Annual Report (2011)

No.23
The NOVARTIS Foundation (Japan)
for the Promotion of Science

平成 23 年度

財団年報 第 23 号

Contents
Ⅰ. Introduction

はじめに ......................................................................................... 5

Akimichi Kaneko, MD, PhD
Chairman of the Board of Trustees

Ⅱ. Reports from the Recipients of Novartis Research Grants
研究報告 ................................................................................................................ 7
1. The roles of FoxO1 in the regulation of glucose and lipid metabolism
via O-glycosylation of ChREBP ............................................................................ 8
Yukari Kitamura
Institute for Molecular and Cellular Regulation, Gunma University

2. The analysis of the functional linkage between genetically-disrupted cardiac
transcriptional network and the onset of cardiomyopathy: Approach from the
HOP gene mutation causing human dilated cardiomyopathy ............................... 11
Hiroyuki Morita
Department of Translational Research for Healthcare and Clinical Science,
University of Tokyo

3. Elucidation of lipid chaperones, inflammation, and endoplasmic reticulum
stress response in cardiovascular and metabolic diseases .................................... 14
Masato Furuhashi
Second Department of Internal Medicine,
Sapporo Medical University School of Medicine

4. Molecular mechanisms of host inflammatory responses mediated by
Nod-like receptor NLRP3 .................................................................................... 17
Toshihiko Suzuki
Department of Molecular Bacteriology and Immunology
Graduate School of Medicine, University of the Ryukyus

5. Study of olfactory background canceling system that controls courtship behavior . 19
Aki Ejima
Kyoto University

6. Molecular Basis for microtubule plus-end tracking protein CLASP2 ..................... 21
Ikuko Hayashi
Department of Supramolecular Biology, Graduate School of Nanobioscience,
Yokohama City University, Japan

7. Engineered Biosynthesis of Steroidal Antibiotics .................................................... 24
Ikuro AbeThe University of Tokyo

8. Physiological function of the active zone protein CAST/ELKS family .................. 29
Ohtsuka Toshihisa
Department of Biochemistry, Interdisciplinary Graduate School of Medicine andEngineering, University of Yamanashi-1-9. The role of Sox17 in gastrointestinal tumorigenesis ................................................ 31
Masanobu Oshima
Cancer Research Institute, Kanazawa University

10. 4D imaging analysis of insulin exocytosis ............................................................... 34
Mica Ohara-Imaizumi
Department of Biochemistry, Kyorin University School of Medicine

11. Mechanism for development and homeostatic maintenance of human dendritic
cell subsets. .......................................................................................................... 36
Nobuyuki Onai
Department of Biodefense Research, Medical Research Institute,Tokyo Medical and Dental University

12. Research of tumorigenesis and invasion/metastasis mediated by small GTPase Ral 39
Hisanori Horiuchi
Institute of Development, Aging and Cancer, Tohoku University

13. Hepatic fatty acid profile relates to inflammation and fibrosis in nonalcoholicsteatohepatitis ....................................................................................................... 40
Tsuguhito Ota
Frontier Science Organization, Kanazawa University

14. Analysis of the role of CD317/tetherin, inhibitor of virus budding,in the whole body ................................................................................................. 42
Shoichiro Miyatake
Tokyo Metropolitan Institute of Medical Science

15. Driving mechanisms for axon guidance: studies on asymmetric membrane
dynamics in the growth cone ............................................................................... 44
Hiroyuki Kamiguchi
RIKEN Brain Science Institute

16. Mechanism of pathogenesis by human T-cell leukemia virus type 1 ...................... 47
Masao Matsuoka
Institute for Virus Research, Kyoto University

17. Development of chemical fragmentation method for glycan sequencing ................ 51
Shunji Natsuka
Niigata University

18. Development of the direct arylation of C-H bonds in nitrogen heteroaromatics ..... 53
Mamoru Tobisu
Graduate School of Engineering, Osaka University

19. Self-Defence Mechanism for Natural Toxin-Containing Species ............................ 55
Keiichi Konoki
Graduate School of Agricultural Science, Tohoku University

20. Palladium-Catalyzed Enantioselective Synthesis of Highly Substituted Epoxides . 57
Hideki Yorimitsu
Kyoto University-2-

21. Generation of a new common marmoset glioma model .......................................... 62
Tomotoshi Marumoto
Division of Molecular and Clinical Genetics, Medical Institute of Bioregulation,
Kyushu University

22. Revealing olfactory processing algorithm by the study of insects ........................... 65
Makoto Mizunami
Hokkaido University

23. Human RNA dependent RNA polymerase and cancer stem cell ............................. 68
Kenkichi Masutomi
National Cancer Center Research Institute, Division of Cancer Stem Cell

24. Pattern formation played by growth factors signaling in the mammalian embryo .. 70
Isao Matsuo
Osaka Medical Center and Research Institute for Maternal and Child HealthOsaka Prefectural Hospital Organization

25. Inhibition of USAG-1, a BMP antagonist, ameliorates hereditary kidney diseaseof glomerular basement membrane ...................................................................... 73
Motoko Yanagita
Hakubi Project, Kyoto University Graduate School of Medicine

26. Molecular analysis of social behavior using a humanoid model mouse of autism .. 77
Toru Takumi
Hiroshima University

27. Novel signaling pathway from DNA damage repair to RNAi ................................. 82
Hitoshi Nakayashiki
Kobe University

28. Identification of autoimmune susceptible genes using murine models of
autoimmune diseases. ........................................................................................... 85
Taku Okazaki
Institute for Genome Research, The University of Tokushima

29. The Mechanisms How Myocardial Ischemia-reperfusion Initiates Inflammatory
Response in the Myocardium ............................................................................... 89
Masafumi Takahashi
Division of Bioimaging Sciences,
Center for Molecular Medicine, Jichi Medical University

30. Novel strategy towards therapeutic approach for lissencephalyusing calpain inhibition. ..................................................................................... 92
Hirotsune Shinji
Osaka City University Graduate School of Medicine-3-

Ⅲ. Reports from the Recipients of Grants for International Meetings
研究集会報告 ...................................................................................................... 96
1. The 5th International Symposium on Filoviruses (Filo2010) ................................... 97
Yoshihiro Kawaoka
Institute of Medical Science, The University of Tokyo

2. The International Symposium on Protein Community ............................................ 98
Toshiya Endo
Nagoya University

3. The 17th International RUNX Workshop ................................................................. 99
Ichiro Taniuchi
President of the organizing committee for the 17th International RUNX Workshop ,Team Leader, Laboratory for Transcriptional Regulation, RIKEN, RCAI

4. 2010 International Polyamine Conference:
Progress in Medicine and Life Sciences ............................................................ 101
Senya Matsufuji
The Jikei University School of Medicine

5. The 16th International Conference of the International Society ofDifferentiation .................................................................................................... 102
Naoto Ueno
National Institute for Basic Biology

Ⅳ.

The 24th (fiscal year 2010) Promotion Report
第 24 期（2010 年度）助成事業報告 ............................................................... 103

Ⅴ.

The 24th (fiscal year 2010) Financial Report
第 24 期（2010 年度）財務報告 ....................................................................... 106

Ⅵ.

List of the Board Members
役員名簿 .......................................................................................................... 107

Ⅶ.

Information from the Secretariat
事務局便り ....................................................................................................... 110-4-Introduction“The Disaster in Eastern Japan”

Akimichi Kaneko, MD, PhD
Chairman of the Board of Trustees
This annual report includes research and meeting reports written by the 2010
grantees. Their tireless effort is the driving force to promote and to keep the highstandard of the Japanese life science. I strongly believe that the aim of the Foundation isto help keeping their activity and we will do our best toward this goal. I sincerely
appreciate the assistance and warm encouragement extended by the members of theBoard of Trustees, the auditors, the Board of Councilors and the Selection Committee.
The powerful support by the Novartis Pharma KK enabled us to sustain our activity
without interruption.
In March this year, a huge scale earth quake accompanying a frightening tsunami hit
Eastern Japan. In addition the complete damage and melt down of the nuclear reactorsof the Fukushima First Nuclear Electric Plant heavily affected the life of victims andscared them by the unforeseen future when the damaged nuclear reactors could be put
under control. I hereby extend my sincere sympathy to the victims of the present
disaster and hope that everything will recover as soon as possible.
I learned that the land of the area has slid by several meters, but it may not be sosurprising in billion-year-scales in which the Japanese islands drifted apart from theEurasian Continent. During this drift many quakes must have occurred repeatedly.
Unfortunately we are unable to predict exactly when such change will occur. The
technology of earthquake prediction is still in immature stage.
There are quite a few scientists whose research facilities were damaged or who havelost their valuable materials by the quake. We, at the Foundation, wish to extend someassistance to suffered scientists. At the June meeting of the Board of Trustees we
decided to reserve a part of our research grant of the coming year to scientists in Tohoku
and Northern Kanto areas who are victims of the quake and tsunami. Our support ismodest and late coming, but I strongly hope that it will help, at least partially, toreestablish their research activities.

