Annual Report (2016)

No.28
The NOVARTIS Foundation (Japan)
for the Promotion of Scienceॶଁ '2 ॷ঎ᢧ܎ॷ‫ݬ‬ፀ '2 ‫ش‬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. Effect of dendritic cell based vaccine against highly virulent cryptococcal infection .. 9
Yuki Kinjo
National Institute of Infectious Diseases

2. Nanoscale analysis reveals agonist-sensitive and heterogeneous pools ofphosphatidylinositol 4-phosphate in the plasma membrane .................................... 12
Akikazu Fujita
Kagoshima University, Joint Faculty of Veterinary Medicine

3. Extended use of the retinal proteins to control the biological activities by light ........ 15
Yuki Sudo
Okayama University

4. Positive feed-forward loop of mTOR complex links cellular metabolism totargeted therapy resistance ....................................................................................... 18
Kenta Masui
Department of Pathology, Tokyo Women's Medical University

5. Analysis of molecular mechanisms for cell fate determination of retinal
interneurons ............................................................................................................. 21
Rikako Sanuki
Institute for Protein Research, Osaka University

6. Mechanism of meiosis in triploid planarians: difference in the chromosomal
elimination timing between the sexes ...................................................................... 24
Midori Matsumoto
Keio University

7. Roles of chemokine system in peritoneal adhesion formation .................................... 27
Yuko Ishida
Wakayama Medical University

8. Mapping and manipulating neuronal circuits that regulates sleep/wakefulness states 29
Takeshi Sakurai
Kanazawa University-1-

9. Developmental and physiological analyses of spinal neuronal circuits thatgenerate rhythmic locomotor activities ................................................................... 32
Shin-ichi Higashijima
Natural Institutes of Natural Sciences, Okazaki Institute for Integrative Bioscience

10. Identification of the roles of SIRT7 in brown adipocytes............................................ 34
Kazuya Yamagata
Department of Medical Biochemistry, Faculty of Life Sciences, Kumamoto University

11. A novel mechanism of regulation of melanocortin receptor signaling mediatedby glycolipids in hypothalamic inflammation ......................................................... 36
Kei-ichiro Inamori
Tohoku Pharmaceutical University

12. Analysis of epigenetic gene expression mechanism of DDI2/3 by DNA damage ....... 39
Masaya Oki
University of Fukui

13. Drug discovery for neuropathic pain from processed aconite root ................................... 42
Toshiaki Makino
Graduate School of Pharmaceutical Sciences, Nagoya City University

14. Research on genome system regulating bone morphogenesis .................................... 45
Koji Tamura
Tohoku University, Graduate School of Life Sciences

15. Research on neural circuits for the control of water- and salt-intake behaviors ......... 48
Takeshi Y. Hiyama
National Institute for Basic Biology

16. Development of a comprehensive synthetic strategy and study of biologicalactivity of yuzurimine alkaloids .............................................................................. 51
Ichiro Hayakawa
Okayama University

17. Safe and efficient direct cardiac reprogramming for heart repair................................ 53
Masaki Ieda
Department of Cardiology, Keio University School of Medicine

18. A novel mechanism of temperature compensation on mammalian circadian clocks .. 57
Yoshinori Hiraoka
Kobe Gakuin University

19. Induction of anti-tumor innate immune responses by tumor-associated molecularpatterns..................................................................................................................... 59
Hiroaki Ikushima
Institute of Industrial Science, University of Tokyo-2-

20. Functional mechanism of a novel signaling molecule that regulates
differentiation and migration in immune cells......................................................... 62
Yoshihiko Tanaka
Fukuoka Dental College, Department of Functional Bioscience, Section of Infection
Biology

21. Molecular basis of zinc signaling on lymphocyte homeostasis and malignancy ........ 65
Toshiyuki Fukada
Tokushima Bunri University

22. Establishment of experimental systems for monitoring endoplasmic reticulum
stress in planta ......................................................................................................... 68
Yuji Iwata
Osaka Prefecture University

23. Targeting macrophage pinocytosis in atherosclerosis ................................................. 71
Takuro Miyazaki
Showa University School of Medicine

24. Elucidation of the molecular mechanism of DNA damage response andcarcinogenesis mediated by chromatin remodeling................................................. 75
Kiyoshi Masuda
Dept. of Human Genetics, Inst. of Biomedical Sciences,Tokushima University Graduate
School25. Development of a novel fluorescent probe to catch a “dimerization arm” of the
EGF receptor............................................................................................................ 80
Takaaki Mizuguchi
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University

26. Development of novel therapy for peripheral artery disease using ApoA-I
mimetic peptide ....................................................................................................... 84
Satoshi Imaizumi
Department of Cardiology, Fukuoka University School of Medicine

27. Regulation mechanisms of retroelements by RNA virus ............................................ 88
Tomoyuki Honda
Osaka University

28. Single molecule study on ion-channel gating.............................................................. 91
Toru Ide
Okayama University

29. The lack of mucosal barrier in specialized epithelial M cells as a portal for
infectious agents ...................................................................................................... 94
Koji Hase
Graduate School of Pharmaceutical Science, Keio University

30. Epigenetic regulation of Sry by histone H3K9 methylation ....................................... 97
Shunsuke Kuroki
Institute for Enzyme Research, Tokushima University-3-31. Crucial role of plasmacytoid dendritic cells for the TLR-7-mediated autoimmne
inflammation .......................................................................................................... 100
Hideaki Takagi
Division of Immunology, Department of Infectious Diseases, Faculty of Medicine,
University of Miyazaki

32. Development of asymmetric 1,3-alkyl migration reaction for synthesis of drugcandidate compounds ............................................................................................ 104
Norie Momiyama
National Institutes of Natural Sciences, Institute for Molecular Science

33. NAD metabolism as a novel target for anti-cancer therapy ...................................... 106
Takashi Nakagawa
Frontier Research Core for Life Sciences,University of Toyama

34. Creating methods for in vitro amplification of skeletal muscle satellite cells withregenerative myogenic potential ............................................................................ 109
Atsuko Sehara
Kyoto University, Institute for Frontier Medical Sciences

35. Role of protein phosphatases in robustness of mammalian circadian clock ............. 111
Makoto Akashi
Yamaguchi University

36. Development of novel cyclization reaction with SmI2 and application tosynthesis of natural products ................................................................................. 114
Taiki Umezawa
Faculty of Environmental Earth Science, Hokkaido University

37. Genome-wide screening of endocytosis of GPCR .................................................... 118
Junko Toshima
Tokyo University of Technology School of Health Science

38. Detection and analysis of cell chirality in primary cultured cells ............................. 121
Takeshi Sasamura
Osaka University

39. Dissection of neural computation in single cell networks ......................................... 124
Fumitaka Osakada
Nagoya University

40. Studies on the underlying mechanisms for the integration of cortical information .. 127
Makoto Sato
Osaka University

41. Targeting the muscle-liver-fat signaling axis essential for central control ofadaptive adipose remodeling for treatment of metabolic syndrome...................... 130
Hirotoshi Tanaka
Division of Rheumatology, Center for Antibody and Vaccine, IMSUT Hospital, Instituteof Medical Science, University of Tokyo-4-

42. Total synthesis of ustiloxin D via the aryl ether formation of tert-alcohol ............... 133
Itaru Sato
Ibaraki University

Ⅲ . Reports from the Recipients of Grants for International Meetings
研究集会報告 ....................................................................................................... 136
1. The 12th International Workshop on Microbeam Probes of Cellular Radiation
Response (IWM2015) ............................................................................................... 137
Hideki Matsumoto
University of Fukui, Biomedical Imaging Research Center / Associate Professor

2. Oocyte maturation and fertilization IV...................................................................... 139
Kazuyoshi Chiba
Ochanomizu University, Department of Biological Sciences/ Professor

3. 24th Symposium on Environmental Chemistry ........................................................... 141
Mayumi Ishizuka
Graduate School of Veterinary Medicine, Hokkaido University / Vice Director

4. 8th International Tunicate Meeting ............................................................................. 143
Kazuo Inaba
Shimoda Marine Research Center, University of Tsukuba / Professor

5. GIW/InCoB2015 - Joint Conference of the 26th International Workshop
on Genome Informatics (GIW) and the 14th International Conference on
Bioinformatics ....................................................................................................... 145
Paul Horton
National Institute of Advanced Industrial Science and Technology / Director

6. Satellite Symposium by the 40th Annual Meeting of Japanese Society for
Microcirculation .................................................................................................... 147
Toyotaka Yada
Department of Clinical Engineering, Kawasaki University of Medical Welfare / Professor .

Ⅳ . The 29th (fiscal year 2015) Promotion Report
第 29 期 （2015 年度） 助成事業報告 ............................................................... 149
Ⅴ . The 29th (fiscal year 2015) Financial Report
第 29 期 （2015 年度） 財務報告 ....................................................................... 155
Ⅵ . List of the Board Members
役員名簿 .............................................................................................................. 157
Ⅶ . Information from the Secretariat
事務局便り ........................................................................................................... 161-5-

Introduction

Akimichi Kaneko, MD, PhD
Chairman of the Board of Trustees
This booklet includes research and meeting reports written by the 2014 grantees. The
Foundation was originally established on September 4, 1987 with basic assets of JPY 1
billion donated by Ciba-Geigy AG, Switzerland for the purpose of “contributing to academic
development and thus improving public health and welfare by means of promoting creative
research and international exchange in the field of life science and related chemistry”. Since
then, the Foundation has granted approximately JPY 1.9 billion to more than 1,600 researches
and international exchange activities in 28 years. The research funding we provide may be an
extremely small portion of the expenses required for the entire research. However, we havebeen giving grants, hoping to make contributions to the development of excellent researches asmuch as possible. We are glad to feel the joy of grantees when they express their gratitude inan acknowledgment to our Secretariat in their research papers and realize that we are making
certain contributions.
Our Foundation gives grants to innovative researches in the areas of bio/life science, related
chemistry and information science. Multiple members of the Selection Committee review theapplication forms applied by many researchers in a fair and equitable manner and only a few
excellent proposals become subject to grants. We welcome innovative ideas. We expect that new
theories and attempts will bear fruit some day and lead to create a new industry or new therapy
in the future.
This report is a collection of essays including wonderful research conducted by grantees.
The fruit of each researcher‘s labor is the driving force that promotes Japan’s academic
standards. I sincerely appreciate all who support activities of the Foundation including the
Selection Committee, which has selected these outstanding researches.

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はじめに
代表理事 金 子 章 道
ここに 2014 年度にノバルティス科学振興財団研究助成金を受けられた方々の研究報告を収録いたしました。 当財団は 1987 年 9 月 4 日、 スイス、 チバガイギー社からの 10 億円のご寄附をもとに、 「生物・生命科学および関連する化学の領域において、創造的な研究ならびに国際交流への助成を行うことにより、 学術の振興を図り国民の健康と福祉の向上に寄与する」 ことを目的に設立されました。 爾来 28 年間に 1,600件余り、 金額にしておよそ 19 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極く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。 多くの研究者から応募のあった申請書は、 複数の選考委員により公正かつ公平に審査され、 数少ない優れた提案が助成対象となっています。 私達は、 独創的なアイデアを歓迎しています。 新しい理論や試みはいつの日か実を結び、 将来の新規産業や新規治療法に結びついていくことを期待しています。
この年報は受賞者の皆様の素晴らしい研究がまとめられたエッセイ集です。 研究者
お一人おひとりの努力の結晶は我が国の学術水準を発展させていく原動力です。 こ
れらの優れた研究を選考していただいた選考委員の皆様をはじめ、 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。

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II.
Reports from the Recipients ofNovartis Research Grants-8-Effect of dendritic cell based vaccine against highly virulent
cryptococcal infection
Yuki Kinjo

National Institute of Infectious Diseases
ykinjo@niid.go.jp

Abstract
Cryptococcus gattii is an emerging highly virulent fungal pathogen that can evade immune
responses. Our data demonstrate that the DC vaccine is effective to inhibit fungal growth after
pulmonary infection with highly virulent C. gattii. The data suggested that an increase in cytokineproducing CD4T lymphocytes and the development of MGC that engulfed fungal cells wereassociated with the protection against pulmonary infection with highly virulent C. gattii.
Key words : Cryptococcus gattii, Cryptococcus neoformans, dendritic cell, DC vaccine, GXM

Introduction
Cryptococcus gattii is an emerging highly virulent fungal pathogen that infects immunocompetenthumans. This pathogen can colonize the lungs and often disseminates to the brain, causing meningitis
with a mortality rate of 8–20%. It is a key feature of highly virulent strains of C. gattii that theyevade immune responses. This evasion is mediated by glucuronoxylomannan (GXM), a major
component of capsular polysaccharides. In this study, we analyzed the effect of dendritic cell based
vaccine (DC vaccine) against highly virulent C. gattii infection.

Results
Previous studies indicated that highly virulent C. gattii strain R265 isolated from the Canadianoutbreak had immune-evasion capabilities. However, any protective immunity against C. gattii has
not been identified. We used a gain-of-function approach to investigate the protective immunity
against C. gattii infection using a DC vaccine. Mouse bone marrow-derived dendritic cells (BMDC)
efficiently engulfed an acapsular C. gattii mutant, CAP60∆, which resulted in their expression of
co-stimulatory molecules and inflammatory cytokines. This was not observed for BMDC that were
cultured with encapsulated strains (Ref 1).
To analyze the effect of dendritic cell based vaccine (DC vaccine) against infection with highly
virulent C. gattii, we transferred CAP60∆-pulsed BMDC to mice at 14 days and 1 day before
intratracheal R265 infection and analyzed survival rate, fungal burden in lung and lung pathology.
The mice transferred with DC vaccine exhibited significant amelioration of pathology, fungal
burden, and the survival rate compared with controls. The histological analysis showed that multinucleated giant cells (MGC) that engulfed fungal cells were significantly increased in the lungs ofimmunized mice. We also analyzed cytokine production by CD4 T lymphocytes in spleens and lungs-9-

of mice immunized with DC vaccine. IL-17A, IFNγ, and TNFα-producing CD4 T lymphocytes weresignificantly increased in the spleens and lungs of immunized mice. Furthermore, we compared
fungal burden in lungs between DC vaccine treated WT mice and DC vaccine treated IFNγ knockout
mice. The protective effect of this DC vaccine was significantly reduced in IFNγ knockout mice (Ref1).
To elucidate the difference in pathogenicity of C. neoformans and C. gattii, we investigated theinteraction of these two strains with mouse macrophages by confocal laser microscopy. Only
thin-capsulated, and not thick-capsulated C. neoformans and C. gattii were phagocytosed bymacrophages. Preactivation with interferon-γ increased the phagocytic rate of thin-capsulated C.
neoformans, but did not promote phagocytosis of thin-capsulated C. gattii. Lipopolysaccharide
preactivation or Aspergillus fumigatus conidia co-incubation had no effect on internalization ofthin-capsulated C. neoformans or C. gattii by macrophages. Phagocytosis of live thin-capsulated C.
neoformans, but not that of live thin-capsulated C. gattii, induced IL-12 release from macrophages.
However, phagocytosis of heat-killed or paraformaldehyde-fixed thin-capsulated C. neoformans did
not increase IL-12 release, showing that the internalization of live yeast is important for initiating
the immune response during C. neoformans-macrophage interactions. These data suggest thatmacrophage response to C. gattii is limited compared with that to C. neoformans and that theseresults may partially explain the limited immune response and the greater pathogenicity of C. gattii(Ref 2).
To understand the mechanisms of immune evasion by highly virulent strain of C. gattii, we
extracted GXM from C. neoformans and C. gattii and measured cytokine production by mouse DC
stimulated with GXM. The levels of inflammatory cytokines from DC treated with C. gattii GXM
were markedly lower than those induced by C. neoformans GXM. Then, we performed structural
analysis of C. gattii GXM and C. neoformans GXM with high performance liquid chromatography(HPLC) and nuclear magnetic resonance (NMR). Structural analysis of GXM indicated that thehighly virulent strain of C. gattii altered one of two O-acetyl groups detected in the C. neoformans
GXM. Deacetylated GXM lost the ability to induce inflammatory cytokine release from DCs,
implicating these O-acetyl groups in immune recognition. These data indicate the highly virulent
C. gattii processes a structural alteration in GXM that allows this pathogen to evade the immune
response and therefore elimination (Ref 3).

Discussion & Conclusion
Our results demonstrated that DC vaccine induces CD4T cells producing IL-17A, IFNγ, and TNFαin lungs and spleens. And, our results suggested that an increase in cytokine-producing CD4T
lymphocytes and the development of MGC that engulfed fungal cells were associated with the- 10 -

protection against pulmonary infection with highly virulent C. gattii. The data also suggest that IFNγ
may have been an important mediator for this vaccine-induced protection. To get insight into thereasons of high virulence of C. gattii, we compared the macrophage response between C. neoformans
and C. gatti. Our data suggest that macrophage response to C. gattii is limited compared with thatto C. neoformans and that these results may partially explain the limited immune response and thegreater pathogenicity of C. gatti. We also analyzed structural difference on GXM between a standard
strain of C. neoformans and a highly virulent strain of C. gattii. Our data indicate the highly virulent
C. gattii processes a structural alteration in GXM that allows this pathogen to evade the immune
response and therefore elimination. Collectively, our data show that the DC vaccine is effective toinhibit fungal growth after highly virulent C. gattii infection.

References
1. Ueno K, Urai M, Ohkouchi K, Miyazaki Y, Kinjo Y. Dendritic cell-based vaccine against fungal
infection. Methods Mol Biol, 1403:537-49, 2016.
2. Ikeda-Dantsuji Y, Ohno H, Tanabe K, Umeyama T, Ueno K, Nagi M, Yamagoe S, Kinjo
Y, Miyazaki Y ： Interferon-γ promotes phagocytosis of Cryptococcus neoformans but not
Cryptococcus gattii by murine macrophages. J Infect Chemother, 21(12): 831-836, 2015.
3. Urai M, Kaneko Y, Ueno K, Okubo Y, Aizawa T, Fukazawa H, Sugita T, Ohno H, Shibuya K,
Kinjo Y, Miyazaki Y. Evasion of Innate Immune Responses by the Highly Virulent Cryptococcus
gattii by Altering Capsule Glucuronoxylomannan Structure. Front Cell Infect Microbiol,
5:101, 2016.

一般の皆様へクリプトコックスは土壌などの環境中に存在する真菌で、吸入により肺に病巣を形成します。免疫低下症例では、中枢神経系など他の組織に播種し、髄膜炎などの播種性クリプトコックス症をおこします。近年、北米を中心に、健常人にも播種性クリプトコックス症をおこす高病原性クリプトコック
ス感染症が問題となっています。私達は、樹状細胞ワクチンを開発し、高病原性クリプトコックス感染の制御機構を解析しました。樹状細胞ワクチンにより、高病原性クリプトコックスの増殖抑制効果を認め、その免疫学的機序を明らかにしました。

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Nanoscale analysis reveals agonist-sensitive and heterogeneous poolsof phosphatidylinositol 4-phosphate in the plasma membrane
Akikazu Fujita

Kagoshima University, Joint Faculty of Veterinary Medicine
afujita2854@gmail.com

Abstract
The level of the PtdIns(4)P pool in the plasma membrane is sensitive and the distribution of
PtdIns(4)P dramatically changes by agonist stimulation, and there are active sites of production or
replenishment of PtdIns(4)P at undifferentiated membrane and caveolar areas.
Key words : cell membrane, caveolae, G protein-coupled receptor, freeze-fracture, electron
microscopy

Introduction
A substantial pool of PtdIns(4)P resides in the plasma membrane. It has been predictedthat PtdIns(4)P is essential and plays critical roles in the plasma membrane as a key
molecule in some cell phenomena. Further studies with sufficient spatial resolution
for small scale detection are needed to implicate the veritable role of PtdIns(4)P in theplasma membrane. In the present study, we employed the quick-freezing and freezefracture replica labeling (QF-FRL) method, which enables nanoscale membrane lipid
labeling by physically fixing membrane molecules. We demonstrate spatial-temporal
heterogeneity of PtdIns(4)P in the plasma membrane.

Results
To confirm the binding specificity of the anti-PtdIns(4)P antibody to PtdIns(4)P, weprepared liposomes by mixing 95 mol % PtdC and 5 mol % of either PtdIns, one of the sevenphosphoinositides, or one of the three glycerophospholipids (PA, PtdS and PtdE).

The intenselabeling of the anti-PtdIns(4)P antibody was observed in the PtdIns(4)P-containing liposome replica.
We labeled PtdIns(4)P inside Hela cultured cells with the anti-PtdIns(4)P antibody after blocking
with the PH domain of PLC-d1, and the specific labeling at the P face or cytoplasmic leaflet of
the Golgi membrane was detected. The anti-PtdIns(4)P antibody specifically labels PtdIns(4)P at
the biological membrane using our QF-FRL method. The labeling density of PtdIns(4)P in the flat
undifferentiated membrane of human fibroblast cell was quite variable; 4 and 71 gold particles per
2

mm in the lowest and highest density areas, respectively. The average density of the PtdIns(4)P label
in the replica was 23 gold particles per mm2. The L(r)-r curve of anti-PtdIns(4)P antibody labeling
only reached a low level of clustering and showed that the average radius of the labeled cluster
was 52 nm. Previously, we have found that the clustering of PtdIns(4,5)P2 labeling decreased and- 12 -

the average labeling density increased when human fibroblasts were treated with either methyl-bcyclodextrin (MbCD), to extract free cholesterol, or latrunculin A (LatA), to depolymerize actin. The
treatment of MbCD and Lat A significantly reduced the free cholesterol concentration on the cellsurface and in the total cell extract, and depolymerized actin filaments in cells, respectively. Boththe clustering and labeling densities observed by the anti-PtdIns(4)P antibody were not significantly
affected by pretreatment of human fibroblasts with either MbCD or Lat A. Using the replica labelingtechnique, the labeling of PtdIns(4)P slightly, but significantly, decreased to 64.3% of unstimlated
control level at 10 s after the Ang II stimulation. However, the labeling density began to increase at
30 s and dramatically increased to 341.9% at 40 s, and finally returned to the initial level at 130 s.
Using the labeling density in the liposome replica as a scale, we estimated the PtdIns(4)P content
in the plasma membrane of control and the Ang II stimulation at 40 s to be <0.2% and >0.5%,
respectively. Regarding PtdIns(4,5)P2, we have previously reported that intense labeling occurred
around caveolae; however, here no PtdIns(4)P labeling at caveolae was observed in control cells;
the PtdIns(4)P labeling density was comparable to or even lower than that in the undifferentiatedmembrane. In contrast to the undifferentiated membrane, the labeling density of PtdIns(4)P aroundcaveolae slightly increased at 30 and 40 s, and dramatically increased at 70 and 130 s after Ang II
stimulation, but at 130 s the labeling in the undifferentiated membrane returned to the control level
as described above.

Discussion & Conclusion
What is the physiological role of the caveolar PtdIns(4)P pool during Ang II stimulation? It has been
well known that PtdIns(4,5)P2 is a key molecule to regulate the activity of many ion channels andtransporters. Also PtdIns(4)P itself has been reported to modulate the activities of some ion channels
and transporters. Some of ion channels require both PtdIns(4)P and PtdIns(4,5)P2 for regulating theiractivities. Interestingly, many of them are concentrated in caveolae. Our previous report showedthat PtdIns(4,5)P2 in caveolae decreases at 40 sec and recovers to the initial level at 130 sec afterAng II stimulation, but PtdIns(4)P in caveolae is shown to begin to increase at 30 sec and extremely
increased at 130 sec after Ang II stimulation in the present study. The sequential down- and upregulation of ion channels and transporters in the caveolar membrane may generate specific signals
at the cell surface. Analysis with aid of mathematical modeling may be required to understand its
physiological consequence. Sequential regulation of each level of PtdIns(4)P and PtdIns(4,5)P2 in
noncaveolar and caveolar membranes may be important to complete the cellular response to agonists.
To further elucidate the physiological importance of individual PtdIns(4)P pools, their distribution
needs to be studied at the nanometer scale. Freeze-fracture immunoelectron microscopy should be auseful tool for such analyses.

References
A. Fujita, J. Cheng, K. Tauchi-Sato, T. Takenawa, T. Fujimoto, A distinct pool of phosphatidylinositol- 13 -

4,5-bisphosphate in caveolae revealed by a nanoscale labeling technique, Proc Natl Acad Sci U S
A. 106 (2009) 9256-9261.
A. Fujita, J. Cheng, T. Fujimoto, Quantitative electron microscopy for the nanoscale analysis ofmembrane lipid distribution, Nat Protoc. 5 (2010) 661-669.
A. Fujita, J. Cheng, M. Hirakawa, K. Furukawa, S. Kusunoki, T. Fujimoto, Gangliosides GM1 andGM3 in the living cell membrane form clusters susceptible to cholesterol depletion and chilling,
Mol Biol Cell. 18 (2007) 2112-2122.

一般の皆様へ細胞は主に蛋白質、脂質、糖および遺伝子から構成されている。分子生物学の飛躍的な発展により、DNA・RNA と蛋白質についての知見は急速に増加している。しかしながら、生体膜を構成する主成分である膜脂質（脂質二重層）に関しては有力な解析技術が少ないために、まだまだ未知の点が多く残されている。私たちは脂質を観る新たな技術を開発し、生体膜における微細構造レベルでの膜脂質の分布解析を可能にした。この技術は膜脂質の機能解明に大きく貢献し、このアプローチ
によりもたらされた知見は、脂質代謝異常をはじめ様々な疾患の原因や病態の解明に役立つものと期待している。

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Extended use of the retinal proteins to control the biological activities
by light
Yuki Sudo

Okayama University
sudo@pharm.okayama-u.ac.jp

Abstract
Rhodopsins are photoactive membrane-embedded proteins having a retinal as a choromophore.
Here we performed three research projects: i) discovering novel proteins from the nature, ii)
analyzing the molecular properties by biophysical techniques, and iii) applying them for controlling
biological activities by light. As shown in references, we obtained some interesting results.
Key words : retinal, photoreceptor, membrane, bioenergetics, optogenetics

Introduction
One of the major topics in life science is to understand and utilize biological functions using
various advanced techniques. Taking advantage of the photoreactivity of the seven-transmembranerhodopsin protein family has been actively investigated by a variety of methods. Additionally,light is one of the most essential factors for most organisms. On Earth, photoreceptor proteins havecontinuously evolved to use solar energy efficiently in organisms over the past 4.6 billion years.
Rhodopsins serve as models for membrane-embedded proteins, for photoactive proteins and as afundamental tool for optogenetics, a new technology to control biological activity with light. In thisstudy, we performed microbial rhodopsin research from the viewpoint of their distribution, diversity
and potential.

Resultsi) Novel molecules: Although a large number ofgenes encoding microbial type-1 opsins have been
identified in archaea, no opsin gene from bacteria
or eukarya has been identified until recently.
Here we found and characterized several novel
microbial rhodopsins, including a light-driven
proton pump from the eubacterium Pantoea
vagans (PvR) [1] and a light-gated anionchannel-2 from the eukaryote Guillardia theta (ACR2) [2],on the basis of environmental genomics. Thus today, a variety of microbial rhodopsin genes havebeen identified from all domains of life (i.e., archaea, eubacteria and eukarya), indicating the great
physiological significance of the widely distributed microbial rhodopsins for organisms [3].

- 15 -

ii) Functional and structural analysis: The
wide distribution of microbial rhodopsins has also
revealed their rich functional diversity. Here wecharacterized two rhodopsins, channelrhodopsin(ChR) and thermophilic rhodopsin (TR).
ChR is a light-gated cation channel responsible
for motility changes upon photo-illumination. Here we concluded that the N- and/or C-terminal
extended modules of ChR are necessary for the maintenance of the suitable photochemical
properties and the light-gated ion channeling function [4].
TR is a photoreceptor protein with an extremely high thermal stability and the first characterized
light-driven electrogenic p roton pump derived from the extreme thermophile Thermus
thermophilus JL-18. Here we determined the crystal structure of TR at 2.8 Å resolution [5]. Several
distinct structural characteristics of TR, including a decreased surface charge, a larger numberof hydrophobic residues and aromatic-aromatic interactions, were clarified. Based on the crystal
structure, the structural changes of TR upon thermal stimulation were investigated by molecular
dynamics simulations. The simulations revealed the presence of a thermally-induced structural substate, in which an increase of hydrophobic interactions in the extracellular domain, the movementof extracellular domains, the formation of a hydrogen bond and the tilting of transmembrane helices
were observed. From the computational and mutational analysis, we propose that an extracellular
LPGG motif between helices F and G plays an important role in the thermal stability as a “thermal
sensor”. These findings will be valuable for understanding retinal proteins with regard to highprotein stability and high optogenetic performance.
iii) Potential for controlling the biologicalactivities: Here, we successfully produced blueshifted color variants for neural activation [6].
In addition, we first expressed TR as a fusion
construct with green fluorescent protein (GFP) inneurons of Caenorhabditis elegans (C. elegans).
Then we observed the locomotion paralysis
upon illumination. Finally we repor ted therobust neural silencing activity of TR as the firsteubacterial rhodopsin available for optogenetics[5]. In addition, TR is assumed to have a putative
carotenoid binding cavity in the 5th helix.
Because the carotenoid has a broad and blue-shifted absorption spectrum with a large molecularextinction coefficient, the TR-carotenoid complex would be useful for the optogenetics as a moleculethat absorbs a wide range of the visible region with high efficiency.

- 16 -

Discussion & Conclusion
In this report, we summarized our recent progress of microbial rhodopsin research. In short, wediscovered novel rhodopsins, characterized their structures and functions, and applied them to
optogenetics. In addition to the results presented here, we also obtained some unpublished data
regarding the distribution, diversity and potential of microbial rhodopsins. Several papers are
now under construction. The newly identified, optimized and modified rhodopsins are applicablefor controlling not only the neural activity but also a variety of biological functions in the near
future. Finally, I wish to thank collaborators, especially Profs. Takeshi Murata (Chiba University),
Shigehiko Hayashi (Kyoto University) and Shin Takagi (Nagoya University) for structural studies,
MD-simulations and optogenetic measurements.

References[1] *Sudo, Y. & *Yoshizawa, S. (2016) Photochem. Photobiol. in press.
[2] Doi, S., Tsukamoto, T., Yoshizawa, S., & *Sudo, Y. (2016) to be submitted.
[3] Kurihara, M. & *Sudo, Y. (2015) Biophys. Physicobiol. 12, 121-129. [review][3] *Sudo, Y. (2016) YAKUGAKU ZASSHI, 136, 185-189. [review][4] Doi, S., Mori, A., Tsukamoto, T., Reissig, L., Ihara, K. & *Sudo, Y. (2015) Photochem.
Photobiol. Sci. 14, 1628-1636.
[5] Tsukamoto, T., Mizutani, K., Hasegawa, T., Takahashi, M., Honda, N., Hashimoto, N., Shimono,K., Yamashita, K., Yamamoto, M., Miyauchi, S., Takagi, S., Hayashi, S., *Murata, T. & *Sudo, Y.
(2016) J. Biol. Chem. in press.
[6] Kato, H.E., Kamiya, M., Sugo, S., Ito, J., Taniguchi, R., Orito, A., Hirata, K., Inutsuka, A.,
Yamanaka, A., Maturana, A.D., Ishitani, R., Sudo, Y., *Hayashi, S. & *Nureki, O. (2015) Nat. Commun.
6, 7177.

一般の皆様へロドプシンと呼ばれるタンパク質は、ビタミン A を発色団とする光受容体で、様々な生物における
光エネルギー変換にかかわっています。本研究では、（1）自然界から新しいロドプシンを探すこと、(2) それらを様々な手法により調べること、(3) その知見に基づき、様々な生命現象を光で操作するツールを開発すること、の３点を目的としました。実際に、幾つかのロドプシンの発見、形や昨日の決定、
光神経制御を達成しました。

- 17 -

Positive feed-forward loop of mTOR complex links cellular metabolism
to targeted therapy resistance
Kenta Masui

Department of Pathology, Tokyo Women’s Medical University
masui-kn@research.twmu.ac.jp

Abstract
Cancer cells adapt their signaling in response to nutrient availability. We discovered that external
nutrients including glucose or acetate promoted growth factor receptor signaling through acetylCoA–dependent acetylation of Rictor, a core component of the mTORC2 signaling complex, forming
an auto-activation loop of mTORC2 in a deadly brain cancer glioblastoma. A surprising implicationof this study is that nutrient can contribute to targeted therapy resistance by maintaining signaling
through downstream components of the growth factor receptor signaling cascade.
Key words : mTORC2, metabolic reprogramming, targeted therapy resistance, glioblastoma

Introduction
Cancer cells reprogram their metabolism, which is a consequence of upstream mutations in thegrowth factor receptor to activate mechanistic target of rapamycin (mTOR) signaling. Understanding
how cancer cells harness cellular metabolism and its metabolites for their survival may yield insights
into cancer pathogenesis and the ways that tumor cells use to resist targeted therapies. To uncover
the mechanisms regulating cancer metabolism and its functional consequences, we interrogatedcell lines, mouse tumor models and clinical samples of glioblastoma (GBM), the highly lethal brain
cancer.

Results
We recently identified an unexpected Aktindependent role for mTORC2 in GBM metabolic
reprogramming through its ability to control
c-Myc protein level [1]. Indeed, transcriptomic
and metabolomic analyses demonstrated
that mTORC2 regulates the central carbon
metabolism including glycolytic metabolism andthe subsequent metabolic processes to produce
intermediary metabolites. Given the importance of mTORC2-dependent metabolic reprogrammingin GBM pathogenesis, we asked whether mTORC2 signaling itself, could be responsive to theexternal nutrient microenvironment. We added exogenous glucose and acetate to U87 GBM cells
and measured mTORC2 activation [p-AKT S473 and p-NDRG1 T346] and made the surprising- 18 -

discovery that glucose or acetate, two “fuelsources” that are widely available in the brain andreadily taken up by tumor cells are required toactivate oncogenic EGFR-mTOR signaling andpromote tumor growth (Fig. 1A) [2]. Specifically,
we found that glucose or acetate is metabolized
into acetyl coenzyme A (acetyl-CoA) via pyruvate
dehydrogenase (PDH) and acetyl-CoA synthetase
2 (ACSS2) to activate mTORC2 signaling in
GBM cells.
Next we assessed how intermediate metabolites, specifically acetyl-CoA, promote mTORC2
oncogenic signaling. Biochemical analyses demonstrated that glucose and acetate promote theacetylation of Rictor protein through acetyl-CoA to activate mTORC2, linking nutrient availability
and metabolism to oncogenic mTORC2 signaling (Fig. 1B). Additionally, bioinfomatic and genetic
analyses suggested that specific, evolutionarily conserved lysine residues within the Rictor protein(K1116, K1119 and K1125) are important in the glucose-dependent acetylation of Rictor. These
results demonstrate that glucose promotes Rictor acetylation to activate mTORC2.
Protein acetylation, including the acetylation of Rictor, can be controlled through the balance
between histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities [3]. We
demonstrated that one of the AGC subfamily kinases, PKCα phosphorylates and inactivates class IIa
HDACs downstream of mTORC2 signaling, and Rictor is in turn physically associated with class IIa
HDACs and deacetylated by them. This signaling cascade forms an auto-activation loop of mTORC2
and promotes the activity of mTORC2 (Fig. 2). Importantly, the circuitry of mTORC2 signaling,
inactivating phosphorylation of class IIa HDACs, and Rictor acetylation is coordinately up-regulated
in human GBM patients and may be involved in the GBM pathogenesis in the clinic. Together,
the results indicate that mTORC2 forms an auto-activation loop through acetyl-CoA- and HDACmediated Rictor acetylation, which underlies the mechanism of mTORC2’s response to nutrient
availability and metabolic reprogramming in EGFR-mutant GBMs.
We then hypothesize that an acetylation-dependent auto-activation loop maintains mTORC2
signaling independent of upstream stimulation. Therefore, GBM cells with activated mTORC2 in thepresence of glucose would be expected to be resistant to therapies that target its upstream elements
including EGFR and PI3K. We found that GBM cells maintain mTORC2 signaling even under the
suppression of its upstream stimulator, EGFR, through an acetylation-mediated auto-activation
loop (Fig. 1C). Further, using genetic and pharmacological approaches, we showed that glucosedependent acetylation of Rictor makes tumor cells resistant to therapies targeting key components
of a growth factor receptor signaling pathway commonly hyper-activated in cancer by maintaining
persistent downstream signaling (Fig. 2). The observations make the surprising prediction that GBM
cells may use nutrients to escape targeted therapies.

- 19 -

Discussion & Conclusion
Cancer cells reprogram their cellular metabolism to meet the biosynthetic and energetic demands
imposed by rapid tumor growth, and this process seems to be central to GBM pathogenesis. In GBM,
mTORC2 appears to be a central node regulating this process, contributing to tumor growth and drug
resistance. Our results have a number of potentially important and unanticipated implications. First,
extracellular nutrients can maintain oncogenic signaling in GBM cells, and the environment of GBM
patients such as hyperglycemia would affect the therapeutic efficacy. Second, these works raise
the question of how lifestyle changes, including diet, can potentially shift tumor cell metabolism
and promote cancerous growth by linking oncogenic signaling driven by genetic mutations, theextracellular environment and tumor metabolism [5]. Unraveling these important questions may
point the way toward the more effective targeted cancer treatments, including for patients with GBM.

References
1. Masui K, et al. mTOR complex 2 controls glycolytic metabolism in glioblastoma through FoxO
acetylation and upregulation of c-Myc. Cell Metab 18:726-39, 2013.
2. Masui K, et al. mTORC2 in the center of cancer metabolic reprogramming. Trends Endocrinol
Metab 25:364-373, 2014.
3. Masui K, et al. Glucose-dependent acetylation of Rictor promotes targeted cancer therapy
resistance. Proc Natl Acad Sci U S A 112:9406–9411, 2015.
4. Glidden EJ, et al. Multiple site acetylation of Rictor stimulates mammalian target of rapamycincomplex 2 (mTORC2)-dependent phosphorylation of Akt protein. J Biol Chem 287:581–588,
2012.
5. Masui K, et al. mTORC2 and Metabolic Reprogramming in GBM: at the Interface of Genetics
and Environment. Brain Pathol 25:755-9, 2015.

一般の皆様へわれわれが一貫して研究している脳腫瘍の一つである膠芽腫 ( グリオブラストーマ ) は、様々な治療に抵抗性を示す非常に悪性度の高い脳腫瘍です。本研究の結果、悪性脳腫瘍において近年注目されている細胞代謝の異常活性化が、治療抵抗性につながっているという新しい病態を見出すことができました。すなわち、脳腫瘍患者さんにおける栄養状態などの環境因子が、がんの成長や治療効
果に大きな影響をおよぼすことが考えられます。今後は、これらのがんの特徴を逆に利用し、薬剤や食事などを介して、がん細胞の代謝活動へ介入することで、悪性脳腫瘍の代謝を標的とする新規の治療戦略開発に繋げていきたいと考えています。

- 20 -

Analysis of molecular mechanisms for cell fate determination of retinal
interneurons
Rikako Sanuki

Institute for Protein Research, Osaka University
rikako.sanuki@protein.osaka-u.ac.jp

Abstract
Amacrine cells, which are highly diversified interneurons, play critical roles in the processingof retinal visual information. However, molecular mechanisms underlying subtype specificationof retinal amacrine cells have been poorly understood. In this study, we found that Prdm13 (PRDomain-Containing 13) transcription factor is strongly expressed in the amacrine subpopulation of
the mouse retina and Prdm13 is essential for differentiation of GABAergic and glycinergic amacrine
cells. The Prdm13-deficient mice exhibited elevated visual sensitivities. Thus, the current study
showed that Prdm13 regulates subtype specification of retinal amacrine cells and Prdm13-positive
amacrine cells modulates visual sensitivity.
Key words : Retina, Interneuron, Transcription factor, Vision

Introduction
The vertebrate retina consists of five types of neurons and one type of glial cell. Among these cell
types, amacrine cells are interneurons modulating retinal visual information just prior to ganglion
cells which transmit processed signals to the brain. Although amacrine cells are divided into ~30
subtypes, neither subtype specification mechanism nor subtype function are well understood. We
found Prdm13 transcription factor is significantly expressed in a subpopulation of amacrine cells in
the retina. We examined a molecular and in vivo function of Prdm13 in the retina using the mice withtargeted disruption of the Prdm13 gene.

