESCs in a Tetdependent manner. Intriguingly, when Vitamin-C was supplemented in mouse ESC culture the levelsof several germline genes were elevated. This promoted us to test Vitamin-C’s direct effect on freshly
isolated in vivo PGCs. To our surprise, while there was no visible colony observed in control culture,
Vitamin-C supplementation alone led to the emergence of alkaline phosphatase positive colonies andsubsequently the derivation of stable cell line from freshly isolated PGCs. We designated this newly
derived cell line as VcPGCs. To our surprise, these cells can be propagated indefinitely in vitro anddemonstrated pluripotency using teratoma formation assay. More interestingly, the pluripotent celllines generated with Vitamine-C share distinct features compared to conventional EGCs and thus
may represent a distinct pluripotent state arising from the germline. VcPGCs are pluripotent asdemonstrated by successful formation of teratomas comprising cells from all three primary germ

The Novartis Foundation(Japan)
for the Promotion of Science49

The Novartis Foundation(Japan)
for the Promotion of Sciencelayers. Strangely, after injected into blastocysts, unlike EGCs, VcPGCs couldn’t form chimeras,
indicating they exist in a distinct pluripotent state than EGCs. This chimeric-incompetency, however,
could be overcome by culturing VcPGCs in a culture media contained another chemical substances,suggesting distinct epigenetic forces contribute to the new pluripotent state.

Discussion & Conclusion
This goes well with the trending concept that pluripotency is not discrete but rather continuous
states in early embryos and there are many different pluripotent states and each may harbor uniquefeatures and advantages. Our study will be the first of its kind to demonstrate: 1) a simple epigenetic
modifier (VitC) could help derive a stable cell line from the developing PGCs 2) distinct pluripotent
state can arise from the germline, in addition to “naïve state” in mouse ES/ iPS cells and the “Primed
state” in conventional mouse EpiSCs (Epiblast Stem Cells)

一般の皆様へ
我々はこの研究を通じて、小分子化合物 L-Ascorbic Acid（ビタミン C）の添加により、将来

精子や卵子の元となる ” マウス始原生殖細胞（PGCs: primordial germ cells）” から、新規多能性幹細胞が樹立することを見出しました（vcPGC 細胞）。ビタミン C は、DAN 脱メチル化に機能する Tet ファミリーと協調的に機能することが広く知られており、マウス始原生殖細胞は DNA
脱メチル化によるエピジェネティック修飾の変化により、新規多能性幹細胞として再プログラ
ム化されていると考えられます。本研究の成果は、細胞の初期化プロセスにおけるエピジェネ
ティック修飾の役割を解明する、非常に有用なモデルとなることが期待されます。

The Novartis Foundation(Japan)
for the Promotion of Science

50

The Novartis Foundation(Japan)
for the Promotion of Science

Analysis of T2DM pathogenic mechanisms caused by susceptibility
genes using human iPS cells
Shun-ichiro Asahara
asahara@med.kobe-u.ac.jp
Kobe University Graduate School of Medicine

Summary Abstract
We have previously shown that a mutation at the Kcnq1 locus reduces pancreatic β cell mass inmice via epigenetic modulation. We found that disruption of Kcnq1 results in reduced Kcnq1ot1
expression as well as increased expression of p57. However, these results were obtained frommice. Therefore, to confirm that these mechanisms correspond to human, we tried to induce thedifferentiation from hiPSCs into pancreatic β cells using reported methods. As a result, each marker
was expressed in the cells and glucagon was expressed in the final stage, which suggested that
hiPSCs differentiated into pancreatic endocrine cells. In addition, KCNQ1OT1 expression was
significantly increased in the differentiated Glucagon+ cells. This may suggest that pathogenic
mechanism of diabetes which is due to KCNQ1OT1 is specific to pancreatic islets.
Key Words：hiPSCs, KCNQ1, pancreatic beta cell

Introduction
Recently, susceptibility genes for Japanese type 2 diabetes (T2DM), such as KCNQ1, have been
identified. However, most mutations, those may be related to T2DM onset, are on the intron andthus T2DM pathogenic mechanisms haven’t been understood well. We have reported that increased
expression of p57 in pancreatic islets significantly reduced pancreatic beta cell mass and induced
abnormal glucose tolerance in Kcnq1 mutant mice in whose islets the expression of non-coding RNA
Kcnq1ot1 was decreased. However, these results were obtained from mice. Therefore, to confirm thatthese mechanisms correspond to human, we decided to induce the differentiation into pancreatic beta
cells from human iPS cells (hiPSCs) and analyze the effects of each SNP on gene expressions.

Results
At first, we analyzed mutations of KCNQ1 in 11 hiPSC lines including 201B7 by sequencing.
As a result of analyzing mutations on 11 hiPSC lines, 8/11 had a KCNQ1 mutation. Fromthese cell lines, we selected 201B7 cell line that had a KCNQ1 gene mutation and then tried toinduce the differentiation into pancreatic beta cells. When the cells were cultured with ActivinA
and CHIR99021, the expressions of SOX17 and FOXA2 were confirmed by RT-PCR and
immunostaining. Moreover, cell clusters were in suspension culture with NOGGIN, KGF and
EGF. PDX1 and NKX6.1 were expressed in the cells, which suggested that hiPSCs differentiatedinto pancreatic bud. We confirmed that mutations of KCNQ1 didn’t inhibit the differentiationfrom hiPSCs into pancreatic bud. Next, we treated the differentiated cells with forskolin and Alk5
inhibitor. In the treated cells, the expression of Neurogenin3 was found by quantitative real-time
PCR and immunofluorescence. Furthermore, MAFA and MAFB expressions were shown in the
differentiated cells. To confirm the differentiation into pancreatic endocrine cells, we examined the

The Novartis Foundation(Japan)
for the Promotion of Science

51

The Novartis Foundation(Japan)
for the Promotion of Science

expressions of insulin and glucagon by real-time PCR and immunofluorescence. These examination
resulted in that glucagon expression, but not insulin expression, was confirmed both by real-time
PCR and immunofluorescence. These results suggested that hiPSCs differentiated into pancreatic
endocrine cells.
Our previous report has shown that Kcnq1ot1 expression in pancreatic beta cells plays an importantrole in the maintenance of pancreatic beta cell mass in mice. Thus, we believe that KCNQ1OT1
expression is dependent on each tissues. To confirm the importance of KCNQ1OT1 in pancreatic
beta cells, we compared the expression of KCNQ1OT1 between undifferentiated hiPSCs anddifferentiated glucagon positive cells. Real-time PCR showed that KCNQ1OT1 expression wasincreased in differentiated glucagon positive cells compared to hiPSCs. This result suggested thatKCNQ1OT1 expression was expectedly important in pancreatic endocrine cells.

Discussion & Conclusion
We have achieved the differentiation from hiPSCs into pancreatic endocrine cells and clarified thatKCNQ1OT1 expression was up-regulated in those cells. However, the challenge of differentiationfrom hiPSCs into insulin positive cells was unfortunately failed. We have to change the protocol
about the differentiation into pancreatic β cells. Hereafter, we try to induce the differentiation from
hiPSCs with SNP mutations into pancreatic β cells and analyze the effects of KCNQ1 SNP on gene
expressions.

References
1. Asahara S, Etoh H, Inoue H, Teruyama K, Shibutani Y, Ihara Y, Kawada Y, Bartolome A,
Hashimoto N, Matsuda T, Koyanagi-Kimura M, Kanno A, Hirota Y, Hosooka T, Nagashima K,
Nishimura W, Inoue H, Matumoto M, Higgins MJ, Yasuda K, Inagaki N, Seino S, Kasuga M,
Kido Y. Paternal allelic mutation at the Kcnq1 locus reduces pancreatic β cell mass via epigenetic
modification of Cdkn1c. Proc Natl Acad Sci USA. 112(27):8332-8337, 2015
2. Toyoda T, Mae S, Tanaka H, Kondo Y, Funato M, Hosokawa Y, Sudo T, Kawaguchi Y, Osafune
K. Cell aggregation optimizes the differentiation of human ESCs and iPSCs into pancreatic budlike progenitor cells. Stem Cell Res. 14(2):185-197, 2015

一般の皆様へ
日本人2型糖尿病患者では、膵β細胞の脆弱性が問題であることが明らかになっており、その原因の一つとして KCNQ1遺伝子の変異が注目されています。我々は、Kcnq1変異マウスでは

Kcnq1ot1という分子の量が減ることによって膵β細胞量が減少することを報告しましたが、ヒトでも同様のことが起こっているかは不明です。そこでヒト iPS 細胞を膵β細胞に分化させ、マウスの実験結果と比較することにしました。今回の研究では膵内分泌細胞への分化を達成しましたが、膵β細胞への分化誘導には至りませんでした。今後はプロトコールを改良し、さらに実験を進めていく予定です。

The Novartis Foundation(Japan)
for the Promotion of Science

52

The Novartis Foundation(Japan)
for the Promotion of Science

Molecular mechanism of plant cell growth via NIMA-related kinases
Hiroyasu Motose
motose@cc.okayama-u.ac.jp
Okayama University

Summary Abstract
NIMA-related protein kinase 6 (NEK6) promotes directional cell growth through phosphorylationof tubulin and the resulting destabilization of cortical microtubules in the model angiosperm
Arabidopsis thaliana. The basal land plant Marchantia polymorpha, has a single NIMA-related
protein kinase (MpNEK1), which directs tip growth of rhizoids (the early rooting system of land
plants) through microtubule depolymerization. Thus, NEK-dependent mechanism of directional cell
growth might be evolutionarily conserved and contributes to the early rooting system during thecolonization of the terrestrial environment by plants.
Key Words：Directional cell growth, Cell polarity, microtubule, kinase

Introduction
Plant cortical microtubules align perpendicular to the growth axis to determine the direction ofcell growth. However, it remains unclear how plant cells form well-organized cortical microtubule
arrays in the absence of a centrosome. In this study, we investigated the functions of Arabidopsis
NIMA-related kinase 6 (NEK6), which regulates microtubule organization during anisotropic cell
expansion. To obtain insight into the roles of NIMA-related kinases in land plant evolution, we
also analyzed the function of NIMA-related protein kinase 1 (MpNEK1) in the basal land plant(liverwort), Marchantia polymorpha.

Results
The nek6 mutant shows the defects in growth directionality of epidermal cells and variousorgans (Fig. 1). Quantitative analysis of cell growth in the nek6-1 mutant demonstrated that NEK6
suppresses ectopic outgrowth and promotes cell elongation in different regions of the hypocotyl of
Arabidopsis. Because microtubules regulate growth direction of plant cells, we analyzed microtubulesin nek6 mutant. Loss of NEK6 function led to excessive microtubule waving and distortion (Fig. 2).
These distorted microtubules were generated by detachment of microtubules from the cell cortex.
The depolymerization of detached microtubules was significantly decreased in the nek6-1 mutant,demonstrating that NEK6 depolymerizes distorted microtubules after detachment from the cortex.
Live cell imaging showed that NEK6 localizes to themicrotubule lattice and to the shrinking plus and minus
ends of microtubules. Especially, NEK6 localized to theshrinking ends of microtubules detached from the cellcortex. To analyze gain-of-function phenotypes caused
by NEK6 overexpression, we used an estradiol induction
system (an estrogen receptor-based XVE transactivatorsystem, Fig. 3). In transgenic plants harboring estrogenFig.1. Growth polarity defect in nek6 mutant

The Novartis Foundation(Japan)
for the Promotion of Science

53

The Novartis Foundation(Japan)
for the Promotion of Science

inducible NEK6, estradiol treatment induced the expression of NEK6 approximately 100- to 1000fold. The induced overexpression of NEK6 destabilized cortical microtubules and suppressed cell
elongation. This result support our hypothesis that NEK6 depolymerizes cortical microtubules.

Results
Furthermore, we identified five phosphorylation sites in eta-tubulin that serve as substrates
for NEK6 in vitro. Non-phosphorylatable alanine substitution of the phosphorylation site Thr166
promoted incorporation of mutant eta-tubulin into microtubules (Fig. 4). This result suggests thatThr166 as a major regulatory site, which strongly affects microtubule localization of TUB4. To
determine whether the kinase activity of NEK6 is indispensable for its function in directional cell
expansion, the wild type and kinase-dead mutant NEK6-GFP fusion proteins were expressed in
the nek6-1 mutant under the control of NEK6 promoter. The wild type NEK6-GFP complemented
the ectopic outgrowth phenotype of nek6-1, whereas the kinase-dead mutant NEK6-GFP did notcomplement the nek6-1 phenotype. This result demonstrates that NEK6 kinase activity is essential
for directional cell expansion. Taken together, these results indicate that NEK6 promotes directional
cell growth throughphosphorylation of
eta-tubulin and theresulting destabilizationof cortical microtubules.
Fig. 2. Microtubule distortion in the nek6 mutant
Tip growth, an essential process for plant morphogenesis
and reproduction, is mediated by polarized accumulationof cellular materials coupled with turgor-pressure-driven
growth. However, it remains to be solved how a single
polarized growth site is determined and maintained. Here,
we analyzed the function of NIMA-related protein kinase
Fig. 3. Effect of NEK6 overexpression
1 (MpNEK1) in the liverwort, Marchantia polymorpha.
on plant growth
We e m p l o y e d g e n e d i s r u p t i o n b y h o m o l o g o u srecombination and found that Mpnek1 knockouts showeda defect in growth directionality of rhizoid cells. In the wild type, rhizoid cells
differentiate from the ventral epidermis and elongate by tip growth to form hairlike protrusions. In Mpnek1 knockout mutants, rhizoids frequently changed
their direction of growth and developed into a twisted and spiral morphology.
MpNEK1-Citrine complemented the growth polarity defect and localized to theapical growing region in rhizoids. In the apical dome of rhizoids, the wild typeexhibited fine microtubule meshwork, whereas tick microtubule cables extended
into the apical dome of Mpnek1 knockouts. Treatment with the microtubuleFig. 4. T166A
stabilizing drug taxol led to a phenocopy of Mpnek1 knockouts. These results promotes microtubuledemonstrate that MpNEK1 directs tip growth of rhizoids through microtubule localization of tubulin
destabilization. Furthermore, MpNEK1 could rescue ectopic outgrowth of epidermal cells in
Arabidopsis nek6 mutant, strongly supporting evolutionarily conserved NEK-dependent mechanismof directional cell growth, which may contribute to the early rooting system during the colonizationof the terrestrial environment by plants.

The Novartis Foundation(Japan)
for the Promotion of Science

54

The Novartis Foundation(Japan)
for the Promotion of Science

Discussion & Conclusion
In this study, we showed that Arabidopsis NEK6 maintains polar plant growth by promoting
longitudinal cell expansion and suppressing ectopic outgrowth. Both of these functions are mediatedby NEK6-dependent microtubule disassembly. Our results demonstrate that NEK6 coordinates
directional cell growth predominantly through depolymerization of cortical microtubules. NEK6 may
participate in microtubule surveillance system to reduce excessive distortion of microtubules after
detachment from the cortex. In the molecular level, NEK6 phosphorylates beta-tubulin incorporated
into microtubules to destabilize existing microtubules.
Our functional analysis of MpNEK1 indicates its crucial role in directional growth of rhizoids.
Mpnek1 knockouts exhibited a defect in rhizoid growth directionality, but not in rhizoid elongation
per se, implying specific function of MpNEK1 in the determination and maintenance of growthpolarity. This is consistent with the role of AtNEK6 in directional expansion of epidermal cells.
Actually, MpNEK1 could rescue outgrowth phenotype of Arabidopsis nek6 mutant, clearly indicating
the evolutionarily conserved NEK-dependent mechanism of cellular growth polarity. Therefore, land
plant NEKs fundamentally regulate directional cell expansion and this primary function may havealready been acquired during the early phase of land plant evolution.

References
1. Takatani S, Otani K, Kanazawa M, Takahashi T, Motose H (2015) Structure, function, andevolution of plant NIMA-related kinases: Implication for phosphorylation-dependent microtubule
regulation. J. Plant Research 128, 875-891.
2. Takatani S, Hirayama T, Hashimoto T, Takahashi T, Motose H (2015) Abscisic acid induces
ectopic outgrowth in epidermal cells through cortical microtubule reorganization in Arabidopsis
thaliana. Scientific Reports 5:11364.
3. Motose, H., Takatani S, Ikeda T, Takahashi T (2012) NIMA-related kinases regulate directional
cell growth and organ development through microtubule function in Arabidopsis thaliana. Plant
Signaling & Behavior 7, 1552-1555.

一般の皆様へ植物は分裂組織から根や葉・花などの様々な器官を形成します。このプロセスでは、個々の細胞がどの方向にどれくらい成長するかが重要になります。植物細胞の成長方向は、細胞内の骨格である微小管が一定の方向に並ぶことで規定されます。しかし、微小管がどのようなしくみで一定の方向にきれいに並ぶのかわかっていません。私たちは、NEK というチューブリンタ
ンパク質をリン酸化する酵素が、余分な微小管を除去して整理整頓することで、微小管がきれいに並び、細胞の成長方向が決定されることを見いだしました。このしくみは陸上植物の進化の初期に獲得されたと考えられます。

The Novartis Foundation(Japan)
for the Promotion of Science

55

The Novartis Foundation(Japan)
for the Promotion of Science

Molecular mechanism of overeating regulated
by cellular stress response
Takao Iwawaki
iwawaki@kanazawa-med.ac.jp
Kanazawa Medical University

Summary Abstract
This study aims to identify the mechanism of bulimia induced by endoplasmic reticulum (ER)stress. Bulimia behavior began to appear within several hours after the administration of an ER stressagent and continued for approximately one day. The blood glucose level was slightly increased, thateating inhibitory hormone levels tended to be increased, and that eating acceleratory hormone levels
were low after the ER stress loading. Based on these results, we suggest that ER stress-induced
bulimia may not be caused by an abnormality of the hormones that regulate eating.
Key Words：ER stress & bulimia

Introduction
The detailed regulatory mechanism of feeding behavior on a full or an empty stomach has
been elucidated by earlier research. However, much remains unknown about the mechanisms ofbulimia and anorexia. We noticed an unexpected phenomenon: When a certain ER stress agent
was administered to mice, bulimic behavior was induced. In our previous studies, we have alreadyestablished our original tools for analyzing ER stress in live mice and at a molecular level. In thisstudy, using these tools, we aim to discover a new possible role of cellular stress response moleculesin the regulation of feeding behaviors.

Results
In this study, we aim to elucidate the relationship between the cellular stress response and feeding
behavior at a molecular level. To this end, it is essential to investigate the dynamics of cellular stressresponse molecules in the brain area regulating feeding behavior (which has been studied very little
so far). First, we need to compare the activation of important cellular stress molecule responses
between bulimic model mice and normal mice. In essence, we have been performing conventional
histological analysis using slices of part of the brain, including the feeding behavior center, andspecific antibodies. We have also been approaching this problem by live imaging analysis in several
kinds of mice using our original technology for visualizing cellular stress. At the same time, wehave been examining whether bulimia is induced when an ER stress agent is injected in mice thatlack cellular stress response specifically in the bulimic behavior center. By doing this, molecules
responsible for inducing bulimia will be identified. Various knockout mice have either been produced
in-house or purchased from other institutions and are already in use.
We have been investigating the activation status of neurons and molecules involved in feeding
behavior in bulimic model mice, in parallel with previous experiments. Essentially, we have been
comparing the functions of neurons in the hypothalamic paraventricular nucleus and the arcuate
nucleus and the dynamics of leptin and ghrelin between bulimic model mice and normal mice. In

The Novartis Foundation(Japan)
for the Promotion of Science

56

The Novartis Foundation(Japan)
for the Promotion of Science

addition we have been investigating whether bulimic behavior of the relevant model mice is inhibitedby stimulating leptin per se or leptinergic POMC/CART neurons. On the other hand, bulimia isreported to resemble narcotic addition; thus, functions of neurons involved in reward and pleasure
should not be neglected. Therefore, we have also been analyzing the relevant bulimic model mice,focusing on dopamine receptors.
In our initial plan of experiments, we listed establishment of an ER stress-induced bulimic mousemodel and investigation of dynamics of ER stress-related molecules in the feeding behavior
center. Necessary data have been obtained from these experiments without problems, although not
sufficiently. For example, with regard to the relationship between ER stress and bulimia, bulimic
behavior caused by stress loading not only in mice of the single strain of C57BL/6 but also in several
other strains of mice has been investigated to the level that warrants publication in journals. In
addition, blood hormone levels and activation levels of ER stress-related molecules when bulimic
behavior is induced have been investigated to a sufficient level to determine the next analyticalstrategy. Incidentally, the size of the stomach of mice to which an ER stress agent was administered
6 hours before testing is larger than that of mice to which physiological saline was administered.
The stomach content of the former mice weighs approximately 3 times more than that of the latter.
This phenomenon is fairly well reproducible. In fact, we have repeated this experiment more than
one hundred times, and the result has never been reversed. In addition, sex-related differences
have hardly been observed. Some may suggest that abnormalities of digestive functions are thecause of the enlargement of the stomach; however, for the time being, we have been focusing on
feeding abnormalities as the cause of this phenomenon. The reason for this is that the mice that areadministered ER stress agents fill their stomach with bedding material and feces in the breeding cage,even when they are not fed.

Discussion & Conclusion
We have primarily been conducting a study on ER stress to date, and have identified and analyzed
the function of a molecule that senses ER stress, termed IRE1, in mammals (Iwawaki T. et al,Nat. Cell. Biol.), and developed an ER stress visualization technology in live mice (Iwawaki T. et
al, Nat. Med.). Furthermore, recently, we have also been focusing on analyzing the regulation ofphosphorylation/dephosphorylation of eIF-2α in pancreatic beta cells (Akai R. et al, Genes Cells.).
Cellular stress responses represented by the regulation of phosphorylation / dephosphorylationof eIF-2α have been studied in detail at the cellular and molecular levels. The mechanisms forregulation of feeding behavior on a full or an empty stomach have also been well understood tothe level of being described in physiology textbooks. However, findings regarding molecules thatfunction in cellular stress responses associated with the behaviors of mammals, except the findingof “rejection of nutritionally unbalanced feed,” have not been reported (Hao S. et al, Science andMaurin AC. et al, Cell Metab.). We believe that this interdisciplinary study will allow to link themolecular biology of cellular stress and research on feeding behavior regulation in the future.