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はじめに

「東日本大震災」
理事長

金子章道

本年度もここに 2010 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。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。こうした方々の研究に多少なりとお力添えをすることが本財団の事業の基本と考え、今後も努力を続けて行きたいと思っております。これらの優れた研究を選考していただいた選考委員の皆様をはじめ、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。とりわけノバルティスファーマ社の継続した強力な経済的支援に御礼申し上げた
いと思います。
去る 3 月、東日本は未曾有の大震災と津波に襲われました。また津波の影響で引き起こされた東京電力福島第一原子力発電所の大事故は、その後も被災者の生活に計り知れない影響を及ぼしており、またその収束が見えない不安が被災地の方々を悩ませています。この地震では陸地が数メートルも移動したと聞きましたが、ずっと昔、日本列島がユーラシア大陸から離れて出来上がってきたことを考えると、陸地が移動することも、それに伴って大地震が起きることも、何億年というスケールからすれば驚くには値しないのでしょう。しかし、それが何時、どの程度の規模で起きるかを予知する技術は未熟です。このような大災害に遭遇された方々には心からご同情申し上げ、一日も早い復興を祈ります。
今回の大災害に直面して、財団として何が出来るかを考えてみました。震災によって研究設備が破損した方、貴重な資料や材料を失われた方、またご自身やご家族が被災された方々などがおられると思います。去る 6 月の理事会では、
今年募集する研究助成金の一部を東北や北関東地方で研究室が被災された方に優先的に配分することを決議いたしました。ささやかな助成であり、また遅きに失した感もないわけではありませんが、この研究助成が研究室の再建に幾ば
くかのお役にたてればと考えております。被災された研究者の皆様の研究が早急に復旧することを願っております。

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II.
Reports from the Recipients ofNovartis Research Grants-7-

The roles of FoxO1 in the regulation of glucose and lipid metabolism
via O-glycosylation of ChREBP
Yukari Kitamura
Institute for Molecular and Cellular Regulation, Gunma University
yukariam@showa.gunma-u.ac.jp

Abstract
We show that carbohydrate response element binding protein (ChREBP), a glucose-regulatedtranscription factor, is O-glycosylated by OGT, leading to increased ChREBP levels, owing todecreased ubiquitination. Conversely, expression of O-GlcNAcase (GCA) in hepatocytes decreases
ChREBP O-glycosylation, thus reducing its protein levels. Moreover, ChREBP O-glycosylationis regulated by fasting (low) and re-feeding (high) in mouse liver. The nutritional regulation of
ChREBP O-glycosylation is impaired in livers of high fat high sucrose diet fed mice. FoxO1, a
downstream effector of insulin signaling, has been known to regulate glucose metabolism in liver.
We show here that FoxO1 overexpression inhibits ChREBP O-glycosylation in hepatocytes, thus
reducing ChREBP levels. Conversely, conditional FoxO1 knockout in liver results in increased
levels of O-glycosylated ChREBP, even in the fasted state. Chromatin immunoprecipitation revealthat recruitment of ChREBP to the L-PK promoter is suppressed by FoxO1, and increased in liverspecific FoxO1 knockout mice in both fasted and fed conditions. Taken together, our results suggestthat O-glycosylation is an important mechanism to regulate ChREBP function, and that FoxO1
is required for ChREBP O-glycosylation. We propose that FoxO1 is the shared signaling element
linking glucose- and insulin-activated pathways to regulate hepatic glucose metabolism.
Keywords: ChREBP, FoxO1, O-glycosylation, Diabetes

Introduction
ChREBP is a major mediator of glucose action in the control of glycolysis and lipid synthesis inliver. ChREBP regulates the transcription of liver-pyruvate kinase (L-PK), one of the rate-limiting
enzymes of glycolysis, and lipogenic genes, such as acetyl-CoA carboxylase (ACC) and fatty acidsynthase (FAS). However, the molecular mechanism of ChREBP regulation has not been fully
elucidated. Glucose taken into hepatocytes is mainly converted to pyruvate or glycogen to produce
or store energy. However, excess glucose enters into hexosamine biosynthetic pathway (HBP),
which produce UDP-N-acetylglucosamine (UDP-GlcNAc). By using UDP-GlcNAc as the donor
substrate, O-GlcNAc transferase (OGT) catalyzes O-glycosylation modification of the proteins on
Ser/Thr residues. It is known that hyperglycemia causes the increase in glucose flux into HBP and
subsequent O-glycosylation of various proteins. It is suggested that the increase in O-glycosylation
modification is associated with pathophysiology of diabetes. FoxO1 is a member of the forkhead box
containing protein of the O subfamily, which regulates metabolism as well as cellular proliferation,-8-

apoptosis, differentiation and stress resistance. FoxO1 transcriptional activity is regulated byinsulin through phosphorylation by Akt and following nuclear exclusion. We previously reported
that FoxO1 plays important roles in regulating glucose metabolism in liver through the regulationof gluconeogenic genes. We also previously showed that FoxO1 controls glycolytic (L-PK) andlipogenic (ACC) genes, two critical ChREBP targets.

Results
ChREBP protein level is regulated by nutrients via ubiquitination.
We found that the protein level but not mRNA level was significantly increased by high glucose
in primary hepatocytes. Consistent with these results, ChREBP ubiquitination was decreased inhigh glucose compared to low glucose condition in MG132 treated hepatocytes. We also obtained
similar observations in mouse liver. ChREBP ubiquitination was decreased by refeeding, and was
dramatically decreased in the liver of HFHSD fed mice.
ChREBP is O-glycosylated by high glucose or refeeding in primary hepatocytes and mouse
liver.
We found that high glucose significantly increased O-glycosylation of ChREBP in primary
hepatocytes. DON, an inhibitor of GFAT (L-glutamine fructose-6-phosphate amino transferase),clearly inhibited O-glycosylation of ChREBP. In contrast, glucosamine treatment of the cells
induced O-glycosylation of ChREBP even in low glucose condition. These results indicatedthat ChREBP is O-glycosylated by high glucose via hexosamine biosynthetic pathway (HBP) in
hepatocytes. We also found that OGT interacts with ChREBP, and which was enhanced by HFHSD
feeding.
ChREBP O-glycosylation and transcriptional activity are regulated by OGT and GCA.
When we transfected primary hepatocytes with OGT, ChREBP O-glycosylation was significantly
increased in low glucose condition. Importantly, we observed that OGT was co-immunoprecipitatedwith ChREBP, and which was enhanced by high glucose. Conversely, when we knockdown OGT
in primary hepatocytes using specific siRNA for OGT, ChREBP O-glycosylation was decreased
in both high glucose and glucosamine treated conditions. Moreover, when we overexpressed GCA,ChREBP O-glycosylation was significantly decreased.
FoxO1 suppresses ChREBP O-glycosylation, decreases protein level and transcriptional
activity of ChREBP.
When we overexpressed constitutively active FoxO1 mutant in primary hepatocytes, ChREBP
target gene L-PK mRNA levels were significantly decreased, despite that ChREBP mRNA levels
were not changed. FoxO1 expression significantly decreased O-glycosylation and protein levelsof ChREBP. Conversely, when we knockdown FoxO1 by using specific siRNA for FoxO1 inprimary hepatocytes, ChREBP O-glycosylation was increased. FoxO1-ADA expression enhanced
ubiquitination of both endogenous and exogenously expressed ChREBP, which should account forthe decrease in protein levels of ChREBP.

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ChREBP O-glycosylation, protein level, and recruitment to the L-PK promoter wereincreased in the liver of FoxO1 knockout mice.
We generated liver specific FoxO1 knockout mice (L-FoxO1-KO) by crossing albumin-cre mice(reference) with FoxO1 flox mice and isolated livers from L-FoxO1-KO. In the liver of L-FoxO1KO mice, ChREBP O-glycosylation and protein levels were significantly increased, and OGTinteraction with ChREBP was enhanced. Consistent with the increase in ChREBP protein levels,
ubiquitination of ChREBP was apparently decreased in the liver of L-FoxO1-KO mice.

Discussion & Conclusion
In this study, we shoed that O-glycosylation is an important mechanism to regulate ChREBP
function, and that FoxO1 is required for ChREBP O-glycosylation. We propose that FoxO1 isthe shared signaling element linking glucose- and insulin-activated pathways to regulate hepatic
glucose metabolism. FoxO1 inhibition may increase glucose effectiveness.

