Annual Report (2019)

No.31
The NOVARTIS Foundation (Japan)
for the Promotion of Science
令和元年度

財団年報 第31号

The Novartis Foundation(Japan)
for the Promotion of Science

Contents
Ⅰ．Introduction はじめに ……………………………………………………………………… 6
Akimichi Kaneko, MD, PhD
Chairman of the Board of Trustees

Ⅱ．Reports from the Recipients of Novartis Research Grants

研究報告 ………………………………………………………………………………………… 8
1. Epigenetic and metabolic changes on transdifferentiation of white adipocytes ……… 9
Tekeshi Inagaki
Institute for Molecular and Cellular Regulation, Gunma University

2. Development of super resolution chemical imaging microscopy ………………………… 12
Yasufumi Takahashi
Kanazawa University

3. Elucidation of molecular bauplan to determine the positioning of the posterior bodystructure along the anteroposterior axis …………………………………………………… 14
Takayuki Suzuki
Nagoya University

4. Neural circuits that regulate glucose metabolism between hypothalamus andperipheral organs. ……………………………………………………………………………… 16
Kunio Kondoh
National Institute for Physiological Sciences

5. Exploratory analysis to evaluate mutation spectrum of circulating tumor DNA
from metastatic breast cancer treated with aromatase inhibitors ………………………… 19
Siew-Kee LOWCancer Precision Medicine Center, Japanese Foundation for Cancer Research

6. Investigation of the molecular mechanism of osteoblastogenesis. ……………………… 22
Yoshinori Matsumoto
Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences

7. Heart Cell Molecular Atlas Project to dissect the regulatory mechanisms ofcirculatory homeostasis ……………………………………………………………………… 24
Seitaro Nomura
The University of Tokyo

8. Development of the new organ lack model………………………………………………… 28
Ayako Isotani
Nara Institute of Science and Technology

9. Molecular mechanism for the initial assembly of human procentriole. ………………… 30
Satoko Yoshiba
Graduate School of Pharmaceutical Sciences, The University of Tokyo

10. Structural basis for inactivation of Na+,K+-ATPase by β-amyloid ……………………… 33
Ryuta Kanai
The University of Tokyo

The Novartis Foundation(Japan)
for the Promotion of Science

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

11. Evolutionary development of fluorescent RNA sensors by droplet technology ……… 36
Shigeyoshi Matsumura
University of Toyama

12. Functional Analysis of Chk1 Targets for Breast Cancer Treatment …………………… 38
Midori Shimada
Yamaguchi University

13. Revealing a new red light signaling pathway for resetting the circadianclock in green algae ………………………………………………………………………… 41
Takuya Matsuo
Center for Gene Research, Nagoya University

14. Elucidation of the role of fatty acid elongase Elovl6 in the pancreatic β-cell………… 44
Takashi Matsuzaka
Faculty of Medicine, University of Tsukuba

15. Development of regenerative therapy and novel therapeutic drugs for renal anemia
using human iPS cell-derived erythropoietin-producing cells ………………………… 46
Kenji Osafune
iPS Cell Research and Application (CiRA), Kyoto University

16. Mechanism that controls the neuronal survival/death in the developing brain ……… 49
Kazunori Nakajima
Department of Anatomy, Keio University School of Medicine

17. Direct reprogramming into cardiac pacemaker cells for a novel regeneration therapy
for arrhythmias ……………………………………………………………………………… 51
Shu Nakao
Ritsumeikan University

18. Structure determination of formosalide A by the combined technology of the NMR
calculation and total synthesis ……………………………………………………………… 54
Daisuke Urabe
Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University

19. Development of apoptosis induction method with spatiotemporal control using
microRNAs. ………………………………………………………………………………… 56
Kazunori Watanabe
Okayama University

20. Study on the enantioselective total synthesis of dragmacidin E: a natural productwith high potential for medicinal chemistry researches ………………………………… 58
Tetsuhiro Nemoto
Graduate School of Pharmaceutical Sciences, Chiba University

21. Understanding the neural mechanism underlying flexible decision making by the
combination of neurophysiology and deep neural networks …………………………… 61
Hironori Kumano
University of Yamanashi

The Novartis Foundation(Japan)
for the Promotion of Science

2

The Novartis Foundation(Japan)
for the Promotion of Science

22. Drug development of DYRK1A inhibitors: targeting excess phosphorylation in
Alzheimer’s disease ………………………………………………………………………… 63
Masashi Asai
Tokyo University of Science

23. Development of combination therapy targeting on tumor endothelial cells …………… 67
Takahisa Murata
The University of Tokyo

24. Molecular mechanism of store-operated Ca2+ entry signalosome andits physiological significance ……………………………………………………………… 69
Yoshihiro Baba
Division of Immunology and Genome Biology, Department of Molecular Genetics Medical
Institute of Bioregulation, Kyushu University

25. Decoding the epigenome program which defines normal andtumor endothelial cells ……………………………………………………………………… 72
Hiromu Suzuki
Department of Molecular Biology, Sapporo Medical University School of Medicine

26. Elucidation of long non-coding RNA family induced by molecular targeting anticancer drugs ………………………………………………………………………………… 75
Shigetada KONDO
Graduate School of Comprehensive Rehabilitation, Osaka Prefecture University

27. Development of anti-metastatic therapy based on the regulation ofvascular integrity. …………………………………………………………………………… 77
Takayuki Shindo
Shinshu University School of Medicine

28. Mechanisms underlying commitment to thymic epithelial cells preventing onset of
autoimmune diseases ………………………………………………………………………… 81
Taishin Akiyama
RIKEN Center for Integrative Medical Sciences

29. Regulation of cellular functions by the nucleolus as a stress sensor …………………… 84
Keiji Kimura
University of Tsukuba

30. Transomics approach to elucidate cellular signaling pathways in vascular development,
morphogenesis and disease ………………………………………………………………… 87
Osamu NAKAGAWA
National Cerebral and Cardiovascular Center Research Institute

31. Studies on formation and function of extracellular DNA fibers ………………………… 90
Hisato Saitoh
Faculty of Advanced Science and Technology (FAST), Kumamoto University

32. Serotonin 5-HT2A receptor agonist in treatment to psychiatric disorders ……………… 93
Daisuke Ibi
Meijo University, Department of Chemical Pharmacology

33. RopGEF in the regulation of planar cell polarity in plants ……………………………… 96
Teh Ooi Kock
Hokkaido University

The Novartis Foundation(Japan)
for the Promotion of Science

3

The Novartis Foundation(Japan)
for the Promotion of Science

34. A novel oxytocin neuronal system for regulating lactating behavior in mice ………… 98
Yoshikage Muroi
Obihiro University of Agriculture and Veterinary Medicine

35. Molecular mechanisms for the bifurcation of thymic epithelial cells ……………… 100
Izumi Ohigashi
Division of Experimental Immunology, Institute of Advanced Medical Sciences,
University of Tokushima

36. The role of Heat shock protein 40 Member C1 in hepatic lipid metabolism ……… 103
Atsuko Nakatsuka
Okayama University Hospital.

37. Novel strategy targeting neutrophils in the tumor microenvironment of colorectal
cancer ……………………………………………………………………………………… 107
Kenji Kawada
Kyoto University, Department of Surgery,

38. Evolution of sex determination systems ………………………………………………… 110
Yusuke Takehana
Nagahama Institute of Bio-Science and Technology

39. Design principle for specific chemical probes toward 3D neuropathology ………… 112
Kazuki Tainaka
Brain Research Institute, Niigata University

40. Molecular basis of osmoregulatory ionocytes ………………………………………… 115
Hiroshi Miyanishi
University of Miyazaki

41. Clarification of molecular mechanisms underlying the maintenance of satellite cellstemness and its therapeutic application for Muscular Dystrophy …………………… 118
Shinichiro Hayashi
Department of Neuromuscular Research, National Institute of Neuroscience,
National Center of Neurology and Psychiatry

Ⅲ．Reports from the Recipients of Grants for International Meetings
研究集会報告………………………………………………………………………………… 121
1. Symposium on Molecular Chirality 2018 ………………………………………………… 122
Masami Sakamoto
Chiba University

2. The 2nd research conference for the Research Society for Cancer 3-D culture ……… 123
Koji Okamoto, MD, PhD., Chief
National Cancer Center Research Institute, Division of Cancer Differentiation

3. International Consortium on Hallucination Research and Related Symptoms

Kyoto SatelliteMeeting (ICHR 2018 Kyoto)…………………………………………………
Jun Miyata
Kyoto University

124

The Novartis Foundation(Japan)
for the Promotion of Science

4

The Novartis Foundation(Japan)
for the Promotion of Science

4. International Tuberous Sclerosis Complex Research Conference 2018 ……………… 126
Okio Hino, M.D., Ph.D.
Professor, Juntendo University Faculty of Medicine

5. Workshop on Frontiers in Phosphatase Research and Drug Discovery (ICPP13)…… 128
Takeshi Tsubata
Tokyo Medical and Dental University

Ⅳ．The 32nd (fiscal year 2018) Promotion Report

第32期（2018年度）助成事業報告 ……………………………………………………… 130

Ⅴ．The 32nd (fiscal year 2018) Financial Report

第32期（2018年度）財務報告 …………………………………………………………… 138

Ⅵ．List of the Board Members

役員名簿 ……………………………………………………………………………………… 140

Ⅶ．Information from the Secretariat

事務局便り …………………………………………………………………………………… 144

The Novartis Foundation(Japan)
for the Promotion of Science

5

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 FY2017. 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 1,764 researchcases in 32 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.
As usual every year, the timing we are preparing this annual report is the timing of release
for Nobel Prize in Physiology or Medicine. Last year 2018, Prof. Tasuku Honjo at Kyoto
University was awarded the Prize. I have a big dream that some of younger researchers follow
him, and I hope that our Foundation helps them. With such a dream we are continuing ourresearch funding services.
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

6

The Novartis Foundation(Japan)
for the Promotion of Science

はじめに

代表理事

金子

章道

ここに第31回 ノバルティス科学振興財団の研究助成金を受けられた方々の研究
報告（2018年４月～ 2019年３月の研究）を収録いたしました。（研究奨励金 41件、研究集会 5件）
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32年間に1,764件、金額にしておよそ20.5億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が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考えております。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。
この巻頭言を準備している頃、毎年ノーベル生理学・医学賞の発表時期を迎えます。
昨年は京都大学の本庶佑先生がノーベル賞を受けられました。我が国から次々と受賞者が選ばれますことを祈り、また、これらの受賞者に続く若い研究者を輩出する
お手伝いができればと大それた願いを持ちつつ財団の助成事業を続けている次第です。
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年間という限られた時間の中で達成され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

7

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

8

The Novartis Foundation(Japan)
for the Promotion of Science

Epigenetic and metabolic changes on transdifferentiation ofwhite adipocytes
Tekeshi Inagaki
Inagaki@gunma-u.ac.jp
Institute for Molecular and Cellular Regulation Gunma University

Summary Abstract
It is considered that the quantitative changes in the levels of intracellular metabolites are recorded
as forms of epigenetics which will contribute for developing the life-style related diseases including
obesity and diabetes. In this project, we sought to measure intra-cellular concentrations of Fe(II) andα‑ketoglutarate which are indispensable metabolites for activating JmjC domain-containing histone
demethylases and other epigenetic enzymes. Additionally, we tried to establish a system to determine
cellular histone modifications comprehensively. By establishing and performing these systems, we
elucidated the changes in metabolites and histone marks during adipocyte differentiation.
Key Words：Histone demethylase, Metabolite, Adipocyte, Epigenetics

Introduction
We recently presented a two-step working mechanism in which histone demethylase JMJD1A
is recruited to the target genomic regions by being phosphorylated at S265 (Step 1) and thus
demethylases methylated histones which locate in the vicinity regions of its biding (Step 2) 1,2.
Because Fe(II) and α‑ketoglutarate (αKG) are indispensable metabolites for activating JmjC domaincontaining histone demethylases including JMJD1A3,4,5, we speculate that the transition from Step
1 to Step 2 would be mediated by the concentration of these metabolites. Therefore, we sought toestablish the novel systems to determine the cellular concentrations of Fe(II) and αKG as well as
various histone modifications.

Results
In this project, we have performed experiments for establishing probes to detect the intra-cellularconcentrations of Fe(II) and αKG which are indispensable metabolites for activating epigenetic
enzymes including JmjC domain-containing histone demethylases3,4,5. We additionally sought toestablish a comprehensive analysis protocol of histone modifications.
The sensing system of nuclear αKG was established based on a fluorescence resonance energy
transfer (FRET) system. We inserted the functional αKG-binding domain of the nitrogenasetranscriptional regulator protein NifA between the yellow fluorescent protein (YFP) and the cyan
fluorescent protein (CFP) as a FRET probe. The established plasmids were transduced into thepreadipocytes by transient transfection or retrovirus infection. Transient transfection of the FRET
prove enables us to observe intra-cellular concentration of αKG, while retrovirus infected cells
showed reduced fluorescent level. Although the exact reason for the reduction of fluorescent level
was not clear, it is considered that a recombination between CFP coding sequence and YFP coding
sequence occurred during the reverse transcription step in retrovirus. Therefore, we employed
PiggyBac transposon-mediated system for establishing the preadipocytes which stable express the

The Novartis Foundation(Japan)
for the Promotion of Science

9

The Novartis Foundation(Japan)
for the Promotion of Science

FRET probe during their differentiation. On the other hand, the concentration of intracellular Fe(II)was measured using its specific chemical probe SiRhonox which was kindly provided by the group of
Dr. Hideko Nagasawa and Dr. Tasuku Hirayama. Using these measuring probes, we revealed that thecellular concentration of both Fe(II) and αKG changes during adipocyte differentiation. In addition,
the cellular iron concentration affected the levels of adipocyte differentiation with mediating histone
methylation levels. Thus, these data presented an important link between the adipocyte differentiationand the epigenetic demethylation enzymes which require both Fe(II) and αKG for its activity.
We also sought to establish a protocol to determine the intra-cellular histone modifications
comprehensively. Although chromatin immunoprecipitation (ChIP) is widely used for examiningchanges in histone modifications during cell differentiation, ChIP is not appropriate method for
examining comprehensive histone modifications. Because the specific antibody against a certain
histone modification is required for each ChIP analysis, ChIP requires as many numbers of antibodies
as the number of histone modifications we target. Therefore, we established a novel method todetermine intra-cellular histone modifications comprehensively using a mass spectrometer. First, wehave purified histones from pre- and post-differentiated adipocytes and the purified histones werealkylated at lysine residues and digested by trypsin to cut at arginine residues. Next, the fragments ofhistone tails were applied to mass spectrometry and the obtained data were analyzed by the MASCOT
search engine, Progenesis software, and a statistical software. This novel protocol enables to present
novel combinations of histone modifications on a single histone tail. As a consequence of the primary
analysis, we found that the several combinations of histone modification marks in preadipocytes
were resolved following adipocyte differentiation, which indicates their potential role to regulate
adipogenic gene expression. Although this protocol for analyzing multiple histone modifications
using mass spectrometer is a powerful tool to elucidate histone modifications comprehensively,
an additional minor modification was required. Because the pretreatment step of histone tail withpropionic acid in our protocol made a pair of multiple modifications indistinguishable. To avoid thisproblem, we employed an isotope labeling. By performing the modified mass spectrometric analysis
of histone modifications using an isotope labeling, we successfully determined novel combinationsof posttranslational modifications which occur on a histone tail.

Discussion & Conclusion
In this project, we have established the systems to determine intra-cellular concentrations of Fe(II)and α‑ketoglutarate which are indispensable metabolites for activating multiple epigenetic enzymes.
We also succeeded to establish a system to determine cellular histone modifications comprehensively.
Establishment of the protocols required multiple modifications such as employing PiggyBac system
for stably expressing the FRET probe in preadipocyte and using an isotope labeling for enabling
determine indistinguishable modifications. By performing the established protocol, we elucidated
a link between adipocyte differentiation and epigenetic demethylation enzymes which require
both Fe(II) and αKG for its activity. In addition, we determined novel combinations of histone
modifications which changes during adipocyte differentiation. Collectively, these data indicate thatthe concentrations of intra-cellular metabolites mediate epigenetic memory by mediating histone
modifications, while further investigations would be required to reach such a consequence.

The Novartis Foundation(Japan)
for the Promotion of Science

10

The Novartis Foundation(Japan)
for the Promotion of Science

References
1) Abe, Y, Fujiwara, Y, Takahashi, H, Matsumura, Y, Sawada, T, Jiang, S, Nakaki, R, Uchida, A,
Nagao, N, Naito, M, Kajimura, S, Kimura, H, Osborne, TF, Aburatani, H, Kodama, T, Inagaki, T,
Sakai, J. Histone demethylase JMJD1A coordinates acute and chronic adaptation to cold stress via
thermogenic phospho-switch. Nat Commun, 9: 1566, 2018
2) Abe, Y, Rozqie, R, Matsumura, Y, Kawamura, T, Nakaki, R, Tsurutani, Y, Tanimura-Inagaki,
K, Shiono, A, Magoori, K, Nakamura, K, Ogi, S, Kajimura, S, Kimura, H, Tanaka, T, Fukami,
K, Osborne, TF, Kodama, T, Aburatani, H, Inagaki, T, Sakai, J. JMJD1A is a signal-sensing
scaffold that regulates acute chromatin dynamics via SWI/SNF association for thermogenesis. Nat
Commun, 6: 7052, 2015
3) Inagaki, T, Sakai, J, Kajimura, S. Transcriptional and epigenetic control of brown and beige
adipose cell fate and function. Nat Rev Mol Cell Biol, 17: 480-495, 2016
4) Inagaki, T. Histone demethylases regulate adipocyte thermogenesis. Diabetol Int, 9: 215-223,
20185) Tanimura, K, Suzuki, T, Vargas, D, Shibata, H, Inagaki, T. Epigenetic regulation of beige
adipocyte fate by histone methylation. Endocr J, 66: 115-125, 2019

一般の皆様へ
肥満症や糖尿病といった生活習慣病は、遺伝要因と環境要因によって発症する。しかしながら、生体がさらされてきた外部環境がどのように細胞に記憶されているかの分子機構は不明な点が多い。エピゲノムはゲノム配列によらない遺伝子発現の制御機構であり、可塑性が高いシステムであるため、外部環境を記憶するシステムとして優れていると考えられている。今回、エピ
ゲノム酵素の働きの制御に関わる細胞中の代謝物濃度を測定する方法を立ち上げ、さらにヒストン修飾というエピゲノムマークを網羅的に解析する手法を立ち上げることで、代謝環境のエ

ピゲノム記憶を制御する機構に迫った。その結果、肥満症と関連が深い脂肪細胞分化の過程で、代謝物濃度とエピゲノムの変化が見いだされた。

The Novartis Foundation(Japan)
for the Promotion of Science

11

The Novartis Foundation(Japan)
for the Promotion of Science

Development of super resolution chemical imaging microscopy
Yasufumi Takahashi
yasufumi@se.kanazawa-u.ac.jp
Kanazawa University

Summary Abstract
We have developed scanning probe microscopy based chemical imaging tool for visualizing thechemical concentration profile around the cell.
Key Words：Scanning probe microscopy, super resolution microscopy, live cell imaging

Introduction
Scanning ion conductance microscopy (SICM) uses a less than 50 nm radius nanopipette fordetecting ion current and is an effective tool for non-contact live cell nanoscale topography imaging,
local chemical delivery, and cytosol collection. We have developed miniaturized electrochemicalsensor and mounted the sensor on SICM probe to achieve 3D chemical concentration profile
imaging.

Results
The permeation properties of cell membranes
play a crucial role in intracellular and cellsurface biochemical reactions. Scanningelectrochemical microscopy (SECM) uses
ultramicroelectrode for detecting faradaic current
and is a potentially effective tool for evaluating
membrane permeability; however, conventional
S E C M d o e s n o t h a v e s u ff i c i e n t s p a t i a lresolution to image inhomogeneous membrane
permeability on a single cell surface. The
curvature of a cell surface is an important factor
for accurately evaluating permeability, and most Fig.1
previous SECM studies evaluated permeability
based on numerical simulation wherein thecell membrane was assumed to be flat. Various
micro/nanostructures, such as surface microvilli,
are present on actual cell surfaces, and therefore, one should not ignore such structures in numericalsimulation when evaluating local cell permeability. SECM-SICM hybrid probe system can provide
a 3D electrochemical image at nanometer resolution, providing real features of the local membrane
permeability and the ion concentration distribution. To demonstrate a local membrane permeability
measurement, we used the system for high-resolution imaging of 3T3-L1 fixed cells, which havelarge lipid droplets. In the measurements, we defined the setpoint for stopping probe approach in
hopping mode at a 6.0% decrease from bulk ion current to bring the electrode very close to the

The Novartis Foundation(Japan)
for the Promotion of Science

12

The Novartis Foundation(Japan)
for the Promotion of Science

substrate. Fig. 1 shows the SECM–SICM images of 3T3-L1 in PBS containing 0.5 mM FcCH2OH.
The SECM image is based on the oxidation of FcCH2OH, a hydrophobic mediator which cancross the cell membrane. The SICM images represent the topography, whereas the SECM images
measure the flux of FcCH2OH. Therefore, the SECM provides information about the local membrane
permeability of FcCH2OH and thus the local structure of the cell.

Discussion & Conclusion
We performed 3D electrochemical and ion current imaging for measuring cell-membrane
permeability and ionic strength using SECM–SICM. The pyrolytic carbon SECM–SICM probe iseffective for such experiments because the small electrode size and RG bring the electrode close to
the sample surface, without contact. In adipocyte imaging, 3Delectrochemical imaging visualizes
inhomogeneous permeability. Even with a fixed cell, we could observe the ionic strength difference
on the cell surface. Hopping-mode SECM–SICM breaks the limit of microscale spatial resolutionof SECM and opens up new possibilities for special chemical and ion strength profile imaging. Thismethod will allow for the visualization of inhomogeneous chemical distribution and permeability on
single cells, such as neurotransmitter concentration profiles or respiration activity.

References
Zhou, Y. S.; Saito, M.; Miyamoto, T.; Novak, P.; Shevchuk, A. I.; Korchev, Y. E.; Fukuma, T.;
Takahashi, Y., Nanoscale Imaging of Primary Cilia with Scanning Ion Conductance Microscopy.
Anal Chem 2018, 90 (4), 2891-2895.
Matsumae, Y.; Takahashi, Y.; Shiku, H.; Matsue, T., Quantitative Real-Time Monitoringof Antibody-Induced Internalization of Epidermal Growth Factor Receptor on Single Living
Mammalian Cells Using Scanning Electrochemical Microscopy. Chemelectrochem 2018, 5 (20),3096-3101.
Takahashi, Y.; Kumatani, A.; Shiku, H.; Matsue, T., Scanning Probe Microscopy for Nanoscale
Electrochemical Imaging. Anal Chem 2017, 89 (1), 342-357.
Takahashi, Y.; Ida, H.; Matsumae, Y.; Komaki, H.; Zhou, Y. S.; Kumatani, A.; Kanzaki, M.; Shiku,
H.; Matsue, T., 3D electrochemical and ion current imaging using scanning electrochemical-scanningion conductance microscopy. Phys Chem Chem Phys 2017, 19 (39), 26728-26733.

一般の皆様へ細胞―細胞間のコミュニケーションや細胞の代謝により、細胞近傍の化学物質の濃度プロファ

イルは時々刻々と変化している。そのため、この細胞表面の化学物質の濃度プロファイルを捉えることは、非常に重要である。しかし、一般的に利用されている化学物質のセンシング技術は、
位置情報と時間情報が失われた状態で計測が行われている。その一つの理由として、化学セン

サーの感度と、非常に柔らかい細胞近傍にセンサーを配置することが困難なためである。そこで、走査型プローブ顕微鏡のプローブとして、微小な電極やナノピペットに化学修飾を施したケミ
カルセンサーを用いることで、細胞近傍の化学物質の濃度プロファイルを形状方法とともに３次元的に取得した。

The Novartis Foundation(Japan)
for the Promotion of Science

13

The Novartis Foundation(Japan)
for the Promotion of Science

Elucidation of molecular bauplan to determine the positioningof the posterior body structure along the anteroposterior axis
Takayuki Suzuki
suzuki.takayuki@j.mbox.nagoya-u.ac.jp
Nagoya University

Summary Abstract
In this study, we examined the molecular mechanism how posterior body structure is formed basedon the initiation timing of GDF11 to identity molecular bauplan establishing our body structure.
We identified common enhancer sequence of GDF11 which is involved in the determination of theposition of the posterior body.
Key Words：Tetrapods, morphological diversity, vertebral formula, gene expression, heterochrony

Introduction
Tetrapods including human has vertebral bones at the center of our body. Our organs develop
along the antero-posterior axis based on the vertebral formula. Recently, we found that one of the
TGF-β signaling molecules, GDF11, expressing at the posterior side of the developing presomitic
mesoderm, PSM, coordinates the position of the hindlimb to the sacral vertebrae. Interestingly,
initiation timing of GDF11 differs among tetrapod embryos, frog, chick, mouse, gecko, and snake.
These results strongly support the idea that specifies-specific hindlimb/sacral vertebra position
was brought about by unique initiation timing of GDF11. In GDF11 knockout mice, hindlimb
position together with sacral vertebra and other organs located at posterior body structure wasshifted posteriorly. Therefore, in this study, we examined the molecular mechanism how posterior
body structure is formed based on the initiation timing of GDF11 to identity molecular bauplan
establishing our body structure.

Results
To identify the molecular mechanisms involving species-specific initiation timing of GDF11, wethought two hypotheses. One is each specie has unique enhancer sequence to induce unique initiation
timing of GDF11. Second hypothesis is that tetrapods have common enhancer sequences to induce theexpression, however, initiation timing of transcriptional factors that bind to common enhancer sequence
would be different. To explore these hypotheses, first, we looked for enhancer sequence of mouse GDF11.
We compared mouse GDF11 locus with birds, alligator, and lizard, and found that there were only 3
short-conserved sequences between exon1 and exon2 at GDF11 locus. Thus, non-cording sequence near
GDF11 locus was not conserved well. Next, we performed in vivo reporter assay to analysis whether
these sequences has enhancer activity where GDF11 is expressed. We tandemly inserted expectedenhancer sequences upstream of tk-EGFP. We electroporated these reporter constructs with pCAGGSmCherry vector as control for electroporation. We found that only 1 sequence showed enhancer activity
at the posterior side of presomitic mesoderm where Gdf11 is expressed. In this region, we found putativetranscriptional factor binding sites, ETS and TCF4, downstream of FGF and WNT signaling, respectively.
These results indicate that FGF and WNT signaling is involved in the initiation of GDF11 expression.

The Novartis Foundation(Japan)
for the Promotion of Science

14

The Novartis Foundation(Japan)
for the Promotion of Science

Therefore, next, we treated chick embryos with FGF signaling antagonist, PD173074, and WNT signaling
antagonist XAV939. We found that GDF11 expression was strongly down-regulated. In contrast, when weelectroporated constitutive active MEK1 which is downstream target of FGF signaling, GDF11 expression
was up-regulated. As well, when chick embryos were treated with WNT agonist, Chiron, GDF11
expression was also up-regulated. There results indicate that both FGF and WNT signaling up-regulate
GDF11 expression in vivo. Next, we examined whether putative transcriptional factor binding sites of
EST and TCF4 are necessary for its enhancer activity. We made mutant reporter constructs that ETS or
TCF4 binding sites were mutated. After electroporation of these constructs where GDF11 is expressed,enhancer activity was strongly down-regulated in the case of only one transcriptional factor bindingsite was mutated. These results indicate that ETS or TCF4 binding sites on this enhancer sequence arenecessary for enhancer function to induce GDF11 expression. Next, we examined whether this enhancer
sequence is involved in the determination of the positioning of the posterior body structure. We made theenhancer knockout mice by Crisper/Cas9, and found that position of the hindlimb and sacral vertebrae
were shifted posteriorly by 1 vertebra level (right panel). This phenotype indicates that this enhancer
sequence is involved in the position of the posterior body structure through GDF11
expression. However, in GDF11 knockout mice, both hindlimb and sacral vertebrae
were shifted more posteriorly by 7-9 vertebral level, implying that there should be other
GDF11 enhancer sequences in the mouse. Then, we looked for other GDF11 enhancer
sequences by in silico analysis using SMC1-ChIAPET signals that show open chromatin.
Now we are testing whether expected enhancer sequences has enhancer activity where
GDF11 is expressed. Thus, we are focusing on long-range enhancer of GDF11 thatcoordinates initiation timing of GDF11 in the mouse. We are also looking for speciesspecific enhancer element in the turtle and snake by ATAC-seq. We are going to compare
these enhancer sequences in this year.

Discussion & Conclusion
In this study, we identified common enhancer sequence of GDF11 which is involved in thedetermination of the position of the posterior body. From reporter assay, it was thought thatdifference of WNT and FGF signaling activity at the posterior side of the presomitic mesoderm mightbring about positional difference of the positioning of the posterior body structure among tetrapods.
Our results also imply the existence of other enhancer element of GDF11, especially as long-range
enhancer. In conclusion, to establish common body structures as bauplan, both common enhancer
sequence and species-specific enhancer sequence would be necessary to induce species-specificinitiation timing of GDF11. In tetrapods, common posterior structure of our body is developed
downstream of GDF11 which initiates species-specific timing during developmental stages.

一般の皆様へ
私たち人を含む脊椎動物は体の中心に背骨を持っており、頭から頸椎、胸椎、腰椎、仙椎、尾椎に分けられる。興味深い事に、私たちの体の下半身には、仙椎の位置に後ろ足や生殖器、腎臓などの臓器があり、これらの器官の場所はどの動物においても同じである。今回我々は、このよ

うな下半身の位置がそれぞれの動物で異なりながらも、何故下半身に位置する器官の位置関係が保存されているのか、そのメカニズムを明らかにすることを試みた。その結果、GDF11という１つの遺伝子が働くタイミングを生み出す機構がそれぞれの動物で異なることが、ヘビのような特異な体の形を生み出していることが明らかとなった。

The Novartis Foundation(Japan)
for the Promotion of Science

15

The Novartis Foundation(Japan)
for the Promotion of Science

Neural circuits that regulate glucose metabolism between
hypothalamus and peripheral organs.
Kunio Kondoh
kkondoh@nips.ac.jp
National Institute for Physiological Sciences

Summary Abstract
The purpose of this funded study is to elucidate neural circuits that transmit leptin signals fromthe hypothalamus to peripheral tissues. I infected neurons in the skeletal muscle and brown adipose
tissues, major peripheral regulators of glucose uptake, with pseudorabies viruses that can travel
across synapses retrogradely. I found virus-infected neurons in the hindbrain and the hypothalamicareas, suggesting that these areas play an important role in transmitting signals from brains toperipheral tissues involved in glucose uptake.
Key Words：Hypothalamus, Glucose metabolism, Skeletal muscle, Brown adipose tissue

Introduction
Glucose is a major energy source in the body, and the brain maintains glucose levels in the bodyat a proper level. Leptin, which is secreted from white fat, acts on the hypothalamus of the brain
to promote muscle and fat glucose uptake and plays an important role in lowering blood glucose
levels1). Neurons in the hypothalamus then control glucose levels in the body by regulating glucose
utilization in peripheral tissues including skeletal muscle and brown adipose tissues as well as
glucose production in the liver2). However, the neural circuits that transmit leptin signals from the
hypothalamus to peripheral tissues are poorly understood.

Results
In this study, to investigate mechanisms by which leptin regulates glucose metabolism, I analyzed
the anatomical organization of neural circuits that transmit signals from the hypothalamus to skeletal
muscle (soleus) or brown adipose tissues (BAT).
The Bartha strain pseudorabies virus (PRV) is a neurotropic virus that can infect neurons, replicate,and retrogradely travel to upstream neurons through synaptic connections3) (Figure 1). Using PRV,
it is possible to comprehensively identify
upstream neurons that transmit signals to
specific neurons. In addition, by analyzing
virus-infected cells every day after virus
injection, one can estimate the numberof synaptic connections between eachneuron and primarily infected neurons4).
To investigate neural circuits that transmit
leptin signals from the hypothalamus toperipheral tissues, I infected soleus and
BAT with PRVs that express green and

The Novartis Foundation(Japan)
for the Promotion of Science

16

The Novartis Foundation(Japan)
for the Promotion of Science

red fluorescent proteins, respectively (Figure 2).
I observed PRV infected neurons in different brain areas on different days after virus injection.
Interestingly, I also observed many infected neurons that express both green and red fluorescentproteins, suggesting that these neurons send signals to both soleus and BAT. Since the only functionthat is shared by soleus and BAT is energy expenditure including glucose uptake, it is possible thatthe double-labeled neurons play an important role to send glucose uptake-related signals from thebrain to both soleus and BAT.
Two days after infection (possibly
two synapses upstream of soleus andBAT), virus infected neurons wereobserved only in the spinal cord. In the
spinal cord, the infected neurons wereaccumulated in the intermediolateral
nucleus (IML), where neurons thatconsist of the autonomic nervous
systems are located. This result is
consistent with previous findings
suggesting that sympathetic nervous systems are involved in the hypothalamic regulation of glucose
uptake in skeletal muscle and BAT.
Three days after infection (possibly three synapses upstream of soleus and BAT), virus infected
neurons were observed in several central brain areas. These brain areas included several areasin the medulla oblongata, and one area in the hypothalamus, the paraventricular nucleus of the
hypothalamus (PVH). Since I did not observe any infected neurons in the brain two days after virus
injection, the neurons infected three days after infection could send direct inputs to neurons in the
IML. Therefore, these results suggest that the medulla oblongata and PVH is the two downstream
brain area that integrates signals in the central brain.
Four days after infection, virus infected neurons were found in several hypothalamic brain areasthat are known to express leptin receptors and receive leptin signaling. They include the ventromedial
nucleus (VMH) and the arcuate nucleus (ARC). Therefore, the leptin signals may be received in the
VMH and/or ARC and be transmitted to soleus and BAT through brain areas infected with PRVs
three days after infection. These results, taken together, suggest the possible organization of neuralcircuits from hypothalamus to soleus and BAT that regulate glucose uptake. Leptin signals that arereceived in the ARC or VMH may be transmitted first to PVH and/or medulla oblongata, secondly to
the IML in the spinal cord, and then to sympathetic nerves in the soleus and BAT.

Discussion & Conclusion
The results from this study revealed the anatomical organization of neural circuits from the
hypothalamus to two peripheral tissues, skeletal muscle (soleus) and BAT, major regulators of
glucose uptake in the body. The results suggest that leptin sends signals to peripheral tissues throughtwo brain areas, PVH and medulla oblongata. In addition, I found many neurons in many brain
areas that are upstream of both skeletal muscle and BAT. Since glucose uptake is a shared function
between skeletal muscle and BAT, it is possible that the neural pathway that regulates both skeletal
muscle and BAT regulate glucose uptake by leptin. The next step is whether leptin actually activates
brain areas identified in this study.

The Novartis Foundation(Japan)
for the Promotion of Science

17

The Novartis Foundation(Japan)
for the Promotion of Science

References
1) Minokoshi Y, Haque MS, Shimazu T. Microinjection of leptin into the ventromedial hypothalamus
increases glucose uptake in peripheral tissues in rats. Diabetes (1999) 48, 287–291
2) Toda C, Shiuchi T, Lee S, Yamato-Esaki M, Fujino Y, Suzuki A, Okamoto S, Minokoshi Y.
Distinct effects of leptin and a melanocortin receptor agonist injected into medial hypothalamic
nuclei on glucose uptake in peripheral tissues. Diabetes (2009) 58, 2757–2765
3) Li J, Liu T, Dong Y, Kondoh K, Lu Z. Trans-synaptic Neural Circuit-Tracing with Neurotropic
Viruses. Neurosci Bull. (2019) [Epub ahead of print]
4) Kondoh K, Lu Z, Ye X, Olson DP, Lowell BB, Buck LB. A specific area of olfactory cortex
involved in stress hormone responses to predator odours. Nature (2016) 532, 103-106.

一般の皆様へ
脳の視床下部は体内の血糖値を一定の値に保つために重要な働きをしています。そのために視床下部は、白色脂肪から分泌されるレプチンなどの様々な体内・体外のシグナルを受け取り、末梢組織の糖の産生・消費をコントロールしています。今回私は、視床下部から糖の消費を担う２つの末梢組織（骨格筋・褐色脂肪）へシグナルが送られる際に使われる神経回路網の構造を明らかにしました。今回明らかになった神経回路の機能を解明することで、将来的に糖尿病や肥満の治療法の開発につなげていきたいと考えています。

The Novartis Foundation(Japan)
for the Promotion of Science

18

The Novartis Foundation(Japan)
for the Promotion of Science

Exploratory analysis to evaluate mutation spectrum of circulatingtumor DNA from metastatic breast cancer treatedwith aromatase inhibitors
Siew-Kee LOW
siewkee.low@jfcr.or.jp
Cancer Precision Medicine Center, Japanese Foundation for Cancer Research

Summary Abstract
Liquid biopsy, particularly circulating tumor DNA (ctDNA), detected from plasma could be used
to monitor breast cancer patients who develop resistance to aromatase inhibitors (AI) therapy. In
this study, we used ultra-deep targeted next generation sequencing (NGS) to detect mutations from
ctDNA. We detected mutations in TP53, PIK3CA, KRAS, FGFR3, and ESR1, frequently mutatedgenes in breast cancer patients on aromatase inhibitors. Our results suggest that ctDNA has potential
to be applied for monitoring of resistance to AI therapy.
Key Words：Liquid biopsy, circulating tumor DNA, next generation sequencing, drug resistance,
aromatase inhibitor.

Introduction
Aromatase inhibitors (AI), which block the peripheral conversion of androgen to estrogen, arefrequently used as treatment for postmenopausal ER-positive breast cancer patients. ESR1 mutations
are rarely detectable in the primary breast tumor, but are found at significant frequency (25-30%)
after secondary to AI exposure and develop endocrine resistance. Detection of ESR1 mutations inliquid biopsy from metastatic breast cancer, whom tissue biopsies are hard to access, might provide
earliest indication of AI drug resistance.

Results
This exploratory study aims to evaluate mutation detected from liquid biopsy of metastatic breast
cancer by using next generation sequencing (NGS) technology.
A total of 14ml of blood samples from 26 metastatic breast cancer patients were collected. Aftercentrifugation, cell-free DNA/RNA (cfDNA/RNA) were extracted from the plasma using magneticbased total nucleic acid extraction method. The concentration of the cfDNA/RNA ranged from 4.81
to 83.52 ng/ml of plasma, with average and median of 20.18 and 10.55ng/ml, respectively. The sizeof the cfDNA/RNA ranged from 105-197bp.
To perform NGS, Oncomine Pan-Cancer Cell-free assay that include 52 genes that covered >900
mutation hotspots and indels, 12 copy number variations (CNV) and 96 fusions, was used. We
selected this panel as it targets many known mutation hotspots across different types of cancer. Thismight improve ctDNA mutation detection in this sample population. In addition, the Oncomine PanCancer assay are amplicon-based panels, in which molecular coverage of amplicons is expected

The Novartis Foundation(Japan)
for the Promotion of Science

19

The Novartis Foundation(Japan)
for the Promotion of Science

to be more uniform across all targeted regions. This amplicon-based NGS technology has also
integrated molecular barcode system that are known to reduce PCR error and could accurately
detects low-frequency variants from 15-20 ng of input cfDNA/RNA. In brief, the samples were
amplified to generate libraries for targeted sequencing. The libraries of the samples will be pooled
for templating on the Ion Chef System and subsequently sequenced using the Ion S5 Prime System.
Variant calling was performed by using Ion Reporter 5.10 system. The median overall and moleculardepth was 48,172 and 4,284, respectively. As a result, out of 26 samples, mutations were detected in
20 patients, with detection rate of 77%. A total of 44 mutations with mutant allele frequency ranged
from 0.066-14.91%. The distribution of the mutations is summarized in the pie-chart below (Figure1). The most frequently mutated genes are TP53, PIK3CA, KRAS, FGFR3, and ESR1. The mutations
detected were dominated by TP53 and PIK3CA mutations. Mutations in both genes amount to 73%
of all mutations detected. Among the 44 mutations, 42 are missense mutations and 2 are frameshiftdeletion. Two CNVs, FGFR1 (2.85 copies) and MET (2.73 copies) were also detected.
Close to half of the patients (11/26 - 42%) carried multiple mutations. Presence of multiple
mutations suggest the possibility of heterogenous clones being detected. In one patient, we detected
6 mutations (2 TP53, 2 PIK3CA, 1 ESR1 and 1 KRAS). This suggests possible polyclonal mutations
being detected.
Previous study indicated that patients with ESR1 mutations detected in the plasma had a markedly
shorter progression-free survival when treated with AIs (Schiavon G et al., 2015). Two mutations,p.E380Q and p.Y537S, on ESR1 gene were detected from cfDNA/RNA. Both of these mutations
are located on the ligand binding domain of ESR1. The detection rate of ESR1 mutations is about
8% (2/26), which is lower compare to previously reported 25-30% owing to the small sample size
and these patients are yet develop progression disease after AI treatment. Hence, monitoring of thesepatients with repetitive sampling is greatly essential to detect drug resistance of AI.