Results
We expressed Prdm13 into retinal progenitor cells by in vivo electroporation and analyzed their celldifferentiation. We found that the forced expression of Prdm13 promoted amacrine cell differentiationof retinal progenitor cells. Then, we overexpressed Ptf1a, an upstream gene of Prdm13, into retinal
progenitor cells and compared with Prdm13-overexpressed cells by immunohistochemistry. We
immunostained the retina with antibodies against AP-2α and AP-2γ which mainly mark GABAergic
and glycinergic amacrine cells, or Islet1 which marks cholinergic amacrine cells. Whereas 48.5
± 3.2% of Prdm13-electroporated cells were positive for AP-2α, only 17.0 ± 2.4% of Ptf1aelectroporated cells were positive for AP-2α. AP-2γ amacrine cells were induced at a similar levelin both Prdm13- or Ptf1a-electroporated retinas (Prdm13: 23.6 ± 2.0% and Ptf1a: 25.5 ± 1.9%). In
contrast to relatively weak AP-2α induction, Ptf1a stimulated the differentiation of Islet1-positive- 21 -

cholinergic amacrine cells much more strongly than Prdm13 did (Prdm13:9.6 ± 1.9%, Ptf1a: 35.0
± 4.7%, p < 0.01). These data suggested that Prdm13 preferentially promoted GABAergic andglycinergic amacrine cells, but Ptf1a induced GABAergic, glycinergic, and cholinergic amacrine
cells.
Next we produced and examined Prdm13 knockout (KO) mice. We immunostained the Prdm13null retina with an anti-Calretinin antibody and found the loss of sub-layer-segregating nerve fibers
between the sub-layer 2 (S2) and sub-layer 3 (S3) of the inner plexiform layer (IPL) of the Prdm13
KO retina due to a reduced number of Calretinin-positive amacrine cells (Fig. A). We identified
that these Calretinin-positive amacrine cells were GABAergic and/or glycinergic cells. In order toclarify functions of Prdm13-positive amacrine cells, we analyzed retinal light responses of Prdm13
KO mice. By recording electroretinograms (ERGs), we found totally normal ERG amplitudes of thePrdm13 KO mice, including a-waves, b-waves, and oscillatory potentials (Fig. B). To investigate
the effect of the Prdm13 defect, we next measured OKRs in WT and Prdm13 KO mice. OKR is areflect eye movement observed in responding to moving objects. We compared initial and late OKRs
between WT and Prdm13 KO mice. Initial OKRs are observed in a very short period within 500 ms
after visually sensing a moving object, whereas late OKRs are a series of eye movements alternating
slow tracking and quick resetting to the initial eye position. We quantitatively measured OKRs in
WT and Prdm13 KO mice by plotting mean amplitudes for each visual stimulus in the coordinate
system of spatial and temporal frequencies, and visualized quantified OKR responses of WT and
Prdm13 KO mice as a heat-map (Fig. C and D). In the initial OKRs, Prdm13 KO mice showed OKR
responses to a broader range of SF and TF than WT mice (Fig. C, SF: WT 0.06–0.25 cycles/deg,
Prdm13 KO 0.03– 0.25 cycles/deg; TF: WT: 0.75–12 Hz, Prdm13 KO 0.375–12 Hz). Consistent with
the initial OKRs, in the late OKRs, Prdm13 KO mice displayed OKR responses to a broader range ofSF and TF compared with WT mice (Fig. D, SF: WT 0.12– 0.25 cycles/deg, Prdm13 KO 0.03– 0.25
cycles/deg; TF: WT: 0.75–3 Hz, Prdm13 KO 0.187–12 Hz). These data suggest that Prdm13 KOmice show significantly elevated sensitivities to visual stimuli.

- 22 -

Discussion & Conclusion
In the current study, we investigated the functions of Prdm13 in retinal amacrine cells. Whereas
forced expression of Ptf1a promoted the specification of both cholinergic amacrine cells (Islet1positive cells) and GABAergic/glycinergic amacrine cells (AP-2α- and AP-2γ-positive cells), forced
expression of Prdm13 preferentially induced generation of GABAergic/glycinergic amacrine cells.
These observations demonstrate that Prdm13 regulates the specification of more limited amacrine
subtypes than Ptf1a, and suggest that Ptf1a up-regulates Prdm13 to subdivide Ptf1a-lineage amacrine
cells. In OKRs, we observed higher temporal and spatial sensitivities in Prdm13 KO mice comparedwith those in WT mice. Prdm13-positive amacrine cells projecting to the CALBs-positive S2/S3
border bundle in the IPL play important roles in visual processing in the retina. Although amacrineinterneurons, which are mainly inhibitory neurons, have been thought to be crucial for visual
information processing in the retina, their subtype complexity makes our understanding on the exact
function and mechanism of each amacrine subtype extremely difficult. The current study may shedlight on a functional role of the Prdm13-positive amacrine subtype, which specifically project to the
S2/S3 border bundle, in vision.

References
Watanabe S, Sanuki R, Sugita Y, Imai W, Yamazaki R, Kozuka T, Ohsuga M, Furukawa T.
Prdm13 regulates subtype specification of retinal amacrine interneurons and modulates visual
sensitivity.
J Neurosci. 2015 May 20;35(20):8004-8020.

一般の皆様へ眼の奥にある網膜は外界からの光情報を受容し、電気信号に変換して情報処理を行った後に脳へ視覚情報を送り出すユニークな神経組織です。今回の研究で、私たちは網膜の光情報処理に重要だと考えられているアマクリン細胞という神経細胞に注目しました。一言にアマクリン細胞といっても、
30 種類以上もの異なるタイプが存在し、各タイプが作りだされるメカニズムや機能はほとんど知られていませんでした。私たちは、Prdm13 というたんぱく質が特定のタイプのアマクリン細胞を作り出すのに必須であることを発見しました。興味深いことに、Prdm13 によって作りだされるアマクリン細胞を欠失した遺伝子改変マウスでは、視覚情報の時間的、空間的な認知感度が上昇する、つまり視覚機能が良くなることを見出しました。視覚機能が悪くなる遺伝子改変マウスは今まで数多くの例がありますが、視覚機能が良くなった遺伝子改変マウスはこれが世界で初めての例です。このことから網膜には視覚機能を抑制するタイプのアマクリン細胞が存在することが明らかとなりました。

- 23 -

Mechanism of meiosis in triploid planarians:
Difference in the chromosomal elimination timing between the sexes
Midori Matsumoto

Keio University
mmatsumo@bio.keio.ac.jp

Abstract
Triploid planarians reproduce truly bisexually, and they succeed in meiosis by chromosomal
elimination. Female germline cells remained triploid until prophase I; however, male germline cellschanged from triploid to diploid until germ cell differentiation. Meiotic recombination protein Rec8,
an important component of the meiotic cohesion complex, plays a key role in the diplotene stage forchromosome pairing, however, not for chiasmata formation.
Key words : planarian, triploid, meiosis

Introduction
Dugesia ryukyuensis, belonging to the phylum Platyhelminthes, are populations with triploidkaryotypes, normally found in nature as both fissiparous and oviparous planarians. We examined
the genotypes of the offspring obtained by breeding sexualized triploids and found that they
reproduced truly bisexually. Furthermore, meiotic chromosomal behavior in triploid sexualized
planarians differed significantly between male and female germlines; hence, female germline cells
remained triploid until prophase I, whereas male germline cells appeared to have become diploid
before entry into meiosis.
In this study, we have identified the timing at which the male germline cells become diploid from
triploid and the function of the meiosis-specific cohesion protein, Rec8.

Results
Timing of the chromosome elimination of the male germline stem cell
The male germline cells in triploid planarians reduce one set of chromosomes and become diploid
prior to meiosis. The pluripotent stem cells in planarians, termed neoblasts can differentiate
into germline stem cells. In spermatogenesis, spermatogonia enter meiosis and develop into
spermatocytes, and then sperms.
To understand when one set of chromosomes was deleted during the differentiation process ofmale germline stem cells, the ploidy of male germline stem cells was first determined. The specificmarker for germline stem cells is Dr-nanos, a nanos homolog.
Fluorescence in situ hybridization (FISH) was performed using Dr-nanos against the dissociatedcells of the testicular region, and DNA quantity was measured by propidium iodide (PI) staining.

- 24 -

The DNA quantity ratio of Dr-nanos-negative cells and Dr-nanos-positive cells was 1.5.
Meiosis-specific cohesin homolog Rec8
We identified four potential cohesion genes from the RNA-Seq transcriptome data. These genes
were acquired during the sexualization process of D. ryukyuensis, which coded for proteins withsignificant homology to yeast SMC1 and Rad21, and mouse Rec8. As the phylogenetic analysis
demonstrated that the two cohesion genes were more similar to SMCs, we named them Dr-SMC1
and Dr-SMC3, respectively. Since the other two genes were more similar to Rad21/Rec8-like family,
we named them Dr-Rad21 and Dr-Rec8, respectively. Dr-Rec8 was predicted to encode a 631-aminoacid protein, which was very much similar to the Rec8 homolog found in Xenopus tropicalis,(E-value = 7e-07). Dr-Rad21 was predicted to encode an 835-amino acid protein, which was similar
to the Rad21 homolog in Drosophila melanogaster (E-value = 2e-59).
Expression of Dr-Rec8 in sexual worms
To examine the expression pattern of Dr-Rec8, we performed whole-mount in situ hybridization(WISH) for the sexualized planarians. Dr-Rec8 was highly expressed in the ovaries and testes.
After sexualization, the testis developed in the upper part at first and later on, enlarged to the lower
part. A clear signal for Dr-Rec8 transcripts was seen in the upper portion of the testis of the sexual
worm which had just matured; however, the signal shifted to the lower portion following complete
maturation of the testis. When WISH was performed for the asexual D. ryukyuensis OH strain, it
indicated that Dr-Rec8 was not expressed throughout the planarian body.
Functions of Dr-Rec8 genes in germ cells and sex organs
To analyze the Dr-Rec8 acting as a functional homolog of Rec8 in D. ryukyuensis, we performed
RNAi of Dr-Rec8 during the regeneration of sexual worms. On hematoxylin and eosin (HE)staining of Dr-Rec8 (RNAi), no difference could be observed in the somatic sexual organs. The
ovaries tended to become small. However, as Dr-Rec8 was produced at low levels in the asexual
worms, we confirmed that the Dr-Rec8 was responsible for their regeneration. Dr-Rec8 (RNAi) did
not affect regeneration, even when the worm was amputated a second time.
Functions of Dr-Rec8 genes in meiosis
To elucidate the role of Dr-Rec8 in meiosis, we studied chromosomal behavior in oocytes. Cells
at different meiotic stages can be discriminated based on their nuclear morphological features.
Chromosomal synapses appeared to occur in a similar manner in the pachytene cells and the earlydiplotene cells of Dr-Rec8 (RNAi) and control worms. However, in the diplotene cells of DrRec8 (RNAi) worms, all the chromosomes were unpaired, whereas bivalents were formed in all of
the oocytes of control animals. However, meiotic synapsis in testis appeared to occur in Dr-Rec8(RNAi) worms as well as in control worms, and the chiasmata were observed in diplotene cells of
Dr-Rec8 (RNAi) testis.

- 25 -

Discussion & Conclusion
In this study, we have shown that chromosomal elimination from male germ cells of the triploidplanarian occurred when it became the germ stem cell, at the first step of differentiation to the germcell. However, it should be noted that chromosomal elimination from female germ cells occurred at
the end of meiosis I, which emphasized the fact that the timing of chromosomal elimination of germ
cells is different between males and females. This system of sex-specific chromosomal elimination
enables their sexual reproduction in triploid planarians.
Moreover, the action of the meiosis-specific cohesin homolog, Rec8, is important in chromosomal
pairing in the diplotene stage.

References
1.

DrRad51 is required for chiasmata formation in meiosis recombination but not in mitotic
recombination in triploid planarians． Ayako Chinone, Midori Matsumoto, Molecular
Reproduction & Development 81: 409-421(2014)2.

Triploid planarian reproduces truly bisexually with euploid gametes produced through adifferent meiotic system between sex. Ayako Chinone, Hanae Nodono, Midori Matsumoto,
Chromsoma 123: 265-272 (2014)3.

Production of asexual and sexual offspring by inbreeding of the triploid sexual planarian
Dugesia ryukyuensis. Kazuya Kobayashi, Sachiko Arioka, Motonori Hoshi, Midori
Matsumoto. Integrative Zoology, 4:265-271 (2010)

4.

Kobayashi K, Ishizu H, Arioka S, Cabrera JP, Hoshi M, Matsumoto M (2008) Production of
diploid and triploid offspring by inbreeding of the triploid planarian Dugesia ryukyuensis.
Chromosoma 117(3): 289–296.

一般の皆様へ
プラナリアにはゲノムセットを２セットもつ２倍体と３セットもつ３倍体がいます。生物学の世界では３倍体の生物は減数分裂ができないために、無性生殖により自切で殖えるか、または単為生殖で殖えると考えられてきました。しかし、私たちは３倍体のプラナリアが有性生殖により殖えることを発見し、卵と精子が減数分裂の途中で染色体をセットで削減することを明らかにしました。本研究では、雌雄の削減時期が異なり、卵形成では減数分裂第一分裂期まで３倍体の状態で存在するのに対して、精子形成では雄性生殖幹細胞に分化したときにはすでに２倍体に染色体の削減が起こっていることを明らかにしました。

- 26 -

Roles of chemokine system in peritoneal adhesion formation
Yuko Ishida

Wakayama Medical University
iyuko@wakayama-med.ac.jp

Abstract
Our study demonstrated that CX3CL1-CX3CR1 axis may play a role in intraperitoneal adhesion
formation. Administration of CX3CL1 may help to prevent adhesions.
Key words : Adhesion formation, CX3CR1, chemokines

Introduction
Surgical adhesions are a common and often severe complication resulting from abdominal or
pelvic injury. Numerous postsurgical treatment and devices have been tested for their ability toabrogate adhesions or reduce their severity, but to date have been of limited effectiveness and are
not widely used. Little is known in detail about the cellular and subcellular mechanisms underlying
adhesion formation, although it has been hypothesized that reduced fibrinolytic activity, whichcorrelates with chemokine systems, plays a key role.

Results
To understand the cellular mechanism underlying cauterization-induced intestinal adhesion,
we examined CX3CR1 knock-out (KO) mice for their capacity to develop adhesion. We induced
intestinal adhesion by cecal cauterization using the coagulation mode of bipolar forceps. A timecourse experiment revealed that this treatment induced progressive inflammatory and fibroticchanges in CX3CR1 knock-out (KO) mice, compared with wild-type (WT) mice. Adhesions
strongly connected the cecum to the large bowel, the abdominal wall or both at day 7 in CX3CR1
KO mice. Wild-type (WT) mice formed significantly reduced adhesion with plantar attachment(Figure. 1). Mice that underwent control laparotomy without cecal cauterization had no adhesion.
We next examined whether surgical adhesion formation rapidly induced accumulation of
inflammatory cells, such as macrophages, T cells, and/or NK cells, in the cecal wall. We found amarked increase in the number of F4/80-positive macrophages, T cells, and NK cells at 7 days after
operation, further substantiating the idea that these cells are responsible for CX3CR1 expression
after surgical treatment (Figure 2).

- 27 -

Figure 1

Figure 2

Figure 2

Discussion & Conclusion
Our data suggested that treatment of CX3CL1, ligand for CX3CR1, seems to be a promising
way for preventing adhesion. Of course, extensive basic studies are needed for the prevention of
intentional adhesion formation in human cases.

References

一般の皆様へ
術後の臓器癒着は、胃や腸の開腹手術や帝王切開手術では90％以上の確率で生じるとされている。
癒着が形成される原因として、手術の侵襲による臓器表面の損傷、感染や縫合糸等に対する異物反応などが挙げられる。癒着はこうした組織傷害に伴う炎症反応による組織傷害を修復する過程で、フィブリンが析出して形成されることが示唆されている。一方、ケモカインは、白血球走化能を有する
生理活性物質であり、特に炎症の場において重要な役割を担っている。すなわち、ケモカインは臓器癒着に対する新規治療ターゲットとして極めて有望である。このような背景から、申請者は、癒着形成の分子メカニズムをケモカインシステムに着目して行い、その役割を解明することによって、ケモ
カインシステムを人為的に制御することができれば癒着形成の抑制・防止が期待できるのではないかと考えた。

- 28 -

Mapping and Manipulating Neuronal Circuits that regulates Sleep/
wakefulness States
Takeshi Sakurai

Kanazawa University
Takeshi.sakurai@gmail.com

Abstract
We identified neurons that make monosynaptic inputs to orexin neurons using a recombinantrabies virus-mediated trans-synaptic retrograde tracing in mice. Neurons in the many brain regions
implicated in emotion, reward system, and sleep were found to make direct synaptic input to orexin
neurons. By combination of the cell type-specific tracing the relationship between input and output(cTRIO) analysis and anterograde tracing we found many GABAergic neurons in the POA, which
send projections to orexin neurons, receive monosynaptic input by neurons in the nucleus accumbens(NAc) and BST. Pharmaco- or optogenetic activation of BST GABAergic neurons during NREM
sleep made immediate transition from NREM sleep to wakefulness. These observations suggestthat the BST and NAc regulate orexin neurons by inhibiting POA GABAergic neruons to modulateemotion-related arousal.
Key words : orexin, sleep, wakefulness, connectome

Introduction
Wakefulness and vigilance levels are required for maintaining purposeful activities and motivatedbehaviors, which are usually triggered by external cues. Hypothalamic neurons that contain theneuropeptide orexin (hypocretin) are known to be activated by emotionally salient contexts andcues [1]. We hypothesize this activation plays an important role in regulation of vigilance levels andemotion-related behavior.

Results
To reveal the mechanisms by which orexin neurons are regulated by salient cues and/or contexts,
we identified neurons that make monosynaptic inputs to orexin neurons using a recombinant rabies
virus-mediated trans-synaptic retrograde tracing in mice. We identified positive cells in the many
brain regions implicated in emotion, reward system, and sleep. These regions include the septal
regions, bed nucleus of the stria terminalis (BST), the nucleus accumbens (NAC), ventral pallidum(VP), preoptic area (POA), anterior hypothalamus (AHA), lateral hypothalamic area (LHA), basal
hypothalamic regions, posterior hypothalamus, and periaqueductal gray (PAG) of the brain stem(Fig). Cell type-specific tracing the relationship between input and output (cTRIO) analysis suggested
that many GABAergic neurons in the POA, including the VLPO, which send projections to orexin
neurons receive monosynaptic projections by neurons in the central nucleus of the amygdala (CeA)- 29 -

and BST (Saito et al. unpublished). By combination of the cTRIO analyses and anterograde tracings,
we have identified neuronal pathways that connect between the limbic structures and orexin neurons(Fig). We also functionally examined the some of these pathways. For example, pharmaco- or
optogenetic activation of BST GABAergic neurons during NREM sleep made immediate transition
from NREM sleep to wakefulness in a phase-locked manner, while stimulation during REM sleep
did not show any effects. These observations suggest GABAergic neurons in the BST send excitatory
influences to arousal systems including orexin neurons to make transition from NREM to wake,presumably through inhibition of GABAergic neurons in the POA.
Secondaly, we examined the function of orexinergic projections in the regulation of emotionrelated behavior. We previously found that targeted restoration of orexin receptor expression innoradrenergic neurons of the locus coeruleus (LC) and in serotonergic neurons of the dorsal raphe(DR) in OX1R-/-;OX2R-/- mice, which display a severe narcoleptic phenotype, differentially inhibited
fragmentation of wakefulness and cataplexy, respectively[2]. We further examined the effect of
optogenetic stimulation of serotonergic fibers arising form the DR in various brain regions on
the cataplexy in narcoleptic orexin/ataxin-3 mice (Hasegawa et al. unpublished). Stimulation ofserotonergic fibers in the lateral amygdala almost completely inhibited cataplexy, which was inducedby chocolate feeding in the mice. Optogenetic stimulation of serotonergic fibers in the PAG, SLD
and PPT had no effect on the frequency of cataplexy in these mice.
We also examined roles of the orexin neurons→LC-NA neurons→lateral amygdala (LA) pathway
in the fear-related behavioral responses [3]. After fear conditioning in the particular context,
optogenetic stimulation of orexinergic fibers in the LC, or LC-NA fibers in the LA induced anapparent freezing behavior even in the in alternative context which did not induce freezing when thestimulation was not applied (Soya et al. manuscript in preparation). Pharmacogenetic or optogenetic
inhibition of LC-NA neurons reduced the freezing in the fearful context. These results suggest thatorexin neurons activate the amygdala projecting-LC-NA neurons to modulate fear-related behavior.
This pathway is likely to increase arousal against emotional cues, and modulate fear-induced
behavior.

Fig. A schematic drawing that shows interaction between the limbic system andarousal system. Green circles indicate regions that send monosynaptic input to orexin- 30 -

neurons identified by the viral tracing. Blue and red lines suggest inhibitory andexcitatory projections, respectively, which were examined and identified by functional
studies.

Discussion & Conclusion
Our observations suggest that the limbic system plays an important role in regulation of
wakefulness by modulating orexin neurons and monoaminergic systems, and resulting activationof the monoaminergic systems in turn influences amygdala function to modulate emotion-related
behavior. This reciprocal interaction between the limbic system and arousal system including orexin
neurons might play an import role in the regulation of sleep/wakefulness states as well as emotional
memory and emotion-related behavior.

References
1. Sakurai T (2014) The role of orexin in motivated behaviours. Nat Rev Neurosci 15:
719-731.
2. Hasegawa E, Yanagisawa M, Sakurai T, Mieda M (2014) Orexin neurons suppress
narcolepsy via 2 distinct efferent pathways. J Clin Invest 124: 604-616.
3. Soya S, Shoji H, Hasegawa E, Hondo M, Miyakawa T, et al. (2013) Orexin receptor-1
in the locus coeruleus plays an important role in cue-dependent fear memory
consolidation. J Neurosci 33: 14549-14557.

一般の皆様へオレキシンは、1998 年に私たちのグループが発見した脳内物質です。オレキシンは、私たちヒトを含む動物が何らかの行動をとるために必要な覚醒を維持するために必須の分子です。今回、オレキシンを作る神経細胞がどのような機構で活性化されるのかを解明するために、この神経細胞に入力する神経細胞群を明らかにしました。その結果、感情を司る部分からたくさんの制御を受けていることが明らかになりました。オレキシンを作る神経細胞は感情が高ぶった時に活性化され、覚醒を支え、適切な行動をとるために働くことが推測されます。さらに、オレキシンを産生するニューロンの活性化は、覚醒を高めるだけではなく、ノルアドレナリンという別の脳内物質の作用を介して、感情や感情と結びついた記憶の制御にも関与していることをあきらかにしました。オレキシン受容体の拮抗薬はすでに不眠症治療薬として使われていますが、今後、PTSD など不安や恐怖と関連する症状の改善にもオレキシン系に作用する薬が用いられる可能性があります。

- 31 -

Developmental and physiological analyses of spinal neuronal circuitsthat generate rhythmic locomotor activities
Shin-ichi Higashijima

Natural Institutes of Natural Sciences, Okazaki Institute for Integrative Bioscienceshigashi@nibb.ac.jp

Abstract
Rhythmic movements of pectoral fins during swimming in larval zebrafish is an attractive model forthe investigation of coordinated rhythmic movements of limbs. We performed electrophysiologicalrecordings from fin motoneurons (MNs), and found that both abductor (Ab) MNs and adductor (Ad)MNs show spiking activities in particular phases during the swimming cycle. Both Ab MNs and Ad
MNs received alternating excitatory and inhibitory inputs in a swimming cycle, suggesting that thesealternating inputs are the basis for the rhythmic firings of fin MNs.
Key words : Spinal cord, locomotion, rhythm generation

Introduction
Limbed vertebrates exhibit coordinated rhythmic movements of forelimbs and hindlimbs duringlocomotion. Neuronal circuits that control rhythmic limb movements in mammals have been
investigated for decades, but our knowledge is still limited because of the complexity of their limb.
Rhythmic movements of pectoral fins during swimming in larval zebrafish is an attractive model.
Pectoral fins of larval zebrafish show left and right alternated rhythmic movements, and they are
actuated only by two types of muscles, i.e., abductor (Ab) and adductor (Ad). Due to the simplicity
of pectoral fins, we expect that we well be able to characterize neuronal circuits that control rhythmic
pectoral fin more deeply.

Results
We performed electrophysiological recordings of Ab motoneurons (MNs) and Ad MNs during
fictive swimming. Both Ab MNs and Ad MNs show spiking activities in particular phases during theswimming cycle. Activities of Ab MNs and Ad MNs on the same side essentially alternated. BothAb MNs and Ad MNs received alternating excitatory and inhibitory inputs in a swimming cycle,
and excitations mainly occurred in their preferential firing phase, and inhibition mainly occurred
for the rest of the period. To obtain insights into the source of these inputs, we investigated timingsof spiking activities in possible premotor interneurons. The firing phase of neurons that express atranscription factor, En1, coincided with the phase that Ab MNs received inhibitory inputs. Given
that En1-positive neurons are ipsilaterally-projecting inhibitory neurons, the results suggest that En1positive neurons may provide direct monosynaptic inhibitory inputs onto Ab MNs. We investigatedwhether such monosynaptic connections exist, using channelrhodopsin-mediated photo-stimulation.

- 32 -

Upon brief photo-stimulations to larvae that expressed channelrhodopsin in En1-positive neurons,
time-locked IPSPs were recorded in Ab MNs. This strongly suggests that En1-positive neurons makemonosynaptic inhibitory connections onto Ab MNs.

Discussion & Conclusion
The alternating excitatory and inhibitory inputs are likely to be the basis for the rhythmic firingsof pectoral fin MNs. We now have evidence that En1-positive neurons provide direct inhibitions
onto Ab MNs. We are now investigating other types of interneurons with respects to their firingproperties and synaptic connections onto fin MNs. Future studies will reveal a near-complete pictureof neuronal circuits that generate rhythmic pectoral fin movements.

References
一般の皆様へ
脊椎動物は、移動の際にリズミックな運動（哺乳類では歩行運動）を行います。このリズム運動を司る神経回路は脊髄内に存在していますが、その詳細は、50 年以上の研究にも関わらずいまだに明らかになっていません。私は、ゼブラフィッシュという小型魚類を用いて、この課題の完全解明に迫っていきたいと考えています。シンプルなゼブラフィッシュを用いることにより、このリズム運動を司る神経回路の動作様式の基本原理に迫っていけるものと思います。基礎科学者の立場ではありま
すが、得られる成果は、脊椎動物全般に通用するものとなり、脊髄損傷からの回復等の応用研究に対しても有意義なものになると信じています。

- 33 -

Identification of the roles of SIRT7 in brown adipocytes
Kazuya Yamagata

Department of Medical Biochemistry, Faculty of Life Sciences, Kumamoto University
k-yamaga@kumamoto-u.ac.jp

Abstract
The metabolic roles of SIRT7 are largely unknown. We generated adipocyte specific SIRT7
knockout mice and found that the body temperature of the mice is increased. In addition, Ucp1 gene
expression in the brown adipose tissue (BAT) of the knockout mice was significantly increased.
These results suggest that SIRT7 in BAT plays an important role in the energy metabolism in mice.
Key words : SIRT7, brown adipocyte, Ucp1, energy metabolism

Introduction
Sirtuin (SIRT1-7) is an NAD dependent deacetylase and plays important roles in metabolism,
inflammation, DNA repair, and longevity. However, the roles of SIRT7 in metabolism are largely
unknown. We recently identified that the body temperature of SIRT7 knockout mice was higher
than that of control mice and Ucp1 gene expression in brown adipose tissue (BAT) in the SIRT7
knockout mice was increased (Cell Metabolism 2014). These results suggest that SIRT7 plays a rolein BAT. In the present study, we examined the role of SIRT7 in BAT.

Results(1) Identification of SIRT7 interacting proteins.
To clarify the roles of SIRT7 in BAT, we searched SIRT7 interacting proteins in BAT byperforming affinity chromatography (HaloTag pull-down assay) of mouse BAT extract, followed byliquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified that SIRT7 interacts
with RNA splicing factors and RNA helicases. The impact of the interaction between SIRT7 andthese RNA editing proteins on BAT function is now under investigation.
(2) Adipocyte specific SIRT7 knockout mice.
To identify the roles of SIRT7 in BAT, we generated adipocyte
specific SIRT7 knockout mice by crossing SIRT7 flox/flox miceand adiponectin-Cre transgenic mice (kindly provided from Prof.
Evans). As was observed in systematic SIRT7 knockout mice
(Cell Metabolism 2014), the body temperature of the adipocyte
specific SIRT7 knockout mice was higher than that of thecontrol mice. In addition, the expression of temperature related
genes such as Ucp1 and Dio2 was significantly increased in the- 34 -

adipocyte specific SIRT7 knockout mice (Figure 1), indicating the importance of SIRT7 in BAT inthe control of temperature.
(3) The effect of SIRT7 knock-down in vitro.
We generated SIRT7 knock-down brown adipocyte cell line by retrovirus infection of SIRT7shRNA. In contrast the phenotype in adipocyte specific SIRT7 knockout mice, gene expression
levels of Ucp1 and Dio2 in the knock-down cells were unchanged. These data may suggest that theexternal stimulation (e.g. activation of the sympathetic nerve) is also required for the altered gene
expression in the BAT of SIRT7 knockout mice.

Discussion & Conclusion
Previously, we found that the gene expression related to thermogenesis was increased in SIRT7
knockout mice and SIRT7 knockout mice exhibited increased body temperature. However, it was
unclear whether SIRT7 in BAT itself or SIRT7 in other tissues (e.g. neuron) is important for thephenotypes. To clarify the question, we generated adipocyte specific SIRT7 knockout mice andfound that SIRT7 in adipocyte plays an important role for the thermogenesis. Inhibition of SIRT7
could be beneficial for the treatment of metabolic syndrome and type 2 diabetes by increasingenergy expenditure. Since SIRT7 knock-down in brown adipocyte cell line did not affect Ucp1
and Dio2 gene expression, further studies will be necessary to clarify the detailed mechanism of
regulation of BAT function by SIRT7.

References
Yoshizawa T, Karim MF, Sato Y, Senokuchi T, Miyata K, Fukuda T, Go C, Tasaki M, Uchimura
K, Kadomatsu T, Tian Z, Smolka C, Sawa T, Takeya M, Tomizawa K, Ando Y, Araki E, Akaike T,
Braun T, Oike Y, Bober E, Yamagata K: SIRT7 controls hepatic lipid metabolism by regulating theubiquitin-proteasome pathway. Cell Metab. 19: 712-721, 2014

一般の皆様へサーチュインは代謝や寿命を制御する酵素であり、ヒトやマウスでは７種類のファミリー分子（SIRT1-SIRT7）が存在しています。これまで SIRT7 の代謝作用は全く不明でしたが、我々は SIRT7の欠損マウスは高脂肪食で誘導される糖尿病に抵抗性を示すことを見出しました（Cell Metabolism
2014）。
本研究の結果、褐色脂肪組織における SIRT7 が熱産生やエネルギー代謝に深く関与していることが明らかになりました。褐色脂肪組織における SIRT7 の抑制薬はエネルギー代謝を亢進させること
で糖尿病やメタボリックシンドロームの治療に有用である可能性が明らかになりました。

- 35 -

A novel mechanism of regulation of melanocortin receptor signaling
mediated by glycolipids in hypothalamic inflammation.
Kei-ichiro Inamori

Tohoku Pharmaceutical University
kinamori@tohoku-mpu.ac.jp

Abstract
GM3, a precursor for the synthesis of a- and b-series gangliosides, has been implicated in insulin
signaling and inf lammation. We generated GM3 synthase knockout mice on the backgroundof obese model KKAy (KKAy/GM3SKO) and interestingly, found that while KKAy mice are
hyperphagic and develop severe obesity, the KKAy/GM3SKO mice showed marked improvementin the body weight and food intake. Hypothalamic response to leptin administration was wellpreserved in the KKAy/GM3SKO mice, suggesting that hypothalamic GM3 and/or its related
gangliosides play an important role in the leptin signaling.
Key words : ganglioside, GM3, hypothalamus, leptin, melanocortin

Introduction
Gangliosides are the group of glycosphingolipids containing sialic acid and are known as
fundamental components of membrane microdomains, where they participate in many biologicalevents including cell growth, adhesion, and signal transduction. GM3, a precursor for the synthesisof a- and b-series gangliosides, has been shown to be increased in conditions of obesity andinflammation. Obesity-induced inflammation causes both insulin resistance and leptin resistance,
resulting in type 2 diabetes and the metabolic syndrome. Although there is growing evidence that
this response occurs in the hypothalamus as well as in peripheral tissues by high-fat feeding, thedetailed molecular mechanism remains unclear.

Results
In this study, we attempted to investigate the role of GM3 and related ganglioside species in thepathogenesis of obesity by generating a mouse model in which GM3 synthase gene is disrupted in
the yellow obese KKAy mouse. KKAy mice have been established by introducing Ay mutation into
KK mice, resulting in ectopic expression of agouti signaling protein (ASP) that causes perturbed
melanocortin signaling and the mice develop more severe and earlier onset of obesity and diabetic
pathology than in KK mice.
We generated GM3 synthase knockout mice in the background of KK and KKAy (KK/GM3SKO
and KKAy/GM3SKO, respectively). Interestingly, we found that the KKAy/GM3SKO mice havegrayish coat color while the heterozygotes remain yellow. The yellow Ay mutation (mutated in the
agouti gene) causes ectopic expression of the gene product, ASP, directing follicular melanocytes to- 36 -

switch from the synthesis of eumelamin (black) to phaeomelamin (yellow) through the inhibition ofmelanocortin 1 receptor (MC1R) signaling as an antagonist by blocking α-melanocyte stimulating
hormone (α-MSH), the agonist for the receptor. Our observation of the coat color change of the
KKAy/GM3SKO mice suggests that GM3 and/or its related ganglioside is involved in the MC1R
signaling.
While KKAy mice are hyperphagic and develop severe obesity, the KKAy/GM3SKO mice
showed marked improvement in the body weight and food intake, which was comparable tothose observed in the control KK and KK/GM3SKO mice. In hypothalamus, which is one of thecentral sites of food intake regulation, melanocortin 4 receptor (MC4R) plays an essential rolefor decreasing food consumption through the receptor activation by binding to its ligand α-MSH,
a pro-opiomelanocortin (POMC)-derived neuropeptide. The agouti-related protein or AgRP,
a homologue of ASP and an endogenous antagonist of MC4R, is produced locally within the
hypothalamus and inhibits the action of α-MSH on MC4R-bearing cells. In the KKAy brain, it has
been know that deregulated overexpression of ASP antagonizes the binding of α-MSH at MC4R
in the hypothalamus, resulting in increased food consumption. Our observation of decreased foodconsumption of the KKAy/GM3SKO mice prompted us to examine the hypothalamic response toleptin, an adipocytokine which acts on leptin-sensing neurons in the hypothalamic arcuate nucleus(ARC). In ARC, AgRP neurons are inhibited by leptin, whereas POMC neurons are stimulated
to release α-MSH, resulting in suppression of food intake through the activation of MC4R. We
administered leptin to KKAy and KKAy/GM3SKO mice intraperitoneally and assessed the responseby measure of c-fos immunostaining in the ARC. The KKAy mice showed very few c-fos staining
cells as indicative of leptin resistance, whereas the expression was well preserved in the KKAy/
GM3SKO mice, indicating that the KO mice retained leptin responsiveness.

Discussion & Conclusion
Considering with the observation of the coat color change in the KKAy/GM3SKO mice, ourresults suggest that GM3 and/or its related gangliosides play an important role in the leptin
and melanocortin signaling. Since it has been shown that GM3 expression is upregulated
by inf lammatory cytokines in adipocytes, our results raise the possibility that hypothalamic
inflammation might alter the ganglioside expression pattern and it might be one of the mechanisms
underlying the dysregulation of feeding and body weight control. Currently we are investigating theexpression pattern of gangliosides in hypothalamus of obese models and the role of gangliosides in
the receptor functions.

References
1) Tagami, S., et al.: J Biol Chem, 277: 3085-3092 (2002)- 37 -

2) Kabayama, K., et al.: Proc Natl Acad Sci USA, 104: 13678-13683 (2007)
3) Yamashita, T., et al.: Proc Natl Acad Sci USA, 100: 3445-3449 (2003)
4) Thaler, J.P., et al.: Diabetes, 62: 2629-2634 (2013)

一般の皆様へ
脂肪細胞がつくるホルモンであるレプチンは、脂肪の蓄積量に比例して分泌も増え、体重のコント
ロールに重要な役割をもちます。しかし、肥満症患者の多くは血中レプチン濃度が高いのにも関わらず、その作用（摂食抑制やエネルギー代謝亢進）が不全であるレプチン抵抗性にあることが知られています。これまでに、ガングリオシドとよばれる糖脂質がインスリン受容体の機能に関わることが示されていましたが、本研究において、脳の視床下部でガングリオシドが、レプチンおよびメラノコルチン受容体が関わる摂食調節やエネルギー代謝機能に役割をもつことが示唆されました。各受容体機能におけるガングリオシドの役割について検討を行っています。

- 38 -

Analysis of Epigenetic gene expression mechanism of DDI2/3 by DNA
damageMasaya Oki

University of Fukui
ma4sa6ya@u-fukui.ac.jp

Abstract
We focused on a novel epigenetic regulation gene by DNA damage and identified DDI2/3 geneusing ChIP on chip and Microarray analysis. DDI2/3 gene expression was regulated by Sir complex
which was important to make Heterochromatin, and the expression increase significantly by DNA
damage.
Key words : Epigenetics, Single cell, DNA damage, S.cerevisiae, Chromatin

Introduction
A heterochromatin region does not spread forever and stop by making a boundary. We had reported
about “Heterochromatin boundary” which moved among individual cell and regulated epigenetic
gene expression by single cell analysis in S.cerevisiae. However biological function of “boundary”
is still unknown. Therefore we focused Sir3 protein that was key factor to make heterochromatinin S.cerevisiae. We performed ChIP on chip analysis using the anti Sir3 antibody and Microarray
analysis using sir3 deletion strain, and we narrowed down the candidate gene DDI2/3 which an
epigenetic gene expression change by fluctuation of boundary by DNA damage.

Results(A) Development of Single cell analysis system
To examine epigenetic gene expression change through the over generations in single cells, wedeveloped a new single cell analysis system that utilizes expression of fluorescent proteins. Using
this methods, a single cell is placed in the center of the field of vision of a microscope and changes
in epigenetic gene expression that occur during cell division are followed using time-lapse imaging.
This experiment result showed Figure 1.