References
1) Kitamura T, Kido Y, Nef S, Merenmies J, Parada LF, Accili D (2001) Preserved pancreatic betacell development and function in mice lacking the insulin receptor-related receptor. Mol Cell Biol
21: 5624-5630
2) Kitamura YI, Kitamura T, Kruse JP, et al. (2005) FoxO1 protects against pancreatic beta cell
failure through NeuroD and MafA induction. Cell Metab 2: 153-163- 10 -

The analysis of the functional linkage between genetically-disrupted
cardiac transcriptional network and the onset of cardiomyopathy:
Approach from the HOP gene mutation
causing human dilated cardiomyopathy
Hiroyuki Morita
Department of Translational Research for Healthcare and Clinical Science,
University of Tokyo
hmrt-tky@umin.net

Abstract
HOP (Homeodomain-Only Protein) is a unique transcriptional regulator, which lacks DNA binding
capacity but functions as the repressor of SRF-induced transcription. To identify the key molecule
which could account for the functional consequences triggered by the HOP missense mutation
identified previously in the patents with familial dilated cardiomyopathy, protein partner of HOP
was screened using yeast 2-hybrid system. In this study, I could identify 14 proteins which canbind with HOP. Until now, however, any of these 14 partner proteins could not explain the drastic
cardiac abnormality triggered by the HOP missense mutation. I will continue to examine in anotherexperimental method the interaction of HOP with these partner proteins identified in this study.
Keywords: heart failure, cardiomyopathy, transcriptional regulator, protein-protein interaction

Introduction
HOP (Homeodomain-Only Protein) is a unique transcriptional regulator, which lacks DNA binding
capacity but functions as the repressor of SRF-induced transcription [1, 2]. Previously, I identified
a missense mutation of HOP in the patient with familial dilated cardiomyopathy (DCM) and heart
failure. To elucidate the functional consequences of this HOP mutation, I created the transgenic miceoverexpressing wild-type HOP and mutant HOP in the heart, and compared their phenotypes. Unlike
mice overexpressing wild-type HOP, transgenic mice of mutant HOP could greatly recapitulate thehuman cardiac phenotype observed in the patient bearing HOP mutation. In the ANF-luciferase
reporter assay, this mutated HOP was found to repress the SRF-induced transactivation in a gainof-function manner, but the extent of which was not remarkable enough to account for the drastic
cardiac abnormality caused by the HOP mutation in human and mice. Therefore, I hypothesized thatthe novel unknown partner of HOP should be the key molecule for modifying the molecular pathway
triggered by HOP. By clarifying this partner (called as protein X), the drastic cardiac abnormality
caused by a missense mutation of HOP can be well-explained. The aim of this study is to identify thenovel partner of HOP and characterize the interaction between HOP and protein X.

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Results
With the yeast 2-hybrid system (Matchmaker Gold Yeast Two-Hybrid System (Clontech)), thebinding partners of HOP were screened. The full-length coding region of human HOP was inserted
into the pGBKT7, which was used as the bait. Pretransformed Human Heart Matchmaker cDNA
Library (Clontech) was used as the prey. Finally, 14 proteins were picked up as the candidate proteinof HOP partner. Here, I describe ATPase Na/K β1 subunit, one of 14 proteins I identified in thisstudy.
ATPase Na/K β1 subunit is a component of the transmembrane protein ATPase Na/K, which iswell-known as the pharmacoceutical target of digitalis. Recently, this protein was demonstrated tobe essential for maintaining the ventricular cardiomyocyte morphology and viability in the cardiac
development [3].
With the immunoprecipitation experiments, binding of ATPase Na/K β1 subunit with HOP could
be shown. However, ATPase Na/K β1 subunit could bind with mutant-type HOP as well as wild-type
HOP, suggesting that the difference of the extent of binding with ATPase Na/K β1 subunit could notaccount for the consequences of the HOP mutation. The 13 other proteins are under investigation.

Discussion & Conclusion
In this study, the partner of transcriptional regulator HOP was screened using yeast 2-hybrid
experiments. As a result, 14 candidate proteins were acquired. The purpose of this study was
to identify the key molecule which could explain the drastic cardiac abnormality caused by the
missense mutation of HOP. With the immunoprecipitation experiments, I could confirm the bindingof HOP with ATPase Na/K β1 subunit, one of the 14 candidate proteins identified here, but the
mutant-type HOP also could bind with this protein in the same manner. At least, from the viewpointof the difference in binding affinity, this protein cannot explain the phenotypic change in the humanand mouse bearing the mutant-type HOP. The 13 proteins else are under investigation. Also, as for
the ATPase Na/K β1 subunit, I will perform the additional experiments on the downstream pathway
followed by the binding with HOP.
So far, gene mutations of sarcomere proteins, Z-disc related proteins (muscle LIM protein,
telethonin, α-actinin 2, cypher/ZASP), metavinculin, ion-cycling related proteins (phospholamban,
K-ATP channel) were reported to be causative of the isolated DCM without extra-cardiac
involvement [4]. However, few genetic mutations in transcriptional regulator have been reported
to cause human isolated cardiomyopathy [5, 6]. Given that mutation in the structural or metabolic
proteins is sufficient to cause inappropriate cardiac remodeling, it is surprising that defects intranscriptional regulation of these same proteins have not also been identified as primary causesof cardiomyopathy. Several possible explanations may account for this. Transcription factor genemutations may be lethal or may at least substantially impair reproductive fitness so as to be rapidly
lost. The consequences of transcription factor gene mutations may be so pleiotropic that these cause
systemic rather than single-organ disease.

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The abnormality in transcriptional regulators is related to the cardiac remodeling induced bypressure overload or reperfusion injury, therefore the identification of the pathways triggered by
HOP could contribute to the clarification of the general mechanism how heart fails as well as the
specific mechanism how HOP missense mutation causes severe dilated cardiomyopathy. After thesepathways are clarified, the studies on the control of the cardiac remodeling through intervening HOP
protein are warranted.
This study was performed with the aid of Research Grant of the NOVARTIS Foundation (Japan) for
the Promotion of Science.

References
1) Chen F, Kook H, Milewski R, Gitler AD, Lu MM, Li J, Nazarian R, Schnepp R, Jen K, Biben C,
Runke G, Mackay JP, Novotny J, Schwartz RJ, Harvey RP, Mullins MC, Epstein JA.
Hop is an unusual homeobox gene that modulates cardiac development.
Cell 2002;110(6):713-23.
2) Shin CH, Liu ZP, Passier R, Zhang CL, Wang DZ, Harris TM, Yamagishi H, Richardson JA,
Childs G, Olson EN.
Modulation of cardiac growth and development by HOP, an unusual homeodomain protein.
Cell 2002;110(6):725-35.
3) Yuan S, Joseph EM.
The small heart mutation reveals novel roles of Na+/K+-ATPase in maintaining ventricularcardiomyocyte morphology and viability in zebrafish.}
Circ Res. 2004;95(6):595-603.
4) Morita H, Seidman J, Seidman CE.
Genetic causes of human heart failure.
J Clin Invest. 2005;115(3):518-26.
5) Schönberger J, Wang L, Shin JT, Kim SD, Depreux FF, Zhu H, Zon L, Pizard A, Kim JB, Macrae
CA, Mungall AJ, Seidman JG, Seidman CE.
Mutation in the transcriptional coactivator EYA4 causes dilated cardiomyopathy and sensorineural
hearing loss.
Nat Genet. 2005;37(4):418-22.
6) Moulik M, Vatta M, Witt SH, Arola AM, Murphy RT, McKenna WJ, Boriek AM, Oka K, Labeit S,
Bowles NE, Arimura T, Kimura A, Towbin JA.
ANKRD1, the gene encoding cardiac ankyrin repeat protein, is a novel dilated cardiomyopathygene.
J Am Coll Cardiol. 2009;54(4):325-33.