Figure 1: Mutation distribution of 26 metastatic breast cancer
who received aromatase inhibitor treatment.

Discussion & Conclusion
The exploratory study is to evaluate the usefulness of liquid biopsy (blood) as an alternative
biomarker to detect drug resistance in metastatic breast cancer patients who received aromataseinhibitor as first-line treatment. In addition, mutations on other cancer related genes such as TP53,
PIK3CA, KRAS and FGFR3 were detected in this exploratory analysis by using NGS system. Since
the mutation status in ctDNA from plasma is highly concordant with corresponding tumor tissue

The Novartis Foundation(Japan)
for the Promotion of Science

20

The Novartis Foundation(Japan)
for the Promotion of Scienceand the level of ctDNA increase corresponds to the stage of cancer (Bettegowda C et al., 2014),
evaluation of ctDNA could be used an alternative method to detect and monitor disease statusof cancer patient when tissue samples are difficult to access. Importantly, ctDNA has superiorsensitivity compared to conventional biomarkers, such as CA-153, and has a greater dynamic rangethat correlates with changes in tumor burden (Dawson SJ et al., 2013), hence, evaluation of ctDNA
could be a complementary assessment with conventional biomarkers and imaging. The next phase ofthis study is to monitor these cancer patients and detect AI drug resistance at an earlier stage.

References
1. Schiavon G et al., Sci Transl Med. 7(313):313ra182, 2015
2. Bettegowda C et al., Sci Transl Med. 6(224):224ra24, 2014;
3. Dawson SJ et al., N Engl J Med. 368(13):1199-209, 2013;

一般の皆様へこれまで、診断目的のためにがん患者から生検を得る方法として、侵襲的な針による生検が行われることが多かった。しかしながら、この侵襲的な方法は複数回実行することは難しい。また、がんは非常に不均一であり、そして腫瘍のプロファイルは経時的に変化するため、原発腫瘍からの生検は、腫瘍における特定の時点でのゲノムプロファイルのみしか反映されないことになる。
一方で、リキッドバイオプシー、特に ctDNA は、血液、尿、脳脊髄液および唾液から抽出することができる。 ctDNA から得られるゲノムプロファイルは、非侵襲的で迅速かつ複数回入手することができることから、とても重宝されている。

本研究では、先進的である次世代シーケンシング技術を用い、血漿（血液サンプル）からの変異を検出することを目的とした。我々はこの研究から、血漿中に1つ以上の変異を有する20/26

人の進行性乳がん患者を検出した（検出率：77％）。がんに関連する突然変異は全部で44個検出され、このうち ESR1遺伝子の突然変異は薬剤耐性と関連があることが知られている。ctDNA のモニタリングは、病状のモニタリング、薬剤耐性および臨床的再発の早期発見に有用なツールである。

The Novartis Foundation(Japan)
for the Promotion of Science

21

The Novartis Foundation(Japan)
for the Promotion of Science

Investigation of the molecular mechanism of osteoblastogenesis.
Yoshinori Matsumoto
ymatsumoto@okayama-u.ac.jp
Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences

Summary Abstract
The adapter protein 3BP2 is mutated in Cherubism, an autosomal dominant inherited disorderresulting in cranial facial dysmorphia. 3BP2 binds to and activates the non-receptor tyrosine kinase
ABL known from genetic studies to be essential for osteoblast function. However, the molecularmechanism linking 3BP2-ABL to bone synthesis remains unclear. Here we show that nuclear ABL
stabilizes the RUNX2:proteinX master transcription factor complex driving osteoblast differentiation.
Key Words：ABL, Osteoblastogenesis, Tyrosine kinase

Introduction
3BP2 encoded by the SH3BP2 locus was originally identified as an ABL binding protein and binds
directly to the ABL SH3 domain via a diproline motif. 3BP2 triggers increased ABL kinase catalytic
activity required for osteoblast maturation (1). 3BP2 knockout mice are osteoporotic due to defective
osteoblastogenesis (1) and phenocopy ABL-/- mice (2), which demonstrate similar osteoblast
dysfunction suggesting that 3BP2 and ABL are part of a common genetic and biochemical pathway.
However, the molecular mechanism by which ABL regulates bone formation remains unknown.

Results
First, we asked whether ABL links to RUNX2, which is the master transcription factor for
osteoblastogenesis. Previous work, which demonstrated that RUNX2 and proteinX form atranscription factor complex required for BGLAP transcription, prompted us to query whether ABL
could induce BGLAP transcription through the RUNX2-proteinX complex. We observed that a
constitutively active form of ABL (P242E/P249E, ABL (PP)) but not a kinase dead version of ABL(K290M, ABL (KD)) (Barila and Superti-Furga, 1998) increased the RUNX2-proteinX complex
formation. These results corresponded to increase in the BGLAP transcriptional activity of RUNX2
and proteinX in the presence of active ABL. We next queried whether ABL was a direct componentof the RUNX2-proteinX complex and showed by co-precipitation that ABL bound to both RUNX2
and proteinX, either in two binary complexes or as part of a single ternary complex.
Next we asked whether the formation of the RUNX2-proteinX complex was dependent on tyrosine
phosphorylation of RUNX2 by ABL. Wild-type RUNX2 but not all tyrosine to phenylalanine
mutant variants (YF) was abundantly tyrosine phosphorylated when co-expressed with ABL (PP),demonstrating that RUNX2 is a substrate of ABL. We observed that the RUNX2 (YF) mutant poorly
formed a complex with proteinX compared to wild-type RUNX2 and was transcriptionally inactive.
These data demonstrate that phosphorylation of RUNX2 by ABL was absolutely required for theformation of the ternary active transcription factor complex.
Interestingly, we found that ABL stabilized proteinX. This may be because of inhibition ofphosphorylation of proteinX which is required for its protein degradation.

The Novartis Foundation(Japan)
for the Promotion of Science

22

The Novartis Foundation(Japan)
for the Promotion of Science

Lastly, to investigate whether ABL kinase activity was required for enhancing osteoblast
maturation and mineralization, we used an FKBP chimeric form of ABL whose activity is inducibleby the small molecule FK1012. Expression of FKBP-ABL (WT) but not kinase dead version in Saos2 cells showed enhanced mineralization detected by Alizarin red staining.

Discussion & Conclusion
Mesenchymal commitment towards the osteoblast lineage is essential for embryonic skeletal
development. In our present study, we show that ABL is poised as a critical factor needed to “lock-in”
mesenchymal specification towards the osteoblast lineage.
Further studies will be needed to clear the following questions.
1. How is the proteinX stabilized by ABL?
2. Is 3BP2 involved in this ternary complex of ABL, RUNX2 and proteinX?
3. Do proteinX knockout mice suffer from osteoporosis due to osteoblast defect?
4. Is the effect of ABL on osteoblastogenesis contingent on RUNX2 and proteinX?
5. Is the transcriptional complex of RUNX2 and proteinX is dependent on 3BP2?
This project is now ongoing to investigate these questions.

References(1) Levaot, N., Simoncic, P.D., Dimitriou, I.D., Scotter, A., La Rose, J., Ng, A.H., Willett, T.L.,
Wang, C.J., Janmohamed, S., Grynpas, M., et al. (2011). 3BP2-deficient mice are osteoporotic
with impaired osteoblast and osteoclast functions. The Journal of clinical investigation 121, 32443257.
(2) Li, B., Boast, S., de los Santos, K., Schieren, I., Quiroz, M., Teitelbaum, S.L., Tondravi, M.M.,
and Goff, S.P. (2000). Mice deficient in Abl are osteoporotic and have defects in osteoblast
maturation. Nature genetics 24, 304-308.

一般の皆様へ
健康で豊かな長寿社会を実現させる為には、骨粗鬆症の予防、改善が急務です。50歳以上の日本人女性の 3 割が骨折や寝たきりの原因となる骨粗鬆症を発症する為、骨形成、骨吸収を担う骨芽細胞及び破骨細胞を制御する機序の解明は骨粗鬆症の克服に必須の研究テーマですが、未だその詳細は不明です。本研究では、骨形成を担当する

骨芽細胞の新たな制御機構を明らかにしました。更なる研究を積み重ね、骨粗鬆症の治療へと応用出来るよう、今後も精進致します。

The Novartis Foundation(Japan)
for the Promotion of Science

23

The Novartis Foundation(Japan)
for the Promotion of Science

Heart Cell Molecular Atlas Project to dissect the regulatory
mechanisms of circulatory homeostasis
Seitaro Nomura
senomura-cib@umin.ac.jp
The University of Tokyo

Summary Abstract
Pressure overload induces a transition from cardiac hypertrophy to heart failure, but its underlying
mechanisms remain elusive. Here we reconstruct a trajectory of cardiomyocyte remodeling andclarify distinct cardiomyocyte gene programs encoding morphological and functional signaturesin cardiac hypertrophy and failure, by integrating single-cardiomyocyte transcriptome with cellmorphology, epigenomic state and heart function. During early hypertrophy, cardiomyocytes activate
mitochondrial translation/metabolism genes, whose expression is correlated with cell size and linked
to ERK1/2 and NRF1/2 transcriptional networks. Persistent overload leads to a bifurcation into
adaptive and failing cardiomyocytes, and p53 signaling is speciﬁcally activated in late hypertrophy.
Cardiomyocyte-speciﬁc p53 deletion shows that cardiomyocyte remodeling is initiated by p53independent mitochondrial activation and morphological hypertrophy, followed by p53-dependent
mitochondrial inhibition, morphological elongation, and heart failure gene program activation.
Human single-cardiomyocyte analysis validates the conservation of the pathogenic transcriptionalsignatures. Collectively, cardiomyocyte identity is encoded in transcriptional programs thatorchestrate morphological and functional phenotypes.
Key Words：Heart failure, single-cell RNA-seq, cardiomyocyte remodeling

Introduction
Organs respond appropriately to external and internal stress to maintain homeostasis, but
excessive stress disrupts the adaptive response, leading to organ dysfunction. Hemodynamic stimuli
such as pressure and volume overload to the heart initially induce cardiac hypertrophy to reducewall stress and prevent cardiac dysfunction. However, sustained overload causes cardiac dysfunction
leading to heart failure. During this process, cardiomyocytes activate various signaling cascades
initially for adaptive morphological hypertrophy, followed by a transition to the failing phenotype
characterized by elongation and contractile force reduction. Yet, it remains elusive how individualcardiomyocytes undergo molecular and morphological remodeling in response to stress, contributing
to cardiac adaptation and dysfunction.

Results
Single-cardiomyocyte transcriptomic profiles in heart failure
We obtained single-cardiomyocyte transcriptomes from mice exposed to pressure overload. In ourmodel, transverse aorta constriction (TAC) in 8-week-old C57BL/6 mice induced cardiac hypertrophy
at 1–2 weeks after the operation and heart failure at 4–8 weeks. We isolated cardiomyocytes fromthe left ventricular free wall after sham operation and at 3 days and 1, 2, 4, and 8 weeks after TAC
using Langendorff perfusion and obtained 396 single-cardiomyocyte transcriptomes (sham, 64
cardiomyocytes; day 3, 58; week 1, 82; week 2, 61; week 4, 73; and week 8, 58) in which >5,000

The Novartis Foundation(Japan)
for the Promotion of Science

24

The Novartis Foundation(Japan)
for the Promotion of Sciencegenes were detected (RPKM > 0.1). We next used Monocle to reconstruct the branched trajectoryof cardiomyocyte remodeling after pressure overload, which consisted of 3 cell states and 1 branch
point. Cardiomyocytes of the early stage after TAC operation (D3 and W1) mainly belonged to State1, suggesting that cardiomyocytes enter into State 1 immediately after TAC operation and werebifurcated into distinct cell fates (State 2 and State 3) after the branch point. Cardiomyocytes near
the branch point were mainly from mice at 2 or more weeks after TAC operation, suggesting that thebifurcation occurs in the late stage of hypertrophy.
Modules and regulators of cardiomyocyte hypertrophy
To identify the gene modules involved in cardiomyocyte hypertrophy, we again isolated
cardiomyocytes from mice at 1 week after TAC, measured their area, obtained their transcriptomes,
and integrated them. Correlation analysis indicated that the expression of mitochondrial ribosome
and oxidative phosphorylation genes is closely linked to the extent of cell hypertrophy, suggestingthat pressure overload-induced mitochondrial activation might participate in mitochondrial
biogenesis to match the increased energy demand during cardiac hypertrophy. We next performed
chromatin immunoprecipitation followed by sequencing for histone H3 acetylated lysine 27(H3K27ac) modification in cardiomyocytes from mice at 1 week after TAC and evaluated theenrichment of transcription factor recognition motifs to infer the regulatory factors for each module.
Recognition motifs of ETS domain-containing protein (ELK1) and nuclear respiratory factors 1
and 2 (NRF1/2) were specifically enriched around M1 genes. Extracellular signal-regulated protein
kinases 1 and 2 (ERK1/2) signaling induces cardiomyocyte hypertrophy via ELK1 phosphorylation
and NRF1/2 signaling enhances mitochondrial biogenesis.
Signaling pathway inducing heart failure
We investigated how cardiomyocytes in the adaptive hypertrophy stage become failingcardiomyocytes. Pathway enrichment analysis revealed that genes involved in the cell cycle andp53 signaling pathways were enriched in late hypertrophy stage-specific gene modules. To assess
whether p53 activation in cardiomyocytes is critical for the transition to failing cardiomyocytes
and development of heart failure, we generated cardiomyocyte-specific p53 knockout (p53CKO)
mice. Echocardiography demonstrated that p53flox/flox and p53CKO mice showed similar cardiac
hypertrophy at 2 weeks after pressure overload. However, at 4 weeks after pressure overload,
cardiac function was impaired in p53flox/flox, but not in p53CKO mice. Single-cardiomyocytetranscriptome analysis of p53flox/flox and p53CKO mice at 2 weeks after pressure overload showedthat failing cardiomyocytes appeared in p53flox/flox mice, but rarely in p53CKO mice, suggestingthat p53 activation is necessary for the induction of failing cardiomyocytes. Epigenomic profilingof murine cardiomyocytes in the heart failure stage revealed the enrichment of binding motifs ofmyocyte enhancer factor-2 (MEF2) and nuclear factor-erythroid 2-related factor 2 (Nrf2) at active
regulatory elements around p53-regulated genes. We performed western blot analysis of heart tissues
from p53flox/flox and p53CKO mice after sham operation and at 8 weeks after TAC operation (heart
failure stage) to confirm that Nrf2 protein expression was increased after TAC operation in p53flox/
flox mice, which was blocked in p53CKO mice. We also conducted chromatin immunoprecipitation
followed by quantitative PCR of TAC cardiomyocytes to show the significant enrichment of Nrf2
at the Mef2a promoter, validating our hypothesis that the p53-Nrf2-Mef2a axis is essential for the
induction of failing cardiomyocytes.

The Novartis Foundation(Japan)
for the Promotion of Science

25

The Novartis Foundation(Japan)
for the Promotion of Science

Discussion & Conclusion
Having established a system for the comprehensive analysis of multilayer cardiomyocyte
responses to hemodynamic overload in vivo at the single-cell level, we elucidated the trajectoryof cardiomyocyte remodeling in response to pathological stimuli, distinguished gene modules for
cardiomyocyte hypertrophy and failure, and revealed the coordinated molecular and morphologicaldynamics of cardiomyocytes leading to heart failure.
Upon pressure overload, cardiomyocytes activated the mitochondrial ribosome and oxidative
phosphorylation module, the activity of which is correlated with the extent of morphologicalhypertrophy and is linked to the ELK1 and NRF1/2 transcriptional networks. This suggests that
ERK1/2-induced ELK1 phosphorylation and NRF1/2 activation cooperatively regulate mitochondrial
biogenesis to generate more ATP, which is required for enhanced protein synthesis to adapt to theenlargement of cell size and for increased contractility to overcome pressure overload. We also
revealed the association of morphological hypertrophy and the oxidative stress response, supporting
the hypothesis that increased mitochondrial biogenesis is the source of oxidative stress.
Sustained stimuli induce the accumulation of oxidative DNA damage, leading to p53 signaling
activation during hypertrophy. Single-cell analysis of cardiomyocyte-specific knockout miceprovided strong evidence indicating that p53 in cardiomyocytes increases cell-to-cell transcriptional
heterogeneity, induces morphological elongation, and drives pathogenic gene programs by disrupting
the adaptive hypertrophy modules and activating the heart failure module, thereby elucidating how
DNA damage accumulation leads to heart failure. Heterogeneous p53 activation in vivo may berelated to recent live-cell imaging results showing stochastic p53 signaling activation for cell fate
determination in vitro. Our single-cell analysis uncovered a small population of hypertrophy-stage
cardiomyocytes with transient activation of p53 signaling. Heart failure stage-specific cells dominate
in the failing heart, suggesting that p53 signaling might be activated in most cardiomyocytes to
induce heart failure. This p53 signaling-induced cellular dysfunction might be associated withfindings that p53 activation at the G2-M phase is necessary and sufficient for cellular senescence.
The p53-induced heart failure gene program is orchestrated by MEF2 and Nrf2. We showed that
p53 is necessary for Nrf2 activation in the heart failure stage, consistent with the previous findingof p21-mediated Nrf2 protection against Keap1-mediated ubiquitination. Bardoxolone methyl, anantioxidant Nrf2 activator, increases the risk of heart failure, suggesting that the p53-induced geneprogram might cause heart failure.

References
1. Nomura S, Satoh M, Fujita T, Higo T, Sumida T, Ko T, Yamaguchi T, Tobita T, Naito AT, Ito M,
Fujita K, Harada M, Toko H, Kobayashi Y, Ito K, Takimoto E, Akazawa H, Morita H, Aburatani
H, Komuro I. Cardiomyocyte gene programs encoding morphological and functional signatures in
cardiac hypertrophy and failure. Nat Commun. 9: 4435, 2018
2. Satoh M*, Nomura S*, Harada M, Yamaguchi T, Ko T, Sumida T, Toko H, Naito AT, Takeda N,
Tobita T, Fujita T, Ito M, Fujita K, Ishizuka M, Kariya T, Akazawa H, Kobayashi Y, Morita H,
Takimoto E, Aburatani H, Komuro I. High-throughput single-molecule RNA imaging analysis
reveals heterogeneous responses of cardiomyocytes to hemodynamic overload. J Mol Cell
Cardiol. 128: 77-89, 2019 *co-first authors

The Novartis Foundation(Japan)
for the Promotion of Science

26

The Novartis Foundation(Japan)
for the Promotion of Science

一般の皆様へ【背景】

心臓に高血圧などの負荷がかかると心臓は肥大してポンプ機能を維持しようとしますが、負荷が持続すると心機能が低下して心不全を発症します。しかし、これまでその詳細なメカニズムはわかりませんでした。

【目的】

心筋シングルセル解析により心不全発症メカニズムを明らかにする。

【結果】

我々は、シングルセル解析・機械学習により世界で初めて心筋リモデリングの過程における分子

プロファイルの挙動を明らかにしました。その結果、心筋細胞の肥大化にはミトコンドリア生合成の活性化が重要であること、肥大心筋細胞は代償性心筋細胞と不全心筋細胞へと分岐すること、不全心筋細胞への誘導には DNA 損傷（DNA に傷が入ること）とがん抑制遺伝子である p53の活性化が重要であることを明らかにしました。さらに、遺伝子発現パターンから患者の予後・治療応答性を予測できることを実証しました。

【今後の発展性】

明らかとなった分子メカニズムを標的として心不全の新しい治療法の開発が進むと考えられます。また本研究の成果は、あらゆる心臓疾患の病態の解明に役立つだけでなく、個々の心不全患者さんの治療方針を決める上で実際の臨床の現場に応用されることが期待され、これにより循環器疾
患の精密医療（個別化医療）が実現すると考えられます。

The Novartis Foundation(Japan)
for the Promotion of Science

27

The Novartis Foundation(Japan)
for the Promotion of Science

Development of the new organ lack model
Ayako Isotani
isotani@bs.naist.jp
Nara Institute of Science and Technology

Summary Abstract
In this study, to generate an organ-deficient animal model, we attempted to create new methodby using the CRISPR/Cas9 genome editing technique. For this purpose, we established the mouseline that has gRNA that could induce cell death in the presence of the Cas9 protein. In future, it isexpected that new organ-deficient animal models will be produced by modifying them with variousorgan-specific Cas9 proteins.
Key Words：organ formation, inducible cell death, genome editing, chimera

Introduction
Several studies have been performed in regenerative medicine research to solve the problem of
the shortage of organs for transplantation, and specifically, to develop interspecies animal chimeras
to generate organs of interest in an animal body. An organ-deficient animal model is essential toproduce an organ in an interspecies chimeric animal body. Therefore, we attempted to develop amethod for producing new organ-deficient animal models.

Results
We focused on the CRISPR/Cas9 system, a genome editing technique, as a method to generate a
new organ-deficient animal model. In this study, we aimed 1) to identify a target sequence of guidRNA (gRNA) that induces cell death in the presence of the Cas9 protein and 2) to express the Cas9
protein in a specific organ to generate an organ-deficient model.
First, we selected candidates for the target sequence of gRNA and transiently expressed each ofthem with the Cas9 protein by transfection into ES cells to examine whether any of them induced
cell death. Thus, we identified two suitable gRNA sequences.
Second, although gRNA is constitutively expressed, it does not work when the Cas9 protein isabsent. Therefore, the expression sequences of the identified gRNAs were knocked-in the Rosa26
locus of mouse ES cells, and an attempt was made to establish ES cells that constitutively expressed
gRNA. In the recent years, the CRISPR/Cas9 system has often been used to increase the efficiency ofknock-in. However, in the present study, this strategy could not be employed because the expressionof the Cas9 protein resulted in cell death. Therefore, knock-in was performed using a similar
CRISPR/Cpf1 system to establish ES cells with gRNA in the Rosa26 region. To confirm if gRNA
was expressed in the established ES cells and could induce cell death, a Cas9 expression vector wastransfected into the ES cells. As expected, the Cas9-expressing ES cells did not survive (Figure 1).
Subsequently, to establish a genetically-modified mouse line using the established ES cells, theES cells were injected into 8-cell-stage mouse embryos, which were then transferred into the uteriof pseudo-pregnant mice. This enabled us to obtain chimeric pups. Once they reached maturity,
the chimeric mice were mated with the wild-type ones to allow germline transmission and the

The Novartis Foundation(Japan)
for the Promotion of Science28

The Novartis Foundation(Japan)
for the Promotion of Scienceestablishment of a mouse line. Finally, we established amouse strain that expressed gRNA and was capable ofinducing cell death in the presence of the Cas9 protein.
Furthermore, we aimed to perform phenotypic analysisof the chimeric mice. Cas9 cDNA was knocked-in behind
Figure 1
the promoter of a gene expressed in a specific organ, using
gRNA-expressing ES cells gRNA successfully obtained frommice. Unfortunately, we could only obtain ES cells knockedin both the alleles and gene-deficient ES cells with a mutationon the other side, even if knocked-in in just one allele. It is predicted that both types mutant ES celllines had the gene knocked-out phenotype. Therefore, even if the organ-deficient phenotype appearedin the chimera following the use of established ES cells, it was difficult to judge whether it was aphenotype due to a gene defect or the system was functioning. Therefore, we decided to establish amouse line and analyze this in the next generation. As described above, the established ES cells were
injected into the mouse embryos and chimeric mice were obtained. Surprisingly, some chimeras
were found to have the gene knocked-out phenotype, whereas some chimeras had no significant
phenotype. This was dependent on which ES cell lines were used.
We are currently waiting for the growth of more chimeric mice to obtain the next generation.

Discussion & Conclusion
In this study, to generate an organ-deficient animal model, we attempted to induce cell death in an
organ-specific manner using the CRISPR / Cas9 genome editing technique. We determined gRNA
sequences that could induce cell death in the presence of the Cas9 protein. Thereafter, we establishedthat gRNA was constitutively expressed in ES cells and confirmed whether cell death was inducedby the expression of the Cas9 protein in established ES cell lines. In addition, using chimeric mice,mouse lines expressing gRNA were established gRNA.
In future, it is expected that new organ-deficient animal models will be produced by modifying
them with various organ-specific Cas9 proteins.

一般の皆様へ近年、再生医療研究において、動物の体内に臓器を作らせる、異種間キメラ動物が注目されつつあります。異種間キメラ動物体内で臓器を作らせるためには、臓器欠損モデルの作出が欠かせないため、本研究において、新たな臓器欠損モデル動物の作製法の開発を試みました。
我々は、Crispr/Cas9システムに着目し、細胞死誘導する sgRNA を同定し、機能を細胞レベルで確認しました。さらに、この sgRNA を恒常的に発現するマウス系統を樹立しました。今後、Cas9を臓器特異的に発現するマウスと組合せる事で、臓器欠損モデルを作製することができると期待しています。

The Novartis Foundation(Japan)
for the Promotion of Science

29

The Novartis Foundation(Japan)
for the Promotion of Science

Molecular mechanism for the initial assembly of human procentriole.
Satoko Yoshibasayoshiba@mol.f.u-tokyo.ac.jp
Graduate School of Pharmaceutical Sciences, The University of Tokyo

Summary Abstract
The role of the cartwheel structure, a universal template of centriole across species, is further
characterized using a combination of super-resolution microscopy and Crispr/Cas9-mediated auxininducible protein degradation (AID) system in human cells.
Key Words：centriole duplication, cartwheel, SAS-6, AID

Introduction
The centrosome is a major microtubule-organizing center of animal cells and comprises a pair ofcentrioles surrounded by pericentriolar material. The centriole duplicates once in a cell cycle, and its
assembly is strictly regulated with cell cycle progression. At the onset of procentriole formation, astructure called the cartwheel is formed adjacent to pre-existing centriole. SAS-6 proteins are thought
to constitute the hub of the cartwheel structure. In this study, we focused on the roles of humanSAS-6 (HsSAS-6), and tried to elucidate its unveiled functions, using conditional protein knockout
system.

Results
Establishment of HsSAS-6-AID human cell line. The cartwheel/SAS-6 is known to be
essential for the initiation of procentriole formation. However, whether it has an additional role inthe subsequent processes of procentriole formation remains to be elucidated. Therefore, to further
investigate the possible functions of the cartwheel, we generated an HsSAS-6-AID human cell line,
which enabled swifter removal of endogenous HsSAS-6 proteins than the conventional reversegenetic approach using RNAi. We applied a combination of genome editing by CRISPR/Cas9 and the
AID-based protein degradation systems to human HCT116 cells. In the HCT116 cells constitutively
expressing osTIR1, which is an essential component of SCF/E3 ligase for AID-based degradation
with the addition of auxin, both endogenous HsSAS-6 alleles were AID-tagged at their C-terminal
ends. First we tested the degradation properties of HsSAS-6-AID proteins in these cells, and foundthat after addition of auxin, cytoplasmic HsSAS-6-AID proteins were quickly reduced within 1 h,
whereas it took 6 h to completely remove HsSAS-6-AID proteins from the centrioles. Next, we
examined whether centriole duplication occurs when HsSAS-6-AID is degraded before centriole
formation starts in the late G1 phase. HsSAS-6-AID cells were synchronized in the G2/M phase bytreatment with nocodazole, released and then simultaneously treated with auxin until the S phaseor the next G1 phase. The recruitment of HsSAS-6-AID to the centriole was inhibited as a resultof the addition of auxin, and centriole duplication was effectively blocked. These results confirmedthat HsSAS-6 is required for the initiation of centriole formation. HsSAS-6-AID removal from thecentriole in the process of procentriole formation. Next, we sought to analyze whether HsSAS-6
is dispensable after the early stages of procentriole assembly, or alternatively whether it is essential

The Novartis Foundation(Japan)
for the Promotion of Science

30

The Novartis Foundation(Japan)
for the Promotion of Sciencefor further elongation. Given that it normally takes 6 h to remove HsSAS-6-AID proteins from thecentrioles in asynchronous cells, we arrested cells in the early S phase, and then treated them with
auxin to remove HsSAS-6-AID from centrioles for the last 6 h prior to analysis. Unfortunately, we
failed to completely remove HsSAS-6 from centriole, even in the S-phase arrested cells after 6 hof auxin treatment. We speculated this is presumably because continuous loading of HsSAS-6 at
centrioles during the extended S phase. Centriolar loading of HsSAS-6 is known to be dependent on
Plk4 kinase activity. Therefore, to completely remove HsSAS-6 from the centrioles, we combined
auxin treatment with simultaneous transient Plk4 inactivation via the addition of centrinone, a Plk4
specific inhibitor. We found that the addition of auxin and centrinone for 3 h in the early S phase orfor 4 h in mid-S phase, was sufficient to remove HsSAS-6 from the centrioles. We found that in thecells treated with auxin and transiently with centrinone in the early S phase, the number of cells with
duplicated centrioles was dramatically decreased in the next G1 phase (24% duplicated). By contrast,in the cells treated in mid-S phase, the number of duplicated centrioles in the next G1 phase wassignificantly increased (83% duplicated) compared with that in the case of HsSAS-6 removal in early
S phase. These data suggest that cartwheel/HsSAS-6 is necessary for the integrity of procentriole
intermediates at least up to the early S phase, but are thereafter dispensable for subsequent centriole
elongation and maturation. Testing the loading of centriolar proteins through early S phase toG2 phase using super-resolution microscopy. Cep295 is a conserved protein that is not required
for the initiation of cartwheel assembly, but is necessary for the proper elongation and maturationof centrioles through recruitment of Cep135, Cep192, Cep152 and other PCM proteins. Amongthese proteins, we found that Cep135, which is known to be required for well-organized stackingof cartwheel, was frequently observed to start to localize at the procentriole in the mid-S phase,suggesting Cep135 loading may be a key event for stabilizing the procentriole structure.

Discussion & Conclusion
In this study, we characterized the novel function of the cartwheel/HsSAS-6 in centriole formation,
by developing a system for inducible HsSAS-6 degradation in human cells. We demonstrated that
HsSAS-6 is essential not only for the initiation of procentriole formation, but also for the stabilizationof the proximal half of the procentriole. However, it seems that the cartwheel structure is dispensablefor the subsequent steps for full elongation and maturation of centrioles. Such requirement of the
cartwheel structure implies a stepwise procentriole assembly in the following way. Several layersof cartwheel structures that are stacked on the mother centriole wall establish the overall geometryof a procentriole. Thereafter, some critical components are incorporated into the intermediate, andfacilitate arrangement of centriolar triplet microtubules and further procentriole elongation. In ourprevious study, we showed that Cep295 is required for the recruitment of Cep135 to the proentriole.
Together with the findings in this study, it is possible that the acquisition of Cep135 leads to thestabilization of the centriole intermediate, and it would allow further elongation and maturation of
the procentriole. Overall, our results experimentally demonstrated for the first time the importance of
HsSAS-6 until the procentriole structure is stabilized for subsequent maturation.

References
Yoshiba, S., Tsuchiya, Y., Ohta, M., Gupta, A., Shiratsuchi, G., Nozaki, Y., Ashikawa, T.,
Fujiwara, T., Natsume, T., Kanemaki, M., Kitagawa, D. (2019). HsSAS-6-dependent cartwheel
assembly ensures stabilization of centriole intermediates. Journal of Cell Science, in press.

The Novartis Foundation(Japan)
for the Promotion of Science

31

The Novartis Foundation(Japan)
for the Promotion of Science

Ran, F., Hsu, P., Wright, J. and Agarwala, V. (2013). Genome engineering using the CRISPRCas9 system. Nat. Protoc. 8, 2281–2308
Natsume, T., Kiyomitsu, T., Saga, Y. and Kanemaki, M. T. (2016). Rapid Protein Depletion in
Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors. Cell Rep. 15, 210–
218.
Tsuchiya, Y., Yoshiba, S., Gupta, A., Watanabe, K. and Kitagawa, D. (2016). Cep295 is aconserved scaffold protein required for generation of a bona fide mother centriole. Nat. Commun. 7,12567.

一般の皆様へ細胞のなかにある１組の中心体は、細胞が分裂し２つに分かれるときにそれぞれの極となって分裂を正しく導く重要な役割を持っています。中心体の数の増加や機能の異常は、がんの発症やさまざまな病気と密接に関係していることがわかっています。中心体がどのように形成されるのかについて研究することで、細胞のがん化のメカニズムや関連する病気の原因が明らかになり、将来的には治療に役立つことが期待されます。

The Novartis Foundation(Japan)
for the Promotion of Science

32

The Novartis Foundation(Japan)
for the Promotion of Science

Structural basis for inactivation of Na+,K+-ATPase by β-amyloid
Ryuta Kanai
ryuta-kanai@iam.u-tokyo.ac.jp
The University of Tokyo

Summary Abstract
Amylospheroid (ASPD) composed of amyloid β-proteins causes neurodegeneration correlatingwith Alzheimer’s disease and inhibits the ATPase activity of the α3 isoform of Na+,K+-ATPase(NKAα3). In order to elucidate the mechanism of NKA inactivation by ASPD, we performed
crystallographic analysis of NKA, constructed mammalian expression system for NKAα3, andcarried out isothermal calorimetry experiments to determine thermodynamic parameters for theinteraction between NKA and ASPD. (64 wrds)
Key Words：Na+,K+-ATPase, Amyloid β-protein, crystallography

Introduction
Amyloid β-proteins form neurotoxic oligomers correlating with Alzheimer’s disease.
Recently, ASPD consisting of amyloid β-proteins have been reported to target neurospecific
NKAα3 and eventually to cause neurodegeneration (Ohnishi, et al. (2015) PNAS). NKAα3
pumps three Na+ ions out and two K+ ions into the cell per molecule of ATP hydrolysed. Structural
information about NKAα3 and ASPD is necessary for designing a drug to block the interaction
between NKAα3 and ASPD but is not available yet.

Results
NKA consists of three polypeptides; the α- andβ-subunits and a FXYD protein. ASPD is known tobind an extracellular loop L7/8 of the α subunit thatis not the ATPase catalytic site or the transmembrane
ion binding sites. However, as L7/8 appears close toouterleaflet of membrane and the ectodomain of theβ subunit, we thought ASPD binding L7/8 results Fig. 1. ASPD would interfere with a rigid motion of NKA βsubunit necessary to form an unphosphorylated E1 state.
in destabilizing the interaction between the α and β (left) the E2∙P∙2K state of NKA (right) the E1~P∙ADP∙3Na
subunits, or phospholipids. As a precise membrane state inclined toward membrane plane by 18.4˚ like E1∙2Ca of
Ca -pump. Cyan and yellow, α subunit; violet, β subunit; green
position was unknown for NKA, we re-visited the circle, ASPD binding site; orange spheres, phosphorous atoms of
crystal structures of NKA in E1~P∙ADP∙3Na+ and phospholipid membrane. The membrane model is constructed byoverlapping the membrane structure experimentally determined for
E2∙Pi∙2K + reported previously, and built atomic Ca -pump in its equivalent state (E1~P∙ADP∙2Ca ) onto the partial
models for missing parts of NKA and phospholipids phospholipid membrane determined in this study. Please note theM1-2 helices in an unphosphaorylated E1 state would move down
surrounding NKA by re-refining the structure toward the extracellular side to avoid such energetically unfavorable
conformation and to open the cytoplasmic gate.
including more low resolution reflections.
We have succeeded in identifying 12-13 phospholipids (PLs) and a new glycosylation site ofthe β-subunit as well as extending N-terminal cytoplasmic region of the β-subunit. These partial
membrane models in two states enabled to predict a membrane position in the crystals and raised a
+

+

2+

2+

2+

2+

The Novartis Foundation(Japan)
for the Promotion of Science

33

The Novartis Foundation(Japan)
for the Promotion of Science

possibility that the ectodomain of the β subunit in an unphosphorylated E1 state rigidly moves close
to outerleaflet membrane and the closed extracellular gate composed of M1-4 helices of the α subunit(Fig. 1b). Such rigid motion of the β subunit indicates that the ectodomain contributes to close theextracellular gate by interacting with M1-2 helices and/or outerleaflet membrane. Therefore, ASPD
binding to L7/8 may interfere with these interactions of the β subunit and the extracellular gate and/or outerleaflet membrane by blocking such movement of the β subunit.
For further structural studies for an inhibition mechanism of NKAα3β1 by ASPD, we areestablishing mammalian expression system for human NKAα3β1 which atomic structure has notbeen determined yet. Here, we constructed a recombinant adenovirus to express human NKAα3β1,
and tested several cell lines for optimal virus infection and NKA expression. A549 and AD293
cells showed better expression than the others. ATPase assay of the purified microsome including
NKA showed that NKA from AD293 is active but not from A549. As AD293 cells are not suitablefor protein production using the recombinant adenovirus, we are trying constructing a recombinantbaculovirus to express NKA α3β1 in AD293 cells.
In addition, we are trying to determine thermodynamic parameters of the interaction between
NKA and ASPD by using isothermal calorimetry (ITC). As ASPD is too large to stably besolubilized, truncated amyloid β-protein (Aβ1-42), which forms oligomers like ASPD, was usedin this experiment. As an initial attempt, we titrated the purified membrane fraction containing
NKA with Aβ1-42 solubilized in DMSO. However, the titration curves were very unstable because
thermodynamic effect from DMSO appeared much larger than that of Aβ1-42 even when carefullymeasured the background using DMSO. Next, to minimize such noise effect, DMSO was added tothe membrane fraction as same concentration as in the Aβ1-42 solution. This adjusting backgrounddramatically improved the baseline but the signal occurred by titration was too weak to determine
reliable Kd. Because, a thermal change caused in association of Aβ1-42 with NKA may be verysmall or Aβ1-42 used in this experiment may be inactive because of a solubilization problem. In
fact, preparation of Aβ1-42 solution needs special cares like using DMSO with the highest grade,
low humidity when solubilizing, and immediate use. Currently, we are carefully analyzing thesolubilization condition of Aβ1-42.

Discussion & Conclusion
In this study, we re-constructed crystal structures of NKA in E1~P∙ADP∙3Na+ and E2∙Pi∙2K+ topredict inhibition mechanism of NKA by ASPD and suggested that ASPD appears to interfere withinteractions of the β subunit with M1-2 helices and/or outerleaflet membrane.
For further structural study, we are establishing mammalian expression system for human NKAα3and β1 and found that AD293, a substrain of HEK293T cells, is the best cell line for expression andactivity of NKA.
We are performing ITC experiments for biochemistry of the interaction between NKA and ASPD
but got some technical problems. We are trying to solve them but also planning of other biochemical
tools like surface Plasmon resonance.

The Novartis Foundation(Japan)
for the Promotion of Science

34

The Novartis Foundation(Japan)
for the Promotion of Science

References
Ohnishi, et al. (2015) PNAS, 112, E4466-E4474. Na, K-ATPase α3 is a death target of Alzheimer
patient amyloid-β assembly.

一般の皆様へアルツハイマー病は脳内でアミロイドβの凝集・蓄積により発症します。近年、アミロスフェ

ロイド (ASPD) と呼ばれる、アミロイドβの特定の凝集体が NKA α3と呼ばれる神経細胞に特異的な膜蛋白質に結合し、強い神経毒性を示すことが分かってきました。そこで、私達は将来的にアルツハイマー病の治療薬創出を目指して、ASPD がどのようにして NKA α3に結合し、その機能を破壊するのかを研究しました。その結果、ASPD は NKA α3が機能する際に生じる構造変化を阻害しているらしいことが分かりました。実際の創薬にはまだ遠いですが、これからも研究を続けていきたいと思います。

The Novartis Foundation(Japan)
for the Promotion of Science

35

The Novartis Foundation(Japan)
for the Promotion of Science

Evolutionary development of fluorescent RNA sensors
by droplet technology
Shigeyoshi Matsumura
smatsumu@sci.u-toyama.ac.jp
University of Toyama

Summary Abstract
We have established the directed evolution system for a fluorescent RNA using droplet
microfluidics. Based on the developed technology, we are currently running the directed evolution
experiments of the RNA. This effective method to engineer the fluorescent RNAs would lead muchbroader applications for life science.
Key Words：Fluorescent RNA, directed evolution, droplet microfluidics

Introduction
Recently a fluorescent RNA called “Spinach” was developed, binding with a fluorophore DFHBI
to activate its GFP-like green fluorescence 1). This enables any cellular RNAs to be labelled andvisualized in vitro and in vivo. An effective method to engineer such fluorescent RNAs is desiredfor much broader applications. Our objective is establishment of such methodology by combining
directed evolution strategy and droplet microfluidic technology.