- 39 -

Fig1: Time-lapse imaging of single cell through multiple generations.
(B) Analysis of Epigenetic gene expression by DNA damage
We focused DDI2 and DDI3 gene in this candidate that expression was known to be induced at thetime of DNA damage induction and report the result that became clear by single cell analysis. We
constructed yeast strain for single cell analysis that left a promoter and the terminator which were
important to expression control and replace ORF to encode fluorescence protein (EYFP).
We used MMS (Methyl Methanesulfonate) for DNA damage induction and started division from
1 cell and added MMS after progress for eight hours and chased the change of the expression state
without MMS and continued culture. Images showed left panel in Fugure2 after 8h, 16h, and 30hfrom the start of division of single cell. The expression of DDI3 was always OFF, and m-cherry(Red) was internal control. Images showed right panel in Fugure2, cells cultured without MMS until
8hours and added 0.03% MMS after 8hours. After 16hours, we changed again fresh medium without
MMS and continued culture. Images showed after 8h, 16h, and 28h from the start of division of
single cell. When DDI3 expressed, EYFP (Yellow) appeared.

Fig2. Image of time-lapse experiment by MMS- 40 -

Discussion & Conclusion
Expression state of every cell was OFF until we added MMS, but we added MMS, and an
expression state became “ON” was different speed compared with an individual cell, and when
we removed MMS, expression state again became “OFF”, this timing also different among theindividual cell. The boundary expansion and contraction speed was different in individual cells, andthis result suggested the possibility that a boundary change controlled the epigenetic gene expression
state.

References[1]

K. Kamata, G. Goswami, S. Kashio, T. Urano, R. Nakagawa, H. Uchida and M. Oki. J.
Biochem.,155 (2014) 159.

[2]

Y. Mano, TJ Kobayashi, J Nakayama, U. Uchida and M. Oki.
PLOS Biology, 11 (2013) e1001601[3]

K. Kamata, A. Hatanaka, G. Goswami, K. Shinmyozu, J. Nakayama, T. Urano, M. Hatashita,
H. Uchida and M. Oki. Genes to cells, 18 (2013) 823.

[4]

A. Hatanaka, B. Chen, JQ. Sun, Y. Mano, M. Funakoshi, H. Kobayashi, Y. Ju, T. Mizutani, K.
Shinmyozu, J. Nakayama, K. Miyamoto, H. Uchida and M. Oki. Genes Genet. Syst., 86 (2011)
305.

[5]

JQ. Sun, A. Hatanaka, M. Oki. Genes Genet.Syst., 86 (2011) 73.

一般の皆様へ
DNA 配列に依存しないエピジェネティックな発現制御機構は、癌をはじめとする様々な疾患への関与が報告され注目を集めています。従来までのエピジェネティックス研究は主に細胞集団を対象にして行われてきましたが、エピジェネティックな発現は個々の細胞によって異なっていることが明らかとなり１細胞での解析の重要性が指摘されています。本研究では独自に１細胞の世代を越えたエピジェネティクな発現状態変化を追跡するシステムを確立し、DNA 損傷を受けたときにのみエピジェネテ
ィックに発現状態が変化する

DDI2/3 遺伝子を見出しました。今後、どのようなタンパク質がどのようなメカニズムで DDI2/3 遺伝子の発現を調節しているかを明らかにすることにより、新たな転写調節機構の発見が期待できます。

- 41 -

Drug discovery for neuropathic pain from processed aconite root
Toshiaki Makino

Graduate School of Pharmaceutical Sciences, Nagoya City University
makino@phar.nagoya-cu.ac.jp

Abstract
Processed aconite root (PA, the root of Aconitum carmichaeli) is a crude drug used in traditional
Chinese or Japanese kampo medicine to generate heat in the body and to treat pain associated withcoldness. Oxaliplatin (L-OHP) is a platinum-based anticancer drug that frequently causes acuteand chronic peripheral neuropathies, including cold and mechanical hyperalgesia. I investigated theeffects of PA on L-OHP-induced peripheral neuropathies and identified the active ingredient within
PA extract. L-OHP was intraperitoneally injected into mice, and PA boiled water extract was orally
administered. Cold and mechanical hyperalgesia were evaluated using the acetone test and the vonFrey filament method, respectively. Dorsal root ganglion (DRG) neurons were isolated from normal
mice and cultured with L-OHP with or without PA extract. Cell viability and neurite elongation
were evaluated. PA extract significantly attenuated cold and mechanical hyperalgesia induced by
L-OHP in mice. In cultured DRG neurons, L-OHP reduced neurite elongation, and PA extract
significantly alleviated it. From PA extract, I isolated neoline as active ingredient. Subcutaneous
injection of neoline attenuated cold and mechanical hyperalgesia in L-OHP-treated mice. Neoline ispromising agents to alleviate neuropathic pain.
Key words : Processed aconite root; oxaliplatin; peripheral neuropathies; dorsal root ganglion;
neolineIntroduction
Oxaliplatin (L-OHP) is commonly used to treat colorectal cancer. L-OHP frequently induces acuteand chronic peripheral neuropathy, with this side effect potentially being dose limiting, leading todiscontinuation of chemotherapy. I evaluated the effects of processed root of Aconitum carmichaeli(PA) on cold and mechanical hyperalgesia in mice treated with L-OHP, and develop the active
ingredient for drug discovery for neuropathic pain.

Results
Mice were evaluated for mechanical hyperalgesia by von Frey test 3 days after receiving asingle intraperitoneal (i.p.) injection of L-OHP (10 mg/kg). The mechanical threshold value wassignificantly lower compared with the vehicle-treated group 3 days after L-OHP treatment andremained significantly lower from day 3 to day 7. When PA powder was administered orally
to L-OHP-treated mice at a dose of 1 g/kg/day, the induction of mechanical hyperalgesia wassignificantly inhibited on days 5–7. When PA powder was orally administered to mice treated with- 42 -

5% glucose (vehicle), the mechanical threshold was not significantly changed. A single i.p. injectionof L-OHP (10 mg/kg) also produced hypersensitivity to acetone stimulation in mice 4 days after the
L-OHP injection, and this L-OHP-induced cold hyperalgesia lasted up to 7 days after the treatment,
indicating a significant main effect of L-OHP and PA treatment, time, and interaction between
L-OHP and PA treatment and time. Oral administration of boiled water extract of PA significantly
reduced the L-OHP-induced hypersensitivity to cold stimulus on days 4–7.
I prepared primary cultures of DRG neurons from mice. L-OHP inhibited neurite elongation inmurine DRG neurons, and the administration of boiled water extract of PA significantly preventedit.
I attempted to isolate the active ingredients from PA extract. First, we partitioned AL and non-AL
fractions from boiled water extract of PA. DRG neurons were cultured with L-OHP (1 µM) plus AL
or non-AL at a concentration corresponding to 1.0 mg/ml of PA extract. The alkaloidal fraction (10µg/mL) significantly prevented the shortening of neurite elongation induced by L-OHP. Both AL
and PA extract significantly improved L-OHP-induced hyperalgesia on day 7, and the mechanicalthresholds exhibited equivalent levels after treatment with either AL or PA, indicating a significant
main effect of L-OHP, PA or AL treatment, and time, but not interaction between L-OHP, PA or AL
treatment and time.
Next, AL was further separated into five fractions using silica gel preparative TLC. Toxic
alkaloids, aconitine, and mesaconitine were contained in fraction 1, and benzoylaconine, the marker
compound of PA documented in the Japanese Pharmacopoeia XVI, was contained in fraction 2.
When each fraction was added into the medium at 2 µg/ml, fractions 1, 2, and 5 did not exhibit
a significant preventive effect but fraction 3 and 4 significantly reduced the inhibition of neurite
elongation of DRG neurons induced with L-OHP. A single common compound was identified in
fraction 3 and 4 on TLC. I isolated compound 1 from fraction 4. Based on 1H- and 13C-NMR and EIMS analysis, compound 1 was identified as neoline. Neoline significantly prevented the inhibitionof neurite elongation in DRG neurons in a concentration-dependent manner, and 2 µg/ml of neoline
negated the effect of L-OHP on neurite length.
I examined the effect of neoline on the development of L-OHP-induced neuropathic pain.
Subcutaneous injection of neoline (10 mg/kg) increased the lowered mechanical threshold in
L-OHP-treated mice, indicating a significant main effect of L-OHP and neoline treatment, time, andinteraction between L-OHP and PA treatment and time. This increase was evident 3 days after the
L-OHP treatment and lasted for 5 days after treatment. The effect of neoline on cold hyperalgesia
was evident at 4 days after the L-OHP treatment, indicating a significant main effect of L-OHP andneoline treatment, and time, but not interaction between L-OHP and neoline treatment and time.

- 43 -

Discussion & Conclusion
I could isolate neoline as an active ingredient of PA. There is only one pharmacological study
reporting that neoline has a protective action against sodium pentobarbital-induced damage to
cardiomyocytes by restoring beating rhythm and increasing the cell viability. Neoline is not themarker compound used to control the quality of PA, and it is likely that the content of neoline amongcommercially available PA products varies considerably. When physicians recommend PA to prevent
neuropathic pain in L-OHP-treated patients, it could be beneficial that pharmacists suggest the useof PA products with higher content of neoline. In the future, new drugs to treat neuropathic pain
could be developed from neoline, and these agents could be potentially used to treat neuropathic
pain caused by other chemotherapeutic agents, such as paclitaxel or vincristine or in Seltzer model.

References
The Society of Japanese Pharmacopoeia, 2012. The Japanese Pharmacopoeia Sixteenth Edition.
Yakuji-Nippo, Tokyo, Japan.
Xiong, L., Peng, C., Xie, X.F., Guo, L., He, C.J., Geng, Z., Wan, F., Dai, O., Zhou, Q.M., 2012.
Alkaloids isolated from the lateral root of Aconitum carmichaelii. Molecules 17, 9939-9946.

一般の皆様へオキサリプラチンという抗がん薬は、副作用として神経障害性疼痛を高頻度で発症し、それが用量や治療継続の制限因子となっています。本研究では、マウスにオキサリプラチンを投与した時に生じる神経障害性疼痛に対する加工ブシ（キンポウゲ科ハナトリカブトの根を減毒処理した生薬で、さまざまな漢方薬に配合されます）の緩和作用について検討し、その有効成分として neoline を発見しました。本化合物はこれまで鎮痛活性は知られておらず、神経障害性疼痛の予防、治療のための新薬のシーズとして利用が可能と考えられます。

- 44 -

Research on genome system regulating bone morphogenesis
Koji Tamura

Tohoku University, Graduate School of Life Sciences
tam@m.tohoku.ac.jp

Abstract
An animal group-specific regulation of bone morphology and sizing were examined in somevertebrate model systems. Or results revealed some molecular mechanisms on group-specificmorphogenesis of the bone.
Key words : bone morphogenesis, genome, hippo pathway, bone size

Introduction
Bone morphogenesis, how appropriate bone morphology and size are established, is an important
information on vertebrate evolution because the bone is the main component of vertebrate
fossils. Bone morphogenesis is also a key for organ regeneration such as limb regeneration. Bonemorphogenesis should be regulated by certain genome information, and an animal group-specific(a group of vertebrates such as fishes, birds, and mammals) genome information would regulate
the group-specific morphology of the bone. In this study, we investigate a group-specific genome
information which is involved in the group-specific morphology of the bone. We also investigate
molecular mechanism of bone sizing during bone regeneration.

Results
We selected 100 candidate genes from chicken
genome, regulatory sequences of which containsome avian group- specific sequences. Our gene
expression screening, in which we compared
expression pattern of those candidate genes
among chicken, mouse, and gecko embryos,
revealed that some of the 100 candidates show

Figure1. Expression pattern of candidate genes in the developing limb.

avian-specific expression in the developing limb.
Boc and Pax9, which have chicken-specificexpression in the developing limb (Fig. 1),
are good candidates for regulators of bonemorphogenesis because they are expressedin the first digit (thumb) region that isvery short specifically in the bird group.
We also studied molecular mechanism of- 45 -

Figure2. Effect of dn TEAD on regenerating bone size.

bone sizing (control of bone length) during limb regeneration. Hippo signaling pathway
is known to regulate organ size, and TEAD is an intra-cellular component of the hippo
pathway (Ref. 1). Dominant-negative TEAD, which down-regulates the hippo pathway,significantly shorten the bone size during limb regeneration in amphibian (Fig. 2).
Further investigations on molecular interaction between the above factors, Boc, Pax9and Hippo pathway, are waiting for more new insights into morphogenesis for a groupspecific bone morphology.

Discussion & Conclusion
Our results on avian-group specific gene expression showed a possibility that regulatory sequences
specific to the avian genomes may regulate specific gene expression of those genes in the birds. Boc
and Pax9 are expressed in the first digit-forming region in the chicken forelimb, and it is possiblethat they are involved in bird-specific morphology of the first digit bone. Further studies on thesegenes such as functional assay and reporter assay are interesting, and we are now preparing theseexperiments.
Regarding bone sizing, we showed that down regulation of this pathway decreased the length ofregenerating limb bone, suggesting that hippo signaling pathway is involved in bone sizing during
limb regeneration. We will further investigate the function of this signaling pathway on boneregeneration, with using constitutive active form of TEAD.
These molecular mechanisms (Boc, Pax, and Hippo) are so far independent in terms of bonemorphogenesis and sizing, but some studies (Ref 2 and 3) have suggested they interact each otherand coordinate some developmental events, and further investigation on these pathways will givenew insights into coordination of a group-specific bone morphology and size.

References
1. Hayashi et al., 2015. Roles of Hippo signaling pathway in size control of organ regeneration.
Development Growth and Differentiation, 57, 341-351.
2. Allen et al., 2011. Overlapping roles and collective requirement for the coreceptors GAS1, CDO,
and BOC in SHH pathway function. Developmental Cell, 20, 775-787.
3. Manderfield et al., 2014. Pax3 and Hippo signaling coordinate melanocyte gene expression inneural crest. Cell Reports, 9, 1885-1895.

一般の皆様へ
骨は脊椎動物の化石記録にも残る貴重な構造であり、また哺乳類の四肢などの器官再生を考える
上で無くてはならない存在でもあります。ある動物群に特徴的な骨の形態（たとえばヒトを含め霊長- 46 -

類は 5 本指だが鳥類はみな 3 本指）や長さ（たとえばクジラの前腕骨は短いがフラミンゴの大腿骨は長い）がどのようなメカニズムで制御され作られるのかは、進化を考える上で、また定まった形態を再生させるために、重要な情報となります。本研究では、ゲノム情報と遺伝子機能の面からいくつかの分子（遺伝子）に着目し、ある動物群に特有の骨形態や骨の長さを生み出すメカニズムを解析しました。今後いくつかのメカニズムの関わり合いを調べていくことで、動物に固有の骨形態の形成機構を明らかにできるものと期待します。

- 47 -

Research on neural circuits for the control of water- and salt-intake
behaviors
Takeshi Y. Hiyama

National Institute for Basic Biology
hiyama@nibb.ac.jp

Abstract
Animals continuously monitor body-fluid conditions in the brain, and thereby feel thirst or salt
appetite to maintain the homeostasis; plasma angiotensin II (Ang II), which induces water andsalt intakes, is known to be increased regardless of water-depleted or Na-depleted conditions. We
revealed that thirst and salt appetite were driven by distinct groups of Ang II receptor positive
neurons in the subfornical organ (SFO) that project to distinct nuclei. The SFO is a brain region,
where Na+-levels in body fluids are sensed by Nax channels. Optical control of the activities of therespective pathways specifically modulated respective behaviors.
Key words : Homeostasis, circumventricular organs, body fluid, thirst, salt

Introduction
Since Na+ is the main cation in body fluids (such as plasma and the cerebrospinal fluid) and themain determinant of osmolality, Na+ levels ([Na+]) in body fluids are continuously monitored in thebrain in order to strictly maintain them within physiological ranges. We have already shown that the
SFO, a brain region devoid of a blood-brain barrier, is the sensing site of Na+-levels in body fluids
to control salt appetite, and that the Nax channel is the sensor for it. [Na+]-dependent activation of
Nax in the SFO leads to suppression of salt appetite (J. Neurosci., 2000: Nature Neuroscience, 2002;
Neuron, 2007; Neuron 2010; Cell Metab, 2013; Pflügers Arch, 2015).

Results
We employed a highly efficient retrograde gene-transfer virus system to achieve the neuralpathway specific gene-expression. We injected the retrograde virus encoding Cre recombinase to
the mice, in which the portion of the Ang II receptor gene can be excised with Cre recombinase. In
these mice, Ang II receptors were deleted specifically in the neurons innervating the injected nuclei.
We found that the Ang II receptor positive neurons in the SFO can be divided into several subgroups
depending on the targeting nuclei. When the Ang II receptors were deleted from a subgroup, thewater-intake amount after the diuretics injection was markedly reduced. On the other hand, deletionof Ang II receptors from the other subgroup resulted in the reduction of salt intake under the same
condition.
We next injected the retrograde virus encoding the channelrhodopsin, a light-gated ion channel,
to the target nuclei of Ang II receptor positive neurons in the SFO. Exogenous expression of the- 48 -

channelrhodopsin in neurons provides a means of activating distinct populations of neurons withan unprecedented degree of spatial and temporal precision. After the injection of the virus, we
implanted stainless steel cannulas to the mice head aiming at the SFO. To achieve in vivo photo
illuminations of freely moving mice, a blue laser light was delivered through plastic optic fibers
inserted into the cannulas. The laser output (20 Hz) for optical activation with channel rhodopsin
was maintained at 5–10 mW as measured at the tip of the fiber. When we started the opticalexcitation of the neural pathway that was critical for the water intake in the deletion experimentsof Ang II receptors, the mice rushed to the water and drank much. When the light stimulation was
terminated shortly after the initiation of drinking, most mice immediately stopped drinking andleft the spout, indicating that the neural stimulation is the motivation for drinking. It is noteworthy
that salt intake was not affected by the optical excitation of this condition. After the opticalexcitation, intensive Fos expression, a marker of neural excitation, was observed at the target nucleiof the optically manipulated neurons, indicating that the optical excitation of the neural pathway
successfully evoked synaptic transmission, and activated the post synaptic neurons in the target
nucleus.
We next tried to optically silence this neural pathway using archaerhodopsin, a yellow-lightdrivable proton pump. We injected the retrograde virus encoding archaerhodopsin into the same
nuclei as the channel rhodopsin experiments. We connected the optical fibers to the mice as thesame way and the fibers were connected to a yellow light laser. In order to achieve the opticalinhibition with archaerhodopsin, the laser output was maintained at 7–10 mW as measured at the
tip of the fiber. The light stimulation markedly reduced water intake after dehydration. We also
succeeded to modulate the appetite for salt by stimulating the other neural pathway.

Discussion & Conclusion
To maintain a physiological level of Na/osmolality in body fluids, the control of Na/water intake
and excretion are of prime importance. Since Buggy et al. reported the central roles of Ang II inwater and Na dual intakes (Buggy and Fisher, Nature 1974), the dipsogenic and natriorexigenic
functions of Ang II have been important targets in the research field of body-fluid homeostasis. In
the present study, we demonstrated that different groups of Ang II receptor-positive SFO neurons
separately encode thirst and salt appetite. We herein referred to them as “water neurons” and“salt neurons”, respectively. The results of the present study provide an insight into the central
mechanisms by which blood [Na+] and Ang II regulate thirst and salt appetite in a coordinatedmanner. Our study thus shed light on neural mechanisms to generate appropriate water/salt intake
behaviors depending on the body condition.

References
1.

Noda, M. & Hiyama, T.Y. (2015) Sodium sensing in the brain. Pflugers Arch. 67, 465-74.

2.

Matsumoto, M., Hiyama, T.Y., Kuboyama, K., Suzuki, R., Fujikawa, A. & Noda, M. (2015)- 49 -

Channel properties of Nax expressed in neurons. PLOS ONE 10, e0126109.
3.

Noda, M. & Hiyama, T.Y. (2015) The Nax Channel: What It Is and What It Does.
Neuroscientist 21, 399-412.

一般の皆様へ
ヒトを含む哺乳動物は、脳の感覚性脳室周囲器官において、体液のナトリウムレベルを監視するとともに、アンジオテンシン II などの血中ホルモンを介して末梢からの情報を受け取っています。この情報に基づいて、飲水 / 塩分摂取の制御と腎臓における排泄／再吸収の制御を統合的に行っています。我々は、脳のナトリウムレベルセンシング機構を明らかにしてきました。本研究成果は、そのような体液情報に基づいた水分／塩分摂取行動の制御を司る神経回路の全容解明につながるものです。

- 50 -

Development of a comprehensive synthetic strategy and study of
biological activity of yuzurimine alkaloids
Ichiro Hayakawa

Okayama University
ichiro.hayakawa@okayama-u.ac.jp

Abstract
Synthetic study of all the yuzurimine-type alkaloids was investigated. We achieved theconstruction of the [6-7-5-5] tetracyclic core of all the carbocyclic frameworks of yuzuriminetype alkaloids by using a unique in situ intramolecular Wittig reaction and samarium-mediatedcyclization as key steps. Furthermore, we designed and prepared the tricyclic iminium compound as
a common intermediate of heterocyclic portion of all the yuzurimine-type alkaloids, featuring the
Staudinger reaction-aza-Wittig reaction and intramolecular SN2 reaction.
Key words : Yuzurimine-type alkaloids, intramolecular Wittig reaction, Samarium-mediatedcyclization, Staudinger reaction, aza-Wittig reaction

Introduction
Yuzurimine-type alkaloids possess a complex polycyclic condensed ring-system, and various
analogues have been reported so far. The skeleton of yuzurimine-type alkaloids is similar to that ofgalantamine, which is known drug of Alzheimer’s disease. However, synthesis of yuzurimine-type
alkaloids has never been reported. Thus, we planned to develop a comprehensive synthetic strategy
and study of biological activity of yuzurimine alkaloids.

Results
Fo r t h i s p u r p o s e , W i t t i g r e a c t i o n o faldehyde with (methoxycarbonylmethylene)
tributylphosphorane was examined (see below).
However, we could not obtain the desired a,bunsaturated ester; only the unexpected sevenmembered enone was formed.
Wit h t he BCD r i ng of t he y u z u r i m i ne type alkaloid in hand, we next examined theconstruction of the A-ring portion. Treatmentof aldehyde with SmI 2 , LiCl, and t-BuOH
afforded lactone (21%) and tetracyclic compound(58%), which are diastereomeric at C4. These
compounds contain the [6-7-5-5] tetracarbocyclic- 51 -

cores of yuzurimine-type alkaloids. Thus, we have established the construction of the [6-7-5-5]
tetracyclic core of all the carbocyclic frameworks of yuzurimine-type alkaloids by using a unique insitu intramolecular Wittig reaction and samarium-mediated cyclization as key steps.
Furthermore, we designed and prepared the tricyclic iminium compound as a common
intermediate of heterocyclic portion of all the yuzurimine-type alkaloids, featuring the Staudinger
reaction-aza Wittig reaction and intramolecular SN2 reaction (unpublished result).

Discussion & Conclusion
A plausible reaction pathway for the formation of the unexpected seven-membered enone isshown in above. Alkylation of iodide with phosphorane followed by deprotonation with an excessof phosphorane afforded ylide. Ylide was then cyclized by intramolecular Wittig reaction to afford
the seven-membered enone. In this hypothesis, the intermolecular alkylation of the phosphorane
occurs faster than an intermolecular Wittig reaction, possibly because of steric hindrance around thealdehyde that prevents the latter from occurring.
Also, we designed and prepared the tricyclic iminium compound as a common intermediate ofheterocyclic portion of all the yuzurimine-type alkaloids. Construction of the heterocyclic portionof yuzurimine-type alkaloids from the tricyclic iminium compound is currently underway in ourgroup.

References
Construction of the [6–7–5–5] tetracyclic core, all the carbocyclic framework of yuzurimine-type
alkaloids
Hayakawa, I.; Niida K.; Kigoshi, H. Chem. Commun. 2015, 51, 11568–11571.

一般の皆様へアルツハイマー型認知症の患者様には「アリセプト ®」のようなアセチルコリンエステラーゼ阻害剤が処方されますが、現在販売されているアルツハイマー型認知症の処方薬はわずか 3 種類程度しか選択肢がなく、新たな薬のリード化合物が求められています。私達は、未だ詳細な生物活性の報
告例のないユズリミン類が、既存の処方薬であるガランタミンと 3 次元構造が類似していることに着目し、ユズリミン類の合成を検討しました。これまでにユズリミン類の高度に縮環した全炭素骨格と、4 つに分類される複素環部分構造を全て合成できる共通中間体を合成しました。今後、ユズリミン類をリード化合物としたアセチルコリンエステラーゼ阻害剤の開発を目指します。

- 52 -

Safe and Efficient Direct Cardiac Reprogramming for Heart Repair
Masaki Ieda

Department of Cardiology, Keio University School of Medicine
mieda@z8.keio.jp

Abstract
Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpressionof cardiac transcription factors, including Gata4, Mef2c, and Tbx5. We developed new Sendai
viral vectors expressing Gata4, Mef2c, and Tbx5 for efficient and safe cardiac reprogramming. We
could generate transgene-free, safe beating cardiomyocytes by SeV-GMT in this study, which canfacilitate future applications in regenerative medicine.
Key words : Heart, reprogramming, regeneration, Sendai virus

Introduction
Heart disease remains a leading cause of death worldwide, and new therapies are highly demanded.
As cardiomyocytes are terminally differentiated cells, regenerative therapy has emerged as an
attractive approach for the treatment of heart failure. Direct cardiac reprogramming approach might
be a powerful strategy toward this treatment. We reported that a combination of three cardiacspecific transcription factors, Gata4, Mef2c, and Tbx5 (GMT), could directly reprogram fibroblasts
into cardiomyocyte-like cells in vitro and in vivo.

Results
The aim of this study is to develop new Sendai viral vectors (SeVs) which overexpress
reprogramming factors without genomic integration, and to determine the efficiency and safety ofcardiac reprograming with SeVs.
1. Generation of SeV-GMT and infection of SeV
We investigated whether SeV could generate iCMs from MEFs, which have a distinct embryonicorigin compared to CFs. We used mouse embryonic fibroblasts (MEFs) from αMHC-GFP transgenic
mice, in which no cardiomyocytes or cardiac progenitor cells were detected by immunofluorescence,
fluorescence activated cell sorting (FACS), and quantitative RT-PCR (qRT-PCR) analyses. We first
determined the transduction efficiency of SeV in fibroblasts by FACS and fluorescent microscope
analyses. We found that infection of SeV-GFP into mouse fibroblasts revealed that infection
efficiency was more than 90% without harmful effects in fibroblasts (Fig 1). We determined
the appropriate MOI for SeV infection by changing MOI for SeV-GFP and found that MOi 30
was sufficient. We confirmed that the SeV-GFP alone did not induce cardiac troponin T (cTnT)
expression in MEFs after 1 week of infection. We next developed Gata4 (G), Mef2c (M), and- 53 -

Tbx5 (T) SeVs in collaboration with DNAVEC corp. We then infected SeV-GMT into MEFs, andconfirmed that GMT was expressed in MEFs by QRT-PCR (Fig 2).

Fig1. Infection efficiency by SeVMore than 90% of MEFs expressed

Fig2. Expression of GMT by SeV andretroviral vectors

GFP
2. Optimal condition for cardiac reprogramming using SeV-GMT into mouse fibroblasts
To test the optimal condition for cardiac reprogramming, we changed the appropriate temperaturefor SeV infection into fibroblasts. We investigated cardiac reprogramming efficiency by FACS
for αMHC-GFP and cTnT expression, specific markers for cardiomyocytes, after 1 week. We
used mouse embryonic fibroblasts (MEFs) from αMHC-GFP transgenic mice, in which only
cardiomyocytes expressed GFP, to determine the cardiac induction. We found that 35 degree culture
for 12 h during infection was better than 37 degree for cardiac induction by FACS. Induction of
αMHC-GFP expression was 7.6% (Fig 3), which was comparable with that of the original condition,using retroviral vector-GMT.
Fig 3. Induction of αMHC-GFP by SeV-GMT
Induction of αMHC-GFP expression was 7.6%

3. Analyses of induced cardiomyocytes by immunohistochemistry, gene expression, and functional
assaysWe analyzed cardiac reprogramming efficiency by immunohistochemistry (IHC), gene expression,and functional assays by calcium transient and counting the number of spontaneously contracting
iCMs. SeV-GMT or retroviral-GMT transduced MEFs were cultured with Dulbecco’s modified
Eagle medium (DMEM)/M199/10% fetal bovine serum (FBS) or StemPro-34 SF medium (SF) with
FGF2, FGF10, and VEGF (Yamakawa H, Stem Cell Reports 2015). The number of contracting iCMs
was counted using an All-in-One fluorescence microscope system. The uninfected MEFs did notcontract, suggesting that no starting MEFs contaminated the cardiomyocyte population.
We found that GMT-transduced MEFs cultured with FFV started to contract earlier, and a- 54 -

greater population of αMHC-GFP+ cells contracted in FFV-containing medium than in FBS.
Immunostaining revealed that the iCMs induced with SeV-GMT expressed multiple cardiac
markers, including αMHC-GFP, α-actinin, and cardiac troponin T (cTNT) and had well-definedsarcomeric structures (Fig 4). Genomic DNA analyses revealed that the SeV transgenes were notintegrated into the host genome, and no tumors were detected in culture after 2 months of SeV-GMT
transduction.

Fig 4. Induction of cardiac proteins by SeV-GMT
Induction of αMHC-GFP (left), α-actinin (middle), and merged picture (right) by SeV-GMT.

Discussion & Conclusion
We developed new Sendai viral vectors expressing Gata4, Mef2c, and Tbx5 for efficient andsafe cardiac reprogramming. We identified optimal conditions for cardiac induction, and analyzed
induced cardiomyocytes by SeV-GMT. The mouse fibroblasts transduced with SeV-GMT changed
the cell morphology from spindle to polygonal shape, expressed multiple cardiac-specific proteins,and increased a broad range of cardiac genes.
Moreover, the iCMs also concomitantly suppressed fibroblast genes, exhibited spontaneous Ca2+
oscillations, and the cells matured to contract spontaneously. These findings demonstrated thatfibroblasts could be directly converted to iCMs by defined factors without genomic integration,
which may facilitate future applications in regenerative medicine.

- 55 -

References
1. Yamakawa H, Muraoka N, Miyamoto K, Sadahiro T, Isomi M, Haginiwa S, Kojima H, Umei T,
Akiyama M, Kuishi Y, Kurokawa J, Furukawa T, Fukuda K, Ieda M. Fibroblast Growth Factors and
Vascular Endothelial Growth Factor Promote Cardiac Reprogramming under Defined Conditions.
Stem Cell Reports. 5(6):1128-42, 2015. doi: 10.1016/j.stemcr.2015.10.019.
2. Sadahiro T, Yamanaka S, Ieda M. Direct Cardiac Reprogramming: Progress and Challenges
in Basic Biology and Clinical Applications. Circ Res. 116(8):1378-1391, 2015. doi: 10.1161/
CIRCRESAHA.116.305374.
3. Muraoka N, Ieda M. Stoichiometry of transcription factors is critical for cardiac reprogramming.
Circ Res. 116(2):216-8, 2015. doi: 10.1161/CIRCRESAHA.114.305696.

一般の皆様へ
我々はこれまでに心筋リプログラミング遺伝子をレトロウイルスベクターで遺伝子導入することで直接心筋作製可能であることを示してきた。しかしながら臨床応用には挿入変異のない安全なベクターの開発が必要と考えて、センダイウイルスベクターによる安全な心筋誘導確立を目指して本研究を行った。これまでに心筋リプログラミング遺伝子を発現するセンダイウイルスベクターを作製して、このベクターを用いることでマウス線維芽細胞を直接心筋細胞にリプログラミングすることに成功した。

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A novel mechanism of temperature compensation on mammalian
circadian clocks
Yoshinori Hiraoka

Kobe Gakuin University
yhiraoka@pharm.kobegakuin.ac.jp

Abstract
In order to evaluate temperature compensation in the mammalian circadian clock, we measurecircadian rhythms in nardilysin-deficient (NRDc-/-) mice by housing at standard ambient
temperature (23 °C) and at low ambient temperature (10-15 °C). However, it is difficult to keep theirbody temperature stable for a long period at low temperature, so that we cannot measure circadianrhythms in NRDc-/- mice at low temperature.
Key words : Circadian clock, Temperature compensation

Introduction
Circadian rhythms are daily oscillations in gene expression and protein concentration that regulate
diverse physiological and metabolic processes. One of the most striking features of circadianclock is “temperature compensation”; that is, the endogenous period of the oscillation is relatively
insensitive to temperature. This characteristic is evolutionarily conserved in organisms ranging
from photosynthetic bacteria to warm-blooded mammals. Temperature compensation, however,remains poorly understood.

Results
Body temperature homeostasis in mammals is governed centrally through the regulationof shivering and non-shivering thermogenesis and cutaneous vasomotion. Non-shivering
thermogenesis in brown adipose tissue (BAT) is mediated by sympathetic activation, followed by
PGC-1a induction, which drives UCP1. We previously reported NRDc-/- mice show hypothermia(at 23 °C) and severe cold intolerance (at 4 °C), which are attributed to impaired cold-induced
BAT thermogenesis. Our studies also revealed that NRDc and PGC-1a interact and co-localized
in the UCP1 enhancer where NRDc regulates PGC-1a activity and UCP1 transcription. These
results indicate that NRDc critically regulates body temperature homeostasis, in part, through themodulation of PGC-1a activity.
As mentioned above, NRDc-/- mice could not keep their body temperature at low ambient
temperature (4 °C). In other words, the body temperature of NRDc-/- mice can change according
to the ambient temperature. In this study, we evaluate temperature compensation in mammalian
circadian clocks by housing NRDc-/- mice at standard ambient temperature (23 °C) and at low
ambient temperature (10-15 °C).

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After cold exposure (4 °C), the body temperature of NRDc-/- mice dropped below 30 °C within
2h and below 15 °C within 3h. Prolonged exposure to cold caused potentially lethal hypothermia.
Therefore, we first examined whether NRDc-/- mice can keep their body temperature under mild
cold stress (10-15 °C). The core body temperature of NRDc-/- mice was 36.5 °C at standard ambient
temperature (23 °C). When NRDc-/- mice were exposed to mild cold (10-15 °C), the core bodytemperature of them dropped to 35 °C. NRDc-/- mice can keep their body temperature around 35°C for 2 weeks, but prolonged exposure to mild cold caused lethal hypothermia. It is necessary forone month to measure circadian rhythms in NRDc-/- mice at different ambient temperature. NRDc/- mice were entrained initially to a 12 h:12 h light-dark (LD) cycle for 2 weeks and then released
into a 12 h:12 h dark-dark (DD) environment. However, it is difficult to keep their body temperaturestable for one month, so that we cannot measure circadian rhythms in NRDc-/- mice under mild
cold stress (10-15 °C).

Discussion & Conclusion
Standard housing temperature for laboratory mice in research facilities is maintained to bearound 23 °C. However, this temperature causes mild chronic cold stress to mice, activating
thermogenesis to maintain normal body temperature. Although NRDc-/- mice could not keep theirbody temperature under severe cold stress (4 °C) as previously reported, they can keep their bodytemperature under mild cold stress (10-15 °C). However, it is difficult to keep their body temperaturestable for a long period at these temperatures. One possibility is that NRDc-/- mice have a highsurface area to body mass ratio and lose heat rapidly because NRDc-/- mice are much smaller thanwild-type (NRDc+/+) mice.

References1.

Hiraoka Y, Matsuoka T, Ohno M, Nakamura K, Saijo S, Matsumura S, Nishi K, Sakamoto J,
Chen PM, Inoue K, Fushiki T, Kita T, Kimura T and Nishi E. Critical roles of nardilysin in
the maintenance of body temperature homeostasis. Nat. Commun. 2014; 5:3224. doi: 10.1038/
ncomms4224.

2.

Ohno M, Hiraoka Y, Matsuoka T, Tomimoto H, Takao K, Miyakawa T, Oshima N, Kiyonari
H, Kimura T, Kita T and Nishi E. Nardilysin regulates axonal maturation and myelination in
the central and peripheral nervous system. Nat. Neurosci. 2009; 12: 1506-1513.

一般の皆様へ生物には概日時計が備わっており、約 24 時間周期のリズム（概日リズム）を刻んでいる。概日リ
ズムは温度変化に関わらず、ほぼ一定の周期を保つ “ 温度補償性 ” があり、バクテリアから哺乳類培
養細胞にまで認められている生物時計の特徴の一つであるが、その詳しいメカニズムは明らかにされていない。したがって、我々は遺伝子 X をノックアウトしたマウスを用いて、生物時計の温度補償性のメカニズム解明を目指した研究を行っている。

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Induction of anti-tumor innate immune responses by tumor-associatedmolecular patterns
Hiroaki Ikushima

Institute of Industrial Science, University of Tokyo
hikushima-tky@umin.ac.jp

Abstract
The eradication of tumor cells requires communication to and signaling by cells of the immune
system. Here, we show that the innate immune receptor Dectin-1 expressed on dendritic cells andmacrophages is critical to NK cell-mediated killing of tumor cells that express N-glycan structuresat high levels. Consistent with this, we show exacerbated in vivo tumor growth in Dectin-1-deficientmice. The critical contribution of Dectin-1 in the recognition of and signaling by tumor cells may
offer new insight into the anti-tumor immune system with therapeutic implications.
Key words : anti-tumor immunity, innate immunity

Introduction
In harnessing the adaptive immune system, cancer immunotherapy is emerging as a successfultreatment approach for patients with several types of advanced cancers, and has the potential to
become the standard of care. The discovery of signal-transducing innate immune receptors and how
they instruct the adaptive immune system has been a pillar of Immunology for the past two decades.
Thus far, our understanding of how the innate immune receptors functions has primarily focusedto their recognition of the components of invading pathogens such as bacteria and viruses. As such,whether and how these receptors recognize tumor cells remained totally unknown.

Results
We examined the in vivo contribution of Dectin-1 to anti-tumor innate immune responses bychallenging wild-type (WT) and Dectin-1-deficient mice with the B16F1 cell lung metastasis model.
Fourteen days after tail vein injection of B16F1 cells, a marked enhancement of metastasis of B16F1
cells was seen in the lungs of Dectin-1-deficient mice as compared with those of WT mice. Loss oftumor growth control also occurred when Dectin-1-deficient mice were inoculated subcutaneouslywith B16F1 cells. In addition, the control of lung metastasis of B16F1 cells is equally lost between
WT and Dectin-1-deficient mice when NK cells were depleted prior to tumor challenge. It is worth
noting that no overt difference was observed between WT and Dectin-1-deficient mice in terms of(i) the proportion of splenic NK cells, (ii) production of IFN-γ by NK cells in vitro, and (iii) NK
cell-dependent clearance of murine cytomegalovirus in vivo. Therefore, NK cell-dependent immune
responses are not globally impaired by the absence of Dectin-1; impairment is selective to the antitumor response.