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Elucidation of lipid chaperones, inflammation,and endoplasmic reticulum stress response
in cardiovascular and metabolic diseases
Masato Furuhashi
Second Department of Internal Medicine, Sapporo Medical University School of Medicine
furuhasi@sapmed.ac.jp

Abstract
Increased adiposity is associated with a wide range of chronic metabolic disorders, including
insulin resistance, type 2 diabetes, and cardiovascular disease. During the development of obesity,
several inf lammatory and stress responses occur in metabolically active tissues, leading tochronic, low grade inflammation which plays a central role in the inhibition of insulin action anddisruption of systemic metabolic homeostasis. In recent years, organelle dysfunction emerged asa key mechanistic component in obesity and in the emergence of these metabolic abnormalities4.
In this study, we focused on fatty acid-binding proteins (FABPs) and endoplasmic reticulum (ER)stress which can activate inflammatory responses and metabolic deterioration, resulting in thedevelopment of cardiovascular diseases.
Keywords: Fatty acid-binding protein, endoplasmic reticulum stress, inf lammation, insulin
resistance, cardiovascular disease

Introduction
Obesity is characterized by an increase in adiposity, and it is well known that obesity is associatedwith several aspects of metabolic diseases, such as insulin resistance, type 2 diabetes, fatty liverdisease, hypertension, dyslipidemia, and cardiovascular disease. In the adipose tissue, it has beensuggested that the adipocyte and macrophage interact each other and regulate both metabolic andinflammatory responses. In these adipocyte and macrophages, several cytokines and lipids activate
key regulators of metabolic and inflammatory signaling, which are c-Jun N-terminal kinase (JNK)
and inhibitor of kappa kinase (IKK). Other intracellular stresses such as endoplasmic reticulum(ER) stress, oxidative stress, and some kinds of lipid chaperones, fatty acid-binding proteins(FABPs), can also activate JNK and IKK, resulting in enhanced inflammatory responses andmetabolic deterioration.
FABPs are a family of about 14-15 kDa lipid chaperones that bind hydrophobic ligands such as
long chain fatty acids and eicosanoids with high affinity. At least 9 FABPs have been alreadyidentified. Among them, aP2 (FABP4/A-FABP) and mal1 (FABP5/E-FABP) are expressed in both
adipocytes and macrophages. We have demonstrated that both aP2 and mal1 play a significant rolein many aspects of metabolic syndrome, and aP2/mal1-deficient mice protect form obesity, diabetes,
hepatosteatosis, and atherosclerosis. Furthermore, we recently developed a specific inhibitor of aP2- 14 -

and showed that it can treat diabetes and atherosclerosis in several animal models in a target specificmanner. Interestingly, it has been shown that aP2 is secreted from adipocytes although there are no
typical secretion signal peptides. Several clinical studies have demonstrated that serum aP2 levels
are associated with obesity, type 2 diabetes, and risk for cardiovascular diseases. However, there
is a lack of analysis in subjects without any medications. Furthermore, it is still unknown whether
secreted aP2 might have a physiological function as an adipokine.
The endoplasmic reticulum (ER) is a vital organelle that serves as the quality control device forproteins and coordinates the synthesis, folding, and trafficking of proteins. The proper functionof ER is critical for metabolism. ER stress and inappropriate adaptation through the unfolded
protein response (UPR) is a feature of obesity, insulin resistance, and type 2 diabetes. The principal
branches of UPR signaling are mediated through three molecules: inositol-requiring enzyme 1(IRE1), PKR-like endoplasmic-reticulum kinase (PERK) and activating transcription factor 6(ATF6). It has been indicated that ER stress is critical to the initiation and integration of pathwaysof inflammation and several diseases. However, the role of each canonical UPR sensor in metabolic
regulation is not yet clearly understood.

Results
In FABP projects, we first investigated the detection of aP2 in conditioned medium (CM) fromwild-type adipocytes. In the CM, aP2 and adiponectin as a control of secretary protein, but not actin
as a non-secretary protein, were detected, suggesting that release of aP2 from adipocytes is not due
to apoptosis or necrosis of cells. As another negative control, in aP2 deficient adipocytes, aP2 was
not detected in cell lysates and CM.
We are now conducting an epidemiological cohort, the Tanno-Sobetsu Study, which has been
continuing for more than 30 years. We measured aP2 concentration using ELISA kits in subjects
without any medications. The concentration of aP2 was significantly higher in females than inmales, which is similar to other adipokines, adiponectin and leptin. Serum levels of aP2 werepositively correlated with HOMA-R, an index of insulin sensitivity, and adiposity such as bodymass index (BMI) and waist circumference. Multiple regression analysis showed that aP2 was anindependent predictor of insulin resistance independent of adiposity.
In ER stress projects, we examined the role of the PERK pathway in obesity and peripheral insulin
sensitivity. Since PERK knockout mice are lethal, we used PERK heterozygous (Perk+/-) mice.
Perk+/- mice on a high fat diet had higher levels of glucose levels during glucose tolerance andinsulin tolerance tests than did the controls, indicating that Perk+/- mice are more insulin resistant.
Compromise of the PERK branch led to activation of JNK and inhibition of insulin receptor
signaling.

- 15 -

Discussion & Conclusion
In FABP studies, we confirmed that aP2 is secreted from adipocytes although there are
no secretary signal peptides. Non-classical pathway of secretion might be involved. Further
investigations need to be done. It is still unknown whether circulating aP2 has a physiological
function. In such a case, there might be receptors for aP2, developing for further studies. The small
chemical molecules for blocking aP2 receptors might result in new treatments for obesity, diabetes,
and atherosclerosis. We are now considering experiments of obesity and diabetes models using
recombinant aP2 protein to elucidate whether aP2 has a physiological function in vivo.
In ER stress projects, our results indicate that roles of PERK and the consequent UPR activationin tissues are important for the development of insulin resistance and type 2 diabetes. To elucidate
this regulation in each tissue under several conditions help us how to address these metabolic
diseases for therapeutic purpose. Specific modulations of UPR branches would be a new therapeutictarget for wide range of metabolic and cardiovascular diseases. Modulating ER function might be aparadigm-shifting approach for several diseases as a novel “organelle therapy”.

References
1) Maeda K, Cao H, Kono K, Gorgun CZ, Furuhashi M, Uysal K, Cao Q, Atsumi G, Malone H,
Krishnan B, Minokoshi Y, Kahn BB, Parker RA, Hotamisligil GS. Adipocyte/macrophage fatty
acid binding proteins control integrated metabolic responses in obesity and diabetes. Cell Metab
1: 107-119, 2005
2) Ozcan U, Yilmaz E, Ozcan L, Furuhashi M, Vaillancourt E, Smith RO, Gorgun CZ, Hotamisligil
GS. Chemical chaperones reduce ER stress and restore glucose homeostasis in a mouse model oftype 2 diabetes. Science 313: 1137-1140, 2006
3) Wellen KE, Fucho R, Gregor MF, Furuhashi M, Morgan C, Lindstad T, Vaillancourt E, Gorgun
CZ, Saatcioglu F, Hotamisligil GS. Coordinated regulation of nutrient and inflammatory
responses by STAMP2 is essential for metabolic homeostasis. Cell 129: 537-548, 2007
4) Furuhashi M, Tuncman G, Görgün CZ, Makowski L, Atsumi G, Vaillancourt E, Kono K, Babaev
VR, Fazio S, Linton MF, Sulsky R, Robl JA, Parker RA, Hotamisligil GS. Treatment of diabetes
and atherosclerosis by inhibiting fatty acid-binding protein aP2. Nature 447: 959-965, 2007
5) Furuhashi M, Fucho R, Görgün CZ, Tuncman G, Cao H, Hotamisligil GS. Adipocyte/
macrophage fatty acid-binding proteins contribute to metabolic deterioration through actions inboth macrophages and adipocytes in mice. J Clin Invest 118: 2640-2650, 2008
6) Vallerie SN, Furuhashi M, Fucho R, Hotamisligil GS. A predominant role for parenchymal c-Jun
Amino Terminal Kinase (JNK) in the regulation of systemic insulin sensitivity. PLoS ONE 3:
e3151, 2008
7) Nakamura T, Furuhashi M, Li P, Cao H, Tuncman G, Sonenberg N, Gorgun CZ, Hotamisligil GS.
Double-stranded RNA dependent protein kinase links pathogen sensing with stress and metabolic
homeostasis. Cell 140: 338-348, 2010- 16 -

Molecular mechanisms of host inflammatory responses
mediated by Nod-like receptor NLRP3
Toshihiko Suzuki
Department of Molecular Bacteriology and Immunology
Graduate School of Medicine, University of the Ryukyus
t-suzuki@med.u-ryukyu.ac.jp

Abstract
Aeromonas are Gram-negative bacteria that cause serious infectious disease in humans. Aeromonas
spp. have been shown to induce apoptosis in infected macrophages, yet the host responses triggeredby macrophage death are largely unknown. Here, we demonstrate that the infection of mouse bonemarrow derived macrophages with A. hydrophila or A. veronii triggers activation of caspase-1 withthe ensuing release of IL-1β and pyroptosis. Our results indicated that multiple factors from both thebacteria and the host play a role in eliciting caspase-1 activation during infection with Aeromonas.
Keywords: caspase-1, inflammasome, infection

Introduction
The genus Aeromonas is responsible for a significant number of animal and human infections.
Aeromonas are opportunistic human pathogens that cause intestinal infections, of which the mostcommon is gastroenteritis with diarrhea. Aeromonas also cause extraintestinal infections, including
wound, soft tissue, skin, and blood infections and septicemia. Recently, it has been reported thatthe infection with A. hydrophila and A. veronii biotype sobria (A. veronii) induces apoptosis inmacrophages. While the cytotoxicity by the bacteria is thought to enable the bacteria to evade
eradication and clearance by the macrophages, the host proinflammatory responses triggered by thedeath of the macrophages remain unknown. We focused the activation of caspase-1, a key moleculefor processing pro-IL-1β and IL-18 to bioactive forms.