Results
Droplet microfluidics is a technology which provides precise handling of tiny water droplets(emulsions) on microfluidic chips 2). To evolve fluorescent RNAs, this technique is essential toencapsulate and evaluate each species of the RNA library. We have developed two microfluidic
devices for encapsulation and sorting of the fluorescent RNAs. The one is for making droplets whose
size is around 1 pL, which is 10 times smaller than that of the droplets prepared with a previous
device. The other is for analyzing and sorting the 1 pL droplets. The new design of this device
provides low false positive (miss-sorting), achieving higher throughput of the sorting (2000 events /sec.).
Next, a single-molecule fluorescent RNA needs to be encapsulated into one droplet, and be
detected. But it is impossible since the RNA concentration under such condition (1 molecule /droplet) is too low. To enhance the fluorescent signal in droplets, we employed an RNA amplification
technique called Nucleic Acid Sequence-Based Amplification (NASBA). In this way, transcription
from DNA to RNA and reverse transcription from RNA to DNA proceed simultaneously in solution,
enabling exponential amplification of RNA under isothermal condition 3). The first test of the
NASBA with the fluorescent RNA showed amplification, but its efficiency was quite low when the
RNA template concentration is less than 10 pM. Improvement of the reaction condition including
buffer compositions increased the yield at low template concentration. It was also verified that thefluorescent RNA is functional under the NASBA conditions.
We subsequently introduced the reaction above into the tiny droplets. The template, the NASBA
enzymes and components, and the fluorophore DFHBI were compartmentalized into the 1 pL

The Novartis Foundation(Japan)
for the Promotion of Science

36

The Novartis Foundation(Japan)
for the Promotion of Science

droplets, and then incubated at 37 °C. After incubation, the droplets were re-injected into thesorting device, and then the individual fluorescence was analyzed on the chip. We have succeeded
in detecting the green fluorescence originating from the fluorescent RNA, indicating that theamplification of the functional fluorescent RNA works nicely even in the tiny droplets, not only inthe bulk solution.
To proceed into next evolution experiments, we constructed an initial library of the fluorescentRNA. Point mutations were introduced into the RNA sequence by mutagenic PCR. Furthermore, therandomness of the resulting library was analyzed and evaluated by next-generation sequencing. The
data indicates that the mutations are uniformly introduced into the entire region of the fluorescent
RNA sequence, which is likely suitable as a library for directed evolution experiment.

Discussion & Conclusion
As described above, we have established the directed evolution system for the fluorescent RNA
using droplet microfluidics. We already achieved: (1) new microfluidic system and devices, (2)exponential amplification method for the fluorescent RNA, (3) the same reaction in droplets, (4)initial library construction by mutagenic PCR. Based on the above, we are currently running theevolution experiments of the RNA. If the enrichment of the active species is observed, we are goingto analyze the enriched pool of RNA by next-generation sequencing (Illumina MiSeq), and thenisolate the improved (evolved) variants. The NGS analysis would reveal the evolutionary dynamics
of the RNA during the selection, which should be helpful to understand what kind of selection
conditions are useful or not for successful evolution.

References
1) Paige, J. S. et al., RNA mimics of green fluorescent protein. Science, 333, 642-646 (2011)
2) Matsumura, S. et al., Transient compartmentalization of RNA replicators prevents extinction due
to parasites. Science, 354, 1293-1296 (2016)
3) Compton, J., Nucleic acid sequence-based amplification. Nature, 350, 91-92 (1991)

一般の皆様へ近年、蛍光を発する人工 RNA の開発が急速に進んでおり、細胞内の任意の RNA の蛍光標

識および可視化、細胞内 RNA 動態のリアルタイム解析などへの応用が期待されている。本研

究は、これらの蛍光 RNA を細胞サイズのマイクロドロップレットへ封入し、人為的に「進化」させることで、望みの特性をもつ蛍光 RNA を自在に創製する方法論を確立し、ライフサイエ
ンスにおける RNA イメージングなどへの貢献を目指すものである。

The Novartis Foundation(Japan)
for the Promotion of Science

37

The Novartis Foundation(Japan)
for the Promotion of Science

Functional Analysis of Chk1 Targets for Breast Cancer Treatment
Midori Shimada
shimada@yamaguchi-u.ac.jp
Yamaguchi University

Summary Abstract
Triple-negative breast cancer (TNBC) is associated with poor prognosis and a high risk of relapse.
A previous report showed that the calcineurin/NFATc pathway was activated in TNBC cells, andhas an essential role in the tumorigenic and metastatic properties. The effect of FK506 has not been
totally analyzed in TNBC cells. In this study, we demonstrate that expression of Chk1 was reduced
and G1/S progression, but not G2/M, was inhibited in TNBC cells by FK506 treatment.
Key Words：Cell cycle, triple negative breast cancer, calcineurin

Introduction
FK506 (tacrolimus) is a major immunosuppressant agent widely used for the prevention of graft
rejection in organ transplantation and the treatment of autoimmune diseases [1]. FK506 inhibits
calcineurin protein phosphatase by binding to various FK506 binding proteins (FKBPs) [2].
Calcineurin activates the nuclear factor of activated T cell (NFATc) transcription factors, which
induces T cell activation via expression of cytokines [3]. It has become increasingly apparent
that two other macrolide immunosuppressants, cyclosporine A and rapamycin, as well as FK506,
induce apoptosis in a wide variety of cancer cells [4-6]. Conversely, one study shows that a low
concentration of FK506 promotes proliferation in vascular smooth muscle cells. Owing to theirantiproliferative effects, these immunosuppressant agents are expected to provide new potential
therapeutic approaches for cancer treatment. A recent study demonstrated that cyclosporine A andrapamycin decreased mRNA expression of cyclin D1 and showed a defect in G1/S progression [7].
However, it is largely unknown whether FK506 has any effect on cell cycle progression.

Results
Given that the CN/NFATc pathway is activated in the ER-PR-HER2-triple-negative breast
cancer (TNBC) subgroup [8], we investigated the effect of a CN/NFATc inhibitor, FK506, on cellproliferation in the TNBC, Hs578T, and MDA-MB-231 cell lines. FK506 (50 μM) efficiently
inhibited TNBC cell growth, which was consistent with a previous report showing that FK506
induced apoptosis in the Jurkat human T cell line and other non-T cell lines [4-6]. In contrast,
treatment of TNBC cell lines with FK506 (10 μM) did not produce growth defects. At 24 h after
treatment with FK506 (50 μM), cells became rounded and detached from culture dishes. FACS
analysis revealed that FK506 (50 μM) did not increase mitotic cells but increased the populations of
G1 phase cells and the sub-G1 fraction. A low dose of FK506 (10 μM) did not change the cell cycle
profile. To confirm that 50 μM FK506 stimulates apoptosis, we monitored two apoptotic markers,
the cleaved form of caspase 3 and PARP1. We found that both caspase 3 and PARP1 were efficiently
cleaved in the FK506-treated cells, suggesting that FK506 induced apoptosis in TNBC cells. Our
results showed that FK506 induced G1/S arrest and apoptosis at a concentration of 50 μM.

The Novartis Foundation(Japan)
for the Promotion of Science

38

The Novartis Foundation(Japan)
for the Promotion of Science

The data above indicate that FK506 affected G1/S cell cycle progression. Next, we synchronizedcells at the G1/S boundary using a double thymidine block and examined the effect of FK506. We
added FK506 at 2 h just before the release from the thymidine block and the cells were releasedin the presence of FK506. FACS analysis revealed that cells treated with FK506 progressed to
S phase more slowly than the control cells. At 8 h after release, about 70 % of the control cells
entered into the G2/M phase, whereas most of the cells treated with FK506 were still in the G1/S
or middle S phase, and less than 30 % of the cells showed progression into the G2/M phase. These
results indicate that FK506 induces a delay in G1/S progression. Progression from the G1 to the Sphase, and the S phase of the cell cycle is regulated by cyclins D and E, which are essential for theactivation of cyclin-dependent kinases. A previous report showed that cyclin A2 is important forthe continuation of the S phase, B1 for the G2/M phase, E for the S phase initiation, and D1 mainly
for G1/S progression [9,10]. Next, we investigated the expression of these key cyclins. Treatmentof cells with FK506 led to decreased cyclin D1 expression, whereas FK506 had no effect on
cyclin E. Decreased expression of cyclin A2 and B1 by FK506 may be due to the altered cell cycle
populations, because they were not changed in synchronized cells at G2. In addition, expression of
Chk1 was reduced in FK506 treated cells.
Next we investigated whether FK506 post-translationally regulated cyclin D1. Inhibition ofproteasome activity with MG132 reversed the FK506-induced reduction in cyclin D1, suggestingthat FK506 induces the proteasomal degradation of cyclin D1. To investigate other possible effectsof FK506, with potential influence on cyclin D1 mRNA expression, we performed quantitative realtime PCR analysis and found that cyclin D1 mRNA expression was not affected by treatment with
FK506 or FK506 combined with MG132. Taken together, FK506 facilitates proteasomal degradationof cyclin D1, which may lead to delayed G1/S progression.

Discussion & Conclusion
In summary, we found that calcineurin inhibition promoted cyclin D1 degradation by impairing
dephosphorylation, resulting in G1/S delay. It is worthy to note that overexpression of cyclin D1
is detected in more than 50% of human invasive breast cancers 32, and this may be partly due toderegulated proteasomal degradation. In TNBC as well, overexpression of cyclin D1 is relates
to poor prognosis 33. In addition, it is reported that the calcineurin/NFAT pathway is frequently
activated in TNBC subgroups, and has an essential role in the tumorigenic and metastatic properties
4. Similarly, mutation on T286 has been reported in esophageal and endometrial cancers 34. We
therefore propose that therapeutic targeting of cyclin D1 could be useful for the prevention andtreatment of cancers, such as TNBC, where cyclin D1 is overexpressed.

References[1] J.R. Azzi, M.H. Sayegh, S.G. Mallat, Calcineurin inhibitors: 40 years later, can't live without, J
Immunol, 191 (2013) 5785-5791.
[2] M.W. Harding, A. Galat, D.E. Uehling, S.L. Schreiber, A receptor for the immunosuppressant
FK506 is a cis-trans peptidyl-prolyl isomerase, Nature, 341 (1989) 758-760.
[3] G.P. Mognol, F.R. Carneiro, B.K. Robbs, D.V. Faget, J.P. Viola, Cell cycle and apoptosis
regulation by NFAT transcription factors: new roles for an old player, Cell Death Dis, 7 (2016)
e2199.

The Novartis Foundation(Japan)
for the Promotion of Science

39

The Novartis Foundation(Japan)
for the Promotion of Science[4] S.J. Choi, H.S. You, S.Y. Chung, Tacrolimus-induced apoptotic signal transduction pathway,
Transplantation proceedings, 40 (2008) 2734-2736.
[5] Y. Hashimoto, N. Matsuoka, A. Kawakami, M. Tsuboi, T. Nakashima, K. Eguchi, T. Tomioka, T.
Kanematsu, Novel immunosuppressive effect of FK506 by augmentation of T cell apoptosis, Clin
Exp Immunol, 125 (2001) 19-24.
[6] K. Migita, K. Eguchi, Y. Kawabe, T. Tsukada, A. Mizokami, S. Nagataki, FK506 augments
activation-induced programmed cell death of T lymphocytes in vivo, J Clin Invest, 96 (1995)
727-732.
[7] M. Weischer, M. Rocken, M. Berneburg, Calcineurin inhibitors and rapamycin: cancer protection
or promotion?, Experimental dermatology, 16 (2007) 385-393.
[8] C.T. Quang, S. Leboucher, D. Passaro, L. Fuhrmann, M. Nourieh, A. Vincent-Salomon, J.
Ghysdael, The calcineurin/NFAT pathway is activated in diagnostic breast cancer cases and isessential to survival and metastasis of mammary cancer cells, Cell Death Dis, 6 (2015) e1658.
[9] E.A. Musgrove, C.E. Caldon, J. Barraclough, A. Stone, R.L. Sutherland, Cyclin D as atherapeutic target in cancer, Nat Rev Cancer, 11 (2011) 558-572.
[10] P. Hydbring, M. Malumbres, P. Sicinski, Non-canonical functions of cell cycle cyclins andcyclin-dependent kinases, Nat Rev Mol Cell Biol, 17 (2016) 280-292.

一般の皆様へ
乳癌は日本人女性が罹患する悪性腫瘍の第１位であり、その罹患数ならびに死亡数は増加の一途を辿っています。ホルモン療法および抗 HER2 療法では効果がないトリプルネガティブ乳癌に対しては治療法は限定されており、予後は極めて不良であることから、有効な分子標的薬の創出が緊急に迫られています。本研究では、トリプルネガティブ乳癌で高発現している

Calcineurin 脱リン酸化酵素を阻害する FK506が乳癌の増殖を抑制できることがわかりました。

The Novartis Foundation(Japan)
for the Promotion of Science

40

The Novartis Foundation(Japan)
for the Promotion of Science

Revealing a new red light signaling pathway for resetting
the circadian clock in green algae
Takuya Matsuo
t-matsuo@gene.nagoya-u.ac.jp
Center for Gene Research, Nagoya University

Summary Abstract
In this study, a gene involved in a light response mechanism of circadian clock in the green alga
Chlamydomonas reinhardtii has been identified. This gene provides a clue to understand an unknown
light response mechanism in algae.
Key Words：alga, light response, circadian clock

Introduction
Most organisms are characterized by an endogenous clock that generates a ~ 24-hour rhythmof various processes from sleep/wake cycles in mammals, leaf movement in plants to phototaxis
and chemotaxis in algae. This rhythm can be reset by signals such as light and temperature. In caseof light resetting in C. reinhardtii, the light induced degradation of ROC15, a DNA binding clock
protein, was identified as a crucial event. Here, by a forward genetics approach, we identified a green
algae specific gene that plays a crucial role in the light induced degradation of ROC15.

Results

1. Screening for mutants: For the isolation of light response mutants, the ROC15-luciferase
bioluminescence levels of the insertional mutants were monitored in response to red and blue
light. From approximately 5000 mutants monitored, two mutants (M1 and M2) showing abnormal
bioluminescence phenotypes were isolated. Both failed to show acute decrease in ROC15luciferase bioluminescence levels in response to red and blue light, unlike in the WT, whichshows an acute decrease in ROC15-luciferase bioluminescence level in response to both red andblue light. Therefore, M1 and M2 were selected as light response mutants and were pursued.
2. Genetic linkage Analysis: To confirm the linkage of hygromycin resistance and abnormal
response to light, M1 and M2 were backcrossed with WT strain of opposite mating type. In caseof M1 backcross, almost all progenies that were hygromycin resistant did not show an acute

The Novartis Foundation(Japan)
for the Promotion of Science

41

The Novartis Foundation(Japan)
for the Promotion of Sciencedecrease in bioluminescence after red light exposure. On the other hand, in case of M2 backcross,not all individuals that were hygromycin resistant had a decreased bioluminescence level in
response to red light. In only M1, the cause of the abnormal bioluminescence response could bedirectly attributed to the insertion of the hygromycin resistance gene. The mutant in which almost
all progeny were both hygromycin resistant and showed abnormal response to light in other words
failed to show acute decrease in ROC15-luciferase bioluminescence levels in response to light
were pursued. Hence, only M1 was pursued.
3. Identification of disrupted gene: In order to identify the insertion locus, TAIL-PCR andsequencing analysis was performed on M1. The secondary and tertiary downstream PCR products
were used for sequencing. The resulting sequence was searched against the genome database
and was found in the gene Cre07.g357500 on chromosome 7. This indicated that the insertion
occurred in the gene Cre07.g357500 on chromosome 7 of the Chlamydomonas genome. To
confirm the result of the TAIL- PCR, genomic PCR was performed around the insertion locus.
The mutant band was heavier than the WT band by 1.7kb, thus confirming the result of TAILPCR.
4. Complementation with Cre07.g357500 WT allele: To clarify that Cre07.g357400 was thegene responsible for the abnormal bioluminescence response, M1 was transformed with WT
Cre07.g357500 gene, and with only spectinomycin cassette were used as controls. ROC15luciferase bioluminescence levels of 660 transformants (244 controls and 416 transformed with
WT gene) were measured in response to red light and blue light. As expected, none of the 244
control were complemented, in other words, they still showed abnormal response to light, while
6 of 416 colonies transformed with WT gene were complemented, in other words, the ROC15bioluminescence levels in response to both red and blue light were restored to WT levels. These
results indicate that the Cre07.g357500 gene was responsible for the abnormal bioluminescence
response.
5. Protein Sequence analysis: Cre07.g357500 was found to code for a protein of 2386 amino acids.
The functional annotation tool in Phytozome 12.1 however did not reveal any known functional
motifs. Conserved domains were also searched for (against all databases) (CD search service,
NCBI) though no domain of significant similarity was obtained. On the other hand, a homology
search (BLASTp, NCBI) revealed two possible homologs, XP_002956981.1 (Volvox carteri) and
KXZ53772.1 (Gonium pectorale). The Cre07.g357500 peptide sequence was then aligned withthe peptide sequences of the homologs (Clustal Omega, EMBL-EBI). From the alignment, theprotein appears to have conserved regions in green algae.

Discussion & Conclusion
Cre07.g357500 which codes for a protein of 2298 amino acids was found to be disrupted as a resultof insertional mutagenesis in the mutant M1. The disruption of this gene appears to affect the acutelight induced degradation of ROC15. By analyzing the phosphorylation of ROC15 and interactionof this protein with other identified components of the light signaling pathway for resetting thecircadian clock, the significance of this protein in the pathway can be further elucidated.

The Novartis Foundation(Japan)
for the Promotion of Science

42

The Novartis Foundation(Japan)
for the Promotion of Science

References
1. Kondo T, Johnson CH, Hastings JW .1991. Action spectrum for resetting the circadian phototaxis
rhythm in the cw15 strain of Chlamydomonas. I. Cells in darkness. Plant Physiol 95: 197–205.
2. Niwa Y, Matsuo T, Onai K, Kato D, Tachikawa M, Ishiura M. 2013. Phase-resetting mechanismof the circadian clock in Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A.;110: 13666–71.
3. Kinoshita A, Niwa Y, Onai K, Yamano T, Fukuzawa H, Ishiura M, Matsuo T. 2017. CSL encodes
a leucine-rich-repeat protein implicated in red/violet light signaling to the circadian clock in
Chlamydomonas. PLoS Genetics, 13(3), e1006645-e1006645.

一般の皆様へ
私はクラミドモナスという緑藻の一種を使って体内時計を研究しています。私たち人間と同様、緑藻の体内時計も光によってリセットされます。しかしその分子メカニズムはよく解っていませんでした。私はこの研究により、その未知のメカニズムに関わる遺伝子を明らかにしました。その遺伝子の解析から、緑藻は他の生物には見られない独特な光応答メカニズムを持つことが見えてきました。この研究成果は、生物の光応答メカニズムの進化の理解につながると考えています。

The Novartis Foundation(Japan)
for the Promotion of Science

43

The Novartis Foundation(Japan)
for the Promotion of Science

Elucidation of the role of fatty acid elongase Elovl6in the pancreatic β-cell
Takashi Matsuzaka
t-matsuz@md.tsukuba.ac.jp
Faculty of Medicine, University of Tsukuba

Summary Abstract
To investigate the role of Elovl6 in pancreatic β-cell function and T2D, we generated and analyzed
pancreatic β-cell specific Elovl6 deficient mice. We found that β-cell specific Elovl6 deficiency
significantly improved hyperglycemia and increased plasma insulin concentration in diabetic db/db
mice.
Key Words：Fatty acid composition, Type 2 diabetes, Conditional knockout mouse

Introduction
Elovl6 is a microsomal enzyme that elongates saturated- and monounsaturated-fatty acids (FAs)with 12, 14, and 16 carbons to FAs with 18 carbons such as stearic acid (C18:0), vaccenic acid
(C18:1n-7), and oleic acid (C18:1n-9). Loss of Elovl6 function reduces stearate and oleate levels but
increases palmitate, palmitoleate and vaccinate levels. We assessed the effects of Elovl6 deletion in
leptin receptor-deficient db/db mice, a model of type 2 diabetes (T2D) and reported that db/db;Elovl6
KO mice showed improved glycemic control, an adaptive increase in insulin, and had a markedly
increased β-cell mass. To further investigate the role of Elovl6 in pancreatic β-cell function and T2D,
we generated pancreatic β-cell specific Elovl6 deficient mice and analyzed its effects in this study.

Results
Elovl6 lox/lox (Flox) mice were crossed to an insulin-1 promoter-Cre transgenic mice, which
express Cre exclusively in β cells, to obtain Elovl6 lox/+;Cre+ mice. Subsequently, these mice werecrossed to Flox mice to yield Elovl6 lox/lox;Cre+ (βE6KO) mice. The genotype of mice harboring
the null allele was confirmed by PCR using tail genomic DNA. To confirm the deletion of Elovl6 in
βE6KO mice, we analyzed mRNA levels of Elovl6 in various tissues by qPCR. The result indicatedthat the Elovl6 gene deletion was restricted to the pancreatic islet in βE6KO mice.
Oral glucose tolerance testing of a chow-fed βE6KO and Flox mice indicated that plasma glucose
levels of Flox and βE6KO mice after overnight fasting or an oral glucose gavage was similar.
Pancreatic islets isolated from βE6KO mice were morphologically compared with those from Flox
mice. The number, size, and morphology of isolated islets were similar between chow-fed Floxand βE6KO mice. Isolated islets were tested for insulin secretion. Basal (low glucose) and glucosestimulated (high glucose) insulin secretion was almost similar between Flox and βE6KO islets.
Insulin content of βE6KO islets was also indistinguishable from that of Flox islets.
To investigate the role of β-cell Elovl6 in T2D development and progression, we next generated
db/db;βE6KO mice. db/db and db/db;βE6KO mice both gradually gained body weight (BW) without
any significant differences. Although obesity in db/db mice was not improved, Elovl6 deficiency in
β-cell significantly improved hyperglycemia, increased plasma insulin concentration, and reduced

The Novartis Foundation(Japan)
for the Promotion of Science

44

The Novartis Foundation(Japan)
for the Promotion of Science

HbA1c levels. Immunostaining ofpancreatic sections from 16-week-old
animals with antibodies to insulin andglucagon revealed that β-cell Elovl6
deficiency in db/db mice resulted in asignificant increase in β-cell mass.

Discussion & Conclusion
Current results indicated that Elovl6 deficiency in β cells did not affect the development of βcells and capacity of the insulin production and secretion in β cells under the normal chow diet fed
condition. These results are consistent with the phenotype of Elovl6 global knockout mice.
In contrast, we demonstrated that β-cell-specific Elovl6 deletion in db/db mice significantly
reduced circulating blood glucose levels without affecting obesity. This result suggests that Elovl6mediated modulation of FA composition in β cells was essential in preserving proper β-cell function
and preventing the development of T2D in obesity.

References
Zhao H, Matsuzaka T, Nakano Y, Motomura K, Tang N, Yokoo T, Okajima Y, Han SI, Takeuchi
Y, Aita Y, Iwasaki H, Yatoh S, Suzuki H, Sekiya M, Yahagi N, Nakagawa Y, Sone H, Yamada N,
Shimano H. Elovl6 Deficiency Improves Glycemic Control in Diabetic db/db Mice by Expanding
β-Cell Mass and Increasing Insulin Secretory Capacity. Diabetes. 2017 Jul;66(7):1833-1846. doi:
10.2337/db16-1277.

一般の皆様へ
本研究では、パルミチン酸（C16:0）からステアリン酸（C18:0）への伸長を触媒する酵素

Elovl6に着目し、2型糖尿病モデル db/db マウスの膵β細胞でこの酵素を欠損させると、2型糖尿病の発症・進展が抑制される可能性があることが示唆されました。本研究をさらに進めることにより、膵β細胞における Elovl6の阻害や脂肪酸バランスの管理が、2型糖尿病の新規治療法となることが期待されます。

The Novartis Foundation(Japan)
for the Promotion of Science

45

The Novartis Foundation(Japan)
for the Promotion of Science

Development of regenerative therapy and novel therapeutic drugs
for renal anemia using human iPS
cell-derived erythropoietin-producing cells
Kenji Osafune
osafu@cira.kyoto-u.ac.jp
iPS Cell Research and Application (CiRA), Kyoto University

Summary Abstract
In this study, we identified one small molecule that increase the mRNA expression and protein
secretion of erythropoietin (EPO) from human iPS cell-derived EPO-producing cells (hiPSC-EPO
cells). This compound may contribute to the development of cell therapy and new therapeutic drugs
for renal anemia.
Key Words：iPS cell, erythropoietin, EPO, small molecule, screening

Introduction
An increasing number of patients with end-stage renal failure (ESRF) start dialysis therapy, whichcause medical and economical problems. Renal anemia in ESRF have been treated with recombinanthuman erythropoietin (rhEPO) agents. However, physiological control of renal anemia is still
difficult. To overcome the problem, we previously developed the differentiation method for hiPSCEPO cells and demonstrated that cell therapy using hiPSC-EPO cells ameliorates renal anemia inmice (Hitomi H. et al., 2017). In this study, we aimed to identify small molecules that facilitate
the production of EPO from hiPSC-EPO cells towards the development of cell therapy and drug
discovery against renal anemia.

Results(1) Screening of chemical compounds that increase EPO secretion from hiPSC-EPO cells.
We had developed the directed differentiation method to induce hiPSCs into EPOproducing cells by modifying the previously reported hepatocyte differentiation
protocols (Hitomi H. et al., 2017). In our differentiation method for EPO cells, hiPSCs
were first induced to differentiate into definitive endoderm cells expressing SOX17 and
HOXA2 by the combination treatment with activin A and a glycogen synthase kinase(GSK)3 inhibitor, CHIR99021, and then treated with dimethyl sulfoxide (DMSO) togenerate AFP(+) hepatoblasts. We found that the hepatoblasts also expressed EPO andthat the prolonged treatment with DMSO more efficiently generates hiPSC-EPO cells.
We then set up the screening system for chemical compounds, in which AFP(+)EPO(+)
hepatoblasts differentiated from an hiPSC line, 585A1, were split into 96-well plates andindividual compounds were added to different wells. The effects of individual compounds
were analyzed by enzyme-linked immunosorbent assay (ELISA) for EPO protein in the culture
supernatants. By using the unbiased screening system for chemical compounds, we haveidentified one hit compound that increase the secretion of EPO protein from hiPSC-EPO cells.

The Novartis Foundation(Japan)
for the Promotion of Science

46

The Novartis Foundation(Japan)
for the Promotion of Science(2) Evaluation of hit compound.
We next confirmed the reproducibility of effects of the hit compound in 24-well platesettings that increased the EPO mRNA expression and protein secretion from hiPSC-EPO
cells and found that the hit compound increases the EPO mRNA expression and protein
secretion in a concentration dependent manner. We also optimized the treatment protocol
using the hit compound to maximize the EPO expression and secretion from hiPSC-EPO cells.
The main regulators of EPO expression in our body are known to be hypoxia, hypoxia inducible
factors (HIFs) and prolyl hydroxylase domain-containing protein (PHD). We thus examined
whether the hit compound increases the secretion of EPO synergistically with hypoxia andconfirmed that the compound additionally increases the EPO production from hiPSC-EPO cells
under hypoxic conditions.
We had previously established the renal anemia mouse model induced by oral additionof adenine (Hitomi H. et al., 2017). We are currently examining the in vivo effects of the hit
compound by intraperitoneally injecting the hit compound to the renal anemia mice.
(3) Elucidation of the mechanisms of action of hit compound.
We purchased several analogs of the hit compound and examined the effects of the analogs on
the EPO expression and secretion from hiPSC-EPO cells. The results showed that the analogs
also increased the EPO expression and secretion, which help providing the information of target
molecules of the hit compound.
In an attempt to elucidate the mechanisms of action by which the hit compound increases the
EPO production from hiPSC-EPO cells, we performed RNA sequencing analyses and compared
the expression profiles between hiPSC-EPO cells treated with or without the hit compound.
Consequently, we identified several molecules whose expression levels are upregulated by the hit
compound. We are currently examining the knockdown experiments for the molecules by usingsiRNAs to see which molecules work downstream of the hit compound. Based on the data, wewill eventually elucidate the mechanisms of action of the hit compound.

Discussion & Conclusion
Although the rhEPO agents have been successfully used to treat renal anemia in ESRF patients,
the physiological control of renal anemia is still difficult and the cost of rhEPO therapy is becoming
huge burden on health care system. In this study, we have identified one small molecule thatfacilitate the EPO expression and protein secretion from hiPSC-EPO cells. This small molecule may
be used to develop new therapeutic drugs for renal anemia, which facilitate EPO secretion from
EPO-producing cells in our body. From basic research aspects, our small molecule may elucidate thedetailed mechanisms of EPO production or secretion from EPO-producing cells. It is well known
that EPO production in our body is regulated by HIF-PHD axis. Future studies should elucidate therelationship between our small molecule and the axis.

The Novartis Foundation(Japan)
for the Promotion of Science

47

The Novartis Foundation(Japan)
for the Promotion of Science

References
Hitomi H, Kasahara T, Katagiri N, Hoshina A, Mae SI, Kotaka M, Toyohara T, Rahman A,
Nakano D, Niwa A, Saito MK, Nakahata T, Nishiyama A, Osafune K. Human pluripotent stem
cell-derived erythropoietin-producing cells ameliorate renal anemia in mice. Science Translational
Medicine, 2017; 9(409). pii: eaaj2300.

一般の皆様へ
現在、慢性腎臓病の進行によって慢性腎不全となり透析療法を受けている多くの患者さんがいます。慢性腎不全の合併症として腎性貧血があり、遺伝子組換えヒトエリスロポエチン
製剤投与による治療が行われておりますが、貧血の生理的なコントロールは依然として困難です。本研究において、我々はヒト iPS 細胞からエリスロポエチンを産生する細胞を作る方法を開発し、さらに本細胞からのエリスロポエチン分泌を促進する活性を有する化合物を発見しました。今後、本化合物を用いた腎性貧血に対する再生医療と新規治療薬の開発が期待されます。

The Novartis Foundation(Japan)
for the Promotion of Science

48

The Novartis Foundation(Japan)
for the Promotion of Science

Mechanism that controls the neuronal survival/
death in the developing brain
Kazunori Nakajima
kazunori@keio.jp
Department of Anatomy, Keio University School of Medicine

Summary Abstract
We found a unique pattern of neuronal cell death that occurs when cerebral hemispheres arecultured under a certain condition. The neuronal cell death progresses from the lateral cortex to thedorsomedial cortex in a very reproducible manner. Interestingly, we found that a very potent “cellsurvival factor” with a relatively small size is endogenously produced and secreted from the cerebral
hemispheres and can completely prevent this neuronal cell death.
Key Words：neuronal cell death, cerebral cortex, cell survival factor

Introduction
We previously established a whole-mount culture system of mouse developing cerebral
hemispheres. Using this system, we can cultivate the entire cerebral hemispheres for 1-2 days invitro. Under a certain condition, however, we found that some developmental events were disturbed
and massive neuronal cell death was induced in an interesting and unusual sequence. In this study,
we have analyzed the detailed profile of this cell death.

Results
Cerebral hemispheres including cerebral cortex and ganglionic eminences were dissected out
from embryonic day (E) 15.5 ICR mouse brain, and cultured individually in a glass culture tube
attached to a rotation disk of the whole embryo culture system (Ikemoto Rika Kogyo, Tokyo, Japan)
containing a 2 ml DMEM/F12 medium supplemented with N2 supplement (Invitrogen, Carlsbad,
CA) and several other components. The rotation disk was maintained in a 37 ℃ incubator and rotated
at 20 rpm. As a result, we found that culturing the brain in this condition resulted in bumpy brain
surface in all the analyzed hemispheres. Immunohistochemical analyses showed ectopic invasionof cells into the marginal zone, retraction and disorganization of radial glial fibers, abnormal layer
formation in the cortical plate, malalignment of Cajal-Retzius cells, and reduction of the MAP2 andReelin immunoreactivity. Since the massive reduction of MAP2 and Reelin immunoreactivity andsevere disruption of layer structures suggested cell death of cortical plate neurons and marginal zone
cells, we then evaluated the cell viability using a membrane-impermeant chromatin dye Propidium
Iodide (PI) as a fluorescent viability indicator: cells with intact membranes (live cells) would exclude
PI; cells with damaged membranes (dead cells) would be stained with PI. After 2 days in the rotation
culture (days in vitro, DIV), coronal slices of the cerebral hemispheres were prepared and stainedwith PI. As the result, we observed widespread and massive cell death in the cortical plate andmarginal zone. Interestingly, there was a clear border between the PI-negative region and the PIpositive region. Furthermore, whole mount staining of the cerebral hemispheres using PI revealed
that the PI-positive dead cells formed island-like clusters, separated by PI-negative linear zones that

The Novartis Foundation(Japan)
for the Promotion of Science49

The Novartis Foundation(Japan)
for the Promotion of Scienceappeared along the rostro-caudal axis with relatively constant intervals. These unique features of acobblestone-like brain surface were observed in all the analyzed hemispheres. To determine when
and where the cell death begins, the time course of the cell death was then examined. Each cerebral
hemisphere was collected at 0.5, 1, or 2 DIV and their viability was analyzed using PI. At 1 DIV, PIpositive dead cells were observed only in the lateral region of the telencephalon, and by 2 DIV the
PI-positive region extended to reach the dorsomedial region of the telencephalon, suggesting that thecell death progressed in a lateral-to-medial direction with a clear boundary between the dead region
and the live (intact) region. Interestingly, this massive neuronal cell death was almost completely
rescued when multiple cerebral hemispheres were co-cultured in a single culture tube. To examine
whether there is a “secretory cell survival factor”, we next put a single hemisphere into a dialysis
membrane tube and cultured with several other hemispheres that were placed outside of the dialysis
membrane. As the result, neuronal cell death in the hemisphere in the dialysis membrane tube wasalso clearly rescued, suggesting that this “cell survival factor” is indeed secreted from the cultured
hemispheres. We have then repeated this coculture experiments using dialysis membranes that hadvarious molecular weight cut-off. The results indicated that this endogenous “cell survival factor” isa relatively small molecule.

Discussion & Conclusion
In the whole-mount cerebral hemisphere cultures, we found a unique pattern of neuronal cell
death that progresses from the lateral cortex to the dorsomedial cortex in a very reproducible manner.
Interestingly, although almost all neurons died in the “dead” region, a “live (intact)” region exists
next to the “dead” region and is clearly demarcated from the “dead” region. The border between the“dead” and “live” regions moves medially until almost the entire cortex became dead eventually.
The putative “cell survival factor” that is secreted from the cerebral hemispheres seems to havea very potent function to prevent this neuronal cell death that occurs in the whole-mount cerebral
hemisphere cultures. The identification of this molecule would be important, especially because it isan endogenous and small size molecule with a very strong preventive effect on neuronal cell death.

一般の皆様へマウス胎生期の大脳半球をある条件下で培養すると、神経細胞死が外側から背内側に向かって進行し最終的に全体が死滅することを見出した。その際、死んだ領域とまだ生き残っている領域の境界は極めて明瞭であり、かつ細胞死が起こらない筋状の領域が前後方向にほぼ一定間隔で現れた。複数の脳を半透膜で包んで共培養する実験から、この神経細胞死を完全に防ぐことのできる強力かつ低分子量の内在性因子が脳から分泌されていることを発見した。
この因子を今後の研究で同定できれば、発生期の脳に内在する低分子量の強力な神経細胞死
抑制因子として、臨床応用も視野に入ると期待される。

The Novartis Foundation(Japan)
for the Promotion of Science

50

The Novartis Foundation(Japan)
for the Promotion of Science

Direct reprogramming into cardiac pacemaker cells
for a novel regeneration therapy for arrhythmias
Shu Nakao
snakao@fc.ritsumei.ac.jp
Ritsumeikan University

Summary Abstract
Severely slow heart rate requires transplantation of a mechanical pacemaker, which has
disadvantage in infection, mechanical failure, and medial economy. This proposal aims to develop analternative heart rhythm therapy using reprogrammed cells by gene introduction of some moleculesfrom somatic cells of the patients. In a whole genome expression analysis, the cardiac pacemakertissue in arrhythmia model mice exhibited decreased expression of more than 4,000 genes by themodelling. In particular, 42 transcription factors were downregulated that suggests these factors are
possibly important to maintain the expression of pacemaking ion channels. We are now working
on in silico analysis to figure out what transcription factors really regulate heart rate throughmodification of ion channel functions. This work will provide insights into cardiac regenerative
medicine.
Key Words：Cardiac pacemaker, cell reprogramming, transdifferentiation, arrhythmias

Introduction
Heart rate is decreased age-dependently, and severe bradycardia causes respiratory distress andsyncope, which requires pacemaker implantation. Mechanical pacemaker is broadly available over
the world, however, there are a risk of infection and mechanical failure in those millions of patientsand an impact on medical economy, so that an alternative therapy has been sought.

Results
1. Transcriptomic analysis of the pacemaker tissue in arrhythmia model mice
We first established a mousemodel of pacemaker dysfunction
characterized by slow heart rate.
The model was induced by swim
training which designated twice
60 min sessions/day, 6 days/week
for 4 weeks (Fig. 1). We nextcollected the sinoatrial node region(the primary pacemaking site in
the heart) and the neighbouring right atrial wall tissue for the transcriptomic analysis using a next
generation sequencing, which can measure the expression level of 41,195 genes. 19,679 genes wereexpressed in the sinoatrial node of sedentary mice. There were 655 genes upregulated (> 2-fold)
and 4,306 genes downregulated (< 1/2) by exercise(Fig. 2). We particularly focused on the result oftranscription factors as candidates of the reprogramming factor, and found 42 transcription factor
genes downregulated.

The Novartis Foundation(Japan)
for the Promotion of Science

51

The Novartis Foundation(Japan)
for the Promotion of Science

2. In silico analysis of binding motif of the transcription factors to pacemaking molecules.
As these 42 transcription factors
could be involved in the regulation oftranscription and/or functions in cardiac
ion channels, we have been working
on the bioinformatics analysis to figureout theoretical binding sites of thetranscription factors in the genomic regionof ion channels such as HCN pacemakerchannels and voltage-gated Ca2+ channels.
Further analyses including luciferase reporter assay in vitro and reprogramming experiments using
viral vectors encoding those transcription factors will be performed.
3. Built a platform for reprogramming somatic cells into pacemaker-like cells
Direct reprogramming of somatic cells to cardiomyocytes
was first reported by Ieda et al. in 2010. They introduced
Gata4, Mef2c, and Tbx5 (GMT), known as the master
regulator genes of cardiac development, into fibroblasts toconvert to cardiomyocyte-like cells. As for developmentof the cardiac conduction system, there are several key
transcription factors as seen in cardiac development. So
far, we have tried to induce pacemaker-like cells usingtransduction of these transcription factors in combination
with GMT. Fig. 3 shows the result that little changes of theexpression level of pacemaker channel HCN4 were observedin contrast to upregulation of a cardiac marker gene cardiac
α-actin. This tells us the necessity to further find novel transcription factors to produce pacemakerlike cells.

Discussion & Conclusion
RNA sequencing of the whole genome from the sinoatrial node region in sedentary and trained
mice revealed that transcription factors downregulated by swim exercise would be candidates of the
reprogramming factor for production of pacemaker cells. We are now working on identification of
the reprogramming factors with an in silico approach. To pursue this project, other mouse models of
pacemaker dysfunction such as heart failure models may be required to find common transcription
factors changed by pathology. Similar to GMT, typical transcription factors for development of the
cardiac conduction system may also be important for cell reprogramming into pacemaker cells, so weare seeking novel molecules regulating pacemaker development. Induced pacemaker cells developed
from this project have potential to restore the heart rate as a regenerative therapy. This study will also
gain understanding of the bradycardia pathophysiology and sinoatrial node development.