References
Akai R, Hosoda A, Yoshino M, Iwawaki T “Constitutive role of GADD34 and CReP in cancellationof phospho-eIF2α-dependent translational attenuation and insulin biosynthesis in pancreatic β cells”
Genes Cells, 20, 871-886, 2015.

The Novartis Foundation(Japan)
for the Promotion of Science

57

The Novartis Foundation(Japan)
for the Promotion of Science

Hao S, Sharp JW, Ross-Inta CM, McDaniel BJ, Anthony TG, Wek RC, Cavener DR, McGrath BC,
Rudell JB, Koehnle TJ, Gietzen DW “Uncharged tRNA and sensing of amino acid deficiency inmammalian piriform cortex” Science, 307, 1776-1778, 2005.
Iwawaki T, Akai R, Kohno K, Miura M “A transgenic mouse model for monitoring endoplasmic
reticulum stress” Nat. Med., 10, 98-102, 2004.
Iwawaki T, Hosoda A, Okuda T, Kamigori Y, Nomura-Furuwatari C, Kimata Y, Tsuru A, Kohno
K “Translational control by the ER transmembrane kinase/ribonuclease IRE1 under ER stress” Nat.
Cell Biol., 3, 158-164, 2001.
Maurin AC, Jousse C, Averous J, Parry L, Bruhat A, Cherasse Y, Zeng H, Zhang Y, Harding
HP, Ron D, Fafournoux P “The GCN2 kinase biases feeding behavior to maintain amino acidhomeostasis in omnivores” Cell Metab., 1, 273-277, 2005

一般の皆様へ
満腹時および空腹時における摂食行動の制御機構は詳細なところまで明確になっている。しかしながらストレスが引き起こすとされる過食や拒食のメカニズムは殆ど分かっていない。私たちはマウスを用いた実験で次の意外なことに気が付いた。細胞の健康状態を悪化させるストレスを与えると過食行動が引き起こされるのである。この現象は「やけ食い」のような心的ス

トレス性の摂食異常を分子レベルで解明する糸口になるかもしれない。本研究を通じて私たちはストレス応答反応が果たしているであろう摂食行動制御に関する新たな役割を理解しつつある。

The Novartis Foundation(Japan)
for the Promotion of Science58

The Novartis Foundation(Japan)
for the Promotion of Science

Analysis of a signal through ER required for the inductionof axon regeneration
Naoki Hisamoto
i45556a@cc.nagoya-u.ac.jp
Graduate school of Science, Nagoya University

Summary Abstract
The ability of specific neurons to regenerate their axons after injury is governed by cell-intrinsic
regeneration pathways. However, the signaling pathways that orchestrate axon regeneration are notwell understood. Here we show that DDR-2, an RTK containing a discoidin domain that is activatedby collagen, and EMB-9 collagen type IV regulate the regeneration of neurons following axon injury
in C. elegans. Further analyses suggest that DDR-2 modulates the SVH-2 - JNK MAP kinase cascade
pathway via SHC-1 in the regulation of axon regeneration.
Key Words：axon regeneration, C. elegans, collagen

Introduction
Axon regeneration after nerve injury is a conserved biological process in many animals, including
humans (1). The nematode Caenorhabditis elegans has recently emerged as a genetically tractablemodel for studying regenerative responses in neurons (2). Extensive studies over several years using
this organism have revealed that a number of intrinsic and extrinsic signal transduction cascades,
which are conserved from worms to humans, regulate axon regeneration (3).

Results
We found that DDR-2, a receptor tyrosine kinase (RTK) containing a discoidin domain, is required
for efficient axon regeneration in C. elegans (4). At 24 hr after axon injury, the frequency of axon
regeneration in ddr-2 deletion mutant animals was reduced. We also monitored severed axons at alater time point (72 hr) after ablation. We observed that the frequency of axon regeneration in ddr2 mutant was increased at 72 hr after axon injury to a level, comparable to that of the wild type.
Thus, in ddr-2 mutants, initiation of axon regeneration after laser surgery is delayed. DDR-2 isexpressed in several tissues including neurons, and regulates axon regeneration in a cell-autonomous
manner. In mammals, DDRs are unique members of the family of RTKs in that they bind to and are
activated by triple-helical collagen, the major component of the extracellular matrix (ECM). For
this reason, we thought that the basement membrane collagens are likely to be candidates as ligands
for DDR-2. In C. elegans, three collagens are associated with the basement membrane: the type IV
collagens EMB-9 and LET-2 and the type XV/XVIII collagen CLE-1. We found that emb-9, but
not cle-1, is required for the axon regeneration. The ddr-2; emb-9 double mutant showed no further
enhancement of axon regeneration defect, and the regeneration defect of emb-9 could be suppressedby overexpression of DDR-2, indicating that emb-9 acts upstream of ddr-2 in axon regeneration.
Genetic epistatic analysis also revealed that ddr-2 acts upstream of svh-2, a Met-like growth factor
RTK that acts through the JNK MAPK pathway (5). We also executed biochemical analysis using
mammalian cultured cells and found that the cytoplasmic domain of DDR-2 can interact with SHC-

The Novartis Foundation(Japan)
for the Promotion of Science

59

The Novartis Foundation(Japan)
for the Promotion of Science

1, an adaptor protein required for efficient axon regeneration in C. elegans (6). The SHC-1 protein
has two phopshotyrosine-binding domains, SH2 and PTB. Interestingly, interaction between DDR2 and SHC-1 is independent of both the tyrosine kinase activity of DDR-2 and the phosphotyrosinebinding activities of SH2 and PTB domains in SHC-1. Further immunoprecipitation analysis
revealed that SHC-1 also associates with the cytoplasmic domain of the tyrosine kinase SVH-2. In
contrast to the case of DDR-2, a catalytically inactive mutant form of SVH-2 was unable to associatewith SHC-1, suggesting that the interaction between SVH-2 and SHC-1 is dependent on the Tyrautophosphorylation of SVH-2. We next investigated the functional significance of the SHC-1–SVH2 interaction in the rescue of the ddr-2 defect by shc-1 overexpression. The axon regeneration defectcaused by the ddr-2 mutation was suppressed by overexpression of SHC-1 protein. However, amutant SHC-1 protein that is unable to interact with SVH-2 failed to suppress the axon regeneration
defect in ddr-2 mutants. These results suggest that the binding of SHC-1 with SVH-2 is important for
SVH-2 activity in the axon regeneration pathway.
We further found that a series of ER/Golgi glycosylation enzymes, including a sugar transporter,
Galactose transferase and Fucose transferase, are required for efficient regeneration in C. elegans.
Further analysis revealed that these factors regulate axon regeneration via N-glycosylation of theextracellular domain of DDR-2 protein.

Discussion & Conclusion
In this study we examined the role of DDR-2 in the regeneration of injured axons in C. elegans. We
found that the ddr-2 mutation delayed initiation of regeneration in D-type motor neurons after laser
surgery. Similarly, the emb-9 mutation, defective in the type IV collagen, affected the initiation ofaxon regeneration. Further data indicate that emb-9 regulates axon regeneration via ddr-2.
Our genetic data also suggest that DDR-2, the C. elegans Shc adaptor protein SHC-1, and the HGFlike RTK SVH-2 function in the same genetic pathway regulating axon regeneration. When DDR2 binds to collagen, SHC-1 acts as a bridging molecule linking SVH-2 to the DDR-2 cytoplasmic
domain. This triggers SVH-1 growth factor-mediated activation of SVH-2 in a specific region, which
in turn leads to the activation of the JNK pathway. Although collagens have been used in the nerve
conduit in the treatment of nerve injury in humans (7), the molecular action of the collagen in axon
regeneration remained obscure. Thus, our data may provide a cue to understand the role of collagenand downstream signaling in axon regeneration of other organisms including human.

References
1. Chen ZL, Yu WM, Strickland S (2007) Peripheral regeneration. Annu Rev Neurosci 30: 209–233.
2. Yanik MF, Cinar H, Cinar HN, Chisholm AD, Jin Y, et al. (2004) Neurosurgery: functional
regeneration after laser axotomy. Nature 432: 822.
3. Hisamoto N, Matsumoto K (2017) Signal transduction cascades in axon regeneration: insightsfrom C. elegans. Curr. Opin. Genet. Dev. 44: 54-60.
4. Hisamoto N, Nagamori Y, Shimizu T, Pastuhov SI, Matsumoto K (2016) The C. elegans
discoidin domain receptor DDR-2 modulates the Met-like RTK-JNK signaling pathway in axon
regeneration. PLoS Genet. 12, e1006475.
5. Li C, Hisamoto N, Nix P, Kanao S, Mizuno T, et al. (2012) The growth factor SVH-1 regulates
axon regeneration in C. elegans via the JNK MAPK cascade. Nat Neurosci 15: 551–557.
6. Pastuhov SI, Fujiki K, Nix P, Kanao S, Bastiani M, et al. (2012) Endocannabinoid-Goα signaling

The Novartis Foundation(Japan)
for the Promotion of Science

60

The Novartis Foundation(Japan)
for the Promotion of Science

inhibits axon regeneration in Caenorhabditis elegans by antagonizing Gqα-PKC-JNK signaling.
Nat Commun 3: 1136.
7. Muheremu A, Ao Q (2015) Past, present, and future of nerve conduits in the treatment of
peripheral nerve injury Biomed. Res. Int. 2015: 237507.

一般の皆様へ
神経細胞は、軸索という長い突起を介して電気信号を伝達しており、外傷などで軸索が切断されると神経として機能できなくなります。しかし、多くの神経は軸索を再生する能力を潜在的に持っています。今回、モデル動物である線虫 C. エレガンスを用いた解析により、コラーゲ

ンが DDR と呼ばれるコラーゲン受容体を介して切断された軸索再生を促進することを新たに発見し、その分子メカニズムを解明しました。本成果で得られた基礎的知見は、神経再生促進技
術の改善・改良の手助けになることが期待されます。

The Novartis Foundation(Japan)
for the Promotion of Science

61

The Novartis Foundation(Japan)
for the Promotion of Science

Development of molecular delivery system using cell membrane
penetrating DNA nanostructures
Masayuki Endo
endo@kuchem.kyoto-u.ac.jp
Institute for Integrated Cell-Material Sciences, Kyoto University

Summary Abstract
We develop a DNA nanostructure having a molecular needle to penetrate lipid membrane fordelivering target molecules into the cell. We created the DNA nanostructure to introduce the target
molecule to a liposome containing DNA amplification system and successfully initiated the reaction.
The system will be employed for molecular delivery for control of the cellular functions.
Key Words：DNA nanostructure, delivery system, lipid membrane, cell function control

Introduction
Development of a new molecular delivery system is a fundamental issue for control of thecellular functions and responses. In this research, we develop basic technology for designing andfunctionalizing a DNA nanostructure, which can penetrate cell membranes using a nano-sized needle
molecule, and applying them for delivery of specific molecules and control of cellular functions.
Based on our DNA molecular technology, we designed and constructed DNA nanostructures forexpression of various functions depending on the purpose. First, we develop a molecular systemthat can work in a liposome containing a biochemical reaction system. Then the system will beapplied for delivery of target DNA, RNA and biomolecules for control of the cellular functions.
By using these functionalized DNA nanostructures, we intend to develop a new molecular systemfundamentally different from the conventional molecular delivery approach to the cells.

Results
We designed a cross-shaped DNA nanostructure with a four-helix bundled nano-sized DNA
needle which can penetrate cell membranes. We used DNA origami technology for constructionof the nanostructure to create a system to control binding and release of target molecules. We
first investigated the interaction between the cross-shaped DNA nanostructure and lipid bilayer
membrane, and visualized the behavior of the cross-shaped DNA nanostructure and their assemblyon the lipid bilayer surface using high-speed atomic force microscopy (AFM).
After investigating the fundamental properties, we examined the interaction between the DNA
nanostructure and liposome. First, in order to prove that the needle part penetrates lipid membrane,
we prepared a liposome containing DNA amplification system which can be activated by specific
DNA strand (trigger). We tested the incorporation of this trigger DNA strand by attaching to the endsof the needle. When the needle part with the trigger molecule penetrates the lipid membrane, theDNA amplification system inside should be initiated to start the reaction. We added the fluorescencelabelled cross-needle DNA nanostructure to the liposome, and observed the attachment of the
DNA nanostructure onto the surface of the liposome using a confocal microscope. Then we used
the trigger-attached cross-needle DNA nanostructure, and examined the fluorescence-based DNA

The Novartis Foundation(Japan)
for the Promotion of Science

62

The Novartis Foundation(Japan)
for the Promotion of Science

amplification in the liposome. Without the trigger strands, the amplification was not observed within
the expected period. On the other hand, using the trigger-attached cross-needle DNA nanostructure,
we clearly observed the amplification of the DNA inside the liposome. These results indicate thatthe needles penetrated the lipid membrane and consequently the trigger strand incorporated into theliposome initiated the DNA amplification reaction.
For increasing the efficiency of attachment onto the liposome surface, we incorporated cholesterol
molecules to the cross-needle nanostructure for increase of the interaction with the lipid membrane.
We also introduced photo-cleavable molecule to rapidly release the trigger strand inside theliposome. These will improve the efficient molecular delivery and reactions, and help creation of a
new molecular system that incorporates target molecules into the liposome. In the next step, we will
also examine the lattice assembly of the cross-needle structures for more efficient delivery of thetarget molecules into the liposome.
This cross-needle DNA nanostructure and delivery system will be applied to carry out target
molecule delivery to the cells. We plan to examine whether this DNA nanostructure can attach to thecell surface, and confirm whether the needle structure can penetrate the cell membrane and introduce
the target molecule into the cytoplasm.

Discussion & Conclusion
We have developed the cross-shaped DNA nanostructure having a four-helix bundled DNA needle,
which attaches onto the liposome surface and penetrate the lipid bilayer membrane. Using theliposome containing the DNA amplification system, the system inside the liposome was activatedby the addition of the cross-needle DNA nanostructure having the target DNA strand for initiationof the reaction. These results indicate that the target molecule can be incorporated via the needle
part, and the molecule delivery and subsequent reaction was controlled using this developed DNA
nanostructure. Using our DNA molecular technology, we successfully designed and constructed
DNA nanostructures for the initiation of the reaction in the liposome. In addition, the DNA latticeassembly to attach onto the lipid membrane was created, and their formation was visualized by highspeed AFM. For cellular applications, we are planning to adopt the cross-needle DNA nanostructure
and molecular system to deliver target DNA, RNA and biomolecules for control of the cell functions
and responses such as induction of apoptosis (cell death) and suppression of gene expression.

References
1. Single-Molecule Imaging of Dynamic Motions of Biomolecules in DNA Origami Nanostructures
Using High-Speed Atomic Force Microscopy. M. Endo, H. Sugiyama, Acc. Chem. Res. 47, 16451653 (2014).
2. Lipid bilayer-supported two-dimensional self-assembly of DNA origami nanostructures. Y.
Suzuki, M. Endo, H. Sugiyama, Nature Communications, 6, 8052 (2015).
3. Photoresponsive DNA Nanocapsule Having an Open/Close System for Capture and Releaseof Nanomaterials. T. Takenaka, M. Endo, Y. Suzuki, Y. Yang, T. Emura, K. Hidaka, T. Kato, T.
Miyata, K. Namba, H. Sugiyama, Chem. Eur. J. 20, 14951-14954 (2014).

The Novartis Foundation(Japan)
for the Promotion of Science

63

The Novartis Foundation(Japan)
for the Promotion of Science

一般の皆様へ
本研究では、DNA ナノ構造体の構築技術を利用し、ターゲット分子を細胞内に送り込む分子

輸送システムを開発する。DNA 鎖からなる分子針を持つ DNA オリガミ構造体を用いて、脂質二重膜との相互作用を高速原子間力顕微鏡 (AFM) によって観察した。次に、生化学反応系を導入したリポソーム（脂質膜）に、DNA 構造体を結合し、ターゲット分子が脂質膜を貫通し、リ
ポソーム内の生化学反応を開始できることを見出した。これら構築した技術を使って、細胞膜を貫通し、細胞内に分子を送り込む新たな分子システムの構築を検討する。最終的に細胞死の誘導、遺伝子発現の抑制、免疫増強など細胞機能制御や細胞応答を見る。

The Novartis Foundation(Japan)
for the Promotion of Science

64

The Novartis Foundation(Japan)
for the Promotion of Science

Studies toward total syntheses of biologically active, bridged
polycyclic meroterpenoids
Seiichi Nakamura
nakamura@phar.nagoya-cu.ac.jp
Nagoya City University

Summary Abstract
A stereoselective synthesis of C3-dehydrated berkeleytrione has been achieved through a polyene
cyclization to construct the [7.3.1.02,7] tricyclic ring system and ring closing metathesis to fashion thesix-membered ring as key steps.
Key Words：meroterpenoids, bridged polycyclic compounds, polyene cyclization

Introduction
Berkeleyones are a family of meroterpenoids isolated from the extremophilic fungus Penicillium
rubrum by Stierle and co-workers. Berkeleytrione, isolated along with berkeleydione in 2004, is theprominent member of the family and is reported to inhibit matrix metalloproteinase-3 and caspase-1.
Having developed a method for the construction of [7.3.1.02,7] tricyclic ring system by a biomimetic
polyene cyclization reaction,1 we addressed the synthesis of berkeleytrione.

Results
Epoxyallylsilane 1 was chosen as the key synthetic intermediate to most likely lead to thecompleted synthesis, since the siloxy group at C8 would be disposed equatorially in the chair−chair−

The Novartis Foundation(Japan)
for the Promotion of Science

65

The Novartis Foundation(Japan)
for the Promotion of Science

chairlike transition state of the crucial polyene cyclization reaction. The synthesis of the cyclizationprecursor 1 was initiated with known alcohol 2 with >99% ee. A six-step sequence involving TBS
protection of the hydroxyl group at C8, ozonolysis, Horner−Wadsworth−Emmons reaction under
Masamune conditions, reduction with DIBALH, phenyl sulfide formation and Mo(VI)-catalyzed
oxidation provided phenyl sulfone 3, alkylation of which with allyllic bromide 4 was followedby removal of the phenyl sulfonyl and TBDPS groups and acid hydrolysis of the cyclic acetal to
give ketone 5. After interim protection of the hydroxyl group as a THP ether and installation of anester functionality at C11 with Mander’s reagent, (trimethylsilyl)methyl group was uneventfully
introduced by a sequence involving enol triflate formation and Kumada−Tamao−Corriu coupling
using (trimethylsilyl) methylmagnesium bromide. Selective liberation of the allylic hydroxyl groupin allylsilane 6, followed by Sharpless asymmetric epoxidation, Dess−Martin oxidation and Wittig
olefination completed the synthesis of epoxyallylsilane 1.
With epoxyallylsilane 1 in hand, we then proceeded to investigate the key cyclization reaction.
Although the anticipated reaction proceeded to give desired tricyclic compound 8 upon treatment of
epoxyallylsilane 1 with Et2AlCl in dichloromethane at –78 °C, monocyclized product 7 was obtained
as a major product under these conditions. After considerable experimentation, we found that theallylsilane 7 could be cyclized with the aid of a Lewis acid, providing tricyclic compound 8 inacceptable yield. The resultant alcohol was oxidized in preparation for the A ring construction.
While a propargyl group could be introduced at C15, attempts to convert the resultant enyne tothe desired tetracyclic compound met with little success. After a survey of nucleophiles and ring
construction methods, we found that the six-membered ring could be formed by the ring-closing
metathesis of the diene obtained by the addition of an organostannane reagent. The hydroxyl groupat C15 could be eliminated upon mesylation, and a two-fold methylation almost completed thefunctionalization of the A ring.
To functionalize the D ring, deprotection of the C8 TBS ether was followed by oxidation to givethe corresponding C8-ketone. After installation of a methyl group at C9 by the enolate alkylation,
successive oxidations furnished triketone 10, which only needs to be hydrated for the total synthesisof berkeleytrione.

Discussion & Conclusion
While introduction of a siloxy group at C8 made the concerted cyclization somewhat difficult
to occur, it was found that desired tricyclic compound 8 could be formed by a stepwise approach.
It is speculated that monocyclized product 7 adopted the suitable conformation required for thecyclization due to the steric preference of the siloxy group for the equatorial position.
We have also demonstrated that the product 8 can serve as a key intermediate for the stereoselective
synthesis of berkeleyone family meroterpenoids. Efforts to convert advanced intermediate 10 toberkeleytrione and its derivatives are in progress and results will be reported in a future.

References
1 Suzuki, K.; Yamakoshi, H.; Nakamura, S. Construction of Bridged Polycyclic Systems by
Polyene Cyclization Chem. Eur. J. 2015, 21, 17605−17609.

The Novartis Foundation(Japan)
for the Promotion of Science

66

The Novartis Foundation(Japan)
for the Promotion of Science

一般の皆様へ
バークレーオン類は、米国の銅鉱山跡にできた湖で採取された菌から見つけられた化合物群

ですが、バークレージオンが抗がん作用を示すことで知られているなど、創薬のリード化合物として注目を集めています。本研究では、菌がこれらの化合物を作り出す方法を模倣することで、これらの化合物群を化学合成することに挑戦しました。本研究は、当該天然物はもちろんのこと、類縁構造を持つ化合物群の供給に道を拓くものであり、将来的な医薬品開発につながることが期待されます。

The Novartis Foundation(Japan)
for the Promotion of Science

67

The Novartis Foundation(Japan)
for the Promotion of Science

Functional significance of chandelier cell in cortical microcircuit
Yoshiyuki Kubota
yoshiy@nips.ac.jp
National Institute for Physiological Sciences

Summary Abstract
Inhibitory synapse function in the rat frontal cortex are investigated using cell attached recordingwith GABA uncaging method. The GABA may effectively hyperpolarize the membrane potentialof the postsynaptic cell dependent on its excitability, but almost likely it does not induce membrane
depolarization in mature layer 5 cortical pyramidal cells.
Key Words：neural network, neocortex, microcircuit, GABA, electron microscopy

Introduction
Cortical microcircuits are very complex and a detailed understanding may be impossible withpresent techniques. A single neuron receives thousands of synaptic inputs onto their dendrites
and somata, and the synaptic inputs are integrated on dendritic trees to initiate spikes at the soma.
Cortical inhibitory synapses innervate different sites on target pyramidal cell: soma, proximal
dendrite, distal dendrite, spines or axon initial segments. The inhibition is known to counter somatic
EPSPs and control spike initiation. However the role of inhibitory inputs on the axon initial segment
is in controversial either excitatory or inhibitory. We pursued this question in GABAergic inhibition
effect on layer 5 pyramidal cell in rat frontal cortex, which possibly due to GABAergic chandelier/basket cells.