Results
We investigated the cell death of macrophages infected with A. hydrophila or A. veronii, anddemonstrated that cell cells cause pyroptosis with caspase-1 activation and IL-1β release. Caspase-1
activation with the infection of A. hydrophila was dependent on inflammasome activation by
NLRP3, one of the member of nucleotide-binding oligomerization domain (NOD)-like receptors(NLRs). Aerolysin, hemolysin and multifunctional repeat-in-toxin produced by A. hydrophila are
required to elicit the NLRP3 inflammasome and necrotic cell death.
We also investigated the role of NLR inflammasome activation in macrophages during A. veronii
infection and demonstrate that aerolysin and T3SS are required to elicit caspase-1 activation andthe subsequent secretion of IL-1β and pyroptosis. Aerolysin triggers the activation of the NLRP3- 17 -

inflammasome. By contrast, T3SS of A. veronii triggers both NLRP3 and NLRC4 inflammasome
activation. Our data indicated that multiple factors from both the bacteria and the host are involvedin caspase-1 activation during proinflammatory responses.

Discussion & Conclusion
We identified the essential bacterial and host factors for eliciting caspase-1 activation and theproinflammatory responses upon Aeromonas infection. Our data indicated that multiple factors
from both the bacteria and the host are involved in caspase-1 activation during proinflammatory
responses.

References
1) McCoy A, Koizumi Y, Higa N, Suzuki T. (2010) Differential regulation of caspase-1 activation
via NLRP3/NLRC4 inflammasomes mediated by aerolysin and type III secretion system during
Aeromonas veronii infection. J Immunol 185: 7077-7084.
2) McCoy A, Koizumi Y, Toma C, Higa N, Dixit V, Taniguchi S, Tschopp J, Suzuki T. (2010)
Cytotoxins of the human pathogen Aeromonas hydrophila trigger, via the NLRP3 inflammasome,
caspase-1 activation in macrophages. Eur J Immunol 40: 2797-2803.

- 18 -

Study of olfactory background canceling systemthat controls courtship behavior
Aki Ejima
Kyoto University
aki@cp.kyoto-u.ac.jp

Abstract
The excitatory feedback into the sensory neurons mediates the signal equilibrium between two
pheromone pathways and contributes to background canceling.
Keywords: behavioral neurobiology

Introduction
In Drosophila melanogaster, cis-vaccenyl acetate (cVA), a product of the male ejaculatory bulb,
works as a courtship inhibitory pheromone. Recently I found that a male was capable to detect as
low cVA on a target fly as one thousandth of its own and exhibited a reduced level of courtship. But
the mechanisms that allows a male to detect such a tiny amount of cVA without being distracted byhis own cVA was largely unknown.

Results
In order to examine how pheromonal information is processed in the olfactory system, we
performed physiological analysis using an activity-sensitive fluorescent markers, synaptopHluorin(spH) and GCaMP in two groups of pheromone odorant receptor neurons (ORNs), one expressing
OR47b which respond to fly odors and the other expressing cVA-responsive OR65a.
The “naked brain” expressing the markers
showed that both Or65a and Or47b OR Ns
received cholinergic excitatory input to theirpresynaptic terminals. Blocking expressionof the acetylcholine receptor in either Or65aor Or47b ORNs diminished cVA-responsive
courtship suppression, suggesting that lateral
feedback modification of ORNs is critical for pheromone coding. Intriguingly, however, cosuppression of the excitatory feedback in both ORNs rescued the cVA-response defect, indicatingthat the behavioral output is determined by thesignal equilibrium between the Or65a courtshipinhibitory pathway and the Or47b courtshipstimulatory pathway.
It was also found that prolonged exposure- 19 -

to purified cVA caused an enhanced level of subsequent courtship activity, indicating that thebasal balance between the two groups of ORNs is maintained at neutral level and this adjustingmechanism contributes to the background-noise canceling mechanism that allows a male to ignore
his own smell and lead to an appropriate courtship decision.

Discussion & Conclusion
In this study, the neural-activity imaging and behavioral analysis revealed the role of acetylcholine
input at the ORN presynaptic terminals for pheromone coding and behavior control in Drosophilamelanogaster. It was found that the basal activity levels of two pheromone pathways are maintainedby the homeostatic control to adjust constant background activities, which allows a male to detect
novel stimuli and lead to appropriate behavior decision-making. Further analysis would uncover thedetailed physiological mechanism that underlies the lateral interaction of the multiple ORN groups.

- 20 -

Molecular Basis for microtubule plus-end
tracking protein CLASP2
Ikuko Hayashi
Department of Supramolecular Biology, Graduate School of Nanobioscience,
Yokohama City University, Japanihay@tsurumi.yokohama-cu.ac.jp

Abstract
Crystal structures of CLASP2 microtubule-binding domains
Keywords: Crystallography, microtubule, cell migration

Introduction
Microtubule (MT) is essential for many cellular functions, such as cell polarity, cell divisionand cell migration. The dynamic behavior of MTs is highly regulated by MT-associated proteins(MAPs): they stabilize or destabilize MT structure, which leads to MT two alternating phases ofgrowing or shortening, called dynamic instability. CLASPs are a member of a conserved class of
MAPs which specifically bind to MT plus-ends to stabilize MT filament. We determined the crystal
structures of CLASP2 MT binding domains and showed that they possess known MT-associated
TOG domains. Furthermore, our biochemical analyses showed CLASP2 cooperatively promotes
MT assembly with another MT plus-end associated protein EB1 in vitro.

Results
1. Crystal structures of MT-binding domains in CLASP2
We expressed, purified and crystallized the MT binding domains of CLASP2 using bacterial
expression system. Crystal structures of two CLASP2 MT-binding domains were determined byselenomethionine multiwavelength anomalous diffraction method (Fig. 1). These domains bothcomprise six HEAT-repeat helices stacked side by side, belonging to the MT-binding TOG domains
Fig. 1 Crystal structures of TOG domains.
CLASP2 possesses two TOG domains with six HEAT-repeat modules seen in XMAP215. Trptophan and basic
residues essential for MT recognition are shown.
(a) CLASP2 N-terminal TOG
Trp106

Lys191

Arg271(b) CLASP2 C-terminal TOG
Trp687 Lys744- 21 -

Arg838(c) XMAP215 TOG
Trp292

Lys373

Arg446

found in MT stabilizing factor XMAP215. The Trp and basic residues identified as MT-interacting
elements in XMAP215 TOG domains are also conserved in the ternary structure of CLASP2 MTbinding domains, indicating that CLASP2 has a similar mechanism to stabilize MTs. We mutatedconserved residues in CLASP2 TOG domains and found that the mutations cause the defect in
TOG’s binding to MTs.
2. CLASP2 and EB1 cooperatively promote MT assembly in vitro
Although CLASP family proteins are conserved throughout the eukaryotes, there are significant
differences between the species. Human CLASPs are monomeric, on the other hand yeast CLASP
homologue (Cls1p) dimeric; CLASPs associates with the plus-end MAP EB1, whereas Cls1p doesnot. EB1 binding to CLASPs Ser-rich region leads to the specific localization of CLASPs to MT
plus-ends (ref. 1). By the use of isothermal titration calorimetry, we determined the Kd value
for CLASP2 and EB1 binding (3x10 -6 M). MT polymerization effects of EB1 and CLASP2 were
examined by light scattering analyses. CLASP2 increases the turbidity of MTs by itself, and thecooperative MT stabilization effect between CLASP2 and EB1 is observed.
3. GCC185 associates with C-terminal domain of CLASP2
C terminus of CLASP2 is shown to interact with trans-Golgi network (TGN) protein GCC185 (ref.
2). The interaction is essential for Golgi-derived MT growth in a migrating cell. By yeast two-hybrid
assay using C terminal region of CLASP2 as a bait, we identified approximately 100 a.a. CLASP2
binding region in GCC185.
With fluorescent microscopic technique, we examined if the region affects the localization of
CLASPs in the cell. This GCC185 fragment removed CLASPs from the Golgi apparatus, revealing
the dominant-negative effects (Fig. 2). Mass spectroscopy and gel filtration analyses showed that
this GCC185 mutant forms a dimer. Now we are investigating the structural analyses of GCC185
and CLASP2 complex by x-ray crystallography.
Fig. 2 Overexpression of dominant-negative GCC185 mutant causes mislocalization of CLASPs.
(left) Overexpression of GCC185 mutant in Hela cells. GFP labeled GCC185 localizes to the Golgi.
(middle) CLASPs do not localize to the Golgi because of dominant-negative GCC185. Asterisks show CLASP
localization at MT plus-ends.
(right) Merged image

GFP-GCC185

CLASP

merge

Discussion & Conclusion
Multiplicity of the MT-binding domains gives a wide variety of MT dynamics. Here we showedthat CLASPs MT-binding domains possess TOG-domain fold – one of the typical MT-binding- 22 -

domains seen in XMAP215. CLASPs’ MT-binding ability is also regulated by EB1 binding sites.
The region possesses serine/arginine-rich stretches that can affect CLASPs’ MT-lattice binding aswell as EB1 binding. Although CLASPs are shown to be one of the members of XMAP215/Dis1
family proteins, MT-recognition depends not only on the divergence of TOG domain characters
but also on the franking region of TOGs. The serine/arginine-rich region can be phosphorylated by
GSK3β, which is critical to the localization of CLASPs in the cell (ref. 3). GSK3β is not active at
the cell periphery, where non-phosphorylated CLASPs can localize at MTs. The effect of GSK3βphosphorylation is now examined in vitro to see how the interactions between CLASP2 and MTs/
EB1 can be affected. We further investigated CLASPs C-terminal domain’s interaction with
GCC185. CLASPs C-terminal domain is responsible for cellular localization such as Golgi, cellcortex and kinetochore by interacting with its binding partners. We believe that CLASP association
with GCC185 will shed light on the structural basis of CLASP2 recognition, eventually explain thecellular localization mechanisms between CLASPs and its binders.