The Novartis Foundation(Japan)
for the Promotion of Science

52

The Novartis Foundation(Japan)
for the Promotion of Science

References
D’Souza A et al. Circ Res 2017;121:1058-1068
Ieda M et al. Cell 2010;142:375-386
Wiese C et al. Circ Res 2009;104:308-397

一般の皆様へ
加齢に伴う心拍数の低下（徐脈）は、ときに息切れや動悸、失神を生じるほどに進行し、その主な治療法である機械式ペースメーカーの移植には、感染、故障、高額などの問題が残されている。本研究では、徐脈性不整脈に対する新たな再生医療の開発を展望として、心臓ペー

スメーカー細胞を体細胞から作り出す方法の確立を目的とする。この方法は、iPS 細胞などの未分化細胞へ脱分化させる過程をスキップできることから、時間と費用を大きく節約できる特長がある。また、ペースメーカー細胞の性質を獲得させるための因子を特定するための手掛かりとして、病気モデルや胎児期の心臓形成に着目し、データの集積・解析を進めている。

The Novartis Foundation(Japan)
for the Promotion of Science

53

The Novartis Foundation(Japan)
for the Promotion of Science

Structure determination of formosalide A by the combined
technology of the NMR calculation and total synthesis
Daisuke Urabe
urabe@pu-toyama.ac.jp
Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University

Summary Abstract
We have applied computational techniques to predict the structure of a bioactive polyketide,
formosalide A. An exhaustive conformational search was performed using molecular mechanics,
semi-empirical MO, and DFT methods on model compounds of formosalide A. NMR chemical shiftsof the model compounds were simulated to predict the gross structure of formosalide A. Synthetic
approaches to the C9-C18 fragment and the C19-C30 side chain have also been established toward acomplete structure determination of formosalide A.
Key Words：Natural products, structure determination, NMR calculation, total syntheiss

Introduction
Marine dinoflagellates are a rich source of bioactive secondary metabolites. Formosalide A is acytotoxic macrolide, which was isolated from the cultured dinoflagellate Prorocentrum sp. by Lu andcoworkers.1) The unique structural features including tetrahydrofuran and pyrane moieties embedded
in a 17-membered ring and the C19-C30 tetraene side chain were elucidated by extensive NMR
studies, while the stereochemical relationship among the anti-C2,C5, syn-C16,C18 and C8-C12
pyrane residue remained to be determined. In this study, we aimed at a complete determination of thestructure of formosalide A by using both computational and experimental approaches.

Results
anti
Since formosalide A has 3 structural motifs
O
whose stereochemical relationships are unknown,
2
we must consider the possibility of 4 isomers OH
O 5
OH O
18
16
HO
for determination of the gross structure of 30
19
O
OH
formosalide A. Thus, we planned to predict thesynstructure by NMR calculations before startingOH
the total synthesis. To simplify the calculations,formosalide A
4 isomeric model compounds lacking the C19-C30 acyclic side chain were designed. The procedure
for calculating the NMR chemical shifts of an isomer was as follows. The geometry of the modelcompound was optimized by molecular mechanics (MMFF/MCMM), semi-empirical MO (PM6)and DFT (pcm/M062X/6-31g(d)) methods, giving a number of conformers within 2.0 kcal/mol fromthe minimum energy conformer. Clustering of conformers with RMSDs of 0.1 Å removed similargeometries from the library and then the selected conformers were subjected to a DFT-based NMR
calculation (pcm/mPW1PW91/6-31+g(d,p)). The calculated NMR chemical shifts were Boltzmannweighted to afford the simulated NMR chemical shifts for the model compound. This computational
procedure was applied to the other 3 isomers. DP4+ comparison of the simulated NMR chemical

The Novartis Foundation(Japan)
for the Promotion of Science

54

The Novartis Foundation(Japan)
for the Promotion of Science

shifts for the 4 model compounds with the experimental NMR spectrum of formosalide A
successfully predicted the stereochemical relationship for the 3 structural motifs to determine the true
structure of formosalide A with 100% probability.2) The results of the NMR calculations prompted
us to embark on the total synthesis of the predicted structure of formosalide A. A series of catalystcontrolled asymmetric syntheses enabled us to prepare the C9-C18 fragment in enantiopure form.
Furthermore, an efficient approach to the C19-C30 acyclic side chain featuring SN2 displacement
and partial reduction of multiple alkynes to Z-alkenes was established.

Discussion & Conclusion
A challenge in the present study was how to simulate NMR chemical shifts for a conformationally
flexible natural product. Formosalide A possesses a 17-membered ring and a C19-C30 tetraeneside chain, which generate a number of conformational isomers, and thereby complicate the
NMR calculation. We used a sequential approach to select the important conformers for the
NMR calculations. Specifically, MMFF was first used to perform an exhaustive conformational
search, followed by optimization with semi-empirical and DFT methods. Subsequent clusteringof the optimized geometries allowed us to generate the important conformers with reasonable
computational costs. Consequently, we succeeded in predicting the gross structure of formosalide
A. We expect that the stereoselective total synthesis of the predicted structure of formosalide A willcomplete the structure determination.

References
1) Grimblat, N.; Zanardi, M. M.; Sarotti, A. M. J. Org. Chem. 2015, 80, 12526.
2) Lu, C.-K.; Chen, Y.-M.; Wang, S.-H.; Wu, Y.-Y.; Cheng, Y.-M. Tetrahedron Lett. 2009, 50, 1825.

一般の皆様へ天然には、抗がん活性や抗菌活性など創薬において魅力的な生物活性を有する有機化合物が数多く存在します。一方で、天然から得られる量が限られている希少天然物の基礎研究は進展していません。本研究では、渦鞭毛藻から単離された希少天然物ホルモサリド A の初期段階の基礎研究として、その構造決定に取り組みました。現在までに、コンピューターを使ったホルモサリド A の構造推定に成功し、有機合成による推定構造の確認に着手しています。近い将来
ホルモサリド A の構造が決定できると考えています。

The Novartis Foundation(Japan)
for the Promotion of Science

55

The Novartis Foundation(Japan)
for the Promotion of Science

Development of apoptosis induction method with spatiotemporalcontrol using microRNAs.
Kazunori Watanabek_watanabe@okayama-u.ac.jp
Okayama University

Summary Abstract
The methods of cell functions regulated by light have been developed. Previously, we developed
photo-dependent RNA delivery method called PCDR (1. 2). In addition, we identified a novel premiR-XX inducing apoptosis. In this study, we attempted to develop a spatiotemporally controllable
apoptosis-inducing method using pre-miR-XX and PCDR.
As a result, we have demonstrated the efficient and selective induction of apoptosis by the
combination of pre-miR-XX-U1A and PCDR.
Key Words：photo-regulation, apoptosis, miRNA

Introduction
The methods of cell functions regulated by light have been developed. For instance, photo
dynamic therapy using photosensitizer can induce photo-dependently apoptosis.
It is reported that microRNAs (miRNAs) regulate apoptosis. We identified a novel pre-miR-XX
inducing apoptosis (prepared paper). In addition, we developed photo-dependent RNA delivery
method called PCDR (1. 2). In this study, we attempted to develop a spatiotemporally controllable
apoptosis-inducing method using pre-miR-XX and PCDR.

Results
Apoptosis-inducing method using miR-XX and PCDR has one problem. The RNA carrier used in
PCDR recognizes RNA having hairpin structure which contains specific sequence. Pre-miRNA has
hairpin structure. Therefore, pre-miRNA-U1A which was replaced with specific sequence recognizedby the RNA carrier was prepared. In this study, pre-miR-XX-U1A identified as a novel apoptosisinducing miRNA was prepared. In addition, pre-miR-16-U1A and pre-miR-218-U1A which induce
apoptosis were also prepared. We attempted to induce apoptosis by the combination of pre-miR-XX
and PCDR. As a result, all pre-miRNA-U1A could
induce apoptosis, especially apoptosis induction
efficiency by pre-miR-XX was highest. In addition,
PCDR could induce apoptosis more efficiently
than lipofection method.
We attempted to control region specificapoptosis induction by the combination of premiR-XX-U1A and PCDR. Pre-miR-XX-U1A
entered into the cells only within the photoirradiated area, and then apoptosis was induced
only within the irradiated area (Fig 1, lower panel).

The Novartis Foundation(Japan)
for the Promotion of Science

56

The Novartis Foundation(Japan)
for the Promotion of Science

In contrast, while pre-miRNA-U1A control, which is non-targeting, entered into the cells only within
the photo-irradiated area, apoptosis was not induced (Fig 1, upper panel). These results indicate that
apoptosis can be spatially controlled by the combination of pre-miR-XX-U1A and PCDR.
PCDR can transiently deliver RNA into the cells (3). Therefore, the cells delivered miR-XXU1A by PCDR might be induced apoptosis faster than the cells delivered by lipofection method.
We validated this hypothesis. As a result, the cells delivered miR-XX-U1A by PCDR were induced
apoptosis faster than the cells delivered by lipofection method.

Discussion & Conclusion
In conclusion, we have demonstrated the efficient and selective induction of apoptosis by the
combination of pre-miR-XX-U1A and PCDR. PCDR is not observed cytotoxicity (1, 2). Therefore, thismethod using pre-miR-XX-U1A and PCDR has the potential to develop as a treatment with lower
side effect than photo dynamic therapy.
Target mRNAs of pre-miR-XX has not been clarified. Thus, to understand the apoptosis induction
mechanism by pre-miR-XX, further studies will be necessary.

References(1) T. Endoh, M. Sisido and T. Ohtsuki, “Cellular siRNA delivery mediated by a cell-permeant RNAbinding protein and photoinduced RNA interference” Bioconjug. Chem. 19, 1017-1024, (2008).
(2) Y. Matsushita-Ishiodori, M. Morinaga, K. Watanabe and T. Ohtsuki, “Near-infrared light-directed
RNAi using a photosensitive carrier molecule” Bioconjug. Chem. 24, 1669-1673, (2013).
(3) K. Shiraga, T.H. Soe, S. Matsumoto, K. Watanabe and T. Ohtsuki, “Red and near-infrared lightdirected cytosolic delivery of two different RNAs using photosensitive RNA carriers” Bioconjug.
Chem. 29, 3174-3179, (2018)

一般の皆様へ
国内外の研究者により光による細胞機能制御法が開発されている。本研究では、申請者らが同定したアポトーシスを誘導するマイクロ RNA である pre-miR-XX と、光依存的に RNA を細胞内に輸送できる PCDR 法を組み合わせることで、アポトーシスを時空間的に制御することを目的とした。

その結果、PCDR 法で pre-miR-XX を細胞内で導入することで、アポトーシスを高効率に誘導できるだけでなく、アポトーシスを時空間的に制御できることも明らかになった。本手法は副作用の小さいがん治療法として発展していく可能性を秘めている。

The Novartis Foundation(Japan)
for the Promotion of Science

57

The Novartis Foundation(Japan)
for the Promotion of Science

Study on the enantioselective total synthesis ofdragmacidin E: a natural product with high potential
for medicinal chemistry researches
Tetsuhiro Nemoto
tnemoto@faculty.chiba-u.jp
Graduate School of Pharmaceutical Sciences, Chiba University

Summary Abstract
Synthetic studies of dragmacidin E have been performed. A strategy for constructing the core
structure of this natural product was developed. In addition, a novel synthetic method for the 3,4-fused
tricyclic indoles and benzofurans was developed based on a radical cascade cyclization strategy.
Key Words：dragmacidin E, total synthesis, heterocycles, catalysis

Introduction
Dragmacidin E was isolated from an Australian marine sponge by Capon et al. in 1998. The
structure features a 7-membered ring-fused tricyclic indole skeleton functionalized with an indolyl
pyrazinone motif and two contiguous stereocenters. Because of its unique structure, dragmacidin
E is an attractive target for synthetic organic and medicinal chemists. It is noteworthy that thisnatural product exhibits serine-threonine phosphatase inhibitory activity. Dragmacidin E cannot be
efficiently obtained from natural sources (0.01% yield from the natural source), however, hampering
detailed investigation into its biological activities. This background led us to initiate synthetic studiesof this natural product.

Results
We already reported a synthetic study of dragmacidin E, which
provided an access to the core skeleton of this natural product (ref 1).
To extend the present method to the total synthesis, we examined the
key reaction for constructing the 3,4-fused tricyclic indole skeleton
using a densely functionalized substrate. The requisite tetra-substituted
aromatic ring substrate was prepared using a regioselective nitration
reaction. After condensation of the corresponding aldehyde withdimethyl malonate, a methyl group was introduced by Cu-mediatedconjugate addition. Coupling with an allenyl tosylate with the malonate derivative provided a
substrate for the Pd-catalyzed cascade cyclization. The key reaction was performed using 10 mol% of Pd catalyst and 24 mol % of phosphine ligand and the corresponding tricyclic product was
obtained in 70% yield, revealing that the present Pd catalysis could be applied to the synthesis of asuitably functionalized synthetic intermediate.

The Novartis Foundation(Japan)
for the Promotion of Science58

The Novartis Foundation(Japan)
for the Promotion of Science

We next examined the construction of the contiguous stereocenters in an asymmetric fashion.
Asymmetric hydrogenation of tri- and tetra-substituted olefin substrates was first investigated.
However, high asymmetric induction could not be accomplished when substrates with a tetrasubstituted aromatic ring were utilized. In addition, we examined catalytic asymmetric allenylation of
benzophenone imine type prochiral nucleophiles to construct the tetra-substituted carbon center using
chiral phase transfer catalysis. Products with high enantiomeric excess were not obtained as well. To
overcome the current situation, biocatalytic processes will be examined as a collaborative study with
Prof. Harald Gröger in Bielefeld University in Germany. We have already started discussions for thisproject and the reactions in the below scheme are under investigation.

In addition to the synthetic method based on transition metal catalysis, we tried to develop analternative synthetic method for 3,4-fused tricyclic indoles and related benzofuran derivatives using
a radical cascade cyclization strategy. As shown in the scheme below, when O-allyl iodophenol type
substrates were heated in toluene in the presence of HSnBu3 (hydride source) and AIBN (radical
initiator), the radical cascade cyclization proceeded smoothly to give the corresponding tricyclic
product in excellent yield. Acid-promoted isomerization proceeded well to give benzofuran typecompound. This method was also applicable to the synthesis of 3,4-fused tricyclic indoles by using
N-allyl iodoanilines as substrates. These results will be reported in due course (ref 2).

In addition to the experimental works, we wrote a review article on the synthesis of 3,4-fused
tricyclic indoles (ref 3).

Discussion & Conclusion
Pd-catalyzed cascade cyclization developed by our group could be applied to the synthesisof suitably functionalized tricyclic ring system of dragmacidin E. We thus concentrated on theconstruction of contiguous stereocenters on the seven-membered ring. Initial trials using chemocatalytic asymmetric reactions were not successful. Therefore, enzymatic enantioselective processes
are currently investigated. A collaborator: Prof. Harald Gröger possesses a series of enzymes libraries
for this purpose. Enzyme screening for a couple of candidate transformations will be examined.
A radical cascade cyclization for synthesizing 3,4-fused tricyclic indoles and related benzofuran
derivatives was also developed.

The Novartis Foundation(Japan)
for the Promotion of Science

59

The Novartis Foundation(Japan)
for the Promotion of Science

References
1) Synthetic Study of Dragamcidin E: Construction of the Core Structure Using Pd-Catalyzed Cascade
Cyclization and Rh-Catalyzed Aminoacetoxylation
Naoya Inoue, Shun-ichi Nakano, Shingo Harada, Yasumasa Hamada, Tetsuhiro Nemoto
J. Org. Chem. 2017, 82, 2787–2793.
2) Radical Cascade Cyclization for Synthesizing 3,4-Fused Tricyclic Benzofuran Derivatives
Masaya Nakajima, Yusuke Kondo, Shun-ichi Nakano, Yusuke Adachi, Robert Franzén, Tetsuhiro
NemotoManuscript in preparation.
3) Synthetic Methods for 3,4-Fused Tricyclic Indoles via Indole Ring Formation
Tetsuhiro Nemoto, Shingo Harada, and Masaya Nakajima
Asian J. Org. Chem. 2018, 7, 1730–1742.

一般の皆様へ
ドラグマシジン E は、生体内で重要な役割を果たすセリン - スレオニンホスファターゼの阻害作用を示す化合物として知られているが、天然から得られる量はごく微量であり、詳細な研究には合成化学的な供給法の開発が不可欠となっている。私たちは、本天然有機化合物の合成法を開発し、医薬品開発への可能性を調査するために研究を進めている。これまでに、本化合物の主要骨格の構築法は開発したものの、鏡像異性体の作りわけに関しては一層の検討を要する。標的分子骨格の新規合成法開発を含めた、網羅的な研究を進めることで、本天然有機化合物の供給法を今後開発していきたい。

The Novartis Foundation(Japan)
for the Promotion of Science

60

The Novartis Foundation(Japan)
for the Promotion of Science

Understanding the neural mechanism underlying
flexible decision making by the combination ofneurophysiology and deep neural networks
Hironori Kumano
hkumano@yamanashi.ac.jp
University of Yamanashi

Summary Abstract
Flexible behavior based on multiple rules is a hallmark of human cognition. To understand therole of the prefrontal cortex in flexible task switching, we recorded electrocorticogram signals inwide areas of the prefrontal cortex while a monkey performed task switching. Representation of taskrules was first observed in the orbitofrontal cortex. We suggest that rule signal is first encoded in theorbitofrontal cortex and propagates in wide areas of the prefrontal cortex.
Key Words：Macaque monkey, Electrocorticogram

Introduction
Switching behavior based on multiple rules is a fundamental ability of flexible behavior. We havepreviously shown that neurons in the lateral intraparietal area (LIP) integrate relevant informationpreferentially depending on rule (Kumano et al., 2016). We hypothesized that the prefrontal cortex(PFC) control the speed of integration depending on task rule. In this study, We examined neuralactivity of a wide range of PFC by recording electrocorticogram (ECoG) signals while a monkey
performed the reaction time task switching.

Results
A Japanese macaque (Macaque fuscata) was trained to switch between discriminating motion
direction and stereoscopic depth of a random-dot stimulus. In each trial, the color of the fixation point(FP) indicated which discrimination task the monkey had to perform. In the direction discriminationtask, the monkey was required to saccade to the lower target when the dots moved down and to theupper target when the dots moved up. In the depth discrimination task, the monkey was required
to saccade to the lower target when the dots were nearer than the plane of fixation and to the uppertarget when the dots were farther. The two tasks were randomly interleaved from trial to trial so thatthe monkey was unable to predict the task rule of the upcoming trial. The monkey was allowed tomake a saccade as soon as a decision was made. Difficulty of the tasks was varied by changing thepercentage of coherently moving and binocularly correlated dots in the random-dot stimulus.
We first customized ECoG electrodes that can cover all surfaces of the prefrontal cortex including
the lateral PFC (64 channels), the medial PFC (32 channels), and the orbitofrontal cortex (OFC; 32
channels). After implanting these electrodes using aseptic procedures, we recorded ECoG signals
while the monkey performed the reaction time task switching.
We analyzed how well the monkey switched between the two tasks by calculating the switch ratio(SR, Sasaki and Uka, 2009). If the monkey switched between the two tasks perfectly, then the SR

The Novartis Foundation(Japan)
for the Promotion of Science

61

The Novartis Foundation(Japan)
for the Promotion of Sciencewould be 1, whereas if the monkey was ignoring the task cue and solving the task the same way
on each trial, then the SR would be 0. The mean SR for the direction discrimination task was 0.85
and the mean SR for the depth discrimination task was 0.91 (n = 53 sessions). The SRs were inagreement with our previous studies (Sasaki and Uka, 2009; Kumano et al., 2016) and indicated thatthe monkey switched successfully between the two tasks, although not perfectly.
During the task, visual response and saccade related response were observed in various areas
within the PFC. We explored whether neural activity in the PFC represented task-related signals
using a decoding-based approach. We examined whether task rule can be decoded from the event
related potentials (ERPs). In our task switching paradigm, the color of the FP indicates which task
the monkey should perform. We thus used a linear Support-Vector-Machine (SVM) to decode taskrule by classifying the ERP in a 20-ms window that slid every 10ms from 100 ms before FP onset to
400 ms after FP onset. We found that ERP in all areas of the PFC encoded the task rule (significantly
above the pre-FP period). Peak classification accuracy was observed as early as 80 ms after the FP
onset in the OFC. Medial PFC exhibited peak accuracy at around 100 ms after the FP onset. The
peak accuracy in the lateral PFC was delayed compared to other areas and was observed at 200 - 300
ms after the FP onset.

Discussion & Conclusion
The results suggest that information about task rules is first represented within the OFC andpropagates in wide areas of the PFC. Statistical analysis of neural signals such as Granger Causality
analysis and coherence measurement between electrode channels would be needed to test thishypothesis. Examining whether the manipulation of rule-selective activity in the PFC alter thedecision related activity in area LIP is essential for understanding the role of the PFC in flexible
decision making.
Neural activity in the PFC, including ECoG signals examined in this study, is known to havecomplex and diverse response properties. Trained neural network models may provide insights into
complex neural representations in the PFC.

References
Kumano H, Suda Y, Uka T. Context-dependent accumulation of sensory evidence in the parietal
cortex underlies flexible task switching. J Neurosci 36, 12192-12202, 2016.
Sasaki R, Uka T. Dynamic readout of behaviorally relevant signals from area MT during task
switching. Neuron 62, 147-157, 2009.

一般の皆様へ
ヒトは環境に応じて柔軟に判断を切り替え、そのときの環境に応じた行動をとることができる。これまでに我々は、判断を切り替える際には、判断をくだすのに必要な情報を収集する過程が環境に応じて異なることを見出した。本研究では、前頭前野がどのようにダイナミックに

情報収集過程を制御しているかを理解するために、前頭前野の広い範囲から皮質脳波を計測した。その結果、環境に関する情報は眼窩前頭皮質で最初に現れ、前頭前野の広い領域に伝播する可能性が示唆された。今後は、前頭前野に存在する環境情報がどのように判断をつかさどる

神経回路に作用し柔軟な行動が可能になるのかを、神経生理学的手法と深層学習を併用して明らかにしたい。

The Novartis Foundation(Japan)
for the Promotion of Science

62

The Novartis Foundation(Japan)
for the Promotion of Science

Drug development of DYRK1A inhibitors:
targeting excess phosphorylation in Alzheimer’s disease
Masashi Asai
euterpeoleracea@rs.tus.ac.jp
Tokyo University of Science

Summary Abstract
Alzheimer’s disease (AD) is the most common cause of dementia in elderly people. Currently,
there is no disease-modifying drug for AD. Dual-specificity tyrosine-phosphorylation-regulated
kinase 1A (DYRK1A), encoded in chromosome 21 Down syndrome critical region, is involved in
the progression of AD through the phosphorylation of AD-related molecules. Therefore, DYRK1A
inhibitors would be a potent anti-AD drug. Indeed, harmine and epigallocatechin gallate, natural
products, led to upregulated the activity of neprilysin, the dominant amyloid-β peptide (Aβ)-degrading
enzyme in the brain.
Key Words：Alzheimer’s disease, Down syndrome, Chromosome 21, DYRK1A, Neprilysin

Introduction
Alzheimer’s disease (AD) is the most common cause of dementia in elderly people [1]. Despite
the increasing prevalence of AD, no effective therapeutic agent is available [2]. Dual-specificity
tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) is a member of the protein kinase family
that plays a role in several signaling pathways for developmental processes and cell homeostasis inphysiological and pathological conditions [3-5]. It has been reported that DYRK1A phosphorylated
tau and APP, leading to development of AD pathology. Since DYRK1A is an excellent target toeffectively treat AD, we focused the relationship between DYRK1A and amyloid-β peptide (Aβ), andtried to develop its inhibitors.

Results
First, to determine whether the expression of genes located on chromosome 21 is increased in twocell lines of fibroblasts form DS patients (DS fibroblasts), we performed quantitative real-time PCR
analysis. The mRNA levels of APP and DYRK1A were significantly higher in the DS fibroblasts
than in fibroblasts from healthy controls (control fibroblasts) (Fig. 1A). Also, the protein level of
DYRK1A was significantly elevated in the DS fibroblasts, compared with the control fibroblasts (Fig.
1B).

Fig. 1. Expression levels of APP and DYRK1A
in fibroblasts from healthy controls and people with DS.

The Novartis Foundation(Japan)
for the Promotion of Science

63

The Novartis Foundation(Japan)
for the Promotion of ScienceAβ is currently the primary therapeutic target in attempts to arrest AD. In the brain, secreted Aβ is
rapidly degraded by neprilysin (NEP), the dominant Aβ-degrading enzyme in the brain [6]. Although
DYRK1A promotes to produce Aβ through phosphorylation of APP by DYRK1A [7], it is unclear
the effect of DYRK1A on the Aβ-degrading system, especially NEP.
Next, we analyzed the protein level and activity of NEP in cell lysates of control and DS
fibroblasts using the quantitative western blot method and an indirect coupled enzyme assay,
respectively. The protein levels of NEP were remarkably decreased in the DS fibroblasts, comparedwith the control fibroblasts (data not shown). The peptidase activity of NEP in DS fibroblasts issignificantly lower than that in control fibroblasts (Fig. 2).

Fig. 2. NEP activity in fibroblasts from healthy controls and people with DS.
Then, to rescue NEP activity, we treated the two DS fibroblast cell lines from different DS
patients with a DYRK1A inhibitor, harmine, to inhibit the catalytic activity of DYRK1A (Fig. 3).
The pharmacological inhibition of DYRK1A at a concentration of at least 5 μM enhanced NEP
activity by 1.5–2.0-fold in both DS fibroblasts. It has been reported that harmine is capable ofinhibiting recombinant monoamine oxidase A (MAO A) and DYRK1B, a DYRK1A homologue[8]. In a test-tube, the IC50 values for MAO A and DYRK1B are 2 nM and 166 nM, respectively,
whereas for DYRK1A it is 33 nM. To exclude the involvement of these molecules in the modulationof NEP activity, we evaluated the knockdown effects of DYRK1A in the two DS fibroblast lines
using a specific siRNA (data not shown). We confirmed using western blot analysis that DYRK1A
expression in the DS fibroblasts was suppressed by the knockdown. The knockdown of DYRK1A
significantly increased NEP activity and protein amount by 1.5–2.0-fold in both DS fibroblasts.
The level of increase was almost the same as that observed by the harmine treatment, of which theeffective dosage was much higher than the IC50 value for DYRK1A in the test-tube.

Fig. 3. Rescue of NEP activity by a DYRK1A inhibitor in fibroblasts from people with DS.

The Novartis Foundation(Japan)
for the Promotion of Science

64

The Novartis Foundation(Japan)
for the Promotion of Science

Moreover, to determine the phosphorylation sites of NEP by DYRK1A, we performed an invitro kinase assay with recombinant DYRK1A and synthetic NEP intracellular domain peptide as a
substrate. The phosphorylations by recombinant DYRK1A were detected by dot blot analysis using
specific anti-phosphorylated
NEP antibodies. As a result of this kinase assay, DYRK1A is capable of catalyzing thephosphorylation of NEP at Thr11, Thr15, and Thr25 in a direct way.
Epigallocatechin gallate (EGCG), a key member of the family of catechins originating from tea,
has many biological activities beneficial for human health, including anti-cancer, anti-diabetes, antiinfectious, and neuroprotective effects [9,10]. It has been reported that EGCG had an inhibitory
effect on DYRK1A [11]. Finally, our in vitro kinase assay was performed to determine whether
EGCG can inhibit the phosphorylation of NEP by DYRK1A in a similar way. EGCG inhibitedthe phosphorylation of NEP at Thr25 by DYRK1A (data not shown). Interestingly, NEP activity
in neuronal cultured cells treated with EGCG was significantly increased, compared with the cells
treated with vehicle (data not shown).

Discussion & Conclusion
DYRK1A phosphorylates APP as well as tau, leading to an increase in Aβ levels. Taken together
with our results, DYRK1A appears to have multiple sites of action and to exacerbate AD pathology
at multiple stages of the disease development. Thus, DYRK1A may act as a triple modulator,
affecting both production and degradation of Aβ, and tau phosphorylation. Inhibition of DYRK1A
may be a promising target for the development of drugs against AD and DS.
Nevertheless, in vivo studies using mouse model treated with a DYRK1A inhibitor [12,13] andclinical trials for administration of EGCG [12,14] have been successful. Because of increased
expression of DYRK1A in the brains of not only people with DS but also AD patients [15] andinduction of DYRK1A expression by Aβ [15], drugs targeting DYRK1A could address the cause of
exacerbation of AD with few adverse effects. Although further studies are required, future researchshould be devoted to the development of DYRK1A inhibitors for the treatment of AD.

References[1] Alzheimer’s Association. 2017 Alzheimer’s disease facts and figures. Alzheimers Dement. 2017;
13: 325–373.
[2] Agatonovic-Kustrin S, Kettle C, Morton DW. A molecular approach in drug development for
Alzheimer's disease. Biomed Pharmacother 2018; 106: 553–565.
[3] Aranda S, Laguna A, de la Luna S. DYRK family of protein kinases: evolutionary relationships,
biochemical properties, and functional roles. FASEB J 2011; 25: 449–462.
[4] Tejedor FJ, Hämmerle B. MNB/DYRK1A as a multiple regulator of neuronal development.
FEBS J 2011; 278: 223–235.
[5] Wegiel J, Gong CX, Hwang YW. The role of DYRK1A in neurodegenerative diseases. FEBS J
2011; 278: 236–245.
[6] Nalivaeva NN, Turner AJ. Targeting amyloid clearance in Alzheimer’s disease as a therapeuticstrategy. Br J Pharmacol in press.

The Novartis Foundation(Japan)
for the Promotion of Science

65

The Novartis Foundation(Japan)
for the Promotion of Science[7] Ryoo SR, Cho HJ, Lee HW, Jeong HK, Radnaabazar C, Kim YS, Kim MJ, Son MY, Seo H,
Chung SH, Song WJ. Dual-specificity tyrosine(Y)-phosphorylation regulated kinase 1A-mediatedphosphorylation of amyloid precursor protein: evidence for a functional link between Down
syndrome and Alzheimer’s disease. J Neurochem 2008; 104: 1333–1344.
[8] Brierley DI, Davidson C. Developments in harmine pharmacology--implications for ayahuasca
use and drug-dependence treatment. Prog Neuropsychopharmacol Biol Psychiatry 2012; 39:
263–272.
[9] Mandel SA, Amit T, Weinreb O, Reznichenko L, Youdim MB. Simultaneous manipulation ofmultiple brain targets by green tea catechins: a potential neuroprotective strategy for Alzheimer
and Parkinson diseases. CNS Neurosci Ther 2008; 14: 352–365.
[10] Pae M, Wu D. Immunomodulating effects of epigallocatechin-3-gallate from green tea:mechanisms and applications. Food Funct 2013; 4: 1287–1303.
[11] Bain J, McLauchlan H, Elliott M, Cohen P. The specificities of protein kinase inhibitors: an
update. Biochem J 2003; 371: 199–204.
[12] De la Torre R, De Sola S, Pons M, Duchon A, de Lagran MM, Farré M, Fitó M, Benejam
B, Langohr K, Rodriguez J, Pujadas M, Bizot JC, Cuenca A, Janel N, Catuara S, Covas MI,
Blehaut H, Herault Y, Delabar JM, Dierssen M. Epigallocatechin-3-gallate, a DYRK1A
inhibitor, rescues cognitive deficits in Down syndrome mouse models and in humans. Mol Nutr
Food Res 2014; 58: 278–288.
[13] Neumann F, Gourdain S, Albac C, Dekker AD, Bui LC, Dairou J, Schmitz-Afonso I, Hue N,
Rodrigues-Lima F, Delabar JM, Potier MC, Le Caër JP, Touboul D, Delatour B, Cariou K, Dodd
RH. DYRK1A inhibition and cognitive rescue in a Down syndrome mouse model are inducedby new fluoro-DANDY derivatives. Sci Rep 2018; 8: 2859.
[14] de la Torre R, de Sola S, Hernandez G, Farré M, Pujol J, Rodriguez J, Espadaler JM, Langohr
K, Cuenca-Royo A, Principe A, Xicota L, Janel N, Catuara-Solarz S, Sanchez-Benavides G,
Bléhaut H, Dueñas-Espín I, Del Hoyo L, Benejam B, Blanco-Hinojo L, Videla S, Fitó M,
Delabar JM, Dierssen M; TESDAD study group. Safety and efficacy of cognitive training plus
epigallocatechin-3-gallate in young adults with Down’s syndrome (TESDAD): a double-blind,randomised, placebo-controlled, phase 2 trial. Lancet Neurol 2016; 15: 801–810.
[15] Kimura R, Kamino K, Yamamoto M, Nuripa A, Kida T, Kazui H, Hashimoto R, Tanaka T, Kudo
T, Yamagata H, Tabara Y, Miki T, Akatsu H, Kosaka K, Funakoshi E, Nishitomi K, Sakaguchi G,
Kato A, Hattori H, Uema T, Takeda M. The DYRK1A gene, encoded in chromosome 21 Down
syndrome critical region, bridges between beta-amyloid production and tau phosphorylation in
Alzheimer disease. Hum Mol Genet 2007; 16: 15–23.

一般の皆様へ
認知症患者は、団塊世代が75歳以上となる2025には700万人に達すると見積られており、その最大の原因疾患はアルツハイマー病である。アルツハイマー病の根本的治療薬は存在せず、多くの化合物の治験が中止となっている。本研究では、治療薬開発の着眼点を変え、早期からアルツハイマー病を発症するダウン症からヒントを得て、21番染色体に存在するリン酸化酵素に辿り着いた。この酵素はアルツハイマー病に増悪的に作用し、患者脳で発現が亢進していることから、その阻害剤は複数の作用点を有し、副作用が少ない治療薬になり得る。茶カテキン等が阻害活性を有していることから、さらなる開発が望まれる。

The Novartis Foundation(Japan)
for the Promotion of Science

66

The Novartis Foundation(Japan)
for the Promotion of Science

Development of combination therapy targetingon tumor endothelial cells
Takahisa Murata
amurata@mail.ecc.u-tokyo.ac.jp
The University of Tokyo

Summary Abstract
We found that the inhibition of a lipid mediator synthase increases the sensitivity against anticancer drugs of vascular endothelial cells in tumor.
Key Words：Tumor endothelial cell, Angiogenesis, Drug resistanceIntroduction
Tumor cells produce cytokines and/or growth factors to stimulate the formation of new vessels, bya process called angiogenesis. Since tumor vessels are indispensable for tumor growth, normalizing
and reducing tumor vessels can be developed into a potential anti-tumor therapy.
Tumor vessels mainly comprise vascular endothelial cells (ECs), which possess completely
different characteristics compared to normal ECs. For example, tumor ECs have fenestrations andthey are widened intercellular junctions. They also obtain drug resistance against anti-cancer drugs.
In the preliminary study using comprehensive analysis of mRNA expression, we found that tumor
ECs strongly express one type of lipid mediator synthase. In this project, we attempted to reveal its
function especially focusing on drug resistance in tumor ECs.

Results
1. We performed gene expression analysis and found a marked increase of one type of lipid synthase(PYP2) expression in tumor ECs derived from mice melanoma.
2. Immunostaining of mice melanoma revealed protein expression of PYP2 in the tumor ECs. In
situ hybridization also showed PYP2 mRNA expression in the ECs of human melanoma and oral
squamous cell carcinoma .
3. We generated systemic or endothelial specific PYP2 deficient mice and examined the anti-cancer
drug sensitivity of tumor grown on these mice. Administration of low dosage (1 mg/kg) of
doxorubicin or 5-FU was not effective against the growth of lung carcinoma or melanoma on WT
mice. In contrast, these regimens were beneficial against tumor growth on PYP2 deficient mice.
4. We also found that pharmacological inhibition of PYP2 also significantly increased the sensitivityof tumor against anti-cancer drugs in WT mice.

The Novartis Foundation(Japan)
for the Promotion of Science

67

The Novartis Foundation(Japan)
for the Promotion of Science

5. Morphological studies showed tumor on PYP2-/- mice possessed more hyper-permeable andapoptotic ECs suggesting increased extravasation of anti-cancer drugs into tumor mass.
6. In vitro studies showed PYP2 inhibition decreased while stimulation of its signaling pathway
increased the mRNA expressions of transporters which eliminate drugs in ECs. The inhibition of
PYP2 significantly attenuated the elimination of doxorubicin and increased the apoptosis of ECs.

Discussion & Conclusion
Taken together, 1) inflammatory stimuli in tumor microenvironment seems to increase PYP2
expression specifically in ECs, 2) the inhibition or deficiency of PYP2 makes ECs hyper-permeable
and increased the sensitivity against anti-cancer drugs by inhibiting the expressions of transporters intumors.

References
Hematopoietic prostaglandin D synthase-derived prostaglandin D2 ameliorates adjuvant-induced
joint inflammation in mice. Tsubosaka Y, Maehara T, Imai D, Nakamura T, Kobayashi K, Nagata N,
Fujii W, Murata T. FASEB J. 2019 Feb 27:fj201802153R.
L-PGDS-derived PGD2 attenuates acute lung injury by enhancing endothelial barrier formation.
Horikami D, Toya N, Kobayashi K, Omori K, Nagata N, Murata T. J Pathol. 2019 Feb 8.
Inhibition of stromal cell-derived factor-1α/CXCR4 signaling restores the blood-retina barrier inpericyte-deficient mouse retinas. Omori K, Nagata N, Kurata K, Fukushima Y, Sekihachi E, Fujii N,
Namba-Hamano T, Takabatake Y, Fruttiger M, Nagasawa T, Uemura A, Murata T. JCI Insight. 2018
Dec 6;3(23).
Epithelial cell-derived Prostaglandin D2 inhibits Chronic Allergic Lung Inflammation in mice.
Maehara T, Nakamura T, Maeda S, Aritake K, Nakamura M, Murata T. FASEB J 2019 in press.

一般の皆様へ
がん細胞と同様に、がんに伸びて酸素や栄養を届ける血管細胞も抗癌剤耐性能を獲得することが知られている。私たちは、このがんの血管に特異的に発現している酵素を発見し、この酵素の阻害ががん血管の薬剤耐性を解除することを発見した。この酵素の阻害剤と抗癌剤を同時に投与することで、がんに伸びる血管を効率よく抑えて、がんの増殖をとめることが可能となる可能性がある。

The Novartis Foundation(Japan)
for the Promotion of Science68

The Novartis Foundation(Japan)
for the Promotion of Science

Molecular mechanism of store-operated Ca2+ entry
signalosome and its physiological significanceYoshihiro Babababay@bioreg.kyushu-u.ac.jp
Division of Immunology and Genome Biology,
Department of Molecular Genetics Medical Institute of Bioregulation, Kyushu University

Summary Abstract
A chief Ca2+ entry pathway in immune cells is store-operated Ca2+ (SOC) influx, which is triggered
by depletion of Ca2+ from the endoplasmic reticulum (ER). STIM1 functions as the chief ERresident protein to activate SOC influx by detecting ER Ca2+ depletion in many cells. However, theactivation mechanism of STIM1 and the identity of SOC influx signalsome remain elusive. Here, wehave identified a novel STIM1-binding protein, STBP1, as a component of SOC influx signalsome.
Furthermore, our data suggest that STBP1 regulates STIM1 activity and cell viability.
Key Words：Store-operated calcium influx, B cells

Introduction
SOC influx is essential for a wide variety of physiological responses. Previous studies haveidentified STIM1, a Ca2+-binding membrane protein localized in the ER, as the ER Ca2+ sensor andcritical SOC influx activator (1-5). Subsequent studies have identified Orai1 as a calcium channel.
The interaction of STIM1 with Orai1 is essential to induce SOC influx. In addition to Orai1, STIM1
has been reported to make signalsome by interacting with several proteins. However, the molecularmachinery modulating STIM1 activity remains poorly understood. Here, we aimed to identify novel
binding partners of STIM1 that can regulate SOC influx.

Results
To identify novel regulators of SOCE, affinity purification for STIM1 was conducted by using
DT40 B cells stably expressing Flag-STIM1. We used in vivo cross-linking of cells to preserve
STIM1 protein complexes and to validate possible STIM1-interacting proteins. This cross-linker
is reactive with primary amines to one another and is easily reversible, which is used to preserve
protein – protein complexes. To identify the binding proteins of Flag-STIM1, the STIM1 complexesin cross-linked cells were immunoprecipitated with anti-FLAG mAb and eluted with the FLAG
peptide. Then, multidimensional protein identification technology (MudPIT) analysis was performed
to identify proteins containing STIM1 complexes. This analysis identified many STIM1- associatedproteins and we focused on a novel protein and named it as STBP1, (STIM1-binding protein 1).
To further investigate the interaction between STBP1 and STIM1, immunoprecipitation was
conducted in 293T cells transiently transfected with plasmids encoding STBP1 and STIM1. We
found that STBP1 was co-immunoprecipitated with STIM1 and its association was not affected whenstimulated with TG, an ER calcium pump inhibitor.
Next, we investigated where STBP1 is localized in cells. After STBP1-YFP was transfectedtransiently in HeLa cells, the images of STBP1-YFP were obtained by confocal fluorescence

The Novartis Foundation(Japan)
for the Promotion of Science69

The Novartis Foundation(Japan)
for the Promotion of Science

microscope. We found that STBP1 was localized in the ER. To assess the spatial relationship between
STIM1 and STBP1, the distribution of these proteins was investigated in HeLa cells transiently
expressed with STBP1-YFP and mCherry-STIM1. At steady state, a large portion of STBP1-YFP
colocalized with mCherry-STIM1 in the ER. Store depletion induced by histamine and TG resulted
in co-redistribution of mCherry-STIM1 and STBP1-YFP into puncta in the immediate vicinity of the
ER near the PM. Thus, these results strongly suggest that STBP1 binds with STIM1 in the ER.
To examine the molecular function of STBP1, we established conditional STBP1-deficient
DT40 B cells by using CreERT2 system. At first, we searched chicken homolog of stbp1 using theexpressed sequence tag database and obtained the genomic clone by PCR using oligonucleotides, andDT40 genomic DNA was used as a template. The targeting vectors were constructed and transfectedby electroporation. A floxed stbp1 construct was transfected at the same time. Homologous
recombination was identified by PCR. This tamoxifen-inducible method allows us to delete STBP1
gene when the cells was treated with tamoxifen. Deletion of STBP1 was verified by genomic PCR
and RT-PCR in tamoxifen-dependent manner. After tamoxifen treatment, we examined the cell
viability. Unexpectedly, we found that the deletion of STBP1 in DT40 B cells induced cell death.
Furthermore, to determine the effects of STBP1 on calcium signal, we monitored cytosolic Ca2+ inwild-type and STBP1-deficient DT40 B cells after tamoxifen treatment. Conditional STBP1-deficient
DT40 B cells showed a significant increase of cytosolic Ca2+ concentration compared with STBP1intact cells. These results collectively suggest that STBP1 regulates cell survival, death or growth,and may modulate calcium mobilization.
Now we are establishing the B cell specific STBP1 deficient mice to understand the physiological
function of STBP1.