Results
GFP construct was injected into lateral ventricle of rat embryonic brain using in utero
electroporation method to label layer 5 pyramidal cells in the rat frontal cortex. Brain slices of 300µm thick were obtained from the rat at postnatal 21 - 28 days. Some layer 5 pyramidal cells weresuccessfully showed the GFP. Membrane potential of the GFP labeled layer 5 pyramidal cells wasmeasured using cell attached electrode and ramp current injection (Verheugen JAH, Fricker D, Miles
R, J Neuroscience (1999) 19:2546-2555). We found the membrane potential of the pyramidal cells
was between -85 and -90 mV in control, and was not changed at GABA uncaging. The axon initial
segment of the pyramidal cells was uncaged, but no significant difference in the membrane potential
was found. This indicates that the GABA effect is probably neither excitatory nor inhibitory at thepostsynaptic cell of the resting state. To depolarize the membrane potential, we applied glutamate
puff on the pyramidal cell, then GABA uncaging was applied. We found the depolarized membranepotential observed under the glutamate puff condition was hyperpolarized after GABA uncaging
dependent on the location. The GABA hyperpolarizing effect by the uncaging was only found insoma or the dendritic tree where the glutamate puff was applied. These results indicate that the
GABA inhibition is only effective on neuronal compartments where the membrane potential isdepolarized by excitatory signal. We assume the membrane potential of the axon initial segmentof the pyramidal cell is probably quite similar as its somatic membrane potential, so the GABAThe Novartis Foundation(Japan)
for the Promotion of Science68

The Novartis Foundation(Japan)
for the Promotion of Science

release on the axon initial segment by the chandelier cell can act as an efficient inhibition at only
postsynaptic pyramidal cells in highly active state.
The functional significance of GABA action can be considered thoroughly with morphologicalevidence of excitatory/inhibitory synapse distribution on the neuron. To understand the synaptic
inputs distribution on the layer 5 pyramidal cell, we investigated synapse density on somata anddendrites of neurons stained with nickel-DAB after whole cell recording of slice preparation of
the rat frontal cortex using combined three-dimensional reconstructions from light and electron
microscopic observations. Somata and dendrites with identified spines and synaptic inputs werereconstructed three-dimensionally (Fig. 1), and synapse distributions were analyzed and quantified.
Spine density of the apical dendrite of the pyramidal cells were low in the proximal dendrites andincreased gradually to an apical dendritic bifurcation, and decreased gradually to the tips of the tuft
dendritic trees. The basal dendritic trees also showed the similar spine distribution at the peak in
about 100 µm from the soma. The synapse also showed the similar distribution as the most spine
receiving one synaptic input. GABAergic/non-GABAergic synapse distributions on these neurons
were analyzed using post-embedding GABA immunohistochemistry with 15 nm colloidal goldlabeled secondary antiserum using serial ultrathin sections of large volume electron micrograph data
set obtained with automated tape-collecting ultramicrotome (ATUM)/scanning electron microscope(SEM) system. We reconstructed dendritic trees
3 dimensionally from the serial thin sections andfound GABAergic terminal contacted on the spine
head for about 3 %, on dendritic shaft for about 4%
among all synapses. The non-GABAergic terminal
innervated mostly spine head (88 %) and only rarely
the dendritic shaft (5 %). We estimated that a layer
5 pyramidal cell in rat frontal cortex receives about
20,000 excitatory and 2000 inhibitory synapses.

Discussion & Conclusion
These results indicate that inhibition of the somatic/axon initial segment by GABA release frombasket cell/chandelier cell axon terminals can hyperpolarize the membrane potential only when
the postsynaptic target cell is in depolarized state due to intense excitatory synaptic inputs. The
postsynaptic layer 5 pyramidal cell receives and integrates a huge number of excitatory/inhibitory
inputs on its dendritic branches, axon initial segment and soma elaborately. They may effectively
hyperpolarize the membrane potential of the postsynaptic cell dependent on its excitability, butalmost likely GABA release does not induce membrane depolarization in mature layer 5 corticalpyramidal cells.

References
1. Villa KL, Berry KP, Subramanian J, Cha JW, Oh WC, Kwon HB, Kubota Y, So PT, Nedivi E.
Inhibitory synapses are repeatedly assembled and removed at persistent sites in vivo. Neuron.
89(4):756-69 (2016)
2. Shigematsu S, Ueta Y, Mohamed AA, Hatada S, Fukuda T, Kubota Y, Kawaguchi, Y. Selective
thalamic innervation of rat frontal cortical neurons Cerebral Cortex, 26, 2689-270 (2016)
3. Kubota Y, Karube F, Nomura M, Kawaguchi Y. The diversity of cortical inhibitory synapses.

The Novartis Foundation(Japan)
for the Promotion of Science69

The Novartis Foundation(Japan)
for the Promotion of Science

Frontiers in Neural Circuits. 10:27 (2016)
4. Kubota, Y
Morphological and neurochemical characterization of electrophysiologically identified cells.
"Receptor and Ion Channel Detection in the Brain: methods and protocols." Eds: Luján, R.,
Ciruela, F., Springer, New York, U.S.A. Volume 110, pp277-309 (2016)

一般の皆様へ
大脳皮質は多くの領域から構成され，それぞれが機能分担をすることで知覚，運動，思考といった我々の知的活動を支えています。大脳皮質の局所神経回路は、多種類の神経細胞と他の領域からの入力線維から構成されており、非常に複雑な情報処理をしていると考えられています。この仕組みを知るためには、皮質内神経回路の構造と機能を明らかにする必要があります。
本研究では、シャンデリア細胞やバスケット細胞と呼ばれている大脳皮質の抑制性神経細胞の機能的役割に焦点をあて、この局所神経回路の機能構造を解析しました。これらの神経細胞は、ターゲットの錐体細胞の活動に強力な抑制効果をもたらしているという結果を得ました。

The Novartis Foundation(Japan)
for the Promotion of Science70

The Novartis Foundation(Japan)
for the Promotion of Science

Characterization of T-follicular regulatory cells in human bloodand tumor tissues
WING, Jamesjbwing@ifrec.osaka-u.ac.jp
Immunology Frontier Research Center (IFReC), Osaka University

Key Words：Immune regulation, Regulatory T-cells (Tregs).

Introduction
Regulatory T-cells (Tregs) are vital for the maintenance of immune homeostasis. T-follicularhelper (Tfh) cells are responsible for the generation of high affinity antibody by B-cells. As aresult control of these cells is critical to vaccination, antimicrobial immunity and antibody driven
autoimmunity such as SLE. Tregs can also undergo a similar pathway of differentiation as Tfh tobecome T-follicular regulatory cells (Tfr). In mice it has been demonstrated that Tfr are critical
for the control of Tfh but Tfr are still poorly understood, particularly in humans. We have made animportant discovery of a new population of Foxp3 expressing regulatory T-cells (Tregs) present in
human blood and organs.

Results
Naïve regulatory T-cells can be identified by their expression of markers such as CD45RA, CCR7
and lack of CD45RO. The chemokine receptor CXCR5 is expressed by memory and effector T-cells,
most notably by T-follicular helper cells. As expected Foxp3- Naïve T-cells do not express CXCR5.
However, we have discovered a novel population of CXCR5+CD45RA+CCR7+Foxp3+ “Naïve”
Tregs. These cells make up a significant fraction (around 50%) of CD45RA+ Naïve Tregs in adulthuman blood. These cells were also found in the liver, spleen and tonsils, but not in tumor samples.
Additionally CXCR5+CD45RA+ Tregs are completely absent from cord blood and enriched in
the blood of older donors suggesting that this is not a truly naïve population but possibly an earlyprecursor of CXCR5+CD45RA- T-follicular regulatory cells. RNA sequencing reveals that CXCR5+
and CXCR5- Naïve Tregs are extremely similar but that CXCR5+ Naïve Tregs have enhanced
expression of the suppressive molecule TIGIT. In vitro suppression assays confirm that CXCR5+
Naïve Tregs have increased suppressive activity in comparison to CXCR5- Naïve Tregs.

Figure 1: CXCR5 staining of
CD45RA+Foxp3+ Tregs from
PBMC. Left panel is staining
control

Further to this, I also completed work assessing CXCR5+ CD45RA- effector Tfr, described in

The Novartis Foundation(Japan)
for the Promotion of Science71

The Novartis Foundation(Japan)
for the Promotion of Science

human blood. Here I found that these cells termed circulating Tfr (cTfr) gain a transcriptional signatureequidistant between circulating Tfh (cTfh) and effector Tregs (eTregs).

Figure 2: RNA-sequencing of circulating Tfh, Tfr and effector Tregs from human PBMCs.
A) Euclidean distance between gene expression profiles. B) Heatmaps of most differentiallyexpressed gene between cTfh and eTregs. cTfr show a mixed expression pattern.

Discussion & Conclusion
This work has yielded two main findings.
1) The discovery of a Novel subset of CXCR5 expressing CD45RA+ regulatory T-cells found in
human. This work is ongoing but the data thus far is compelling. We now plan to carry out further
experiments to better define the functional role of these cells and determine if they are precursors
or activated Tfr cells or another type of memory Treg.
2) The characterization of circulating CXCR5+ CD45RA- Tfr in human blood. These cells have agene expression pattern
Characterization of Tfr is likely to be an important step to better defining their role inautoimmunity. This data has been combined with my other work on Murine Tfr cells and has
been recently accepted for publication in PNAS (Wing et al. 2017).
Additionally, it is currently believed that CD45RA+ Tregs are a naïve population and clinical
trials to expand Tregs are using these cells on this basis. It now seems possible that a large
proportion of these cells are not truly naïve but may instead represent a novel memory population
with some naïve characteristics.

References
Wing JB, Kitagawa Y, Locci M, Hume H, Tay C, Morita T, Kidani Y, Matsuda K, Inoue T, Kurosaki
T, Crotty S, Coban C, Ohkura N, Sakaguchi S.
A distinct subpopulation of CD25 negative T-follicular regulatory cells localizes in the germinal
centers.
Accepted for publication in PNAS, 2017.

The Novartis Foundation(Japan)
for the Promotion of Science72

The Novartis Foundation(Japan)
for the Promotion of Science

一般の皆様へ
制御性 T 細胞（Treg）は、免疫恒常性を維持する上で重要な役割を果たしている。 Treg のサブセットである Tfr は、抗体反応を制御することに特化しているが、特にヒトにおいてはよ

く理解されていない。 本研究では、ヒト血液中の Tfr の詳細な特徴を明らかにした。この成果は、SLE および関節炎などの自己免疫疾患における役割を理解する上で重要である。 さらに、以前はナイーブな Treg であると考えられていた細胞の大多数が、実際には新しいメモリー Treg 集
団である可能性があることを示し、これらの細胞の免疫学的役割およびその制御の理解と応用に重要である。

The Novartis Foundation(Japan)
for the Promotion of Science73

The Novartis Foundation(Japan)
for the Promotion of Science

Conformational plasticity of JRAB regulates transition between
mesenchymal and amoeboid invasiveness in cancer cells.
Ayuko Sakane
sakane@tokushima-u.ac.jp
Tokushima University Graduate School of Medical Sciences, Department of Biochemistry

Summary Abstract
During cancer metastasis and invasion, either MAT (Mesenchymal Amoeboid Transition) or AMT(Amoeboid Mesenchymal Transition) is often observed. The dramatic morphological changes basedon actin cytoskeletal dynamics are involved in the switching of mode of cancer cell migration. We
recently found that JRAB (Junctional Rab13-binding protein)/MICAL-L2 (molecules interactingwith CasL-like 2), which is an effector protein of Rab13 GTPase, spatiotemporally regulates actin
cytoskeletal reorganization and also identified that JRAB changes its conformation via Rab13,
followed by interplay with several actin-binding proteins. Here we provided a new concept thatthe conformational plasticity of JRAB plays a pivotal role in the morphological changes leading
to the switching of mode of cell migration on the basis of our data derived from a robust approachcombining computational biology, bioinformatics, biomechanics, biochemistry, live imaging, andcell biology.
Key Words：cancer metastasis and invasion, morphological change, conformational plasticity of
JRAB, actin cytoskeletal reorganization

Introduction
During cancer metastasis and invasion, cancer cells change their morphology to circumventthe effects from the extracellular environment. Dramatic morphological change, such as MAT(Mesenchymal Amoeboid Transition) or AMT (Amoeboid Mesenchymal Transition), is basedon actin cytoskeletal dynamics and regulates the switching of the mode of cancer cell migration.
Some kinds of cancer cells form cluster and move as a single unit. The morphological change
would be occurred in such type of cell migration, termed collective cell migration, especially at thefront of the cell cluster. We have identified JRAB (Junctional Rab13-binding protein)/MICAL-L2(molecules interacting with CasL-like 2) as an effector protein of Rab13, a member of the Rab
family small GTPases. Our previous studies have shown that JRAB may spatiotemporally regulate
the actin dynamics. In this study, we focused on the conformational plasticity of JRAB in collective
cell migration, to explain the mechanism of the switching of mode of cell migration based on themorphological change of the cells located at the leading edge.

Results
We have previously found that JRAB has the intramolecular interaction between its N-terminal
and C-terminal regions, and that the binding of Rab13 releases this interaction, resulting in theconformational change; i.e., the closed to the open form, but we have obtained no solid evidence.
In this study, we successfully generated a structural model of JRAB and thereby provided the firm
evidence for a JRAB conformational change induced by Rab13 using an approach that combines

The Novartis Foundation(Japan)
for the Promotion of Science74

The Novartis Foundation(Japan)
for the Promotion of Sciencebioinformatics and biochemistry. We thereby revealed that Rab13 and JRAB-N compete for binding
with JRAB-C and that this competition leads to a release of the intramolecular interaction. We also
developed a JRAB/MICAL-L2 fluorescence resonance energy transfer (FRET) probe and foundusing cells expressing this FRET probe that JRAB changes its conformation depending on theposition of the cell within the population. Previously we prepared JRAB mutants fixed in a specificconformation (i.e., the open form; JRAB∆CC or the closed form; JRAB∆CT). Our present results
demonstrated that JRAB∆CT and JRAB∆CC adopt a closed and open form, respectively, based on
results from several methods (pull-down assay, ANS analysis, and gel filtration chromatography). In
addition, we found that cells expressing JRAB∆CT or JRAB∆CC exhibit distinctive morphology at
the leading edge of each cell group. Formation of thick actin bundles at the free edge is enhanced by
JRAB∆CT, but inhibited by JRAB∆CC. We next examined the detailed structure of cells at the freeborder using super-resolution microscopy. Super-resolution images revealed the fine meshwork offilamentous actin (F-actin) generated by JRAB∆CC. In cells expressing JRAB∆CT, several F-actinsradiated from the thick bundle and seemed to project onto the growth surface adjacent to the cell.
Therefore, we analyzed the structures involved in the cell–matrix adhesion, that is, focal adhesions(FAs). Cell clusters expressing JRAB∆CT exhibited prominent staining of long, vinculin-positive
structures at the free edge, whereas those expressing JRAB∆CC exhibited punctate staining of
shorter structures. Long and short vinculin-positive structures indicate mature and immature FAs,
respectively. These results suggest that closed JRAB may form mature FAs at the tip of F-actinsradiating from the thick F-actin bundle. Moreover, we tried to reveal the relationship between
morphological change regulated by conformational plasticity of JRAB and the switching of modeof cell migration. For that, we performed the wound healing assay using the cell groups expressing
JRAB mutants and then the obtained results were subjected to computational analysis using opticalflow followed by kurtosis and PCA analysis. From these, we found that JRAB∆CT and JRAB∆CC
exhibit different mode of cell migration. Groups of cells expressing JRAB∆CT migrated strongly in a
fixed direction. By contrast, in groups expressing JRAB∆CC, the cells formed ruffles one by one andmoved in random directions; former is akin to precision dancing, latter resembles awa-odori, likea Rio Carnival in Japan. We also showed by new methods based on biomechanics that JRAB∆CT
causes excessive rigidity, whereas JRAB∆CC causes a loss of directionality due to lack of rigidity.
Thus, our a robust approach combining cell biology, live imaging, computational biology, andbiomechanics brings a novel concept that the conformational plasticity of a single molecule, JRAB,
regulates morphological change of the cells leading to the switching of the mode of cell migration.

Discussion & Conclusion
In this study, we demonstrated that the dynamic morphological change followed by switchingof mode of cell migration is regulated through the conformational plasticity of a single molecule,
JRAB/MICAL-L2. We do not know the detailed molecular mechanism allowing the morphologicalchange depending on the conformation of JRAB. However, there are several possibilities. First, wefound several actin-binding proteins (actinin-1, actinin-4, and filamins) preferentially interacting withthe open form of JRAB. Such interplay between them may be the key to solve the question. Second,
linkage between JRAB and Rho family protein, which is one of the representative regulatory systemsof actin cytoskeletal rearrangement, should not be excluded. Actually, MAT is occurred followed by
activation of RhoA signaling pathway, whereas activation of Rac signaling pathway causes AMT.
Thus, there may be switching of the activation of Rho family protein under the conformational

The Novartis Foundation(Japan)
for the Promotion of Science75

The Novartis Foundation(Japan)
for the Promotion of Sciencechange of JRAB; the closed form of JRAB may generate a thick actin bundle with radial F-actins
leading to the generation of traction force via activated RhoA, whereas the open form of JRAB may
generate a ruffle under the effect of Rac. These possibilities need to be pursued elsewhere. Finally,
these results will be applied to the switching of the mode of cell migration in the case of MAT or
AMT.

References
1. Sakane, A., Yoshizawa, S., Nishimura, M., Tsuchiya, Y., Matsushita, N., Miyake, K., Horikawa,
K., Imoto, I., Mizuguchi, C., Saito, H., Ueno, T., Matsushita, S., Haga, H., Deguchi, S.,
Mizuguchi, K., Yokota, H. and Sasaki, T. Conformational plasticity of JRAB/MICAL-L2
provides ‘law and order’ in collective cell migration. Mol. Biol. Cell, 27(20), 3095-3108 (2016)
2. Sakane, A., Abdallah, A.A.M., Nakano, K., Honda, K., Kitamura, T., Imoto, I., Matsushita, N.
and Sasaki, T.
Junctional Rab13-binding protein (JRAB) regulates cell spreading via filamins. Genes Cells.,
18(9), 810-822 (2013)
3. Sakane, A., Abdallah, A.A.M., Nakano, K., Honda, K, Ikeda, W., Nishikawa, Y., Matsumoto,M., Matsushita, N., Kitamura, T., and Sasaki, T. Rab13 small G protein and Junctional Rab13binding protein (JRAB) orchestrate actin cytoskeletal organization during epithelial junctional
development. J. Biol. Chem., 287(51), 42455-42468 (2012)
4. Sakane, A., Honda, K. and Sasaki, T.
Rab13 regulates neurite outgrowth in PC12 cells through its effector protein, JRAB/MICAL-L2.
Mol. Cell. Biol., 30(4), 1077-1087 (2010)

一般の皆様へ
最近、癌細胞が転移・浸潤していく上で、細胞の形態および運動能の変換が重要な役割を担っていることが明らかになりつつある。したがって、癌の転移・浸潤過程で認められる細胞形態・運動能の変換の分子機構を理解し、その変換を自由に調節することが可能となれば、癌の転移・浸潤に対する新たな治療法の開発や創薬にもつながることが期待される。これまで本分子機構には多くの分子が関与し、それらの分子間相互作用が複雑に絡み合って成立すると考えられていたが、本研究では JRAB というたった1分子の構造変化に依存したアクチン細胞骨格の再編成
が細胞の形態・機能の変換を時空間的に制御しているという独創的な成果が得られた。

The Novartis Foundation(Japan)
for the Promotion of Science76

The Novartis Foundation(Japan)
for the Promotion of Science

Development of apoptosis inhibitor targeting mitochondria
Kenji Matsumoto
kmatsumoto@ph.bunri-u.ac.jp
Tokushima Bunri University, Faculty of pharmaceutical sciences

Summary Abstract
Bongkrekic acid is an inhibitor of adenine nucleoside translocator and also known as an inhibitorof apoptosis. In order to develop novel potent apoptosis inhibitor based on our total synthesis of
BKA, we developed the stereocontrolled allene-Claisen rearrangement affording multisubstituted
1,3-dienes. Furthermore, we applied to synthesize BKA analogues for the structure-activity
relationship.
Key Words：mitochondria, apoptosis, structure-activity relationshipIntroduction
Bongkrekic acid (BKA) was discovered as the content responsible for fatal food poisoning from thefermented coconut product. Among the numerous types of bioactivity reported for BKA, it strongly
binds to the adenine nucleotide translocator (ANT) residing in the mitochondrial inner membrane,
which results in the inhibition of apoptosis. In order to develop apoptosis inhibitor, we examined
the synthesis of BKA analogues for the structure-activity relationship based on our total synthesis of
BKA.

Results
We previously reported the total synthesis of BKA via an efficient convergent strategy basedon successive three-component coupling reactions, including a Julia-Kocienski olefination and a
Suzuki-Miyaura coupling [1]. However, the final steps to construct the (2Z,4E)-3-substituted-2,4dienoic acid structure of BKA, also known as the 1,3-diene moiety, remained problematic due to theinstability and facile isomerization. As such, the development of practical and efficient methods forthe stereoselective synthesis of 1,3-dienes is necessary.
The Claisen rearrangement provides a convenient method for the preparation of a wide rangeof substituted alkenes with reliable positional selectivity and generally high levels of geometricalcontrol. As a number of synthetic approaches to 1,3-dienes have been developed via the Claisen
rearrangement, we propose that an allene-Claisen rearrangement approach can be used toconstruct 1,3-dienes possessing the substitution patterns and functional groups present in BKA
in a stereoselective manner. As shown in Scheme 1, starting from allene A, the [3,3]-sigmatropic
rearrangement of B would procced in opposite direction from the allene substituent (R’) topreferentially furnish 1,3-diene C due to steric repulsion between the R’ group and the ester enolate.
OAc
C

R
A

R'

H
R

O

OM
H

OH

R'

O

H
R

B

E

Z
C R'

Scheme 1 The allene-Claisen rearrangement.