References
1) Mimori-Kiyosue Y et al. (2005) CLASP1 and CLASP2 bind to EB1 and regulate microtubuleplus-end dynamics at the cell cortex. J Cell Biol, 168, 141-53.
2) Efimov A et al. (2007) Asymmetric CLASP-dependent nucleation of noncentrosomal
microtubules at the trans-Golgi network. Dev Cell 12, 917-30.
3) Kumar P et al. (2009) GSK3beta phosphorylation modulates CLASP-microtubule association andlamella microtubule attachment. J Cell Biol 184, 895-908.

- 23 -

Engineered Biosynthesis of Steroidal Antibiotics
Ikuro AbeThe University of Tokyo
abei@mol.f.u-tokyo.ac.jp

Abstract
Oxidosqualene:protostadienol cyclase (OSPC) from the fungus Aspergillus fumigatus, catalyzes
the cyclization of (3S)-2,3-oxidosqualene into protosta-17(20)Z,24-dien-3b-ol which is the precursorof the steroidal antibiotic helvolic acid. To shed light on the structure-function relationship between
OSPC and oxidosqualene:lanosterol cyclase (OSLC), we constructed an OSPC mutant in which the
C-terminal residues 702APPGGMR708 were replaced with 702NKSCAIS708, as in human OSLC. As
a result, the mutant no longer produced the protostadienol, but instead efficiently produced a 1:1
mixture of lanosterol and parkeol. This is the first report of the functional conversion of OSPC into
OSLC, which resulted in a 14-fold decrease in the Vmax/KM value, whereas the binding affinity forthe substrate did not change significantly. Homology modeling suggested that stabilization of the
C-20 protosteryl cation by the active-site Phe701 through cation-π interactions is important for the
product outcome between protostadienol and lanosterol.
keywords: oxidosqualne cyclase; lanosterol synthase; protostadienol synthase; steroidal antibiotics;
helvolic acid

Introduction
Oxidosqualene cyclases (OSCs) are pivotal enzymes in the biosynthesis of sterols and triterpenes[1-4]. Oxidosqualene:lanosterol cyclase (OSLC) catalyzes the cyclization of (3S)-2,3-oxidosqualene
into lanosterol, which is the key step in the biosynthesis of sterols in animals and fungi (Fig.
1). The formation of lanosterol is initiated by the oxirane ring opening of oxidosqualene foldedin the chair-boat-chair conformation; the sequential ring forming reaction first produces thetetracyclic protosteryl C-20 cation, which then undergoes backbone rearrangement (H-17a→20 ,
H-13a→17a, CH3-14b→13b, CH3-8a→14a) followed by removal of H-9 to generate the D8 doublebond of lanosterol. On the other hand, oxidosqualene:protostadienol cyclase (OSPC) (AfuOSC3
or Afu4g14770) from the fungus Aspergillus fumigatus is a recently reported novel OSC which is
involved in the biosynthesis of the fusidane-type steroidal antibiotic helvolic acid [5,6]. OSPC is an
Mr 83,086 membrane bound protein with 735 amino acids, sharing 40% amino acid identity withhuman OSLC. A recombinant OSPC heterologously expressed in an OSLC-deficient mutant GIL77
strain of Saccharomyces cerevisiae catalyzed the cyclization of (3S)-2,3-oxidosqualene into a 3:1
mixture of protosta-17(20)Z,24-dien-3b-ol and (20R)-protosta-13(17),24-dien-3b-ol (Fig. 1) [5]. In
this case, the cyclization of (3S)-2,3-oxidosqualene proceeds without the backbone rearrangement,
and the C-20 protosteryl cation immediately undergoes proton elimination from H-17a or H-13a toform a double-bond between C-17/C-20 or C-13/C-17, respectively.

- 24 -

Fig. 1. Proposed mechanism of cyclization of (3S)-2,3-oxidosqualene into lanosterol by OSLC andprotosta-17(20)Z,24-dien-3b-ol and (20R)-protosta-13(17),24-dien-3b-ol by OSPC.

To clarify the structure-function relationship between OSLC and OSPC, and to understand theintimate structural details that govern the stabilization of the C-20 protosteryl cation and the
backbone rearrangement reactions, we constructed a series of A. fumigatus OSPC mutants andinvestigated the effects of the mutagenesis on the enzyme activity. Here we report the functional
conversion of A. fumigatus OSPC into OSLC by replacing the C-terminal residues 702APPGGMR708,
located just downstream of the active-site Phe701, with 702NKSCAIS708, as in human OSLC (Fig.
2). The active-site Phe701 is considered to be important for stabilization of the positive charges at
the C-20 protosteryl cation through cation-p interactions, which would be crucial for the formationof lanosterol via the backbone rearrangement reaction [7]. The results suggested that the swappingof the residues neighboring the active-site Phe701 caused changes in the position of Phe701,
thereby affecting the cation-p interactions and the stabilization of the C-20 cation, which led to the
production of lanosterol by the OSPC mutant.
#
Human OSLC693

AGV F N K SCA I S Y T S Y R N I F P I WA L GR F SQ L Y P E R A L AGHP

Saccharomyces OSLC

696

E GV F NH SCA I E Y P S Y R F L F P I K A L GMY S R A Y E T H T L

Candida OSLC692

E GV F NH SCA I E Y P S Y R F L F P I K A L G L Y K N K Y GD K V L V

Cephalosporium OSLC

721

E GV F N K SCM I S Y P N Y K F I F P I T A L GMF GNR Y P D E K I D L N

Afu OSLC (Afu4g12040)

681

E G I F N K SCA I T Y P N Y K F I F P I L A L GK F GR K Y P H L V

732

731
728
760

715

Afu OSLC (Afu5g04080)

715

E GV F NQSCM I S Y P N Y K F YWP I R A L G L Y S R K F GN E E LM

Afu OSPC (Afu4g14470)

698

E GV F A P P GGMR Y P N Y K F H F T LMA L GR Y V A I HGN E C L A I

751
735

# * * * * * * *Fig. 2. Comparison of the amino acid sequences of the C-terminal regions of the A. fumigatus OSPC andthe OSLCs from a mammal, yeast and fungi: Human, Homo sapiens; Saccharomyces, Saccharomyces
cerevisiae; Candida, Candida albicans; Cephalosporium, Cephalosporium caerulens; Afu, Aspergillus
fumigatus. The active-site Phe701 (numbering in A. fumigatus OSPC) is marked with #. The residues
702
APPGGMR708 are marked with *.

Results
A comparison of the primary sequences of the A. fumigatus OSPC and OSLC enzymes from a
mammal, yeast and fungi revealed that the moderately conserved C-terminal residues of human
OSLC, 697 NKSCAIS703, located just downstream of the active-site Phe696 (numbering in humanOSLC), are characteristically replaced with 702APPGGMR708 in A. fumigatus OSPC (Fig. 2) [5]. In
the X-ray crystal structure of human OSLC, the active-site Phe696 (which corresponds to Phe701
in A. fumigatus OSPC) is located near the C-13/C-20 of the bound lanosterol. This Phe residue is- 25 -

considered to be important for stabilization of the positive charges at the C-13 anti-Markovnikov
tricyclic cation and also at the C-20 tetracyclic protosteryl cation through cation-p interactions,
thereby guiding the stereochemical course of the enzyme reaction [7]. Indeed, the F696T mutant
of S. cerevisiae OSLC (ERG7) (numbering in human OSLC) was recently shown to produce atruncated rearrangement product, protosta-13(17),24-dien-3b-ol, which was formed by hydride
shift from C-17 to C-20 followed by proton elimination of H-13a [8,9]. We therefore surmisedthat the residues located around Phe701 might be participating in the fate of the C-20 cation, andthe difference between OSLC around this region might be responsible for the different productoutcomes between the two enzymes. Thus, we first constructed a mutant of A. fumigatus OSPCin which the residues 702APPGGMR708 were replaced with 702NKSCAIS708, as in human OSLC, andinvestigated the effects of the mutagenesis on the enzyme activity.
As in the case of the wild-type A. fumigatus OSPC, the gene encoding the mutant enzyme wascloned into the yeast expression vector pESC(Ura) and heterologously expressed in the OSLCdeficient sterol auxotrophic yeast mutant strain GIL77 (erg7, ura3-167, hem3-6, gal2) under thecontrol of the GAL10 promoter [5]. After induction, the transformed cells were harvested and therecombinant membrane-bound enzyme was solubilized with Triton X-100 and partially purified byhydroxylapatite column chromatography as described previously [10].
An in vitro enzyme assay revealed that the A. fumigatus OSPC mutant in which the residues
702