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Does Dectin-1 recognize a molecular structure(s) on B16F1 cells? To address this question, we
generated a soluble form of Dectin-1 conjugated to human IgG1 Fc (termed sDectin-1) to detectbinding of Dectin-1 to the cell surface. Interestingly, substantial binding of sDectin-1 to B16F1 cells
was detected, whereas binding was almost undetectable to mouse embryonic fibroblasts (MEFs) andother primary, non-transformed cells.
Since β-glucans, known ligands of Dectin-1, are not expressed by mammalian cells, wehypothesized the presence of other types of glycosylated structure(s) on B16F1 cells thatare mediating the recognition by Dectin-1. In this context, it has been shown that enhanced
glycosylation levels provide growth advantages to many if not all tumor cells. Interestingly, we
found that sDectin-1 binding to B16F1 cells was markedly reduced upon N-glycosidase treatment,
while treatment by O-glycosidase or neuraminidase showed only a marginal effect, suggesting
there is a major requirement for N-glycan structures to Dectin-1 binding. Consistent with this,a marked reduction of tumoricidal activity of splenocytes was observed when B16F1 cells werepretreated with N-glycosidase, whereas O-glycosidase treatment did not affect this activity. Further,N-glycosidase treatment of B16F1 cells did not affect the in vitro killing activity of purified NK
cells, indicating in toto there is a critical role of Dectin-1 recognition of and signaling by N-glycan
structures on tumor cells by dendritic cells and macrophages. We then investigated the nature of
N-glycan structures on B16F1 cells by subjecting the supernatant generated after N-glycosidase
treatment to mass spectrometric (MS) analysis. As reported previously, N-glycan structures, suchas N-glycans with β1,6-GlcNAc branching, the expression of which is increased in tumor cells, weredetected. Of note, pretreatment of the supernatant by sDectin-1 did not alter the MS peak pattern.
This observation suggests that N-glycan structures, highly expressed in tumor cells, need to bebound to proteins for the Dectin-1 recognition.
To what extent the Dectin-1-induced anti-tumor immune responses account for other types oftumor cells? To address this issue, we first examined the binding capacity of sDectin-1 for othertumor cell lines. The binding capacity was variable in that the binding was strong for 3LL (lungcarcinoma), YAC-1 (lymphoma), and Meth-A (fibrosarcoma) cell lines, but was significantly
weaker for some tumor cells such as SL4 (colon carcinoma) and B16F10 (melanoma) cell lines. In
addition, chemical cross-linking of sDectin-1, followed by immunoblot analysis, generated multiple
smear bands of similar pattern using B16F1 and 3LL cells, whereas bands were barely detectablewith B16F10 cells or MEFs. Further, these bands are abolished by the pretreatment of cells with
N-glycosidase. Thus, these data suggest that similar or identical N-glycan structures are expressedon multiple proteins that function as Dectin-1 ligands in these, and probably other, Dectin-1 binding
tumor cells.

Discussion & Conclusion
In this study, we demonstrated that the innate immune receptor Dectin-1 expressed on dendritic
cells and macrophages is important to NK cell-mediated killing of tumor cells. Our results indicate- 60 -

that NK cells are required to orchestrate with dendritic cells and macrophages for cell killing,wherein activation of the IRF5 transcription factor by Dectin-1 signaling instigated by receptor
recognition of N-glycan structures on tumor cells is critical (data not shown). This notion is
supported by an excessive growth of tumors with sDectin-1 binding in Dectin-1 deficient mice invivo. To our knowledge, this is the first demonstration that an innate immune receptor contributes toanti-tumor recognition and signaling through orchestration of innate immune cells. Our study offers
new insight into the NK cell-mediated anti-tumor activity of the innate immune system.

References
Chiba S, Ikushima H, Ueki H, Yanai H, Kimura Y, Hangai S, Nishio J, Negishi H, Tamura T, Saijo
S, Iwakura Y, Taniguchi T. Recognition of tumor cells by Dectin-1 orchestrates innate immune cells
for anti-tumor responses. eLife. 2014;3:e04177.
Ikushima H, Negishi H, Taniguchi T. The IRF family transcription factors at the interface of innate
and adaptive immune responses. Cold Spring Harb Symp Quant Biol. 2013;78:105-16.

一般の皆様へ古くから体内の免疫系ががん細胞を認識し排除することが分かっていましたが、その機構としては、獲得免疫系によるがん細胞の認識機構がよく知られており、免疫系のもう一つの軸である自然免疫系がどのようにしてがん細胞を認識し、その排除を促しているのか、という点については不明な点が多く残されていました。本研究成果は、自然免疫受容体ががん細胞を認識し、その排除を促していることを世界に先駆けて明らかにするものであり、今後、自然免疫系の活性化を通した、新しいがんの治療法や予防法の開発へとつながると期待されます。

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Functional mechanism of a novel signaling molecule that regulates
differentiation and migration in immune cells.
Yoshihiko Tanaka, M.D., Ph.D.

Fukuoka Dental College, Department of Functional Bioscience, Section of Infection Biology
tanakayo@college.fdcnet.ac.jp

Abstract
T helper cells especially Th2 cells play a triggering role in the allergic response. However, themechanism underlying the differentiation of T helper cells into Th2 cells has not been completely
elucidated. We found a novel signaling molecule, which was expressed specifically in Th2 cells
during the differentiation of T helper cells. In this study, we investigated the function of thesignaling molecule in T cells.
Key words : Immunology, T helper cells, Signaling molecule

Introduction
Th2 cells play a central role in the allergic responses, including asthma and pollen allergies.
However, the mechanism underlying the differentiation of T helper cells into Th2 cells has notbeen completely elucidated. Our group has studied various signaling molecules controlling thedifferentiation of T helper cells. We found a novel signaling molecule, which was expressed
specifically in Th2 cells during the differentiation of T helper cells. We investigated thecharacterization and function of the signaling molecule.

Results
1) Isolation of a novel signaling molecule in T helper cell differentiation.
To examine a novel signaling molecule during T helper cell differentiation, murine CD4+ T cells
were differentiated in vitro in Th2- or Th1-inducing conditions. Western blotting analysis using
antibodies for previously known signaling molecules revealed that an unexpected small size of thesignaling molecule was found in Th2 cells, but not in Th1 cells. We focused on the unexpected small
size of the signaling molecule. RT-PCR was performed using primers for the signaling molecule. In
addition of the entire PCR product, a smaller PCR product was found. The smaller PCR product wascloned and sequenced, succeeded in isolating as a novel signaling molecule.
2) Expression of a novel signaling molecule.
To determine the expression of the signaling protein, murine CD4 + T helper cells weredifferentiated and restimulated under several conditions, such as Th2-, Th1-, Th17-, and Treginducing conditions. The protein was expressed in Th2 cells, particularly in restimulated in Th2
cells, but not in other types of T helper cells.
3) Effect of cytokine production by a novel signaling molecule.

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To investigate the effect of cytokine production by the signaling molecule, we examined its effecton the activity of IL-4 or IFN-g promoter-luciferase reporter genes in human Jurkat cells. Comparedto the control empty vector-transfected cells, overexpression of the signaling molecule could notaffect on both of the cytokine-luciferase reporter genes in the basal and stimulated conditions.
To establish more physiological conditions, we analyzed the effect of retrovirus-mediatedectopic the signaling molecule expression in murine T helper cells. We generated a retroviral thesignaling molecule expression vector, which was used to infect Th2-inducing murine CD4+ T
cells. Transduction of primary T cells with the signaling molecule increased the fraction of IL4-producing cells compared to the noninfected population. We also examined the effect of thesignaling molecule transduction on IFN- g-producing cells in Th1-inducing cells but found noconsistent effects.
4) Role of cellular migration by a novel signaling molecule.
To study the potential role of the signaling molecule in cellular migration, we analyzed chemotactic
response of Jurkat cells overexpressed the signaling molecule. In the transwell chemotaxis assay,
overexpression of the signaling molecule did not affect motility of T cells in the response to
CXCL12.

Discussion & Conclusion
We report the isolation of a novel signaling molecule expressed in Th2 cells. It is expressed inTh2 cells but not in other types of T helper cells, and might positively regulate the Th2 cytokines,
such as IL-4 production. Activation of T helper cells under Th2-inducing conditions upregulates
expression of a couple of transcription factors, such as GATA-3 and c-Maf. It is reported that several
pivotal signaling molecules play important roles for the regulation of these transcription factors.
Therefore, the novel signaling molecule could have some potential roles in cytokine production
through the transcription factors in Th2 cells. Future work should be aimed at defining the precise
mechanisms of the role in cytokine production and allergy-specific differentiation of T helper cellsby this signaling molecule.

References
1) Hashimoto, M., Nagao, J., Tasaki, S., Narita, Y., Cho, T., Yuasa, K., Tanaka, Y. Functional analysis
of a novel immune signaling molecule involved in Th2-mediated allergic response. The 44th Annual
Meeting of The Japanese Society for Immunology. Sapporo, Nov. 18-20th (18th), 2015.

2) Hashimoto, M., Nagao, J., Tasaki, S., Narita, Y., Cho, T., Yuasa, K., Tanaka, Y. The identificationand characterization of a novel immune signaling molecule involved in allergic response. The 57th
Annual Meeting of Japanese Association for Oral Biology. Niigata, Sep. 12nd -13th (12nd), 2015.

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一般の皆様へアレルギーの病態には T 細胞による免疫応答が重要な役割を果たすことが知られていますが、疾患を引き起こす分子メカニズムについては不明な点が多く残されています。我々はアレルギー反応に関する研究を進める過程で、T 細胞に新しいシグナル伝達分子が発現することを見出しました。この分子を標的としてヘルパー T 細胞における発現パターンとサイトカイン産生に及ぼす影響などを解析しました。その結果、アレルギー応答の制御に重要な役割をもつ可能性が認められました。今後、ヘルパー T 細胞の分化の制御機構を明らかにするとともに、アレルギーの病態解明と新しい治療法の開発へと研究を展開していく予定です。

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Molecular basis of zinc signaling on lymphocyte homeostasis andmalignancy
Toshiyuki Fukada

Tokushima Bunri University
fukada@ph.bunri-u.ac.jp

Abstract
Zinc deficiency causes decrease of lymphocytes leading to immunological defects, while zincconcentration is frequently increased in cancer cells like leukemia, although the mechanisms how
zinc involves in their fate decision have been unknown. We demonstrated that zinc transporter
ZIP10 in B cells is required for their survival, and is overexpressed in lymphoma. This study aimed
to explore the mechanisms how ZIP10 controls B cell homeostasis and related cancer cells, so that
we performed molecular screening to isolate ZIP10-binding proteins by using yeast two hybrid
system. Further investigation for the binding proteins to ZIP10 will uncover how zinc contributes
lymphocyte homeostasis and malignancy.
Key words : zinc, zinc transporter, B cell, cancer cell

Introduction
Zinc is an essential trace element and its deficiency causes defects in immune systems associatedwith decrease of lymphocytes, but the mechanisms how zinc involves in lymphocyte homeostasis
have remained unclear. We have demonstrated that zinc transporter-mediated zinc ion acts as
signaling factor called “zinc signal” (Ref 3), and that B cell-specific conditional knockout mouse ofzinc transporter ZIP10 (Zip10-cKO) exhibited abnormal B cell homeostasis. We also found ZIP10
was overexpressed in B cell lymphoma (Ref 1-2). In this study, we performed yeast two hybrid
system to identify ZIP10-binding and functional proteins to address the molecular mechanisms how
ZIP10 regulate B cells’ fate.

Results
1: Isolation of ZIP10-binding proteins
To investigate how ZIP10-mediated zinc signaling regulates B cellhomeostasis and functions, we performed yeast two hybrid system(Y2H) by using cDNA library derived from mouse spleen, and theunique intracellular region of ZIP10 as the bait (Fig 1.1, unpublished).
We cloned candidate genes as ZIP10-binding molecule involved either
in immunity, cell cycle, cancer, human disease, signaling, transport, or
metal homeostasis. We have selected genes with some criteria including
the biological and pathological relevance, and considering the potential- 65 -

Fig.1 Experimental strategy of
isolation and characterizationof ZIP10-binding proteinsrelationship with pathogenic phenotypes of Zip10-cKO mice. We are analyzing these molecules
whether they could directly bind to ZIP10 by using immune precipitation (Fig 1.1).
2: Role of ZIP10 in cancer cells
ZIP10 expression was upregulated in leukemia cells and B cell lymphoma tissues. In order toassess the role of ZIP10 in cancer cells, we downregulated Zip10 gene in 293T cells and Hela cellsby siRNA, which reduced cell numbers and colony formation ability (Fig 1.2, unpublished). We are
currently investigating if lymphoma-related cell lines might exhibit similar abnormalities,using theloss of ZIP10, by using Raji (lymphoblast-like cell), Daudi (Burkitt’s lymphoma-derived cell), andJurkat cells (leukemic T-cell lymphoblast).

Discussion & Conclusion
We have shown that zinc transporter ZIP10 is required for anti-apoptosis of B cell, and is highly
expressed in human leukemia and B cell lymphoma cells, indicating that ZIP10-mediated zinc signal
might be therapeutic target to restore abnormalities occurred in B cells and B cell-derived cancer
cells (Ref 1-2). The molecules identified as ZIP10-binding protein in this study include both known
molecules that are involved in B cell functions, and molecules that have no reports on B cells.
Further investigation of the roles of these proteins on the functions of ZIP10 will allow us to uncover
the biological and clinical relevance of zinc signaling for B cell homeostasis and malignancy.

References
1: Hojyo, S., T. Miyai, H. Fujishiro, M. Kawamura, T. Yasuda, A. Hijikata, BH. Bin, T. Irié, J.
Tanaka, T. Atsumi, M. Murakami, M. Nakayama, O. Ohara, S. Himeno, H. Yoshida, H. Koseki, T.
Ikawa, K. Mishima, and *T. Fukada.
Zinc transporter SLC39A10/ZIP10 controls humoral immunity by modulating B cell receptor signal
strength.
PNAS. 111:11786-91, 2014 (*corresponding author)
2: Miyai, T., S. Hojyo, T. Ikawa, M. Kawamura, T. Irié, H. Ogura, A. Hijikata, BH. Bin, T. Yasuda,
H. Kitamura, M. Nakayama, O. Ohara, H.o Yoshida, H. Koseki, K. Mishima, and *T. Fukada.
Zinc transporter SLC39A10/ZIP10 facilitates antiapoptotic signaling during early B-celldevelopment.
PNAS. 111:11780-85, 2014 (*corresponding author)
3: Hojyo S, Miyai T, *Fukada T.
B-cell receptor strength and zinc signaling: Unraveling the role of zinc transporter ZIP10 in humoral
immunity.

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Receptor and Clinical Investigation 2: e387; 2015 (*corresponding author)

一般の皆様へ亜鉛は健康維持に必須な微量元素であり、亜鉛の輸送体が体内の亜鉛量を一定に保っています。亜鉛輸送体が調節している亜鉛は情報の運び屋と機能しており、その働きは「亜鉛シグナル」と呼ばれています。亜鉛シグナルは健康と病気を統御す
る重要なシステムであり、生命科学における新しい研究分野です ( 図 .1)。
亜鉛輸送体に関する申請者らの研究結果は、亜鉛シグナルの異常が免疫機能を司るリンパ球の一つである B 細胞の機能破綻と血液腫瘍に関与することを示すものです。B 細胞の運命決定に関わり、がんへの関与が示唆される亜鉛シグナルに関する
私達の研究結果は、免疫応答と発がんの新たなメカニズムの解
明と、免疫制御剤や制がん剤等の新たな薬の開発につながる可能性を有していると思われます。今後もさらに本研究を推進して、亜鉛の健康と病気への関わりを追求する所存です。

- 67 -

図 .1：亜鉛輸送体と亜鉛シグナルの健康と病気における役割
亜鉛の輸送体が調節する亜鉛は情報伝達の担い手として機能し、亜鉛シグナルと呼ばれています。亜鉛シグナルは健康と病気を統御する新たなシステムとして注目されています。

Establishment of experimental systems for monitoring endoplasmic
reticulum stress in planta
Yuji Iwata

Osaka Prefecture University
yuji.iwata@plant.osakafu-u.ac.jp

Abstract
In order to understand the physiological functions of the endoplasmic reticulum (ER) stressresponse in plants, I designed a construct expressing functional yellow fluorescent proteins in
response to ER stress. I successfully generated transgenic Arabidopsis plants in which tissues andcells that undergo ER stress exhibit yellow fluorescence.
Key words : Arabidopsis, endoplasmic reticulum stress, unfolded protein response

Introduction
The endoplasmic reticulum (ER) stress response is widely conserved among eukaryotic cells. In
plants, the ER stress response has been implicated in a number of developmental processes andstress responses. Molecular components of the ER stress response has also been identified using themodel plant Arabidopsis (Arabidopsis thaliana). However, the detailed molecular understanding has
been lacking due to the difficulty in identifying ER stressed-cells in high resolutions.

Results
In the Arabidopsis ER stress response, cytoplasmic splicing of bZIP60 mRNA mediated by the
ER-localized kinase/ ribonuclease Inositol requiring 1 (IRE1) is the key mechanism to activate
the downstream ER stress-responsive genes [1,2]. Cytoplasmic splicing removes the intron of 23
nucleotide in length that causes a frame shift, resulting in the functional bZIP60 transcription
factor. I utilized this mechanism to identify the tissues and cells that undergo ER stress in highresolution. I designed construct expressing functional yellow fluorescent proteins in response to
ER stress. The construct harbors a partial fragment of Arabidopsis bZIP60 gene and fragments ofyellow fluorescent protein (YFP). It expresses functional YFP in response to ER stress-triggered
cytoplasmic splicing of bZIP60 mRNA.
I first expressed the active form of proteins expected to be expressed under ER stress conditions inbacterial expression system to see whether the resulting protein is capable of exhibiting fluorescence.
I introduced the construct under T7 promoter, which can be activated by addition of isopropyl b-D1-thiogalacto- pyranoside (IPTG). The bacterial colonies exhibited YFP fluorescence under blue
light only when IPTG was added in the Luria Bertani (LB) medium, showing that the protein is able
to exhibit fluorescence if produced in plants.
Next, I generated the binary vector construct with authentic bZIP60 promoter or strong- 68 -Cauliflower mosaic virus 35S promoter, and introduced it into the wild type Arabidopsis plants
by Agrobacterium tumefaciens-mediated floral dip method [3]. After the selection by bialaphos, I
obtained a number of independent T1 transformants and confirmed the presence of the introduced
construct in the Arabidopsis genome by polymerase chain reaction (PCR). I next isolated severaltransgenic Arabidopsis plants with homozygous, single locus insertion. For the construct withbZIP60 promoter, the reverse transcription (RT)-PCR showed that the mRNA derived fromthe introduced construct underwent cytoplasmic splicing in response to the ER stress inducer
tunicamycin and dithiothreitol (DTT). The more detailed analysis using quantitative real time RTPCR demonstrated that tunicamycin- and DTT-induced cytoplasmic splicing of the introducedmRNA occurred in a very similar kinetics to that of endogenous bZIP60 mRNA. The immunoblot
analysis detected the proteins derived from spliced mRNA by treatment with tunicamycin and
DTT. Most importantly, when transgenic seedlings were treated with tunicamycin and DTT, YFP
fluorescence was observed. in contrast, plants with 35S promoter unexpectedly exhibited YFP
fluorescence even without ER stress inducer, although stronger fluorescence was observed by ER
stress treatment.

Discussion & Conclusion
Our results clearly showed that the transgenic plants harboring the bZIP60 promoter-driven
construct exhibited ER stress-dependent fluorescence and therefore can be used to identify tissues
and cells that experience ER stress in a single cell resolution. The transgenic Arabidopsis plants
generated in this study will facilitate our understanding of the roles of the ER stress response
in a physiologically relevant circumstances, such as development and stress responses in plants.
The study also suggests that the system would be more useful if the construct is improved to givestronger fluorescence. Unexpectedly, the Cauliflower mosaic virus 35S promoter-driven constructexhibited fluorescence even without treatment by ER stress inducers. It is conceivable that the
overproduction of the expressed proteins under the strong promoter affects the ER membranes andresults in stressed ER, triggering the ER stress response.

References[1] Nagashima Y, et al. (2011) Arabidopsis IRE1 catalyses unconventional splicing of bZIP60 mRNAto produce the active transcription factor. Sci Rep. 1: 29.
[2] Iwata, Y., and Koizumi, N. (2012) Plant transducers of the endoplasmic reticulum unfolded
protein response. Trends Plant Sci. 17: 720-7.
[3] Clough, S.J., and Bent, A.F. (1998) Floral dip: A simplified method for Agrobacterium-mediatedtransformation of Arabidopsis thaliana. Plant J. 16: 735-43.

- 69 -

一般の皆様へ本研究では、植物における環境ストレス応答・耐性において重要な役割を果たしている小胞体スト
レス応答について、シロイヌナズナを用いて研究を行った。具体的には、小胞体ストレスが起こっている組織・細胞の高い解像度での可視化を試みた。その結果、ストレスを受けている組織・細胞を蛍光顕微鏡により特定することができる形質転換シロイヌナズナを作出することが出来た。本研究による成果は、植物における小胞体ストレス応答の分子メカニズムの理解やストレス耐性作物の作出へ向けた基礎的知見に繋がると考えられた。

- 70 -

Targeting macrophage pinocytosis in atherosclerosis
Takuro Miyazaki

Showa University School of Medicine
taku@pharm.showa-u.ac.jp

Abstract
Pinocytotic deposition of native low-density lipoprotein (LDL)-derived cholesterol in macrophages
in the vascular wall has a considerable impact on atherosclerosis. However, how this process ispotentiated in the atheroprone vasculature remains unknown. We report that calpain-6 (CAPN6),
an unconventional non-proteolytic calpain, confers hyperpinocytosis on macrophages during
atherogenesis. Mechanistically, CAPN6 is induced in macrophages by tumor necrosis factor(TNF)- a stimulus, thereby suppressing the induction of Rac1. CAPN6 deficiency in Ldl receptornull mice reduces pinocytotic activity in macrophages, generating an atheroprotective phenotype in
the aorta. Consistently, bone marrow transplantation experiments in mice showed the contributionof myeloid CAPN6 to atherogenesis. Furthermore, CAPN6 induction was evident in macrophages inadvanced human atheromas. Thus, CAPN6 acts as a pathogenic cue for inducing hyperpinocytosis
in inflamed macrophages during atherogenesis.
Key words : Calpain-6; Atherosclerosis; Macrophage; Fluid-phase pinocytosis

Introduction
Cholesterol deposition in vascular walls is mainly due to foam cell formation within monocytederived macrophages. Recent advances have shown that scavenger receptor-independent uptake ofnative LDL in macrophages can drive foam cell formation. This phenomenon is known as fluidphase pinocytosis. Although pinocytosis is mediated through Rho GTPase cytoskeletal dynamics,
little is known about its regulatory mechanisms. In macrophage biology, growing evidence suggeststhat the post-translational processing of functional proteins, in addition to their transcriptional
regulation, defines their physiological and pathophysiological behavior. Calpain (CAPN), an
intracellular Ca2+-sensitive protease, plays a pivotal role in this process. We herein investigated theimpact of calpain family members on LDL uptake in pro-atherogenic macrophages.

Results
To examine the involvement of CAPNs in macrophage regulations, we investigated the expressionof CAPN family members in murine bone marrow-derived macrophages in LDL receptor deficient(Ldlr−/−) BMMs. Capn6 mRNA was selectively induced by supplementing the culture medium with
TNF- a. The uptake of oxidized LDL in M-CSF/TNF- a-primed Ldlr−/− BMMs was not influenced by
Capn6 deficiency; conversely, the pinocytotic uptake of native LDL in these cells was significantly
downregulated by Capn6 deficiency. We then sought to determine the mechanisms underlying- 71 -

the changes in pinocytotic activity in Capn6 −/yLdlr−/− BMMs. RhoA/Rac1 GTPases and theirmodulators, rho/rac guanine nucleotide exchange factor 2 (GEF-H1) and Rho GDP dissociation
inhibitor a (RhoGDIa) were induced during differentiation in both Capn6+/yLdlr−/− and Capn6−/yLdlr−/− BMMs in the absence of TNF- a stimulus. The induction of Rac1 and GEF-H1 in Capn6+/yLdlr−/− BMMs, but not of RhoA and RhoGDIa, was abolished by TNF- a stimulus, whereas theinduction of Rac1 and GEF-H1 was rescued by Capn6 deficiency. Pinocytotic activity in Capn6+/yLdlr−/− BMMs was potentiated by TNF- a stimulus and attenuated by Capn6 deficiency. This

reduced pinocytotic activity in Capn6−/yLdlr−/− BMMs was rescued by NSC23766-induced inhibitionof Rac1 but not by Y27632-induced inhibition of RhoA/Rho kinase signaling. To evaluate pinosome
maturity, we visualized endosome-related markers on dextran-labeled pinosomes. We identified that
intensity of the early endosome marker Rab5 and lysosomal labeling with LysoTracker on pinosomes
was reduced by Capn6 deficiency; conversely, that of Clathrin, a marker for Clathrin-coatedvesicles, was unchanged by Capn6 deficiency. We next compared the aortic expression of catalytic
subunits of the calpain family in chow-fed and high-fat diet (HFD)-fed Ldlr−/− mice. We found thatCapn6 was induced in the aortas of HFD-fed mice. Capn6 deficiency in Ldlr−/− mice significantly
inhibited the development of atherosclerotic lesions in the aorta without ameliorating plasma
dyslipidemia. Immunohistochemistry showed CAPN6 expression in macrophages in atherosclerotic
lesions but not in other vascular component cells, including endothelial cells and smooth muscle
cells. Next, we evaluated CAPN6 expression in human atheromas. CAPN6 expression was negligiblein adventitial macrophages in the normal aorta and in macrophages in mild carotid lesions.
Conversely, CAPN6 was abundant in macrophages in severe aortic and coronary lesions. We further
assessed the contribution of myeloid Capn6 to atherogenesis through bone marrow transplantation
experiments in mice. Transplantation of Capn6−/yLdlr−/− bone marrow cells significantly decreased
HFD-induced aortic atherosclerotic lesions compared with transplantation of Capn6+/yLdlr−/− bonemarrow cells independent of the recipient genotype. Macrophage recruitment into atherosclerotic
lesions and expression of the macrophage marker CD68 were reduced by Capn6 deficiency, whereas
the expression of the inflammatory molecules Icam1, Vcam1, Sele, Tnfa, Il1b, Il6, and Ccl2, as well
as of the lymphocyte markers Cd4 and Cd8a1, remained unchanged. Filipin III-based cholesterol
staining in macrophages in murine atheromas showed that Capn6 deficiency reduced the amountof cholesterol in individual macrophages, whereas it did not affect expression of genes related to
the receptor-mediated uptake of oxidized LDL, including Msr1, Cd36, Abca1, Abcg1, and Acat1. We
assessed the in vivo uptake of fluorescent nanoparticles in macrophages as an index of pinocytotic
activity. Pinocytotic activity in macrophages in Capn6−/yLdlr−/− atheromas was significantly lower
than those in Capn6+/yLdlr−/y lesions. Immunohistochemical and immunoblot analysis demonstrated
that Capn6 deficiency upregulated Rac1 protein expression in macrophages in atherosclerotic
plaques.

- 72 -

Discussion & Conclusion
Our data disclosed that induction of CAPN6 confers hyperpinocytosis on macrophages during
atherogenesis. Mechanistically, CAPN6 abolishes Rac1 induction in macrophages during thedifferentiation. Capn6 deficiency in Ldl receptor-null mice reduces pinocytotic activity inmacrophages and generating an atheroprotective phenotype in the aorta. Consistently, bone marrowtransplantation experiments in mice showed the contribution of myeloid calpain-6 to atherogenesis.
Furthermore, CAPN6 induction was evident in macrophages in advanced human atheromas. Thus,
CAPN6 acts as a pathogenic cue for inducing hyperpinocytosis in inflamed macrophages during
atherogenesis. Targeting CAPN6 may represent an efficient approach to suppress pro-atherogenic
pinocytosis because CAPN6 is specifically expressed in inflamed macrophages, and calpain-6
induction may be translatable into humans with atherosclerosis.

References
Miyazaki T, ＊ Taketomi Y, Saito Y, Hosono T, Lei XF, Kim-Kaneyama JR, Arata S, Takahashi
H, Murakami M, Miyazaki A. Calpastatin counteracts pathological angiogenesis by inhibiting
suppressor of cytokine signaling 3 degradation in vascular endothelial cells. Circ Res. 116, 11701181, 2015.
Kigawa Y, Miyazaki T, ＊ Lei XF, Nakamachi T, Oguchi T, Kim-Kaneyama JR, Taniyama M,
Tsunawaki S, Shioda S, Miyazaki A. NADPH oxidase deficiency exacerbates angiotensin II-induced
abdominal aortic aneurysms in mice. Arterioscler Thromb Vasc Biol. 34, 2413-20, 2014.
Miyazaki T, ＊ Koya T, Kigawa Y, Oguchi T, Lei XF, Kim-Kaneyama JR, Miyazaki A. Calpain andatherosclerosis. J Atheroscler Thromb. 20, 228-237, 2013.（Review article）
Koya T, Miyazaki T, ＊ Watanabe T, Shichiri M, Atsumi T, Kim-Kaneyama JR, Miyazaki A.
Salusin-β accelerates inflammatory responses in vascular endothelial cells via NF-κB signaling in
LDL receptor-deficient mice in vivo and HUVECs in vitro. Am J Physiol Heart Circ Physiol. 303,
H96-H105, 2012.
Miyazaki T, ＊ Taketomi Y, Takimoto M, Lei XF, Arita S, Kim-Kaneyama JR, Arata S, Ohata H,
Ota H, Murakami M, Miyazaki A. m-Calpain induction in vascular endothelial cells on human andmouse atheromas and its roles in VE-cadherin disorganization and atherosclerosis. Circulation.
124, 2522-2532, 2011.
Miyazaki T, ＊ Kimura Y, Ohata H, Hashimoto T, Shibata K, Hasumi K, Honda K. Distinct effectsof tissue-type plasminogen activator and SMTP-7 on cerebrovascular inflammation following
thrombolytic reperfusion. Stroke. 42, 1097-1104, 2011.
(*Corresponding author)- 73 -

一般の皆様へ
現行の動脈硬化症の病態生理学は、マクロファージがスカベンジャー受容体依存的に酸化 LDLを取込み、その結果コレステロールが血管壁に蓄積する、いわゆる「酸化 LDL 仮説」に基づく。
しかし、生体内に存在する LDL の多くは受容体が認識するほど高度に酸化されておらず、同仮説だけで血管壁のコレステロール貯留を合理的に説明することは難しい。近年提唱されている飲作用（pinocytosis）モデルでは、LDL の取込みが酸化度に依存せず、酸化 LDL 仮説に修正を迫るものとして注目を集めているが、その分子機構は明らかになっていない。本研究では、細胞内タンパク質・
カルパイン -6 の消去により、飲作用を介したマクロファージ泡沫化が抑制可能であることを解明した。
これは動脈硬化症に対する極めてユニークな介入方法と期待される。

- 74 -

Elucidation of the molecular mechanism of DNA damage response andcarcinogenesis mediated by chromatin remodeling.
Kiyoshi Masuda

Dept. of Human Genetics, Inst. of Biomedical Sciences,Tokushima University Graduate School
kiyoshim@tokushima-u.ac.jp

Abstract
DNA damage response (DDR) provides a potent innate barrier against cellular transformation andtumorigenesis. The aim of this study was to identify novel functions of Homeodomain-interacting
protein kinase 2 (HIPK2) in DNA damage repair. HIPK2-knockdown human colon cancer cells
could not remove γH2A.X after irradiation with a sublethal dose of UV-C, resulting in apoptosis.
Proteomic analysis of HIPK2-associated proteins identified HP1γ as a novel target for HIPK2.
HIPK2 specifically associated with HP1γ through its chromo-shadow domain and phosphorylated
it. Overexpression of HIPK2 increased HP1γ binding to H3K9me3, rescued the UV-C-induced
phosphorylation of HP1γ, triggered release of HP1γ from histone H3K9me3 and suppressed γH2A.X
accumulation. Our results suggest that HIPK2-dependent phosphorylation of HP1γ may participate
in the regulation of dynamic interaction between HP1γ and histone H3K9me3 to promote DNA
damage repair.
Key words : HIPK2, DNA repair response, HP1

Introduction
Genome integrity is constantly threatened by environmental agents and by metabolic products thatcan cause DNA damage. To counteract these threats, eukaryotic cells rely on a coordinated seriesof events termed the DNA damage response (DDR), which allows DNA damage detection, cellcycle checkpoint activation and DNA damage repair.DDR provides a potent innate barrier against
cellular transformation and tumorigenesis. Homeodomain- interacting protein kinase 2 (HIPK2) is
a DNA damage-responsive kinase that phosphorylation dependently activates the apoptotic program
through interacting with diverse downstream targets including tumor suppressor p531,2. Here, we
found that HIPK2 specifically bound to and phosphorylated HP1γ in response to sublethal UV-C
irradiation in human colon cancer cells (HCT116).

Results
1. Reduction in HIPK2 induces apoptosis after sublethal UV-C irradiation
Exposure of HCT116 cells to 10J/m2 UV-C irradiation did not induce phosphorylation of
p53 at Ser46 and did not activate caspase-3; this dose was thus considered to be sublethal. In
HCT116 cells treated with HIPK2 siRNA, 10 J/m2 UV-C increased the numbers of terminal
deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL)- positive cells in association- 75 -

with processing of caspase-9 and caspase-3. Consequently, the irradiation significantly decreased
viability of HIPK2-knockdown cells. After sublethal UV-C irradiation, HIPK2 protein werestabilized without caspase-dependent cleavage in its inhibitory C-terminal domain. These results
suggested an important role of HIPK2 in protection against damage caused by sublethal UV-C
irradiation.
2. Reduction in HIPK2 impairs DNA damage repair
UV light forms three major classes of DNA lesions; (6-4) photoproducts (6-4PPs), cyclobutane
pyrimidine dimers and their Dewar isomers, which are removed by the nucleotide excision repair
system. Control siRNA-treated cells removed about 80% of the initial 6-4PPs within 3 h after
finishing UV-C irradiation, while HIPK2-knockdown cells removed only 10% of 6-4PPs within
3 h after UV-C irradiation, and significantly higher levels of 6-4PPs remained to be accumulated.
HIPK2 knockdown significantly enhanced the UV-C- induced accumulation of γH2A.X, a marker
for kinetics of DNA repair. We also examined whether p53 was needed for the HIPK2- mediatedprotection against sublethal UV-C irradiation using p53-KO HCT116 cells. The absence of p53 did
not cause accumulation of γH2A.X or activation of caspase-3 and did not change cell viability after
irradiation with 10 J/m2 UV-C. Moreover, apoptosis was induced even in the absence of p53.
3. HIPK2 associates with HP1γ
HIPK2 interacts with numerous target proteins in a variety of biological processes. We surveyed
HIPK2 targets reported and focused on the 22-kDa protein band, since any interacting partnerwith a molecular mass of 22 kDa has not been reported. Analysis of the 22-kDa protein band using
liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified HP1γ as a possible
downstream effector of HIPK2. HP1 partners possess a conserved PxVxL/M/V pentapeptide motif
called HP1box, which is necessary and sufficient for their interaction with HP1.30 HP1γ possesses
two HP1box motifs (525- PFVTM-529 and 883-PTVSV-887). Mutation of the 883- PTVSV-887(HIPK2mt2), but not the 525-PFVTM-529 (HIPK2mt1), blocked the interaction between HIPK2 andHP1γ. Using HCT116 cells co-transfected with the FLAG-HIPK2- encoding vector and the vector
encoding HA-HP1γ, HA-HP1γ lacking the chromo domain (HA-ΔCD) or HA-HP1γ lacking thechromo-shadow domain (HA-ΔCSD), we confirmed that the chromo-shadow domain of HP1γ was
responsible for the association between HIPK2 and HP1γ.
4. HIPK2 phosphorylates HP1γ
To test the possibility that HIPK2 might mediate the phosphorylation of HP1γ, first we employedin vitro kinase assays. Wild-type FLAG-HIPK2, but not FLAG-HIPK2- KD (kinase-dead HIPK2
mutant), was able to phosphorylate HP1γ and HIPK2 itself. Phos-tag SDS-PAGE demonstrated
that sublethal UV-C irradia- tion phosphorylated HP1γ. Using cells overexpressing HA-HP1γ ortruncated HA-HP1γ lacking the chromo domain or the chromo-shadow domain, we showed thatthe chromo-shadow domain was likely to possess the phosphorylation sites. We also confirmed that- 76 -

overexpression of FLAG-HIPK2 mutant, which could not bind to HP1γ, did not stimulate the UV-Cinduced phosphorylation of HP1γ. HP1γ at Ser83 was already identified as the phosphorylation siteof protein kinase A and Aurora kinase A, while sublethal UV-C irradiation did not phosphorylate
HP1γ at Ser83.
5. HIPK2 regulates association between HP1γ and histone H3K9me3
Immunoprecipitation experiments with an anti-histone H3K9me3 showed that HA-HP1γ did not
significantly associate with histone H3K9me3 before and after UV-C exposure when endogenous
HIPK2 was reduced with HIPK2 siRNA. Overexpression of FLAG-HIPK2 increased HA-HP1γ
binding to histone H3K9me3, and that UV-C triggered the release of HA-HP1γ from histone
H3K9me3. Overexpression of the HP1box mutant or the kinase-dead mutant of HIPK2 did not
trigger the interaction between HA-HP1γ and histone H3K9me3. The chromatin fractionation assay
showed that overexpressed FLAG-HIPK2 increased the distribution of HP1γ in soluble fractions in
response to UV-C.
6. HIPK2 modulates the expression of cell cycle regulator and DNA repair factor
Exhaustive gene expression analysis using microarray showed that overexpression of FLAGHIPK2 increased the expression of cell cycle regulators, such as CDKN1B and CEBPA, and DNA
repair factors including FTH1 and GZMA. Knockdown of HIPK2 repressed the induction of thesefactors after sublethal UV-C irradiation.

Discussion & Conclusion
We identified that HIPK2 was stabilized in response to sublethal DNA damage. HIPK2 upregulates
the expression of a p53-inducible DNA repair factor p53R2 after sublethal UV irradiation or
Adriamycin treatment3. p53R2 is required for maintenance of mitochondrial DNA and for optimal
DNA repair after UV damage, suggesting a possible role of HIPK2 in DNA repair responses4.
However, the RRM2B gene encoding p53R2 is inactivated due to a point mutation in HCT116 cells.
Furthermore, using p53-KO HCT116 cells, we revealed that the absence of p53 did not cancel the
HIPK2-dependent protection against damage after sublethal UV-C irradiation. Recently, it has
been shown that HIPK2 constitutively phosphorylates WIP1 (wild-type p53-induced phosphatase
1) and facilitates the proteasomal degradation of WIP1 in unstressed cells5. This HIPK2-mediateddownregulation of WIP1 levels is crucial for the initiation of the DSB repair signaling pathway;
reduction in HIPK2 inhibits ionizing radiation-induced γH2A.X foci formation, cell-cycle
checkpoint activation and DNA repair signaling. In our experiments, however, HIPK2-knockdown
cells failed to remove UV-C-induced 6-4PPs and rather enhanced γH2A.X foci formation, leading
to p53-independent apoptosis. Besides the p53R2- or WIP1-mediated regulation, our results suggest
the presence of another HIPK2-dependent, p53- independent pathway for DDR through interacting- 77 -

with HP1γ.
In response to DNA damage, chromatin becomes remodeled to facilitate access of the DDR
machinery to the sites of damage. However, the chromatin rearrangements taking place in response
to DNA damage are not fully understood. It is also unknown whether and how HIPK2 affects
chromatin remodeling, although several epigenetics-related proteins have been identified as
HIPK2-interacting partners6. Recently, several lines of evidence have revealed that HP1 proteinsdynamically associate and dissociate with chromatin to play an active role in DNA damage repair
processes7. Our results suggest that HIPK2-dependent phosphorylation of HP1γ may at least in part
regulate the dynamic interaction between HP1γ and histone H3K9me3 for DNA damage repair.
To definitively prove that HP1γ is an important downstream effector of HIPK2 in the response to
UV-C, the specific sites by which HIPK2 phosphorylates HP1γ should be identified. The chromoshadow domain seems to possess the phosphorylation site(s). Further studies are needed to identify
the site(s).