The Novartis Foundation(Japan)
for the Promotion of Science

77

The Novartis Foundation(Japan)
for the Promotion of Science

To evaluate the feasibility of the allene-Claisen rearrangement strategy, we examined the
Eschenmoser-Claisen rearrangement of both syn- and anti-allenyl alcohols (1, 3), which wereprepared by the known protocol in a stereoselective manner (Scheme 2). When syn-allenyl
alcohol 1 was heated with N,N-dimethylacetamide dimethylacetal, (1E,3E)-1,3-dienyl amide
2 was predominantly obtained in 60% yield as a 76:24 mixture of stereoisomers. Furthermore,
the reaction of anti-allenyl alcohol 3 gave an 89:11 mixture of 1,3-dienes (1E,3Z)-1,3-diene 4
and other stereoisomer in quantitative yield. Notably, the (1E,3Z)-1,3-diene possessing the samestereochemistry as BKA was obtained from the anti-allenyl alcohol in a high yield and a highly
H

OH
C

1

4

C4H9

+

Ph
sy n-allene 1

OMe
OMe
NMe2
Me

1

∆

CONMe23

C4H9+ stereoisomers

Ph
1E ,3E -diene 260%
1E,3E : others = 76 : 24

CONMe2

H

OH
C

1

4

C4H9

Ph
anti -allene 3

+

1

OMe
OMe
NMe2
Me

∆
quant.

3

C4H9+ stereoisomers

Ph
1E ,3Z -diene 4

1E,3Z: others = 89 : 11

selective manner.
Scheme 2 The stereocontrolled allene-Claisen rearrangement of syn- and anti- allenyl alcohols.
We assume that the major stereoisomers can be likely produced via the chair-like transition states.
In the case of the syn-allenyl alcohols, the chair-like transition state, which bears a pseudo-equatorial
R-substituent at the C-1 position, is more favorable than other transition state, which bears a pseudoaxial R-substituent due to 1,3-diaxial repulsion. This ultimately results in the preferential formationof (1E,3E)-1,3-dienes. Similarly, anti-allenyl alcohols proceed through the chair-like transition state,
which bears a pseudo-equatorial R-substituent, to selectively afford (1E,3Z)-1,3-dienes.
We next examined the feasibility of applying this reaction in the synthesis of BKA analogs (Scheme
3). Initially, TBS ether of 1,3-diene 5 was deprotected by treatment with tetrabutylammonium
fluoride (TBAF) to produce dienyl alcohol 6 in quantitative yield. Subsequent oxidation of the
primary alcohol of 6 under mild conditions with MnO 2, followed by Pinnick oxidation, gave
carboxylic acid 7 in a modest yield without isomerization. As we previously reported that the dienoic
acid-containing partial structure of BKA inhibited mitochondrial functions [2], our developed
method is expected to provide useful and important insights into the structure-activity relationship of
CONMe2

CONMe2TBAF

1. MnO2
2. NaClO2

CONMe2

E

Z

THF, rt
Ph

5

OTBS

Ph

6

OH

Ph

CO2H
7

BKA.
Scheme 3 The synthesis of BKA analogues via allene-Claisen rearrangement.

The Novartis Foundation(Japan)
for the Promotion of Science78

The Novartis Foundation(Japan)
for the Promotion of Science

Discussion & Conclusion
In conclusion, we successfully developed an efficient and stereocontrolled synthetic method for the
preparation of multisubstituted 1,3-dienes through a stereospecific allene-Claisen rearrangement. This
novel route is expected to address the issues surrounding the preparation of substituted 1,3-dienes,
such as their inherently instability under oxidation conditions. In addition, it is noteworthy that
(1E,3Z)-1,3-dienes possessing the same stereochemistry as BKA were obtained from anti-allenesin high stereoselectivity. Further studies will expand this approach to the total synthesis of natural
products possessing highly functionalized conjugated dienes. Furthermore, our present work
provides important insights into the SAR of BKA and a useful biological tool for the mechanistic
investigations of apoptosis.

References[1] Matsumoto, K.; Suyama, M.; Fujita, S.; Moriwaki, T.; Sato, Y.; Aso, Y.; Muroshita, S.; Matsuo,
H.; Monda, K.; Okuda, K.; Abe, M.; Fukunaga, H.; Kano, A.; Shindo, M. Efficient Total Synthesis
of Bongkrekic Acid and Apoptosis Inhibitory Activity of its Analogues, Chemistry A European
Journal 21, 11590-11602 (2015).
[2] Yamamoto, A.; Hasui, K.; Matsuo, H.; Okuda, K.; Abe, M.; Matsumoto, K.; Harada, K.;
Yoshimura, Y.; Yamamoto, T.; Ohkura, K.; Shinohara, Y.; Shindo, M., Bongkrekic acid analogue,
lacking one of the carboxylic groups of its parent compound, shows moderate but pH-insensitive
inhibitory effects on the mitochondrial ADP/ATP carrier, Chemical Biology & Drug Design 86,
1304-1322 (2015).

一般の皆様へアポトーシスは、生理的な細胞死とも呼ばれ、癌、エイズ、アルツハイマー病など多くの疾病に関わっており、アポトーシス阻害剤はこれら難治性疾患の治療薬として期待されています。
ボンクレキン酸（BKA）は、ミトコンドリアに作用するアポトーシス阻害剤として知られていますが、非常に高価で大量入手が難しいために詳細な作用機序は未だ十分には解明されていません。本研究では、我々の開発したボンクレキン酸の全合成を足掛かりに、大量供給法の確立
および高活性な新規アポトーシス阻害剤の創製を目指しています。

The Novartis Foundation(Japan)
for the Promotion of Science

79

The Novartis Foundation(Japan)
for the Promotion of Science

Cortical synaptic plasticity for coexistence of chronic pain andemotions
Kohei Koga
kkoga@hotmail.co.jp
Department of Neurophysiology, Hyogo College of Medicine

Summary Abstract
Chronic pain is an unpleasant sensation and produce synaptic plasticity in the CNS. The anterior
cingulate cortex (ACC) is a critical area for nociception and chronic pain. Here, we studied if chronic
pain model could produce plastic changes in inhibitory synaptic transmission in the mice ACC.
Using whole-cell patch-clamp recording, we found that chronic inflammatory pain model reduced
the frequency of spontaneous inhibitory postsynaptic currents. Using western blot analysis, we foundthat vesicular GABA transporter was reduced in the model. These results suggest that chronic pain
model mainly produced GABAergic synaptic plasticity in the presynapse of the ACC.
Key Words：Chronic pain, anterior cingulate cortex, GABA, synaptic plasticity

Introduction
The ACC is a critical cortical area for nociception, chronic pain and emotions such as fear andanxiety. Cortical area including the ACC is composed of excitatory and inhibitory neurons andproduce neural network. Chronic pain models show short-term or long-term excitatory plasticity inexcitatory neurons in the ACC. However, the synaptic mechanisms of GABAergic transmission forchronic pain are not fully understood, yet.

Results
In this study, we examined three major experiments.
First, we made a major chronic inflammatory pain model and confirmed the mechanicalhypersensitivity 2-3 days after the injection.
Next, we performed whole-cell patch-clamp method, and recorded spontaneous and miniature
inhibitory postsynaptic currents (sIPSCs & mIPSCs) on pyramidal neurons in layer II/III of the adultmice anterior cingulate cortex (ACC).
Finally, we studied the possible alterations of GABAergic transmission related proteins in thepresynapse and postsynapse of the mice ACC. We found that vesicular GABA transporter (vGAT)
was reduced in the presynpase of CFA model.
In contrast, GABAA receptors subunits including α1, α5, β2, γ2 and δ did not change between
control and CFA groups.
Chronic inflammatory pain model reduced inhibitory synaptic transmitter release in the ACC
We first made chronic inflammatory pain model using adult mice (male C57BL/6, 8-12 week-old).
Complete freund adjuvand (CFA) is a major inflammatory pain model so that we injected the CFA
in the left hindpaw (Koga et al. 2015, Zhao et al. 2006). The 2-3 days after the injection of CFA, we
confirmed the hypersensitivity on mechanical stimulation using von Frey filaments in CFA-induced

The Novartis Foundation(Japan)
for the Promotion of Science80

The Novartis Foundation(Japan)
for the Promotion of Science

inflammatory pain model and control group. CFA-induced inflammation mice significantly reduced
mechanical thresholds compared with control group.
After the assessment of the mechanical hypersensitivity, we prepared a coronal brain slice forwhole-cell patch clamp recording method. We performed whole-cell patch-clamp recording fromlayer II/III pyramidal neurons and recorded sIPSCs under voltage-clamp mode at 0 mV in thepresence of CNQX, an alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid/GluK (AMPA/
KA) receptors antagonist, AP-V, a N-methyl-D-aspartic acid (NMDA) receptors antagonist and
CGP35845, a γ(gamma)-aminobutyric acid B (GABAB) receptors antagonist in CFA model andcontrol group (Koga et al. 2017). We analyzed the averaged frequency and amplitude of sIPSCs
between two groups. We performed statistics and found that the averaged frequency of sIPSCs in
CFA model was significantly reduced compared with control group. In contrast, averaged amplitudes
of sIPSCs were not altered between the two groups. These results suggest that chronic pain model
reduced inhibitory synaptic transmitter release in the ACC.
CFA-induced inflammatory pain model inhibited the frequency of mIPSCs
We next recorded mIPSCs in the presence of tetrodotoxin (TTX, 1 µM) to block sodium channels.
CFA induced inflammatory pain model reduced the averaged frequency of mIPSCs in the layer II/III
pyramidal neurons from the ACC compared to the control group. However, the averaged amplitude
of mIPSCs did not change between two groups. Taken together, these results suggest that chronic
pain model reduced synaptic transmitter release in the ACC.
Chronic inflammatory pain model decreased the protein expression of vesicular GABA
transporter in the ACC
We further studied how the animal model of chronic pain could alter inhibitory synaptictransmissions in the ACC area. Vesicular GABA transporter (vGAT) is expressed in GABAergic
neurons, and is responsible for vesicular storage and subsequent exocytosis of these
inhibitory amino acids. We tested if chronic pain model could alter the protein
expression of vGAT in the ACC area. Interestingly, chronic pain model reduced theexpression of vGAT in the ACC.
We examined whether GABAA receptors subunits including α1, α5, β2, γ2 and δ and GABARAP
were changed or not in the ACC of CFA-induced inflammatory pain group. The protein expressionsof GABAA receptors subunits including α1, α5, β2, γ2 and δ and GABARAP did not change
between inflammatory and control groups. These results suggest that chronic pain model produced
presynaptic plasticity by altering vesicular GABAergic transporter in the ACC.

Discussion & Conclusion
In this study, we recorded transient GABAA receptors mediated inhibitory synaptic transmission in
pyramidal neurons from layer II/III of the ACC. We found that a major type of chronic inflammation
model induced by CFA reduced the frequency of both sIPSCs and mIPSCs. Western blotting analysis
showed that vGAT was reduced in CFA treated group. On the other hands, GABAA receptors subunits
did not change.
Electrophysiological experiments suggest that chronic inflammatory pain model mice produced
synaptic plasticity. Especially, the model reduced the transmitter release of GABA in the ACC.
Western blotting analysis showed that chronic pain model decreased the protein expression of

The Novartis Foundation(Japan)
for the Promotion of Science81

The Novartis Foundation(Japan)
for the Promotion of SciencevGATs. Since vGAT express in the presynaptic terminal, chronic pain model may reduce the releaseof GABA via vGAT. We need further studies how chronic pain model can alter vGAT in the future.

References
Koga, K., Descalzi, G., Chen, T. et al. (2015) Co-existence of two forms of LTP in ACC provides asynaptic mechanism for the interactions between anxiety and chronic pain. Neuron, 85, 377389.
Koga, K., Matsuzaki, Y., Honda, K., Eto, F., Furukawa, T., Migita, K., Irie, K., Mishima, K. andUeno, S. (2017) Activations of muscarinic M1 receptors in the anterior cingulate cortex
contribute to the antinociceptive effect via GABAergic transmission. Mol Pain, 13,
1744806917692330.
Zhao, M. G., Ko, S. W., Wu, L. J. et al. (2006) Enhanced presynaptic neurotransmitter release in theanterior cingulate cortex of mice with chronic pain. J Neurosci, 26, 8923-8930.

一般の皆様へ
慢性疼痛は侵害受容と心因性の要因からなり、前帯状回はこれら２つの要因に関わる重要な大脳皮質である。本研究では、前帯状回の抑制性シナプス伝達の可塑性に着目し、慢性疼痛が形成する大脳皮質の局所神経回路を理解することを目的に研究を行った。その結果、慢性疼痛

モデルは前帯状回の抑制性シナプス伝達に可塑的な変化を示すことが明らかとなった。特に、GABA の放出が抑制される仕組みとして、GABA をシナプス小胞に貯蔵するトランスポーターである小胞 GABA トランスポーターのタンパク質の発現量が関与する可能性が示された。

The Novartis Foundation(Japan)
for the Promotion of Science82

The Novartis Foundation(Japan)
for the Promotion of Science

Functions and expression of synaptotagmin 13 driving peritoneal
metastasis of gastric cancer cells
Mitsuro Kanda
m-kanda@med.nagoya-u.ac.jp
Department of Gastroenterological Surgery (Surgery II),
Nagoya University Graduate School of Medicine

Summary Abstract
We identified synaptotagmin 13 overexpressing specifically in gastric cancer tissues with peritoneal
metastasis from global expression profiling of 57751 molecules. Inhibition of synaptotagmin 13
led to decreased invasion and migration ability of gastric cancer cells, and formation of peritoneal
metastasis in mouse xenograft models. Overexpression of synaptotagmin 13 serves as diagnostic andpredictive biomarker for peritoneal metastasis from gastric cancer
Key Words：Gastric cancer, peritoneal metastasis, synaptotagmin 13

Introduction
Advanced gastric cancer frequently recurs because of undetected micrometastases even after
curative resection. Peritoneal metastasis has been the most frequent recurrent pattern after
gastrectomy and is incurable.

Results
We conducted a recurrence pattern-specific transcriptome analysis in an independent cohort of
16 patients with stage III gastric cancer who underwent curative gastrectomy and adjuvant S-1 forscreening candidate molecules specific for peritoneal metastasis of gastric cancer. The effects ofsiRNA-mediated knockdown on phenotype and fluorouracil sensitivity of gastric cancer cells were
evaluated in vitro, and the therapeutic effects of siRNAs were evaluated using a mouse xenograftmodel.
Synaptotagmin 13 was identified as a candidate biomarker specific to peritoneal metastasis.
Inhibition of synaptotagmin 13 expression by gastric cancer cells correlated with decreased invasion,
migration.
Intraperitoneal administration of synaptotagmin 13-siRNA inhibited the growth of peritoneal
nodules and prolonged survival of mice engrafted with gastric cancer cells. High synaptotagmin
13 levels were significantly and specifically associated with peritoneal metastasis, and served as anindependent prognostic marker for peritoneal recurrence-free survival of patients with stage II/III
gastric cancer.

Discussion & Conclusion
Synaptotagmin 13 is a promising diagnostic and predictive biomarker for peritoneal metastasisof gastric cancer. Inhibition of SYT13 may represent a key of treatment strategy to overcome
uncontrolled peritoneal metastasis from gastric cancer. Since anti-SYT13 treatment is based on quite

The Novartis Foundation(Japan)
for the Promotion of Science83

The Novartis Foundation(Japan)
for the Promotion of Science

different mechanisms of action from existing molecularly targeted therapies, it can open new medical
frontiers in the field of gastric cancer treatment, and possibly other malignancies involving peritoneal
metastasis.

References
1: Shimizu D, Kanda M, Tanaka H, Kobayashi D, Tanaka C, Hayashi M, Iwata N, Niwa Y, Takami
H, Yamada S, Fujii T, Nakayama G, Fujiwara M, Kodera Y. GPR155 Serves as a Predictive
Biomarker for Hematogenous Metastasis in Patients with Gastric Cancer. Sci Rep. 2017 Feb
6;7:42089.
2: Kanda M, Shimizu D, Sueoka S, Nomoto S, Oya H, Takami H, Ezaka K, Hashimoto R, Tanaka
Y, Kobayashi D, Tanaka C, Yamada S, Fujii T, Nakayama G, Sugimoto H, Koike M, Fujiwara
M, Kodera Y. Prognostic relevance of SAMSN1 expression in gastric cancer. Oncol Lett. 2016
Dec;12(6):4708-4716.
3: Kanda M, Shimizu D, Tanaka H, Tanaka C, Kobayashi D, Hayashi M, Iwata N, Niwa Y, YamadaS, Fujii T, Sugimoto H, Murotani K, Fujiwara M, Kodera Y. Significance of SYT8 For the
Detection, Prediction, and Treatment of Peritoneal Metastasis From Gastric Cancer. Ann Surg.
2016 Dec 6. [Epub ahead of print]
4: Tanaka H, Kanda M, Shimizu D, Tanaka C, Kobayashi D, Hayashi M, Iwata N, Yamada S, Fujii
T, Nakayama G, Sugimoto H, Fujiwara M, Niwa Y, Kodera Y. FAM46C Serves as a Predictor of
Hepatic Recurrence in Patients with Resectable Gastric Cancer. Ann Surg Oncol. 2016 Oct 21.
[Epub ahead of print]
5: Kanda M, Tanaka C, Kobayashi D, Tanaka H, Shimizu D, Shibata M, Takami H, Hayashi M,
Iwata N, Niwa Y, Yamada S, Fujii T, Nakayama G, Fujiwara M, Kodera Y. Epigenetic suppressionof the immunoregulator MZB1 is associated with the malignant phenotype of gastric cancer. Int J
Cancer. 2016 Nov 15;139(10):2290-8.

一般の皆様へ57751分子の網羅的遺伝子発現解析から、進行胃癌の転移再発形式の中で最も高頻度かつ難治性である腹膜播種に特異的発現する synaptotagmin 13を発見しました。synaptotagmin 13を阻害することで、胃癌細胞の活動性を低下させ、腹膜播種形成を抑制できることが明らかになりました。

synaptotagmin 13は、すでに腹膜播種のある胃癌だけでなく、治癒切除術の後、腹膜播種を再発した胃癌でも高発現していることが分かりました。難治性の胃癌腹膜播種の新
たな診断、治療への応用を目指しています。

The Novartis Foundation(Japan)
for the Promotion of Science84

The Novartis Foundation(Japan)
for the Promotion of Science

Epigenetic and biological analysis of human cancer-associatedfibroblasts
Masayuki Shimoda
shimoda@a2.keio.jp
Keio University School of Medicine

Summary Abstract
Cancer-associated fibroblasts (CAFs) are composed of heterogeneous populations of cells andit is an important issue to understand the unique roles of the different subpopulations of CAFs. In
this study, we evaluated various CAF cell lines by analyzing both their biological tumor-promoting
properties and gene expression patterns. Our findings provide gene signatures characterizing
functionally active CAFs, which may contribute to developing combinatorial therapies that can target
such active CAFs besides neoplastic cells.
Key Words：Cancer-associated fibroblast, microarray, epigenome, lung cancer

Introduction
Tumors are complex tissues composed of carcinoma cells and surrounding stroma including various
different types of mesenchymal cells and an extracellular matrix. Carcinoma-associated fibroblasts(CAFs) are frequently observed in the stroma of human carcinomas, and their presence in large
numbers is often associated with the development of high-grade malignancies and poor prognoses(1). Although accumulating evidence suggests that it is conceivable to therapeutically target CAFs topromote anti-tumor responses, finding an agent that specifically targets CAFs remains elusive.

Results
CAFs within the tumor stroma are known to acquire activated phenotype and have been shown tostimulate tumor cell proliferation, elevate tumor cell invasion and boost angiogenesis by secreting
various growth factors and cytokines (1). However, CAFs are also thought to derive from multiple
origins and make up a heterogeneous population of cells (2), it is an important question to understandthe unique roles of the different subpopulations of CAFs. In this project, we evaluated various CAF
cell lines by analyzing both their biological tumor-promoting properties and gene expression patterns,
and sought to identify gene signatures characterizing functionally active CAFs. First, we biologicallyassessed tumor-promoting activities of human CAFs in vivo. Human CAF cell lines were establishedfrom surgically resected tumor tissues of lung cancer patients. We co-injected CAFs with human lungcancer cell line, PC9 cells, into immunodeficient mice subcutaneously. As controls, PC9 cells alone
were injected. Tumor growth kinetics over 5 weeks showed that several CAF cell lines significantly
accelerated growth of PC9 xenografts to varying degrees, whereas the kinetics of PC9 xenografts
were unaffected by some CAF cell lines. CAFs from different cancer patients appear to exhibit
different biological effects on tumor growth, suggesting that CAFs are biologically heterogeneous
cell populations. Based on their biological tumor growth-promoting abilities, we classified these
CAF cell lines into 2 groups: CAF cell lines with functionally tumor-promoting activities (fCAFs)
and those without tumor-promoting activities (nCAFs). Mice bearing mixed PC9 cells and fCAFs

The Novartis Foundation(Japan)
for the Promotion of Science85

The Novartis Foundation(Japan)
for the Promotion of Science

also tend to have increased microscopic lung metastases compared to those bearing cancer cells
alone or with nCAFs. We next performed comprehensive gene expression analysis of these CAFs
to acquire gene signatures characterizing functionally tumor-promoting fCAFs as well as to further
gain insights into mechanisms by which activated tumor-promoting fCAFs contribute to cancer
microenvironments. Gene expression profiling was carried out using SurePrint G3 Human GE 8x60K
ver.3.0 and analyses were done using Genespring GX14.5 and IPA (Ingenuity Pathways Analysis).
In these analyses, we identified multiple genes related to cancer progression increased in fCAFs
compared to nCAFs, and are now further confirming these data as well as identifying molecularpathways induced in fCAFs. For example, we detected several metalloproteinases dysregulated
between fCAF and nCAF groups. Metalloproteinases including MMPs (matrix metalloproteinases)
and ADAMs (a disintegrin and metalloproteinases) are known to play a pivotal role in tumor growth
and the multistep processes of invasion and metastasis, including proteolytic degradation of ECM,
alteration of the cell-cell and cell-ECM interactions, migration and angiogenesis. We have previously
shown that dysregulated metalloproteinase activity is involved in the induction of CAF-like activatedfibroblast state in fibroblasts (3). We also evaluated the significance of tumor stroma-associatedmetalloproteinase expression by using publicly available gene expression data sets representing
tumor stroma from human breast cancer patients, and found that several metalloproteinases such as
ADAM10 were significantly upregulated in human tumor stroma. Thus these findings suggest thatfuncitionally active CAFs may exhibit a specific gene expression pattern and the gene signature
characterizing such active CAFs will contribute to establishing novel therapies targeting tumor
stroma.