APPGGMR708 were replaced with the conserved 702NKSCAIS708 sequence no longer produced

protostadienols, but instead efficiently produced a 1:1 mixture of lanosterol and parkeol (Fig.
3). Indeed, the mutant complemented the erg7 deficiency of the yeast GIL77 strain, since thetransformed cells could grow on media lacking ergosterol, in sharp contrast to the wild-type OSPC.
The steady-state kinetic analysis revealed that the mutant had an apparent KM = 16.5 mM, Vmax =0.32 mM min-1, and Vmax/KM value of 0.019 min-1, which are comparable to the values of the wild-type
OSPC, which showed an apparent KM = 14.4 mM, Vmax = 4.08 mM min-1, and Vmax/KM value of 0.283
min-1. Thus, the swapping of the residues resulted in a 14-fold decrease in the Vmax/KM value whereas
the binding affinity for the substrate did not change significantly.

Fig. 3. GC-MS trace of the enzyme reaction products of (A) the wildtype A. fumigatus OSPC and (B) the OSPC mutant in which the residues
702
APPGGMR708 were replaced with the conserved 702NKSCAIS708.

- 26 -

Discussion & Conclusion
This is the first demonstration of the functional conversion of OSPC into OSLC and the formationof lanosterol by the OSPC mutant. Here the proposed mechanism of (3S)-2,3-oxidosqualene
cyclization to parkeol is essentially the same as that for lanosterol; the only difference is the final
proton elimination from H-11 to produce the double-bond between C-9/C-11 of parkeol, whereas
proton elimination from H-9 generates the double-bond between C-8/C-9 of lanosterol (Fig. 1).
To further evaluate the importance of the conserved residues, we also created a set of four point
mutations (A702N, P703K/P704S, G705C, and M707I) in A. fumigatus OSPC. However, none of
the three mutants (A702N, G705C, and M707I) affected the enzyme reaction product pattern while
the P703K/P704S mutant lost its activity, which clearly demonstrated that a single amino acidsubstitution is not sufficient for the functional conversion of OSPC into OSLC.
It was thus confirmed that these residues located just downstream of Phe696 of human OSLC
play important roles for the formation of lanosterol via the stabilization of the C-20 protosterylcation and the backbone rearrangement reaction. Presumably, the swapping of the residues
neighboring the active-site Phe701 caused changes in the position of Phe701, thereby affecting thecation-p interactions and the stabilization of the tetracyclic C-20 cation, which led to the formationof lanosterol and parkeol by the OSPC mutant. Indeed, a homology model based on the human
OSLC crystal structure [7] suggested that in A. fumigatus OSPC, the active-site Phe701 is locateda bit farther from the presumed C-20 cationic center of the protosteryl cation as compared withthat in human OSLC. This increased distance would reduce both the cation-p interactions and thestabilization of the C-20 cation, and hence would interrupt the backbone rearrangement, resulting
in termination of the enzyme reaction by
proton elimination of H-17a or H-13a to
produce a 3:1 mixture of protosta-17(20)Z,24 - d ien-3b - ol a nd (20R) -protost a13(17),24-dien-3b-ol (Fig. 1). Alternatively,
an activated water molecule might belocated in a cavity formed between the
C-20 cation and Phe701, and abstract a
proton from C-17 or C-13 to terminate thereaction at the protosteryl cation stage.
The relocation of the Phe701 residue in the
mutant might have prevented the watermolecule from entering this cavity, thus
Fig. 4. (A) A homolog y model of A. fumigatus
OSPC complexed with lanosterol. (B) The X-ray
crystal structure of human OSLC complexed withlanosterol. Only the active-site Phe701 and the residues
702
APPGGMR708 (numbering in A. fumigatus OSPC)
are indicated. Lanosterol is shown in magenta and theposition of the C-20 indicated.

- 27 -

precluding the early termination of thereaction to produce lanosterol and parkeol.
In conclusion, this is the first report of thefunctional conversion of OSPC to OSLC

by changing the conserved C-terminal residues 702APPGGMR708 into 702NKSCAIS708. The results
suggested that stabilization of the C-20 protosteryl cation by the active-site Phe701 through cation-πinteractions is important for the product outcome between protostadienol and lanosterol. To further
clarify the structural details of the enzyme reactions, crystallization trials of both the wild-type andmutant A. fumigatus OSPCs are now in progress in our laboratories.

References
1) I. Abe, M. Rohmer, G.D. Prestwich, Enzymatic cyclization of squalene and oxidosqualene tosterols and triterpenes, Chem. Rev. 93 (1993) 2189-2206.
2) I. Abe, Enzymatic synthesis of cyclic triterpenes, Nat. Prod. Rep. 24 (2007) 1311-1331.
3) K.U. Wendt, G.E. Schulz, E.J. Corey, D.R. Liu, Enzyme mechanisms for polycyclic triterpene
formation, Angew. Chem. Int. Ed. 39 (2000) 2812-2833.
4) T. Kushiro, Y. Ebizuka, Triterpene, in: L.N. Mander, H.-w. Liu (Eds.), Comprehensive Natural
Products II: Chemistry and Biology, Vol. 1, Elsevier, Oxford, 2009, in press.
5) H. Mitsuguchi, Y. Seshime, I. Fujii, M. Shibuya, Y. Ebizuka, T. Kushiro, Biosynthesis of steroidal
antibiotic fusidanes: functional analysis of oxidosqualene cyclase and subsequent tailoring
enzymes from Aspergillus fumigatus, J. Am. Chem. Soc. 131 (2009) 6402-6411.
6) S. Lodeiro, Q. Xiong, W.K. Wilson, Y. Ivanova, M.L. Smith, G.S. May, S.P.T. Matsuda,
Protostadienol biosynthesis and metabolism in the pathogenic fungus Aspergillus fumigatus, Org.
Lett. 11 (2009) 1241-1244.
7) R. Thoma, T. Schulz-Gasch, B. D’Arcy, J. Benz, J. Aebi, H. Dehmlow, M. Hennig, M. Stihle, A.
Ruf, Insight into steroid scaffold formation from the structure of human oxidosqualene cyclase,
Nature 432 (2004) 118-122.
8) T.-K. Wu, H.-Y. Wen, C.-H. Chang, Y.-T. Liu, Protein plasticity: a single amino acid substitution
in the Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase generates protosta-13(17),24dien-3b-ol, a rearrangement product, Org. Lett. 10 (2008) 2529-2532.
9) T.-K. Wu, C.-H. Chang, Y.-T. Liu, T.-T. Wang, Saccharomyces cerevisiae oxidosqualenelanosterol cyclase: a chemistry-biology interdisciplinary study of the protein’s structure-functionreaction mechanism relationships, Chem. Rec. 8 (2008) 302-325.
10) I. Abe, M. Bai, X.-y. Xiao, G.D. Prestwich, Affinity labeling of vertebrate oxidosqualne cyclaseswith a tritiated suicide substrate, Biochem. Biophys. Res. Commun. 187 (1992) 32-38.

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Physiological function of the active zone protein
CAST/ELKS family
Ohtsuka Toshihisa
Department of Biochemistry, Interdisciplinary Graduate School of Medicine and Engineering,
University of Yamanashi
tohtsuka@yamanashi.ac.jp

Abstract
To explore the physiological roles of the CAST/ELKS family, we have tried to produce the double
KO mice.
Keywords: Active Zone, CAST, ELKS

Introduction
Our higher brain functions such as learning and memory, emotion, and consciousness rely on thecomplicated neural network in the brain. The fundamental unit of this network is called “synapse”,
which is composed of the three parts: the presynaptic terminal, synaptic cleft, and postsynaptic
compartment. In the presynaptic nerve terminals, a specialized structure, active zone, plays a pivotal
role in neurotransmitter release and possibly in synaptic plasticity. We have recently isolated andcharacterized the active zone-specific protein, named CAST (cytomatrix at active zone -associatedstructural protein) and its family member ELKS. While CAST is mainly expressed in the brain,
ELKS is ubiquitously expressed in the body. And, unfortunately, the KO mice for ELKS wasembryonic lethal. To overcome this difficulty, we attempted to produce the double KO mice andexamine the effect on synaptic structure and function.