References
1. Sombroek D, Hofmann TG. How cells switch HIPK2 on and off. Cell Death Differ 2009; 16: 187–
194.
2. Puca R, Nardinocchi L, Givol D, D’Orazi G. Regulation of p53 activity by HIPK2: molecularmechanisms and therapeutical implications in human cancer cells. Oncogene 2010; 29: 4378–4387.
3. Nardinocchi L, Puca R, Sacchi A, D’Orazi G. HIPK2 knock-down compromises tumor cell
efficiency to repair damaged DNA. Biochem Biophys Res Commun 2007; 361: 249–255.
4. Pontarin G, Ferraro P, Bee L, Reichard P, Bianchi V. Mammalian ribonucleotide reductase subunit
p53R2 is required for mitochondrial DNA replication and DNA repair in quiescent cells. Proc Natl
Acad Sci USA 2012; 109: 13302–13307.
5. Choi DW, Na W, Kabir MH, Yi E, Kwon S, Yeom J et al. WIP1, a homeostatic regulator of the
DNA damage response, is targeted by HIPK2 for phosphorylation and degradation. Mol Cell 2013;
51: 374–385.
6. Hofmann TG, Glas C, Bitomsky N. HIPK2: a tumour suppressor that controls DNA damageinduced cell fate and cytokinesis. Bioessays 2013; 35: 55–64.
7. Luijsterburg MS, Dinant C, Lans H, Stap J, Wiernasz E, Lagerwerf S et al. Hetero- chromatinprotein 1 is recruited to various types of DNA damage. J Cell Biol 2009; 185: 577–586.

一般の皆様へ
本研究では、これまで知られていない HIPK2 スイッチによる context-dependent な DNA 損傷スト
レス応答機構と、HIPK2 が損傷領域の動的クロマチン構造変換を介して DNA 修復に関わる分子機構を明らかにすることを目的とした。本研究の結果から、DNA 損傷ストレス時における HIPK2 遺伝
子の新しい細胞内機能を見いだし、HIPK2 が大腸がん細胞株においてクロマチン構造制御を介して広範囲の遺伝子発現を制御している可能性があるという重要な知見を得ることができた。本研究の- 78 -

結果をさらに発展させることにより、DNA 修復機構とアポトーシス誘導機構を切り替える分子スイッチとしての HIPK2 の役割とその下流の分子機構を明らかにできれば、DNA 損傷にストレスに対する細胞の DNA 修復、がん化抑制反応とその障害によるがん化促進機序について新たなモデルを提唱でき、この分子スイッチを修飾する低分子化合部の探索によって、画期的ながん予防法開発のための基盤となる知見を与えると考えられる。

- 79 -

Development of a novel fluorescent probe to catch a “dimerizationarm” of the EGF receptor
Takaaki Mizuguchi

Institute of Biomaterials and Bioengineering,Tokyo Medical and Dental University
mizuguchi.mr@tmd.ac.jp

Abstract
To develop novel f luorescent probes to catch a β-hairpin loop structure, which would be
responsible for the dimerization of the EGF receptor ectodomains, we designed and synthesized
several peptides with or without a fluorescein moiety, based on the crystal structures of the receptor.
Some of them showed inhibitory activity against the receptor autophosphorylation on the intact
carcinoma cells.
Key words : epidermal growth factor receptor; dimerization; anticancer; fluorescent probe; peptidic
inhibitor

Introduction
Epidermal growth factor (EGF) receptor is a medicinal target for discovery of anticancer drugs
because its overexpression or unregulated activation would cause canceration in many cells. The
binding of its ligands to the ectodomains induces the dimerization, resulting in promotion of theactivity of intracellular kinases. A β-hairpin loop structure (residues 242–259) of the “dimerizationarm” is known to be responsible for both the dimerization and the autoinhibition of the receptor.
In this study, we tried to develop a novel fluorescent probe to catch the dimerization arm as a tool
molecule in finding out new types of inhibitors against the dimerization.

Results
Design and synthesis of candidate peptides to catch a dimerization arm
The extracellular structure of the dimerized EGF receptor has been elucidated by X-ray
crystallographic studies1,2. The receptor’s extracellular region consists of four domains (I–IV), withmost of the interface region of the dimer composed of domain II. The dimerization arm in domain II
is thought to be essential to maintaining the dimeric structure. Tyr246, Thr249, Tyr251, and Gln252of the arm head form hydrogen bonds with Cys283, Asn86, Arg285, and Ala286 of the counterpart,respectively, to tightly hold together the active dimer. The arm head also has hydrophobic interaction
with the counterpart. On the other hand, according to the other crystallographic study3, the arm
head is known to interact with the domain IV in the inactivated state. Because of the key functionof the ‘dimerization arm’ to hold together the receptor, peptide sequences of the arm have beenused as a template to design novel compounds with inhibitory activity toward the functional dimer.

- 80 -

Previously, we have developed a cyclic decapeptide which mimics the loop structure of the arm, andshowed that it inhibits the dimerization of the EGF receptor4,5. In the present study, several candidate
peptides, which are potential to catch the dimerization arm, were designed based on three crystal
structures. Some of them were cyclic and the others were liner. The formers were cyclized with an
intramolecular disulfide bridge between two cysteine residues.
Each peptide chain was elongated by conventional Fmoc-based solid-phase peptide synthesison 2-chlorotrityl resin. The linear peptides were cleaved from the resin by treatment with a
TFA cocktail (TFA/EDT/H 2O/TIS), and then precipitated with excess cold diethyl ether. An
intramolecular disulfide bridge was constructed by air oxidation in 0.1 M ammonium bicarbonate
buffer (pH ~8.0) under a condition of which the peptide was distilled at the high level to avoid
the formation of intermolecular disulfide bridges. The cyclization reactions were monitored byanalytical reversed-phase HPLC (RP-HPLC). The crude peptides were purified by preparative RPHPLC, followed by lyophilization to give the desired peptides as white powders. The homogeneityof each peptide was confirmed by analytical RP-HPLC and mass spectrometry.
Inhibitory effects of synthetic peptides on EGF receptor autophosphorylation
The inhibitory effect of the synthetic peptides on autophosphorylation of the EGF receptor was
evaluated using intact human epidermoid carcinoma cell line (A431), which abundantly expresses
the receptor on the cellular surface. For comparison, the effect of erlotinib, which is a potent
tyrosine kinase inhibitor, was also examined in parallel. The cell-based assay was performedby immunoblotting as described in our previous studies5,6 with minor modifications. In thisassay, an anti-EGFR [pY1068] ABfinityTM antibody, which specifically recognizes the receptor
phosphorylated at Tyr1068, was used as a primary antiboby. Tyr1068 at the C-terminal tail of theintracellular domain is an autophosphorylation site that allows binding of Grb2 and activation of
the Ras-Raf-ERK1/ERK2 signaling pathway, which is deeply related to the cell proliferaction anddifferentiation7.
After the cells were stimulated with EGF in the presence of an appropriate amount of each peptide,
the receptor autophosphorylation was observed by immunoblotting with an anti-phosphotyrosine
antibody. Two of synthetic peptides inhibited the receptor autophosphorylation on the intact
cells. Quantitative densitometric analyses of the immunostained bands revealed that the residual
autophosphorylation level of the EGF receptor was suppressed to approximately 50% by the additionof 10 μM of the peptides to the culture medium.

Discussion & Conclusion
In this study, several peptides which would be able to catch the dimerization arm were designed
based on the elucidated crystal structures1-3 of the EGF receptor ectodomain. Two of them showed
the inhibitory activity against the receptor autophosphorylation. To label a fluorescent moiety at an
optimal site of the peptides, further investigation of the key residues for their binding to the receptor- 81 -

is currently in progress. In addition, we synthesized several candidates for our desired probe which
are labeled with a fluorescein moiety based on the two peptides. Now, we continue to investigate
their further functions such as their affinity to the dimerization arm and their effects on the EGF
receptor-positive carcinoma cells.
Recently, the dimerization arm of the EGF receptor has become a more attractive target with which
to develop anticancer drugs. Since our cyclic decapeptide which mimics the dimerization arm4,
other research groups have found useful candidates for new EGF receptor-targeting drugs basedon the structures of the dimerization arm or the binding site on the dimer interface8–11. In addition,a dimerization arm-catching-peptide has also been discovered by examination of a phage display
library12. Our two peptides would also contribute to the development of a novel fluorescent probe tocatch the dimerization arm of the receptor.

References
1. Ogiso, H.; Ishitani, R.; Nureki, O.; Fukai, S.; Yamanaka, M.; Kim, J.-H.; Saito, K.; Sakamoto, A.;
Inoue, M.; Shirouzu, M.; Yokoyama, S. Cell 2002, 110, 775.
2. Garrett, T. P.; McKern, N. M.; Lou, M.; Elleman, T. C.; Adams, T. E.; Lovrecz, G. O.; Zhu, H. J.;
Walker, F.; Frenkel, M. J.; Hoyne, P. A.; Jorissen, R. N.; Nice, E. C.; Burgess, A. W.; Ward, C. W.
Cell 2002, 110, 763.
3. Ferguson, K. M.; Berger, M. B.; Mendrola, J. M.; Cho, H.-S.; Leahy, D. J.; Lemmon, M. A. Cell
2003, 11, 507.
4. Mizuguchi, T.; Uchimura, H.; Kakizawa, T.; Kimura, T.; Yokoyama, S.; Kiso, Y.; Saito, K.
Bioorg. Med. Chem. Lett. 2009, 19, 3279.
5. Mizuguchi, T.; Ohara, N.; Iida, M.; Ninomiya, R.; Wada, S.; Kiso, Y.; Saito, K.; Akaji, K. Bioorg.
Med. Chem. 2012, 20, 5730.
6. Toyama, K.; Mizuguchi, T.; Nomura, W.; Tamamura, H. Bioorg. Med. Chem. 2016, in press (doi:
10.1016/j.bmc.2016.05.026).
7. Yamazaki, T.; Zaal, K.; Hailey, D.; Presley, J.; Lippincott-Schwartz, J.; Samelson, L. E. J. Cell Sci.
2002, 115, 1791.
8. Xu, R.; Povlsen, G. K.; Soroka, V.; Bock, E.; Berezin, V. Cell Oncol. 2010, 32, 259.
9. Yang, R. Y. C.; Yang, K. S.; Pike, L. J.; Marshall, G. R. Chem. Biol. Drug Des. 2010, 76, 1.
10.Hanold, L. E.; Oruganty, K.; Ton, N. T.; Beedle, A. M.; Kannan, N.; Kennedy, E. J. PLoS One
2015, 10, e0118796.
11.Hanold, L. E.; Watkins, C. P.; Ton, N. T.; Liaw, P.; Beedle, A. M.; Kennedy, E. J. Bioorg. Med.
Chem. 2015, 23, 2761.
12.Choi, Y. S.; Yoon, S.; Kim, K. L.; Yoo, J.; Song, P.; Kim, M.; Shin, Y. E.; Yang, W. J.; Noh, J. E.;
Cho, H. S.; Kim, S.; Chung, J, Ryu, S. H. PLoS One 2014, 9, e92513.

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一般の皆様へ
ヒト上皮成長因子（EGF）受容体は、多くのがん細胞で過剰発現が観測されている細胞膜タンパク質です。この受容体の無秩序な活性化が、がん細胞の異常増殖や増悪化を引き起こします。本研
究テーマでは、EGF 受容体の「構造変化阻害」や「二量体化阻害」といった新しい抗がん薬リードを創出するための新しい道具となる蛍光分子の開発を目指しています。また、この蛍光分子は、既存の抗がん薬を選択する際の診断薬の開発にも繋がる可能性があります。

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Development of novel therapy for peripheral artery disease using
ApoA-I mimetic peptide
Satoshi Imaizumi

Department of Cardiology, Fukuoka University School of Medicine
Imachan1005@gmail.com

Abstract
FAMP promoted recovery from hindlimb ischemia through a nitric oxide (NO)-related pathway
by activation of a PI3K / Akt pathway. FAMP may become a new therapeutic agent for the future
clinical treatment of critical limb ischemia (CLI).
Key words : apolipoprotein A-I mimetic peptide, high-density lipoprotein, endothelial nitric oxide
synthase

Introduction
HDL has various atheroprotective functions and improves endothelial function. Apolipoprotein
A-I (apoA-I) is a major protein of HDL and plays a crucial role in HDL functions. We developed anovel apoA-I mimetic peptide, FAMP (Fukuoka University ApoA-I Mimetic Peptide). It is unclear
whether an apoA- I mimetic peptide can promote neovascularization in vivo. Here, we investigatedthe effect of FAMP on endothelial nitric oxide synthase (eNOS) activation and angiogenesis in amurine hindlimb ischemia model.

Results（図は論文を御参照ください）
1 FAMP enhanced blood flow recovery in HCD mice
To examine whether FAMP enhances blood flow recovery, we induced acute hindlimb ischemia
in wild-type mice, which were put on either a chow diet or a high-cholesterol diet (HCD). Although
the ratio of blood perfusion decreased after the induction of operative-induced acute ischemia, thisratio did not differ among the 4 groups, indicating that the severity of induced-ischemia was nearly
equivalent in the groups. Blood flow recovery was significantly impaired in mice fed a HCD diet,compared with that in mice fed a chow diet (Fig. 1A).
Treatment with 1mg/kg and 10mg/kg FAMP was associated with significant improvements inblood perfusion recovery, whereas there were no changes in mice treated with PBS (Fig. 1A, 1B).
Figure 1C shows representative images of gastrocnemius (GC) muscles stained with the endothelial
marker CD31. Treatment with 1mg/kg and 10mg/kg FAMP significantly increased capillary density
in the ischemic muscle at 5 weeks after surgery, compared with that in PBS-treated mice (Fig. 1D).
2 Effect of FAMP treatment on functional recovery- 84 -

At postoperative day 7, motor functions were assessed using the CatWalk System. Body speed was
calculated by dividing the distance that the mouse’s body traveled from an initial contact of a paw to
the next by the time required to travel that distance (Fig. 2A). Significant differences in body speed
in the ischemic hindlimb were observed, as shown in Fig. 2B (also see supplemental video 1). Thisobservation reflects the functional recovery of ischemic limbs in the FAMP-treated group.
3 Effects of FAMP treatment on HDL functionality: HDL-mediated cholesterol efflux capacity and
HDL inflammatory index (HII)To examine the effects of FAMP on HDL functionality, we measured both HDL-mediatedcholesterol efflux capacity and HII using plasma samples obtained at 5 weeks after surgery. As
shown in Fig. 3A and 3B, intramuscular administration of FAMP did not improve HDL function inmice fed a HCD.
4 Effect of FAMP treatment on muscle NOX2 levels
To assess the effect of FAMP on the NADPH oxidase pathway, we measured the NOX2 level in theischemic hindlimb. The NOX2 level in the 10mg/kg FAMP group was lower than that in the grouptreated with PBS (Supplemental Fig. 1).
5 Effects of FAMP treatment on EPC mobilization
The effect of FAMP on EPC mobilization from bone marrow to peripheral blood was investigatedusing hindlimb ischemia-induced mice at postoperative day 2. The basal numbers of EPCs did not
differ among these groups (data not shown). EPC mobilization induced by FAMP was not confirmed
compared to that in the control (HCD-PBS), as shown in Fig. 3C and 3D.
6 FAMP activated phospho-Akt / ERK / eNOS pathways in HAECs
We investigated the effect of FAMP treatment on the signal transduction pathways in human aortic
endothelial cells (HAECs). The activation of eNOS by FAMP peaked at 30min (data not shown).
FAMP treatment significantly activated eNOS at serine residue 1177, Akt at serine residue 473, and
ERK, as shown in Fig. 4A. To confirm the signal transduction from FAMP to eNOS, we incubated
HAECs with the PI3K / Akt inhibitor LY294002 (10µM) and the ERK inhibitor PD98059 (50µM).
As shown in Fig. 4B, LY294002, but not PD98059, significantly inhibited the upregulation ofphospho-Akt and phospho-eNOS induced by FAMP treatment. These results indicate that FAMP
treatment mainly activates eNOS at serine residue 1177 through activation of a PI3K / Akt pathway.
7 FAMP significantly promoted the migratory function of HAECs
After serum starvation to stop cell growth, FAMP significantly improved the migratory functionof HAECs (Fig. 5A). This effect was inhibited by L-NAME (NOS inhibitor), which indicates that
FAMP treatment improved the migratory function of HAECs through a NO-related pathway.
With regard to the migratory function of HAECs under the addition HCD-HDL, which was HDL- 85 -

collected from HCD fed mice, cells that were coincubated with HCD-HDL and FAMP for 1h at
37℃ showed superior migration compared to cells incubated with HCD-HDL alone(Fig. 5B).
8 FAMP treatment failed to enhance blood flow recovery in eNOS-/- mice
To investigate the role of eNOS in blood flow recovery induced by FAMP, we evaluated the effectsof FAMP on eNOS-/- mice. As shown in Fig. 6A and 6B, FAMP treatment had no beneficial effects
in eNOS-/- mice. This result indicates that FAMP treatment enhances blood flow recovery throughthe activation of a NO-related pathway.

Discussion & Conclusion
Our study demonstrated that FAMP enhanced blood flow recovery and increased capillary density
in acute ischemic muscle in HCD mice. Furthermore, FAMP ameliorated functional recovery, which
was assessed in terms of an unforced gait. Although it has been reported that HCD feeding brings
about endothelial dysfunction, FAMP improved the impaired endothelial function through theactivation of PI3K /Akt /eNOS pathways. These findings indicate that FAMP may support biological
HDL function as a small molecule that mimics apoA-I. We previously reported that FAMP
was taken up by atherosclerotic tissues in the blood vessels and aorta in an animal model withatherosclerosis using radiolabeled 68Ga-DOTA FAMP and positron emission tomography (PET).
Therefore, FAMP may be especially useful for treating CLI patients who have severe atherosclerotic
lesions. In addition, FAMP has strong anti-atherogenic activity and suppressed aortic plaqueformation in apo-E-/- mice fed a high-fat diet. These effects are an attractive advantage in future
clinical applications for the treatment of patients with severe atherosclerotic lesions and CLI.

References
1. Uehara Y, Ando S, Yahiro E, Oniki K, Ayaori M, Abe S, et al. FAMP, a novel apoA-I
mimetic peptide, suppresses aortic plaque formation through promotion of biological
HDL function in ApoE-deficient mice. Journal of the American Heart Association.
2013;2:e000048.

2. Kawachi E, Uehara Y, Hasegawa K, Yahiro E, Ando S, Wada Y, et al. Novel molecularimaging of atherosclerosis with gallium-68-labeled apolipoprotein A-I mimetic peptide
and positron emission tomography. Circulation journal : official journal of the Japanese
Circulation Society. 2013;77:1482-9.

一般の皆様へ
下肢の動脈が動脈硬化により閉塞する閉塞性動脈硬化症などの患者さんは、日本に約 600 万人
以上いると推測されています。本研究では、善玉コレステロールをもとに我々が人工的に合成した薬（FAMP）の、閉塞性動脈硬化症に対する効果を調べました。その結果、FAMP が閉塞性動脈硬化症の改善効果を有し、全く新しい治療薬となる可能性があることを発見しました。この研究をさらに- 86 -

発展させ、動脈硬化性疾患に苦しむ患者さんのお役に立てればと思っております。

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Regulation mechanisms of retroelements by RNA virus
Tomoyuki Honda

Osaka University
thonda@virus.med.osaka-u.ac.jp

Abstract
Animal genomes contain endogenous viral sequences, such as endogenous retroviruses. Recently,
we have reported that ancient bornavirus, a non-retroviral RNA virus, has also endogenized andleft endogenous RNA viral elements in many vertebrate genomes. Bornaviruses do not encode
reverse transcriptase gene, raising the question of how bornaviruses produce their DNA fossils inhost genomes. Several features of integrated sequences of bornaviruses suggest that integrationof bornavirus segments is likely to be mediated by long interspersed nuclear element 1 (LINE1), which is one of retrotransposons constituting 17% of the human genome. We can detect DNAs
containing bornavirus sequences in infected cell lines. However, it is still unknown how bornavirus
and LINE-1 interact in detail. In this study, we analyzed the possible crosstalk between Borna
disease virus (BDV), a current mammalian bornavirus, and LINE-1. We identified LBP-1 as a novel
LINE-1 and BDV RNP-binding protein. LBP-1 might play central roles in LINE-1-mediated reverse
transcription of BDV RNAs.
Key words : RNA virus, retrotoransposon

Introduction
Animal genomes contain endogenous viral sequences, such as endogenous retroviruses. Recently,
we have reported that ancient bornavirus, a non-retroviral RNA virus, has also endogenized and left
endogenous RNA viral elements in many vertebrate genomes (ref. 4). Bornaviruses do not encode
reverse transcriptase gene, raising the question of how bornaviruses produce their DNA fossils inhost genomes. Several features of integrated sequences of bornaviruses suggest that integrationof bornavirus segments is likely to be mediated by long interspersed nuclear element 1 (LINE1), which is one of retrotransposons constituting 17% of the human genome. We can detect DNAs
containing bornavirus sequences in infected cell lines (ref. 4). We call this phenomenon “transcript
reversion”. However, it is still unknown how bornavirus and LINE-1 interact in detail. In this study,
we analyzed the possible crosstalk between Borna disease virus (BDV), a current mammalian
bornavirus, and LINE-1. Furthermore, we analyzed the significance of transcript reversion using
one of DNA fossils derived from ancient bornavirus in the human genome, endogenous bornaviruslike elements from nucleoprotein 1 (EBLN-1), as a model.

Results
LINE-1 is transcribed into LINE-1 mRNA, which encodes two open reading frames (ORFs), ORF1

- 88 -

and ORF2. The ORF1 protein (ORF1p) is a major component of LINE-1 ribonucleoprotein complex(RNP) that encapsidates LINE-1 mRNA. The ORF2 protein (ORF2p) is a reverse transcriptase
and also is incorporated into LINE-1 RNP. LINE-1 RNP enters the nucleus and produces DNA
copies in the genome. BDV RNP consists of BDV genomic RNA, the N protein, the P protein andthe L protein. BDV RNP is a replication unit of BDV. Because we can detect BDV DNA in BDVinfected cells, it is speculated that BDV mRNA might be a favorable substrate for LINE-1. Given
BDV mRNA is a favorable substrate for reverse transcription mediated by LINE-1, there might besome physiological interactions between BDV and LINE-1 RNP. Therefore, we first evaluated if
there are any physiological interactions between BDV and LINE-1. To this end, we isolated LINE1 RNP by immunoprecipitation with ORF1p of LINE-1. Using real-time RT-PCR analysis, we
detected BDV mRNA in LINE-1 RNP. We also demonstrated that the N protein of BDV, a marker of
BDV RNP, was co-precipitated with ORF1p. These results suggest that LINE-1 RNP incorporated
both BDV RNP and BDV mRNAand that this interaction might be a reason for LINE-1-mediatedreverse transcription of the BDV sequences. To identify host candidate molecules involved in the
crosstalk between BDV and LINE-1, we further searched LINE-1-binding proteins (LBPs) andisolated LBP-1 in the LINE-1 RNP complex. The interaction of LBP-1 with ORF1p was confirmed
by Western blot. Co-distribution of LBP-1 and ORF1p in the nucleus was observed using proximity
ligation assay. Knockdown of LBP-1 reduced retrotransposition activity of LINE-1, suggesting theimportance of LBP-1 in LINE-1 retrotransposition. We then evaluated if LBP-1 interacts with BDV
RNP. Immunoprecipitation of LBP-1 revealed that LBP-1 binds to both LINE-1 RNP and BDV
RNP. However, BDV infection did not affect the interaction between LBP-1 and LINE-1 RNP. We
therefore examined the possibility that LINE-1 has an impact on BDV infection. We found that
ORF1p overexpression affects BDV transcription activity. Collectively, LBP-1 plays a critical rolein both the efficient LINE-1-meditated retrotransposition and the interaction between LINE-1 andBDV.
To reveal the significance of transcript reversion mediated by LINE-1, we focused on EBLN1 in the human genome. Expression profiling of EBLN-1 showed that EBLN-1 expression wasrestricted in somatic tissues except for testis. This restriction was mediated by histon deacetylationand methylation. Based on these observantions, we induced EBLN-1 expression in somatic cells
using a histon deacetylation inhibitor. When we induced EBLN-1 expression, the expression of the
COMMD3 gene, a neighboring gene of EBLN-1, was downregulated. This downergulation wascanceled by knockdown of EBLN-1 RNA, suggesting that EBLN-1 RNA function as an RNA andregulate the COMMD3 gene. These results indicate that transcript reversion might provide a novel
mechanism that regulates gene expression (ref. 3).

Discussion & Conclusion
Our results suggest that the LBP-1 is a potent bridging molecule between BDV and LINE-1,
which may regulate the incorporation of BDV mRNA into LINE-1 RNP. LBP-1 enhances LINE-- 89 -

1 retrotranscription activity. On the other hand, LBP-1 recruits BDV RNP to LINE-1 RNP and
LINE-1 enhances BDV transcription. In this situation, abundant BDV mRNA is available forretrotransposition-potentiated LINE-1 RNPs. Therefore, LBP-1 may play a central role in theinteraction between BDV and LINE-1.
We further found that EBLN-1 regulates the expression of a neighboring gene, the COMMD3
gene. Because the COMMD3 gene negatively regulates NF-kB signaling, EBLN-1 might regulate
immune system indirectly. Transcript reversion mediated by LINE-1 might play important roles in
the control of immune systems (ref. 2 and 3).

References
1. Honda T. (**) Links between human LINE-1 retrotransposons and hepatitis virus-related
hepatocellular carcinoma. Frontiers in Chemistry 4, 21, 2016
2. Honda T. (**), Tomonaga K. Endogenous non-retroviral RNA virus elements evidence a novel
type of antiviral immunity. Mobile Genetic Elements 6(3), e1165785, 2016
3. Sofuku K., Parrish NF., Honda T. (**), Tomonaga K. Transcription profiling demonstrates
epigenetic control of non-retroviral RNA virus-derived elements in the human genome. Cell Reports
12, 1-7, 2015
4. Horie M. (*), Honda T. (*), Suzuki Y., Kobayashi Y., Daito T., Oshida T., Ikuta K., Jern P.,
Gojobori T., Coffin JM., Tomonaga K. Endogenous non-retroviral RNA virus elements in
mammalian genomes. Nature 463(7277), 84-87, 2010(*) equal contribution(**) corresponding author

一般の皆様へRNA ウイルスは、一般的に RNA のみでその生活環を完結させると考えられてきた。しかし、最近
私たちは、RNA ウイルス感染細胞において、ウイルス配列由来の DNA が存在することを見出した。
この現象は、LINE-1 と呼ばれるレトロトランスポゾンにより制御されている可能性が示唆されている。
本研究では、RNA ウイルスの一つであるボルナ病ウイルス (BDV) と LINE-1 との相互作用のメカニズ
ムを解明することを目的とした。その結果、両者を結ぶ新規宿主因子を同定することができた。本
研究の成果は、今後さらに RNA ウイルス感染細胞内に存在するウイルス配列 DNA の意義を検証していく上で、基盤となる成果となった。

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Single molecule study on ion-channel gating.
Toru Ide

Okayama University
ide@okayama-u.ac.jp

Abstract
We have developed a measurement system for simultaneous optical and electrical recording of
single ion-channel proteins by combining a single channel recording apparatus and a single molecule
imaging microscope. Several types of Ion-channel proteins immobilized on a solid substrate wereincorporated into an artificial bilayer membrane and the single channel currents were measured.
KcsA, BK, P2X4 channels were successfully incorporated into artificial bilayers.
We found that KcsA channels labeled with a f luorescent dye showed significantly higher
fluorescence at acidic pH than at neutral pH. This suggests the channel gating is accompanied bystructure change in the cytoplasmic domain.
Key words : Ion-channel, single molecule imaging, single channel recording

Introduction
Molecular mechanism of ion-channel gating is yet elusive. The structure-function relationships
have been discussed by combining information from structural and electrophysiological studies.
However, there is no direct evidence showing the determined structure corresponds to a certain
functional state of the channel. There has been need for methods to measure structure changes
simultaneously with the ionic current. Here, we developed an apparatus for simultaneous optical andelectrical recordings of single ion-channel proteins.

Results
1)Ion-channel incorporation into artificial bilayer membranes.
The artificial planar bilayer single channel recording method has been applied to various typesof channel proteins for nearly 40 years and revealed their properties. It is difficult to image single
molecules in the artificial bilayers because of thermal
diffusion of the molecules within the bilayers, whichsignificantly lowers the measurement accuracy. We havetherefore developed the technologies to immobilize proteinsin bilayer membranes. In this study, we incorporated
ion-channel proteins immobilized on the surface of asolid substrate directly into previously formed artificial
bilayer membranes (Fig. 1a). We investigated conditionsof ion-channel incorporation and have found the optimal- 91 -

conditions for material and shape of the supporting substrates for individual type of channel.
The substrates were sharpened by electro-chemical etching and the surface was modified with
polyethyleneglycol to make the surface hydrophilic. Channel proteins were fixed at the tip of thesubstrates via histidine-tag. Using this technique, we measured single channel currents of several
types of channel proteins such as KcsA, BK, and P2X4. Fig. 1b shows single KcsA channel current
trace recorded by the technology developed in this study.
2)Single channel imaging (Fig. 1b).
We developed a microscope to image single f luorophors in artificial bilayer membranes bycombining the fluorescence microscope designed to see single molecules and aforementioned
protein immobilizing technology. As shown in the figure, artificial bilayer membranes are formed
across a small pore in a thin plastic sheet. Ion-channel proteins fixed at the tip of the fine supporting
substrate are mechanically incorporated into the membrane. The vertical motion of supporting
substrate is manipulated by a stepping motor. Current across the membrane is measured with apatch-clamp amplifier. Fluorescent dye molecules attached to channel proteins are excited by light
introduced through an optical fiber. The fluorescence is detected with a high-sensitivity camera or
photodetection device.
When we use a metal needle as a supporting substrate, excitation light incidents from below
through the objective lens, and the fluorescence is enhanced by local plasmon at the metal tip.
We measured single molecule fluorescence attached to KcsA channel proteins, which is known
to be activated only at acidic pH. We found that the steady state intensity was much higher atacidic pH than at neutral pH. This indicates that the KcsA channel gating is accompanied by astructure change within the protein and that we could detect the change in structure as the change
in fluorescence at the single molecular level. We have proposed a model for KcsA channel gating, inwhich the cytoplasmic part of the protein changes its structure and moves toward the membrane at
the channel opening.

3)Application to sensor technology (see reference).
The channel incorporation technology developed in this study is applicable to developing a highthroughput screening device for channel drugs. Single channel recording techniques with high drug
sensitivity and high temporal resolution are gold standard in the pharmacological investigation.
However, the efficiency of these measurements are very low. We developed a novel artificial bilayer
single channel recording technique in which bilayers are made and channels are reconstituted into

- 92 -

the membranes by contacting a metal electrode to a lipid-solution interface. Using this technique,we measured the single channel currents of several types of channels, KcsA, MthK and P2X4,
which were immobilized on the tip of the metal electrode, along with those of two channel-forming
peptides, gramicidin and alamethicin, and a channel-forming protein, α-hemolysin.

Discussion & Conclusion
Here we developed an apparatus for simultaneous electrical and optical recordings of single ionchannel proteins. Our final goal is to elucidate structure-function relationship of ion-channel
proteins. Structure changes in a channel protein are optically detected simultaneously withmeasuring its function as an ionic current fluctuation.
We measured KcsA channel, a bacterial potassium channel, and found that we could detect steadychanges in its gating as differences in fluorescence. However, we have not yet seen rapid changes
in fluorescence that synchronize current changes. We should improve the method for fluorescencelabelings.
The main characteristic of our technology is that channel proteins immobilized on the solid surface
are directly reconstituted into bilayer membranes. The further improvement of this process isneeded to enhance the versatility.
This reconstitution technique is applicable to developing high-efficiency sensor device. Using
this technique, we could increase the channel current measurement efficiency compared to theconventional methods in 10-100 folds. This allows the technique to potentially be combined withhigh-throughput screening devices for channel-drugs.

References
特許・出願番号 2015-154014、「人工生体膜を製造するデバイス及び製造方法」、発明者：平野美奈子、
井出徹、平成 27 年 8 月 4 日

一般の皆様へイオンチャネル蛋白は、生体膜のイオン透過性を制御している蛋白質で、生命活動に於いて大変重要なものであることが分かっています。ところが、この蛋白質が働く詳しいメカニズムは未だに分
かっていません。そこで、私達は、蛋白がどのように動いたときに、活性がどのように変わるかを測る装置を開発しようと考えました。人工的に作った膜にこの蛋白質を組み込み、１個の蛋白質を透過する電流を計測することに成功しました。また、蛋白質１つずつの動きを見ることも出来るようになりました。この方法で神経細胞のチャネル蛋白質の動作メカニズムを解明しようと考えています。

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The lack of mucosal barrier in specialized epithelial M cells as a portal
for infectious agents
Koji Hase

Graduate School of Pharmaceutical Science, Keio University
hase-kj@pha.keio.ac.jp

Abstract
The specialized epithelial microfold cells (M cells) in the gastrointestinal tract actively transport
luminal antigens into the organized lymphoid follicles for induction of antigen-specific IgA response
in mucosa. However, the biological significance of M cells still remains unknown. We here foundthat M cells paly a significant role in protection against mucosal infection.
Key words : M cells, IgA, Th17, mucosal infection

Introduction
The mucosal immune system forms the largest part of the entire immune system, containing about
three-quarters of all lymphocytes and producing grams of secretory IgA daily to protect the mucosal
surface from pathogens. To evoke the mucosal immune response, antigens on the mucosal surface
must be transported across the epithelial barrier into organized lymphoid structures such as Peyer’s
patches. This function, called antigen transcytosis, is mediated by specialized epithelial M cells 1.
To better characterize M cells, we performed transcriptome analysis of M cells. We also exploredbiological significance of M cells by taking advantage of Spi-B-deficient mice that lack M cells.

Results1)Transcriptome analysis of M cells
To understand the molecular basis of functionality and development of M cells, we attempted toperform transcriptome analysis. We initially established a experimental protocol to dissociate andisolate epithelial cells by flowcytometory. M cells are a minor cell population among intestinal
epithelial cell lineages, because this cell type is only located in follicle-associated epithelium, but
not in the villous epithelium that occupy most of intestinal surface. A recent study suggested thattreatment with Receptor activator of nuclear factor kappa-B ligand (RANKL) induces M cells
throughout the villi 2. Following this observation, we treated the mice with RANKL and thenprepared epithelial cell suspension. We successfully isolated an epithelial cell population positive forGP2 (a mature maker of M cells). We compared that gene expression profile among several epithelial
cell populations, namely, M cells, enterocytes and Paneth cells. We identified approximately ahundred genes highly enriched in M cells. These M-cell-signature genes included transcription
factors, membrane-bound molecules, intercellular signaling molecules, and membrane trafficking
factors. We are currently investigating functions of these M-cell-specific genes.

- 94 -

2)Analysis of the biological significance of M cells
Spi-B is a master transcription factor of M cell differentiation. Spi-B-deficient mice lackmature M cells3. The size of gut-associated lymphoid tissues (GALTs) such as Peyer’s patch andisolated lymphoid follicles was lower in Spi-B-deficient mice than in Spi-B-sufficient mice. Thisobservation underscores the importance of M cells in maturation of GALTs. Spi-B-deficient mice
showed a defect in the development of gut-associated To examine biological significance of M
cells in protection against mucosal infection, Spi-B-deficient and –sufficient mice were infectedwith Citrobacter rodentium, a mouse model of Enteropathogenic Escherichia coli (EPEC). C.
rodentium causes attaching effacing lesion and potently induces Th17 response. We found that
Spi-B-deficiency exacerbated infectious colitis, as evidenced by increased clinical scores andexcessive infiltration of macrophages and neutrophils. Several hematopoietic cell lineages also
express Spi-B. Therefore we performed bone marrow chimeric experiments, which confirmed thatthe exacerbation of infectious colitis is ascribable to the absence of Spi-B in non-hematopoietic M
cells. Spi-B-deficient mice also displayed splenomegaly, suggesting that Spi-B is essential to protect
against systemic translocation of C. rodentium. We observed that the number of Th1 and Th17 cells
decreased in the colonic lamina propria of Spi-B-deficient mice compared to Spi-B-sufficient control
mice at the early phase of infection. In contrast, under the physiological conditions, there was no
significant difference in colonic Th17 cell populations between Spi-B-deficient and -sufficient mice.
Furthermore, the composition of intestinal microbiota, which potentially influences C. rodentium
colonization, was similar between the two groups. Together, these results suggested that M-celldependent antigen uptake plays a significant role in the induction of Th17 response to mucosal
infection.

Discussion & Conclusion
Our transcriptome analysis revealed M-cell-specific gene expression network. The characterizationof the M-cell-specific signature genes will shed a light on functionality and differentiation of thisspecialized epithelial cell. Molecular functions of the M-cell-specific genes are currently underinvestigation.
Furthermore, we found that M cells are indispensable for maturation of GALTs. M cell-dependent
antigen uptake is also critical for protection against mucosal infection. In the absence of M cells,
bacterial translocation into body is increased, which causes severe inflammation during mucosal
infection. Based on these observations, we reason that M cell is essential for barrier functions aswell as the maintenance of immune homeostasis on the mucosal surface.

References
1. Hase, K. et al. Uptake through glycoprotein 2 of FimH(+) bacteria by M cells initiates mucosal
immune response. Nature 462, 226–230 (2009).
2. Knoop, K. A. et al. RANKL is necessary and sufficient to initiate development of antigen-- 95 -

sampling M cells in the intestinal epithelium. J. Immunol. 183, 5738–5747 (2009).
3. Kanaya, T. et al. The Ets transcription factor Spi-B is essential for the differentiation of intestinal
microfold cells. Nat Immunol 13, 729–736 (2012).

一般の皆様へ
腸管の粘膜は、食餌とともに摂取される病原菌やウイルス、さらには 100 兆個にも及ぶとされる腸内常在細菌に曝されており、常に感染の危険と隣り合わせにある。これら大量の外来抗原に対応するため、粘膜には粘膜免疫系と呼ばれる特殊な免疫系が発達している。腸管免疫系に存在する M細胞は、粘膜面の抗原の一部をサンプリングし、免疫応答を促す。本研究では M 細胞は粘膜面の感染排除と炎症の抑制に重要な役割を果たすことを明らかとした。今後は M 細胞に積極的に抗原を取り込ませることで免疫力を高める M 細胞標的ワクチンの開発が期待される。

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Epigenetic regulation of Sry by histone H3K9 methylation
Shunsuke Kuroki

Institute for Enzyme Research, Tokushima University
skuroki@tokushima-u.ac.jp

Abstract
Expression of sex-determining gene Sry is stage- and cell type-specifically regulated toactivate male-developmental pathway. We previously reported that the histone H3 lysine 9(H3K9) demethylase enzyme Jmjd1a plays an essential role for Sry expression through histone
demethylation of the Sry locus in mouse by-potential gonads. Here we show that one of the H3K9
methyltransferase GLP plays an antagonistic role on Jmjd1a-induced Sry expression, suggesting theimportance of fine-tuning of H3K9 methylation level on mouse sex determination.
Key words : Epigenetics, Sex determination

Introduction
Covalent modification of histone tails plays an important role for transcriptional regulation.
Among variety of histone modifications, H3K9 methylation is epigenetic mark of transcriptionallysilenced heterochromatin. Mammalian sex is determined in embryonic gonad, where the sexdetermining gene Sry has a pivotal role for male development. We previously showed that XY micelacking H3K9 demethylase Jmjd1a exhibit male-to-female sex reversal and loss of Jmjd1a leads tothe increased H3K9 methylation level on Sry locus followed by reduced Sry expression. This factsuggests the existence of H3K9 methyltrandferase counteracting Jmjd1a on the Sry loucus. However
it is remains to be known.