Discussion & Conclusion
In this study, we evaluated both biological properties and gene expression patterns of CAF celllines established from human lung cancer patients, and identified multiple genes related to cancer
progression increased in functionally active CAF cell lines. Although the mechanisms by which thesegenes are upregulated in activated tumor-promoting CAFs remain unknown, epigenetic changes,
such as global DNA hypomethylation frequently seen in neoplastic cells, may be also involved in thisprocess and further studies are needed. Nevertheless, our study will contribute to classification andprecise molecular definition of subtypes of fibroblasts in tumor stroma on the basis of their biological
functions, which may offer additional insights into CAF biology and inform clinical opportunities for
new cancer therapies.

References(1) Shimoda M, Mellody KT and Orimo A: Carcinoma-associated fibroblasts are a rate-limiting
determinant for tumour progression. Semin Cell Dev Biol 21:19-25, 2010.
(2) Öhlund D, Elyada E and Tuveson D: Fibroblast heterogeneity in the cancer wound. J Exp Med
211:1503-23, 2014.
(3) Shimoda M, Principe S, Jackson HW, Luga V, Fang H, Molyneux SD, Shao YW, Aiken A,
Waterhouse PD, Karamboulas C, Hess FM, Ohtsuka T, Okada Y, Ailles L, Ludwig A, Wrana JL,
Kislinger T and Khokha R: Loss of the Timp gene family is sufficient for the acquisition of the
CAF-like cell state. Nat Cell Biol 16:889-901, 2014.

The Novartis Foundation(Japan)
for the Promotion of Science86The Novartis Foundation(Japan)
for the Promotion of Science

一般の皆様へ
がん組織は、がん細胞とそれを支える間質と呼ばれる成分から構成されています。がん間質に出現する活性化した線維芽細胞は「がん関連線維芽細胞（cancer-associated fibroblast：CAF）」と呼ばれ、新たな治療標的の一つとして注目されています。本研究では、網羅的解析手法および生物学的機能解析手法を用いて CAF の層別化を行うとともに、機能的な CAF の細胞特性を明らかにすることにより、CAF を標的とした新規診断法および治療法の開発を目指した
基礎的データの取得を行っていきたいと考えています。

The Novartis Foundation(Japan)
for the Promotion of Science87

The Novartis Foundation(Japan)
for the Promotion of Science

Study on the RNA splicing checkpoint mechanism to prevent theaccumulation of pre-mRNA
Daisuke Kaida
kaida@med.u-toyama.ac.jp
University of Toyama

Key Words：mRNA, splicing, transcription

Introduction
It was reported that an anti-cancer compound, spliceostatin A (SSA) binds to spliceosome to inhibit
splicing very potently. Treatment with SSA or another splicing inhibitor pladienolide B also causes
transcription elongation defect and dephospholyration of the second serine residues in the C-terminal
domain (CTD) of RNA polymerase II (Pol II), which consists of multiple heptad repeat (YSPTSPS)
and is important for transcription elongation, suggesting that splicing inhibition causes transcriptional
elongation defect. However, the detailed molecular mechanism of transcription elongation defect bysplicing inhibition remains unknown.

Results
To reveal the molecular insight of transcription elongation defect caused by splicing inhibition, we
performed western blotting after SSA treatment to investigate the effect of splicing inhibition on theprotein levels of kinases and phosphatases of Pol II, in addition to activators and repressors of Pol IItranscription. However, none of the protein was affected after SSA treatment. In addition, to identify
the responsible factor for the transcription elongation defect by splicing inhibition among the kinases
and phosphatases of Pol II and activators and repressors of Pol II transcription, we subcloned thegenes coding the proteins. We transfected cells with the genes, and treated the transfected cells with
SSA to test whether overexpression of these factors suppresses the transcription elongation defectby SSA. However, over expression of any proteins did not rescue the transcription elongation defectby SSA and phosphorylation status of Pol II. In addition, we knocked down these factors usingsiRNA and observed transcription elongation efficiency. Knocking down of the factors did not affecttranscription elongation efficiency. We will try to identify the responsible factor in a future study.
We also performed an immunoprecipitation experiment using an antibody against the largest
subunit of Pol II. Several proteins which are related to transcriptional activity were coimmunoprecipitated with Pol II. We will perform another immunoprecipitaion experiment using
SSA-treated cells to test whether splicing inhibition affect protein-protein interaction between Pol II
and co-immunoprecipitated proteins.
It is well known that splicing and transcription are temporally and spatially coordinated. Therefore,
RNA binding proteins, especially splicing factors, might affect transcriptional activity. To investigate
the relationship between RNA binding proteins and transcriptional activity, we searched for
RNA binding proteins which is able to suppress the transcription elongation defect caused by
SSA treatment. As we expected, overexpression of several RNA binding proteins suppressed thetranscriptional elongation defect by SSA and some of them increased the basal transcriptional

The Novartis Foundation(Japan)
for the Promotion of Science88The Novartis Foundation(Japan)
for the Promotion of Science

activity. Since some of these proteins were splicing factors, it is possible that overexpression ofthese splicing factors rescued the splicing defect caused by SSA, as a consequence, the transcription
elongation defect by SSA was suppressed. However, we did not observe any recovery of splicing
activity after overexpression of these RNA binding proteins. Therefore, these RNA binding proteinssuppressed the transcriptional elongation defect by SSA independent of splicing activity. To
understand the molecular mechanism that RNA binding proteins suppress transcriptional elongation
defect, we checked whether overexpression of the RNA binding proteins affect phosphorylationlevel of Pol II, protein levels of Pol II kinases and phosphatases, and protein levels of transcriptional
activators and repressors. Out of the RNA binding proteins, several proteins affected phosphorylation
status of second serine residues within heptad repeat of Pol II CTD, which are important fortranscription elongation, and fifth serine residues of the repeat, which are important for transcription
initiation. In addition, protein levels of some transcription factors were affected by the overexpression. We will investigate whether these RNA binding proteins affect the protein-protein
interaction between Pol II and transcription factors and Pol II kinases and phosphatases. In addition,
it will be also studied whether these RNA binding proteins interact with Pol II.

Discussion & Conclusion
We found that some RNA binding proteins affected transcription elongation efficiency. However,these RNA binding proteins did not affect the splicing efficiency. These results suggest thatthese RNA binding proteins affect the expression level of transcription factors and/or kinases
and phosphatases of Pol II. Because RNA binding proteins recognize nascent RNA just after
transcription, and numerous RNA binding proteins have physical interaction with transcription
factors including Pol II, it is also possible that these RNA binding proteins regulate transcription
activity directly.
Considering the results, we propose a model in which the transcription elongation defect after SSA
treatment functions as a novel check point mechanism to prevent accumulation of unspliced premRNAs and to protect the integrity of the gene expression when splicing is inhibited.

一般の皆様へ
本研究は、ヒトをはじめとした真核生物の DNA 上にある遺伝情報が、どのようにして正確に読み取られ、タンパク質へと翻訳されるかという、生物学的に非常に本質的な質問の答えを探すものです。本研究の結果から、スプライシングの異常時には異常な pre-mRNA の蓄積を防ぐ
ため、転写伸長を止めてしまうという一種のチェック機構が働いていると考えられます。

The Novartis Foundation(Japan)
for the Promotion of Science89

The Novartis Foundation(Japan)
for the Promotion of Science

Sensing ECM stiffness may trigger the transition from Solid-like to
Liquid-like behaviour in collective cell migrationof cranial neural crest cells
Sei Kuriyama
seikury@med.akita-u.ac.jp
Akita University

Summary Abstract
Unexpectedly we found that cranial neural crest cohorts migrated on the mechano-pattern gel
without any other stimulation. The soft/hard boundary may enhance the directional migration. We
could call this phenomina as collective durotaxis.
Key Words：Collective durotaxis, photocurable gelatin, neural crest cells

Introduction
In previous research, we found that frog cranial neural crest cells dissociate their cell-cell adhesions
intermediately upon going through the in vitro obstacles. The transition from premigratory to
migratory neural crest was similar to solid- to liquid-phase transition1. However, we couldn’t know
if neural crest cells undergo this transition before or after touching the obstacle. Therefore, we started
to use new tool having physiologically appropriate rigidness for testing mechano-sensing ability of
cranial neural crest cells. We found that mechano-pattern gel triggered collective migration of neuralcrest cells without any other external signals.

Results
1. Confirmation of the preliminary results
First, we confirmed if the rigidness changed the collective behaviours. The photocurable gelatinalone couldn’t accommodated the cranial neural crest cells, therefore, we chemically cross-linked
fibronectin to the remaining free-NH2 residues of the photocurable gelatin. We confirmed thatthe surface of the gels were covered with fibronectin uniformly even when the rigidness between
the stripes were very different. Again we confirmed that the soft gel increased the collective cell
behaviour, and the hard gel promoted the cell dissociation.
2. Collective Durotaxis
Durotaxis is the behaviour which the cells migrate toward stiff region from softer region on thegradient of the rigidness. The researches of the durotaxis on the single cell migration have beenwell-established, however, the collective motion on the gradient of the rigidness has not yet been
analyzed. We planned to test if neural crest cells dissociated their cell-cell adhesions upon going
through the mechano-pattern. Before using the chemokines to induce the directional migration
toward vertical direction of the boundaries of stiffness, we performed time-lapse imaging of control
without chemokines. However, unexpectedly, neural crest cells started to migrate along the stripes
without any other stimulation except the jumping of the stiffness. Moreover, the narrower stripe
maintained the collective migration longer as much as in vivo.

The Novartis Foundation(Japan)
for the Promotion of Science

90

The Novartis Foundation(Japan)
for the Promotion of Science

3. Sensing the obstacle
When we put microfluidic tunnel to the path of the neural crest cell migration, the neural crest cells
squeezed their body to go through the tunnel even narrower than the longer axis of single cells andLPAR2 lacking neural crest cells failed to change their shape.
Therefore, we thought neural crest dissociated to cell adhesion before going through the obstacle.
However, when we put neural crest cells lacking LPAR2 gene to mechano-pattern gel, neural crest
cells also failed to migrate and stalled at the middle of stripe because cells can not change theirpositions. Thus, neural crest cells sensed their surrounding circumstance mechanically, and then it
might trigger the cell dissociation. Moreover, when we applied chemokine to this system, neuralcrest cells often ignored the boundaries. It may mean that neural crest cells is parrallely regulated bydurotaxis and chemotaxis.
4. The window of the rigidness
Next, we analyzed which the rigidness or the gap of the rigidness controlled cell migration. To test
that, we designed two types of the gels: The hard (<200 kPa) and the soft (<10 kPa) ; The hard (>400kPa) and the soft (100-200kPa). The gap between soft and hard region was similar but the absoluterigidness was different. As the result, the harder gel couldn’t promote the collective cell migration,
and we also found that the window of the rigidness was very narrow. It might be the rigidness aroundcranial neural crest cells.
5. Cancer metastasis and the rigidness
We also tested if the rigidness controlled cancer cell migration. We made the sub-cell linesof osteosarcoma cell line, which metastasized into knees, lymphocytes, liver and kidney. The
parental cell line (ori) and kidney cell line (kid) were compared. These cell lines didn’t show themuch differences except N-cadherin expression. However, the cells were put on the mechano-gel(hydrogel), the rigidness where each cell preferred to attach was different. We are going to analyze
the correlation between the rigidness and the selectivity of the tissue metastasized.

Discussion & Conclusion
We found a novel system to control the collective cell migration. The local gradient of therigidness triggered the migration without any other external signals. Our preliminary results suggestthat collective durotaxis independently regulated cell migration, however, we haven’t clarify the
relationships between durotaxis and chemotaxis or the other signals. Moreover, we still need to showin vivo requirement of the collective durotaxis. Otherwise it might be an artifact only happened invitro system.

References
1. Kuriyama et al. In vivo collective cell migration requires an LPAR2-dependent increase in tissue
fluidity J Cell Biol. (2014).

The Novartis Foundation(Japan)
for the Promotion of Science91The Novartis Foundation(Japan)
for the Promotion of Science

一般の皆様へ細胞は集団で移動する方がバラバラの細胞が動くよりもはるかに効率が良いことが分かっています。しかし、がん細胞が塊になっていると狭い所で詰まってしまうため生体内でがんが集団移動するとは信じられていませんでした。先の研究でがんに似た神経堤細胞が適度に流動性を持つ集団として移動している事が分かりました。つまり「がんは水の様に器に沿う」のです。
次の疑問として器の形を認識して形を変えるのか、元から水の様なのかがありました。本研究助成から、神経堤細胞が器を触って「硬さ」を感じて形を変えていることが分かってきました。
この制御方法が分かればがんを閉じ込めることができるようになるでしょう。

The Novartis Foundation(Japan)
for the Promotion of Science92

The Novartis Foundation(Japan)
for the Promotion of Science

Functional analysis of a novel silencing mechanism in higher plants
Kei-ichiro Mishibamishiba@plant.osakafu-u.ac.jp
Graduate School of Life and Environmental Science, Osaka Prefecture University

Summary Abstract
Unlike conventional transgene silencing models, 35S promoter hypermethylation in gentian occurs
irrespective of copy number and genomic location. By using transgenic gentian plants having single
copy of some modified 35S promoters, we investigated sequence specificity of DNA methylationof the 35S promoter in gentian. Modified 35S promoter lacking a core (−90) region induced
hypermethylation, whereas substitution of two “GAAGA” motifs within the promoter significantly
reduced methylation in gentian. A 64-bp fragment of the 35S promoter can induce moderate
methylation. These results suggest that the 64-bp region containing the “GAAGA” motif is essential
for the 35S methylation in gentian.
Key Words：transgene, silencing, DNA methylation, gentian

Introduction
We have presented a strict cauliflower mosaic virus (CaMV) 35S promoter-specific silencingof transgene associated with DNA methylation in gentian (Gentiana triflora × G. scabra) [1-3]and lettuce (Lactuca sativa L.) [4]. These studies revealed that the 35S-specific silencing occurs
regardless of the T-DNA copy number and genomic location, and is species-specific. The silencing
phenomenon is distinct from the silencing features previously found in model plants, although themechanism is unclear. The present study aimed to investigate the mechanism of the 35S promoter
silencing in gentian.

Results
1. Hypermethylation of the 35S(Δcore) promoter lacking strong transcriptional activity
To confirm whether the strong transcriptional activity of the 35S promoter is required for theconsistent 35S hypermethylation in gentian, a modified 35S promoter lacking core (−90) region,
designated as 35S(Δcore), was introduced to gentian. Single copy 35S(Δcore) transgenic gentianlines were selected and methylation status of the introduced 35S(Δcore) region was analyzed bybisulfite genomic sequencing. As a result, hypermethylation at CpG/CpWpG sites on the region wasobserved in all the lines analyzed. The methylation level of the 35S(Δcore) region is equivalent tothat of the unmodified 35S promoter.
2. Gentian nuclear factor binding to the highly methylated regions of the 35S promoter
From the results of the previous studies [2, 3] and the methylation analysis of the 35S(Δcore)region, two regions (−148 to −85 and −298 to −241) within the 35S promoter were highly methylated
at CpHpH/CpCpG sites. Moreover, electrophoretic mobility shift assay (EMSA) showed thatgentian nuclear factors bound to the −149 to −124 and −275 to −250 sequences, both of which have
“GAAGA” and “GTGGAAAA” motifs [2, 3]. To confirm the contribution of these motifs for the

The Novartis Foundation(Japan)
for the Promotion of Science

93

The Novartis Foundation(Japan)
for the Promotion of Sciencebinding, EMSA was performed. Consistent with the previous studies, distinct complexes with gentian
nuclear extracts were formed with the −149 to −124 probe. Formation of the complex was competedby excess unlabeled −275 to −250 competitor, but not by −296 to −271 competitor. In addition,
substitution of “GTGGAAAA” to “TCTGAAAA” in the −275 to −250 competitor diminished thecompetition.
3. Substantial methylation reduction by nucleotide substitutions of the 35S promoter
Transgenic gentian plants with a modified 35S promoter designated as 35S(m1), wherein the two“GAAGA” motifs within the promoter were substituted with “GTTCA”, were also produced, andmethylation of the promoter region was analyzed. Most of the single-copy 35S(m1) lines obtained
showed strong transgene expression. The methylation status of the modified 35S promoter region
was low level in all the lines at both CpG/CpWpG and CpHpH/CpCpG sites.
4. Methylation induction by the 64-bp fragment of the 35S promoter
The next objective was to determine whether the highly CpHpH/CpCpG methylated region at−148 to −85 (designated as 64-bp) of the 35S promoter can induce methylation alone. Ten single
copy transgenic lines were produced and methylation status of the introduced transgene region was
analyzed. For the CpG/CpWpG sites, six lines exhibited hypermethylation on the 64-bp region at thesame level as that of the unmodified 35S lines. Other four lines showed moderate or somewhat lower
methylation levels. On the other hand, CpHpH/CpCpG methylation of the 64-bp region in all ten
lines was substantially lower than that in the unmodified 35S lines. A complementary strand of the
153 bp transgene region (consisting 64-bp region, a 39 bp upstream region, and a 50 bp downstream
region) was also analyzed to examine CpHpH/CpCpG methylation in detail. Consequently, a distinct
methylation peak was found within the 64-bp region (−105 to −88) in all ten lines. Nevertheless, themethylation frequency of the peaks was substantially lower than that found in the unmodified 35S
and 35S(Δcore) promoters. Methylation of the outer regions were rare and irregularly dispersed.
Transgenic lettuce plants having single copy of the 64-bp construct also showed methylation withinthe −105 to −88 region.

Discussion & Conclusion
The present study showed that in gentian the 35S methylation status was altered by the modificationof the promoter sequence. Hypermethylation of the 35S(Δcore) suggests that basal transcriptional
activity is dispensable for the 35S methylation in gentian. EMSA analysis suggests that the −149
to −124 and −275 to −250 regions having “GAAGA” and “GTGGAAAA” motifs are targeted by
DNA binding factor(s). Since substitution of the “GTGGAAAA” motif alter the competition of
the complex formation, the motif may be required for the binding of the nuclear factor. Transgenic
gentians having 35S(m1) showed transgene expression and hypomethylation of the modified
promoter. These results suggest that the substituted two “GAAGA” motifs are essential for the
methylation. Although the 64-bp region induced methylation in gentian, its activity is weaker thanthose of the unmodified 35S and 35S(Δcore) promoters. This result suggests that the presence of boththe 64-bp and the other highly methylated region is required for a high level of methylation. Overall,a set of the study using modified 35S promoters implies that DNA binding factor(s) binding to the“GAAGA” and “GTGGAAAA” motifs may participate in the 35S methylation machinery.

The Novartis Foundation(Japan)
for the Promotion of Science

94

The Novartis Foundation(Japan)
for the Promotion of Science

References
1. Mishiba KI, Nishihara M, Nakatsuka T, Abe Y, Hirano H, Yokoi T, Kikuchi A, Yamamura S (2005)
Consistent transcriptional silencing of 35S-driven transgenes in gentian. Plant J 44:541−556
2. Mishiba KI, Yamasaki S, Nakatsuka T, Abe Y, Daimon H, Oda M, Nishihara M (2010) Strict denovo methylation of the 35S enhancer sequence in gentian. PLOS ONE 5:e9670
3. Yamasaki S, Oda M, Koizumi N, Mitsukuri K, Johkan M, Nakatsuka T, Nishihara M, Mishiba KI(2011) De novo DNA methylation of the 35S enhancer revealed by high-resolution methylation
analysis of an entire T-DNA segment in transgenic gentian. Plant Biotechnol 28:223−230
4. Okumura A, Shimada A, Yamasaki S, Horino T, Iwata Y, Koizumi N, Nishihara M, Mishiba KI(2016) CaMV-35S promoter sequence-specific DNA methylation in lettuce. Plant Cell Rep 35:
43−51

一般の皆様へ
遺伝子組換え植物は食料問題や環境問題などの解決に貢献できるものとして、世界中で研究開発が進められています。しかし植物種によっては遺伝子導入が困難なことや、導入した遺伝子が期待通りに働かないことが問題になることがあります。私たちは、組換え植物で広く利用されている35S プロモーターが、園芸植物であるリンドウでは発現抑制が起きることを見出しています。本研究では、この発現抑制を引き起こす配列を明らかにしました。この成果は、組換え植物の発現制御技術の発展に役立つものと考えています。

The Novartis Foundation(Japan)
for the Promotion of Science95

The Novartis Foundation(Japan)
for the Promotion of Science

The origin of animal multicellular system – an approach from genome
editing of unicellular holozoans
Hiroshi Sugahsuga@pu-hiroshima.ac.jp
Department of Environmental Life Sciences, Prefectural University of Hiroshima

Summary Abstract
Unicellular holozoans are the closest unicellular relative to animals. Our recent study revealed thatthe genomes of unicellular holozoans encode a number of proteins that are involved in multicellularorganism-specific mechanisms such as cell-cell adhesion, cell-cell communication, and cellproliferation and differentiation. To understand the function of those genes in unicellular holozoans,
we set up the framework of genome editing using the TALEN technique. We made 10 TALEN
constructs with the intrinsic promoters, and determined the optimal transformation condition to
obtain the maximum number of TALEN-expressing cells, yet the clear footprint of genome editing
has not been identified.
Key Words：Evolution of multicellularity, Unicellular holozoans, Genome editing

Introduction
How did the multicellular animals evolve from a single cellular ancestor? To elucidate the origin
of multicellularity, we use “unicellular holozoans” as model organisms. Unicellular holozoans are
the closest unicellular relatives to animals, presumably maintaining the genetic and cellular featuresof the direct unicellular ancestor of animals. In former studies we had found that the unicellularholozoans have a wide range of “genetic toolkits” that were previously thought to be multicellularspecific, such as intercellular communication genes, developmental transcription factor genes, andcell adhesion genes (Suga et al. 2014). The aim of the study is to understand the functions of such
“multicellular genes” owned by unicellular organisms.