Discussion & Conclusion
Our findings identify a novel STIM1-binding protein STBP1 as a component of STIM1-mediated
SOC influx signalsome. We showed that STBP1 is colocalized and physically associated with STIM1
in the ER. It has been reported that STIM1 interacts with several proteins, but it is unclear how thesemolecules modulate STIM1 activity. Our study by using STBP1-conditional deficient B cells suggestthat STBP1 may be important for calcium-dependent cell survival or death, which is a uniqueeffect among STIM1-binding partners. Whether the binding of STBP1 with STIM1 regulate SOC
influx awaits further studies. In addition, the physiological function of STBP1 will be uncovered byanalysis of our STBP1 conditional knock out mice.

References
1. Parekh, A.B. & Penner, R. Store depletion and calcium influx. Physiol. Rev. 77, 901–930 (1997).
2. Parekh, A.B. & Putney, J.W., Jr. Store-operated calcium channels. Physiol. Rev. 85, 757–810(2005).
3. Roos, J. et al. STIM1, an essential and conserved component of store-operated Ca2+ channel
function. J. Cell Biol. 169, 435–445 (2005).
4. Liou, J. et al. STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx. Curr.
Biol. 15, 1235–1241 (2005).
5. Baba, Y. et al. Coupling of STIM1 to store-operated Ca2+ entry through its constitutive andinducible movement in the endoplasmic reticulum. Proc. Natl. Acad. Sci. USA 103, 16704–
16709 (2006).

The Novartis Foundation(Japan)
for the Promotion of Science70

The Novartis Foundation(Japan)
for the Promotion of Science

一般の皆様へ
本研究の目的はカルシウム流入に必須の分子である STIM1の活性を制御する新規分子を同定することです。免疫細胞の分化や活性においてカルシウム流入は極めて重要であり、免疫不全、自己免疫疾患、アレルギーの発症にも関与します。そこで、本研究成果はこれら疾患に対する新規薬剤の創薬につながる可能性があります。本研究では新規の STIM1結合分子を同定でき、カルシ
ウム流入に関与する可能性が示唆されたことから、今後さらに研究を進めていきたい。

The Novartis Foundation(Japan)
for the Promotion of Science71

The Novartis Foundation(Japan)
for the Promotion of Science

Decoding the epigenome program which defines
normal and tumor endothelial cells
Hiromu Suzuki
hsuzuki@sapmed.ac.jp
Department of Molecular Biology, Sapporo Medical University School of Medicine

Summary Abstract
Angiogenesis plays an essential role in tumor development and is an important therapeutic target.
In this study, we identified that adipocyte enhancer binding protein 1 (AEBP1) promotes tumor
angiogenesis in colorectal cancer (CRC), and that it could be a potential therapeutic target.
Key Words：colorectal cancer, tumor endothelium, AEBP1

Introduction
Tumor angiogenesis plays essential role in the development and progression of cancer.
In colorectal cancer (CRC), clinical effectiveness of anti-VEGF treatment demonstrates thatangiogenesis is a promising therapeutic target. To unravel novel factors involved in angiogenesis
of CRC, we isolated epithelial cells and endothelial cells from primary CRC tissues and adjacentnormal tissues, and performed RNA-sequencing analysis. We identified a series of genes upregulated
in tumor endothelial cells including adipocyte enhancer binding protein 1 (AEBP1). We aimed tounravel the molecular function of AEBP1 in tumor endothelium, and to clarify its usefulness as atherapeutic target.

Results
Elevated expression of AEBP1 in CRC
We first tested the expression of AEBP1 in a series of epithelial cells and endothelial cells isolated
from pairs of CRC tissues and corresponding normal colonic mucosa (n = 10). We found that AEBP1
was specifically upregulated in tumor endothelial cells. By using The Cancer Genome Atlas (TCGA)
dataset, we analyzed the association between AEBP1 expression and clinicopathological features,and we found that higher AEBP1 expression is associated with poorer overall survival of CRC
patients.
Functional analysis of AEBP1 in endothelial cells
We next analyze the function of AEBP1 cells in endothelial cells. Quantitative RT-PCR analysis
revealed that AEBP1 is expressed in human umbilical vein endothelial cells (HUVECs) and humanmicrovascular endothelial cells (HMVECs), while it is undetectable in CRC cell lines. We found thatco-culture of HUVECs with CRC cells or treatment of HUVECs with conditioned medium derived
from CRC cells upregulated AEBP1 expression in HUVECs.
By transfecting HUVECs with siRNAs targeting AEBP1, we found that knockdown of AEBP1
in HUVECs induced cell cycle arrest and suppressed cell proliferation, migration and in vitro
tube formation by HUVECs. We next injected CRC cells with HUVECs with or without AEBP1
knockdown in nude mice subcutaneously. We found that AEBP1 knockdown in HUVECs suppressed

The Novartis Foundation(Japan)
for the Promotion of Science72

The Novartis Foundation(Japan)
for the Promotion of Science

microvascular formation in xenograft tumors. These results suggest that AEBP1 expression inendothelial cells is induced by CRC cells, and that AEBP1 promotes tumor angiogenesis.
AEBP1 is a potential therapeutic target in CRC
To evaluate whether AEBP1 could be a therapeutic target, we next performed further xenograftexperiments. We found that co-transplantation of CRC cells with HUVECs with AEBP1
overexpression promoted xenograft formation in nude mice. We next inoculated CRC cells into nude
mice, and administered intratumoral injection of a siRNA targeting mouse Aebp1 or a control siRNA
every 4 days, and found that Aebp1 siRNA suppressed xenograft tumor growth. We also observedthat treatment with Aebp1 siRNA inhibited microvascular formation in the xenograft tumors.
Downstream targets of AEBP1 in endothelial cells
To clarify the intracellular localization of AEBP1, we performed fluorescent immunocytochemistryin HUVECs, and found that AEBP1 is located in both cytoplasm and nucleus. To unravel
downstream targets of AEBP1, we next assessed the effect of AEBP1 knockdown on gene expression
profiles in endothelial cells. We transfected HUVECs with either one of 2 different siRNAs targeting
AEBP1 or a control siRNA, and harvested total RNA 48h or 72h after transfection. We performed
gene expression microarray analysis and found that AEBP1 knockdown led to significant changes ingene expression profile. Gene ontology analysis suggested that genes associated with cell cycle are
significantly enriched among genes affected by AEBP1 knockdown. Moreover, we found that a seriesof genes reportedly associated with tumor angiogenesis including aquaporin 1 (AQP1) and periostin(POSTN) were downregulated by AEBP1 knockdown in HUVECs. We validate the results by qRTPCR, and found that 2 different siRNAs targeting AEBP1 could suppress expression of the putative
target genes. We also confirmed that overexpression of AEBP1 in HUVECs led to opposite results.
These results suggest that AEBP1 may regulate tumor angiogenesis through inducing downstream
target genes which promote angiogenesis.

Discussion & Conclusion
In this study, we analyzed the role of AEBP1 in CRC. We found that expression of AEBP1 invascular endothelial cells was induced by CRC cells, and that AEBP1 promotes proliferation,
migration and tube formation of endothelial cells. Tumor xenograft experiments suggested that
AEBP1 facilitates tumor angiogenesis, and that AEBP1 is a potential therapeutic target in CRC.
AEBP1 was originally identified as a transcriptional repressor that negatively regulate
adipogenesis. Role of AEBP1 in cancer is not well understood, but recent studies show oncogenic
function of AEBP1. For instance, a study reported that AEBP1 is upregulated in glioblastoma andpromotes cell survival. Another study showed that AEBP1 upregulation confers chemoresistance inmelanoma cells. However, its role in tumor angiogenesis has not been reported.
We also found that AEBP1 may regulate angiogenesis via inducing multiple target genes. AQP1
is a water channel protein expressed in vascular endothelial cells, and that its disruption impairs
angiogenesis. POSTN is reportedly upregulated in various types of tumor, and promotes tumor
angiogenesis and metastasis. Thus, AEBP1 may play as a transcription factor which upregulate
angiogenesis promoting genes in endothelial cells.

The Novartis Foundation(Japan)
for the Promotion of Science73

The Novartis Foundation(Japan)
for the Promotion of Science

References
1. He GP, Muise A, Li AW, Ro HS. A eukaryotic transcriptional repressor with carboxypeptidase
activity. Nature. 1995 Nov 2;378(6552):92-6.
2. Ladha J, Sinha S, Bhat V, Donakonda S, Rao SM. Identification of genomic targets oftranscription factor AEBP1 and its role in survival of glioma cells. Mol Cancer Res. 2012
Aug;10(8):1039-51.
3. Hu W, Jin L, Jiang CC, Long GV, Scolyer RA, Wu Q, Zhang XD, Mei Y, Wu M. AEBP1
upregulation confers acquired resistance to BRAF (V600E) inhibition in melanoma. Cell Death
Dis. 2013 Nov 7;4:e914.
4. Saadoun S, Papadopoulos MC, Hara-Chikuma M, Verkman AS. Impairment of angiogenesis andcell migration by targeted aquaporin-1 gene disruption. Nature. 2005 Apr 7;434(7034):786-92.
5. Zhu M, Fejzo MS, Anderson L, Dering J, Ginther C, Ramos L, Gasson JC, Karlan BY, Slamon
DJ. Periostin promotes ovarian cancer angiogenesis and metastasis. Gynecol Oncol. 2010
Nov;119(2):337-44.

一般の皆様へ
腫瘍血管新生はがんの発生や成長に重要な役割を担っており、有望な治療標的と考えられています。我々は大腸がん組織の遺伝子発現解析から新たな腫瘍血管関連因子として AEBP1を同定しました。我々の研究から AEBP1は大腸がん組織の血管内皮細胞で高発現していること、そして
AEBP1が腫瘍血管新生を促進することが分かりました。マウスを用いた実験で AEBP1の阻害ががん組織の成長を抑制する効果が認められたことから、AEBP1はがん治療標的となりうる可能性が示されました。

The Novartis Foundation(Japan)
for the Promotion of Science74

The Novartis Foundation(Japan)
for the Promotion of Science

Elucidation of long non-coding RNA family inducedby molecular targeting anti-cancer drugs
Shigetada KONDO
kondoshi@rehab.osakafu-u.ac.jp
Graduate School of Comprehensive Rehabilitation, Osaka Prefecture University

Summary Abstract
In this research, we identified novel long noncoding RNAs (lncRNAs), which were induced intumor cells by treatment with anti-VEGF agent (bevacizumab) and VEGFR tyrosine kinase inhibitor.
The lncRNAs (lncRNA-1 and -2) were specifically expressed in colorectal cancer cells of patients
who were treated with bevacizumab. Since the lncRNA-1 silenced tumor suppressor mRNAs (gene-A
and gene-B) expression, induction of this lncRNA may not only promote tumor growth, but also
render tumors more refractory against VEGF/VEGFR-targeted therapies.
Key Words：acquired drug resistance, molecular targeted therapy, long noncoding RNA

Introduction
Molecular targeted therapies are promising novel treatment options for patients with cancer.
Different targeted therapies have achieved various degrees of success in cancer treatment. However,in many cases patient response is limited by acquired resistance to the drugs. The mechanisms ofacquired resistance can be classified as three types: target gene mutation, bypass signaling pathway
activation and down-stream pathway activation (1-3). We recently identified a novel mechanism ofacquired resistance mediated by long noncoding RNAs (lncRNA-1 and -2).

Results
In the present study, we aimed to investigate lncRNAs (lncRNA-1 and -2) expression profiling incolorectal cancer tissues and to identify the molecular target of lncRNAs.
(1) Expression profiles of lncRNA-1 and -2 in human colorectal cancer tissues
Expression of lncRNA-1 and -2 was evaluated by RNA-FISH analysis. In colorectal cancer
tissues from the untreated patients, lncRNA-1 and lncRNA-2 were insignificantly expressed.
By contrast, in colorectal cancer tissues from the bevacizumab-treated patients, lncRNA-1 andlncRNA-2 were highly expressed. However, those lncRNAs were not expressed in the adjacentnormal colorectal epithelial cells.
(2) Identification of target molecule of lncRNA-1 and -2
To identify target molecule of lncRNA-1 and -2 in colon cancer cells, RNA pull-down
experiments using the lncRNA-1 or lncRNA-2 as bait and RNA-seq analysis were performed.
We identified that molecular targets of lncRNA-1 were the tumor suppressor A mRNA andthe tumor suppressor B mRNA. We further demonstrated that lncRNA-1 contained partial
complementary sequence to the mRNAs and that lncRNA-1 could silence the both mRNAs incolon cancer cells.

The Novartis Foundation(Japan)
for the Promotion of Science75

The Novartis Foundation(Japan)
for the Promotion of Science

Discussion & Conclusion
In this research, we identified novel lncRNAs, which were induced in tumor cells by treatment
with bevacizumab and VEGFR tyrosine kinase inhibitor. Since the lncRNA-1 silenced tumor
suppressor genes, induction of this lncRNA may not only promote tumor growth, but also rendertumors more refractory against VEGF/VEGFR-targeted therapies. The gene status of the tumor
suppressor A and B plays important roles in anti-tumor effects of VEGF-targeted therapy. However,
how tumor cells lose the gene products during VEGF-targeted therapy remains unknown. Ourfindings may, at least in part, provide one of the mechanisms by which tumor cells lose theexpression of the tumor suppressors and promotes their malignancy during anti-VEGF/VEGFR
therapy.

References(1) Alexander PB, Wang XF. Resistance to receptor tyrosine kinase inhibition in cancer: molecularmechanisms and therapeutic strategies. Front Med. 2015; 9:134-138.
(2) Mirone G, Shukla A, Marfe G. Signaling mechanisms of resistance to EGFR- and AntiAngiogenic Inhibitors cancer. Crit Rev Oncol Hematol. 2016; 97: 85-95.
(3) Simon T, Gagliano T, Giamas G. Direct Effects of Anti-Angiogenic Therapies on Tumor Cells:
VEGF Signaling. Trends Mol Med. 2017; 23: 282-292.

一般の皆様へ
現在、がん関連分子を標的とした分子標的薬治療は悪性腫瘍の標準化学療法となっている。

しかしながら、がん細胞は分子標的薬に対して抵抗性を獲得することが臨床的に大きな問題となっている。これまでに、３つの分子標的薬耐性メカニズム〔① 標的分子の耐性変異獲得、②

バイパス経路の活性化、③ 標的分子の下流経路の活性化〕が明らかとされている。本研究では全く新しい耐性獲得メカニズムの可能性を見出した。

The Novartis Foundation(Japan)
for the Promotion of Science76

The Novartis Foundation(Japan)
for the Promotion of Science

Development of anti-metastatic therapy basedon the regulation of vascular integrity.
Takayuki Shindotshindo@shinshu-u.ac.jp
Shinshu University School of Medicine

Summary Abstract
Controlling vascular integrity is expected to be a novel therapeutic target of cancers as wellas cardiovascular diseases. Adrenomedullin (AM) and its receptor-modulating protein, RAMP2,
have been identified as essential mediators of cardiovascular homeostasis. In this study, we usedinducible vascular endothelial cell-specific RAMP2 knockout (DI-E-RAMP2-/-) mice to clarify thecontribution made by the endogenous AM-RAMP2 system to angiogenesis and metastasis.
Subcutaneously transplanted melanoma cells showed less growth and angiogenesis in DI-ERAMP2-/- than control mice. On the other hand, after transplantation of B16BL6 melanoma cells
into hindlimb footpads, spontaneous metastasis to the lung was enhanced in DI-E-RAMP2-/- mice.
Early after RAMP2 gene deletion, DI-E-RAMP2-/- mice showed enhanced vascular permeability,
endothelial-mesenchymal transition (EndMT)-like change, and systemic edema. Within the lungs of
DI-E-RAMP2-/- mice, pulmonary endothelial cells were deformed, and inflammatory cells infiltrated
the vessel walls and expressed the chemotactic factors S100A8/9, which attract tumor cells andmediate formation of a pre-metastatic niche. Conversely, overexpression of RAMP2 suppressedtumor cell adhesion to endothelial cells, tumor metastasis, and improved survival.
These findings indicate that the AM-RAMP2 system regulates vascular integrity, whereas
RAMP2 deletion promotes vascular permeability and EndMT-like change within primary lesions
and formation of pre-metastatic niches in distant organs by destabilizing the vascular structure andinducing inflammation. Vascular integrity regulated by the AM-RAMP2 system could thus be ahopeful therapeutic target for suppressing tumor metastasis.
Key Words：Adrenomedullin (AM), RAMP2, tumor metastasis, vascular integrity

Introduction
Controlling vascular integrity is expected to be a novel therapeutic target of cancers as wellas cardiovascular diseases. Adrenomedullin (AM) and its receptor-modulating protein, RAMP2,
have been identified as essential mediators of cardiovascular homeostasis. In this study, we usedinducible vascular endothelial cell-specific RAMP2 knockout (DI-E-RAMP2-/-) mice to clarify thecontribution made by the endogenous AM-RAMP2 system to angiogenesis and metastasis.

The Novartis Foundation(Japan)
for the Promotion of Science

77

The Novartis Foundation(Japan)
for the Promotion of Science

Results
Using tumor transplantation models with DI-E-RAMP2-/- mice and control mice, we analyzed
the effects of deleting the RAMP2 gene from vascular endothelial cells on tumor angiogenesis andgrowth. In the transplantation of B16F10 melanoma cells, tumor weights were significantly lower
in DI-E-RAMP2-/- than control mice. Interestingly, B16F10 melanoma tumors excised from DIE-RAMP2-/- mice showed signs of self-destruction. We thought that the self-destruction of thetransplanted tumor was triggered by a lack of blood flow due to poor angiogenesis.
To assess the effect of RAMP2 deletion on tumor metastasis, B16BL6 melanoma cells weretransplanted into the hindlimb footpads of DI-E-RAMP2-/- mice and control mice, and 3 weeks
later the primary lesions were resected. Spontaneous lung metastasis was then analyzed 4 weeks
after resection of the primary tumor. Contrary to our expectation, the incidence of metastasis and thenumber of metastatic lesions were higher in DI-E-RAMP2-/- mice than control mice.
Endothelial-mesenchymal transition (EndMT) has recently attracted attention as a phenomenon
associated with tumor cell intravasation and metastasis. By immunostaining of primary B16F10
melanoma lesions, we detected abnormal growth of α-smooth muscle actin (αSMA)-positivemesenchymal cells within vascular walls in DI-E-RAMP2-/- mice, whereas the numbers of CD31positive endothelial cells were decreased, which strongly suggests the occurrence of an EndMT-likechange. To further confirm that RAMP2 gene deletion enhanced the EndMT-like change, we isolated
vascular endothelial cells from DI-E-RAMP2-/- and control mice and then assayed EndMT induced
by TGF-β in vitro. Our findings confirmed that among VE-cadherin-positive endothelial cells, cells
also positive for fibroblast-specific protein-1 (FSP-1), a mesenchymal cell marker, were greatly
increased in DI-E-RAMP2-/- mice as compared to control mice.
Recently, it is suggested that a favorable environment within a distant organ increases thelikelihood that cancer metastasis will occur. This is the so-called pre-metastatic niche. We speculated
that changes to the lung vasculature caused by RAMP2 deletion leads to formation of a premetastatic niche. Electron microscopic observation revealed the pulmonary endothelial cells in DIE-RAMP2-/- mice to be deformed and to extend into the vascular lumen. Moreover, we detected
prominent invasion of the lungs by macrophages, which were detected mainly along vascularlumens, in DI-E-RAMP2-/- mice. In addition, oxidative stress indicated by the accumulation of
4HNE, an unsaturated fatty acid peroxide, was enhanced in the lungs of DI-E-RAMP2-/- mice.
Recently, S100A8 and S100A9 have been identified as tumor chemoattractant factors enhancing
tumor metastasis to distant organs, and their expression was elevated in the lungs of DI-E-RAMP2-/mice. These observations indicate that RAMP2 deletion from vascular endothelial cells enhances
pre-metastatic niche formation in the lung, through invasion of the vasculature by inflammatory cellswith increases in oxidative stress, and production of tumor chemoattractant factors.
Finally, to determine whether tumor cell metastasis could be suppressed by stimulating the AMRAMP2 system, we generated endothelial cell-specific RAMP2-overexpressing transgenic mice
(E-RAMP2 Tg). As expected, after transplantation of B16BL6 melanoma cells into the hindlimb
footpads, there was less spontaneous lung metastasis in E-RAMP2 Tg mice than WT mice.
Moreover, E-RAMP2 Tg mice showed greater survival in a spontaneous lung metastasis model

The Novartis Foundation(Japan)
for the Promotion of Science78

The Novartis Foundation(Japan)
for the Promotion of Science

Discussion & Conclusion
Our findings indicate that deletion of RAMP2 from endothelial cells suppresses growth of locallytransplanted tumors by reducing tumor angiogenesis. Contrary, metastasis was exacerbated in DI-ERAMP2-/- mice. To elucidate the mechanism by which this process is enhanced in DI-E-RAMP2-/mice, we examined the vascular structure within primary tumors to determine whether it possesses
features. Within primary B16F10 melanomas in DI-E-RAMP2-/- mice, abnormal overgrowth of
αSMA-positive mesenchymal cells in the vascular walls was accompanied by reduced numbers of
CD31-positive endothelial cells, which suggests the occurrence of EndMT-like change.
Cancer metastasis depends on the microenvironment in distant organs as well as the primarytumor. We examined the effect of RAMP2 deletion on the formation of a pre-metastatic niche in
the lung, and found S100A8/9 expression was elevated in the lungs of DI-E-RAMP2-/- mice. The
elevated S100A8/9 expression is induced in distant organs prior to metastasis, which may enhance
the recruitment of tumor cells from the primary lesion.
Conversely, endothelial cell-specific RAMP2 overexpression in E-RAMP2 Tg mice reduced lungmetastasis and promoted survival in a lung metastasis model. We previously reported that stable
overexpression of RAMP2 in endothelial cells enhances their barrier function through formation ofintercellular tight-junctions. Thus, activation of the AM-RAMP2 system in endothelial cells enhances
vascular integrity, which may suppress tumor cell adhesion and invasion.
AM is a peptide with a very short half-life in the bloodstream, which limits its usefulness fortreatment of chronic diseases. It is noteworthy that we are able to modulate AM function in thevasculature by modulating RAMP2. RAMP2 could be a useful therapeutic target to suppress
metastasis by controlling vascular integrity.

References
Shindo T, Tanaka M, Kamiyoshi A, Ichikawa-Shindo Y, Kawate H, Yamauchi A, Sakurai T.
Regulation of cardiovascular development and homeostasis by the adrenomedullin-RAMP system
Peptides. 111:55-61. 2019
Xian X, Sakurai T, Kamiyoshi A, Ichikawa Y, Tanaka M, Koyama T, Kawate H, Yang L, Liu T, Imai A,
Zhai L, Hirabayashi K, Dai K, Tanimura K, Liu T, Cui N, Igarashi K, Yamauchi A, Shindo T.
Vasoprotective activities of the adrenomedullin-RAMP2 system in endothelial cells.
Endocrinology 158(4): 1–14, 2017
Tanaka M, Koyama T, Sakurai T, Kamiyoshi A, Ichikawa-Shindo Y, Kawate H, Liu T, Xian X, Imai A,
Zhai L, Hirabayashi K, Owa S, Yamauchi A, Igarashi K, Taniguchi S, Shindo T.
The endothelial adrenomedullin-RAMP2 system regulates vascular integrity and suppresses tumor
metastasis
Cardiovasc Res. 111(4):398-409. 2016
Koyama T, Ochoa-Callejero L, Sakurai T, Kamiyoshi A, Ichikawa-Shindo Y, Iinuma N, Arai T,
Yoshizawa T, Iesato Y, Yang Lei, Uetake R, Okimura A, Yamauchi A, Tanaka M, Igarashi K,
Toriyama Y, Kawate H, Adams R.H, Kawakami H, Mochizuki N, Martínez A, Shindo T.
Vascular endothelial adrenomedullin-RAMP2 system is essential for vascular integrity and organ
homeostasis
Circulation. 127(7) 842-53. 2013

The Novartis Foundation(Japan)
for the Promotion of Science

79

The Novartis Foundation(Japan)
for the Promotion of Science

一般の皆様へ
血管の恒常性は、液性因子とその受容体システムによって制御されています。私達は、アドレノメデュリン (AM) が主として血管から分泌され、様々な生理作用を有することを明らかとしてきました。AM とその受容体活性調節タンパクである RAMP2は、血管恒常性維持に重要な働きをしています。この研究において、私達は、血管内皮細胞特異的な RAMP2ノックアウト

マウスや、RAMP2過剰発現マウスを用いて、AM-RAMP2系の癌転移における意義をはじめて明らかとしました。

注目すべきは、血管内皮細胞に RAMP2を過剰発現したマウスでは、癌の転移が抑制され、生存率が改善したことです。この結果から、血管の恒常性維持に注目することで、癌転移を抑制する新しい治療法につながることが期待されます。

The Novartis Foundation(Japan)
for the Promotion of Science80

The Novartis Foundation(Japan)
for the Promotion of Science

Mechanisms underlying commitment to thymic epithelial cells
preventing onset of autoimmune diseases
Taishin Akiyama
taishin.akiyama@riken.jp
RIKEN Center for Integrative Medical Sciences

Summary Abstract
Medullary thymic epithelial cells (mTECs) are critical for preventing onset of autoimmune
diseases by promiscuously expressing tissue-specific antigens. mTECs differentiate from stem/
progenitor cells that have a capability of differentiating into cortical TECs. To data, mechanisms
determine the fate of the common stem/progenitor of TECs into mTECs and cTECs are largely
unknown. We asked possible role of interferon regulatory factor 6 (IRF6) in mTEC differentiation.
Although regulating epithelial cell differentiation of skin, IRF6 does not have non-redundant function
in differentiation and gene expression of TECs
Key Words：Thymus, autoimmunity, epithelial cells, transcription factor

Introduction
Medullary thymic epithelial cells (mTECs) are critical for preventing onset of autoimmunity(ref1). mTECs express many kinds of tissue specific antigens ectopically, which in part regulatedby transcriptional regulator Aire. Previous several studies suggested that both mTECs and cortical
TECs differentiate from common stem/progenitor cells (ref1). However, mechanisms regulating
differentiation of mTECs remain to be determined. In this study, we aimed to find transcriptional
regulators that determines fate of common stem/progenitor cells into mTECs.

Results
We previously reported two different precursor cells of mTECs expressing Aire (ref2). Then we
performed microarray analysis of gene expressing these precursor cells and identified transcription
factors expressing in all of mTECs and their precursor cells, but not in cTECs and their precursor
cells. We obtained three candidate transcription factors, IRF6 and other two transcription factors withsimilar domain structure (tentatively named MTF1 and MTF2). We next asked if these transcription
factors are necessary for mTEC differentiation.
We first analyzed role of IRF6 in mTEC differentiation. IRF6 is reportedly necessary for skinepithelial cells differentiation (ref3). Deficiency of IRF6 in whole body caused death just after birth
due to defect of skin epithelium development. Therefore, we used IRF6 flox/flox mouse line, which
are kindly provided by Dr. Kinoshita in Nagasaki University. We crossed Foxn1-Cre, which allows
deletion of flox allele in thymic epithelial cells and hair follicles (IRF6 cKO mice). IRF6 cKO mice
were born normally and showed a hair loss after around 3 weeks old age. Body weight of IRF6 cKO
mouse are normal. Weight of thymus and spleen was also normal in IRF6 cKO mice as comparedto littermate control mice. We further detailed development of thymocytes and TECs in IRF6 cKO.
Thymocytes are largely classified by expression of surface markers CD4 and CD8 by flow cytometer.

The Novartis Foundation(Japan)
for the Promotion of Science81

The Novartis Foundation(Japan)
for the Promotion of Science

The IRF6 deletion in TECs did not influence cellularity and ratio of CD4+CD8+, CD4+CD8-, CD4CD8+, CD4-CD8- thymocytes, suggesting that IRF6 in TECs is not required for TEC function
regulating differentiation and development of thymocytes. We performed flow cytometric analysis
of TECs. EpCAM-positive TECs were separated by expression of UEA-1 lectin ligand, CD80, andLy51 in CD45-negative TER119-negative cell fraction. Cell number and frequency of mTECs andcTECs were not changed in IRF6 cKO, indicating that IRF6 is not essential for differentiation of
mTECs. We further analyzed gene expression of mTECs and cTECs in IRF6 cKO mice. EpCAM+
cells were separated into mTECs (UEA-1+ Ly51-) and cTECs (UEA-1- Ly51+) and sorted by flow
cytometry. Expression of mRNA in mTECs and cTECs of IRF6 cKO and control mice was evaluatedby high-throughput sequencing (RNA-seq). Gene expression level was determined by using CLC
Genomics Workbench. Unexpectedly, RNA-seq showed that mRNA expression in IRF6 deficient
mTECs and cTECs is almost identical with that in control. Taken together, we concluded thatIRF6 does not have non-redundant role in TEC differentiation and gene expression even if IRF6 isexpressed in mTECs.
We next tried to address role of MTF1 and MTF2 on mTEC differentiation. Because MTF1 deficient mice had never been established yet, we first prepared MTF-1 deficient mice. Because
public database suggest that MTF-1 is expressed in epithelial cells of several organ, we chose toestablish MTF-1 flox/flox conditional mice of which MTF-1 gene is deleted in TECs by crossing
with Foxn1-Cre. Moreover, MTF-1 expression can be detected by beta-galactosidase (LacZ) activity
in these reporter mice. We first checked MTF-1 expression in murine organs by monitoring LacZ
activity. We found that MTF-1 is expressed in mTECs, intestinal epithelial cells, and lung epithelial
cells. In mTECs, MTF-1 is highly expressed in a part of mTECs. We are currently analyzing TEC
differentiation of MTF1 cKO, MTF2 cKO, and double cKO of MTF1 and MTF2.

Discussion & Conclusion
In this study, we are trying to identify “master regulator” for mTEC differentiation. IRF6 isreportedly critical for epithelial cell differentiation in skin (ref3). However, we found that IRF6 does
not have non-redundant role in differentiation of both mTECs and cTECs. IRF family consists of
9 members. Database analysis and previous study suggested that, beside IRF6, several other IRF
family members are expressed in TECs. It is possible that other IRF family member compensate
the deletion of IRF6. For instance, a previous study suggested that IRF7 partially contribute todevelopment of mTECs (ref4). It is important future work to address such possible redundant role of
IRF family proteins in differentiation and function of mTECs.

References
1. Abramson J and Anderson G., Annu. Rev. Immunol. 35:85-118 (2017)
2. Akiyama N., J. Exp. Med. 213:1441-1458 (2016)
3. Nat. Genet. 38:1335-1340 (2006)
4. J Immunol. 190:3289-3298 (2013)

The Novartis Foundation(Japan)
for the Promotion of Science82

The Novartis Foundation(Japan)
for the Promotion of Science

一般の皆様へ
免疫応答の司令塔である T リンパ球のほとんどが胸腺で産生されます。胸腺で T 細胞が産生する際、自己の組織に応答する T リンパ球も生じます。そのほとんどは胸腺であらかじめ除かれますが、それらがうまく除かれない場合、自己免疫疾患の原因になると考えられています。
本研究では、胸腺で自己組織応答性 T リンパ球を除去し、自己免疫疾患の発症を抑制する上皮細胞に着目し、それらの細胞がどのような機構で発生するのか調べます。本研究の成果は、将来的に自己免疫疾患の発症予防や治療に貢献することが期待されます。

The Novartis Foundation(Japan)
for the Promotion of Science83

The Novartis Foundation(Japan)
for the Promotion of Science

Regulation of cellular functions by the nucleolus as a stress sensor
Keiji Kimura
kekimura@tara.tsukuba.ac.jp
University of Tsukuba

Summary Abstract
The nucleolus is a dynamic nuclear body whose structure changes in response to cellular stresses.
The nucleolar structure is also changes during mitosis. We found that a novel nucleolar protein
complex, which we called the NWC complex, translocates from interphase nucleoli to the peripheryof mitotic chromosomes during mitosis. The NWC complex regulates centromeric function of
mitotic chromosomes by regulating centromeric enrichment of Aurora B.
Key Words：nucleolus, cellular stress, mitosis, nucleolar disruption, chromosome periphery

Introduction
The nucleolus is the largest nuclear body and is the site of ribosome biogenesis. In addition, recent
studies have revealed that the nucleolus in involved in various cellular processes (1). In response
to various stresses, the nucleolar structure changes and many of the nucleolar factors (proteins andRNAs) are released from the nucleolus and induce stress response (2-4). The nucleolar structure isalso changes during mitosis in higher eukaryotes, which leads to translocation of nucleolar proteinsto the cytoplasm or periphery of mitotic chromosomes. In this project, we have characterized the roleof the novel nucleolar protein complex involved in mitotic progression.

Results
In the previous study, we found that depletion of a nucleolar protein called nucleolar protein
11 (NOL11) or induced nucleolar disruption during interphase, which in turn delayed activationof cyclin-dependent protein kinase 1 (cdk1) and mitotic entry (5). We also noticed that NOL11
depletion increased mitotic index, suggesting that NOL11 has the functions in the progression ofmitosis (5).
In this project, we first examined the subcellular localization of NOL11 to test the mitotic functionof NOL11. We found that NOL11 localized at the periphery of mitotic chromosomes (PR). Then we
tested whether NOL11 forms a protein complex because NOL11 has no functional motif. By tandem
affinity purification approach, we found that NOL11 formed a novel protein complex with WDR43
and Cirhin, both of which are nucleolar component. This complex, which we call the NWC (NOL11WDR43-Cirhin) complex, localized in the nucleolus during interphase and translocated to the PRof condensed mitotic chromosomes during mitosis when the nucleolus disassembles. Treatment of
RNase A led to dissociation of the NWC complex from the PR. Furthermore, the NWC complex
interacted with pre-rRNA in mitotic cells. These result shows that the NWC complex localized the
PR pre-rRNA, which is consistent with the report that newly synthesized pre-rRNA associates with
the PR and act as a binding scaffold of several nucleolar proteins.
To address the roles of the NWC complex, we depleted of each component of the NWC complex
using siRNAs. Depletion of the NOL11, WDR43, or Cirihin (The NWC component) spent longer
time progressing prophase to metaphase. Consistent with this results, depletion of the NWC complex

The Novartis Foundation(Japan)
for the Promotion of Science84

The Novartis Foundation(Japan)
for the Promotion of Science

severely compromised chromosome alignment on the metaphase plate, showing activation of
spindle assembly checkpoint and metaphase arrest. Because chromosome alignment depends on theregulated interaction between microtubules and kinetochores (MT-KT), we tested whether the NWC
complex were involved in the process by monastrol release assay. We found that regulated MT-KT
interaction was defective in the cells depleted of the NWC complex.
Mitotic phenotypes induced by the NWC complex depletion are similar to those induced byinhibition of Aurora B, a kinase involved in multiple mitotic events and the centromeric localization
is essential. Therefore, we tested the effects of the NWC depletion on the localization and activityof Aurora B. We found that the NWC depletion had little influenced the activity and total amount of
Aurora B but severely reduced Aurora B signals at centromere, showing that the NWC complex isessential for centromeric enrichment of Aurora B. In addition, we found that depletion of the NWC
complex markedly reduced centromeric localization of histone H3 phosphorylated at threonine 3(H3T3ph) that is the histone mark to recruit Aurora B at centromere. However, depletion the NWC
complex little affected total levels of H3T3ph. Taken together, we speculate that the NWC complex
prevents H3T3ph from spreading from centromeres to chromosome arms, thereby contributes tocentromeric enrichment of Aurora B.
Finally, we tested the effect of NWC depletion on sister chromatid cohesion, which depends
on centromeric localization of Aurora B. We found that depletion of the NWC complex caused
defects in mitotic chromosome cohesion, showing the NWC complex is essential for centromere
organization and the maintenance of sister chromatid cohesion.

Discussion & Conclusion
In this project, we identified a novel nucleolar protein complex, consists of NOL11, WDR43, andCirhin, which plays an essential role in regulating centromeric function of mitotic chromosomes.
The NWC complex existed in nucleoli during interphase and associated with the PR of mitotic
chromosomes. The NWC complex was required for centromeric enrichment of Aurora B throughthe phosphorylation of histone H3 at threonine 3, thus contributing to regulated interaction between
microtubules and kinetochores and establishment of sister chromatid cohesion.
There are several proteins that dynamically shuttles between nucleoli and the PR of chromosomes
in a cell cycle dependent manner and are considered to be involved in regulation of mitosis. In thisproject, we have characterized that the NWC complex, one of these nucleolar-perichromosome
shuttling proteins, regulates chromosome function in mitosis through facilitating centromeric
concentration of Aurora B.

References
1) F. M. Boisvert, S. van Koningsbruggen, J. Navascues, A. I. Lamond. The multifunctional
nucleolus.
Nat. Rev. Mol. Cell Biol. (2007) 8, 574-585.
2) Nishimura K, Kumazawa T, Kuroda T, Katagiri N, Tsuchiya M, Goto N, Furumai R, Murayama A,
Yanagisawa J, and Kimura K. Perturbation of ribosome biogenesis drives cells into senescencethrough 5S RNP-mediated p53 activation.
Cell Rep. (2015) 10:1310-1323

The Novartis Foundation(Japan)
for the Promotion of Science85

The Novartis Foundation(Japan)
for the Promotion of Science

3) Katagiri N, Kuroda T, Kishimoto H, Hayashi Y, Kumazawa T, and Kimura K. The nucleolar
protein nucleophosmin is essential for autophagy induced by inhibiting Pol I transcription.
Sci. Rep. (2015) 5:8903
4) Kumazawa T, Nishimura K, Katagiri N, Hashimoto S, Hayashi Y, and Kimura K. Gradualreduction in rRNA transcription triggers p53 acetylation and apoptosis via MYBBP1A.
Sci. Rep. (2015) 5:10854
5) Hayashi Y, Fujimura A, Kato K, Udagawa R, Hirota T, Kimura K. Nucleolar integrity during
interphase supports faithful Cdk1 activation and mitotic entry. Science Adv. (2018) 4:eaap7777

一般の皆様へ
核小体は核内に存在する多種類のタンパク質と RNA から成る構造体で、リボソーム生合成やストレス応答など多くの細胞機能に関与します。細胞が種々のストレスに細胞がさらされた際には、核小体が崩壊しそれに伴って局在を変化させた核小体因子が種々の細胞機能に関与します。
また、細胞分裂に伴って核小体が崩壊し多くの核小体因子が局在を変えることから、核小体と細胞分裂の関連性が示唆されてきましたが、その実態は分かっていませんでした。本研究で我々は、核小体因子である NWC 複合体が、分裂期の染色体周辺に結合し動原体の機能を制御することにより正常な細胞分裂に寄与することを見出しました。

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

Transomics approach to elucidate cellular signaling pathways
in vascular development, morphogenesis and disease
Osamu NAKAGAWA
osamu.nakagawa@ncvc.go.jp
National Cerebral and Cardiovascular Center Research Institute

Summary Abstract
Proper formation and function of cardiovascular network are essential for embryonicdevelopment, and mutations of the genes involved in cardiovascular signaling often cause intractable
human diseases. We have performed various experiments that examine transcriptome, proteome andphosphoproteome profiles in endothelial cells to clarify the influences of vascular signal activation
or inhibition upon downstream signaling pathways and endothelial functions. These studies provide
a clue to understand mechanisms of normal cardiovascular development and etiologies of humandiseases.
Key Words：Vascular development, Cellular signaling, Bone morphogenetic protein, ALK1 receptor
complex, protein kinase

Introduction
Formation of cardiovascular network is essential for embryonic development, andneovascularization is associated with various adult diseases such as ischemic heart diseases,
retinopathy and cancer (1). Vasculogenesis occurs as a de novo organization of endothelial cell
plexus, followed by a series of processes for vascular remodeling and maturation, including
endothelial cell migration, proliferation and tubular reorganization, collectively called angiogenesis.
In addition, the structure of blood vessels is established by the stabilization of endothelial cells,
acquirement of arterial or venous identity, and recruitment of mural cells to vascular walls (2).