Results
We previously showed that H3K9 methylase GLP and G9a form a heterodimer complex andthis is responsible for H3K9 methylation. So we considered the GLP/G9a is a candidate for thecounteracting enzyme to Jmjd1a-mediated H3K9 demethylation and Sry expression. As we havedemonstrated that GLP is the limiting factor in GLP/G9a complex we decided to test the possibilityof GLP being the contracting enzyme to Jmjd1a.
First, we test the possible epistasis between GLP and Jmjd1a in mouse sex determination. Miceheterozygous for a GLP mutation (GLP ∆/+) combined with a Jmjd1a-null (Jmjd1a ∆/∆) backgroundwere generated. As previously reported, XY Jmjd1a ∆/∆ mice were frequently sex reversed (40%were male, 40% were female and 20% were intersex). In contrast, XY Jmjd1a ∆/∆; GLP ∆/+ mice
were all male (n=7). These finding shows that GLP mutation is epistatic to Jmjd1a-null mutation inmouse sex determination.

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Next, to address the role of GLP-mediated H3K9 methylation in Sry expression, we examined theexpression of Sry in E11.5 gonads by co-immunofluorescence analysis. The numbers of Sry-positive
cells was reduced to approximately 25% in XY Jmjd1a ∆/∆ gonads. On the other hand, the numberof Sry-positive cells were significantly increased in XY Jmjd1a ∆/∆; GLP ∆/+ gonads, indicating that
GLP heterozygous mutation is epistatic to Jmjd1-null mutation also in Sry transcriptional activation.
Finally, to address the contribution of GLP-mediated H3K9 methylation to Jmjd1a-mediated Srytranscriptional regulation, chromatin immunoprecipitation (ChIP) assay was performed using
MACS magnetically purified gonadal somatic cells from E11.5 embryios. As previously reported,
the H3K9 methylation level of Sry locus in Jmjd1a-null gonadal somatic cells was significantly
increased compared to those of control cells. Importantly, the H3K9 methylation level was
significantly reduced by GLP mutation, suggesting that GLP and Jmjd1a are counteracting enzymes
for H3K9 methylation and epigenetic regulation on Sry.

Discussion & Conclusion
Epigenetic regulation of Sry is an important, yet poorly understood factor for achieving sex
determination and differentiation. We demonstrated that that the opposing activity of GLP against
Jmjd1a contributes to the fine-tuning of the H3K9 methylation level on Sry to ensure is accurate
expression.
To our knowledge, this is the first report identifying the combination of histone methylase anddemethylase, which is responsible for the stage- and cell type-specific gene expression patternduring mammalian development.

References
Kuroki S, et al., “Epigenetic regulation of mouse sex determination by the histone demethylase
Jmjd1a” Science 341, p1106–1109 (2013)

一般の皆様へ
ほ乳類の性決定 Sry は同定されて 20 年以上たちますが、その発現を制御する仕組みはほとんど分かっていません。今回わたしたちは、ヒストン脱メチル化酵素のひとつ Jmjd1a とヒストンメチル化- 98 -

酵素 GLP の間で拮抗したヒストンメチル化の微細な制御が、Sry の発現に重要であることを明らかにしました。

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Crucial role of plasmacytoid dendritic cells for the TLR-7-mediatedautoimmne inflammation
Hideaki Takagi

Division of Immunology, Department of Infectious Diseases, Faculty of Medicine, Universityof Miyazakihideaki_takagi@med.miyazaki.ac.jp

Abstract
Plasmacytoid dendritic cells (pDCs) are important for the innate and adaptive immune responses
by producing robust type-I interferon (IFN-I) through the toll-like receptor (TLR)-mediatedsignaling. However, how pDCs control TLR-mediated immune responses that cause autoimmunity
remains unclear. In this study, we demonstrated a critical function of pDCs in the induction of
TLR7-mediated innate and adaptive immune responses that cause autoimmunity using genemodified mice with impaired expression of Siglec-H and selective ablation of pDCs. Our findings
reveal that pDCs provide an essential link between TLR7-mediated innate and adaptive immunity
for the initiation of IFN-I-associated autoimmune inflammation.
Key words : Plasmacytoid cendritic cells (pDCs)
Toll-like receptor (TLR)
Autoimune disease
Inflammation
Type-I interferon (IFN-I)

Introduction
pDCs are known as IFN-I producing cells via TLR7/9-mediated recognition of nucleic acids(NAs). Recent studies suggested that the activation of pDCs by self NAs causes the elevated
IFN-I levels and contributes to the induction of pathogenesis in psoriasis and systemic lupus
erythematosus (SLE). However, how pDCs control immune responses leading to the initiationof these IFN-I-associated autoimmune diseases is poorly understood. In this study, we show that
pDCs are indispensable for the TLR7-mediated development of I-IFN-associated psoriasis-like skininflammation and lupus-like systemic autoimmune disease under the intrinsic Siglec-H-mediatedcontrol of inflammation and T-cell responses.

Results
To clarify the role of pDCs and Siglec-H-mediated pDCs regulation in the TLR-mediated immune
responses for the initiation of autoimmune inflammation, we used genetic modified mice that havethe deficient expression of Siglec-H on pDCs (referred as Siglechkd mice) and allows the conditional
depletion of pDCs mediated by diphtheria toxin receptor knock-in system (referred as pDC-ablated- 100 -

mice).
First, we addressed the role of Siglec-H in the TLR-mediated stimulation in vivo. While purified
wild-type (WT) pDCs produce IFN-α, IFN-β, and interleukin (IL)-12p40 in response to imiquimod(IMQ) known as TLR7 ligand, Siglechkd pDCs exhibited the further enhanced production of thesecytokines after IMQ stimulation, indicating that Siglec-H act as a negative regulatory molecule for
pDCs in response to TLR7 stimulation.
Next, we examined the roles of pDCs in the TLR7-mediated inflammatory response in vivo. Afterthe administration of IMQ and D-galactosamine (D-GalN), Siglechkd mice displayed the increased
levels of serum cytokines, including IFN-I, while pDC-ablated mice exhibited a dramatic reductionof the cytokine production compared with WT mice. Furthermore, Siglechkd mice or pDC-ablated
mice were more susceptible or resistant to the TLR7-mediated lethality than WT mice. These results
indicate that pDCs are required for the initiation of TLR7-mediated systemic inflammatory response
in vivo.
To address the roles of pDCs in the induction of the responses of antigen (Ag)-specific T cells
in vivo, carboxyfluorescein diacetate-succinimidyl ester (CFSE)-labeled OT-II CD4+T cells wereadoptively transferred into mice, and their division was analyzed after systemic injection of soluble
ovalbumin (OVA) protein plus IMQ. When compared with Ag-specific division of CD4+T cells in
WT mice, Siglechkd mice or pDC-ablated mice exhibited the enhancement or the reduction of this
response. We also examined the role of pDCs in the CD8+T-cell response. Upon immunization with
OVA protein plus IMQ, Siglechkd mice and pDC-ablated mice displayed the reduction of Ag-specificdivision of CD8+T cells compared with WT mice. Collectively, these results indicated that pDCs
potentiate Ag-specific T-cell responses under IMQ-induced inflammatory conditions in vivo.
Finally, to clarify the role of pDCs in the development of autoimmune diseases, we examined
the pathological mechanisms of IMQ-induced psoriasiform dermatitis and pristine-induced lupus
like disease. Topical application of IMQ on the back skin of WT mice led to the developmentof psoriasiform dermatitis. Analysis of sections of skin obtained from IMQ-treated WT mice
showed a phenomenon typical of psoriasis skin lesions. On the other hand, Siglechkd mice or pDCablated mice showed the accelerated or the attenuated development of IMQ-induced psoriasiform
inflammation. Following the topical application of IMQ, Siglech kd mice or pDC-ablated mice
exhibited a higher or a lower serum production of IFN-I and inflammatory cytokines than WT mice.
Furthermore, Siglechkd mice or pDC-ablated mice displayed a higher or a lower frequency of IL-17producing T cells and BrdU+ lymphocytes than WT mice after the topical treatment of IMQ. These
results indicate that pDCs also control systemic inflammation, the generation of effector T cells, andthe proliferation of lymphocytes in the development of psoriasiform dermatitis triggered by skinmediated incorporation of IMQ.
We also examined the contribution of pDCs to the pathogenesis of pristane-induced lupus. When
compared with pristane-treated WT mice, pristane-treated Siglechkd mice or pristane-treated pDCablated mice exhibited the enhanced or the reduced serum production of anti-nuclear antibody(Ab), such as anti-single-strand DNA (ss-DNA) Ab, anti-double-strand DNA (ds-DNA) Ab, anti-- 101 -

snRNP Ab, and anti-Sm Ab. Furthermore, pristane-treated pDC-ablated mice exhibited a lower
serum level of creatinine than pristane-treated WT mice and pristane-treated Siglechkd mice. Whilethe histopathologic assessment of the kidneys in pristane-treated WT mice showed an enlarged
hypercellular glomeruli, an increase in the mesangial matrix, and mild peritubular mononuclear cell
infiltrates, the development of the renal pathology was promoted or suppressed in pristane-treatedSiglech kd mice or pristane-treated pDC-ablated mice. Simultaneously, the immunofluorescenceanalysis revealed that the depositions of IgG, IgM, and C3 within the glomeruli were enhanced or
reduced in pristane-treated Siglech kd mice or pristane-treated pDC-ablated mice compared withthose in pristane-treated WT mice. These results indicate that pDCs promote the development ofpristane-induced lupus-like glomerulonephritis.

Discussion & Conclusion
While recent accumulating results suggest that pDCs are linked to the pathogenesis of psoriasis
and SLE, how pDCs control these IFN-I-associated autoimmune diseases remains unclear. In thisstudy, we demonstrated a critical function for pDCs in the induction of TLR7-mediated innate andadaptive immune responses that cause IFN-I-associated autoimmune inflammation. Furthermore,in line with recent reports regarding the role of pDCs in SLE-prone animals, our findings suggeststhat the Siglec-H-mediated control of pDCs function is crucial for the IFN-I-dependent progressionof lupus-like disease as manifested by the impact on the production of anti-self NA Ab andglomerulonephritis. Therefore, our biochemical and genetic results clearly show that Siglec-H acts
as an intrinsic “regulatory receptor” for the TLR7-mediated activation of pDCs that is important for
regulation of the magnitude and quality of inflammation and T-cell responses. In conclusion, ourfindings suggest that pDCs control the initiation and the development of psoriasis and SLE, and thetargeting of the pDC lineage as a viable therapeutic strategy to ameliorate type I IFN-associatedautoimmune disorders.

References
Takagi, H., Arimura, K., Uto, T., Fukaya, T., Nakamura, T., Choijookhuu, N., Hishikawa, Y., andSato, K. Plasmacytoid dendritic cells orchestrate TLR7-mediated innate and adaptive immunity forthe initiation of autoimmune inflammation. Sci. Rep., 6:24477, 2016.

一般の皆様へ
形質細胞様樹状細胞 (pDCs) は多量の I 型インターフェロン (I 型 IFN) を産生する免疫細胞であり、抗ウイルス免疫応答を初めとした様々な免疫反応に重要な役割を果たしています。その一方で pDCsと I 型 IFN は、全身性エリテマトーデス (SLE) や尋常性乾癬といった自己免疫疾患への関与が考えられておりますが、その詳細なメカニズムについてはこれまで不明でありました。本研究では pDCsを生体内で特異的に消失させた遺伝子改変マウスを用いて検討した結果、自己免疫疾患の発症と増悪に pDCs が重要な役割を担っていることを明らかにしました。本研究成果を応用することで、pDCs- 102 -

の機能制御を基にした新たな自己免疫疾患に対する治療法の開発が期待されます。

- 103 -

Development of Asymmetric 1,3-Alkyl Migration Reaction for
Synthesis of Drug Candidate Compounds
Norie MOMIYAMANational Institutes of Natural Sciences, Institute for Molecular Sciencemomiyama@ims.ac.jp

Abstract
Chirality transferred 1,3-alkyl migration of 1,2,2-substituted butenyl amines was discovered in thepresence of trifluoromethyl acetic acid.
Key words : alkyl migration, Brønsted acid

Introduction
Allylation of imines with allylic metal reagents has been one of the most valuable tools tosynthesize enantioenriched homoallylic amines. Due to the inherent nature of allylic metal reagent,
however, regioselective synthesis of linear homoallylic amines has been a long-standing subject inthis area.

Results
To develop the synthetic reaction for enantioenriched linear homoallylic amines, we discoveredchirality transferred 1,3-alkyl migration of 1,2,2-substituted butenyl amines in the presence oftrifluoromethyl acetic acid, and developed it as synthetic method for variety of enantioenrichedlinear homoallylic amines.
The reaction of 1,2,2-substituted butenyl amines was conducted under the microwave irrageation to
give 1,3-dialkyl migration products in quantitative yields with more than 90% enantioselectivities.
To think about the mechanism, control experiments were examined by taking 1H NMR. NMR
studies indicated that the reaction proceeded via imine formation, followed by [3,3]-sigmatropic
rearrangement.

Discussion & Conclusion
Ollis et al. previously reported that chirality was significantly dropped in 1,3-alkyl migration ofN,N-dimethyl-1-substituted-3-buten-1-amine. In sharp contrast, we discovered chirality transferred
1,3-alkyl migration of 1,2,2-substituted butenyl amines in the presence of trifluoromethyl acetic
acid. To the best our knowledge, our discovery is the first example of chirality transferred 1,3-alkyl
migration and the new entry of the synthetic methodology for the linear enantioenriched homoallylic- 104 -

amines.

References

一般の皆様へ
オレフィン末端に置換基を有する光学活性ホモアリルアミンは、医薬品候補化合物の合成中間体として有用な化合物です。今回、私たちは、市販で容易に入手可能な酸触媒を用いることで、良好な
化学および光学収率で、オレフィン末端に置換基を有するホモアリルアミンを合成することに成功しました。

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NAD metabolism as a novel target for anti-cancer therapy
Takashi Nakagawa

Frontier Research Core for Life Sciences, University of Toyama
nakagawa@med.u-toyama.ac.jp

Abstract
Cancer cells have a unique metabolism to support their aggressive proliferation. Especially, aerobic
glycolysis is a character feature of cancer cell metabolism, and known as the Warburg effect. In thisstudy, we examined the role of NAD metabolism pathway in cancer cells. We demonstrated that
NAD synthesis pathway was important to recover the NAD level after DNA damage. These results
indicated that NAD metabolism can be a therapeutic target for cancer cells resistant to DNA damage
inducing anti-cancer drugs.
Key words : NAD, metabolomics, cancer metabolism, DNA damage

Introduction
NAD (Nicotinamide adenine dinucleotide) is an essential co-enzyme mediating various metabolic
enzymes through redox reactions. NAD also serves as a substrate for poly(ADP-ribose)polymerases(PARPs) and sirtuins for poly(ADP-ribosylation) (PARylation) and deacetylation, respectively.
Single-strand DNA damage (SSB) induces the PARylation and depletes NAD pool in cancer cells.
However, NAD level will be recovered if cancer cells can be survived after SSB by anti-cancer drug
or irradiation. Thus, inhibition of NAD metabolism can boost the sensitivity of anti-cancer drug or
irradiation. We focus on PRPS in this study and investigated their role in cancer metabolism.

Results
Result 1: NAD depletion after the treatment of DNA damage reagent
A549 cells (lung cancer cell line) were treated with DNA damage inducing reagents, such ashydrogen peroxide (H2O2) or Methyl methanesulfonate (MMS). We checked the NAD level by LC/
MS at various time points after the treatment. H2O2 treatment induced the PARylation after 30 minof treatment, and PARylation was immediately degraded. Consistent with this result, NAD level also
dropped down at 30 min, and kept the low level until 6 hours. When the cell are treated relatively
high-dose of H2O2, the cell died after 6 hours. However, low dose of H2O2 treatment did not kill thecells, and NAD level was recovered until 24 hours after the treatment. We also observed the similar
results when we used MMS as DNA damage inducing reagent. Thus these results indicated that
NAD is robustly depleted after DNA damage, but it will be recovered when cell can survive.
Result 2: Identifying the pathway to recover the NAD level after DNA damage
In mammalian cells, NAD is predominantly synthesized through the salvage pathway, and- 106 -

nicotinamide phosphoribosyltransferase (Nampt) is a rate-limiting enzyme in this pathway. Nampt
catalyzes the formation of NMN from nicotinamide (NAM) and 5’-phosphoribosyl-pyrophosphate(PRPP), and NMN is subsequently converted to NAD by nicotinamide mononucleotide
adenylyltransferase (Nmnat). After the DNA damage, PARylated NAD was recessively degraded
into ADP-ribose by Poly (ADP-ribose) glycohydrolase (PARG). In NAD synthesis, PRPP is
generated from glucose through pentose phosphate pathway (PPP) or degrade ADP-ribose.
Therefore, we investigated whether the degraded ADP-ribose is re-used to generated NAD. To
distinguish the PRPP generating pathway, we employed 13C-labeled glucose, and measured therecovered NAD by LC/MS after DNA damage treatment. If NAD is synthesized from degraded
ADP-ribose, recovered NAD is not 13C-labelled (i.e. 12C-labelled). In contrast, in case that NAD
is come from PRPP through PPP, NAD was 13C-labelled. We treated A549 cells with low-doseof H2O2 (non lethal concentration) and harvested the cells after 0.5, 3, 6 and 24 hours. As same asprevious results, NAD level dropped down at 0.5 hour. After 3 or 6 hours, 13C-labeled NAD wasslightly increased, but not 12C-laballed NAD. Furthermore, NAD level was fully recovered after 24hours, and most of them are 13C-labelled. These results demonstrated that the pathway to recover
the NAD level after DNA damage was mainly PPP from glucose.
Result 3: Role of PRPS in DNA damage induced cell death
Next, we try to identify the key enzyme in the NAD recovery after DNA damage. We focus on
PRPS (phosphoribosyl pyrophosphate synthetase), which catalyze the phosphoribosylation of ribose
5-phosphate to PRPP. We knocked down the expression of PRPS1 in A549 cells by siRNA, andevaluated the cell death rate after the exposure of sub-lethal dose of H2O2. In control siRNA treatedcells, most of cell could survive and recovered NAD level. However, the cell treated with PRPS1
siRNA died after H2O2 treatment. This result indicated that PRPS1 is critical for the NAD recovery
after DNA damage, and its inhibition could be the therapeutic target for cancer cells resistant toDNA damage inducing anti-cancer drugs.

Discussion & Conclusion
In this study, we revealed that the recovery of NAD after the DNA damage was mediated bypentose phosphate pathway. Particularly, PRPS1 is the key enzyme of this pathway, and inhibition of
PRPS1 in cancer cells resulted to induce the cell death even at the sub-lethal dose of DNA damage
inducing reagent. These results suggested that the inhibition of PRPS1 could cancel the resistanceagainst anti-cancer drug inducing DNA damage or boost the effect of these drugs or irradiation.
Although more detailed study will be desired to establish the role of PRPS1 in DNA damage
induced cell death, we expect PRPS1 can be the target for cancer treatment for future.

References
1.

Yamamoto M, Hikosaka K, Mahmood A, Tobe K, Shojaku H, Nakagawa T. Nmnat3 is not- 107 -

essential for the maintenance of mitochondrial NAD level in vivo. PLOS ONE 11(1): e0147037.
(2016)
2. Hikosaka K, Ikutani M, Shito M, Kazuma K, Gulshan M, Nagai Y, Takatsu K, Konno H, Tobe
K, Kanno H, Nakagawa T. Deficiency of Nicotinamide Mononucleotide Adenylyltransferase 3(Nmnat3) Causes Hemolytic Anemia by Altering the Glycolytic Flow in Mature Erythrocytes.
J Biol Chem. 289(21): 14796-14811. (2014)

一般の皆様へ
がん細胞は、正常細胞と異なった特異的な代謝経路を持つことが知られています。このことは、がん細胞が非常に高い増殖性を持つことと関連があり、これらをターゲットとした抗がん治療の開発が期待されています。今回の研究は、こうしたがん特異的な代謝経路に着目した研究であり、特に今回の研究の標的分子である PRPS を阻害することで、放射線治療や抗がん剤によるがん治療に対して、抵抗性のあるがん細胞を細胞死へと追いやることができると考えています。

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Creating methods for in vitro amplification of skeletal muscle satellite
cells with regenerative myogenic potential
Atsuko Sehara

Kyoto University, Institute for Frontier Medical Sciences
asehara@frontier.kyoto-u.ac.jp

Abstract
It is very important to establish effective methods to amplify skeletal muscle stem cells in vitro.
However, they tend to differentiate once they are cultured in vitro. We have found a method toamplify them keeping their immature states.
Key words : Developmental Biology, Cell Biology

Introduction
Adult skeletal muscle stem cells are kept in quiescent states, that is, arrested their cell proliferation.
Although previous studies revealed efficient incorporation of adult muscle stem cells into
regenerating myofibers of Duchenne muscular dystrophy (DMD) model mice, their regenerative
capacity declines significantly when cultured ex vivo, and this decline is likely associated with theexit from quiescence of muscle stem cells (Montarras et al., Science 2005). Therefore, It is very
important to establish effective methods to amplify skeletal muscle stem cells in vitro keeping
stemness from the therapeutic viewpoint;

Results
Previous studies revealed the involvement of key transcription factors in the establishmentof skeletal muscle stem cells (MuSCs), the paired box transcription factors, Pax3 and its
paralogue Pax7. The Pax3/7-positive progenitors were shown to be the source of adult MuSCs.
The expression of Pax7 is required for the establishment of adult MuSCs and high in immature
MuSC. Based on the level of Pax7, MyoD, and myogenin that is expressed in differentiatedmyocytes, we found a culture condition that maintains high level of Pax7 expression andcapacity of self renew. That is, under the culture condition, Pax7-positive, MyoD-negative
MuSCs proliferate prominently, and they do not differentiate to Pax7-negative myogeninpositive myocytes significantly. We also found a culture method promoting differentiation of
MuSCs.
Interestingly, these two methods were related with each other, and likely involved a couple ofgenes, gene A and B. We are now preparing mice carrying MuSC-specific deletion of the gene
A by crossing Knock-in mice of Pax7/Cre and conditional gene A knockout mice. We will beable to demonstrate potential of muscle regeneration and self renew/ differentiation of MuSCs inknockout mice of these genes soon.

- 109 -

If we can demonstrate involvement of these genes in regulation of the balance between self
renew and differentiation of MuSCs, manipulation of these genes will enable us to amplify
MuSCs keeping stemness of MuSCs in vitro.

Discussion & Conclusion
We established two culture methods, a method to proliferate keeping self renew and a method
to promote differentiation of MuSCs. These two methods have revealed a novel pathway that
regulates the balance of self-renew and differentiation of MuSCs in vivo, and allowed us topredict involvement of genes in that process. By manipulating these genes, we will be able todefine more efficient methods to amplify MuSCs with high regenerative myogenic potential.

References
Sato, T., Yamamoto, T., Sehara-Fujisawa, A.. (2014) miR-195/497 induce postnatal quiescenceof skeletal muscle stem cells. Nat. Cammun., 5: 4597.
( 上記は本研究のアイデアのもととなった研究。本研究は、現時点では未発表です。)

一般の皆様へ幹細胞には、多分化能を持つ組織幹細胞や IPS 細胞が良く知られているが、骨格筋にある幹細胞
は、骨格筋が損傷を受けた時その再生を担う、骨格筋特異的な幹細胞です。もし、この幹細胞を試
験管内で無限に増殖させることができれば、それは癌化を心配することなく骨格筋に移植することができ、筋ジストロフィーなどの疾患治療に役立てることができるはずです。私達の研究は、その培養法を確立しようとするもので、成功に近づいていると確信しています。

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Role of protein phosphatases in robustness of mammalian circadianclock
Makoto Akashi

Yamaguchi University
akashima@yamaguchi-u.ac.jp

Abstract
We investigated the role of two protein phosphatases in robustness of the circadian clock invivo and ex vivo using mice lacking the genes coding these proteins. Mice lacking these two
phosphatases showed impaired circadian phase shift、 unstable free-running period in locomotor
activity and inappropriate circadian phase in the suprachiasmatic nucleus (SCN), the circadian
center of mammals, suggesting their essential role in the molecular clock.
Key words : circadian rhythm; circadian clock; clock gene; protein phosphatase

Introduction
The circadian clock drive diurnal rhythms in physiology and behavior, and enables organisms
to adapt earth’s rotation. Light, the most potent entrainment signal, adjusts circadian phase by
activating intracellular kinase signaling pathways in the suprachiasmatic nucleus, the central
clock. However, the role of protein phosphatases modulating the activity of these kinases remains
unknown.

Results
Intracellular kinases in the suprachiasmatic nucleus are not only essential for robustness of the
mammalian circadian clock but also play roles when light adjusts circadian phase. On the other
hand, although protein phosphatases that negatively regulate the activity of these kinases arethought to have some potential roles in the mammalian circadian clock, the details remain unknown.
In this study, we examined this hypothesis by performing animal experiments in vivo and ex vivo.
All experiments were performed with the approval of the Committee on Animal Care and Use at
Yamaguchi University.
To examine the role of the protein phosphatases X and Y (anonymous for confidential purposes)in the mammalian circadian clock, we obtained mice lacking the genes coding them from ourcollaborators. We first established double knockout (DKO) mice lacking both these phosphatases,
and then these DKO mice were cross-mated with mice carrying the firefly luciferase gene fusedwith the clock gene Period2 (Per2), which is indispensable for the circadian clockwork, in order tomonitor circadian gene expression of PER2 protein as the intensity of bioluminescence in real-timewith a high time-resolution.
We first examined the free-running period of locomotor activity in these mice, the length of which- 111 -

reflects the intrinsic circadian period length of mice. Their behavior was monitored under a constantdark condition in dark boxes equipped with an infrared sensor. The obtained data suggest that theircircadian period length was variable among individuals; some mice showed a short circadian period
in locomotor activity and others a long one, when compared with that of wild-type mice.
Next, we investigated circadian adaptability of these mice to a 6-hr phase shift of light-dark cycles
by monitoring their behavior in dark boxes equipped with an infrared sensor, and found that their
behavioral rhythms phase-shifted faster than those of wild-type mice. This result indicates that micelacking these two phosphatases may have a circadian clock with a decreased robustness against
environmental changes.
Finally, we examined circadian characteristics of PER2-LUC expression by performing ex vivo
slice cultures of the suprachiasmatic nucleus, the center for the mammalian circadian clock in thebrain. Although circadian characteristics of PER2 expression such as amplitude and period were
almost normal in X and Y phosphatase-deficient mice, the circadian phase of the suprachiasmatic
nucleus seemed to be advanced when compared with that of wild-type mice. This result indicatesthat these phosphatases have a role for normal circadian phase adjustment in the suprachiasmatic
nucleus.

Discussion & Conclusion
In this study, we examined whether the protein phosphatases X and Y play a role in circadianbehavior and molecular clockwork in mice, and our data indicate that these molecules may
contribute to robustness of the mammalian circadian clock. Although a few studies implied thatthese phosphatases have some roles for normal circadian rhythms because they modulate the activityof protein kinases regulating robustness of autonomous circadian machinery and light-induced
circadian phase shift, there was no direct evidence that these molecules are essential components inthe circadian clockworks. Our present data may be direct evidence that they play indispensable rolesin the core clock and the input signaling pathway. However, the number of replicates are still small,and further experiments are therefore required to make solid conclusions. Furthermore, we areplanning to perform experiments using ex vivo culture of the suprachiasmatic nucleus to investigate
difference between wild-type and KO mice in the effect of neurotransmitters involving the light
input pathway on circadian characteristics of clock gene expression.

References
一般の皆様へ
２４時間周期の体内時計である概日時計は、生物が地球の自転によって生じる毎日の環境変化に適応するために必須の生体機能である。概日時計が地球の自転から脱同調を起こすと、身体機能が低下するだけにとどまらず多様な疾患の原因にもなるため、太陽光などによる概日時計の調節メカニ
ズムの理解は重要である。これまで、光刺激が概日時計中枢（視交叉上核）に作用すると、タンパ- 112 -

ク質リン酸化酵素が働くことで概日時計が調節されることがわかっていました。今回の研究ではこの酵素の機能を抑制する因子が正常に機能することが、概日時計制御において重要であることを示しました。

- 113 -

Development of novel cyclization reaction with SmI2 and application
to synthesis of natural products
Taiki Umezawa

Faculty of Environmental Earth Science, Hokkaido University
umezawa@ees.hokudai.ac.jp

Abstract
Two intramolecular reactions, aldol type reaction and Barbier type reaction, were tackled in thecurrent study. In the former reaction, it was revealed that the reaction gave unidentified compounds.
The latter reaction successfully afforded the desired cyclization product in a diastereoselective
manner. This could be applied to the total synthesis of kainic acid.
Key words : Samarium diiodide, cyclization reaction, total synthesis

Introduction
SmI 2 has been known as a single electron reducing reagent to induce a radical reaction1.
Many useful reactions have been described with SmI 22 . However, further developments of a
new type of reaction should be developed for more convenient construction of complex carbon
frameworks. In the present studies, two reactions,
1,1-dimethylcyclopropane and① intramolectular aldol type reaction with

② intra- molecular Barbier type reaction between allyl chloride

and a,b-unsaturated ester, have been focused. These reactions are expected to be applied to total
syntheses of Kainic acid3 and Vinigrol4, respectively. The results of these studies are shown below.

Results① intramolectular aldol type reaction
Toward the investigation of the novel aldol type reaction, model substrate 3 was first synthesized
as shown in Scheme 1. The synthesis was started from (+)-3-carene, a cheap optically active
commercially available material including 1,1-dimethylcyclopropane, which was converted to
Weinreb amide 1 through oxidative cleavage of double bond and amide formation reaction of theresultant carboxylic acid in high yield. Baeyer-Villiger oxidation, reduction of the amide and Wittig
reaction in the presence of trifluoroethanol afforded a,b-unsaturated ketone 2. The substrate 3 forthe target reaction was successfully obtained by reduction of olefin with palladium-carbon andoxidation of the primary alcohol.
Toward the desired reaction as shown in dotted arrows, extensive conditions, including addition of
alcohols or HMPA, equivalent of SmI2 or reaction temperature, were examined to find unidentified
compounds were obtained which depends on the reaction conditions. For example, NMR study
suggested that isopropyl group was observed when HMPA was added in the reaction, and that two
methyl groups not on cyclopropane were found in the absence of HMPA. However, the precise- 114 -

chemical structure could not be assigned by NMR and molecular weight. Furthermore, someattempts toward X-ray crystal graphic analysis also have failed to date. Now, the identification of thereaction products and researches with other reaction condition are currently underway.
Scheme 11) mCPBA, 76%2) DIBAL

1) RuCl3.xH2O
NaIO42) Me(MeO)NH
EDCI, Et3N
3-(+)-Carene 94% 2 steps
(
)
SmI2

CHO
nBu

3

O

AdditiveOHO1) H2, Pd/C
99%

O
Me
HO
O
N
3) Bu3P
nBu
OMe
2
CF3CH2OH
1ses
60 % (2 t p )

O
Sm

SmI2

H

nBu

nBu

O nBu OH+
Sm(III)O2) PDCH
O nBu OH

② intramolecular Barbier type reaction
A simple substrate 4 having allyl chloride and a,b-unsaturated ester was optimized toward theintramolecular Barbier type reaction. The results are depicted in Table 1. The efficiency of thecyclization in the absence of HMPA even with water was very low (entries 1-4). It was revealed that
addition of HMPA and water remarkably improved chemical yield (entries 5-8). In these reactions,diastereomeric mixture of 5a and 5b was obtained and trans-product 5b was preferred. Next, theamount of HMPA was increased to find improvement of the selectivity of 5b (entries 9, 10). On theother hand, addition of NiI2 in the presence of HMPA decreased the selectivity for 5b (entry 11). To
our surprise, the selectivity of cis-product 5a became preferred up to 5.0:1 ratio in the conditions ofcatalytic NiI25 and no HMPA when the reaction was carried out at room temperature (entries 12, 13).
These results suggest that kainic acid and a-allo-kainic acid can be prepared selectively by changing
the reaction conditions.
Table 1 Cl

CO2Et

CO2Et

SmI2 (6.0 equiv.), HMPA (x equiv.)+

H2O (y equiv.), NiI2 (z equiv.)
THF, temp.

N
Boc
4
entry

x

y

z

temp. yielda (5a:5b)b

1
2
3
4
5
6
7
8

0
0
0
0
24
24
24
24

0
0
10
10
0
0
10
10

0
0
0
0
0
0
0
0

rt
-78 oCrt-78 oCrt-78 oCrt-78 oC

7% (NDc)
NRd
23% (1:2.0)
NR
34% (1:1.0)
CMe
58% (1:2.6)
61% (1:3.1)- 115 -

CO2Et

N
Boc
5a

N
Boc
5b

entry

x

y

z

temp. yielda (5a:5b)b

9
10
11
12
13

60
60
24
0
0

10
10
10
10
10

0
0
0.3
0.3
0.3

rt
-78 oCrt
rt
0 oC

a so a e

56% (1:4.6)
33% (1:4.5)
21% (1.1:1)
62% (5.0:1)
50% (2.6:1)

l t d yield after column chromatography.
d by NMRc
ND = Not Determined. d NR = No Reaction.
e
CM = Complex Mixture.
Ib ratio was estimate

The above-mentioned conditions were next investigated with substrate 7 for kainic acid which
was synthesized from D-serine methyl ester 6 in 5 steps including reductive amination reaction andWittig reaction (Scheme 2). When the conditions of entry 7 in Table 1 were employed, the cyclization
reaction with SmI2 proceeded to provide the desired product 8 as a mixture of diastereomers in 85%
yield. Although the diastereomers could not be separated at this stage, the diasteromers could be
separated by HPLC after removal of TBS group with TBAF to furnish desired cis-product 9a andtrans-product 9b in 1.2:1 ratio. These were successfully converted into kainic acid and a-allo-kanic
acid by Jones oxidation, hydrolysis with basic condition and deprotection of Boc group. Further
studies toward more selective syntheses of kainic acid or a-allo-kanic acid by using other conditions
are ongoing.
Scheme 2Cl

7

CO2Et SmI2, H2O
HMPA
N
Boc

OTBS

THF, rt
85%

CO2Et

CO2Et
TBAF
8

N
Boc

OTBS

THF

+
9a
37%

N
Boc

H2N

6

OH

3 steps

5 steps
CO2Me
OH

CO2Et
N
9b Boc OH
31%
3 steps

CO2H

CO2H

CO2H
N
H
kainic acid

CO2H
N
H
α-all o-kainic acid

Discussion & Conclusion① Although effective synthesis of the model substrate was developed, the targeted product was not
obtained because different type of products was formed as side reactions. First of all, identificationof the product is essential. On the other hand, the concept of cleavage of cyclopropane by generationof a-radical can be applied to a,b-unsaturated carbonyl compounds which is easily prepared fromthe same model substrate. This idea also should be tried.
② Intramolecular Barbier type reaction between allyl chloride and a,b-unsaturated ester could
be developed. Switching the diastereoselectivity was also achieved by changing the reaction
conditions. This is probably due to the change of reaction mechanism. To make the present reaction
more useful, the mechanism should be revealed. As an application, total synthesis of kainic acid wasachieved with this reaction. Further studies toward the elucidation of the reaction mechanisms andoptimization for selective synthesis of kainic acid are underway.

References
1. P. Girard, J. L. Namy, H. B. Kagan, J. Am. Chem. Soc. 1980, 102, 2693.
2. H. B. Kagan, Tetrahedron 2003, 59, 10351.
3. I. Nitta, H. Watase, Y. Tomiie, Nature 1958, 181, 761.

- 116 -

4. I. Uchida, T. Ando, N. Fukami, K. Yoshida, M. Hashimoto, T. Tada, S. Koda, Y.
Morimoto, J. Org. Chem. 1987, 52, 5292.
5. F. Machrouhi, B. Hamann, J.-L. Namy, H. B. Kagan, Synlett 1996, 633.

一般の皆様へ
本研究では、有用化合物にしばしば含まれる複雑な環状構造を、2 ヨウ化サマリウム (SmI2) を用いて一挙に構築することを目的としており、2 種類の環化反応を検討した。1 つ目の反応は、新しい概念を含むアルドール反応であり、環化前駆体を効率的に合成するとともに、アルドール反応を検討した。現在、生成物の構造を確認している。2 つ目は新規共役付加反応であり、本反応の開発の結果、生命科学分野で有用な分子ツールとして用いられるカイニン酸を効率的に合成できた。

- 117 -

Genome-wide screening of endocytosis of GPCR
Junko Toshima

Tokyo University of Technology School of Health Science
toshimajk@stf.teu.ac.jp

Abstract
In the previous study, we performed the screening of yeast knock down strains for the mutants
that have defects in endocytic internalization or subsequent intracellular transport pathway, andidentified 196 mutants out of 5155 strains. In this study, I characterized these mutants according to
the groups (Group A~C) categorized dependent on transport defects. I specifically focused on SRV2,
which exhibited sever delay in endocytic internalization step. I revealed that Srv2p has the multiple
roles in the regulation of the actin cytoskeleton that are required for normal endocytosis.
Key words : Genome-wide screening, endocytosis, actin

Introduction
GPCRs are seven trans-membrane receptors, and are involved in various important physiologicalprocesses. GPCRs are the target molecules of ~30% of the drugs currently used for the treatmentof a wide range of human diseases. Thus, elucidating the regulatory mechanism of GPCR signalingis essential for the development of more effective and safer therapeutic agents. In the previous
study, I developed the fluorescence marker that could specifically label activated GPCR for yeast(Toshima JY. et al, 2006). Using this marker, I screened yeast knockout strains for the mutants thathave defects in endocytic internalization or subsequent transport process. I have identified 196
mutants, and categorized them into Group A~C, according to their defects. Here, I represent thecharacterization of these mutants.