Results
We aimed to establish a protocol of genome editing on
unicellular holozoans. Although CRISPR/Cas9 systemrepresents the latest trend, we consider TALEN system
would be more applicable to our model protists because of its
simplicity; just two proteins are required to be expressed in thecell. By a collaboration with Hiroshima University, we havemade 10 TALEN constructs against two of our model protists
Capsaspora and Creolimax (Figure 1), both being unicellularholozoans.
We found that the TALEN proteins show some toxicity
to the Capsaspora cells, regardless of the constructs to be
introduced. We therefore first sought for the best amount of
the constructs for transformation (i.e. the maximum construct

Figure 1 Unicellular holozoans and ourmodel protists Capsaspora and Creolimax

The Novartis Foundation(Japan)
for the Promotion of Science96

The Novartis Foundation(Japan)
for the Promotion of Scienceamount that does not drastically impair the survival rate). We also developed the technique to isolate
a single transformant using a micropipette or microinjector).
We first performed a pilot experiment, the SSA (single strand annealing) assay, to confirm thatTALEN in fact functions in Capsaspora and Creolimax cells. Unfortunately the conventional CMV
promoter did not drive any transcription in the cells. Thus we replaced the CMV promoter in theoriginal SSA constructs (from Addgene) by the elongation factor 1α promoter (for Capsaspora) or
the β-tubulin promoter (for Creolimax), which are already known to operate in our model organisms.
As expected, the new constructs expressed the TALEN proteins and the marker mCherry fluorescentprotein. Moreover, the SSA target construct alone did not produce the marker protein EGFP due tothe intentionally introduced stop codon. However, when both are introduced into the cell, the EGFP
expression was not observed either.
In parallel to the SSA assay, we aimed to knock out the endogenous “multicellularity-related”
genes of unicellular holozoans, targeting the Src tyrosine kinases of Capsaspora and Creolimax.
Tyrosine kinases are, in animals, involved in cell-cell communication, cell differentiation, and cellproliferation, which represent essential features of multicellularity, yet the genomes of unicellularholozoans contain many tyrosine kinases including Src (Segawa et al. 2006; Schultheiss et al.
2012, 2014). It remains unclear what are the function of Src proteins in the unicellular context.
We therefore introduced three sets of TALEN constructs designed against two Capsaspora Src
genes and one set of TALEN constructs against a single Creolimax Src gene. We successfully
established six Capsaspora cell lines expressing the marker mCherry protein, indicating the TALEN
proteins are also present. From these cell lines the genomic DNA was extracted, and we amplified
by PCR the Src gene regions, which were subsequently sequenced. However, we could find any
clearly positive evidence supporting the TALEN proteins actually knocked out the target genes.
Two possibly positive lines are still under investigation. On the other hand, we did not obtain any
TALEN-positive Creolimax cell lines due to the low transformation efficiency, which critically went
down in the recent years.
For the knock out experiments, controlling ploidy and sexual stage of the organism is essential.
In some protists, sexual stages are introduced by a severe environmental condition. To knowhow unicellular holozoans react against environmental changes, we investigated the protein
phosphorylation of the cells under either oxidative or oligotrophic conditions. A western blotting anda following mass-spec analysis revealed two proteins that are specifically downregulated at oxidative
condition.

Discussion & Conclusion
The largest question in applying genome editing on unconventional models is at the choice of target
gene. Choosing the target in protist models is particularly difficult because the functions of genes
are mostly unknown. We chose Src genes because 1) Capsaspora has two Src genes with possibly
redundant functions, 2) the function of Src has already been studied well in animal models, and 3)
tyrosine kinase is one of our most important targets for functional analysis. Until today, we have notobtained any clearly positive results. This could account for the choice of target, i.e. losing even one
Src gene may be lethal to Capsaspora. On the other hand, it is also possible that the success rate ofgenome editing is much lower than we expect even if TALEN are expressed in the cell. To obtain the
genome-edited cell lines, we need both to 1) try other target genes that seem less lethal such as very
redundant gene families and to 2) increase the number of experiments.

The Novartis Foundation(Japan)
for the Promotion of Science

97

The Novartis Foundation(Japan)
for the Promotion of Science

References
Schultheiss, K.P., Suga, H., Ruiz-Trillo, I., and Miller, W.T. (2012). “Lack of Csk-Mediated Negative
Regulation in a Unicellular Src Kinase” Biochemistry 51, 8267-8277.
Schultheiss, K. P., Craddock, B. P., Suga, H., and Miller, W. T. (2014) “Regulation of Src and Csk
Nonreceptor Tyrosine Kinases in the Filasterean Ministeria vibrans” Biochemistry 53, 1320-1329.
Segawa, Y., Suga, H. (equally contributed), Iwabe, N., Oneyama, C., Akagi, T., Miyata, T. andOkada, M. (2006). “Functional development of Src tyrosine kinases during evolution from aunicellular ancestor to multicellular animals.” Proc Natl Acad Sci U S A 103:12021-6.
Suga, H., Chen,, Z., De Mendoza, A., Sebé-Pedrós, A., Brown, M., Kramer, M., Carr, M., Kerner,
P., Vervoort, M., Sánchez-Pons, N., Torruella, G., Derelle, R., Manning, G., Lang, B. F., Russ, C.,
Haas, B. J., Roger, A. J., Nusbaum, C & Ruiz-Trillo, I. (2013). “The genome of Capsaspora reveals
a complex unicellular prehistory of animals” Nature Communications 4:2325.

一般の皆様へ動物のからだはたくさんの細胞からできています。細胞同士は連絡を取り合いながら高度な分業体制をとっており、このことは動物が地球のエコシステム上で優位な地位を占めるに至る一因となったと考えられています。しかし一方で、人類が苦しめられている癌は細胞増殖の制御が狂うことが原因で起きており、多細胞生物にしかありえない病気といえます。我々の研究は、動物に最も近縁な単細胞生物「単細胞ホロゾア」と総称される生物を材料に、動物多細胞性の進化の秘密を探り、多細胞システムが本質的に抱える問題にも迫ろうとするものです。

The Novartis Foundation(Japan)
for the Promotion of Science98

The Novartis Foundation(Japan)
for the Promotion of Science

Regulating microtubule arrays for neuronal dendrite growthand for neuronal regeneration
Adrian Walton Moore
adrianm@brain.riken.jp
RIKEN Brain Science Institute

Summary Abstract
We describe two previously unknown and independently organized microtubule nucleation centersin growing dendrite branches. We show that at each of these centers microtubule nucleation occurs
through a differing molecular mechanism. The microtubule nucleation activities we will describe are
critical to promote dendrite outgrowth and branching, we predict they will be mechanism to drive
dendrite regeneration after injury.
Key Words：Neuron Growth, Dendrite, Microtubule

Introduction
Neurons must connect through dendrites to receive input from their appropriate partners; thisis required for correct circuit wiring and nervous system function. Just small alterations in the
differentiation of dendrites can contribute to intellectual disability, which affects 2-3% of populationworldwide1-3. To understand how these disorders arise it is important to first elucidate the molecular
processes of dendrite branching and dendrite pattern formation4. Moreover, dendrites are also
highly susceptible to damage by trauma, seizure, and stroke. For successful regeneration after injury
dendrites must return to a growing state. Therefore, understanding the mechanisms controlling
microtubule dynamics in the growing dendrite is critical not only for understanding neuron
development, but also for discovering strategies to support neuron regeneration

Results
The microtubule cytoskeleton is critical to provide structure and to exert force in order to create,stabilize, and remodel dendrites during development. Both anterograde and retrograde microtubule
polymerization occur within growing dendrites.
We have developed an in vivo imaging protocol to follow the development of Drosophila pupal
sensory neurons from the initial stage of dendrite outgrowth until the branching structure isstabilized5,6. From these analyses the critical dendrite outgrowth and branching events that create
the major branch structures of the dendrite arbor were identified. Next, in order to understand how
microtubule nucleation events are organized with the growing dendrite and how they are used
to pattern the dendrite, the spatial organization of microtubule nucleation events during dendrite
outgrowth will be mapped. To do this, we will assay EB1::GFP comets7. The +Tip protein8 EB1concentrates at the growing end of a microtubule. Therefore an EB1::GFP comet marks the end of apolymerizing microtubule, and the appearance of a comet indicates a site of microtubule nucleation7.
In dendrites the length of microtubules exceeds the diameter of the cell, and therefore microtubules

The Novartis Foundation(Japan)
for the Promotion of Science99

The Novartis Foundation(Japan)
for the Promotion of Science

are necessarily orientated either plus-end-distal and anterograde polymerizing (away from the cellbody), or minus-end distal and retrograde polymerizing (towards the cell body)9. Our results showedthat regulation of the position and polarity of microtubule nucleation events in the growing dendritearbor is essential for dendrite arbor patterning. Spatial control over microtubule nucleation is used toorganize the cellular cytoskeleton. We will show that two spatially separate microtubule organizing
centers exist within the terminal region of a developing dendrite. At the tip of the extending dendrite
microtubules are nucleated an anterograde direction. Within the shaft of the dendrite is a separate
zone where microtubules are nucleated in a retrograde direction.
Abnormalities in dendrite pattern are a central feature in intellectual disability1,10. To understandhow dendrite patterning is controlled, we carried out a pilot screen to analyze a set of candidate
genes centered an on intellectual disability predisposition network2. This was done using in vivo
EB1::GFP live imaging to identify candidates that alter the pattern of microtubule nucleation andpolymerization in the growing dendrite. From this pilot screen we identified MyosinVI and Wee
Augmin (Wac). MyosinVI is best known as a actin-associated motor; however it has also been show
to link to microtubules through recruiting microtubule associated proteins, (in particular the +Tip
Clip170), to its cargo binding domain11. Furthermore cargo binding induces MyosinVI to dimerize,
and in the dimerized form it can act as a non-moving actin filament crosslinker. Wac is associatedwith the Augmin complex, which nucleates new microtubules from the sides of pre-existing
microtubules12. Additional factors were identified in this pilot screen. Including an unstudied factor
with C2 (a calcium-mediated membrane targeting domain) and DH and PH domains (indicating
guanine nucleotide exchange factor (GEF) activity for regulation of the Rho family GTPases). Ourpresent data shows that Myosin VI drives the anterograde directed microtubule nucleation in thedeveloping dendrite tip.

Discussion & Conclusion
We have demonstrated new independent microtubule nucleation centers in the neuron, and showthat they control developmental dendrite arbor patterning; furthermore our data begin to that they
are regulated by different molecular mechanisms. We predict that MyosinVI localizes microtubule
polymerization promoting factors to amplify Augmin-mediated microtubule nucleation. Furthermore,
MyosinVI simultaneously bundles actin into tracks that target polymerizing microtubules in theanterograde direction. This is a new paradigm for how neuron shape can be achieved, and whether asimilar process also controls shape in other cell types is a key follow up area for study. Importantly,
dendrites are highly susceptible to damage, and will next show how these developmental mechanisms
are reused for dendrite regeneration and repair after injury.

References
1. Kaufmann, W.E. & Moser, H.W. Dendritic anomalies in disorders associated with mental
retardation. Cereb Cortex 10, 981-91 (2000).
2. Kaufman, L., Ayub, M. & Vincent, J.B. The genetic basis of non-syndromic intellectual disability:
a review. J Neurodev Disord 2, 182-209 (2010).
3. van Karnebeek, C.D. & Stockler, S. Treatable inborn errors of metabolism causing intellectual
disability: a systematic literature review. Mol Genet Metab 105, 368-81 (2012).
4. Lefebvre, J.L., Sanes, J.R. & Kay, J.N. Development of Dendritic Form and Function. Annu Rev

The Novartis Foundation(Japan)
for the Promotion of Science

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

Cell Dev Biol 31, 741-77 (2015).
5. Satoh, D., Suyama, R., Kimura, K. & Uemura, T. High-resolution in vivo imaging of regenerating
dendrites of Drosophila sensory neurons during metamorphosis: local filopodial degeneration andheterotypic dendrite-dendrite contacts. Genes Cells 17, 939-51 (2012).
6. Yasunaga, K., Kanamori, T., Morikawa, R., Suzuki, E. & Emoto, K. Dendrite reshaping of adult
Drosophila sensory neurons requires matrix metalloproteinase-mediated modification of thebasement membranes. Dev Cell 18, 621-32 (2010).
7. Yalgin, C. et al. Centrosomin represses dendrite branching by orienting microtubule nucleation.
Nat Neurosci 18, 1437-45 (2015).
8. Akhmanova, A. & Steinmetz, M.O. Control of microtubule organization and dynamics: two ends
in the limelight. Nat Rev Mol Cell Biol 16, 711-26 (2015).
9. Delandre, C., Amikura, R. & Moore, A.W. Microtubule nucleation and organization in dendrites.
Cell Cycle 15, 1685-92 (2016).
10. Kulkarni, V.A. & Firestein, B.L. The dendritic tree and brain disorders. Mol Cell Neurosci 50, 1020 (2012).
11. Finan, D., Hartman, M.A. & Spudich, J.A. Proteomics approach to study the functions ofDrosophila myosin VI through identification of multiple cargo-binding proteins. Proc Natl Acad
Sci U S A 108, 5566-71 (2011).
12. Sanchez-Huertas, C. & Luders, J. The augmin connection in the geometry of microtubule
networks. Curr Biol 25, R294-9 (2015).

一般の皆様へ
神経系が正しく機能するためには、神経細胞は神経樹状突起を介して適切な伝達経路からの入力を受け取る必要がある。よって、神経樹状突起の発達に異常が起こると知的障害および神経発達障害を引き起こす可能性がある。本研究では、神経樹状突起の発育時に起こる【2つの】未知かつ独立して存在する微小管核による中心体形成について明らかにした。我々は、神経系の中心体形成過程において重要な役割を果たすことが知られている微小管核形成は、異なる分

子メカニズムを介して起こること示した。また、微小管核形成は神経樹状突起が成長、枝分かれするのを促すことで、神経損傷などが起こった際に神経樹状突起が再生するのに非常に重要な役割を果たすことを示唆した。

The Novartis Foundation(Japan)
for the Promotion of Science101The Novartis Foundation(Japan)
for the Promotion of Science

A novel network between mature adipocytes and endothelial cells that
regulates adipose tissue angiogenesis and homeostasis
Koji Ikeda
Ikedak@kobepharma-u.ac.jp
Kobe Pharmaceutical University

Summary Abstract
Adipose tissue contains highly developed vascular networks and its blood vessel density is closely
associated with adipocyte functions. However, detailed molecular mechanisms underlying adipose
tissue angiogenesis remain unclear. We searched for genes that is involved in the crosstalk between
mature adipocyte and endothelial cells, and identified Nrg4 as one of such genes. We found that
Nrg4 is highly expressed in mature adipocyte and it enhanced angiogenic capacity in endothelial
cells. Gain and loss of function studies using genetically modified mice revealed that Nrg4-medated
adipose tissue angiogenesis play a crucial role in the preservation of metabolic homeostasis,
especially in obesity. Our data established Nrg4 as a novel therapeutic target in the treatment and/or
prevention of metabolic disorders associated with obesity.
Key Words：Obesity; Adipose tissue; Endothelial cell; Angiogenesis

Introduction
Obesity is epidemic worldwide and its deleterious impact on cardiovascular disease makes it asa life-threatening state. Adipocyte dysfunction is causally implicated in obesity-related metabolic
disorders; however molecular mechanisms by which obesity impairs adipocyte functions remain
to be elucidated. Insufficient angiogenesis in adipose tissue during obesity causes hypoxia andconsequently triggers inflammation and dysfunction in adipose tissue; therefore adipose tissue
angiogenesis is a potential pharmacotherapeutic target in the treatment of obesity-related metabolic
disorders.

Results
We searched for genes that are involved in the communication between endothelial cells (ECs)and mature adipocytes (MA) by using DNA microarray, and identified Nrg4 as one of such genes.
Nrg4 was highly expressed in adipose tissues, predominantly in mature adipocytes. Of note,
Nrg4 expression was substantially reduced in adipose tissue of obese mice potentially throughinflammatory cytokines and ER-stress. Nrg4 belongs to EGF family of proteins, and recombinantNrg4 enhanced proliferation and tube-formation in ECs, while it reduced EC apoptosis. In vivo
angiogenesis assessed by Matrigel-plug assay also revealed angiogenic function of Nrg4. These
results indicate that Nrg4 is a novel angiogenic adipokine. On the other hand, we found that ECs
negatively regulated Nrg4 expression in MA. Experiments using inhibitors for various signaling
pathway identified a crucial role of JAK signaling in EC-mediated down-regulation of Nrg4 in MA.
To further elucidate the role of Nrg4 in metabolic homeostasis, we generated mice with a targeted
deletion of Nrg4. Interestingly, Nrg4-KO mice showed modest but significant increase in bodyweight and adiposity even while consuming normal chow. Moreover, loss of Nrg4 caused impaired

The Novartis Foundation(Japan)
for the Promotion of Science

102

The Novartis Foundation(Japan)
for the Promotion of Scienceinsulin sensitivity under normal dietary condition. Blood vessel density in brown adipose tissue wasmodestly reduced and consequently energy expenditure was significantly decreased in Nrg4-KO
mice compared to those in WT mice. Blood vessel density in white adipose tissue was also reduced,
and adiponectin expression significantly decreased in Nrg4-KO mice. When fed a high fat-diet (HFD),
obesity was significantly deteriorated in Nrg4-KO mice. Blood vessel density in brown adipose
tissue substantially reduced and energy expenditure significantly decreased in Nrg4-KO mice fed
a HFD. Blood vessel density in white adipose tissue also substantially reduced, and hypoxia andchronic inflammation was deteriorated in HFD-fed Nrg4-KO mice. In contrast, targeted activationof Nrg4 in adipocytes improved insulin sensitivity even under normal chow diet, although bodyweight and adiposity was unchanged. Blood vessel density in white adipose tissue was significantly
increased in Nrg4-Tg mice. When fed a HFD, Nrg4-Tg mice showed body weight and adiposity
similar to WT mice. Nevertheless, HFD-fed Nrg4-Tg mice exhibited ameliorated insulin resistanceand glucose intolerance compared to those in WT mice fed a HFD. Chronic inflammation in white
adipose tissue was significantly reduced in HFD-fed Nrg4-Tg mice. To further clarify the role ofenhanced adipose tissue angiogenesis in improved metabolic homeostasis in Nrg4-Tg mice, we
administered angiogenesis inhibitor SU-5416 that inhibit VEGF-R2 signaling. Blood vessel density
in white adipose tissue was similar between WT and Nrg4-Tg mice after the treatment with SU-5416.
Of note, administration of SU-5416 abolished the improvements in insulin sensitivity and glucose
tolerance observed in Nrg4-Tg mice.

Discussion & Conclusion
Our data collectively indicate that Nrg4 is a novel angiogenic adipokine that regulates adipose
tissue angiogenesis, and that Nrg4-mediated angiogenesis is crucially implicated in the maintenanceof systemic metabolic homeostasis. Because Nrg4 expression in adipose tissue was down-regulated
during obesity, enhancing and/or preserving Nrg4 expression in adipocytes has an attractive
therapeutic potential in the treatment of obesity-related metabolic disorders.

一般の皆様へ
肥満は世界的に増加を続けており、その健康被害は甚大です。肥満時には脂肪細胞が病的に肥大し、脂肪組織が機能不全に陥る結果、全身のエネルギー・糖代謝が障害されると考えられていますが、その詳細な分子機構は明らかではありません。脂肪組織は非常に発達した血管網を有しており、その血管密度は脂肪細胞機能と密接に関わっています。私たちは脂肪細胞と血管内皮細胞のコミュニケーションに注目し、Nrg4という分子を発見しました。Nrg4は脂肪細胞から分泌され、血管新生を誘導して脂肪組織の血管密度を高めます。肥満時には Nrg4の発現が減少することが明らかとなり、Nrg4の発現を増やすことで肥満時にも脂肪血管密度を維持し、脂肪組織の機能障害を予防できると考えられます。

The Novartis Foundation(Japan)
for the Promotion of Science103

The Novartis Foundation(Japan)
for the Promotion of Science

Investigation of the mechanism of cell fate determination independent
from transcriptional factor
Ai Kotani
aikotani@k-lab.jp
Department of Hematology and Oncology, Tokai University School of Medicine
Department of Hematological Malignancy, Tokai University The Institute of Medical Sciences

Summary Abstract
Trancription factor has been assumed to be critical to determine cell fate.
However, we challenged the canonical notion based on the fact we observed in B cell lineage
commitment. Without EBF1, a crucial transcriptional factor, the single miRNA can induce B celllineage commitment. The mechanistic analysis of the intereting phenomena has been studied in this
proposal.
Key Words：Transcription factor, miRNA, EBF1, B cell

Introduction
In the previous study, we found that miR-126 could cause B cell differentiation in MLL-AF4
acute lymphocytic leukemia (ALL) without any upregulation of EBF1, E2A, and Pax5, which are
indispensable for B cell differentiation. Moreover, we found that miR-126 could partially rescue
EBF deficiency in B cell lineage commitment.