Results
A variety of cellular signaling pathways controlling vascular development involve cytokines andgrowth factors, such as vascular endothelial growth factors (VEGF), angiopoietins, transforming
growth factor β (TGFβ) and bone morphogenetic proteins (BMPs) (3,4). BMP9 and BMP10 mainly
act through the endothelium-specific ALK1 receptor and promote arterial endothelial maturationand quiescence (5-8). Mutations in the genes involved in the ALK1 receptor signaling cause
hereditary hemorrhagic telangiectasia and are implicated in the pathogenesis of pulmonary arterial
hypertension (9-10). ALK1 signal induces SMAD1/5/9-dependent transcription of target genes,
including its own signaling components, transcription factors and membrane ligands/receptors. To
clarify the downstream events of ALK1 signal activation, we performed a transcriptome analysis ofhuman umbilical artery endothelial cells stimulated with BMP9. Various genes showed an increase
in mRNA expression following the ALK1 receptor activation, most of which had SMAD binding intheir putative enhancer regions in the in silico analysis. These genes are likely new target genes thatserve for endothelial differentiation and vascular morphogenesis downstream of the BMP-ALK1SMAD signaling pathway.

The Novartis Foundation(Japan)
for the Promotion of Science87

The Novartis Foundation(Japan)
for the Promotion of Science

Among them, we found that the SGK1 gene encoding Serum/glucocorticoid-regulated kinase 1
showed a rapid and significant increase in mRNA expression by the stimulation with BMP9 (11). At
embryonic day 9.5, the Sgk1 mRNA expression was clearly enriched in vascular endothelial cells,
which was significantly down regulated in Alk1/Acvrl1 null embryos. ALK1 signal was known to
evoke post-translational activation of protein kinases such as TAK1 and ERK as a non-canonicalpathway, but SGK1 was the first example of protein kinases whose expression was controlled by
the SMAD-dependent canonical pathway. SGK1 belongs to the AGC (named after PKA, PKG and
PKC) superfamily of protein kinases often involved in cardiovascular development and disease (12).
Interestingly, the transcriptome analysis demonstrated that only SGK1 showed BMP9-dependent
induction of gene expression among the genes encoding AGC kinases. Sgk1 null mice with aparticular genetic background exhibit severe abnormalities of vascular development and embryoniclethality (13-14), suggesting its role in the mechanisms of angiogenesis defects due to the ALK1
signaling deficiency.
While there were a couple of SGK1 substrates that were implicated in the control of vascularformation and maintenance, such as the Foxo family of transcription factors and the NDRG proteins(12), the detailed mechanisms of SGK1 actions in endothelial cells were yet to be clarified. We
therefore employed a proteomic approach to search for possible endothelial-enriched SGK1
substrates using the mass spectrometry analysis of phosphorylated proteins in cultured endothelial
cells expressing constitutively-activated SGK1. We have identified several proteins as candidates
for novel SGK1 substrates, which show specific phosphorylation of consensus-matched serine or
threonine residues. Further studies are ongoing to examine whether these proteins are functionally
important in endothelial cell differentiation and vascular morphogenesis, and how SGK1-dependent
phosphorylation influences their actions.
We have also started omics analyses using vascular endothelial cells of mouse embryos.
Endothelial cells were isolated using anti-CD31 antibody for the application to RNA sequencing
and protein mass spectrometry. mRNA and protein expression in endothelial cells of wild typeembryos and gene knockout embryos of vascular signaling factors is currently under study. In
addition to the analysis of individual layers of signaling network, such as transcriptome, proteome
and phosphoproteome, the comparison of multiple omics data will be performed to clarify the global
interrelationship of signaling events in endothelial cells.

Discussion & Conclusion
Elucidating cellular signaling pathways involved in endothelial cell differentiation and vascularmorphogenesis is important for the mechanistic dissection of normal embryonic development. In
addition, such information helps understand the pathogenesis of various congenital cardiovasculardiseases. We have studied the regulation of transcriptome, proteome and phosphoproteome
downstream of ALK1 and other vascular signaling pathways in cultured endothelial cells as well as
in mouse embryos. Particularly, identification of SGK1 as a novel downstream target gene of ALK1
signaling is of high interest (11), and characterization of additional SGK1 substrates in endothelial
cells provides a clue to better understanding of endothelial signaling during normal and abnormal
vascular development.

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

References
1. Herbert, S.P. and D.Y. Stainier, Molecular control of endothelial cell behaviour during bloodvessel morphogenesis. Nat Rev Mol Cell Biol, 2011. 12(9): p. 551-64.
2. Potente, M., H. Gerhardt, and P. Carmeliet, Basic and therapeutic aspects of angiogenesis. Cell,
2011. 146(6): p. 873-87.
3. Miyazono, K., Y. Kamiya, and M. Morikawa, Bone morphogenetic protein receptors and signaltransduction. J Biochem, 2010. 147(1): p. 35-51.
4. Cai, J., et al., BMP signaling in vascular diseases. FEBS Lett, 2012. 586(14): p. 1993-2002.
5. Garcia de Vinuesa, A., et al., BMP signaling in vascular biology and dysfunction. Cytokine
Growth Factor Rev, 2016. 27: p. 65-79.
6. Urness, L.D., L.K. Sorensen, and D.Y. Li, Arteriovenous malformations in mice lacking activin
receptor-like kinase-1. Nat Genet, 2000. 26(3): p. 328-31.
7. Oh, S.P., et al., Activin receptor-like kinase 1 modulates transforming growth factor-beta 1
signaling in the regulation of angiogenesis. Proc Natl Acad Sci U S A, 2000. 97(6): p. 2626-31.
8. Li, D.Y., et al., Defective angiogenesis in mice lacking endoglin. Science, 1999. 284(5419): p.
1534-7.
9. Tillet, E. and S. Bailly, Emerging roles of BMP9 and BMP10 in hereditary hemorrhagictelangiectasia. Front Genet, 2014. 5: p. 456.
10. Ma, L. and W.K. Chung, The role of genetics in pulmonary arterial hypertension. J Pathol, 2017.
241(2): p. 273-280.
11. Araki M., et al., Serum/glucocorticoid-regulated kinase 1 as a novel transcriptional target ofbone morphogenetic protein-ALK1 receptor signaling in vascular endothelial cells. Angiogenesis,
2018. 21(2):415-423.
12. Di Cristofano, A., SGK1: The Dark Side of PI3K Signaling. Curr Top Dev Biol, 2017. 123: p.
49-71.
13. Zarrinpashneh, E., et al., Ablation of SGK1 impairs endothelial cell migration and tube formation
leading to decreased neo-angiogenesis following myocardial infarction. PLoS One, 2013. 8(11):p. e80268.
14. Catela, C., et al., Serum and glucocorticoid-inducible kinase 1 (SGK1) is necessary for vascularremodeling during angiogenesis. Dev Dyn, 2010. 239(8): p. 2149-60.

一般の皆様へ
細胞分化・増殖・移動など多様な生命現象の組み合わせにより、複数の起源を有する前駆細胞から心臓・血管の発生・形態形成が行われ、多様な細胞群が協調して働く成熟機能システムを実現するメカニズムは非常に複雑であり、その破綻が先天性心疾患・遺伝性血管疾患のみならず成人の循環器疾患の病因に深く関わることは明らかです。心血管系の発生・形態形成過程において様々な細胞間・細胞内シグナル伝達系が働くことが知られていますが、私たちはそれらシグナル伝達系の活性化や阻害によって内皮細胞の遺伝子発現様式・タンパク質分子発現様式などがどのような影響を受けるかを検討しました。今後も心血管形成におけるシグナル伝達機構の研究を続け、ヒト心血管疾患の病因・病態解明と診断法・治療法開発を目指したいと考えております。

The Novartis Foundation(Japan)
for the Promotion of Science89

The Novartis Foundation(Japan)
for the Promotion of Science

Studies on formation and function of extracellular DNA fibers
Hisato Saitoh
hisa@kumamoto-u.ac.jp
Faculty of Advanced Science and Technology (FAST), Kumamoto University

Summary Abstract
Although the genomic DNA is compacted and sheltered in the nucleus in nearly all eukaryotic
cells, DNAs can be detected in the extracellular spaces in many species, including humans. Suchobservations suggest existence of the mechanism that generates extracellular DNAs in eukaryotic
cells. In this study, using cultured human cells, we investigate formation and function of genomic
DNA which are extruded from the nucleus and are discharged to the extracellular environment.
Key Words：extracellular DNA, chromatin, nucleus, neutrophil extracellular traps (NETs)

Introduction
Eukaryotic cells, including human myeloid cells, are known to extrude their nucleus and DNAsin response to intrinsic or environmental cues. For instances, erythroid precursor cells efficiently
separate the cytoplasmic compartment from the nucleus by squeezing the nucleus to extracellularspaces during the final erythroid maturation step, known as “enucleation” [1, 2]. Other example isobserved in subsets of leucocytes, such as neutrophils and macrophages, undergoing extracellular
trap-cell death (NETosis) [3, 4]. During NETosis, decondensed chromatin fibers are discharged fromthe nucleus to extracellular spaces, resulting in formation of the complex fibrous network which havea potential to capture and immobile pathogens. Mechanisms to generate such extracellular nucleus
and/or chromatin fibers in the myeloid cellular lineages remain largely uninvestigated.

Results
1. The nuclear extrusion to extracellular spaces in TN-exposed HL-60 cell culture
To elucidate the effect of tunicamycin (TN) [5-7] on human myeloid cell lines under the FBSfree conditions, exponentially growing HL-60 cells were cultured in the presence or absence of TN
under the FBS-free medium conditions at 37oC. More than 80% of the TN-exposed cells exhibitedthe ovoid-shape after the 60 min incubation. Our time-lapse analysis of a TN-exposed cell revealed
that the ovoid-shaped object appeared suddenly with no premonitory sign and indicated that suchovoid-shaped objects emerged in a very rapid manner, which was completed within less than 5 min.
Since these ovoid-shaped objects were readily stained by the DNA-staining fluorescent dyes, suchas SYTOX Green and DAPI, without any detergent treatment, we considered that the TN-induced
ovoid-shaped objects might represented the nucleus of HL-60 cells, which were somehow extruded
from the cultured cells to the extracellular spaces under our experimental settings.
2. Generation of extracellular chromatin/DNA fibers
When TN-exposure was continued for 120 min, the numbers of SYTOX Green-positive ovoidshaped objects reduced in the culture dish. Instead, the numbers of SYTOX Green-positive fiberlike structures increased in the culture medium, suggesting that DNA fibers were extruded from thenucleus. We isolated DNA from the fiber-like structures and performed agarose-gel electrophoresis,

The Novartis Foundation(Japan)
for the Promotion of Science

90

The Novartis Foundation(Japan)
for the Promotion of Science

confirming that the fiber-like structures were consisted of DNAs. We also detected that the TNinduced fiber-like structures were susceptible to MNase-digestion, generating 160-200 DNA ladderbands on agarose gel. These results indicated that TN-exposure of HL-60 cells under the FBS-free
conditions resulted in generation of extracellular chromatin fibers, at least, in part of which consistedof MNase-sensitive structural units. This scenario was feasible, since SDS-PAGE analysis of theproteins associated with the fiber-like structure revealed existence of a large amount of histone
proteins on the fiber-like structures.
3. Time-lapse observation of the extracellular chromatin formation
We performed the time-lapse images of representative TN-exposed cell and DMSO-treated control
cells. In the time-lapse images of the TN-exposed cell, it appeared to squeeze large ovoid-shaped
objects to the extracellular space, followed by discharge of fiber-like structures to extracellular spacefrom the nucleus. In contrast, the control cells exhibited neither extrusion of the ovoid-shaped objects
nor fiber-like structures during our time-lapse observation period. Together, these data indicated thesequential order of two events; extrusion of fiber-like structures to extracellular space proceeded
following the nuclear extrusion.
4. TN-induced nuclear and chromatin extrusion in other myeloid cell lines
To elucidate TN-effect on other cell-types besides HL-60 cell line, we tested human
myelomonocytic lymphoma U937 and human acute monocytic leukemia THP-1 cells. We found thatboth U937 and THP-1 cells exhibited a phenotype similar to TN-exposed HL-60 cells at either time
point; they extruded ovoid-shaped objects were detected at 30~60 min-incubation, suggesting that
TN-induced the nuclear extrusion in both cell lines, and the formation of SYTOX Green-positive
extracellular fiber-like structures were discharged to extracellular spaces at 120 min-incubation,
implying the formation of extracellular chromatin fibers in both cell lines.

Discussion & Conclusion
We found a unique effect of TN on a range of cultured myeloid cell lines to induce extracellular
nucleus and chromatin fibers in a previously unappreciated fashion. Given that apoptosis prevents
discharge of genetic and epigenetic materials of the dying cells, our discovery of the TN-induced
cellular event provides quite different concept with respect to fate of genetic and epigenetic materials
after cell death. At the same time, the discovery of this unique phenomenon will open the door to theinvestigation of the mechanism of the nuclear and chromatin discharge to extracellular environment,
which are often observed in myeloid cellular lineage, such as enucleation during erythroid maturationand formation of NETs in leukocytes during pathogen infection. We anticipate that the identificationof candidate regulators and effectors in the TN-induced pathway will enable to elucidate the factors
which regulate 4D structure of chromatin in the nucleus and cells. Additionally, we expect that the
extruded nucleus and chromatin in the TN-exposed cells will be suitable for the in vitro analyses
of DNA replication, transcription, nuclear scaling and mitotic chromosome condensation. The TNinduced extracellular chromatin fibers will be beneficial for researchers studying interaction ofchromatin with pathogens in vitro.

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

References[1] Ji P. New insights into the mechanisms of mammalian erythroid chromatin condensation andenucleation. Int Rev Cell Mol Biol. 2015;316:159-82.
[2] Kalfa TA, Zheng Y. Rho GTPases in erythroid maturation. Curr Opin Hematol. 2014;21:165-71.
[3] Brinkmann V, Zychlinsky A. Neutrophil extracellular traps: is immunity the second function ofchromatin? J Cell Biol. 2012;198:773-83.
[4] Castanheira FVS, Kubes P. Neutrophils and NETs in modulating acute and chronic inflammation.
Blood. 2019;133: 2178-2185.
[5] Oakes SA, Papa FR. The role of endoplasmic reticulum stress in human pathology. Annu Rev
Pathol. 2015;10:173-94.
[6] Prescher JA, Bertozzi CR. Chemical technologies for probing glycans. Cell. 2006;126(5):851-4.
[7] van Galen P, Kreso A, Mbong N, Kent DG, Fitzmaurice T, Chambers JE, et al. The unfolded
protein response governs integrity of the haematopoietic stem-cell pool during stress. Nature.
2014;510:268-72.

一般の皆様へDNA は核内に保持されるだけでなく、細胞外に放出されることがある。例えば、白血球が細胞外に形成する Neutrophil Extracellular Traps（NETs）の DNA は、細菌の侵入とシグナル因子の拡散を調節することで、免疫応答に関わる。しかしながら、細胞外 DNA の形成と機能についての詳細は、不明な点が多い。本研究では、白血病由来細胞 HL-60を小胞体ストレス誘導剤

Tunicamycin で処理することで細胞外 DNA を誘導する実験系を構築した。細胞外 DNA に関する
未踏破の研究領域に踏み込んだことで、遺伝暗号やエピジェネティクス以外の DNA 機能の重要性を指摘し、新たな保健医療技術を開拓するための知的基盤を構築した。

The Novartis Foundation(Japan)
for the Promotion of Science92

The Novartis Foundation(Japan)
for the Promotion of Science

Serotonin 5-HT2A receptor agonist in treatment topsychiatric disorders
Daisuke Ibi
ibid@meijo-u.ac.jp
Meijo University, Department of Chemical Pharmacology

Summary Abstract
Ketamine, a NMDA antagonist, has been available as an antidepressant for treatment-resistantdepression in USA, but it has psychiatric-like symptom and a potential for addiction. Therefore, its
use is restricted. Recent clinical studies demonstrated that psilocybin known as a serotonin 5-HT2A
receptor (5-HT2A) agonist as well as a psycedelic also has a therapeutic effect to treatment-resistantdepression although its mechanisms remains unclear. Therefore, we investigated that mechanisms
involved in antidepressant effect of a 5-HT2A agonist. We demonstrated that a 5-HT2A agonist
showed the anti-depressant effect in mice, which occurred in a dose-dependent manner. Further,
acute injection of a 5-HT2A agonist increased the c-Fos-positive cells in GABAergic neurons in
the lateral septum of mice, which was suppressed by pre-treatment of a 5-HT2A antagonist. These
results suggest that a 5-HT2A agonist may have an antidepressant effect through the activation of
GABAergic neurons in the lateral septum.
Key Words：serotonin 5-HT2A receptor, depression, psychiatric disorder, lateral septum

Introduction
Depression (major depressive disorder or clinical depression) is a common but serious mooddisorder. It causes severe symptoms that affect how you feel, think, and handle daily activities, suchas sleeping, eating, or working. To be diagnosed with depression, the symptoms must be present for
at least two weeks. A various antidepressant have been developed for many decades. However, about
30% of the patients are considered treatment resistant, and will continue to depressive symptoms
despite the optimal use of available antidepressants. Ketamine, a NMDA antagonist, has been
available as an antidepressant for treatment-resistant depression in USA. The antidepressant effectof ketamine occurs rapid-acting and long-lasting, but it also has aversive effect such as psychiatriclike symptom and a potential for addiction. Recent clinical studies reported that 5-HT2A agonist
also has rapid-acting and long-lasting anti-depressant effect1. Further, occurrence frequency of
5-HT2A agonist adverse effect such as hallucination would be less than ketamine 2 although themolecular mechanisms underlying antidepressant effect of 5-HT2A agonist remains unclear. Here,
we investigated about mechanisms of a 5-HT2A agonist antidepressant effect.

The Novartis Foundation(Japan)
for the Promotion of Science

93

The Novartis Foundation(Japan)
for the Promotion of Science

Results
We examined the effect of antidepressant effect of
5-HT2A agonists in mice, for which the forced-swim
test was employed. The immobility time of mice in
FST was shorten after 24 hr after acute treatment ofa 5-HT2A agonist, DOI, which was inhibited by a
5-HT2A antagonist, volinanserin (Fig. 1).
Next, we investigated the brain region involvedin the antidepressant effect of 5-HT2A agonists, forwhich mice with 5-HT2A agonist
treatment were sacrificed for c-Fos
staining 60 min after FST. We
found that increase in the number ofc-Fos-positive cells in GABAergic
neurons of the lateral septum (LS)of mice treated with DOI, which
was also suppressed by volinanserin(Fig. 2).

Discussion & Conclusion
Together with the results as described above, the shorten immobility time in FST and increase in
the number of c-Fos-positive cells in the LS after FST occur dependency of 5-HT2A stimulation,suggesting that antidepressant effect of 5-HT2A agonist is associated with activation of GABAergic
neurons in the LS. Thus, the lateral septum activation may inhibit the brain region projected fromlateral septum, for which further study is required. Ketamine that has therapeutic effect for treatmentresistant depression has some problem such as psychosis-like behavior as well as the limitation of
the route of administration3. So, new therapeutic strategy is required instead of ketamine. Given that
occurrence frequency of 5-HT2A agonist adverse effect such as hallucination would be less thanketamine2, 5-HT2A may be available for a new target of treatment-resistant depression.

References
1. Carhart-Harris RL, Bolstridge M, Rucker J, Day CM, Erritzoe D, Kaelen M, Bloomfield M,
Rickard JA, Forbes B, Feilding A, Taylor D, Pilling S, Curran VH, Nutt DJ. Psilocybin withpsychological support for treatment-resistant depression: an open-label feasibility study. Lancet
Psychiatry 3:619-627, 2016.
2. Reiche S, Hermle L, Gutwinski S, Jungaberle H, Gasser P, Majić T. Serotonergic hallucinogens
in the treatment of anxiety and depression in patients suffering from a life-threatening disease: A
systematic review. Prog Neuropsychopharmacol Biol Psychiatry 81:1-10, 2018.
3. Berman RM, Cappiello A, Anand A, Oren DA, Heninger GR, Charney DS, Krystal JH.,
Antidepressant effects of ketamine in depressed patients. Biol Psychiatry 47:351-4, 2000.

The Novartis Foundation(Japan)
for the Promotion of Science

94

The Novartis Foundation(Japan)
for the Promotion of Science

一般の皆様へ
うつ病患者の30％は既存の抗うつ薬が効きにくい難治性である。最近の臨床研究から幻覚薬

でセロトニン5-HT2A 受容体（5-HT2A）刺激薬のシロシビンが、難治性うつ病患者に対して、即効的かつ持続的な抗うつ作用を示した。そこで我々は、5-HT2A 刺激薬による抗うつ作用についてマウスを用いて調べたところ、5-HT2A 刺激薬は、うつ病モデルマウスにおいて、即効的かつ持続的な抗うつ作用を示した。さらにその作用には外側中隔核における GABA 神経の活性化が関与していることが証明された。本研究によりうつ病治療における5-HT2A 刺激薬の有用性が示されれば、本研究が新たな抗うつ研究の礎となることが期待できる。

The Novartis Foundation(Japan)
for the Promotion of Science95

The Novartis Foundation(Japan)
for the Promotion of Science

RopGEF in the regulation of planar cell polarity in plants
Teh Ooi Kockokteh@oia.hokudai.ac.jp
Hokkaido University

Summary Abstract
This research proposal addresses the involvement of ROP guanine nucleotide exchange factor(RopGEFs) in the regulation of planar cell polarity (PCP). Using homologous recombination
technology, we generated single knock-out mutants for all six RopGEFs (RopGEF1~RopGEF6)
in Physcomitrella patens. We found that ropgef1 showed multiple and misplaced protonema
protrusions. This suggests that RopGEF1 has a direct role in the regulation of PCP. In the future, wewill focus on the molecular mechanisms of RopGEF1 in mediating moss protonema PCP.
Key Words：Physcomitrella patens, RopGEF, planar cell polarity

Introduction
One of the major unanswered questions in developmental biology is how multicellular organisms
take its shapes by coordinating and integrating internal as well as external environmental cues.
An essential feature of multicellular organisms is their ability to undergo cellular differentiationand polarized growth within the plane of a cell sheet-this planar morphogenesis is termed “planarcell polarity (PCP)1”. The underpinning hypothesis of this proposal is: PCP signaling pathways inplants are analogous to that in the animals but involved distinctively different players. We proposethat RopGEFs modulate moss PCP by relaying auxin-dependent signals from upstream receptors todownstream effectors via regulation of membrane trafficking steps.

Results
The moss genome encodes 6 RopGEFs, RopGEF1~ RopGEF6. To determine which RopGEF(s)are involved in the moss PCP, we generated single knock-out mutants for all RopGEFs. We are
surprised to find that only ropgef1 mutant showed compromised PCP, with multiple protrusions
emerged within a single protonema cells. More importantly, the positions of these new protrusions
are misplaced and switched from apical-end to middle- or basal-end. The following figure shows
representative images of protonemata from ropgef1 andropgef2. Arrow heads indicate the apical-end of cells
where protrusions normally emerged in wild-type.
We also attempted to generate yellow fluorescentprotein (YFP)-fused reporter lines for all the
RopGEFs. YFP N-terminal fusions of RopGEFs, YFPRopGEFs driven by an overexpression promoter did
not produce any detectable signal. This hampers our
subsequent effort to perform live cell imaging and coimmunoprecipitation experiments.

The Novartis Foundation(Japan)
for the Promotion of Science96

The Novartis Foundation(Japan)
for the Promotion of Science

Discussion & Conclusion
Although the ROP signaling pathway has been implicated in the polarized tip growth of
Arabidopsis root hair, its roles remain obscure in the basal land plant. Our genetic evidence
demonstrates that RopGEF1 is the only RopGEF necessary to mediate PCP in Physcomitrella
patens. Whether other RopGEFs have redundant or overlapping functions with RopGEF1 remains
to be determined through analyses of double mutants. Failure to observe fluorescence in our
YFP-RopGEFs reporter lines suggests that the N-terminal of RopGEFs may be involved in posttranslational modification for membrane anchorage. Obstructing this anchorage by fusing afluorescent protein may therefore hinder the RopGEF attachment to cell surface and inhibits its
subsequent functions.

References
1. Devenport, D. The cell biology of planar cell polarity. Journal of Cell Biology (2014). doi:10.1083/
jcb.201408039

一般の皆様へ
Our study highlights the importance of ROP signaling pathway in the regulation of body plantdevelopment in early land plants.

（我々の研究は初期陸上植物の植物体の発生調節における ROP シグナル伝達経路の重要性を強調

している。
）

The Novartis Foundation(Japan)
for the Promotion of Science

97

The Novartis Foundation(Japan)
for the Promotion of Science

A novel oxytocin neuronal system for regulating
lactating behavior in mice
Yoshikage Muroi
muroi@obihiro.ac.jp
Obihiro University of Agriculture and Veterinary Medicine

Summary Abstract
Mammalian dams nurture their pups by lactation. In this study, we examined whether oxytocin is
involved in regulating nurturing behavior in a nutritional state-dependent manner. We found a novel
function of oxytocin to facilitate nurturing behavior under negative energy conditions. Oxytocin actsin the dorsal raphe nucleus. In addition, oxytocin may be released from the paraventricular nucleusof the hypothalamus in response to presentation with pups outside the nest under mildly negative
energy conditions. Our data suggest that oxytocin facilitates milk delivery to pups by the behavioral
control as well as milk ejection even under negative energy conditions.
Key Words：oxytocin, nurturing behavior, dorsal raphe nucleus, paraventricular nucleus

Introduction
Mammalian newborns, which can obtain energy only from milk before weaning, are deliveredprematurely. Mothers need to partition their energy for the survivals of themselves and their pups.
Nurturing behavior as well as milk production in mammary gland is suppressed under food-restrictedconditions. These biological responses are understood as a self-defense from energy deficiency.
However, dams do not abandon their pups even under negative energy states. In the present study, we
examined how dams can continue to display nurturing behavior under negative energy conditions.

Results
To examine whether nurturing behavior alters in dependence on energy states of dams, we firstcompared the effect of different period of fasting on nurturing behavior. The fed dams or 3-h-fasted
dams displayed nurturing behavior including nest maintenance, retrieval of pups into the nest,
and crouching over the pups during the behavioral test. In contrast, 4-h-fasted or 8-h-fasted dams
decreased the display of nurturing behavior. Dams obtained 0.61 ± 0.05 g of food pellets for 3 h
under fed conditions. These results means that dams display nurturing behavior, regardless of lacking
energy intake for 3 h. To examine the involvement of oxytocin in the dorsal raphe nucleus (DRN)with nutritional state-dependent regulation of nurturing behavior, dams were treated with an oxytocin
receptor antagonist, L-368899. The injection of 2 pmol L-368899 into the DRN did not affect all of
the parameters for nurturing behavior in fed dams, compared to saline injection. However, L-368899
inhibited retrieving behavior and crouching behavior in 3-h-fasted dams. These results indicate thatoxytocin in the DRN is required for displaying nurturing behavior under the 3-h-fasted, but not
fed, condition. To confirm that oxytocin in the DRN has an antagonistic effect on the inhibition of
nurturing behavior by food deficiency, oxytocin was injected into the DRN in 8-h-fasted dams. The
injection of 100 pmol oxytocin into the DRN recovered nurturing behavior in 8-h-fasted dams. These
results indicate that oxytocin in the DRN counteracts the inhibition of nurturing behavior under

The Novartis Foundation(Japan)
for the Promotion of Science98

The Novartis Foundation(Japan)
for the Promotion of Science

negative energy conditions.
The paraventricular nucleus (PVN) and supraoptic nucleus (SON) in the hypothalamus are themajor nucleus where oxytocin neurons are distributed. We asked whether oxytocin is released
from the PVN and/or SON in response to presentation with pups following 3-h-fasting. In the
immunohistochemistry, presentation with pups decreased the number of oxytocin-positive cells
and the intensity of oxytocin-positive signals in the PVN of 3-h-fasted dams. To determine whether
the decrease in the number of oxytocin-positive cells and the intensity of oxytocin-positive signals
in the PVN, we investigated the changes in the content of oxytocin in the PVN by using enzymelinked immunosorbent assay. Presentation with pups decreased the content of oxytocin in the PVNof 3-h-fasted dams. These results indicate that oxytocin may be released from the PVN in response
to presentation with pups following 3-h-fasting. We investigated how oxytocin is released from the
PVN. Because presentation with pups did not affect the concentration of oxytocin in the serum in
3-h-fasted dams, oxytocin is unlikely to be released as a hormone in response to presentation withpups under the 3-h-fasted condition. We next examined whether oxytocin is transported from the
PVN into the DRN by the synaptic transmission. In the dams that were injected with a retrograde
tracer fluorogold into the DRN, few oxytocin neurons were positive to fluorogold in the PVN, it isunlikely that oxytocin is transported from the PVN into the DRN by synaptic transmission.

Discussion & Conclusion
In the present study, we found that oxytocin in the DRN is required for displaying nurturing
behavior under a mildly negative energy state in lactating female mice. Oxytocin recovered thedisplay of nurturing behavior inhibited by 8-h-fasting. These results suggest that oxytocin in the
DRN counteracts the inhibitory effect of fasting on the display of nurturing behavior. Oxytocin may
be released from the PVN in response to presentation with pups under negative energy conditions.
Oxytocin is likely to reach the DRN by the styles other than endocrine or synaptic transmission.
Oxytocin may be released by volume transmission to diffuse from the PVN into the DRN. Moreresearches are required for clarifying how oxytocin reaches from the PVN into the DRN.

一般の皆様へ
哺乳類の母親は乳で仔を育てます。エネルギーを失うことは自分の生存に不利なことですが、母親は授乳により自分のエネルギーを自ら進んで仔に分け与えます。本研究ではそんな母親の性質を司る仕組みを明らかにすることを目指しました。今回の研究で、低エネルギー状態でもオキシトシンが脳に作用することで母親は授乳し続けることがわかりました。これまでにオキ
シトシンは乳腺に作用し射乳を促すことがわかっていましたが、母親の行動にも影響し授乳を促すことが明らかになりました。オキシトシンが自己犠牲的なお母さんの性質の一面を担っていると考えられます。

The Novartis Foundation(Japan)
for the Promotion of Science99

The Novartis Foundation(Japan)
for the Promotion of Science

Molecular mechanisms for the bifurcation of thymic epithelial cells
Izumi Ohigashi
ohigashi@genome.tokushima-u.ac.jp
Division of Experimental Immunology, Institute of Advanced Medical Sciences,
University of Tokushima

Summary Abstract
To better understand molecular mechanisms that govern the differentiation and development ofcortical thymic epithelial cells (cTECs) and medullary TECs (mTECs) in the thymus, we carried out
omics analysis of these epithelial cells. Here we report the transcriptome and proteome profiling of
TECs. We also report the functional significance of a molecule that is highly expressed in cTECs
than mTECs in the thymus.
Key Words：Thymus, Thymic epithelial cells

Introduction
cTECs and mTECs characterize T cell-producing and -selecting functions of the thymus. We
have previously reported that cTECs and mTECs are derived from bipotent TEC progenitors thattranscribe beta5t, which is a component of thymoproteasome specifically expressed in cTECs andimportant for the positive selection of CD8T cells1-3. However, molecular mechanisms regulating thedevelopment of bipotent TEC progenitors into cTECs and mTECs is unclear. In this study, we aimed
to better understand molecular mechanisms that govern the differentiation and diversification of
bipotent TEC progenitors into cTECs and mTECs.

Results
We carried out transcriptome and proteome analysis of cTECs and mTECs isolated from wild-typemice and compared those profiles. We also compared profiles between wild-type cTECs and beta5tdeficient cTECs. We found that transcriptomic and proteomic profiles were sharp contrast between
cTECs and mTECs, whereas those are similar between wild-type cTECs and beta5t-KO cTECs.
However, proteomic profiles exhibited the reduction of proteasome components in protein amount in
cTECs by beta5t-deficiency. We also confirmed the reduction of proteasome components in beta5tdeficient cTECs by western analysis and flow cytometry analysis. On the other hand, constitutive
stress response, which was reported to be ignited by proteasome deficiency, was not observedin beta5t-deficient cTECs, indicating a highly specific impact of beta5t on proteasome subunit
composition in cTECs, rather than a pervasive effect on cTEC functions (submitted to Cell Reports).
Next, we isolated transcriptional regulators that showed significant difference in the expression
between cTECs and mTECs in omics profiles, to identify the responsible molecules regulating thedifferentiation and development of cTECs and mTECs. We examined thymuses isolated from several
kinds of deficient mice of molecules. Among them, we found that the systemic deficient mice ofa molecule that belongs to homeobox transcription factor and is highly expressed in cTECs thanmTECs exhibit thymus hypoplasia and reduced expression of functional cTEC-molecules. However,
the expression of this transcription factor is ubiquitous. Therefore, to examine the impact of thisThe Novartis Foundation(Japan)
for the Promotion of Science

100The Novartis Foundation(Japan)
for the Promotion of Sciencetranscription factor in TEC specifically, we are currently generating conditional deficient mice.
In addition to this homeobox transcription factor, we focused on Pithd1 that is highly detected in
cTECs than mTECs. It has been reported that Pithd1 regulates the transcription of Runx1 resulting
in the regulation of megakaryocyte differentiation in in vitro4. Furthermore, Pithd1 has a putative
domain capable of interacting with proteasome. However, the functional significance of Pithd1 inin vivo is unknown. We first examined the expression of Pithd1 in the thymus and several othertissues. We confirmed that Pithd1 is highly expressed in cTECs than other thymic cells, including
mTECs, and other tissues. Additionally, highly expression of Pithd1 was detectable also in testis.
We next examined the proteasome interacting capability of Pithd1. Immunoprecipitation followed
by immunoblotting analysis showed that Pithd1 is incapable of interacting with thymoproteasomein cTECs. On the contrary, Pithd1 was able to interact with proteasome expressed in testis. Then,
we next generated Pithd1-deficient mice to examine the role of Pithd1 in in vivo. We found that theorganization of cortical and medullary microenvironment was not disturbed by Pithd1 deficiency. We
also found that the differentiation and development of cTECs as well as T cell development in thethymus was not impaired in Pithd1-deficient mice. These results indicate that Pithd1 is dispensable
in the development and function of cTECs. On the other hand, we noticed that Pithd1-deficient malemice are infertile. Then, we carried out the detail analysis of sperm isolated from Pithd1-deficientmice. Pithd1-deficient sperm exhibited the abnormal morphology and impaired motility. Furthermore,in vitro fertilization assay revealed that Pithd1-deficient sperm are disable to fertilize even in the
cumulus free condition. Finally, we carried out proteome analysis of sperm isolated from wild-typemice and Pithd1-deficient mice. We found that the amount of proteins known to be involved in themale fertility is decreased in sperm from Pithd1-deficient mice. Therefore, these results indicate that
Pithd1 is indispensable for the male reproductive system (manuscript under preparation).

Discussion & Conclusion
The omics analysis we carried out in this study revealed previously unknown quantitative profilesof unbiased proteomes in freshly isolated cTECs and mTECs. In combination of transcriptomic
analysis with proteomic analysis, we could isolate several molecules that are differently expressed
between cTECs and mTECs and are related to the transcriptional regulation. Among them, we are
currently studying the role of a molecule that belongs to homeobox transcription factor. In addition
to this, we are also studying the role of molecule that functions as transcriptional co-activator. Ouromics data from cTECs and mTECs offer a useful resource for further exploration of the biology ofTECs and their subpopulation. In this study, we also examined the functional significance of Pithd1
that was highly detected in cTECs than mTECs in omics analysis. Although no abnormalities weredetected in the thymus, we unexpectedly detected male infertility in Pithd1-deficient mice. Pithd1deficent sperm exhibited morphological abnormality. Especially, the frequency of sperm having multi
heads was significantly increased in Pithd1-deficient mice compared to control mice. Our results
will contribute to elucidating the mechanism for acquisition of male fertilization capability, and thedevelopment of therapeutic tool for male infertility.

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

References
1. Murata S, Sasaki K, Kishimoto T, Niwa S, Hayashi H, Takahama Y, Tanaka K. Regulation of
CD8+T cell development by thymus-specific proteasome. Science 16:1349-1353, 2007.
2. Ohigashi I, Zuklys S, Sakata M, Mayer CE, Zhanybekova S, Murata S, Tanaka K, Hollander GA,
Takahama Y. Aire expressing thymic medullary epithelial cells originate from β5t-expressing
progenitors. PNAS. 110:9885-9890, 2013.
3. Ohigashi I, Zuklys S, Sakata M, Mayer CE, Hamazaki Y, Minato N, Hollander GA, Takahama Y.
Adult thymic medullary epithelium is maintained and regenerated by lineage-restricted cells rather
than bipotent progenitors. Cell Rep. 13:1432-1443, 2015.
4. Lu B, Sun X, Chen Y, Jin Q, Liang Q, Liu S, Li Y, Zhou Y, Li W, Huang Z. Novel function of
PITH domain-containing 1 as an activator of internal ribosomal entry site to enhance RUNX1
expression and promote megakaryocyte differentiation. Cell. Mo. Life. Sci. 72:821-832, 2015.

一般の皆様へ
免疫の中枢器官である胸腺を構成する皮質、および、髄質上皮細胞は、生体防御に必須な T細胞の産生を担います。本研究では、皮質 / 髄質上皮細胞の分化を制御するメカニズムの解明を目指しました。上皮細胞の分化制御メカニズムを解明することは、将来、免疫不全症や自己免疫疾患の根本的治療法開発につながると考えられます。また、本研究を進める中で、胸腺上皮細胞で発現する遺伝子が精巣でも発現しており、生殖能獲得に重要であるという、予想外の結果を得ました。男性不妊発症のメカニズム解明や、治療法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

102

The Novartis Foundation(Japan)
for the Promotion of Science

The role of Heat shock protein 40 Member C1
in hepatic lipid metabolism
Atsuko Nakatsuka
atsuko-n@md.okayama-u.ac.jp
Okayama University Hospital.

Summary Abstract
Recently non-alcoholic fatty liver disease is increasing worldwide. However, mechanisms ofprogression from simple fatty liver to steatohepatitis (NASH) are not well explored. We focused on
Dnajc1, which is one of 40 kDa heat shock proteins. In Dnajc1 null mice fed with high-fat highsucrose diet, fatty liver was exacerbated with upregulation of fatty acid synthase and lipocalin 2
revealed by DNA microarray analysis. In vitro analysis, Dnajc1 activated AMPK, and inhibitedJNK phosphorylation. GRP78/eIF4B and keratin were identified as Dnajc1 interactive molecules,
respectively. Dnajc1 may be one of key molecules, that prevents fatty liver and a progression to
NASH.
Key Words：Non-alcoholic fatty liver disease, Heat shock protein, Dnajc1,

Introduction
Heat shock protein (Hsp)s are known as molecular chaperones, which are involved in protein
quality control, and categorized by their molecular weights. Hsps of 40 kDa (Hsp40s) is also
known as DnaJ, and there are at least 50 homologs of HSP40s/DNAJ in human (1). We focused on
Dnajc1, which we identified as 78 kDa glucose-regulated protein (GRP78)-interactive moleculeon hepatocyte (2). However, roles of Dnajc1 in human pathophysiology and pathogenesis are notwell investigated. In present study, we investigated beneficial effects and molecular mechanisms ofDnajc1 on non-alcoholic fatty liver diseases.