Results
I have performed the characterization of identified mutant cells from the following 4 points of view.
In the project 1, I examined how clathrin coat formation is initiated at plasma membranes. In the
project 2, I examined how clathrin coated pits are transported to early endosomes. In the project 3,
I examined how early endosomes are matured to late endosomes. And, in the project 4, I examinedthe characterization of the identified genes whose functions are not yet known. I gained progresses
for some extents in each project. Here, I report the results from project 2, focusing on SRV2 gene.
Srv2p, also called cyclase-associated protein (CAP), is a highly conserved actin–binding protein thatis required for normal actin organization (Ono, 2013). Although the previous studies have suggested
that potential roles of Srv2p in actin assembly using in vitro analysis, the physiological roles of
Srv2p have remained unclear (Chaudhry et al. 2010,2013, Bertling et al. 2007, Mattilia et al, 2004).
Therefore, I assessed in vivo quantitative analysis for endocytic internalization and actin assembly- 118 -

at clathrin coated pits, in the absence of SRV2 gene. As a result, I found that srv2D mutants havedefects in both assembly and disassembly of actin patches, resulting in severe defects in endocyticinternalization. In addition, srv2D displayed the appearance of aberrant fragmented actin cables(filamentous actin structure, important for endocytic vesicle transport (Toshima JY. et al. 2006))
that frequently moved past the actin patches, sites of endocytosis. We further assessed the detailanalysis using domain truncated mutants (DHFD, DPWP, DCARP), and found that CARP domain,necessary for ADP/ATP exchange of monomer actin, is vitally important for the proper assemblyof actin patches and cables. I also identified that HFD domain is required for Srv2p’s localizationto actin patches specifically to the ADP-actin rich region through an interaction with cofilin, actin
severing factor. These results demonstrate the in vivo roles of Srv2p in the regulation of the actin
cytoskeleton during clathrin-mediated endocytosis. These results were presented in our recent paper
published at Journal of cell Science, 2016.

Discussion & Conclusion
As described above, I investigated the phenotypesof several srv2 mutants in living cells and obtainedin vivo evidences of Srv2p in actin assembly andendocytic internalization. On the basis of our results,
I propose a mechanism of actin assembly that isregulated by Srv2p as shown in the model.
As well as Srv2p, characterizations of other mutant
cells have also been undertaken. By revealing themechanism that mediate endocytic pathway, I aim tounderstand the basic mechanisms that control GPCR
signaling pathway.

References
・ Bertling E. et al.,(2007) Mechanisms and biological role of profiling-Srv2/CAP interactions. J.
Cell Sci. 120, 1225-1234.
・ Chaudhry F. et al.(2010) A central role for theWH2domain of Srv2/CAP in reaching actinmonomers to drive actin turnover in vitro and invivo. Cytoskeleton 67, 120-133.
・ Chaudhry F. et al.(2013) Srv2/ cyclase-associated protein forms hexameric shurikens that directly
catalyze actin filaments severing by cofilin. Mol. Biol. Cell. 24, 31-41.
・ Mattila p. K. et al.(2004) A high affinity interaction with ADP-actin monomers underies themechanisms and in vivo function of Srv2/ cyclase-associated protein. Mol. Biol.Cell 15, 51585171.
・ Ono,S.(2013). The role of cyclase-associated protein in regulating actin filaments dynamics-more- 119 -

than a monomer-sequestraction factor. J. Cell Sci. 126, 3249-3258
・ Toshima JY. et al. (2006) Spatial dynamics of receptor-mediated endocytic trafficking in budding
yeast revealed by using fluorescent alpha-factor derivatives. Proc Natl. Acad.Sci. USA 103, 57935798

一般の皆様へ体を構成する最小単位である「細胞」の表面には「受容体」という装置があり、外界の情報を細胞の中に伝える働きをしています。私が研究の対象とした G タンパク質共役型受容体（GPCR）は、受容体の中でも最も多く、様々な生理作用を調節します。そのため、GPCR を標的とした薬品は全体の 50% 以上もあります。私はこれまで、GPCR が細胞表面から細胞内へと取込まれることで（エンドサイトーシス）、そのシグナルを不活性化する機構について、モデル生物である酵母細胞を用いて調べてきました。本研究で、私は GPCR のエンドサイトーシスに関わる遺伝子を網羅的にスクリーニン
グし、これらの遺伝子が担う役割について明らかにしました。最終的には、GPCR の不活性化を人為的にコントロールする技術を開発し、創薬や治療に生かしたいと考えています。

- 120 -

Detection and analysis of cell chirality in primary cultured cells.
Takeshi Sasamura

Osaka University
sasamura@bio.sci.osaka-u.ac.jp

Abstract
Cell chirality is a recently identified cell polarity that mirror image does not superpose on originalone. Our laboratory found cell chirality has important roles for directional Drosophila hindgut
rotation. However, due to the difficulty of live imaging detection of cell chirality in hindgut cells,
I tried to find a novel cell chirality in Drosophila primary-cultured cells. I checked several type ofcells, and found a novel chirality in Drosophila macrophage. In this cell, centrosome preferentiallymoves to right-forward/left-backward direction. This chirality is the first example that is detected inprimary-cultured cells.
Key words : Cell chirality, centrosome movement, Drosophila

Introduction
During development or homeostasis, cell polarity plays important roles. It is well known deficiency
of cell polarity causes various human diseases. Our laboratory had been identified cell chirality invivo in Drosophila hindgut cell, which is important for directional movement of the hindgut1. Cellchirality is 3D properties, such as cell shape, that mirror image dose not overlap to original one.
Because cell chirality detection by live imaging is very difficult in Drosophila hindgut cell, we tried
to looking for a novel cell chirality in Drosophila primary cultured cells.

Results
First, I was looking for a novel cell chirality using primary cultured cell that was established from
Drosophila embryo. I overexpressed various type of cell markers, such as MyosinII, or F-actin
in only hindgut cells, where cell chirality had been observed. However, due to the difficulties tocomplete one cell separation (usually I could get only cell clumps composed of 5~20 cells), we could
not be looking for cell chirality, effectively. Next we
examined various type of cells, such as epithelial cellor mesoderm. The results were similar to that using
hindgut cells. So, I tried to use larval hemocytes calledplasmatocytes. Drosophila plasmatcytes are involvedin various function such as immunity, phagocytosis,
ECM secretion. Because plasmatocytes primal cell
Figure 1 Quantification methods detecting
centrosome movementthat phagocytose apoptotic cells, plasmatocyte is often
called Drosophila “macrophage”. I also use this term

- 121 -

in this report.
It is reported that mammalian cultured cell, neutrophillike dHL60 has a chirality of centrosome movement2,
so I checked whether Drosophila macrophage has
similar chirality. I expressed centrosome marker, cnnGFP, and nuclear marker RedStinger in Drosophila
macrophage, by using UAS-GAL4 system. I usedhemocyte specific GAL4 line, He-GAL4, to express
these markers. Drosophila macrophages were primarycultured (ex vivo experiment) by bleeding of 3rd instar
larvae, and I took time laps movies 60 minutes by 2
minutes’ interval. Then I determined both centrosome
and nucleus movement direction compared to t=0

Figure 2 Macrophage chirality(Figure 1). I set arrow center of the nucleus to centrosome, and rotate the picture as the arrow direct
to the top (Figure 1). Then I checked centrosome position after 5, 10, and 20 minutes. I found thatthe nucleus moved to centrosome position. This is the same movement as mammalian cultured cell
(Figure 2). Then I checked centrosome movement. The movement is different from dHL60 cell;more centrosomes moved right-forward/left-backward (RfLb) direction compared to left-forward or
right-backward (LfRb) direction (Figure 2).
Next, I checked whether the movement of centrosome is significant or not. I examined 77 (5 min),75 (10 min), and 60 (20 min) centrosome movement and calculated the movement. The number ofcentrosome moved to RfLb/LfRb is 27/29 (5 min), 35/22 (10 min), 28/19 (20 min). By statisticalanalysis (t-test), significantly higher centrosome moved RfLb compared to LfRb after 10 minutes.
Therefore, I concluded that centrosome movement in Drosophila macrophage is a novel cellchirality.
To dissect why centrosome moves to RfLb direction, I examined two types of movement analysis.
Firstly, I tried to determined which time point the centrosome movement shows the clearest
chirality. I checked centrosome movement of shorter time period. I determined the movement ofcentrosome every 1 minutes until 10 minutes. However, I could not find the best time point. Next, I
checked longer period, every 5 minutes until 60 minutes. Again, I could not find good time point toinvestigate centrosome movement chirality. Second analysis is tracking of centrosome movement at
each 1 minutes, and classified into several categories, such as clock-wise turn or straight movement.
However, I could not find any tendency the centrosome moves. I also superimpose all centrosome
movement trajectory and check movement tendency. The movement are biased RfLb region, but I
could not found further information.

- 122 -

Discussion & Conclusion
I was looking for a novel chirality that can be easily detect. Finally, I could find a novel chiralitythat centrosome preferentially moves right-forward/left-backward direction in Drosophila
macrophage ex vivo. Several groups reported cell chirality in cultured cell, but this is the firstexample showing cell chirality in primary cultured cell. By this finding, I can move to next step;investigation of cell chirality generation mechanisms. Now I can use a plenty of Drosophila cellbiological knowledge and sophisticated genetic methods to dissect them. As a next step, I am
planning RNAi screening. Hairpin RNA of each of candidate genes are expressed in Drosophila
macrophage by GAL4-UAS system, and I examine their centrosome chirality. By this experiment, I
expect I can obtain whole gene sets regulating cell chirality.

References
1. Taniguch et al., (2011) Science 333: 339-341
2. Xu et al., (2007) Proc Natl Acad Sci USA 104:9296-9300

一般の皆様へ
我々人間の内臓など、多くの生物は左右非対称な器官を持ち、脊椎動物ではその形成に Nodal シ
グナルが重要です。しかし、これとは別に個々の細胞が立体的な左右非対称性、すなわちキラリティを持つことで左右非対称な器官が形成されるしくみが近年報告されています。私は、ショウジョウバ
エのマクロファージにおいて、新規の細胞キラリティを見出しました。今後、この細胞キラリティの制御機構を研究することにより、多くの生物に共通な新規の左右非対称性形成機構を見出すことができるのではないかと考えています。

- 123 -Dissection of neural computation in single cell networks
Fumitaka Osakada

Nagoya University
fosakada@ps.nagoya-u.ac.jp

Abstract
The goal of our proposal is to understand how neural circuits process and represent neural
information. To dissect neural computation at the level of resolution of single cells in the mousecortex, we have developed a rabies virus tracing system that makes it possible to label single cell
networks and characterize the connectionally-defined networks with in vivo 2-photon imaging.
Key words : virus, neural circuits, vision, single cell, neural computation

Introduction
Understanding how neural circuits process information requires resolving connectivity with highresolution, correlating connectivity with function, and manipulating activity of defined circuitcomponents. Recent advances in the development of molecular, genetic and viral based tools are
now making this possible at the level of resolution of single neurons.
Rabies viruses infect neurons through axon terminals and spread trans-synaptically in a retrograde
direction in the nervous system. Rabies viruses whose glycoprotein (G) gene is deleted from thegenome cannot spread across synapses. Trans-complementation of G, however, allows for labeling
directly-connected, presynaptic neurons. EnvA/TVA allows single-cell-specific targeting ofrabies viruses. It is also possible to combine rabies labelling of connectionally-defined neuronal
populations with studies monitoring or manipulating their activity. Combining the rabies virus
systems with in vivo imaging and optogenetics/pharmacogenetics will facilitate experiments
investigating neural computation of single cell networks.

Results
In the present study, we have developed a single cell tracing system using G-deleted rabies virusesin conjunction with a 2-photon microscope. To visualize and characterize single cell networks inthe visual cortex of mice, we need to introduce rabies virus glycoproteins and TVA, a receptor of
EnvA, an avian virus glycoprotein that is used for pseudotyping of G-deleted rabies viruses, into apyramidal neuron in layer 2/3 of the mouse visual cortex. For that purpose, we first set up a single
cell electroporation system under a 2-photon microscope. Mice were anesthetized with isoflurane,
and a metal frame was attached to the skull and a craniotomy was made over the V1. We introduced
three plasmids expressing rabies virus glycoprotein, TVA, and GFP through a glass pipette onto
pyramidal neurons in the layer 2/3 of the mouse cortex. Three days later, to label presynaptic cellsof the electroporated cell, we applied EnvA-pseudotyped G-deleted rabies virus around the GFP-

- 124 -

expressing neuron. We observed presynaptic neurons that were secondarily infected with G-deleted
rabies viruses around the electroporated cell in the visual cortex. However, the number of labelledcells were small. Thus, to improve the trans-synaptic labeling efficiency and its reproducibility of
the rabies virus tracing, we designed several chimeric glycoproteins for trans-complementation.
The new chimeric glycoprotein increased the tracing efficiency for long-distance input neurons
up to 20-fold compared to the original glycoprotein. In addition, to relate connectivity to circuit
function, we set up a 2-photon calcium imaging system in conjunction with visual stimulus system.
A visual stimulus consisting of drifting square-wave gratings at various orientations was presented
on a computer screen, with 5 presentations of each grating direction in random order. We also madea rabies virus genomic vector that expresses R-CaMP2, a genetically encoded calcium indicator.
After electroporation of DNAs into a single neurons in the layer 2/3 of the mouse cortex and thesubsequent infection with EnvA-pseudotyped rabies viruses, visual responses of head-fixed, virusinfected mice were characterized under the 2-photon microscope. Labelled presynaptic cells were
distributed across layers in the visual cortex. From morphological and histological analysis of
the infected mouse brains, the labelled neurons contained both excitatory neurons and inhibitory
neurons. Receptive fields of the postsynaptic cell and the presynaptic cells were analyzed. Someof the labelled neurons showed clear orientation selectivity. Taken together with previous reports,
our results suggest that excitatory neurons in layers 2/3, 4, 5 and 6 that connect to the post-synaptic
neuron in the layer 2/3 showed orientation selectivity while inhibitory neurons did not. We are
currently analyzing data of 2-photon calcium imaging on single cell networks that were labeled with
G-deleted rabies viruses in a trans-synaptic manner.

Discussion & Conclusion
Neural circuits in the brain operate over a vast range of spatiotemporal and computational scales,from high-level circuits that integrate information across multiple brain regions, to microcircuitsthat perform simple input/output transformations within a specialized brain structure. Pyramidal
neuron output is shaped by thousands of excitatory and inhibitory inputs. How orientation-tuned
neurons connect to other orientation-tuned neurons remains controversial. Several lines of evidence- 125 -

suggests that individual neurons sample heterogeneous input, from neurons with diverse orientationtuning. Models indicate that specific connectivity is not required for orientation tuning. However,other measurements emphasize that neurons prefer to make synapses with neurons sharing similar
orientation tuning, but orientation tuning at the level of subthreshold membrane potential is weak.
The rabies virus tracing of single cell networks that we have developed here in the present study
will contribute to understanding how neural computation generates a specific output from a large
number of inputs.

References
1. Osakada F, Callaway EM. Design and generation of recombinant rabies virus vectors. Nat
Protoc. 8:1583-601. (2013)
2. Osakada F, Mori T, Cetin AH, Marshel JH, Virgen B, Callaway EM. New rabies virus variants
for monitoring and manipulating activity and gene expression in defined neural circuits. Neuron.
71:617-31. (2011)
3. Marshel JH, Mori T, Nielsen KJ, Callaway EM. Targeting single neuronal networks for gene
expression and cell labeling in vivo. Neuron. 67:562-74. (2010)

一般の皆様へ
脳はおよそ 1,000 億個もの膨大な数のニューロンが神経回路を形成することにより情報を伝達・処理し、複雑な高次脳機能を発揮する。その神経回路の破綻は、神経・精神疾患などを引き起こす原因と考えられる。したがって、脳・神経回路の動作原理の解明、それに基づいた神経・精神疾患の病因解明および予防・治療法の開発は極めて重要な研究課題である。近年神経科学領域では、
二光子顕微鏡、光遺伝学、カルシウムや電位に感受性の蛍光タンパク質、ウイルスベクター、脳の透明化、ゲノム編集技術など、大きな技術革新があり、不可能と考えられていた実験アプローチが可能になり、これまで解明できなかった科学的問いに答えることができるようになりつつある。今回開発した狂犬病ウイルスのシステムも神経回路研究に広く使用され、将来的には難治性疾患への治療開発へとつながると期待される。

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Studies on the underlying mechanisms for the integration of corticalinformation
Makoto Sato

Osaka University
makosato@anat2.med.osaka-u.ac.jp

Abstract
The functions of long-range direct neuronal connections between distant functional areas in
cerebral cortex are yet to be clarified. We have started to establish the system to visualize andmanipulate these neural circuits using specific gene promoters. In utero electroporation with oneof them, we successfully visualized the sensorimotor association connection and are now doing
developmental analysis of this connection. We are also in the process of making Cre-driver mouselines for two promoters by genome editing with CRISPR/Cas9 system. We will utilize them for
optogenetic manipulation of activity in this connection.
Key words : Long association fiber, in utero electroporation, tissue clearing, CRISPR/Cas9, mousemodel

Introduction
Cerebral cortex is composed of several dozens of functional areas. Direct connections between
distant areas are believed to be important for higher cortical functions but this is still to beclarified. Because these connections can be found in multiple cortical layers, we hypothesized thatconnections in different layers convey different aspects of information processed in particularfunctional areas. To visualize and manipulate these connections, we screened for genes specific tosuch inter-areal connections by DNA microarray analysis and shortlisted by further screening down
to 5 genes. In addition, we found one of the layer markers was expressed in this connection.

Results
To visualize and manipulate inter-areal connections (long association fibers), we aimed at
identifying genes specific to long association neurons (LANs). We compared gene expression
profiles between LANs and inter-hemispheric callosal neurons, intermingling in mouse primarysomatosensory area (S1), by DNA microarray analysis. For those preferentially expressed in LANs,
we examined their expression in LANs by combining retrograde tracing from LANs’ axonal
target (primary motor area, M1) and in situ hybridization with probes for these candidate genes.
Eventually, we identified 5 double-positive genes in this analysis: 3 for layer 5, and 2 for layer 6b. To
drive reporter genes in LANs using the promoter of these genes, we constructed the plasmid vectors
harboring the EGFP or Cre sequence at the translation initiation site of each gene by recombining
the BAC clones that cover the respective gene locus. The retrieved plasmid vectors (together with- 127 -

Cre-dependent reporter in the case of the Cre constructs) were transfected into developing corticalneurons by means of in utero electroporation (IUE) at embryonic day 15 (E15), and the expressionof EGFP reporter in neurons and axons was verified at postnatal day 21 (P21).
In a complementary approach, we examined whether any of known layer-specific genes was
expressed in LANs with the double labeling experiment described above. Interestingly, one of the
known layer markers was expressed in both layers 2/3 and 5, and in both LANs and callosal neurons.
We established the EGFP expression system using the promoter of this layer marker with Cre/loxP
system in an above-mentioned way. To image the axonal structure in intact tissues, we established
the brain tissue clearing procedures with SeeDB (Ke et al, 2013 Nat Neurosci) and SeeDB2 (Keet al, 2016 Cell Rep) methods to facilitate imaging the entire structure of the connection. By twophoton imaging of cleared brain samples, we found that layer 2/3 neurons in S1 which express
this layer marker actually project their axons to both M1 and contralateral S1 (cS1). While callosal
projection to cS1 developed from embryonic stages, the projection to M1 was collateral branches
emerging later in the postnatal development from the main shaft of axon that projects to cS1. The
advantage of using this promoter is its expression in dual layers, which allows us to introduce thevectors into layer 2/3 or layer 5 just by performing IUE at E15 or E13, respectively, with the same
plasmid vector.
As layer 6b is much harder to target by IUE, we made two mouse lines which express Cre under
the control of the two promoters for layer 6b by genome editing with CRISPR/Cas9 system. These
results enable us to study structure and physiological functions of LAFs from this layer.

Discussion & Conclusion
In addition to the promoters mentioned above, we have further candidates to be tested, especiallyfor layer 2/3. Also, we have just started retrograde tracing experiments from orbitofrontal cortex tolabel neurons in visual cortex as another example of LAFs. The layer marker promoter which weestablished labels relatively large population of neurons in both layers 2/3 and 5. However, layerspecific promoters which we identified through DNA microarray screening seem to label more
restricted subpopulations and must be useful to analyze such small populations possibly dedicated tomore specific functions. Cre-driver mouse lines we made for layer 6b and those we will also makefor layers 2/3 and 5 (when crossed with appropriate effector mice) can be used as animal models
for disorders with LAF abnormalities such as autism spectrum disorders and schizophrenia. Our
work should shed light on to understanding differential information processing mechanisms among
cortical layers and among different LAFs.

References
Ke MT, Fujimoto S, Imai T. SeeDB: a simple and morphology-preserving optical clearing agent forneuronal circuit reconstruction. Nat Neurosci. 2013 Aug;16(8) :1154-61.

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Ke MT, Nakai Y, Fujimoto S, Takayama R, Yoshida S, Kitajima TS, Sato M, Imai T. SuperResolution Mapping of Neuronal Circuitry With an Index-Optimized Clearing Agent. Cell Rep.
2016 Mar 22;14(11):2718-32.
Yuichiro Oka, Tokuichi Iguchi, Makoto Sato: Development of axon collaterals as the interareal connections in the cortical neurons. (The 58th annual meeting of the Japanese Society for
Neurochemistry). 2015.
Yuichiro Oka, Tokuichi Iguchi, Makoto Sato: A new method to amplify the reporter expression in
the Cre/loxP system applicable to the study on cortical development.
21st Biennial Meeting of the International Society for Developmental Neuroscience (Palais desCongres d’Antibes Juan les Pins, France). 2016

一般の皆様へ大脳皮質の異なる頭葉間を結ぶ神経回路（長連合線維）は外界からの情報を統合して価値判断・意思決定を行う過程において重要であると考えられていますが、具体的にどのような役割を果たして
いるかはまだ解明されていません。我々は、マウスをモデルに長連合線維の神経活動を任意にコントロールできるマウスを作製して、その機能を明らかにしようとしています。

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Targeting the muscle-liver-fat signaling axis essential for central
control of adaptive adipose remodeling for treatment of metabolic
syndrome
Hirotoshi Tanaka

Division of Rheumatology, Center for Antibody and
Vaccine, IMSUT Hospital, Institute of Medical Science, University of Tokyo
Hirotnk@ims.u-tokyo.ac.jp

Abstract
A skeletal muscle-liver-fat signalling axis may serve as a target for the development of therapies
against various metabolic diseases, including obesity. Depletion of plasma alanine serves as a cue toincrease plasma levels of fibroblast growth factor 21 (FGF21) and activates liver-fat communication,
leading to the activation of lipolytic genes in adipose tissues. Given this, we developed a novel
screening system to identify FGF21 mimetics.
Key words : skeletal muscle, adipose tissue, obesity, diabetes, FGF21

Introduction
Glucocorticoid receptor (GR) transcriptionally controls expression levels of components in theboth machineries as well as those of key transcription factors for catabolism, FoxOs and KLF15.
We showed that skeletal muscle-specific GR knockout (GRmKO) mice have increased muscle mass
but smaller adipose tissues, accompanied with a drastic shift of gene expression in the muscle, liver
and adipose tissues. The resulting depletion of plasma alanine serves as a cue to increase plasma
levels of fibroblast growth factor 21 (FGF21) and activates liver-fat communication, leading to theactivation of lipolytic genes in adipose tissues. We, therefore, proposed that this skeletal muscleliver-fat signaling axis controls organismal energy distribution and may serve as a target for thedevelopment of therapies against various metabolic diseases, including obesity.

Results
In order to test the effect of alanine on hepatic FGF21 synthesis, the hepatocytes were subjected
to alanine deprivation. In RT-PCR, FGF21 mRNA expression was was shown to be induced. In
the hepatocytes, ATF4 protein level and phosphorylation of eIF2a were increased after alanine
deprivation, as seen after treatment with tunicamycin. The expression and phosphorylation of
AMP-activated protein kinase (AMPK) and S6 kinase (S6K) 1 was not affected in these conditions.
Chromatin immunoprecipitation (ChIP) assays revealed that alanine deprivation enhanced therecruitment of ATF4 and RNA polymerase (RNAP) II but not that of PPARa onto FGF21 genepromoter. Together, decreased alanine supply, serving as a signal from muscle to liver, might
accentuate FGF21 gene transcription via activating ATF4. Subsequently, increased plasma FGF21- 130 -

may act on adipose tissues and liver and stimulate fat usage. To test this scenario, we orally
administered alanine to mice, and showed that hepatic mRNA expression and plasma concentrationsof FGF21 were suppressed exclusively in GRmKO mice after 24 h fasting. Administration of analanine analogue 3-chloro-L-alanine increased plasma alanine concentrations in GRf/f mice, mostpossibly reflecting the inhibition of alanine uptake and/or utilization by the liver. As expected,
hepatic mRNA expression of FGF21 after 24 h fasting was increased in a dose-dependent manner,
showing a clear contrast to alanine administration. It, therefore, is strongly indicated that plasma
alanine depletion and/or inhibition of alanine utilization is a key signal for modulating liver fasting
response represented by increase in FGF21 expression. Finally, we addressed whether decreased
alanine supply to the liver is a cue for systemic alteration in energy metabolism and decrease in fat
mass. For that purpose, we fed those mice with normal chow diet (NCD), high-alanine diet (HAlaD)
and high-fat diet (HFD) for 3 weeks. HAlaD-fed GRmKO mice showed slight decrease in foodconsumption, but increase in plasma alanine compared with NCD- or HFD-fed GRmKO mice.
Notably, plasma FGF21, free fatty acids and 3-hydroxybutyric acid were declined exclusively in
HAlaD-fed GRmKO mice after 24 h fasting. Decreased FGF21 mRNA expression after HAlaD was
not observed in white fat but in the liver. mRNA expression of cpt1a and 1b in the liver and ATGLand HSL in white fat showed identical tendency with liver FGF21 mRNA expression. Moreover,
HAlaD restored fat mass in GRmKO as seen in HFD feeding. However, accumulation of triglyceridein the liver and gastrocnemius muscle caused by HFD-feeding were partially ameliorated in
GRmKO, suggesting accelerated lipid consumption in GRmKO even in HFD feeding. From theseresults, we may conclude that shutdown of muscle-derived alanine supply to the liver is a trigger forhepatic FGF21 induction and lipolysis in adipose tissues, and that restoration of alanine replenishes
lipid deposition in GRmKO mice. Intraperitoneal glucose tolerance test revealed rather decreased
glucose tolerance in GRmKO under HAlaD feeding, indicating that adipogenic effect of HAlaD is
not caused byincreased glucose tolerance. We, then, created the screening system to identify such
substances that influence on hepatic FGF21 gene expression. For that purpose, we used mouse invivo imaging system after infection of the adenovirus conveying FGF21 promoter. We showedthat alanine deoletion or administration of 3-Cl alanine stimulated the hepatic luminescence andconcluded that our screening system worked for identification of substances affecting alanineFGF21 promoter pathway.

Discussion & Conclusion
Our study clearly supports the notion that glucocorticoid-dependent alanine flux from skeletal
muscle modulates hepatic metabolic profile and FGF21 production, and licenses the adipose tissue
to store energy as triglyceride; plasma levels of alanine, once reaching a threshold, may decrease
hepatic FGF21 production and drastically alter metabolic profile of adipose tissues to ‘thrifty’
phenotype. Of interest, hypoalaninemia is seen in pregnancy, in which the shift of energy supply
from muscle to fat might be beneficial for maternal activity and intrauterine child growth. Although- 131 -

precise mechanism remains unknown, epidemiological studies documented that plasma levels of
ALT activity is closely related to future risk of obesity and diabetes. Targeting the skeletal muscleliver-fat signaling axis involving glucose-alanine cycle or hepatic FGF21 gene expression, therefore,
would be a novel approach for treatment of patients with obesity, diabetes and metabolic syndrome.

References
１． Shimizu N, Yoshikawa N, Ito N, Maruyama T, Suzuki Y, Takeda S, Nakae J, Tagata Y, Nishitani
S, Takehana K, Sano M, Fukuda K, Suematsu M, Morimoto C, Tanaka H. Crosstalk between
glucocorticoid receptor and nutritional sensor mTOR in skeletal muscle. Cell Metabolism 2011: 13,
170-182.
2. Shimizu N, Maruyama T, Yoshikawa N, Matsumiya R, Ma Y, Ito N, Tasaka Y, Kuribara-Souta A,
Miyata K, Oike Y, Berger S, Schütz G, Takeda S, Tanaka H.
A Muscle-liver-fat signaling axis is essential for central control of adaptive adipose remodeling. Nat
Commun. 2015;6:6693. doi: 10.1038/ncomms7693

一般の皆様へヒトなどの哺乳動物は、栄養を効率よく、脂質、タンパク質、および糖質として体内に貯蔵します。
主に、脂質は脂肪組織、タンパク質は骨格筋と、別々の臓器に貯蔵され、過剰なカロリーを脂肪として蓄積しやすくなっています。食べ過ぎると骨格筋は増えずに脂肪が増えて肥満につながり、糖尿病、
生活習慣病などの罹患リスクを上昇させます。この過剰なエネルギーが脂肪に貯まりやすい仕組み、
言い換えれば、骨格筋に貯まりにくい仕組み、はいまだにわかっていません。わたしたちは、「骨格
筋と脂肪が肝臓を介して情報のやりとりをし、体内のエネルギーの流れや貯蔵場所と量を制御している」、ことを発見し、肥満や生活習慣病などの予防法や治療法の開発に応用しています。

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Total synthesis of ustiloxin D via the aryl ether formation of tertalcohol
Itaru SATO

Ibaraki University
itaru.sato.sci@vc.ibaraki.ac.jp

Abstract
Synthesis of potent antimitotic agents ustiloxin D, which possesses 13- membered cyclic peptides witha unique chiral tertiary alkyl-aryl ether linkage, are studied. The aryl ether has be constructed via ourdeveloped arylation with triaryl bismuth in the presence of peptide and the total synthesis of ustiloxin D
has accomplished.

Key words : natural product synthesis, aryl ether formation of tertiary alcohol

Introduction
Ustiloxins, isolated as potent antimitotic agents that inhibit microtubule assembly, are 13membered cyclic peptides with a unique chiral tertiary alkyl-aryl ether linkage.1 Groups ofJoullie and Wandless have independently accomplished total synthesis of ustiloxin D.2 However,constructions of the tertiary alkyl-aryl ether linkage in the presence of peptide chains are stillchallenge.

Results
Our synthetic approach includes direct transformation of tert-alcohol to tertiary alkyl-aryl ether.
Up to date, classical Ullmann type arylation should be thefirst choice. Recent progress in palladium chemistry showsthat arylation could be conducted at relatively mild conditions(70~80 degree). However, the high temperature and use ofstrong bases inhibits arylation of the peptides substrates such as
ustiloxins. On the other hand, Barton and Mukaiyama developed
phenylation of alcohols with five valent bismuth with phenyl
funcutionality.3 The reaction proceeds at room temperature. We confirmed arylation of tert-alcohols
with arylbismuth(V), in situ oxidized from aryl bismuth(III), undergoes smoothly at rt.4
First, we studied on the modification of the procedure of aryl ether formation. Under the modified
conditions, the trivalent aryl bismuth itself could used for arylation (eq1). In the presence of
stoichiometric amount of cupper acetate, tertiary amine, and molecular sieves, tertiary alcohols arearylated with 0.5 mol equivalent of triaryl bismuth under oxygen atmosphere (1 atom). Although- 133 -

oxygen atmosphere is required, careful TLC analysis suggested that the oxidized five valent bismuth
species is not formed. We consider the mechanism is as follows: (1) Transmetallation from aryl
bismuth (III) to cupper acetate gives aryl cupper spices; (2) Ligand exchange reaction forms aryl
cupper alkoxide; (3) then the central cupper species oxidized to Cu(III) and reductive elimination ofalkyl aryl ether gives product and Cu(I).

Next, we conducted arylation of tert-alcohols with peptide chains. Alcohols with mono-, di andtripeptide undergoes arylation in moderate to good yield. During examinations, no epimerization ofamino acid could be observed. Thus, we developed modified arylation procedure.
The aryl ether with tripeptide was used as suitable substrate toward the total synthesis. After theoxy-functionalization of aryl group, TES ether was cleaved and transformed into triflate. Chiralbeta-hydroxy amino acid structure was constructed with Heck reaction and followed by Sharplessamino hydroxylation. Standard condensation conditions allows the formation of macrocycle.
N-Monomethylation and grobal deprotection furnished total synthesis of ustiloxin D.

Discussion & Conclusion
First of all, we demonstrated convenient aryl ether formation with triarylbismuth forms aryl etherof tert alcohol under especially mild conditions. No epimerization and degradation of peptides wereobserved. Moreover, we accomplished the total synthesis of ustiloxin D. Our synthetic route hassome advantages from previous reports; (1) tert-alcohol is used directly for aryl ether formation.
Thus, no structure modification of aryl/aryloxy acceptors is required; (2) The peptide could standfor ary ether forming step. These characteristics allows formation of aryl ether in the late stage ofsynthesis. As important bioactive natural products classes, macrocyclic peptides with aryl ether
linkages are known, such as vancomicin families. We believe that synthetic strategy includes
linear peptide synthesis and successive macrocylization at aryl ether provides simple and efficientsynthetic route and enough substrates for SAR studies.

References
1. (a) Koiso, Y.; Natori, S.; Iwasaki, S.; Sato, S.; Sonoda, R.; Fujita, Y.; Yaegashi, H.; Sato, Z.
Tetrahedron Lett. 1992, 33, 4157. (b) Koiso, Y.; Li, Y.; Iwasaki, S. J. Antibiot. 1994, 47, 765. (c)Koiso, Y.; Morisaki, N.; Yamashita, Y.; Mitsui, Y.; Shirai, R.; Hashimoto, Y.; Iwasaki, S. J. Antibiot.
1998, 51, 418.

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2. (a) Wandless, T. J; Sawayama, A. M; Tanaka, H. J. Am. Chem. Soc, 2003, 125, 6864. (b) Li, P.;
Evans, C. D.; Wu, Y.; Cao, B.; Hamel, E.; Joullie,́ M. M. J. Am. Chem. Soc. 2008, 130, 2351. (c)Brown, A. L.; Churches, Q. I.; Hutton, C. A. J. Org. Chem. 2015, 80, 9831.
3. (a) Barton, D. H. R.; Finet, J.-P.; Khamsi, J.; Pichon, C. Tetrahedron Lett. 1986, 27, 3619. (b)Ikegai, K.; Fukumoto, K.; Mukaiyama, T. Chem. Lett. 2006, 35, 612.
4. Harada, S.; Hayashi, D.; Sato, I.; Hirama, M. Synlett, 2012, 23, 405-408.

一般の皆様へ
ウスチロキシン類は稲コウジカビより単離された細胞内チューブリンの集合阻害活性を示す化合物群である。その構造は環状ペプチドであり，光学活性な第３級アルコールとフェノール間にエーテル結合を持つ。誘導体のライブラリの構築も志向してその合成を行おうとした場合，ペプチド鎖のエピ化などの副反応を起こさない極めて穏やかな条件でのエーテル構築が必須になる。我々は開発したトリアリールビスマスをアリール源とする３級アルコールのアリールエーテル化によりペプチの異性化を伴わないエーテル形成に成功した。またさらに合成を進めウスチロキシン D の全合成を達成した。

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III.
Reports from the Recipients of Grants
for International Meetings- 136 -

Report on Research Meeting
1.Date of making report
July 25, 2015.
2. Name of Research Meeting / Conference
The 12th International Workshop on Microbeam Probes of Cellular Radiation Response(IWM2015)
3. Representative
Hideki Matsumoto, Ph.D.
Associate Professor, Biomedical Imaging Research Center, University of Fukui
4. Opening period and Place
30th May ~ 1st June, 2015.
The Wakasa Wan Energy Research Center (WERC)
64-52-1 Nagatani, Tsuruga, Fukui, Japan
5. Number of participants / Number of participating countries and areas
154 participants from 13 countries (Japan, USA, UK, Germany, France, Australia, Italy, Russia,
Canada, Singapore, Poland, China, Thailand)
6. Total cost
￥6,273,024JPY
7. Main use of subsidy
Venue costs, Printing costs, and Financial support for students and postdoctoral fellows
8. Result and Impression
The International Workshop on Microbeam Probes of Cellular Radiation Response (IWM)
has been held regularly once every 2 years since 1993. In the past, these workshops have been
fruitfully gone up in bringing together groups interested in developing and applying nano- tomicro-irradiation techniques to study radiation-induced damage in cells, tissues and organisms
and to treat cancers of patients.
The IWM2015 was one of the satellite meetings of the 15th International Congress of Radiation
Research (May 25~29, 2015, Kyoto, Japan), which had over 2,000 participants from all over
the world. Therefore, the total number of participants of the IWM2015 was also more than the- 137 -

twice that of the last workshop, and was 150 or more persons who were coming from USA,France, China, UK, Germany, Australia, Russia, Canada, Singapore, Italy, Poland and Thailand
as well as Japan. We had 1 special lecture, 2 luncheon seminars, 21 oral presentations, 36
poster presentations with 3 min poster introduction presentation, and 8 late-breaking poster
presentations without poster introduction.
In the IWM2015, many recent results of the studies about the development of microbeam
technology, the mechanisms involved in non-targeted effects (NTE), biological effect ofcytoplasmic irradiation, DNA damage responses (DDRs) and the related signal transductions, andmicrobeam radiotherapy (MRT) were reported. In the development of microbeam technology, thedevelopment of alpha-particle microbeam irradiation system equipping the stimulated emission
depletion (STED) microscope to irradiate targeted organelles, such as mitochondria, werereported. In addition, the focusing heavy-ion microbeam irradiation system, which can irradiatetargeted cells at high speed by microbeam scanning was reported. These technologies will beapplied to microbeam radiotherapy in the near future. In the biological studies, the differenceof DDRs by the chromatin structure, the mechanisms of radiation-induced bystander responses
studied using X-rays and particle microbeams, and the system biological studies of thesebiological responses were reported. In addition, an application of microbeam irradiation to cancer
treatment was also reported. In all sessions, there were heated arguments.
In Japan, various types of microbeam irradiation systems (X-rays, synchrotron radiations,
protons, and heavy ions) are available to the researchers as joined or collaborative studies;
therefore we could assure that the microbeam research institutes in Japan are developing as acenter of the world in the field of microbeam sciences.
9. Additional description
The IWM2015 was broadcast on the news programs of the NHK Fukui, May 31, 2015.
Short report of the IMW2015 will be published in special issue of the Radiation Biology
Research Communications. IWM2017 will be held in Manchester, UK, in 2017.