Results
Then we introduced another miRNA, which is also downregulated in MLL-AF4 ALL into EBF1
deficient hematopoietic progenitor cells (HPCs). This miRNA could induce CD19 expression, whichis the definitive surface marker of B committed cell. Not only CD19 expression but also B cell
related genes such as Pax5, Rag1/2,and lamda5 were upregulated in these cells.
In vivo mouse as well, the miRNA induced CD19 in EBF deficient HPCs.
To investigate why the miRNA causes B cell lineage commitment in EBF1 deficient HPCs, thedirect target genes of the miRNA have been studied by use of the comprehensive gene expression
profiles and in silico prediction of the miRNA by use of the software. As a results, several target
genes were identified and validated by the luciferase reporter assay and overexpression of themiRNA in EBF1 deficient HPCs.
Next, the relationship between EBF1 and the miRNA has been investigated. When overexpressing
EBF1 in Baf3 cells, a pro B cell line, no change of the miRNA expression was observed to show that
EBF1 does not regulate the expression of the miRNA. The other way around, the miRNA deficient
cells showed no change in EBF1 expression, which demonstrated that the EBF1 and the miRNA are
independently regulated.

The Novartis Foundation(Japan)
for the Promotion of Science

104

The Novartis Foundation(Japan)
for the Promotion of Science

Discussion & Conclusion
Canonical notion of cell fate determination is that the master regulator is transcription factor. Then
miRNAs have been assumed to have marginal function, as a fine tuner”. However, we observedthat only single miRNA can rescue transcription factor deficiency. These results suggest that the
canonical notion should be revised and miRNA has more potent function in cell fate determination.

References
Okuyama K, Ikawa T, Gentner B, Hozumi K, Harnprasopwat R, Lu J, Yamashita R, Ha D,
Toyoshima T, Chanda B, Kawamata T, Yokoyama K, Wang S, Ando K, Lodish HF, Tojo A,
Kawamoto H, Kotani A. MicroRNA-126-mediated control of cell fate in B-cell myeloid progenitors
as a potential alternative to transcriptional factors. Proc Natl Acad Sci U S A. 2013;110(33):13410-5.

一般の皆様へ
細胞の運命決定においては転写因子が重要な働きをするというのが従来の一般的な考え方でした。私たちは、その考えに沿わない、新しい現象を見出しました。その機序を本研究課題で解析し、細胞運命決定には転写因子のみならず、小分子 RNA という因子も深く関与することを明らかにしました。

The Novartis Foundation(Japan)
for the Promotion of Science

105

The Novartis Foundation(Japan)
for the Promotion of Science

III.
Reports from the Recipients of Grants
for International Meetings

The Novartis Foundation(Japan)
for the Promotion of Science106

The Novartis Foundation(Japan)
for the Promotion of Science

Report on Research Meeting
170515 Hiroshi Kawamoto Kyoto University
1. Name of Research Meeting / Conference
KTCC 2017:
7th International Workshop of Kyoto T Cell Conference
2. Representative
Organizing committee:
Hiroshi Kawamoto
Kyoto University
Ichiro Taniuchi
Riken IMS
Manami Itoi
Meiji University of Integrative Medicine
Toshinori Nakayama Chiba University
Takehito Sato
Tokai University
Yosuke Takahama
University of Tokushima

Professor
Group Director
Professor
Professor
Associate Professor
Professor

3. Opening period and Place
13th to17th March 2017
Shiran Kaikan, Kyoto University, Kyoto
4. Number of participants / Number of participating countries and areas
Participants: 295 (domestic 179, oversea 116)
Presentations: 178 (oral 124, poster 53)
Participating countries:22(Japan, USA, Canada, Australia, China, Taiwan, India, UK, France, Germany, Portugal, Norway,
Sweden, Austria, Czech Republic, Netherlands, Belgium, Swizerland, Israel, Singapore, Turkey,
Estonia)
5. Total cost
12 million JPY
6. Main use of subsidy
Production cost for color posters (A2 size x 500)
7. Result and Impression
Kyoto T Cell Conference is an international research conference focusing on the development
and function of T lymphocytes. This meeting represents as one of four T cell biology meetings
held in the world; the other threes being held in USA, Europe, and Australia. These meetings
are held in rotation with one meeting per year pace, so the International Workshop of KTCC isheld once in four years. This time, the meeting was held as the 7th meeting. Among these four
meetings, KTCC is usually the biggest, with more than 200 participants.
Topics discussed at the meeting included: thymic microenvironments, lymphoid progenitors
and lineage commitment, selection and differentiation, recognition and activation, immune
response and memory, and immune regulation and diseases, stromal cells for immune function,
aging of immune system. Leading scientists in the filed of T cell biology from all over the world

The Novartis Foundation(Japan)
for the Promotion of Science

107

The Novartis Foundation(Japan)
for the Promotion of Science

got together. On the first day (March 12th) in the evening, the meeting was started with three
keynote lectures: (i) Jonathan Sprent (Garvan Institute, Australia), who talked about the historyof discovery of the thymus, (ii) Shigeo Koyasu (Riken, Japan), who talked about the innate
lymphoid cells, and (iii) Nagahiro Minato (Kyoto Univesity, Japan), who talked about the aging
of immune system. After that, four full days of the meeting followed. During the whole meeting
period, all participants shared the presented topics, which included unpublished findings, andenjoyed active discussion on the topics. This meeting especially encourage presenters to show
unpublished data, and some of such new data were really exciting. In addition, a special lecture
was given by Shimon Sakaguchi (Osaka University, Japan) on March 16th, concerning thebiology of regulatory T cells. In conclusion, the meeting was very successful.
We made a full support only for one speaker as a keynote speaker (J. Sprent). Just partial
supports were given to 30 core scientists for accommodation, and to 20 students/postdocs fortravel based on selection (100,000 JPY for each). Even in such a situation, 116 oversea scientists
came to join this meeting. Such a meeting may be very exceptional in Japan.
8. Additional description
We sincerely appreciate the financial support from Novartis. Thank you very much.

The Novartis Foundation(Japan)
for the Promotion of Science

108

The Novartis Foundation(Japan)
for the Promotion of Science

Report on Research Meeting
1. Name of Research Meeting / Conference
CeNeuro2016 & Nagoya BNC Joint Meeting
2. Representative
Ikue Mori
Professor, Graduate School of Science, Nagoya University
3. Opening period and Place
July 27th – 30th, 2016
Toyoda Auditorium, Nagoya
4. Number of participants / Number of participating countries and areas
230 participants/ 16 countries and areas
5. Total cost
14,842,076 JPY
6. Main use of subsidy
Travel expenses for invited foreign speakers
7. Results and Impression
The main conference, CeNeuro 2016, was the first International C. elegans Neurobiology
Meeting held outside of Western countries. Even though several of large and related conferences(e.g. Eastern Asian C. elegans meeting, The Allied Genetics Coference) were held around thesame time, we still could have as many as 230 participants (138 overseas and 92 domestic)
in total. The four-day meeting included 16 invited talks, 50 oral presentations and 113 poster
presentations. The number of participants and the size of the venue seemed just appropriate:participants could have intense discussion throughout the entire conference while having enoughopportunity for networking with anyone during poster sessions, meals and mixers.
The conference covered a wide range of topics in C. elegans neurobiology, from fundamental
studies, such as neural circuit development, to cutting-edge researches including large-scale
analysis of neuronal activity underlying behavior. In particular, whole-brain imaging, a field of
research which C. elegans neurobiology takes the leadership of, caught attention of participants.
The recent progress in whole-brain imaging technology, as well as advanced mathematics to deal
with big data obtained by it, was introduced and actively discussed.
After CeNeuro 2016, we had a satellite symposium, Nagoya BNC symposium, which was open
to the public. Here, we expanded our discussion to neurobiology of zebrafish and fruit fly, whichshare the strategy of studying small-scale nervous system with C. elegans. The presentations on
sensorimotor coordination across animal species shed light on the future direction of this research
field towards understanding neuronal network dynamics that integrate external and/or internal
information to generate appropriate behavior. We also discussed issues surrounding today’s
research environment of neuroscience. The proposals by the speakers included multidimentional
approach to revise the social systems for publication, funding, carrier development and education.

The Novartis Foundation(Japan)
for the Promotion of Science

109

The Novartis Foundation(Japan)
for the Promotion of Science

8. Additional description
We are grateful to the NOVARTIS Foundation (Japan) for its generous financial support. It
helped us to lower registration fee, particularly for students, as well as to maximize financial
support for students and postdocs.

The Novartis Foundation(Japan)
for the Promotion of Science

110

The Novartis Foundation(Japan)
for the Promotion of Science

Report on Research Meeting
September 12, 2016
1. Name of Research Meeting / Conference
20th International Symposium on Homogeneous Catalysis
2. Representative
Kyoko Nozaki
3. Opening period and Place
July 10, 2016 – July 15, 2016; Kyoto Terrsa
4. Number of participants / Number of participating countries and areas
460 / 27
5. Total cost
28,869,705 JPY
6. Main use of subsidy
Travel expenses of invited speakers from abroad
7. Result and Impression
In this symposium, both leading researchers and young ones in the field of homogeneous
catalysis from Japan and other countries in the world got together for 6 days in Kyoto, andactively discussed on this area of research through 22 invited lectures, 32 oral presentations,and 258 poster presentations. Presentations were made not only on bulk homogeneous catalysis
such as hydrogenation, oxo-reaction, and oxidation, but also on the synthesis of fine chemicals
such as asymmetric catalysis and selective carbon-carbon bond-forming reactions, which are
indispensible for the development of pharmaceuticals. The theoretical research was also the
topic of interest in this symposium in addition to the experimental research. All the lectures were
presented in one lecture hall and enough time was used for the poster presentations, which madeit possible for all the participants to interact well with each other through active discussions.
8. Additional description
Thanks to the financial support from the Novartis Foundation and several other companies andorganizations, the symposium was successfully held.

The Novartis Foundation(Japan)
for the Promotion of Science

111

The Novartis Foundation(Japan)
for the Promotion of Science

Report on Research Meeting
1. Name of Research Meeting / Conference
The 4th International Symposium on Salivary Glands in Honor of Niels Stensen
2. Representative
Masataka Murakami
3. Opening period and Place
2016.11.30 – 12.2, Okazaki Conference Center, National Institute of Natural Sciences
4. Number of participants / Number of participating countries and areas
60 participants from 9 countries. America (4), Europe (4), Middle East (1), East Asia (51)
5. Total cost
JYEN 2,191,565,6. Main use of subsidy
Main use was airfare and accommodation for overseas participants.
7. Result and Impression
The main purpose of the symposium was to facilitate the clinical application from the basic
research in the field of salivary gland. The individual interests of invited speakers were varied
but all participants has some idea to apply for clinical application. Andy Wolff impressed
the audience that the most of medication influence the salivary secretion and he introduce a
stimulator on lingual nerve for clinical use. Jörgen Ekström indicated some anti- psychotic drug
effects on salivatory centers. Massimo Castagnola explained an application of mass spectroscopy
to analyse human saliva effectively and precisely. Shmuel Muallem proposed possibility forderangement of the ductal CFTR to initiate autoimmune disease and Sjögren Syndrome. The
role of P2X7 for carcinogenesis was discussed by Ivana Novak. Matthew Hoffman stressed theimportant role of intermediate cells such as fibloblast to facillitate the organogenesis in addition
to neurogenesis and vascular formation. From the view on Ca2+ homeostasis, Indu Ambudkar
suggested that uncontrolled Ca2+ entry may injure the cells. James Putney reviewed the history of
store operated Ca2+ entry and expanded the possibility of disease induced by gene modification of
Orai and STIM. Masataka Murakami and Akihiko Tanimura stressed the development of in situ
and ex vivo studies will be more important and we have to assess the molecular events in situ for
application of basic study to clinical procedures.
The discussion was held friendly after each talk and poster session and Young Japanese
researchers could follow the recent results of international pioneers. This is very important andhopeful sign of this field. During the symposium, the organizers discussed for future Stensen
symposium and asked the four Japanese scientist to maintein the work of salivary gland and theStensen Symposium.
8. Additional description

The Novartis Foundation(Japan)
for the Promotion of Science

112

The Novartis Foundation(Japan)
for the Promotion of Science

Report on Research Meeting
1. Name of Research Meeting / Conference
5th International Conference for Food and Environmental Virology (ISFEV2016)
2. Representative
Hiroyuki Katayama, University of Tokyo (ISFEV2016 Chair)
3. Opening period and Place
Sep. 13-16, 2016
Hotel Sakurai (Kusatsu 465-4, Agazuma, Gunma 377-1711)
4. Number of participants / Number of participating countries and areas
108/21
5. Total cost
7,581,726 JPY
6. Main use of subsidy
Participant fee for 9 invited participants
7. Result and Impression
5th International Conference for Food and Environmental Virology was successfully held at
Hotel Sakurai, Kusatsu, Japan from Sep. 13th to 16th, 2016 (Conference Chair: Dr. Hiroyuki
Katayama, The University of Tokyo, Japan). In total 108 participants from 21 countries joined
this event. The plenary lecture was given by Prof. Joan Jofre, University of Barcelona, and eight
key note speeches were given by leading researchers. There were 48 oral presentations and 44
poster presentations. These presentations were evaluated from the viewpoints including novelty
and presentation skill, and the 5 presenters were awarded.
All participants were satisfied by the well-organized conference and enjoyed fruitful discussionin all scientific sessions. Japanese participants also joined the discussion, and learned a lot about
the topic of food virology, such as contamination level and detection methods. One of the main
topics was norovirus, which causes a lot of infection cases whole through the world throughcontaminated water and food. The organizing committee invited world-leading researchers forthe norovirus study, and organized a special workshop for norovirus. Presenters showed the latest
information about norovirus studies, and participants got a lot of suggestions and inspirations for
future studies.
Since this conference was held in a hotel where participants can stay, all participants enjoyedcommunications even during the time for meals (lunch and dinner). This style has not suchcommon in Japan, while conferences held in western countries frequently employ this style.
A difficulty was in that the organizing committee had to take care about not only conference
organization but also the hotel reservation, but the satisfaction of participants rewarded the effort.
This was the first international conference of Food and Environmental Virology in Asia (thefirst 4 conferences were held in Europe). The organizing committee invited news paper writers to
the conference, and they introduced this conference in Yomiuri and Jomo News Paper as the first

The Novartis Foundation(Japan)
for the Promotion of Science

113

The Novartis Foundation(Japan)
for the Promotion of Science

conference on this issue in Asia. In total, this conference was so successful and best remembered
one for all participants.
8. Additional description
No additional description
Nov. 18, 2016
Daisuke Sano
Organizing Committee Secretary
ISFEV2016

The Novartis Foundation(Japan)
for the Promotion of Science

114

The Novartis Foundation(Japan)
for the Promotion of Science

30th Grant Report（FY2016）
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 underauthorization of the Ministry of Education, Science, Sports and Culture, followed by a transition to a
public interest incorporated foundation on Apr. 1, 2012. The grants conducted in FY 2016 are as
follows.
30th Novartis Research Grant: 35Researchers (JPY 1 mil.), Subtotal JPY 35 mil.
（*Including Kumamoto earthguate diaster：5）
Research Meeting Grant: 5 Meetings (JPY 0.4 mil.), Subtotal JPY 2.0 mil.
Total JPY 3.70 mil.

30th Novartis Research Grant（FY2016）
The Grant is to aim supporting creative research in Japan in the field of Bio, life science andrelevant chemistry.
#

Name

Institution

1 Hirose Tetsuro

Institute for Genetic
Medicine, Hokkaido
University

Title
Professor

Regulatory function of stress responsive
splicing controlled by long non-coding
RNA

Research Project

2 Kikuchi Kiyoshi

University of Tokyo

Associate
Professor

How is recombination suppression achievedin the first place between sex
chromosomes?

3 Ohta Akane

Konan University

Professor

Identification of novel neural processing
mechanism by using single transcriptome
analysis

4 Matsushima Ryo

Okayama University,
Institute of Plant Scienceand Resources

Associate
Professor

Analysis of starch grain formation using
live imaging and computer simulation

5 Takayama Kenichi

Department of Functional
Biogerontology, Tokyo
Metropolitan Inst

Researcher

Genome-wide analysis of transcriptional
and translational regulation by RNAbinding protein PSF in prostate cancer

6 Mizuseki Kenji

Osaka City University
Graduate School of
Medicine

Professor

Selective information routing in
hippocampal network

7 Yoshimitsu Takehiko Okayama University

Professor

Drug discovery from antitumor marine
alkaloid

8 Minamino Thru

Niigata University
Graduate School Medical
and Dental Sciences

Professor

Elucidation of pathogenic mechanism of
age-related disease and a stem cell aging
controlled by the vascular niche.

9 Iwasaki Yuka

Keio University School of
Medicine

Associate
Professor

Transposon silencing by small
RNA-mediated regulation of the 3D
organization of the genome

10 Mutoh Yuichiro

Tokyo University of
Science

Associate
Professor

Catalytic Synthesis of Spiroketals Using
Water as the Oxygen Source Triggered byRearrangement of Internal Alkynes

11 Shirakawa Hisashi

Kyoto University, Graduate Associate
School of Pharmaceutical
Professor
Sciences

Molecular analysis for thepathophysiological significance of brain
hypoxic response and application for the
CNS diseases

The Novartis Foundation(Japan)
for the Promotion of Science

115

The Novartis Foundation(Japan)
for the Promotion of Science

#

Name

Institution

Title

Research Project

12 Tago Megumi

Keio University

Associate
Professor

Elucidation of the onset mechanism of
myeloproliferative neoplasm through theerythropoietin receptor

13 Hirai Maretoshi

Kansai Medical University,
Department ofPharmacology

Associate
Professor

Induction and visualization of adultcardiomyocytes proliferation throughgrowth factor receptor activation

14 Ohki Rieko

National Cancer Center
Research Institute,
Division of Rare Canc

Group
leaderAnalysis of the function and genomic
abnormality of tumor suppressor gene
PHLDA3 in neuroendocrine tumors Towards development of a novel genetic
diagnosis method for neuroendocrine tumors

15 Ueda Takeshi

Kindai University

Lecturer

Disease specific regulation of the histone
methylation in leukemia development

16 Uchida Hiroaki

The Institute of Medical
Science, The University of
Tokyo

SeniorAssistant
Professor

Treatment of metastatic tumors by systemic
injection of tumor-retargeted oncolytic
herpesviruses

17 Verma Prabhat

Osaka University

Professor

Revealing cell membrane functions throughin-vitro analysis by tip-enhanced Raman
spectroscopy in liquid

18 Manabe Ichiro

Chiba University

Professor

Elucidation of the immune-nerve-metabolic
systems network in cardiac homeostasis andfailure19 Hayashi Yasunori

RIKEN Brain Science
Institute

SeniorTeam
LeaderRoles of synaptic plasticity and replay
during sleep in memory consolidation

20 Tanaka Minoru

Nagoya University

Professor

Study on the Mechanism of Sex
Determination in Germline Stem Cells

21 Yamaguchi Tomoya

Nagoya University Schoolof Medicine

Designated
Associate
Professor

ROR1-mediated caveolae formation andcaveolar endocytosis in lung cancer

22 Matsumura Yoshihiro

The University of Tokyo,
RCAST

Associate
Professor

Regulation of energy metabolism byenvironmental cue and epigenome

23 Koshiba Takumi

Kyushu university

Associate
Professor

Mitochondrial dynamics and antiviral innate
immunity

24 Sasai Noriaki

Nara Institute of Scienceand Technology

Associate
Professor

Analysis of epigenetic regulation toward
establishment of a stable culture method ofneural progenitor cells

25 Hamamoto Ryuji

National Cancer Center
Research Institute

Divisino
Chief

Elucidating the regulatory mechanism ofhistone modification status in colorectal
cancer stem cells to maintain stemness andits application for drug development

26 Katayama Yoshio

Kobe University

Junior
Associate
Professor

Lymphoma progression promoted by thebreakdown of the system for convergenceof inflammation

27 Furuichi Tatsuya

Co-Department of
Veterinary Medical Science Professor
Faculty of Agricultu

Role of CRIM1 as a novel regulator for
osteoblast functions revealed using aosteoporotic mutant mouse

28 Fujiki Yukio

Medical Institute of
Bioregulation, Kyushu
University

Professor

Peroxisome biogenesis disorders: Molecularmechanism of pathogenesis in the
cerebellum

29 Okahisa Yoko

Kyoto Institute ofTechnology

Associate
Professor

Optically transparent
bamboo-cellulose/collagen nanocomposite
having antibacterial action

30 Oinuma Izumi

Graduate School of Life
Science, University of
Hyogo

Professor

Establishment of therapeutic genome
editing techniques applicable forpost-mitotic neurons

The Novartis Foundation(Japan)
for the Promotion of Science

116

The Novartis Foundation(Japan)
for the Promotion of Science

Novartis Research Grant(Kumamoto Earthguake Diaster frame)
#

Name

Title

Research Project

Kumamoto University
IRCMS

Associate
Professor

Vascular intraluminal pressure-dependent
switching machinery of angiogenic mode

2 Nakamura Taishi

Department of
Cardiovascular Medicine,
Kumamoto University

Associate
Professor

Novel therapeutic strategy for heart failure
targeting PKG/TORC signaling and its
intracellular localization

3 Oshiumi Hiroyuki

Kumamoto University

Professor

Virus-mediated suppression of host innate
immune response

4 Mimori

Koshi

Kyushu University Beppu
Hospital

Professor,
MD,PhD

Treg Related Exosomal microRNA
Involving to the Spatial and Chronologic
Evolution of CRC

5 Ohtsubo

KazuakiKumamoto University

Professor

Elucidation of the sialyl-Tn
antigen-mediated survival mechanism ofhypoxic tumor cells

1 Nishiyama

Institution

Koichi

FY2016 Research Meeting Grant(JPY 400 thousand x5 = 2.0 million)#

Meeting

Date(Place)1

7th International Symposium of Kyoto
T Cell Conference

2017.3.13-17(Kyoto)2

CeNeuro2016 & Nagoya BNC Joint
Meeting2016.7.27-30(Nagoya)3

20th International Symposium on
Homogeneous Catalysis

2016.7.10-15(Kyoto)4

The 4th International Symposium on
Salivary Gland in Honor of Niels Stensen

2016.11.30-12.2（Okazaki）

5

The 5th Conference of International
Society for Food and Environmental
Virology

2016.9.13-16(Gunma）

Institution / Title
Institute for Frontier
Medical Sciences,
Kyoto University/ Professor
Nagoya University,
Graduate School of
Science, Division of
Biological Science/ Professor
The University ofTokyo, Graduate
School of Engineering/ Professor
National Institute forPhysiological Society,Department of Cell
Physiology/ Associate Professor

Name
Hiroshi Kawamoto

Ikue Mori

Kyoko Nozaki

Masataka Murakami

Faculty of Engineering,
Hokkaido University
Daisuke Sano
/ Associate Professor

The Novartis Foundation(Japan)
for the Promotion of Science

117

The Novartis Foundation(Japan)
for the Promotion of Science

第30期（2016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。2016年度は、下記の総額3,700万円の助成事業を実施しました。
第30回ノバルティス研究奨励金（※内、熊本震災枠
研究集会助成35件（1件100万円）
3,500万円
5件）
5件（1件 40万円）
200万円
総額 3,700万円

第30回ノバルティス研究奨励金（2016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.