Results
1) Distribution of Dnajc1 protein in various organs of C57BL/6 mice.
We performed western blot analyses using multiple organ samples of C57BL/6 mice, andubiquitous distribution of Dnajc1 was observed. Especially, Dnajc1 was heavily expressed inpancreas and visceral adipose tissues, and they were followed by stomach, liver and kidney. Dnajc1
was also detected in serum samples, suggesting that soluble form of Dnajc1 is secreted into blood.
2) Expression of Dnajc1 in cultured hepatocyte, HepG2 and H4-II-E-C3 cells.
We investigated the expression of Dnajc1 in cultured hepatocyte cell line, HepG2 and H4-IIE-C3 cells. In the presence of 100 nM insulin, 1μg/ml tunicamycin, 1 μM thapsigargin, and 500
μM palmitate, Dnajc1 protein increased. In immunofluorescence staining, insulin, tunicamycin,
thapsigargin and palmitate increased intracellular punctate signals of Dnajc1 (Figure 1). Next, when
H4-II-E-C3 cells were transfected with pcDNA3.1-Dnajc1 vectors, prominent intracellular vacuolar
formations were observed (Figure 2), and Dnajc1 on vacuole was partially co-localized with alysosomal membrane associated protein 2 (lamp2).

The Novartis Foundation(Japan)
for the Promotion of Science103

The Novartis Foundation(Japan)
for the Promotion of Science

3) Fatty liver was exacerbated in Dnajc1-/- under high-fat high-sucrose (HFHS) diet.
Dnajc1 hepatocyte-specific null mice were generated by crossing floxed Dnajc1 mice with
C57BL/6 transgenic mice expressing Cre recombinase under control of the albumin promoter.
Dnajc1-/- and Djanc1+/+ mice were fed with HFHS diet or standard chow (STD), and at 25 weeks
of age we obtained blood and liver samples. There was no difference of body weight gain and blood
glucose levels between Dnajc1-/- and Dnajc1+/+. At 10 weeks of age, we performed intraperitoneal
glucose tolerance test, and an area under the curve (AUC) of blood glucose in Dnajc1-/- was higher
than Dnajc1+/+ under HFHS (40245±5952 vs 33313±2044 mg･hr/dL) with statistically significance,
although there were no differences under STD. In insulin tolerance test, blood glucose levels were
not altered between Dnajc1-/- and Dnajc1+/+. Food intake was also not altered between both groups.
At 25 weeks of age, liver weight of Dnajc1-/- was heavier than Dnajc1+/+ under HFHS diet and fatty
liver was histologically exacerbated in Dnajc1-/-.
In DNA microarray analysis using liver tissues, fatty acid synthase (FAS) was one of the top 5
upregulated genes in Dnajc1-/- compared with Dnajc1+/+. The expression of Lipocalin 2 (LCN2)
also upregulated in Dnajc1-/-. Pathway analysis demonstrated that Dnajc1 was strongly associatedwith three pathways, i.e. metabolic pathway, pathway in cancer, and PI3-Akt pathway.
4) Dnajc1 enhance AMPK activation and inhibited phosphorylation of JNK.
We performed in vitro analysis using cultured hepatic cell line, H4-II-E3. Overexpression of
Dnajc1 increased an expression of phosphorylated AMP-activated protein kinase (AMPK), andsupressed phosphorylation of c-Jun N-terminal kinase (JNK).

The Novartis Foundation(Japan)
for the Promotion of Science

104

The Novartis Foundation(Japan)
for the Promotion of Science5) Identification of Dnajc1 interactive molecules
We generated plasmid expressing p3xFLAG-Dnajc1 and cloned genes were transfected into HK2cells. Soluble proteins were purified by anti-FLAG M2 affinity agarose gel and subjected to SDSPAGE and Ag staining. Visible bands were excised and in-gel digested with trypsin and analyzed
liquid chromatography-tandem mass spectrometry. As a result, several candidate molecules wereidentified as Dnajc1-interactive molecules; heat shock-related 70kDa protein 2 (HSP72), 78kDa
glucose-regulated protein (GRP78), eukaryotic elongation initiation factor 4B (eIF4B), SCY1-likeprotein 2, importin-8, RL40, Ubiquitin-60S ribosomal protein L40, and keratin. We confirmed abinding of Dnajc1 to GRP78/eIF4B and keratin, respectively by immunoprecipitation and western
blot analysis.

Discussion & Conclusion
Fatty liver and insulin resistance were exacerbated in Dnajc1-/- mice under HFHS diet. DNA
microarray analysis showed that deficiency of Dnajc1 is associated with up-regulation of FAS and
LCN2. These findings indicate that Dnajc1 protect against lipid accumulation and inflammation inliver. Now we are going to promote further investigations for insulin signaling, lipid metabolism, andinflammatory signaling.
Dnajc1 forms a complex with GRP78/eIF4B. It is known that eIF4B plays roles on mRNA capdependent translation initiation, and the decrease in eIF4B phosphorylation correlates with a decreasein cap-dependent translation (3). Kose S reported that Hsp70s are transported into nuclei during heatshock stress (4), therefore we speculate that Dnajc1/GRP78 complexes have roles in nulclei under
metabolic or cellular stresses. Further investigations are required to confirm this hypothesis.
Dnajc1 also binds to keratin. Mallory-Denk bodies (MDBs) are cytoplasmic inclusions, foundin various liver diseases. MDBs consist of intermediate filament proteins, keratins 8/18, ubiquitin,
p62 and Hsps (5). Decrease of proteasome activity and/or impairment of autophagy are one ofmechanisms that form MDBs, therefore we would like to investigate the relation of Dnajc1 andintracellular degradation systems, such as autophagy and ubiquitin-proteasome pathway. Other reportdemonstrated that keratin regulate FasR signaling through modulation of acin/ezrin interplay at lipid
rafts in hepatocyte (6). Further investigation for a role of Dnajc1/keratin complex on Fas-induced
apoptosis are needed.

References(1) Kampinga HH, Hageman J, Vos MJ, et al., Guidelines for the nomenclasture of the human heatshock proteins. Cell Stress and Chaperons 14:105-111, 2009.
(2) Nakatsuka A, Wada J, Iseda I, et al., Vaspin is an adipokine ameliorating ER stress in obesity as aligand for cell-surface GRP78/MTJ-1 complex. Diabetes. 61(11):2823-2832, 2012.
(3) Dennis MD, Jefferson LS, Kimball SR. Role of p70S6K1-mediated phosphorylation of eI4B and
PDCD4 proteins in the regulation of protein synthesis. J Biol. Chem 51:42890-42899, 2012.
(4) Kose S, Furuta M, Imamoto N. Hikeshi, a nuclear import carrier for Hsp70s, protects cells fromheat shock-induced nuclear damage. Cell 149(3): 578-589, 2012.
(5) Hanada S, Harada M, Abe M, et al., Aging modulates susceptibility to mouse liver Mallory-Denk
body formation. J Histochem Cytochem. 60(6):475-483, 2012.
(6) Gilbert S, Loranger A, Lavoie JN, et. al., Cytoskeleton keratin regulation of FasR signaling
through modulation of actin/ezrin interplay at lipid rafts in hepatocytes. Apoptosis. 17(8):880894, 2012.

The Novartis Foundation(Japan)
for the Promotion of Science

105

The Novartis Foundation(Japan)
for the Promotion of Science

一般の皆様へ近年脂肪肝や脂肪肝炎が増加していますが、発症・進展のメカニズムについて未解明な部分も多くあります。細胞は生存・機能に必要な蛋白を合成しており、蛋白が正しい構造になるのを手助けする多種類の “ シャペロン分子 ” が存在しています。近年、それぞれが、シャペロン

機能とは無関係の、独自の「固有機能」を持つことが分かってきました。Dnajc1 はいまだ未解

明な点が多い分子ですが、我々が以前の研究で、肝細胞の細胞質のみならず細胞表面にも存在し、糖脂質代謝に関係する可能性を見出したシャペロン分子の一つです。Dnajc1 は肝臓で糖代謝を改善させ、炎症を抑制する結果が得られました。さらに今後研究を進め、脂肪肝・脂肪肝炎の予防や治療に結び付けたいと考えています。

The Novartis Foundation(Japan)
for the Promotion of Science106

The Novartis Foundation(Japan)
for the Promotion of Science

Novel strategy targeting neutrophils
in the tumor microenvironment of colorectal cancer
Kenji Kawada
kkawada@kuhp.kyoto-u.ac.jp
Kyoto University, Department of Surgery,

Summary Abstract
SMAD4 is a key transcriptional factor of TGF-β signaling, and acts as a tumor suppressor incolorectal cancer (CRC). In the present study, we explored the immunological effect of SMAD4 on
the tumor microenvironment.
We have shown that blockade of the CXCL1/8-CXCR2 axis and/or CCL15-CCR1 axis could be anovel therapeutic approach against SMAD4-negative CRC.
Key Words：colorectal cancer, SMAD4, CCR1, myeloid cells

Introduction
SMAD4 acts as a tumor suppressor in colorectal cancer (CRC), and loss of SMAD4 protein is
associated with a poor prognosis. We previously reported that loss of SMAD4 in human CRC cells
resulted in the up-regulation of CCL15, which caused the recruitment of CCR1+-myeloid cells via
the CCL15-CCR1 axis to facilitate tumor invasion and metastasis. In the present study, we exploredthe immunological effect of SMAD4 on the tumor microenvironment.

Results
1. CXCL1/8-CXCR2 axis
Given that myeloid cells express several types of chemokine receptors, we hypothesized that therecruitment of myeloid cells could be taking place in the tumor microenvironment via chemokine
receptors other than CCR1.
Human CRC cell lines, SW837 and Caco2, express wild-type SMAD4. To identify thedeterminants responsible for neutrophil infiltration, we conducted a chemokine array containing
a panel of chemokines using the SMAD4-knockdown cells. We found that loss of SMAD4 fromhuman CRC cells (SW837 and Caco2) resulted in a significant increase of CXCL1 and CXCL8
expression, which was associated with the recruitment of neutrophils via CXCR2. In turn, whenneutrophils were exposed to the supernatant of SMAD4-negative CRC cells (SW837), they
produced a large amount of CXCL1 and CXCL8 in vitro.
In human clinical specimens, we found that neutrophil infiltration into the peritumoral stroma
was more marked in SMAD4-negative CRC compared to that in SMAD4-positive CRC, andthat both CXCL1 and CXCL8 were abundantly expressed in the tumor-infiltrating neutrophils.
We further found that the neutrophils isolated from primary CRC expressed significantly higher
levels of CXCL1 and CXCL8 than did those isolated from peripheral blood. Furthermore, we next
characterized the tumor-infiltrating neutrophils using double immunofluorescence staining, andfound that the majority of tumor-infiltrating neutrophils were CXCR2+, and expressed MMP2 andThe Novartis Foundation(Japan)
for the Promotion of Science

107

The Novartis Foundation(Japan)
for the Promotion of Science

MMP9 in addition to ARG1 and IDO.
We measured the levels of CXCL1 and CXCL8 in serum samples from CRC patients (n= 125) and healthy donors (n = 11) by ELISA. Serum CXCL8 level was significantly higher
in CRC patients that in controls (median, 13.7 vs. 2.9 pg/mL; P < 0.01), whereas the serum
CXCL1 level was significantly lower in CRC patients (median, 8.8 vs 100.4 pg/mL; P < 0.01).
We then investigated the serum CXCL8 level by the stage-based classification, and found it wassignificantly increased at Stage II/III and then decreased at Stage IV. To evaluate the clinicaloutcome, we analyzed the OS and RFS of the 125 CRC patients. Statistical analysis indicatedthat the CRC patients with high CXCL8 level exhibited a shorter overall survival and relapse-freesurvival compared to those with low CXCL8 level, although not a significant difference (P = 0.07
and 0.08, respectively). In particular, the Stage II/III cases with high CXCL8 level exhibited asignificantly shorter OS compared to those with low CXCL8 level (P = 0.04). On the other hand,
there was no association between the CXCL1 concentration and prognosis.
2. CCL15-CCR1 axis
Regarding the CCL15-CCR1 axis, we investigated the effect of CCR1 deletion on stromal andhematopoietic cells in mouse models. We used MC38 cells (for primary CRC model) and CMT93
cells (for liver metastasis model).
First, we injected tumor cells into the syngeneic C57BL/6 mice, and found that tumor
development was strongly impaired into the CCR1-knockout mice compared to control mice inMC38 and CMT93 cells.
Next, to delineate the contribution of CCR1 on myeloid cells compared to non-hematopoietic
cells, we performed bone marrow (BM) chimera experiments in which sublethally irradiated mice
were reconstituted with CCR1-deficient BM. After tumor cell inoculation, CCR1-/- recipient mice
showed significantly decreased tumor burden in MC38 (primary CRC model) and CMT93 cells(liver metastasis model).
Finally, we have investigated the effect of anti-CCR1 antibody in mouse moldels. We developed
a new anti-CCR1 antibody, and so we have tested the effect of this antibody in vivo.

Discussion & Conclusion
Regarding the project of CXCL1/8-CXCR2 axis, our data suggest that blocking the CXCL1/8CXCR2 axis may provide the possibility of a novel therapeutic strategy for SMAD4-negative CRC.
CXCR2 and its ligands are responsible for recruiting neutrophils under physiological conditions
and have been implicated in tumor-associated neutrophil mobilization. Notably, pharmacologicalinhibitors of CXCR1/2 are already used in clinical trials of inflammatory diseases (e.g. rheumatoid
arthritis, asthma, and chronic obstructive pulmonary disease) as well as certain cancers (e.g.
melanoma and breast cancer)
Regarding the project of CCL15-CCR1 axis, our data indicated that CCR1 deletion on
hematopoietic cells could suppress CRC progression in mouse models. CCR1 antagonists could be a
promising therapeutic strategy to prevent CRC.

The Novartis Foundation(Japan)
for the Promotion of Science

108

The Novartis Foundation(Japan)
for the Promotion of Science

References
・Ogawa R, Yamamoto T, Hirai H, Hanada K, Kiyasu Y, Nishikawa G, Mizuno R, Inamoto S, Itatani Y,
Sakai Y, Kawada K.
“Loss of SMAD4 promotes colorectal cancer progression by recruiting tumor-associated neutrophils
via CXCL1/8-CXCR2 axis” Clin Cancer Res. 2019 Jan 31. doi: 10.1158/1078-0432.CCR-18-3684.
[Epub ahead of print]

一般の皆様へ
大腸癌に対する新規治療法として、腫瘍微小環境にある宿主因子である骨髄球、とくに好中球に着目して研究を進めています。マウスモデルや臨床検体を用いて検討してきた結果、ケモ

カイン受容体である CCR1 と CXCR2 がとくに重要なターゲットになり得ることを明らかにすることができました。臨床の現場で役立つところまではもう少し時間がかかるとは思いますが、さらに研究を進めていけたらと考えています。

The Novartis Foundation(Japan)
for the Promotion of Science

109

The Novartis Foundation(Japan)
for the Promotion of Science

Evolution of sex determination systems
Yusuke Takehanay_takehana@nagahama-i-bio.ac.jp
Nagahama Institute of Bio-Science and Technology

Summary Abstract
To understand the molecular mechanism underling the XY-ZW transitions, we analyzed sex
chromosomes and sex-determining genes of medaka species. We found that duplicated copies ofDmrt1 at the Y chromosomes were required for male determination in the XY species, Oryzias
haugiangensis. Although no sex-determining gene was isolated in the ZW species, O. hubbsi or O.
javanicus, Gsdf was not a conserved downstream component of male determination pathway in O.
hubbsi. These results suggest that sex-determining genes have more diverse downstream targets
between XY and ZW systems than previously thought.
Key Words：Sex-determining genes / Sex chromosome / Medaka fishes

Introduction
Sex chromosomes harbor a primary sex-determining signal that triggers sexual development of theorganism. However, independent evolution of sex chromosomes is widespread in non-mammalianvertebrates, suggesting that sex determination mechanisms are regulated by different genes andhave evolved rapidly. Medaka fishes in the genus Oryzias have different sex chromosomes withdifferent systems (XY and ZW), providing ideal condition for investigating the mechanisms that leadto the rapid turnover of sex chromosomes. In this study, we analyzed sex determination genes andpathways in both XY- and ZW-species to understand the molecular mechanism underling the XYZW transitions.

Results
In Oryzias haugiangensis having XY system, we found Dmrt1 gene at its sex-determining locus
on Chr 10. Fluorescence in situ hybridization analysis showed that a BAC clone containing Dmrt1
gave a strong hybridization signal only on the Y chromosome, but not on the X chromosome.
In addition, weak signals were obtained at the subtelomeric region of a chromosome pair that isequivalent to Chr 9 (autosome), suggesting multiple duplicated copies of Dmrt1 only on the Y
chromosome. Quantitative-PCR assay indicated approximately 10 copies of Dmrt1 genes on the Y
chromosome, confirming its multiple array. Furthermore, Dmrt1 shows higher expression exclusively
in XY individuals at hatching day (the sex-determining period). Then we knockout the Dmrt1 using
CRIPSR/Cas system to determine its sex determining function. XY individuals having multiple
mutations on Y chromosomal Dmrt1 developed as female, suggesting that Dmrt1Y was required formale development.
In O. hubbsi and O. javaniucs, both species have ZW system but their sex chromosomes are nothomologous (Chr 5 and Chr 16, respectively). Based on synteny analysis, we found several candidate
genes on their sex chromosomes, and knocked out these genes using CRIPSR/Cas system. However,no sex reversal phenotype was observed in any genes, suggesting unknown genes at their sex-

The Novartis Foundation(Japan)
for the Promotion of Science

110

The Novartis Foundation(Japan)
for the Promotion of Science

determining loci. Then we sequenced whole genome of O. javanicus to obtain a reference genome
sequence in the javanicus species group. Complementary sequencing approaches including long-read
technology and data integration with a genetic map allowed the final assembly of 908 Mbp of the O.
javanicus genome. Further analyses estimate that the O. javanicus genome contains 33% of repeat
sequences and has a heterozygosity of 0.96%. The achieved draft assembly contains 525 scaffolds
with a total length of 809.7 Mbp, a N50 of 6.3 Mbp and a L50 of 37 scaffolds. We identified 21454
expressed transcripts for a total transcriptome size of 57,146,583 bps. To detect sex chromosomespecific region and isolate candidate genes for sex determination, we are now sequencing haploid
individuals having either Z or W chromosome in both species.
In Oryzias, Gsdf is considered to be a key component of the male determination pathway, because
Dmy and Sox3Y have evolved as the master sex-determining gene by activating the downstream Gsdf
in O. latipes and O. dancena respectively. In addition, Gsdf itself has evolved as male determining
gene in O. luzonensis. These findings suggests that Gsdf is the conserved downstream components
and Gsdf itself or its potential transcriptional regulators might have been generated a novel sex
determination pathway. In fact, we obtained sex reversal XY females in homozygous mutants of
Gsdf in the XY species, O. dancena, which was a close relative of O. hubbsi. On the other hand,
we found that Gsdf is not required for male determination in O. hubbsi, having a ZW system. In thehomozygous mutants of Gsdf, ZW fish developed as females having ovary and ZZ as males having
testis although they showed excessive proliferation of germ cells, demonstrating that Gsdf is not
required for male determination in this ZW species. This Gsdf–independent male determination
system suggests other contributors to generate novel sex determination pathway in ZW system.

Discussion & Conclusion
Our data suggest that duplicated copies of Dmrt1 on the Y chromosome is the sex-determining
gene of the XY species, O. haugiangensis. Similar situation is also observed in O. latipes because its
sex-determining gene, Dmy is the Y chromosome-specific duplicated copy of Dmrt1. These findings
suggest that duplications and subsequent functional divergences of Dmrt1 have contributed togenerate new sex-determining genes independently in O. latipes and O. haugiangensis.
As for their sex determination pathway, we found that Gsdf was not a conserved downstream
component of male determination pathway at least in the ZW species, O. hubbsi. This indicates that
downstream target of its sex-determining gene was different from that of other XY species. These
results suggest that sex-determining genes have more diverse downstream targets between XY andZW systems than previously thought.

一般の皆様へほとんどすべての動物にオス・メスがあるのと対照的に、オス・メスを決定する仕組みは動物種によってさまざまです。しかし、この性決定の仕組みがどのように多様化してきたのかは、
ほとんどわかっていません。今回の研究により、メダカ属という近縁なグループ内で、下流遺伝子の重複転座によって異なる性染色体が独立に進化してきたことや、XY 型と ZW 型で性決定遺伝子の作用点が異なることを示すことができました。これらの成果は、性決定の仕組みが（従来考えられてきたよりも）さらに多様である可能性を示唆しています。

The Novartis Foundation(Japan)
for the Promotion of Science

111

The Novartis Foundation(Japan)
for the Promotion of Science

Design principle for specific chemical probes toward
3D neuropathology
Kazuki Tainaka
kztainaka@bri.niigata-u.ac.jp
Brain Research Institute, Niigata University

Summary Abstract
Current biopsy and histology have long depended on sectioned 2D images with various chemical
staining methods and specific immunohistochemistry. Facile 3D visualization of human brain
tissue with high resolution would provide a novel concept of the neuropathological diagnosis andcontribute our understanding of pathological mechanisms based on comprehensive and quantitative
analysis of individual biomarker. In this project, we aimed at establishing a novel 3D neuropathology
by developing a highly efficient clearing protocol for human brain tissue and combining with specificchemical probes to visualize specific targets.
Key Words：Tissue clearing, 3D imaging, Chemical probes, 3D neuropathology

Introduction
Comprehensive analysis of pathological molecules (e.g. Senile plaques, pTau, α-synuclein) in
human brain tissue would accelerate our understanding of molecular bases in neurodegenerative
diseases. Three-dimensional (3D) imaging based on tissue clearing technique that are compatible
with various fluorescent labeling techniques are promising approaches. Recently, we have developed
a whole-brain/body clearing and imaging method, termed CUBIC (Clear, Unobstructed Brain/Body
Imaging Cocktails and Computational analysis)1,2. However, to establish tissue-clearing based 3D
neuropathology, we have to 1) improve clearing performance for human brain tissues and to 2)
discover chemical probes compatible with tissue clearing protocols.

Results
In this study, we applied a strategy for designing hydrophilic chemical cocktails optimized fordelipidation and refractive index (RI) matching step. To achieve the scalable clearing of human brain
tissue samples, we considered a high-throughput chemical screening system for delipidation and RI
matching.
In delipidation, the lipid solubility of a chemical is roughly and inversely correlated with theturbidity of a fixed brain suspension treated with the chemical1. Thus, we obtained lipid solubility
scores from the OD600 of a mixture of brain suspension and each chemical solution. To determine
the effective chemical parameters for delipidation, we conducted comprehensive chemical profilingby using a multivariate linear regression model. Our model revealed logP (octanol/water partitioncoefficient) to be the most influential property. Actually, the LS score of less polar amines waslinearly correlated with their logP. Therefore, water-soluble, relatively hydrophobic, and uncharged
amine derivatives had the potential for high lipid solubility. Next, we measured the residual
phospholipid and cholesterol contents of mouse brains treated with the chemicals, and evaluated the

The Novartis Foundation(Japan)
for the Promotion of Science

112

The Novartis Foundation(Japan)
for the Promotion of Science

relative transmittance of these brains after RI matching. The delipidation efficiency, especially for thephospholipid content, was correlated with the final tissue transparency, indicating that delipidation
is essential for thorough tissue clearing. Importantly, the logP value of these chemicals could predict
the final transparency of the chemically treated brain.
To clarify whether the RI value of the medium itself is important for the final transparency orwhether specific chemical groups are important for RI homogenization, we used a non-biased
approach to determine the best RI-matched chemicals. To develop an alternative high-RI medium
based on hydrophilic chemicals, we selected candidate chemicals that had high water solubility anda high RI value per unit weight, because the RI value of aqueous medium increases according to thesolute concentration. Chemical profiling of the RI values of 10% chemical solutions with an Abbe
refractometer revealed that aromatic groups exhibited higher RI values among the chemical groups.
We chose 21 extremely water-soluble RI-matching candidates, whose RIs ranged from 1.47 to 1.52.
To examine the clearing performance of these chemicals qualitatively, the OD600 of small pieces ofchemically treated mouse lung was measured. The RI values of the mounting medium showed nocorrelation with the tissue transparency, suggesting that the clearing performance does not depend
solely on the RI value of the medium but also reflects some chemical properties of the constituent.
We further applied the multivariate linear regression model to this result. Interestingly, the amidegroup showed a significantly higher clearing performance than the other chemical groups.
Through the combinatorial evaluation by using biological tissues, we identified effective
delipidation cocktail composed of N-butyldiethanolamine and Triton X-100 (CUBIC-L) and RI
matching cocktail composed of nicotinamide and antipyrine (CUBIC-R). Our new CUBIC protocol
combined with CUBIC-L and CUBIC-R enabled sufficient clearing of human brain tissue including
white matter3. However, we still had to 1) overcome optical issues in human brain tissues including
the resulting brownish color of cleared sample and relatively strong autofluorescence from lipofuscin
and other pigments, and to 2) discover chemical probes compatible with tissue clearing protocols.
By further chemical screening of a series of oxidants and reductants for human brain tissues, wedeveloped several bleach-CUBIC protocols enabling efficient anti-browning and photobleaching ofautofluorescence in human brain tissues. Furthermore, we also screened over 200 chemical probes
visualizing specific targets by using bleached brain samples. Finally, we achieved various kinds ofspecific chemical staining protocols including neurons, fibers, vascular structures, and senile plaques.
Cleared brain samples by our protocol showed preserved subcellular microstructures, and most of
the endogenous protein was retained after CUBIC clearing. In addition, our protocol also largely
retained the antigenicity of the proteins.

The Novartis Foundation(Japan)
for the Promotion of Science

113

The Novartis Foundation(Japan)
for the Promotion of Science

Discussion & Conclusion
In this project, we described a strategy for developing hydrophilic chemical cocktails for tissue
delipidation, and RI matching based on comprehensive chemical profiling. The chemical profilingrevealed important chemical factors: salt-free amine with high logP for delipidation and aromatic
amide for RI matching. Amine derivatives with high logP values are expected to be more cellpermeable and more miscible with hydrophobic lipids. Since highly delipidated tissues are regarded
as protein-based biomaterials, it is plausible that hydrophilic aromatic amides contributed to RI
homogenization due to their efficient solvation of protein backbone amides. The strategic integrationof optimal chemical cocktails provided an effective clearing protocol for human brain tissues. This
strategy represents a future paradigm for the rational design of hydrophilic clearing cocktails that canbe used for large tissues.
By combining of bleach step, we finally enabled multicolor imaging of brain tissue samples.
Comprehensive dye screening provided a series of specific chemical probes for neurons, fibers,
vascular structures, and senile plaques. Our clearing and staining protocol would be expected toestablish future 3D neuropathology.

References
1. Susaki EA, Tainaka K, Perrin D, Kishino F, Tawara T, Watanabe TM, Yokoyama C, Onoe H,
Eguchi M, Yamaguchi S, Abe T, Kiyonari H, Shimizu Y, Miyawaki A, Yokota H, Ueda HR:
Whole-brain imaging with single-cell resolution using chemical cocktails and computational
analysis. Cell, 2014,157(3):726-739.
2. Tainaka K, Kubota SI, Suyama TQ, Susaki EA, Perrin D, Ukai-Tadenuma M, Ukai H, Ueda HR:
Whole-body imaging with single-cell resolution by tissue decolorization. Cell, 2014, 159(4):911924.
3. Tainaka K, Murakami TC, Susaki EA, Shimizu C, Saito R, Takahashi K, Hayashi-Takagi A,
Sekiya H, Arima Y, Nojima S, Ikemura M, Ushiku T, Shimizu Y, Murakami M, Tanaka KF, Iino
M, Kasai H, Sasaoka T, Kobayashi K, Miyazono K, Morii E, Isa T, Fukayama M, Kakita A, Ueda
HR: Chemical Landscape for Tissue Clearing Based on Hydrophilic Reagents. Cell Rep., 2018,24(8):2196-2210.e9.

一般の皆様へ
現在の神経病理学では、薄切標本に基づく 2 次元平面の病理学的解析が基盤となっています

が、平面の病理学的解析だけでは対処困難な多くの課題があります。私たちは、この課題を解決するために「立体的な脳組織を立体のままもれなく観察すること」を実現する、組織透明化技術に基づく 3 次元イメージング CUBIC を開発しました。今回の助成研究では、ヒト脳組織の高度な透明化に取り組むと共に、透明化手法に適用可能な種々の染色手法を確立しました。本
手法を用いて、3 次元画像に基づく病理診断技術の実現を目指します。

The Novartis Foundation(Japan)
for the Promotion of Science

114

The Novartis Foundation(Japan)
for the Promotion of Science

Molecular basis of osmoregulatory ionocytes
Hiroshi Miyanishimiyanishi@cc.miyazaki-u.ac.jp
University of Miyazaki

Summary Abstract
The differentiation mechanisms of osmoregulatory ionocyte in gills are not fully understood. We
focused on transcription factor foxi3 and concluded that foxi3 is necessary for differentiation of
all-types of ionocytes in medaka. To identify novel target involved in ionocyte differentiation, we
conducted comparative transcriptome analysis in specific FW- and SW-type ionoctes. According
to the transcriptome analysis, we identified at least 20 genes involved in functional differentiation
between FW- and SW-type ionocytes.
Key Words：Environmental adaptation, Osmoregulation, ionocyte, medaka,

Introduction
Teleost fishes maintain their body fluid osmolality at approximately one-third that of seawater
regardless of whether they inhabit freshwater (FW) or seawater (SW). Then, regulation of Na+ and
Cl- is of primary importance in adaptation of teleost fish to aquatic environments. Gill ionocytes(also referred to as mitochondrion-rich cells or chloride cells) are in maintaining the body fluid
homeostasis, absorbing and excreting Na+ and Cl- in FW and SW, respectively. Therefore, ionocytes
assume an important role for environmental adaptation in fish. However, the mechanisms of ionocyte
differentiation and functional plasticity are largely unknown.

Results
In zebrafish, forkhead box I (foxi)3 is an important transcriptional factor involved indifferentiation of FW-type ionocytes (1). However, zebrafish is stenohaline FW fish. Therefore, ineuryhaline teleosts, little is unknown about the differentiation factor inducing functional expression
to SW-type ionocyte. To clarify the localization of each medaka FOXI3 in all types of ionocytes,
immunohistochecal analysis was appliedin FW- and SW-acclimated medaka
gills. FOXI3 localized in nuclei of all
types ionocytes (Fig. 1). For functional
analysis, we first performed foxi3 geneknockdown. Then approximately 90%
embryos injected antisense oligo weredied within 2-day post fertilization (dpf).
In response to this result, foxi3 mRNA
was injected in one-cell stage embryos
in FW and SW medium. The number of ionocytes were significantly increased by FOXI3 mRNA
over-expression (Fig. 2). For loss-of function analysis, we established in vivo conditional geneknockdown in developing embryos. Almost embryos survived in FW and SW by in vivo conditional

The Novartis Foundation(Japan)
for the Promotion of Science

115

The Novartis Foundation(Japan)
for the Promotion of Science

gene knockdown of foxi3
from 2 dpf (ionocytes
expressing stage). Then
number of ionocytes
significantly reduced byin vivo conditional geneknockdown of foxi3 from
2 dpf to 4 dpf. The body
fluid osmolality of SW
acclimated embryo wassignificantly increased by
foxi3 knockdown although
there were no significant differences between control group and knockdowned group in that of FW
one. These results show that the transcription factor foxi3 is necessary for differentiation of all types
ionocytes.
Foxi family consist foxi1, foxi2, foxi3 in vertebrates. In medaka, foxi1, 2 and 3 also wereidentified with whole genome data and PCR cloning. In medaka foxi family, only foxi3 wasspecifically expressed in gill. According to phylogenetic analysis of foxi family, teleost foxi3 genes
were clustered with mammalian foxi1 genes. Therefore, it is highly possible that teleosts foxi3 washomologue of mammalian foxi1 involved in ion regulation of inner ear.
To identify novel target involved in ionocyte differentiation, we conducted transcriptome analysis
in FW- and SW-type ionocte. We first established live tissue staining with mitobright for detection
ionocytes under fluorescent observation in non-fixed frozen gill section. Thereafter, each FWand SW-type ionocyte was collected using laser capture microdissection. The collected samples
were analyzed by single cell RNA-seq. In total average, 51,029,046 reads and 20547 contigs weresequenced. According to comparative transcriptome analysis, the expression of 350 genes in SWtype ionocytes were more than 8 folds higher than in FW-type ionocytes. Meanwhile, the expressionof 1357 genes in FW-type ionocytes were more than 8 folds higher than in SW-type ionocytes. In the
350 genes in SW-type ionocytes, the expression of at least 20 genes in ionocytes were significantly
higher than control SW acclimated pavement cells in gills.

Discussion & Conclusion
We applied euryhaline medaka, Oryzias latipes, as model for studying mechanisms of inocyte
differentiation. In this study, we concluded that the transcription factor foxi3 (mammalian foxi1
homologue) is necessary for differentiation of all types ionocytes in medaka. To identify novel target
involved in ionocyte differentiation, we conducted comparative transcriptome analysis in specificFW- and SW-type ionoctes. According to the transcriptome analysis, we identified at least 20 genes
involved in functional differentiation between FW- and SW-type ionocytes. Thus, we identified
new target genes in this study. in addition, we established the molecular basis of osmoregulatory
ionocytes for clarifying the mechanism of functional differentiation between FW- and SW-typeionocytes in the future.

The Novartis Foundation(Japan)
for the Promotion of Science

116

The Novartis Foundation(Japan)
for the Promotion of Science

References[1] Guh YJ and Hwang PP (2017) Insights into molecular and cellular mechanisms of hormonal
actions on fish ion regulation derived from the zebrafish model. Gen Comp Endocrinol, 251: 1220.

一般の皆様へ
淡水と海水の両方で生きられる魚は、異なる塩分環境に適応するため、鰓に存在する塩類細胞の機能を、環境に合わせてイオンの取り込む（淡水中）、または排出（海水中）するように切り替えます。しかし、塩類細胞の分化および機能的可塑性に関する機構は分かっていません。
まず我々は、転写因子 FOXI1 が、全てのイオン取り込み型および排出型の塩類細胞の分化に必須であることを証明しました。FOXI1 のさらに下流のシグナリングを明らかにするために、イオン取り込み型および排出型の塩類細胞特有の遺伝子プロファイルを明らかにしました。さらなる解析をとおして、なぜ魚が川や海で生きられるか？を明らかにするとともに、FOXI1 の関与が示唆されるヒトの難治性難聴の発症機序の基礎知見に繋げていきます。

The Novartis Foundation(Japan)
for the Promotion of Science

117

The Novartis Foundation(Japan)
for the Promotion of Science

Clarification of molecular mechanisms underlying
the maintenance of satellite cell stemness and its therapeutic
application for Muscular Dystrophy
Shinichiro Hayashi
shinhayashi@ncnp.go.jp
Department of Neuromuscular Research, National Institute of Neuroscience,
National Center of Neurology and Psychiatry

Summary Abstract
Muscle stem cells (MuSCs) cannot maintain their stemness in vitro. In this study, we focused on
the mechanisms of maintenance of MuSC stemness regulated by a transcription factor Klf5. Klf5
knockout MuSCs showed high stemness properties in differentiation medium. By the retinoic acidtreatment, Klf5 expression in MuSCs was decreased and MuSCs maintained their stemness in longterm culture. In addition, the efficacy of MuSC transplantation was significantly improved.
Key Words：Muscle stem cells, muscle differentiation, retinoic acid

Introduction
Skeletal muscle has a robust regenerative capacity, and the cell responsible for muscle regenerationis the Muscle stem cells (MuSCs, satellite cell). MuSC transplantation after in vitro expansion would
be beneficial for the treatment of any muscle disease, particularly in the replacement of geneticallydefective muscle fibers such as those lacking dystrophin in patients with Duchenne muscular
dystrophy (DMD). However, MuSCs rapidly lose their regenerative potential in culture. Therefore,
in this research, we attempted to understand the molecular mechanisms underlying the maintenanceof MuSC stemness.

Results
We have previously shown that the zinc finger transcription factor Klf5 is expressed temporarily
during muscle regeneration and differentiation, and promotes muscle differentiation in cooperation
with MyoD11. These findings led us to analyze the role of Klf5 in MuSCs in the present study. First,
we crossed Pax3Cre/+ mice with Klf5flox/flox mice to generate muscle progenitor cell specific Klf5
knockout mice (Klf5cKO). Then we isolated MuSCs from Klf5cKO and control (Klf5flox/flox) mice byfluorescence-activated cell sorting (FACS). Three days after induction of differentiation, although
FACS sorted control MuSCs fused to form multinucleated myotubes, 35% of Klf5cKO cells failed todifferentiate and maintained expression of Pax7, a stem cell marker of MuSCs. To further elucidate
the functions of Klf5 in MuSCs, we analyzed transcriptome and epigenome in control and Klf5cKO
MuSCs by RNA-seq and ChIP-seq. As we expected, gene ontology analysis revealed the genes
whose expression was significantly reduced by loss of Klf5 were involved in the muscle structuredevelopment, myogenesis and muscle cell development. On the other hand, the genes associatedwith extracellular matrix organization and epithelial mesenchymal transition were upregulated in
Klf5cKO MuSCs. Based on these findings, we hypothesized that when the expression of Klf5 isinhibited, MuSCs maintains their stemness.

The Novartis Foundation(Japan)
for the Promotion of Science

118

The Novartis Foundation(Japan)
for the Promotion of Science

Previously, it has been reported that retinoic receptor agonist Am80 inhibits Klf5 expression andtranscriptional activity in smooth muscle cells2. To determine whether retinoic receptor agonists had
an impact on Klf5 expression in MuSCs, different concentrations of Am80 or all-trans retinoic acid(atRA) were added to the culture medium. Consistent with the previous report, Klf5 expression was
significantly decreased in MuSCs by retinoic receptor agonists. Immunocytochemistry also revealed
that the proportion of Pax7 positive MuSCs was significantly increased by retinoid treatment.
More than 80% of MuSCs expressed Pax7 even after 20 passages. On the contrary, the number of
Myogenin positive differentiating myocytes was decreased by the treatment. It is known that atRA
acts through binding to the RAR and RXR receptors, and each of which has three isoforms α, β andγ. To determine the role of these two receptor classes in atRA effect on MuSC stemness, we treated
MuSCs with RAR agonists and RXR agonists. Treatment with RAR agonists, but not RXR agonists,
inhibited muscle differentiation. We further investigated the effect of RARα, RARβ, and RARγ
depletion on MuSC stemness by siRNA-mediated knock-down. We found that siRARα, but not
siRARβ or siRARγ induced muscle differentiation when we treated atRA. These results indicate that
atRA promotes MuSC stemness and inhibits differentiation through RARα.
We next investigated whether MuSCs expanded by atRA treatment could contribute muscle
regeneration after transplantation. Human MuSCs were cultured with or without atRA andtransplanted by intramuscular injection into the TA muscle of immuno-deficient mdx mice. Twentyfour hours before transplantation, TA muscles of recipient mice were injected with cardiotoxin
to induce muscle damage. One month after transplantation, we investigated the contributionof transplanted cells to the regeneration of muscle tissue by detection of dystrophin+ fibers.
Transplantation of MuSCs cultured with atRA produced significantly more dystrophin+ fibers
than those cultures without atRA treatment. These data suggest atRA treatment enhances ability tocontribute to muscle regeneration.

Discussion & Conclusion
Therapeutic approaches to both repair myofibers and repopulate the MuSC pool are required
for DMD patients. MuSCs have great potentials in cell-based therapies to treat muscle diseases.
However, conventional culturing condition cannot be utilized because MuSCs differentiate andquickly lost their abilities to regenerate muscles in vivo. In this study, we found that MuSCs from
Klf5cKO showed highly resistance to differentiation. In addition, we found that atRA treatment
inhibits MuSC differentiation and MuSC stemness was maintained through RARα. Transplantationof MuSCs treated with atRA efficiently contributed to the repair of muscle regeneration. Although
atRA inhibits Klf5 expression in MuSCs, the relationship between Klf5 and retinoic acid is still
unclear. Further studies are ongoing. In conclusion, we provide the evidences for an important role of
the retinoic acid signaling in the regulation of maintenance of the stem cell state of MuSCs.

The Novartis Foundation(Japan)
for the Promotion of Science

119

The Novartis Foundation(Japan)
for the Promotion of Science

References
1. Hayashi S, Manabe I, Suzuki Y, Relaix F, Oishi Y. Klf5 regulates muscle differentiation by directly
targeting muscle-specific genes in cooperation with MyoD in mice. Elife. 2016;5. pii: e17462.
2. Fujiu K, Manabe I, Ishihara A, Oishi Y, Iwata H, Nishimura G, Shindo T, Maemura K, Kagechika H,
Shudo K, Nagai R. Synthetic Retinoid Am80 Suppresses Smooth Muscle Phenotypic Modulation
and In-Stent Neointima Formation by Inhibiting KLF5. Circ Res. 2005 ;97(11):1132-41.