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Report on Research Meeting
1. Name of Research Meeting / Conference
Oocyte maturation and fertilization meeting IV
2. Representative
Kazuyoshi Chiba
3. Opening period and Place
June 15-18, 2015
4. Number of participants / Number of participating countries and areas
45 participants / 6 countries
5. Total cost
1,930,000 Yen ($18000)
6. Main use of subsidy
Part-time job bill
7. Result and Impression“Oocyte maturation and fertilization meeting IV” was held at the Asamushi Research Center
for Marine Biology, Tohoku University, located on the inner part of Mutsu Bay, the northernmost
bay on Honshu-island of Japan, in June 15-18, 2015. It was organized by Kazuyoshi Chiba(Ochanomizu University), Ryusaku Deguchi (Miyagi University of Education), Noriyo Takeda(Tohoku University), Keiichiro Kyozuka (Tohoku University) and Takeo Kishimoto (Ochanomizu
University). Participants from UK, Canada, USA, Italy, Germany, and Japan were leaders ofoocyte maturation and fertilization communities and younger researchers. We could compare ourstudies on eggs and sperm in a wide range of animal models such as cnidarians (jellyfish), insects(Drosophila), echinoderms (starfish and sea urchin), urochordates (ascidian) Crustacea (Daphnia)
and vertebrates (Xenopus and mouse). Twenty eight talks were spread between seven sessions
and provided the attendees with the opportunity to learn more about meiotic arrest of oocytes,
meiosis-inducing hormones, CDK1 activation, protein kinases/phosphatases, cortical contraction,
sperm-egg interaction, Ca2+ signaling at fertilization, chromosomes, and spindle formation/
positioning.
Mark Terasaki showed beautiful images of serial section electron microscopy of mouse ovarian- 139 -

antral follicles. Leia C. Shuhaibar and Laurinda A. Jaffe demonstrated that luteinizing hormone
signaling rapidly reduces cyclic GMP levels in mammalian ovarian follicles by dephosphorylationof NPR2 and phosphorylation of PDE5, leading to meiotic resumption. Evelyn Houlistonand Noriyo Takeda talked about hormones to induce meiotic reinitiation of oocytes of jelly
fish. Olivier Haccard and Marco Conti used frog and mouse oocytes to reveal the molecularmechanisms for meiotic G2/M-phase transition. Péter Lénárt talked about spatiotemporal control
of cortical contraction waves of starfish oocytes. Hiroki Nishida showed that the animal-vegetal
axis in ascidians is established during oocyte maturation. Gary M. Wessel showed determination
mechanisms of germ line during the oocyte to embryo transition. The keynote lecture by Stephen
A. Stricker was calcium signals and kinase activities during oocyte maturation and fertilizationin nemertean worms and other marine invertebrates. Using jellyfish oocytes, Ryusaku Deguchi
showed sperm attraction and fusion abilities are established during meiotic maturation.
Luigia Santella showed change of actin distribution at fertilization of starfish eggs. Johné Liutalked about spindle assembly checkpoint, aneuploidy and developmental potential of mouse
oocytes. Kazuyoshi Chiba showed change of ability of paternal asters to form meiotic spindle
during oocyte maturation in starfish. Alex McDougall talked about meiotic spindle orienting
mechanisms during ascidian early embryonic development. In this meeting, we could discussquestions about essential steps of the reproduction and we learned new findings. We reallyappreciate the financial aid by The NOVARTIS Foundation (Japan) for the Promotion of Science.
8. Additional description- 140 -

Report on Research Meeting
9th July 2015
1. Name of Research Meeting / Conference
24th Symposium on Environmental Chemistry
2. Representative
Mayumi Ishizuka
3. Opening period and Place
24–26 June 2015, Sapporo Convention Center
4. Number of participants / Number of participating countries and areas
548 participants / 16 countries
5. Total cost
10,647,112 yen
6. Main use of subsidy
Travel expenses for inviting the overseas researcher
7. Result and Impression
In this symposium, 548 domestic and abroad researchers were participated and 336 research
report about environmental chemistry and environmental toxicology were presented.
We held the International session according to a plan, and about 50 researchers from 16
countries were attended to the International session. In addition to oral presentation including
the International session, we also held poster session, free discussion meeting, special lectures,student award, banquet and so on.
In the free discussion meeting, totally 8 topics were discussed such as “Sharing the international
information about environmental chemistry”, “Newest knowledge for pharmaceutical andpersonal care products”, “What is the Integrated Exposure and Effects Analysis?”, “Future and aview for Environmental chemistry field”, and so on.
In the special lecture, we invited Prof. Yasuyuki Shibata from National Institute forEnvironmental Studies and Prof. Taisen Iguchi from National Institute for Basic Biology. Prof.
Shibata was lectured from the perspective of analytical chemistry, and Prof. Iguchi was lecture
about evaluating toxic effects.

- 141 -

53 students were applied for student award selection, and 12 students won the awards. Seven
international students were also entered for this award, and three participants got the prize. We
are expecting that this student award leads to improve the study for young researchers.
We also invited specialist of mercury researchers from Vietnam, Philippines, Thai, Indonesia,
USA as a special session for Mercury Monitoring Network.
We believed that it was very good opportunities to know new information about environmental
chemistry and ecotoxicology and the latest knowledge of the world trend through this symposium.
Moreover, we were able to building of new network through international exchanges in this time,
and we also believe to extend to the future's research activities.
Finally, it was adopted as foundation NOVARTIS Foundation for the Promotion of Science, andI'm thankful that a symposium has ended in the success.
8. Additional descriptionNone- 142 -

Report on Research Meeting
1. Name of Research Meeting / Conference
8th International Tunicate Meeting
2. Representative
Kazuo Inaba, Professor, Shimoda Marine Research Center, University of Tsukuba
3. Opening period and Place
July 13-17, 2015, Aomori City Cultural Center, Aomori City, Aomori Prefecture, Japan
4. Number of participants / Number of participating countries and areas
119 (Japan 60, France 20, USA 15, Australia 3, Canada 3, Italy 3, Brazil 2, Germany 2, Israel 2,
Norway 2, UK 2, China 1, India 1, Portugal 1, Singapore 1, Spain 1)
5. Total cost
5,528,500 JPY
6. Amount of subsidy
400,000 JPY
7. Main use of subsidy
Stationery items for participants, 154,423 JPY; Rents for microphone, projector, and poster
boards, 118, 360 JPY; Printing cost for meeting abstract, 88,300 JPY; Others 38,917 JPY
8. Result and Impression
The 8th International Tunicate Meeting held in Aomori in July 2015 provided 9 oral sessions,poster session and 2 round table discussion. Total number of participants was 119, including 59
coming from overseas (France, USA, Australia, Canada, Italy and others; 15 countries). In theplenary lecture, Professor Mike Levine, Princeton University, gave a talk on the evolutionary
origins of the vertebrate body plan. He mainly focused on the homeobox genes that he found andemphasized the significance of tunicate research to understand the origins of vertebrate organs.
He also provided recent research topics of gene regulation network in his laboratory. The oral
session was composed of talks on following 9 topics from molecular to ecology levels. Molecular
Ecology, Population Diversity and Invasive Species; Regeneration, Diseases and Self-non selfrecognition; Reproduction and Cell Biology, Physiology and Neurobiology; Developmental
and Evolutionary Genomics; Cell Fate Specification; Morphogenesis; Imaging Analysis;- 143 -

Metamorphosis; Evo-Devo, Epigenetics. The most significant progress in tunicate research,presented in this meeting was the mechanism of cell fate and development at cellular level.
Recent progress in imaging techniques made it possible to visualize the dynamics of molecules
in a cell. One of the advantages to use tunicates in modern biology is their simplicity in bodyplan, which would be a key to understand the evolution of chordates and vertebrates. Many of theresearch topics in the sessions “Reproduction and Cell Biology”, “Physiology and Neurobiology”,“Cell Fate Specification” and “Morphogenesis” provided updated knowledge in these fields
linked to intracellular localization of key molecules and cell polarity. Two round table discussion,“Community-wide efforts on Functional Genomics” and “Taxomony of Ciona sp.” were managed
by Lionel Christiaen (New York University, USA) and Lucia Manni (Padova University, Italy),respectively. In particular the latter round table had heated discussion among molecular biologists
and taxonomists on the taxonomy of Ciona species. The recent comparative analysis of thisspecies gave rise a controversial issue in the conventional system for classifying this group. The
discussion is still going on among the research community by e-mail, and is planned to deal
with again in the coming meeting in New York in 2017. Overall, it was impressive that many
young scientists participated, presented their works and had exciting discussion. This would be agreat tradition of this meeting, making the community of tunicate researchers more informativeand closer. Finally the organizing committee would like to express sincere gratitude to The
NOVARTIS Foundation for the Promotion of Science for supporting the success of this meeting.
9. Additional description- 144 -

Report on Research Meeting
1. Name of Research Meeting / Conference
GIW/InCoB 2015
GIW is short for Genome Informatics Workshop
InCoB is short for International Conference on Bioinformatics
This years joint conference was the 26th annual GIW and the 14th annual InCoB conference.
2. Representative
The conference chair was Paul Horton of AIST ( 産 総 研 , the Japanese National Institute forAdvanced Industrial Science and Technology), acting under the guidance of two academic
societies: the Japanese Society for Bioinformatics (JSBi), and the Asian Pacific Bioinformatics
Network (aPBioNet)
3. Opening period and Place
GIW/InCoB was held during the week of Sept 7-11, 2015. The main conference was held on Sept
9-11 at the Miraikan ( 日本科学未来館 ) National Museum of Emerging Science and Innovation.
4. Number of participants / Number of participating countries and areas
According to our advance registration system, we had 184 registrants from 18 countries covering
5 continents. Of these 80 were Japanese, the remaining 104 international registrants mostly camefrom Eastern Hemisphere locations such as Taiwan (35), Australia (12), Korea(9), China(9) andSingapore(9); but we also had ten registrants from Europe.
5. Total cost
The toal conference cost was ¥10,409,345. Our main cost was the conference operation, which
we outsourced to a company. The second biggest cost was the cost of the venue. Together thesecompromised just under 70% of the total budget
6. Main use of subsidy
We used the subsidy to support the conference as a whole. Perhaps the most visible benefit ofreceiving the subsidy was that we were able to give travel awards to four students presenting at
the conference.
7. Result and Impression
We were extremely happy with the number and quality of scientific presentations. The
conference program was anchored by six extremely strong keynotes: Arne Elofsson, Mark Baker,
Edward Marcotte, Yana Bromberg, Gil Ast and Annie de Groot; who collectively summarized- 145 -

the frontier of our bioinformatics from structural proteomics and alternative splicing to humandisease and personalized medicine. The keynotes were but the tip of the iceberg however, as thefour track program covered a total of 81 talks including 51 on original research first presented
to the world during the conference. This considerable body of work has now been published as
78 papers, many of them in GIW/InCoB 2015 supplements in BMC Genomics, BMC Medical
Genomics, BMC Systems Biology, BMC Bioinformatics; others in Bioinformatics, IEEE/ACM
Transactions on Computational Biology and Bioinformatics and the Journal of Bioinformatics
and Computational Biology.
8. Additional description
Miraikan was a nice venue and gave us a big discount as an academic conference- 146 -

Satellite Symposium (40th annual meeting of Japanese Society for Microcirculation)
1.Date of creation of report
November 24, 2015
2.Name of research meeting
Satellite Symposium by the 40th Annual Meeting of Japanese Society for Microcirculation
3.Representative
Toyotaka Yada (M.D., PhD.)
Professor, Department of Medical Engineering, Faculty of Health Science and Technology,
Kawasaki University Graduate School of Medical Welfare
4.Opening period and place
September 24th, 2015
Kyoto International Conference Center
Takaragaike, Sakyo-ku, Kyoto 606-0001 Japan
5.Number of participants/Number of participating countries and areas
Number of participants : 119
Number of participating countries and areas
Japan, China, USA, Sweden, Korea, Hong Kong, Netherlands, Australia, Austria, Hungary
6.Total cost
￥3,421,000 JPY
7.Main use of subsidy
Part of the room renting costs
8.Results and Impression
The Satellite Symposium was held in September 24th in Kyoto. This year falls on the 40th
Anniversary of Japanese Society for Microcirculation. For 2015, Kawasaki Medical Schoolof Okayama is the host of this Satellite Symposium, yet we have decided to hold it in Kyoto,
to enable all participants in the World Congress to participate in the Symposium with ease.
Okayama, one of Japan’s major centers for education and culture in the western part of Japan, isjust 1 hour from Kyoto by Shinkansen (bullet train). The previous annual meetings were held in
the middle of winter; however, this time we were happy to welcome you in a more comfortable- 147 -

season, vibrant autumn. In this Symposium, we held total 9 symposia (40 oral presentations) that
focused on various organs (lymph, heart, brain, eye and blood) of microcirculation. The themeof Symposium is frontline of research on organ microcirculation on the Satellite Symposium.
We invite Dr. Hiroaki Shimokawa (Professor, Tohoku University Graduate School of Medicine)and Dr. Paul M Vanhoutte (Professor, University of Hong Kong) as speakers of special lecture.
They speak about Diverse function of endothelial nitric oxide (NO) syntheses system: NOand endothelium-derived hyperpolarising (EDH) and endothelium, Importance of coronary
microvascular dysfunction and Endothelial dysfunction: Regenerate to be old. The purposes ofthese symposia are to discuss the latest and most significant research achievements in our field
and to share the information with leading experts invited from not only throughout Japan but also
around the world. There were active discussions and comments among the participants. We also
selected young researchers for their good presentations and gave them prizes. This symposium
was successful and we believe most participants had good impression and enjoyed Japanese foodand culture.
9.Additional description
The next 41th annual meeting of Japanese Society for Microcirculation will be held in Tokyo, in2016.

- 148 -

29th Grant Report (FY2015)

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 toa public interest incorporated foundation on Apr. 1, 2012. The grants conducted in FY 2015 are as
follows.
29th Novartis Research Grant: 35Researchers (JPY 1 mil.), Subtotal JPY 35 mil.
Research Meeting Grant: 6 Meetings(JPY 0.4 mil.), Subtotal JPY 2.4 mil.
Total JPY 37.4 mil.

29th Novartis Research Grant (FY2015)
The Grant is to aim supporting creative research in Japan in the field of Bio, life science andrelevant chemistry. The 35 grantees are as follows.
#

Name

Institution

Title

1 Hiroshi Harada

Kyoto University Graduate
School of Medicine

Associate
Professor

2 Hiromitsu Hara

Kagoshima University
Graduate School of Medical
and Dental Sciences

Professor

3 Satoshi Goto
4 Kohtaro Takei
5 Goro Sashida
6 Hirotaka Matsui
7 Sayuri Yamazaki
8 Masabumi Minami

9 Yuichiro Miyaoka

10 Tomohiro Suzuki
11 Yasukazu Daigaku
12 Masaharu Kataoka

Rikkyo Universitiy, Collegeof Science
Yokohama City University
Graduate School of Medical
Life Science
Kumamoto University,
International ResearchCenter for Medical Sciences
Kumamoto University,
Faculty of Life Sciences,
Department of Molecular
Laboratory Medicine
Nagoya City University
Graduate School of Medical
ScienceFaculty of Pharmaceutical
Sciences, Hokkaido
University
Regenerative Medicine
Project, Department ofAdvanced Science for
Biomolecules, Tokyo
Metropolitan Institute ofMedical Science
Center for Bioscience
Research and Education,
Utsunomiya University
Tohoku University Frontier
Research Institute forInterdisciplinary Sciences
Keio University School of
Medicine

Associate
Professor

Research Project
Functional and mechanistic crosstalk
between tumor suppressors and hypoxiaregulated genes networks
Control of host immune responses throughinnate pattern-recognition receptors thatrecognize Mycobacterium tuberculosis
virulence lipids.
Research of glycosylation that dynamicallyregulates innate immunity
Research on development of therapy formultuple sclerosis by regulation of LOTUS
expression
Molecular Mechanism of Epigenetic
Dysregulation in Myelofibrosis-Initiating
Stem Cells

Professor

Leukemogenesis caused by a defect of theribosomal RNA processing

Professor

Induction of antigen-specific regulatory T
cells by dendritic cells in human

Professor

Study on the neuronal mechanisms fordepression and anxiety induced by chronic
stress and pain

Project
LeaderPathogenesis of cardiomyopathy by
RBM20 mutations modeled in geneticallymodified human iPS cells

associate
professor

Chemical investigation of the foodpoisoning epidemics caused by themushroom Pleurocybella porrigens.
Developing analysis of genome replicationin single cell to elucidate molecular
dynamics of DNA polymerases
A novel lincRNA,Linc-Heart,regulates
cell cycle and proliferation of adultcardiomyocytes

Professor
Professor

Assistant
Professor
juniorassociate
professor- 149 -

#Name

13 Kensuke Takada
14 Tsutomu Nakada
15 Daiji Okamura
16 Shun-ichiro Asahara

17 Hiroyasu Motose
18 Takao Iwawaki
19 Naoki Hisamoto
20 Masayuki Endo
21 Seiichi Nakamura
22 Yoshiyuki Kubota
23 WING James

24 Ayuko Sakane
25 Kenji Matsumoto
26 Kohei Koga
27 Mitsuro Kanda
28 Masayuki Shimoda
29 Daisuke Kaida
30 Sei Kuriyama
31 Kei-ichiro Mishiba
32 Hiroshi Suga

Institution
University of Tokushima,
Institute for Genome
Research
Shinshu University Schoolof Medicine, Department ofmolecular pharmacology
Kinki University, Departmentof Advanced Bioscience
Division of Diabetes andEndocrinology, Kobe
University Graduate Schoolof Medicine
Okayama University,
Department of Biological
Science, Graduate Schoolof Natural Science &Technology
Gunma University Graduate
School of Medicine
Nagoya university Graduate
school of Science
Kyoto University, Institutefor Integrated Cell-Material
Sciences (WPI-iCeMS)
Graduate School of
Pharmaceutical Sciences,
Nagoya City University
National Institute forPhysiological Sciences
Laboratory of Experimental
Immunology, Immunology
Frontier Research Center,
Osaka University
Tokushima University
Graduate School of Medical
Sciences, Department ofBiochemistry
Tokushima Bunri University,
Faculty of pharmaceuticalsciences
Hirosaki University
Department of
Neurophysiology
Nagoya University
Hospital, Department of
Gastroenterological Surgery
II
Keio University School of
Medicine
University of Toyama,
Frontier Research Core forLife Sciences
Akita University, Dept
Molecular Medicine &Biochemistry, Grad. Sch.
Med.
Graduate School of Lifeand Environmental Science,
Osaka Prefecture University
Prefectural University ofHiroshima, Faculty of Lifeand Environmental Sciences

33 Moore Adrian

RIKEN Brain Science
Institute

34 Koji Ikeda

Kobe Pharmaceutical
University, Department ofClinical Pharmacy

Title

SeniorAssistant
Professor
Assistant
Professor

Research Project
Roles of functional education of T cell
positive selection in immune resnses invivo
Role of junctophilin for the proper function
and intracellular distribution of L-type
calcium channels
Epigenetic regulation in cell
reprogramming and chimera formation

Research
Fellow

Analysis of mechanism of T2DM
pathogenesis caused by susceptibility genes
using human iPS cells

Associate
Professor

Molecular mechanism of plant cell growth
via NIMA-related kinases

Associate
Professor
Associate
Professor

Molecular mechanism of overeating
regulated by cellular stress response
Analysis of a signal through ER required
for the induction of axon regeneration
Development of molecular delivery systemusing cell membrane penetrating DNA
nanostructures
Studies toward total syntheses of
biologically active, bridged polycyclic
meroterpenoids
Functional significance of Chandelier cellin cortical microcircuit

Associate
Professor

Associate
Professor
Professor
Associate
professor
Assistantprofessor(Speciallyappointed)

Characterization of T-follicular regulatory
cells in human blood and tumor tissues

Assistant
Professor

Conformational plasticity of JRAB
regulates transition between mesenchymal
and amoeboid invasiveness in cancer cells.

LectureDevelopment of apoptosis inhibitor
targeting mitochondria

Assistant
Professor

Cortical synaptic plasticity for coexistenceof chronic pain and anxiety neurosis

Assistant
Professor

Functions and expression ofsynaptotagmin 13 driving peritoneal
metastasis of gastric cancer cells

Assistant
Professor

Epigenetic and biological analysis ofhuman cancer-associated fibroblasts
Study on the RNA splicing checkpointmechanism to prevent the accumulation ofpre-mRNA
Sensing ECM stiffness may trigger thetransition from Solid-like to Liquid-likebehaviour in collective cell migration of
cranial neural crest cells

Associate
Professor
Associate
Professor
Associate
Professor

Functional analysis of a novel silencing
mechanism in higher plants

The origin of animal multicellular system– an approach from genome editing of
unicellular holozoans
Regulating microtubule arrays forTeam Leader neuronal dendrite growth and for neuronal
regeneration
A novel network between mature
Associate adipocytes and endothelial cells that
Professor regulates adipose tissue angiogenesis andhomeostasis
Associate
Professor- 150 -#Name

35 Ai Kotani

Institution

Title

Tokai University The
Institute of Medical Sciences

Associate
Professor

Research Project
Investigation of the mechanism of cellfate determination independent fromtranscriptional factor

FY2015 Research Meeting Grant(JPY 400 thousand x 6 = 2.4 million)#

Meeting

Date(Place)Institution / Title

Name

The 12th International Workshop on
1 Microbeam Probes of Cellular Radiation
Response (IWM2015)

2015.5.30-6.1(Fukui)University of Fukui
Biomedical Imaging
Research Center /Associate Professor

Hideki Matsumoto

meeting “Oocyte
2 International
maturation and fertilization IV”

2015.6.15-18(Aomori)

Ochanomizu University,
Department of Biological
Sciences/ Professor

Kazuyoshi Chiba

Symposium on Environmental
3 24thChemistry

2015.6.24-26(Sapporo)

Graduate School of
Veterinary Medicine,
Hokkaido University/ Vice Director

Mayumi Ishizuka

4 8th International Tunicate Meeting2015.7.13-17(Aomori)

Shimoda Marine
Research Center,
University ofTsukuba / Professor

Kazuo Inaba

GIW/InCoB2015 - Joint
Conference of the 26th
Workshop on
5 International
Genome Informatics (GIW) andthe 14th International
Conference on Bioinformatics

2015.9.9-11(Tokyo)National Institute ofAdvanced Industrial
Science and Technology/ Director

Paul Horton

Satellite Symposium by the
6 40th Annual Meeting of Japanese Society
for Miclocirculation

2015.9.24(Kyoto)Department of ClinicalEngineering, Kawasaki
University of Medical
Welfare / Professor

Toyotaka Yada

- 151 -

第 29 期（2015 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。2015 年度は、下記の総額 3,740 万円の助成事業を実施しました。
第 29 回ノバルティス研究奨励金

35 件（1 件 100 万円） 3,500 万円

研究集会助成6 件（1 件 40 万円）

			

総額

240 万円
3,740 万円

第 29 回ノバルティス研究奨励金 （2015 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。2015 年度は 35 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 100 万円）
No

氏名

所属

職位

研究課題

がん抑制遺伝子と低酸素応答遺伝子ネッ
特定准トワークの機能的 ・ 作用機序的クロストー
教授
ク

1

原 田

浩 京都大学大学院医学研究科

2

原

満

3

後 藤

4

竹居 光太郎

横浜市立大学大学院生命医科
学研究科

5

指田 吾郎

特別招
熊本大学国際先端医学研究機
骨髄線維症幹細胞におけるエピジェネ
聘准教構
ティック制御異常の分子基盤
授

6

松井 啓隆

熊本大学大学院生命科学研究部 臨床病態解析学分野

7

博

鹿児島大学大学院医歯学総合研究科

聡 立教大学理学部

教授

結核菌の病原性脂質を認識する自然免疫受容体を介した宿主免疫の制御機構

教授

自然免疫をダイナミックに制御する糖鎖修
飾の研究

教授

神経回路形成因子 LOTUS の発現制御による多発性硬化症の治療法開発

教授

リボソーム RNA プロセシング異常という白血病発症の新しい概念の提示と検証

山崎 小百合 名古屋市立大学医学研究科教授

ヒトにおける樹状細胞による抗原特異的制御性 T 細胞の誘導の研究

8

南

教授

慢性ストレス ・ 慢性疼痛による抑うつ ・ 不安に関わる神経機構の解明

9

東京都医学総合研究所 生体
協力研 遺伝子改変 iPS 細胞を用いた RBM20 変
宮岡 佑一郎 分子先端研究分野 幹細胞プロ究員 異による心筋症発症機序の解明
ジェクト

雅 文 北海道大学大学院薬学研究院

10 鈴 木 智 大

宇都宮大学 バイオサイエンス
教育研究センター

准教授 スギヒラタケ食中毒事件の化学的解明

11 大 学 保 一

東北大学 学際科学フロンティア研究所

助教- 152 -

１細胞を対象としたゲノムワイドＤＮＡ複製解析の開発によるＤＮＡポリメラーゼ挙動
の解明

No

氏名

所属

職位

研究課題

成熟心筋細胞の細胞周期と分裂能を調特任講整する新規 lincRNA(Linc-Heart) の同定
師と昨日解析

12 片 岡 雅 晴 慶應義塾大学医学部准教授

正の選択を介した T 細胞の機能的教育
が生体内免疫応答に果たす役割

勉 信州大学医学部

講師

L 型カルシウムチャネルの正常な細胞内局在 ・ 機能維持におけるジャンクトフィリンの役割

15 岡 村 大 治 近畿大学農学部講師

エピジェネティック修飾による初期化プロ
グラムとキメラ形成分子機構の解明

13 高 田 健 介
14 中 田徳島大学疾患プロテオゲノム研究センター

16 淺原 俊一郎

神戸大学大学院医学研究科 糖尿病 ・ 内分泌内科学

医学研 ヒト iPS 細胞を用いた 2 型糖尿病感受性
究員 遺伝子による糖尿病発症機序の解明

17 本 瀬 宏 康

岡山大学・自然科学研究科 （理NIMA 関連キナーゼによる植物細胞の伸准教授
学部 ・ 生物学科）
長成長制御機構の解明

18 岩 脇 隆 夫 群馬大学大学院医学系研究科

講師

細胞ストレス応答反応の解析から挑む「過食」 の分子メカニズム

19 久 本 直 毅 名古屋大学大学院理学研究科准教授

神経軸索再生誘導に必要な小胞体経由シグナルの解析20 遠 藤 政 幸

特定拠京都大学 物質－細胞統合システ
細胞膜を貫通する DNA ナノ構造体による点准教
ム拠点
分子デリバリーシステムの開発
授

21 中 村 精 一

名古屋市立大学大学院薬学研究科教授

生物活性架橋多環式メロテルペノイドの全合成研究

22 窪 田 芳 之

自然科学研究機構生理学研究所准教授

大脳皮質局所神経回路のシャンデリア細胞の機能的役割23

ウ ィ ン グ 大阪大学免疫学フロンティア研
ジ ェ イ ム ズ 究センター実験免疫学

特任助 ヒト末梢血と腫瘍組織中濾胞性制御性 T
教
細胞の特徴

徳島大学大学院医歯薬学研究24 坂根 亜由子 部医科学部門生理系生化学分
野

助教

癌の浸潤 ・ 転移で認められる細胞形態 ・運動のモード変換を１分子構造変化から解明する

25 松 本 健 司 徳島文理大学薬学部

講師

ミトコンドリアに作用する新規アポトーシス
阻害剤の開発26 古 賀 浩 平 弘前大学大学院医学研究科

助教

慢性疼痛と不安神経症が共存する大脳
神経可塑性の機序解明

助教

新規胃癌腹膜播種責任分子
synaptotagmin13 の発現および機能解析

27 神 田 光 郎

名古屋大学医学部附属病院 消化器外科二

専任講 網羅的エピゲノム解析を用いた機能的が師
ん関連線維芽細胞の探索

28 下 田 将 之 慶應義塾大学医学部
29 甲斐田 大輔
30 栗 山正 秋田大学大学院医学系研究科

31 三柴 啓一郎
32 菅
33

富山大学 先端ライフサイエンス拠
スプライシング異常による未成熟 mRNA の准教授
点蓄積を防ぐチェックポイント機構の解析
准教授

細胞外基質の固さの認知によって惹起される神経堤細胞集団の流動化の研究

大阪府立大学大学院生命環境
高等植物における新規サイレンシング機
准教授
科学研究科
構の解明

裕 県立広島大学生命環境学部准教授

多細胞システムの起源に単細胞ホロゾアのゲノム編集で迫る

ム ー ア エ 理化学研究所脳科学総合研究 チーム 樹状突起の形成と再生における微小管のイ ド リ ア ン センター
リーダー 配列制御のしくみを解明する- 153 -

No

氏名34 池 田35 幸 谷

宏二

所属

職位

研究課題

神戸薬科大学 臨床薬学研究室准教授

成熟脂肪−血管内皮細胞ネットワークによる脂肪組織恒常性維持メカニズムの解明

愛 東海大学総合医学研究所

准教授 単独 miRNA が転写因子をレスキューする2015 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。2015 年度は 6 件の助成を行いました。
（受付順、敬称略、所属・職位は申請時、贈呈額：1 件 40 万円）
No

研究集会名

開催日
（開催地）

1

第 12 回マイクロビーム放射線応
答国際ワークショップ

2015.5.30-6.1（福井）

福井大学高エネルギー医学
松本
研究センター ・ 准教授

英樹

2

第 4 回 「卵成熟と受精」 国際会
議2015.6.15-18（青森）

お茶の水女子大学理学部 ・
千葉
教授

和義

3

第 24 回環境化学討論会

2015.6.24-26( 札幌 )

北海道大学大学院獣医学研究科 ・ 教授

石塚 真由美4

第 8 回国際被囊類学会

2015.7.13-17（青森）

筑波大学下田臨海実験センター ・ 教授稲葉5

GIW/InCoB2015 - Joint
Conference of the 26th
International Workshop on
Genome Informatics (GIW) andthe 14th International
Conference on Bioinformatics

2015.9.9-11（東京）

産業技術総合研究所ゲノム情報研究センター ・ セン Paul HORTON
ター長

6

サテライトシンポジウム （第 40 回
日本微小循環学会主催）

2015.9.24( 京都 )

川崎医療福祉大学医療技術学部 ・ 教授

- 154 -

所属・職位

氏名

矢 田

一男

豊 隆

29th Financial Report

Balance Sheet

As of March 31, 2016
Account(Unit: JP Yen)

I Assets

Account

1. Current Assets
Current Assets Total

13,042,692

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

796,194(3) Other Long-term Assets
Other Long-term Assets Total
Fixed Assets Total
Assets Total

79,971,429
1,180,767,623
1,193,810,315

II Liabilities
1. Current Liabilities
Current Liabilities Total
Liabilities Total

35,455,250
35,455,250

III Equity (Net Assets)
1. Designated Net Assets
Designated Net Assets Total

1,000,796,194(Amount appropriating to Basic Fund)(1,000,000,000)

2. General Net Assets(Amount appropriating to ]AmountI General Net Assets Changes
1. Ordinary Income & Expenditure(1) Ordinary Income(1) Basic Fund
Basic Fund Total

Statement of Net Assets

From April 1st, 2015 to March 31, 2016
(Unit: JP Yen)

Amount157,558,871(100,000,000)

Equity Total (Net Assets)

1,158,355,065

Liabilities & Equity Total

1,193,810,315- 155 -

Donation

43,803,806

Ordinary Income Total

44,260,451(2) Ordinary Expenditure
Project Expenses

47,020,503

Grant Expense

37,400,000

Novartis Research Grant

35,000,000

Research Meeting Grant

2,400,000

Administrative Expense

8,284,115

Ordinary Expenditure Total

55,304,618

Ordinary Balance without Appraisal Profit or Loss

△ 11,044,167

2. Nonrecurring Profit & Loss
Nonrecurring Balance of Current Period

0General Net Assets Ending Balance

157,558,871

II Designated Net Assets Changes
Designated Net Assets Change

796,194

Designated Net Assets Ending Balance 1,000,796,194
III Net Assets Balance Ending Balance

1,158,355,065

第 29 期（2015 年度）財務報告

貸借対照表
2016 年 3 月 31 日現在

正味財産増減計算書（単位：円）

科目

金2015 年 4 月 1 日から 2016 年 3 月 31 日まで（単位：円）

額

Ⅰ資産の部

科

１． 流動資産
流動資産合計13,042,692

２． 固定資産1,100,000,000(2) 特定資産
特定資産合計796,194(3) その他固定資産
その他固定資産合計
固定資産合計
資産合計

79,971,429
1,180,767,623
1,193,810,315

Ⅱ負債の部
１． 流動負債
流動負債合計
負債合計

35,455,250
35,455,250

Ⅲ正味財産の部
指定正味財産合計

1,000,796,194（うち基本財産への充当額）(1,000,000,000)（うち基本財産への充当額）
正味財産合計
負債及び正味財産合計

Ⅰ一般正味財産増減の部
１． 経常増減の部
受取寄付金

43,803,806

経常収益計

44,260,451(2) 経常費用
事業費

47,020,503

支払助成金

37,400,000

ノバルティス研究奨励金

35,000,000

研究集会助成金

2,400,000

管理費

8,284,115

経常費用計

55,304,618

当期経常増減額

157,558,871(100,000,000)
1,158,355,065
1,193,810,315- 156 -

△ 11,044,167

2． 経常外増減の部
当期経常外増減額
一般正味財産期末残高

１． 指定正味財産

２．一般正味財産

決算額(1) 経常収益(1) 基本財産
基本財産合計目

0
157,558,871

Ⅱ指定正味財産増減の部
当期指定正味財産増減額

796,194

指定正味財産期末残高

1,000,796,194

Ⅲ正味財産期末残高

1,158,355,065

List of Board Members[Board of Trustees]

5 trustees, 2 auditors

As of Oct 1, 2016

Post

Name

Chairman

Akimichi KANEKO

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

Shigetaka ASANO

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

Masao ENDOH

Emeritus Professor, MD, Yamagata University

Kuniaki TAKATA

President, Gunma Prefectural College of Health Sciences

Masao TORII

President, Novartis Holding Japan K.K.

Tokuzo NAKAJIMA

Certified Public Accountant

Masanori FUSE

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

Trustee

Auditor[Board of Councilors]
Post

Title

10 councilors

Chairman Tsuneyoshi KUROIWA

Councilor

As of Oct 1, 2016

Name

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

Tadanori MAYUMIEmeritus Professor, Osaka University

Miwako MORI

Visiting Professor, Health Sciences University of Hokkaido;
Emeritus Professor, Hokkaido University

Toyoshi FUJIMOTO

Professor, MD, Graduate School of Med, Nagoya University

Masamitsu IINO

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

Tohru HIROSE

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

- 157 -

[Grantee Selection Committee] 20 members
Post

Name

Chairman Junichi NABEKURA

MemberAs of Oct 1, 2016
Title

Professor, MD, National Institute for Physiological Sciences

Shigeyuki KAWANO

Professor, Graduate School of Frontier Sciences, University ofTokyo

Hiroyuki NAKAMURA

Professor, Chemical Resources Laboratory, Tokyo Institute ofTechnology

Masanori
HATAKEYAMA

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

Tomoko BETSUYAKUProfessor, MD, School of Medicine, Keio University

Tetsuji MIURA

Professor, MD, Sapporo Medical University

Masato YASUIProfessor, MD, School of Medicine, Keio University

Yoshihiro OGAWA

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

Motomu KANAI

Professor, Graduate School of Pharmaceutical Sciences,
University of Tokyo

Koichiro KUWAHARA

Lecturer, MD, Graduate School of Medicine, Kyoto University

Masanobu OHSHIMA

Professor, Cancer Research Institute, Kanazawa University

Yosuke TAKAHAMA

Professor, Institute for Genome Research, Tokushima University

Masafumi TAKIGUCHI

Professor, MD, Center for AIDS Research, Kumamoto University

Eisuke NISHIDA

Professor, Graduate School of Biostudies, Kyoto University

Mitsuyasu HASEBE

Professor, National Institute for Basic Biology

Haruhiko BITO

Professor, MD, Graduate School of Medicine, University ofTokyo

Jun YAMASHITA

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

Motoko YANAGITA

Professor, MD, Graduate School of Medicine, Kyoto University

Masafumi YANO

Professor, MD, School of Medicine, Yamaguchi University

Hirokazu ARIMOTO

Professor, MD, Graduate School of Life Sciences, Tohoku
University- 158 -

公益財団法人ノバルティス科学振興財団

役 員 名 簿2016 年 10 月 1 日現在 ( 順不同、 敬称略 )
職

理

名

事

氏

現

職

就任年月日

常勤･非常勤金子 章道

畿央大学大学院健康科学研究科長･教授
2016 年 6 月 10 日
慶應義塾大学名誉教授

非常勤

浅野 茂隆

神戸大学大学院医学系研究科客員教授
東京大学名誉教授

2016 年 6 月 10 日

非常勤遠 藤 政 夫 山形大学名誉教授

2016 年 6 月 10 日

非常勤高 田 邦 昭 群馬県立県民健康科学大学学長

2016 年 6 月 10 日

非常勤

ノバルティスホールディングジャパン㈱代表取締役社長

2016 年 7 月 1 日

非常勤

中嶋 德三

中嶋德三公認会計士事務所
公認会計士

2016 年 6 月 10 日

非常勤

布施

ノバルティス ファーマ㈱経理・財務統括部長

2016 年 6 月 10 日

非常勤

鳥 居

監

名

正 男

事
正則

評議員名簿
2016 年 10 月 1 日現在 ( 順不同、 敬称略 )
職

名

氏

名

就任年月日

常勤･非常勤2016 年 6月10日

非常勤

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
赤池 紀扶
2016 年 6月10日
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

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

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2016 年 6月10日

非常勤2016 年 6月10日

非常勤2016 年 6月10日

非常勤2016 年 6月10日

非常勤2016 年 6月10日

非常勤2016 年 6月10日

非常勤

評議員長 黒岩 常祥

柴崎 正勝

現

職

日本学士院会員
東京大学名誉教授

東京大学大学院理学系研究科教授
中野明彦
理化学研究所光量子工学研究領域チームリ
評議員ーダー
眞弓 忠範 大阪大学名誉教授
森 美和子

北海道医療大学客員教授
北海道大学名誉教授

藤本 豊士 名古屋大学大学院研究科教授
飯野 正光
廣 瀬

日本大学医学部特任教授
東京大学名誉教授

徹 ﾉﾊﾞﾙﾃｨｽ ﾌｧｰﾏ ( 株 ) 取締役開発本部長- 159 -

選考委員名簿2016 年 10 月 1 日現在 ( 順不同、 敬称略 )
職

名

氏

名

現

職

就任年月日

常勤･非常勤2013 年 6月14日

非常勤

河 野 重 行 東京大学大学院新領域創成科学研究科教授 2013 年 6月14日

非常勤

中 村 浩 之 東京工業大学資源化学研究所教授

2013 年 6月14日

非常勤

畠 山 昌 則 東京大学大学院医学系研究科教授

2013 年 6月14日

非常勤

別 役 智 子 慶應義塾大学医学部教授

2013 年 6月14日

非常勤

三 浦 哲 嗣 札幌医科大学医学部教授

2013 年 6月14日

非常勤

安 井 正 人 慶應義塾大学医学部教授

2013 年 6月14日

非常勤

東京医科歯科大学大学院医歯学総合研究科
2014 年 6月6日
教授

非常勤選考委員長 鍋 倉 淳 一 自然科学研究機構生理学研究所教授

小川 佳宏
金 井

選考委員

求 東京大学大学院薬学系研究科教授

2014 年 6月6日

非常勤桑原宏一郎 京都大学大学院医学研究科講師

2015 年 6月26日

非常勤

大 島 正 伸 金沢大学がん進展制御研究所教授

2015 年 6月26日

非常勤

徳島大学疾患プロテオゲノム研究センター教2015 年 6月26日
授

非常勤

高浜 洋介

滝 口 雅 文 熊本大学エイズ学研究センター教授

2015 年 6月26日

非常勤

西 田 栄 介 京都大学大学院生命科学研究科教授

2015 年 6月26日

非常勤

長谷部光泰 自然科学研究機構生理学研究所教授

2015 年 6月26日

非常勤

尾 藤 晴 彦 東京大学大学院医学系研究科教授

2015 年 6月26日

非常勤

山 下

2016 年 6月10日

非常勤2016 年 6月10日

非常勤

山口大学 医学部医学科器官病態内科学教2016 年 6月10日
授非常勤

潤 京都大学 iPS 細胞研究所教授

柳 田 素 子 京都大学大学院医学研究科教授
矢野 雅文

有 本 博 一 東北大学 生命科学研究所教授- 160 -

2016 年 6月10日

非常勤

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。
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Tel:03-6899-2100 Fax:03-6899-2101
E- メール ： foundation.japan@novartis.com
ホームページ :http://www.novartisfound.or.jp- 161 -