氏

名

所

属

職

位

研究課題

1 廣瀬

哲郎

北海道大学
遺伝子病制御研究所

教授

Long non-coding RNAに よ る ス ト レ ス 応
答性スプライシングの制御機能の解明

2 菊池

潔

東京大学大学院
農学生命科学研究科

准教授

性染色体組み換え抑制の起源

3 太田

茜 甲南大学特別研究員

シングルセルトランスクリプトーム解析による神経情報処理の新規機構の同定

4 松島

良

岡山大学
資源植物科学研究所准教授

ライブイメージングとシミュレーションを用いた澱粉粒の形状決定機構の解明

5 高山

賢一

東京都健康長寿医療センター研究所
老化制御 健康長寿ゲノム探索

研究員

前 立 腺 癌 に お け るRNA結 合 タ ン パ ク 質
PSFを介したゲノムワイドでの転写、翻
訳制御機構

6 水関

健司

大阪市立大学大学院
医学研究科

教授

海馬における経路特異的な情報処理機構
の解明

7 好光

健彦

岡山大学 大学院医歯薬学総合
研究科
（薬学系）
創薬有機化学分野

教授

抗腫瘍活性海洋産アルカロイドをモチー
フとする天然物創薬

教授

血管ニッチによって制御されるステムセル
エイジングと加齢関連疾患発症機序の解明

専任講師

小分子RNAを介したゲノム三次構造制御
によるトランスポゾン抑制機構の解明

助教

アルキンの転位を起点とする水を酸素源とするスピロケタールの触媒的合成法の開発

准教授

脳内低酸素応答の病態生理学的意義およびその介入による中枢神経疾患への応用

12 多 胡 め ぐ み 慶應義塾大学

准教授

エリスロポエチン受容体を介した慢性骨髄増殖性腫瘍の発症機序の解明

関西医科大学
薬理学講座

講師

成長因子受容体を介した成熟心筋細胞の増殖誘導とその分子機構の解明

8 南野
9 岩崎

徹 新潟大学由香

慶應義塾大学医学部
分子生物学教室

10 武 藤 雄 一 郎 東京理科大学
11 白 川13 平 井久志

希俊

京都大学 薬学研究科
生体機能解析学分野

The Novartis Foundation(Japan)
for the Promotion of Science

118

The Novartis Foundation(Japan)
for the Promotion of Science

No.

氏

名

所

属

14 大 木 理 恵 子 国立がん研究センター研究所

職

位

研究課題

グループ 希少がんである神経内分泌腫瘍のがん抑制
リーダー 遺伝子PHLDA3の遺伝子診断による 新し（主任
い予後・治療薬選択の診断法開発〜神経内研究員） 分泌腫瘍患者の個別化医療を目指して〜
講師

病態特異的ヒストンメチル化制御異常の白血病発症における役割

講師

全身投与可能ながん標的化改変ヘルペス
ウイルスによる転移性悪性腫瘍の治療法開発

17 バ ル マ プ ラ 大阪大学
ブ ハ ッ ト教授

液中先端増強超解像ラマン顕微鏡による細胞膜のin vitro観察及びその機能解明

18 眞 鍋一 郎 千葉大学教授

免疫−神経−代謝システム間連携による
心臓恒常性維持と心不全発症機序の解明

19 林

康紀

15 上 田
16 内 田20 田 中健 近畿大学
宏 昭 東京大学医科学研究所

国立研究開発法人理化学研究所
脳科学総合研究センター

実 名古屋大学

21 山 口知也

名古屋大学大学院
医学系研究科22 松 村欣宏

東京大学
先端科学技術研究センター

23 小 柴シニアチーム 睡眠下のシナプス可塑性とリプレー現象
リーダー の記憶固定化における役割
教授

生殖幹細胞の性決定機構の解明

肺腺がんでのROR1によるカベオラ形成と特任講師 カベオラエンドサイトーシス制御機構の解明
助教

環境要因とエピゲノムによるエネルギー代謝制御

琢 己 九州大学

准教授

ミトコンドリア・ダイナミクスと抗ウイルス自然免疫

24 笹 井紀 明 奈良先端科学技術大学院大学（独立） 神経前駆細胞の安定 的培養法の確立に
准教授 向けた、エピゲノム的制御機構の解明

25 浜 本隆 二 国立がん研究センター研究所分野長

大腸がん幹細胞の未分化性維持における
ヒストン修飾調節機構の解明と創薬への応用

26 片 山義 雄 神戸大学

講師

炎症収束機構の破綻によるリンパ腫進展メカニズム

27 古 市達哉

岩手大学
農学部共同獣医学科教授

骨粗鬆症マウスを利用した新規骨芽細胞
機能調節分子CRIM1の同定と機能解析

28 藤 木幸夫

九州大学
生体防御医学研究所

特任教授

ペルオキシソーム欠損症：小脳における病態発症の分子メカニズムの解明

助教

竹由来抗菌セルロースナノファイバー /コラーゲン透明コンポジットの創製

教授

分化後神経細胞への直接的遺伝子治療法確立のための基盤技術開発

29 小林（岡久） 京都工芸繊維大学
陽子
30 生 沼泉

兵庫県立大学大学院
生命理学研究科

The Novartis Foundation(Japan)
for the Promotion of Science

119

The Novartis Foundation(Japan)
for the Promotion of Science

ノバルティス研究奨励金 震災枠
No.

氏

名

所

属

職

位

研究課題

1 西山

功 一 熊本大学国際先端医学研究機構

特任
准教授

2 中村

太志

熊本大学医学部附属病院
循環器内科

客員
助教

PKGのTOR複合体調節機構と細胞内局在化機構を標的とした新たな心不全治療戦略

3 押海

裕之

熊本大学大学院
生命科学研究部免疫学分野

教授

ウイルスが宿主自然免疫応答を抑制する
新規メカニズムの解明

4 三森

功 士 九州大学病院別府病院

教授

大腸がんの時空間的進展にかかわる制御性
T細胞関連のエクソゾーム内包microRNA5 大坪

和 明 熊本大学

教授

sTn糖鎖抗原による低酸素環境下でのがん細胞の生存戦略の解明

外科

血管内腔圧制御による血管新生モードのスイッチング機構

2016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。2016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.

研究集会名

1

京都Ｔ細胞会議第７回国際シンポ
ジウム

開催日
（開催地）
2017.3.13-17
（京都）

所属・職位

氏

京都大学再生医科学研究所・
河 本
教授

名
宏

2016年線虫神経生物学国際集会・
2 名古屋大学神経回路国際シンポジウム合同集会

2016.7.27-30（名古屋）

名古屋大学大学院
理学研究科 生命理学専攻・ 森
教授

郁恵

3 第20回均一系触媒国際会議

2016.7.10-15( 京都 )

東京大学大学院工学系研究科・
野崎
教授

京子

4

第４回ニールス・ステンセン記念 2016.11.30-12.2 生理学研究所細胞器官研究系・
村上
国際唾液腺シンポジウム
准教授
（岡崎）

政隆

5

第５回国際食品・環境ウイルス学会

北海道大学大学院工学研究院・
佐野
准教授

大輔

2016.9.13-16（群馬県）

The Novartis Foundation(Japan)
for the Promotion of Science

120

The Novartis Foundation(Japan)
for the Promotion of Science

30th Financial Report

Balance Sheet

Statement of Net Assets

As of March 31, 2017

From April 1st, 2016 to March 31, 2017
（Unit：JP Yen）

Account（Unit：JP Yen）

AmountⅠ Assets

Ⅰ General Net Assets Changes

1. Current Assets

1. Ordinary income & Expenditure

Current Assets Total

50,535,116

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

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

Fixed Assets Total
Assets Total

Ⅲ

Ordinary Income Total

53,632,169(2) Ordinaty Expenditure
37,000,000

Novartis Research Grant

35,000,000

44,950,051

Research Meeting Grant

2,000,000

1,145,692,245

Administrative Expense

3,988,467

1,196,227,361

Ordinary Expenditure Total

50,784,743

Ordinary Balance without Appraisal Profit or Loss

2,847,426

2. Nonrecurring Profit & Loss
35,078,870

Nonrecurring Balance of Current Period

35,078,870

General Net Assets Ending Balance

160,406,297

Ⅱ Designated Net Assets Changes

Equity（Net Assets）

Designated Net Assets Change

1. Designated Net Assets
Designated Net Assets Total

40,054,000

Grant Expense

742,194

1. Current Liabilities
Liabilities Total

Donation

46,796,276

Ⅱ Liabilities
Current Liabilities Total(1) Ordinary income

Project Expense(3) Other Long - term Assets
Other Long - term Assets Total

AmountAccount

1,000,742,194（Amount Appropriating to basic Fund） 1,000,000,000
2. General Net Assets

160,406,297（Amount Appropriating to）

100,000,000

Equity Total（Net Assets）

1,161,148,491

Liabilities & Equity Total

1,196,227,361△ 54,000

Designated Net Assets Ending Balance 1,000,742,194
Ⅲ Net Assets Balance Ending Balance

1,161,148,491

The Novartis Foundation(Japan)
for the Promotion of Science

121

The Novartis Foundation(Japan)
for the Promotion of Science

第30期（2016年度）財務報告

貸借対照表

正味財産増減計算書

2017 年３月 31 日現在

2016 年 4 月 1 日から 2017 年 3 月 31 日まで（単位：円）

科

目（単位：円）

金 額

Ⅰ資産の部

科
１．経常増減の部

流動資産

合計

50,535,116

⑴

経常収益

２．固定資産

受取寄付金

基本財産

基本財産
⑵

経常収益

合計

1,100,000,000

特定資産

特定資産
⑶

合計

742,194

その他固定資産

その他固定資産
固定資産

合計

合計

資産合計

44,950,051⑵

40,054,000
計

事業費

46,796,276

支払助成金

37,000,000

ノバルティス研究奨励金

35,000,000

研究集会助成金

2,000,000

管理費

1,196,227,361

経常費用

3,988,467
計

当期経常増減額

１．流動負債

53,632,169

経常費用

1,145,692,245

Ⅱ負債の部

50,784,743
2,847,426

２．経常外増減の部

流動負債

合計

負債合計

35,078,870

当期形状外増減額

35,078,870

一般正味財産期末残高

Ⅲ正味財産の部
指定正味財産160,406,297

Ⅱ指定正味財産増減の部

１．指定正味財産

当期指定正味財産増減額
合計（うち基本財産への充当額）

1,000,742,194
1,000,000,000２．一般正味財産

160,406,297（うち基本財産への充当額）

100,000,000

正味財産

金 額

Ⅰ一般正味財産増減の部

１．流動資産⑴目

合計

負債及び正味財産合計

指定正味財産期末残高
Ⅲ正味財産期末残高-54,000
1,000,742,194
1,161,148,491

1,161,148,491
1,196,227,361

The Novartis Foundation(Japan)
for the Promotion of Science

122

The Novartis Foundation(Japan)
for the Promotion of Science

List of Board Members[Board of Trustees] 5 trustees, 2 auditors

Post
Chairman

Trustee

AuditorName

As of Sep 1, 2017

Title

Akimichi KANEKO

Dean, Professor, MD, Graduate School of Health Science, Kio University;
Emeritus Professor, Keio University

Shigetaka ASANO

Visiting Professor, MD, School of Medicine, Kobe University;
Emeritus Professor, University of Tokyo

Masao ENDOH

Emeritus Professor, MD, Yamagata University

Kuniaki TAKATA

President, Gunma Prefectural College of Health Sciences

Masao TORII

President, Novartis Holding Japan K.K.

Tokuzo NAKAJIMA

Certified Public Accountant

Masanori FUSE

Department Head, Region Finance, Novartis Pharma K.K.

[Board of Councilors] 10 councilors

Post
Chairman

Name

Title

Member of the Japan Academy;
Tsuneyoshi KUROIWA Emeritus Professor, University of Tokyo
Norio AKAIKE

Councilor

As of Sep 1, 2017

Head, Kumamoto Kinoh Hospital Clinical Research Center
Visiting Professor, Kumamoto University, Graduate School of Medicine andPharmaceutical Research
Emeritus Professor, Kyushu University

Hiroyuki
KAWASHIMA

Former Professor, Graduate School of Medical & Dental Sciences, Niigata
University

Masakatsu
SHIBASAKI

Director, Microbial Chemistry Research Center, Microbial Chemistry
Research Foundation

Akihiko NAKANOProfessor, University of Tokyo, Science Department; Team Leader, RIKEN(Institute of Physical & Chemical Research)

Tadanori MAYUMIEmeritus Professor, Osaka University

Miwako MORI

Emeritus Professor, Hokkaido University

Toyoshi FUJIMOTO

Professor, MD, Graduate School of Med, Nagoya University

Masamitsu IINO

Specially appointed Professor, MD, Nihon University
Emeritus Professor, University of Tokyo

Tohru HIROSE

Director, Division Head Japan Development, Novartis Pharma K.K.

The Novartis Foundation(Japan)
for the Promotion of Science

123

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

Post
Chairman

Name

As of Sep 1, 2017

Title

Yoshihiro OGAWA

Professor, MD, Graduate School of Medical and Dental Sciences, Tokyo
Medical and Dental University
Professor, MD, Graduate School of Medical Sciences, Kyushu University

Motomu KANAI

Professor, Graduate School of Pharmaceutical Sciences, University of Tokyo

Koichiro KUWAHARA Professor, MD, School of Medicine, Shinshu University
Masanobu OHSHIMA

Professor, Cancer Research Institute, Kanazawa University

Yosuke TAKAHAMA

Professor, Institute for Genome Research, Tokushima University

Masafumi TAKIGUCHI Professor, MD, Center for AIDS Research, Kumamoto University

Member

Eisuke NISHIDA

Professor, Graduate School of Biostudies, Kyoto University

Mitsuyasu HASEBE

Professor, National Institute for Basic Biology

Haruhiko BITO

Professor, MD, Graduate School of Medicine, University of Tokyo

Jun YAMASHITA

Professor, MD, Center for iPS Cell Research and Application, Kyoto University

Motoko YANAGITA

Professor, MD, Graduate School of Medicine, Kyoto University

Masafumi YANO

Professor, MD, School of Medicine, Yamaguchi University

Hirokazu ARIMOTO

Professor, MD, Graduate School of Life Sciences, Tohoku University

Hiroyuki TAKEDA

Professor, Graduate School of Science, University of Tokyo

Eiji HARA

Professor, Research Institute for Microbial Diseases, Osaka University

Makoto HAYASHITeam Leader, Plant Symbiosis Reseach Team, RIKEN Center for
Sustainable Resource Science

Hirokazu HIRAIProfessor, MD, Head of Department of Neurophysiology and Neural Repair,
Gunma University

Kouichi FUKASE

Professor, Graduate School of Science, Osaka University

Michisuke YUZAKI

Professor, School of Medicine, Keio University

Professor, Department of Microbiology and Immunology, Graduate School
Akihiko YOSHIMURA of Medicine, Keio University

The Novartis Foundation(Japan)
for the Promotion of Science

124

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
役 員 名 簿2017 年 9 月 1 日現在（順不同、敬称略）
職

名

理事

監事

氏

名

現

職

就任年月日

常勤･非常勤金子

章道

畿央大学大学院健康科学研究科長･教授
慶應義塾大学名誉教授

2016年 6月10日

非常勤

浅野

茂隆

神戸大学大学院医学系研究科客員教授
東京大学名誉教授

2016年 6月10日

非常勤

遠藤

政 夫 山形大学名誉教授

2016年 6月10日

非常勤

高田

邦 昭 群馬県立県民健康科学大学学長

2016年 6月10日

非常勤

鳥居

正男

ノバルティスホールディングジャパン（株）
代表取締役社長

2016年 7月 1日

非常勤

中嶋

德三

中嶋德三公認会計士事務所
公認会計士

2016年 6月10日

非常勤

布施

正則

ノバルティス ファーマ（株）
経理・財務統括部長

2016年 6月10日

非常勤

評 議 員 名 簿2017 年 9 月 1 日現在（順不同、敬称略）
職

名

氏

評議員長

黒岩

常祥

評議員

就任年月日

常勤･非常勤2016年 6月10日

非常勤

赤池

医療法人社団寿量会熊本機能病院臨床研究センター所長、学術顧問
紀 扶 熊本大学大学院医学薬学研究部客員教授
九州大学名誉教授

2016年 6月10日

非常勤

川島

博 行 元新潟大学大学院医歯学総合研究科教授

2016年 6月10日

非常勤

柴崎

正勝

公益財団法人微生物化学研究会
微生物化学研究所長

2016年 6月10日

非常勤

中野

明彦

東京大学大学院理学系研究科教授
理化学研究所光量子工学研究領域チームリーダー

2016年 6月10日

非常勤

眞弓

忠 範 大阪大学名誉教授

2016年 6月10日

非常勤

北海道医療大学客員教授
北海道大学名誉教授

2016年 6月10日

非常勤

藤本

豊 士 名古屋大学大学院研究科教授

2016年 6月10日

非常勤

飯野

正光

日本大学医学部特任教授
東京大学名誉教授

2016年 6月10日

非常勤

廣 瀬

徹

ノバルティス ファーマ（株）
取締役開発本部長

2016年 6月10日

非常勤

森

名

現

美和子

職

日本学士院会員
東京大学名誉教授

The Novartis Foundation(Japan)
for the Promotion of Science

125

The Novartis Foundation(Japan)
for the Promotion of Science

選 考 委 員 名 簿
2017 年 9 月 1 日現在（順不同、敬称略）
職

名

氏

就任年月日

常勤･非常勤2014年 6月 6日

非常勤

2014年 6月 6日

非常勤

桑 原 宏 一 郎 信州大学医学部教授

2015年 6月26日

非常勤

大島

正 伸 金沢大学がん進展制御研究所教授

2015年 6月26日

非常勤

髙濵

洋 介 徳島大学先端酵素学研究所教授

2015年 6月26日

非常勤

滝口

雅 文 熊本大学エイズ学研究センター教授

2015年 6月26日

非常勤

西田

栄 介 京都大学大学院生命科学研究科教授

2015年 6月26日

非常勤

長 谷 部 光 泰 自然科学研究機構基礎生物学研究所教授

2015年 6月26日

非常勤

尾藤

晴 彦 東京大学大学院医学系研究科教授

2015年 6月26日

非常勤

山下

潤 京都大学 iPS 細胞研究所教授

2016年 6月10日

非常勤

柳田

素 子 京都大学大学院医学研究科教授

2016年 6月10日

非常勤

矢野

雅 文 山口大学医学部医学科教授

2016年 6月10日

非常勤

有本

博 一 東北大学生命科学研究所教授

2016年 6月10日

非常勤

武田

洋 幸 東京大学大学院理学系研究科教授

2017年 6月19日

非常勤

原

英 二 大阪大学微生物病研究所教授

2017年 6月19日

非常勤

理化学研究所環境資源科学センター
チームリーダー2017年 6月19日

非常勤

平井

宏 和 群馬大学大学院医科学系研究科教授

2017年 6月19日

非常勤

深瀬

浩 一 大阪大学大学院理学研究科教授

2017年 6月19日

非常勤

柚﨑

通 介 慶應義塾大学医学部教授

2017年 6月19日

非常勤

吉村

昭 彦 慶應義塾大学大学院医学研究科教授

2017年 6月19日

非常勤

選考委員長 小 川
金 井

選考委員

林

名

現

佳宏

職

九州大学大学院医学研究院教授
東京医科歯科大学大学院医歯学総合研究科教授

求 東京大学大学院薬学系研究科教授

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

126

The Novartis Foundation(Japan)
for the Promotion of Science

事 務 局 便 り

ご寄附のお願い
当財団は、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、国民の健康と福祉の向上に寄与することを目的に公益事業を行っております。
当財団の事業は、基本財産の運用益並びに寄付金によって賄われており、財団では趣旨にご賛同いただける皆様からのご寄付を募っております。
当財団へのご寄付には、下記の税法上の優遇措置が適用されます。
優遇措置の概略
個人：年間寄付金の合計額もしくは年間所得の40％相当額のいずれか低い方から２千円を引いた金額が、所得税の寄付金控除額となります。
法人：支出した寄附金は、通常一般の寄附金の損金算入限度額と同額まで、別枠で損金に算入できます。
ご寄附は、随時受付けております。詳しくは、財団事務局までお問合せ下さい。
（電話：03-6899-2100、E-メール：foundation.japan@novartis.com）

事務局より
本年度もお陰様で、財団年報を発行できることとなりました。これも偏に、助成を受けられた皆様および財団関係者の皆様のご尽力の賜物と心より感謝
申し上げます。
1987年９月の財団設立以来、助成件数は総数で約1,700件、総額約20億円となりました。
事務局は、今後とも財団の設立目的である学術の進展に寄与するべく、研究助成を中心とした公益事業に邁進して参ります。
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

田中

基晴

〒105‑6333 東京都港区虎ノ門1‑23‑1
虎ノ門ヒルズ森タワー 29F
TEL：03‑6899‑2100 FAX：03‑6899‑2101
E‑メール：foundation.japan@novartis.com
ホームページ：http://japanfoundation.novartis.org/

The Novartis Foundation(Japan)
for the Promotion of Science127