一般の皆様へ
遺伝性の筋疾患である筋ジストロフィーの根本的な治療法は未だ確立されていません。筋幹細胞を移植する方法が根治療法の一つとして期待されていますが、生体外で筋幹細胞の未分化性を保持したまま培養することが難しく、筋幹細胞の未分化性維持機構の解明が課題となっています。本研究では筋幹細胞の未分化性をレチノイン酸受容体アゴニストによって維持できること、また、そのシグナルは RAR αを介していることを明らかにしました。本研究をさらに進め、難治性筋疾患治療法開発に役立てたいと考えています。

The Novartis Foundation(Japan)
for the Promotion of Science

120

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 Science

121

The Novartis Foundation(Japan)
for the Promotion of Science

Report on Research Meeting
1. Name of Research Meeting / Conference
Symposium on Molecular Chirality 2018
2. Representative
Masami Sakamoto in Chiba University
3. Opening period and Place
May 12 – 13, 2018
Keyaki-Kaikan, Nishi-Chiba Campus, Chiba University
4. Number of participants / Number of participating countries and areas
200 participants / 6 countries
5. Total cost
¥ 3,160,000
6. Main use of subsidy
Venue setup cost and travel fee
7. Result and Impression
We had good weather during the symposium, and about 200 people participated fromsix countries joined this meeting. A French Professor Coquerel of University of Rouen
Normandie, the famous chemist on crystal chirality, had a lecture for dynamic crystallizationof pharmaceutical materials leading to total optical resolution. The US Professor Sivaguru of
Bowling Green State University introduced their latest results on the photochemical asymmetric
reactions. Six Japanese invited speakers, Professor Tomooka of Kyushu University, Professor
Suginome of Kyoto University, Professor Fujiki of Nara Institute of Science and Technology,
Professor Nagasawa of Tokyo University of Agriculture and Technology, Professor Omatsu of
Chiba University, and Professor Hayashi of Tohoku University, had special lectures for the latest
research results on asymmetric synthesis, chiral supramolecules, CD spectroscopy, chiral laser
science technology, and total synthesis of natural products. Furthermore, we had 16 other oralpresentations and 65 poster presentations. We could provide a special forum for international
information exchange and discussion on all fields related to "Molecular Chirality", such as“pharmacology, toxicity, pharmacokinetics of chiral compounds", "analysis and separationof chiral materials", "chiral detection", "optical resolution", "asymmetric synthesis", "chiralfunctional materials", "origin of chirality on Earth", "chiral compounds”, "Industrial productionof industrial production". We hope that this symposium will contribute to the innovative
development of research on chiral molecular chemistry.
8. Additional description
We would like to express my gratitude for your support from the NOVARTIS Foundation(Japan) for the Promotion of Science.

The Novartis Foundation(Japan)
for the Promotion of Science

122

The Novartis Foundation(Japan)
for the Promotion of Science

Report on Research Meeting
1. Name of Research Meeting / Conference
The 2nd research conference for the Research Society for Cancer 3-D culture
2. Representative
Koji Okamoto, MD, PhD., Chief
National Cancer Center Research Institute, Division of Cancer Differentiation
3. Opening period and Place
2018, November 27, National Cancer Center Research Institute (Tokyo)
4. Number of participants / Number of participating countries and areas
199 people (total)
Japan (198), USA (1)5. Total cost
¥ 1,688,000
6. Main use of subsidy
Travel expenses for the foreign invited speaker, printing costs for poster and programs
7. Result and Impression
The meeting was quite succeful in many aspects. There were many audiences with excellent talks
and discussion. I would like to thank the Novartis foundation for invaluable supports.
8. Additional description

The Novartis Foundation(Japan)
for the Promotion of Science

123

The Novartis Foundation(Japan)
for the Promotion of Science

Report on Research Meeting
1. Name of Research Meeting / Conference
International Consortium on Hallucination Research and Related Symptoms Kyoto
SatelliteMeeting (ICHR 2018 Kyoto)
2. Representative
Jun Miyata (Kyoto University)
3. Opening period and Place
18-19 October, 2018 at Clock Tower Centennial Hall, Kyoto University
4. Number of participants / Number of participating countries and areas
Participants: 71
Countries/areas: 15
5. Total cost
6,442,469JPY
6. Main use of subsidy
Transfer expenses and accommodation cost for invited speakers
7. Result and Impression
We had two keynote lectures, 3 symposia, 1 oral session and two poster sessions in themeeting.
In the first keynote lecture on 18th, Prof. Renaud Jardri, Lille University, summarized current
status of neuroimaging studies of hallucination, as well as latest results from his laboratory. He
also introduced his latest work on circular inference model of psychotic symptoms including
hallucination.
In the second keynote lecture on 19, Prof. Pascal Belin, Institut de Neurosciences de laTimone, first talked about his monumental discovery of the auditory voice area. He then talked
about his continuous work on human / animal voice perception.
In the symposium 1 "Neurophysiology" on 18th, Dr. Yoji Hirano, Kyushu University,
introduced his research on hallucination using EEG. Dr. Michio Koeda, the vice-president of thismeeting from Nippon Medical School, talked about his research on hallucination using functional
magnetic resonance imaging. Prof. Thomas Whitford, University of New South Wales, introduced
his electrophysiological research on the origin of hallucination focusing on inner speech. Prof.
Daniel Mathalon, University of California San Francisco, talked about relationship between
corollary discharge and psychotic symptoms including delusion and hallucination.
In the symposium 2 "Hallucinations and delusions" on 19th, Prof. Todd Woodward introduced
his series of research on the commonality / segregation of neural basis of delusions andhallucinations. Dr. Jun Miyata, the president of this meeting from Kyoto University, talked about
the connectivity analysis of delusion and salience. Dr. Keisuke Takahata, National Institute ofRadiological Sciences, talked about tau neuropathology of late life psychosis, using positron

The Novartis Foundation(Japan)
for the Promotion of Science

124

The Novartis Foundation(Japan)
for the Promotion of Scienceemission tomography. Prof. Philip Corlett, Yale University, introduced his computational modelof hallucination.
In the symposium 3 "Recovery and treatment of approaches" on 19th, Prof. Yong-Sik Kim,
Dongguk Univeristy, addressed the pharmacological treatment of hallucinations. Mr. Wakio
Sato, Hearing Voices Network Japan, talked about first-person experience of hallucination.
Prof. Emmanuelle Peters, King's College London, talked about psychological treatment of
hallucination, specifically cognitive behavior therapy. Prof. Flavie Waters, The University ofWestern Australia, demonstrated the relationship between hallucination and sleep.
In the oral session on 18th, we had 9 presentations. The topics covered broad area from culturalresearch to biological research.
In the poster sessions on 18th and 19th, we had 21 posters mainly by young researchers. We
had exciting discussions here and there.
In summary, this was the first meeting of ICHR in Japan, and top researchers from every cornerof the world gathered together, and had a fruitful discussion. These experience would result ininternational collaborative research in the near future.
8. Additional description
We deeply thank for the support by Novartis Foundation Japan for the Promotion of Science.

The Novartis Foundation(Japan)
for the Promotion of Science

125

The Novartis Foundation(Japan)
for the Promotion of Science

Report on Research Meeting
Dec. 21, 2018
1. Name of Research Meeting / Conference
International Tuberous Sclerosis Complex Research Conference 2018
2. Representative
Okio Hino, M.D., Ph.D. (Professor, Juntendo University Faculty of Medicine)
3. Opening period and Place
Sept. 13 – 15, 2018
JR Tokyo General Hospital and Hotel Sunroute Plaza Shinjuku, Tokyo
4. Number of participants / Number of participating countries and areas
200 participants / 17 countries and areas
Australia, Belgium, China, Germany, Hong Kong, Israel, Italy, Japan,
Netherlands, Poland, Portugal, South Africa, Spain, Sweden,
Switzerland, UK, USA,
5. Total cost
10,433,919 yen
6. Main use of subsidy
Traveling expenses for invited speakers (including a committee member)
7. Result and Impression
The conference was held in mid-September in Tokyo, for the first time in Japan. The theme
was “New Horizons in TSC: Mechanisms, preventive and novel therapies, and improving qualityof life”. From many countries and arias, 200 participants in total got together. Among them, there
were over 70 family members of tuberous sclerosis complex (TSC) patients.
In 10 oral sessions, 39 presentations including 25 invited ones by famous or up-and-coming
researchers and medical were given. Also, in the poster session, 36 presentations were done byyoung and energetic researchers. Various topics from basic sciences as well as clinical trials
were taken up in those oral and poster sessions. An active discussion was seen in each session
and sometimes made program behind schedule. One of the important topics was TAND (TSCassociated neuropsychiatric disorders) and the establishment of its checklist. The Japanese version
of TAND checklist had been completed and was introduced in this conference. It will become auseful tool to develop the examination and treatment of TSC in Japan.
During each break, participants promoted exchanges and friendship. Luncheon seminars were
held and foreign participants enjoyed Japanese-style lunch. There was a reception on the first day
when the ceremony for the travel awards was performed. Four young scientists won the award
and were encouraged. During the poster session, food and drink were served and discussion was
done with a good atmosphere.
On the afternoon of the last day, a special session, planed by Japanese patient’s families, was

The Novartis Foundation(Japan)
for the Promotion of Science

126

The Novartis Foundation(Japan)
for the Promotion of Science

held in a panel discussion style on three clinical topics (seizure, TAND, and lung/kidney). It
aimed to promote exchange between medical doctors and the families and to introduce stateof-the-art knowledge on medical activities for TSC to the families. The families gave manyquestions and comments on them to which medical specialists answered and discussed with allparticipants. Issues to be considered in the future were understood in common.
This conference as a whole was meaningful to develop ideal examinations and therapies for
TSC with a global cooperation. We appreciate the NOVARTIS Foundation for the Promotion of
Science for supporting this conference.
8. Additional description

The Novartis Foundation(Japan)
for the Promotion of Science

127

The Novartis Foundation(Japan)
for the Promotion of Science

Report on Research Meeting
1. Name of Research Meeting / Conference
Workshop on Frontiers in Phosphatase Research and Drug Discovery (ICPP13)
2. Representative
Takeshi Tsubata
3. Opening period and Place
October 23rd ~ October 25th, 2018

Tokyo Medical and Dental University

4. Number of participants / Number of participating countries and areas
100/ Japan, Germany, United States, Canada, Taiwan, Belgium
5. Total cost
3,246,167 Yen
6. Main use of subsidy
Travel expenses for the invited speakers
7. Result and Impression
Protein phosphatase involves in extremely important various enzymatic phenomenon as
intracellular signaling, membranous transport and cellular cycle inhibitor. From recent new tooland basic technology development, it is now established that phosphatases are tightly regulated
and that they show a degree of substrate specificity similar to that of kinases. It has been indicatedthat various phosphatases are important target factors for the new treatment method for nerve,
immune, and metabolic disorder by specifically inhibiting cell growth and its differentiation as
well as metabolism, nerve function, and immune function.
The conference, “Workshop on Frontiers in Phosphatase Research and Drug Discovery”
has been held between October 22nd to 25th, 2018 at Suzuki Memorial hall of Tokyo Medical
and dental university. The presentations and discussions have been held in regards to the roleof protein phosphatase in terms of life phenomenon and drug discovery targeting protein
phosphatase. There was approximately 100 participants for this conference.
Protein phosphatase is distinguished in two different ways, serine/threonine phosphatase andtyrosine phosphatase (PTP). There is also phosphatidylinositol phosphatase as a related molecule.
In this conference, professor Brautigan, the leading researcher of serine/threonine phosphatasefrom United States has participated as well as professor Bollen (Belgium) and Professor Neel
(United States) who is the leading researchers of PTP. Professor Tonks (United States) and
14 international invited speakers (2 female researchers), and 13 Japanese invited speakers (2female researchers) lectured the presentations during the conference. The main themes of thepresentations were the mechanisms and roles of holoenzymes made out of catalytic and inhibitory
subunit of serine/threonine phosphatase, atypical structures and mechanism of phosphatase, andfunctional mechanisms of phosphatase in terms of nerve function, cardiogenic disorders, andimmunology and inflammation. Among others, various kinds of phosphatase are associated withcarcinogenic mechanisms. There were many presentations in regards to the mechanisms and

The Novartis Foundation(Japan)
for the Promotion of Science

128

The Novartis Foundation(Japan)
for the Promotion of Sciencerole of phosphatidylinositol phosphatase in association with PRL family molecules inhibiting ordeveloping the carcinogenic mechanisms as well as protein phosphatase inhibiting carcinogenic
mechanisms.
It is a fact that the development of phosphatase inhibitor has been delay compared tophosphatase. It was because development of targeting active center of compound in phosphatase(PTP) is impossible compared to kinase inhibitor. However, it has been reported that thepromising result has been obtained for the development of phosphatase inhibitor targeting
compound outside of active center. There were also the intriguing presentations lecturing cancer
treatment with the molecule by inhibiting allosteric binding site to inactive SHP-2 and thedevelopment of immunotherapy inhibiting PTP1B and TC-PTP by binding to the molecularpocket close to active center of compound.
There were total of 33 young researchers for the poster presentations (3 participantsfrom foreign counties). During the poster presentation, the opportunities of many active
discussions were held among the young researchers, presenters and invited speakers. Amongthe poster presenters, 8 cases have been picked for short talk presentations which was theexcellent opportunities for the young researchers to present their research achievements to theinternationally known experts. Many participants including the invited speakers and young
researchers participated the reception dinner which provided all participants for international
networking among phosphatase research field.

8. Additional description
None

The Novartis Foundation(Japan)
for the Promotion of Science

129

The Novartis Foundation(Japan)
for the Promotion of Science

32nd Grant Report（FY2018）
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 2018 are as
follows.
32nd Novartis Research Grant: 37Researchers (JPY 1 mil.), Subtotal JPY37.0 mil.
Research Meeting Grant:
5 Meetings (JPY 0.4 mil.), Subtotal JPY 2.0 mil.
Total
JPY39.0 mil.

32nd Novartis Research Grant(FY2018)

The Grant is to aim supporting creative research in Japan in the field of Bio, life science andrelevant chemistry.
#

Name

Institution

Title

Research Project

1 Tanaka Tomoaki

Chiba University, Graduate
School Medicine,)

Fundamental study for GATA3transcriptional complex and new insightProfessor into functional role of type2 innate
lymphoid cells to control life-style relateddisease2 Gotoh Eiji

Faculty of Agriculture,
Kyushu University

Assistant Chloroplast movement optimizes plant
Professor growth through novel signal transduction

3 Arai Yasuyuki

Department of Hematology,
Kyoto University Hospital,

SeniorClinicalStaff

Machine learning- and next generation
sequencing-oriented correlation analysis
between gut microbiota and the risk ofgraft-versus-host disease

4 Iijima Norifumi

National Institutes ofBiomedical Innovation,
Health and Nutriti,

Subproject
leaderImportance of tissue-resident memory
Tcells in mucosal tissue

5 Watanabe Yusuke

Molecular Phys lab,
National Cerebral andCardiovascular Center,

Analysis of cell lineage and transcriptional
Laboratory
regulation during ventricular development
chiefof heart

6 Muguruma Keiko

Dept. of iPS Cell Applied
Medicine, Kansai Medical
University,

Construction of human complex
Professor brain organoids for analysis of brain
development and neurological diseases

7 Shibutani Shusaku

Lab. of Vet. Hygiene,
Joint Faculty of Vet. Med.,
Yamaguchi Univ,

Assistant A Novel Regulatory Mechanism of theProfessor Metabolic Signaling Complex mTORC1

8 Takeuchi Jun

MRI, Tokyo Medical and
Dental University,

Associate Generation of functional-atrialventricular
Professor heart organdies in vitro

9 Sakakura Akira

Grad. Sch. of Natural Scienceand Technology,
Okayama Universtiy,Development of Catalytic Asymmetric
Cycloaddition Reaction of alpha-Keto
Professor
Esters for the Innovative Synthesis of
Tetrahydrofuran Compounds

10 Suganami Takayoshi

Dept. of Molecular Medicine
and Metabolism,
Nagoya University,

Pathophysiologic role of a novel
Professor microenvironment that prolongs tissue
damage in acute kidney injury

The Novartis Foundation(Japan)
for the Promotion of Science

130

The Novartis Foundation(Japan)
for the Promotion of Science

#

Name

Institution

Title

Research Project

11 Okuno Hiroyuki

Dept of Biochem and Mol
Biol, Grad Sch of Med,
Kagoshima Univ,

Role of a novel activity-dependent gene
Professor encoding a lipid modification enzyme onneuronal and cognitive function

12 Ukaji Yutaka

Dept. of Chemistry,Inst. of Sci. and Eng.,
Kanazawa University,

Professor

13 Yachie Nozomu

Yachie laboratory, RCAST,The University of Tokyo,

Associate
Development of new base editor systems
Professor

14 Kitagishi Hiroaki

Faculty of Science andEngineering,
Doshisha University,

Study on the circadian rhythm system
Associate
regulated by intracellular carbon
Professormonoxide

15 Haraguchi Shogo

Department of Biochemistry,
Showa University School of
Medicine,

Assistant Age-related endocrine disruption of skin
Professor leads to senescent alopecia

16 Iijima Takatoshi

Tokai University Instituteof Innovative Science andTechnology,

The RNA-based mechanism underlying
Associate
pathophysiology in the neurologicalprofessor
diseases

17 Nakatogawa Hitoshi

Elucidation of the molecular basis andSch. of Life Sci. and Tech.,
Associate
physiological significance of autophagic
Tokyo Institute of Technology, Professor
degradation of the nuclear pore complex

18 Abe Takayuki

Kobe University Graduate
School of Medicine,Comprehensive understanding of
Associateintracellular innate immune response in
Professor
chronic hepatitis B reactivation

19 Ishimizu Takeshi

Dept. of Biotechnol., Col. ofLife Sci., Ritsumeikan Univ.,

Identification of glycosyltransferases
Associateinvolved in biosynthesis of pectin in plant
Professor
cell wall

20 Kai Toshie

Graduate School of Frontier
Biosciences,
Osaka University,

RNA base modification and RNA
Professor processing regulate the maintenance anddevelopment of germline stem cells

21 Fukuhara Shigetomo

Inst. of Adv. Med. Sci.,
Nippon Medical School,

Professor

22 Tomita Kengo

Dept. of Internal Medicine,National Defense Medical
College,

Development of new treatment for
Associate obesity-related liver cancer, focusing on
Professor a Wnt ligand, ACLP, secreted by hepatic
stellate cells

23 KizakaKondo Shinae

Tokyo Institute of Technology, Professor

24 Yakushiji Nayuta

Ikawa Laboratory,
Tokyo University of Science,

Assistant Novel long-range regulatory mechanisms
Professor controlling B-cell lineage program

25 Miyoshi Keita

Div. of Invertebrate Genetics,
National Institute of Genetics,

Molecular mechanisms of extracellular
Assistantvesicle formations of endogenous
professor
retroviruses

26 Naramto Satoshi

Lab.of Plant Dev.Biol., Grad.
Sch.Life Sci.,
Tohoku university,

Molecular mechanisms of axis formationin plants: Organization, function andAssistantspatiotemporal dynamics of PIN clusters
Professorthat regulate polar localization of auxin
efflux carrier PIN proteins

27 Hashimotodani Yuki

Organization for Research
Initiatives and Development,

Physiological roles and molecular
Associatemechanisms underlying the synaptic
Professor
plasticity in the dentate gyrus

28 Nishikii Hidekazu

Department of Hematology,
University of Tsukuba,

Genomic analysis of human hematopoietic
Associate
stem cell niche in myelodysplastic
Professor
syndrome.

Synthesis of Phytochrome-type
Chromophores Based on Oxidative
Functionalization toward Investigation of
their Functions

Unveiling a novel regulatory mechanismof angiogenesis by intraluminal pressure

Construction of ultra-sensitive
bioluminescence imaging Tg mice

The Novartis Foundation(Japan)
for the Promotion of Science

131

The Novartis Foundation(Japan)
for the Promotion of Science

#

Name

Institution

Title

Research Project

29 SegiNishida Eri

Segi lab, Dept. of Biol. Sci.
and Tech., Tokyo Universityof Sci,The role of desmoplakin in the
Associate
antidepressant-like effects in theprofessor
hippocampus

KimuraYoshida
Chiharu

Dept. Mol Embry, Res. Inst.
Osaka WCH,

Chief
Scientist

31 Suzuki Yoshiro

Div. Cell Signaling, Nat. Inst.
Physiol. Sci.,

Identification and molecular mechanism ofAssistant novel calcium transport disease ;possible
Professor therapeutic target for bone mineralization
defects

32 Akimitsu Nobuyoshi

Isotope Science Center, theUniversity of Tokyo,

Professor

Nuclear noncoding RNA degradation
regulates innate immune response

33 Tagawa Yoshiaki

Dept. Physiology, Facultyof Medicine, Kagoshima
University,

Professor

Projection-specific circuit formation in thedeveloping cerebral cortex

34 Komura Kazumasa

Department of Urology,Osaka
Medical College,

Assistant Uncovering the Biomarker for Treatment
Professor with ATR Inhibitor in Solid Tumor

35 Demizu Yosuke

Division of Organic
Chemistry, NIHS,

Head

Peptide foldamers in drug discovery

36 Maruyama Reo

Project for Cancer
Epigenomics, Cancer Institute,
JFCR,

Project
Leader

Evaluation of Intra-tumor epigenetic
heterogeneity of breast cancer

37 Taniguchi Koji

Department of Microbiology
and Immunology, Keio
University Schoo,

Research on metabolic reprogramming
Associateinduced by cancer development andProfessor
inflammation

30

Functional analysis of the GRHL3 factor
in epithelial fusion.

The Novartis Foundation(Japan)
for the Promotion of Science132

The Novartis Foundation(Japan)
for the Promotion of Science

FY2018 Research Meeting Grant(JPY 400 thousand x5 = 2.0 million)#

Meeting1

Symposium on Molecular Chirality
2018

The 2nd research conference for the
2 Research Society for Cancer 3-D
cultureInternational Consortium on
3 Hallucination Research Satellite
Meeting 2018 Kyoto
4International Tuberous Sclerosis
Complex Research Conference 2018

International Conference on Protein
Phosphatase Workshop on Frontiers
5in Phosphatase Research and Drug
Discovery

Date(Place)Institution / Title

2018.05.11-12 Chiba University(Chiba)/ Professor
2018.12.3(Tokyo)National Cancer Center
Research Institute/ Chief

Name
Sakamoto Masami
Okamoto Koji

Department of Psychiatry,
2018.11.29-30
Kyoto University(Kyoto)/ Senior Lecturer

Miyata Jun

2018.9.13-15(Tokyo)Hino Okio

2018.10(Tokyo)Juntendo University/ Professor
Tokyo Medical and Dental
University Medical ResearchInsti/ professor

Tsubata Takeshi

The Novartis Foundation(Japan)
for the Promotion of Science

133

The Novartis Foundation(Japan)
for the Promotion of Science

第32期（2018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。2018年度は、下記の総額3,900万円の助成事業を実施しました。
第32回ノバルティス研究奨励金

37件（1件100万円）

3,700万円

研究集会助成5件（1件 40万円）

200万円
3,900万円

総額

第32回ノバルティス研究奨励金（2018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

1

田中

知明

千葉大学大学院医学研究院

教授

2

後藤

栄治

九州大学
大学院農学研究院

助教

3

新井

康之

京都大学医学部附属病院
血液内科

医員

4

飯島

則文

医薬基盤・健康・
栄養研究所

5

渡邉

裕介

国立研究開発法人、
国立循環器病研究センター、
分子生理部

6

六車

恵子

関西医科大学 医学部
iPS・幹細胞応用医学講座

7

渋谷

周作

山口大学共同獣医学部獣医
衛生学分野

テニュア
代謝シグナル複合体 mTORC1 の新規制
トラック
御メカニズムの研究
助教

8

竹内

純

東京医科歯科大学

准教授

9

坂倉

彰

岡山大学大学院自然科学
研究科応用化学専攻

教授

10

菅波

孝祥

名古屋大学環境医学研究所
分子代謝医学分野

教授

11

奥野

浩行

鹿児島大学大学院
医歯学総合研究科生化学・分子生物学

急性腎障害において炎症の遷延化をもたらす新規微小環境の病態生理的意義の解
明教授

神経活動依存的発現を示す新しい脂質修
飾酵素による神経・認知機能調節機構

12

宇梶

裕

教授

酸化的官能基化を基盤とするフィトクロ
ム型発色団の合成と機能解明

金沢大学理工研究域
物質化学系

職

位

研究課題

GATA3 転写複合体解析を基盤とした生活習慣病における 2 型 ILC ２細胞の新たな機能的役割植物の生長を司る葉緑体局在変化の新たな制御機構の解明

機械学習と次世代シーケンス技術を用いた、同種造血幹細胞移植後の腸内細菌叢と、腸管 GVHD 発症リスクとの関連解析

サブ粘膜組織における組織局在型メモリー T
プロジェクト細胞の重要性リーダー
室長

胎生期心室筋形成における転写制御機構と細胞系譜の解析

教授

複雑系脳オルガノイドによるヒト脳発生の解明と中枢神経疾患への応用

心筋タイプ別誘導法を用いた in vitro 機
能性心臓組織の構築

テトラヒドロフラン環の構築法を革新するα - ケトエステルの不斉環化付加反応の開発

The Novartis Foundation(Japan)
for the Promotion of Science

134

The Novartis Foundation(Japan)
for the Promotion of Science

No.

氏

名

所

13

谷内江

望

14

北岸

宏亮

15

原口

16

飯島

17

中戸川

18

属

東京大学先端科学技術
研究センター谷内江研究室

職

位

研究課題

准教授

新規塩基編集ツールの開発

同志社大学理工学部
機能分子・生命化学科

准教授

細胞内一酸化炭素によって調節される
サーカディアンリズム機構の解明

省吾

昭和大学医学部生化学講座

助教

崇利

東海大学創造科学技術研究機構医学部門准教授

仁

東京工業大学 生命理工学院

准教授

阿部

隆之

神戸大学大学院
医学研究科 感染制御学分野

准教授

B 型肝炎ウイルスの再活性化・再燃に伴う細胞内自然免疫応答の包括的理解

19

石水

毅

准教授

植物細胞壁ペクチンの生合成に関わる糖転移酵素の同定

20

甲斐

歳恵

教授

21

福原

茂朋

日本医科大学 先端医学研究所 分子細胞構造学分野

生殖幹細胞の維持と分化を制御する RNA
修飾とプロセシングの解明

教授

22

冨田

謙吾

防衛医科大学校
消化器内科学教室

准教授

23

近藤

科江

東京工業大学教授

24

藥師寺那由他

東京理科大学生命医科学研究所免疫生物学研究部門
伊川研究室

助教

25

三好

啓太

国立遺伝学研究所
系統生物研究センター
無脊椎動物遺伝研究室

オープンクロマチン領域解析による B 細
胞特異的エンハンサーの同定とその制御
機構の解明

助教

内在性レトロウイルスによる細胞外粒子
形成の分子機構の解明

26

楢本

悟史

東北大学大学院生命科学
研究科植物発生分野

助教

27

橋本谷祐輝

同志社大学
研究開発推進機構

植物の体軸形成の分子基盤の解明：オーキシン排出担体 PIN の局在を制御する
PIN クラスターの構造とその機能

准教授

28

錦井

筑波大学医学医療系血液内科准教授

東京理科大学 基礎工学部
生物工学科 瀬木研究室

准教授

准教授

秀和

29 瀬木ー西田 恵里
30 木村－吉田 千春

立命館大学生命科学部
生物工学科
バイオエネルギー研究室
大阪大学大学院生命機能
研究科生殖生物学研究室

大阪母子医療センター
研究所 病因病態部門

主任
研究員

加齢に伴う皮膚内分泌環境恒常性維持機構の破綻により生じる老人性脱毛症の分子機構解明

RNA 情報発現系の破綻による脳神経疾患の分子病態解明
オートファジーによる核膜孔複合体の分解の分子基盤と生理的意義の解明

血管内腔圧による血管新生の新たな制御機構の解明

肝星細胞分泌 Wnt リガンド ACLP に着目した、肥満関連肝がんの新たな治療法の開発
超高感度発光イメージング Tg マウスの構築

海馬歯状回の局所回路におけるシナプス
可塑性のメカニズムと生理的役割
骨髄異形成症候群患者における造血幹細胞ニッチの遺伝子発現解析

接着制御因子デスモプラキンによる新規うつ治療シグナルの解明
上皮癒合に着目した GRHL3 因子の機能解析

カルシウム輸送障害による新規骨石灰化不全症の同定と病態メカニズム解明

31

鈴木

喜郎

岩手医科大学医学部
生理学講座統合生理学分野

32

秋光

信佳

東京大学アイソトープ
総合センター

教授

33

田川

義晃

鹿児島大学医学部医学科
神経筋生理学分野

教授

長距離・局所をつなぐ投射特異的な大脳回路構築のメカニズム解明

34

小村

和正

助教

固形腫瘍における ATR 阻害薬の治療効
果予測バイオマーカーの同定

35

出水

庸介

部長

ペプチドフォルダマー創薬研究大阪医科大学泌尿生殖・発達医学講座
泌尿器科学教室

国立医薬品食品衛生研究所
有機化学部

核内ノンコーディング RNA の分解制御による自然免疫応答のコントロールに関する研究

The Novartis Foundation(Japan)
for the Promotion of Science

135

The Novartis Foundation(Japan)
for the Promotion of Science

No.

氏

名

36

丸山

玲緒

37

谷口

浩二

所

属

職

位

研究課題

公益財団法人がん研究会
プロジェクト 乳癌における腫瘍内エピゲノム不均一性
がん研究所リーダー の解明
がんエピゲノムプロジェクト
慶應義塾大学医学部
微生物学・免疫学教室

特任准教授

がん進展と炎症による代謝リプログラミ
ング機構の研究

The Novartis Foundation(Japan)
for the Promotion of Science

136

The Novartis Foundation(Japan)
for the Promotion of Science

2018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。2018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）
開催日
（開催地）

No.

研究集会名

1

シンポジウム モレキュラー・キラ
リティー 20182

第二回がん三次元培養研究会

3

国際幻聴研究コンソーシアム2018
京都

2018.11.29-30（京都）

4

国際結節性硬化症リサーチカン
ファレンス20182018.9.13-15（東京）

5

国際プロテインホスファターゼ
研究集会
プロテインホスファターゼと関連分子についての新たな研究ツールの開発と創薬への展開

2018.05.11-12（千葉）
2018.12.3
（東京）

所属・職位

氏

名

千葉大学／教授

坂本昌巳

国立がん研究センター
研究所／分野長岡本康司

京都大学大学院医学研究科
脳病態生理学講座
精神医学教室／講師

宮田淳

順天堂大学／教授

樋野

興夫

鍔田

武志

2018.10.23-25
東京医科歯科大学／教授（東京）

The Novartis Foundation(Japan)
for the Promotion of Science

137

The Novartis Foundation(Japan)
for the Promotion of Science

32nd Financial Report

Balance Sheet

Statement of Net Assets

As of March 31, 2019

From April 1, 2018 to March 31, 2019
（Unit：JP Yen）

Account（Unit：JP Yen）

AmountⅠ Assets

Ⅰ General Net Assets Changes

1. Current Assets

1. Ordinary income & Expenditure

Current Assets Total

16,270,588

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

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

Fixed Assets Total
Assets Total

634,194

13,556,807

Donation

53,000,000
562,056

Other Income

67,118,863(2) Ordinaty Expenditure
49,368,532

Grant Expense

39,000,000

1,180,626,940

Novartis Research Grant

37,000,000

1,196,897,528

Research Meeting Grant

2,000,000

Administrative Expense

6,220,958

Ordinary Expenditure Total

55,589,490

37,148,627

Ordinary Balance without Appraisal Profit or Loss

11,529,373

37,148,627

2. Nonrecurring Profit & Loss

79,992,746

1. Current Liabilities
Liabilities Total

Interest from basic fund

Project Expense

Ⅱ Liabilities
Current Liabilities Total(1) Ordinary income

Ordinary Income Total(3) Other Long - term Assets
Other Long - term Assets Total

AmountAccount

Ⅲ Equity(Net Assets)

Nonrecurring Balance of Current Period

1. Designated Net Assets

General Net Assets Ending Balance

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

1,000,634,194

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

Designated Net Assets Change

159,114,707
0 1,000,634,194(634,194)

Designated Net Assets Ending Balance

159,114,707

Ⅲ Net Assets Balance Ending Balance

1,159,748,901

100,000,000

Equity Total(Net Assets)

1,159,748,901

Liabilities & Equity Total

1,196,897,528

The Novartis Foundation(Japan)
for the Promotion of Science138

The Novartis Foundation(Japan)
for the Promotion of Science

第32期（2018年度）財務報告

貸借対照表

正味財産増減計算書

2019 年 3 月 31 日現在

2018 年 4 月 1 日から 2019 年 3 月 31 日まで（単位：円）

科

目

金 額

Ⅰ資産の部

科

16,270,588

⑴

経常収益

基本財産

基本財産合計⑵⑶

固定資産合計
資産合計

634,194
79,992,746

計

67,118,863

経常費用
事業費

49,368,532

支払助成金

39,000,000

1,196,897,528

研究集会助成金

2,000,000

管理費
経常費用

6,220,958
計

37,148,627

当期経常増減額

37,148,627

２．経常外増減の部

55,589,490
11,529,373

当期形状外増減額

１ ． 指 定 正 味財 産

一般正味財産期末残高

指定正味財産合計

1,000,634,194（うち基本財産への充当額）(1,000,000,000)（うち特定資産への充当額）(634,194)

２ ． 一 般 正 味財 産

159,114,707（うち基本財産への充当額）

100,000,000

負債及び正味財産合計⑵

562,056

37,000,000

Ⅲ正味財産の部

正味財産合計53,000,000

ノバルティス研究奨励金

１．流動負債
負債合計

受取寄付金

1,180,626,940

Ⅱ負債の部
流動負債合計13,556,807

経常収益

その他固定資産

その他固定資産合計

基本財産運用益
雑収益

1,100,000,000

特定資産

特定資産合計金 額

１．経常増減の部

２．固定資産⑴

目

Ⅰ一般正味財産増減の部

１．流動資産流動資産合計（単位：円）

159,114,707

Ⅱ指定正味財産増減の部
当期指定正味財産増減額
指定正味財産期末残高
Ⅲ正味財産期末残高01,000,634,194
1,159,748,901

1,159,748,901
1,196,897,528

The Novartis Foundation(Japan)
for the Promotion of Science

139

The Novartis Foundation(Japan)
for the Promotion of Science

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

As of Oct 1, 2019

Post

Name

Chairman

Akimichi KANEKO

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
President, Gunma Prefectural Public University Corporation

Trustee

Masao TORII
AuditorTokuzo NAKAJIMA
Masanori FUSE

Title

President, Novartis Holding Japan K.K.
Certified Public Accountant
Financial Advisor, Finance & Administration Div., Novartis Pharma K.K.

[Board of Councilors] 10 councilors
Post

Name

Chairman

Tsuneyoshi KUROIWA

Councilor

As of Oct 1, 2019
Title

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

Norio AKAIKE

Head, Kumamoto Kinoh Hospital Clinical Research Center
Visiting Professor, Kumamoto University, Graduate School of Medicine
and Pharmaceutical 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 NAKANO

Deputy Director, Photonics Engineering Research Center & Team Leader,
Live Cell Super-Resolution Imaging Research, RIKEN

Tadanori MAYUMIEmeritus Professor, Osaka University

Miwako MORI
Toyoshi FUJIMOTO

Emeritus Professor, Hokkaido University
Specially appointed Professor, MD, Research Institute for Diseases of Old
Age, School of Medicine, Juntendo University
Emeritus Professor, 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

140

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

As of Oct 1, 2019

Post

Name

Chairman

Masafumi YANO

Professor, MD, School of Medicine, Yamaguchi University

Jun YAMASHITA

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

Motoko YANAGITA

Professor, MD, Graduate School of Medicine, Kyoto University

Hirokazu ARIMOTO

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

Hiroyuki TAKEDA

Professor, Graduate School of Science, University of Tokyo

Eiji HARA

Member

Title

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

Akihiko YOSHIMURA Professor, Department of Microbiology and Immunology,
Hiroshi ITO

Graduate School of Medicine, Keio University

Takashi OHSHIMA

Professor, Department of Graduate School of Medicine, Keio University

Yoko HAMAZAKI

Professor, Drug Discovery and Development Center, Graduate Schoolof Pharmaceutical Sciences affiliated Company-Academia-Government
collaboration, Kyushu University

Toshiaki OHTEKI

Professor, Graduate School of Medicine, Kyoto University

Yasuyoshi SAKAI

Professor, Medical Research Institute Tokyo Medical and Dental
University

Taisuke TOMITA

Professor, Division of Applied Life Sciences, Graduate School of
Agriculture, Kyoto University

Fumiko TOYOSHIMA

Professor, Graduate School of Pharmaceutical Sciences, The University ofTokyo

Tohru MINAMINO

Professor, Institute for Frontier and Medical Sciences, Kyoto University

Yasuhiro YAMADA

Professor, Niigata University Graduate School of Medical and Dental
Sciences

The Novartis Foundation(Japan)
for the Promotion of Science

141

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2019 年 10 月 1 日現在 ( 順不同、敬称略 )
職

名

代表理事

理事

監事

氏

名

金子

章道

浅野

茂隆

遠藤

政夫

高田

邦昭

鳥居

正男

中嶋

德三

布施

正則

現

慶應義塾大学名誉教授

職

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

山形大学名誉教授

群馬県立県民健康科学大学学長
群馬県公立大学法人理事長

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
中嶋德三公認会計士事務所
公認会計士

ノバルティスファーマ（株）企画管理本部
ファイナンシャルアドバイザー

就任年月日

常勤 ･ 非常勤

2018年6月25日

非常勤

2018年6月25日

非常勤

2018年6月25日

非常勤

2018年6月25日

非常勤

2018年6月25日

非常勤

2016年6月10日

非常勤

2016年6月10日

非常勤

評 議 員 名 簿2019 年 10 月 1 日現在 ( 順不同、敬称略 )
職

名

氏

名

評議員長

黒岩

常祥

赤池

評議員

現

職

就任年月日

常勤 ･ 非常勤日本学士院会員
東京大学名誉教授

2016年6月10日

非常勤

紀扶

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
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

理化学研究所
光量子工学研究センター 副センター長

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

森

美和

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

順天堂大学大学院医学研究科特任教授
名古屋大学名誉教授

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

The Novartis Foundation(Japan)
for the Promotion of Science

142

The Novartis Foundation(Japan)
for the Promotion of Science

選 考 委 員 名 簿
2019 年 10 月 1 日現在 ( 順不同、敬称略 )
職

名

選考委員長

氏

矢野

山下

柳田

有本

武田

原

選考委員

名

雅文
潤

素子

博一

洋幸

英二

林

誠

平井

宏和

現

職

就任年月日

常勤 ･ 非常勤

山口大学大学院医学系研究科教授

2016年6月10日

非常勤京都大学 iPS 細胞研究所教授

2016年6月10日

非常勤

京都大学大学院医学研究科教授

2016年6月10日

非常勤東北大学大学院生命科学研究科教授

2016年6月10日

非常勤

東京大学大学院理学系研究科教授

2017年6月19日

非常勤

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研究センター
チームリーダー2017年6月19日

非常勤

群馬大学大学院医学系研究科教授

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

慶應義塾大学医学部教授

2017年6月19日

非常勤

伊藤

裕

慶應義塾大学医学部教授

2018年6月25日

非常勤

大嶋

孝志

九州大学大学院薬学研究院教授

2018年6月25日

非常勤

濵﨑

洋子

京都大学大学院医学研究科教授

2018年6月25日

非常勤

樗木

俊聡

東京医科歯科大学難治疾患研究所教授

2019年6月10日

非常勤

阪井

康能

京都大学大学院農学研究科教授

2019年6月10日

非常勤

富田

泰輔

東京大学大学院薬学系研究科教授

2019年6月10日

非常勤

豊島

文子

南野

徹

山田

泰広

京都大学ウィルス・再生医科学研究所教授

2019年6月10日

非常勤

新潟大学大学院医歯学総合研究科教授

2019年6月10日

非常勤東京大学医科学研究所教授

2019年6月10日

非常勤

The Novartis Foundation(Japan)
for the Promotion of Science143

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は別枠で、下記の範囲内で損金の算入できます。
（資本金等の額×当期の月数/12×0.375%＋所得の金額×6.25%）÷2
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お力添え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1,764件、総額20億円を超えるものとなりました。
当財団は、自然科学の創造的研究への助成によって、日本の学術発展に寄与することを目指しております。
助成を受けられた研究成果がすぐに応用につながらなくとも、将来、新分野につながることを夢見て、この事業を継続して参ります。
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。
2019年10月吉日
事務局長 原建記〒105-6333 東京都港区虎ノ門1-23-1
虎ノ門ヒルズ森タワー
TEL：03-6899-2100 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 Science144