Results
Because we have already obtained flox-ELKS hetero mice, we first produced the homozygotes
for flox-ELKS. Then, we cross-bred these mice with transgenic mice containing flipase: flipase isan enzyme, which can delete the neomycin site. While we maintain these conditional ELKS mice,
we obtained transgenic mice expressing Cre recombinase, which can be driven by CaMKII. Ourconditional ELKS was bred with CaMKII-cre mice, and we analyzed if ELKS is specifically deleted
in the brain. CaMKKII-cre has been known to be expressed in the fore brain. Thus, we prepared thehomogenates of various brain regions such as olfactory bulb, cortex, hippocampus, and cerebellum.
Western blot analysis using the anti-ELKS antibody showed that ELKS was specifically deleted in
the forebrain containing olfactory bulb, cortex and hippocampus, whereas its expression was intact
in the cerebellum. The expression of CAST was unchanged throughout the brain.

- 29 -

Discussion & Conclusion
The presynaptic active zone is believed to play a crucial role in the docking, priming, and fusion
if synaptic vesicles upon elevation of intracellular concentration of Ca ion. However, especially inmammals, its physiological role or significance of the active zone is still obscure in the whole animalevel. CAST and ELKS forms a complex with the other active zone proteins including Bassoon,
Piccolo, RIM1, and Munc13-1. Accumulating evidence suggest that there were no changes in the
structure of the active zone in the KO mice for Bassoon, Piccolo, RIM1, and Munc13-1, althoughsevere impairments were observed. Therefore, using the conditional KO mice for ELKS producedin this project, we will be able to produce the double KO for CAST and ELKS, and reveal thephysiological role of the CAST/ELKS family in the establishment and maintenance of the active
zone, which will definitely shed new light on the role of active zone in our higher brain function andneuronal disorders such as autism, schizophrenia, and Parkinson diseases.

- 30 -

The role of Sox17 in gastrointestinal tumorigenesis
Masanobu Oshima
Cancer Research Institute, Kanazawa University
oshimam@kenroku.kanazawa-u.ac.jp

Abstract
Sox17 belongs to SRY transcription family members, and plays an important role in definitive
endoderm development. It has been shown that Sox17 functions as a tumor suppressor throughrepression of canonical Wnt signaling. We have investigated expression of Sox17 in gastric andintestinal tumors using mouse models. Importantly, Sox17 expression is induced in the Wntactivated tumor epithelial cells at early stage of tumorigenesis. However, Sox17 expression was
significantly suppressed in human gastric cancer tissues. Consistently, in the malignant mouseintestinal adenocarcinomas, Sox17 expression was rarely detected. These results suggest that
Sox17 plays a protective role against malignant progression at early stage of tumorigenesis, and that
downregulation of tumor suppressor Sox17 contributes to malignant progression.
Keywords: Sox17, Wnt, gastric tumor, intestinal tumor

Introduction
Sox17 is a member of SRY family transcription factors. Disruption of Sox17 gene in mice resulted
in lethal at the early embryogenesis by impaired definitive endoderm formation1. On the other hand,functions of Sox17 in adult tissues have not been understood yet. In human colon cancer tissues,
Sox17 is widely downregulated by DNA methylation 2. Moreover, transfection of Sox17 in coloncancer cells resulted in suppression of colony formation 2. These results suggest that Sox17 functions
as tumor suppressor in colon cancer development. However, molecular mechanisms of Sox17 incancer development have not been elucidated.
Canonical Wnt signaling is important for maintenance of gastrointestinal epithelial stem
cells. Constitutive activation of Wnt pathway by genetic or epigenetic alterations causes tumor
development in the gastrointestinal tract. We have constructed genetically engineered mousemodels, Gan mice and ApcD716 mice, in which Wnt signaling is activated by Wnt1 gene expression3
or Apc gene mutation4, respectively. Gan mice develop gastric tumors, while ApcD716 mice develop
intestinal polyps. Moreover, we previously constructed Apc D716 Smad4 compound mutants
that develop malignant adenocarcinomas in the intestine by mutations in both Apc and Smad4
genes simultaneously5. Here, we have investigated expression of Sox17 during gastrointestinal
tumorigenesis using these mouse models. Importantly, we found that Sox17 is induced at the earlystage of tumorigenesis as a Wnt target gene, but downregulated in the malignant progressed tumors.

- 31 -

Results
We previously examined gene expression profile in Gan mouse tumors by microarray analyses6.
Expression of Sox family member genes were extracted from the data set and compared with that ofnormal gastric mucosa of the wild-type mice. Notably, Sox17 as well as Sox2, Sox4, Sox7, and Sox9
showed elevated expression in gastric tumors7 (Fig. 1A). We thus examined expression of Sox17 in
the primary cultured gastric tumor cells derived from Gan mice. In the primary culture system,
undifferentiated epithelial cells formed spheroid structure (Fig. 1B), whereas surrounding monolayercells showed differentiated morphology. We
confirmed that active form of b -catenin isaccumulated in the spheroid-forming epithelial
cells but not in the surrounding monolayer cells,
indicating Wnt activation only in the spheroid
cells. Importantly, Sox17 expression was
detected only in the nuclei of spheroid-forming
cells (Fig. 1B, right). These results strongly
suggest that Sox17 is induced as one of the Wnt
target genes in the gastric tumor cells.
On the other hand, it has been suggested
that Sox17 functions as a tumor suppressor inhuman colon cancer development through Wnt
suppression. We thus examined expression
profiles of Sox family members in human gastric cancers using public database. Interestingly, themean expression level of Sox17 was significantly decreased in malignant gastric cancer, although
Sox4 and Sox9 are still elevated (Fig. 2A). These results suggest that Sox17 is induced at earlystage of tumorigenesis, but downregulated during malignant progression. We thus examined
S ox17 e x p r e s s i o n i n m o u s ebenign and malignant intestinal
t u mor s d evelo p e d i n Ap c D716
and Apc D716 Smad4 compou nd
mice, respectively (Fig. 2B). In
the Apc D716 mice, strong Sox17
expression was detected in the
adenoma tumor cells but not in thenormal cells. In contrast, Sox17
expression was no longer detectedin the invasive malignant adenocarcinoma of the ApcD716 Smad4 mouse intestines7. These results
support the idea that Sox17 is upregulated at early stage but downregulated at the progression stageof gastrointestinal tumorigenesis.

- 32 -

Discussion & Conclusion
It has been suggested that Sox17 represses Wnt signaling activity through unknown mechanism.
Accordingly, suppression of Sox17 expression in tumors may cause Wnt activation, which
contributes to malignant progression. However, our present results indicate that Sox17 expression
is induced by Wnt activation in the gastrointestinal tumor cells at early stage. These results, taken
together, indicate that Wnt activation triggers tumor development in the gastrointestinal tract,
and that Wnt activation at the same time induces Sox17 expression, which partially represses Wnt
activation level. Accordingly, it is possible that Sox17 plays a protective role against malignant
progression by regulation of Wnt activation level at the early stage of tumorigenesis. When Sox17
expression is suppressed possibly by DNA methylation, Wnt activation level in tumor cells isincreased, resulting in malignant progression. It is therefore possible that Sox17 is a negative
regulator of Wnt signaling pathway.

References
1) Kanai-Azuma M, Kanai Y, Gad JM, et al. Depletion of definitive gut endoderm in Sox17-nullmutant mice. Development, 129: 2367-2379, 2002.
2) Zhang W, Glockner SC, Guo M, et al. Epigenetic inactivation of the canonical Wnt antagonist
SRY-box containing gene 17 in colorectal cancer. Cancer Res 68: 2764-2772, 2008.
3) Oshima H, Matsunaga A, Fujimura T, et al. Carcinogenesis in mouse stomach by simultaneous
activation of the Wnt signaling and prostaglandin E2 pathway. Gastroenterology 31: 1086-1095,
2006.
4) Oshima M, Oshima H, Kitagawa K, et al. Loss of Apc heterozygosity and abnormal tissuebuilding in nascent intestinal polyps in mice carrying a truncated Apc gene. Proc Natl Acad Sci
USA 92: 4482-4486, 1995.
5) Kitamura T, Kometani K, Hashida H, et al. SMAD4-deficient intestinal tumors recruit CCR1+
myeloid cells that promote invasion. Nat Genet, 39: 467-475, 2007.
6) Itadani H, Oshima H, Oshima M, Kotani H. Mouse gastric tumor models with prostaglandin E2
pathway activation show similar gene expression profiles to intestinal-type gastric cancer. BMC
Genomics, 10: 615, 2009.
7) Du YC, Oshima H, Oguma K, et al. Induction and down-regulation of Sox17 and its possible roles
during the course of gastrointestinal tumorigenesis. Gastroenterology, 137: 1346-1357, 2009.

- 33 -

4D imaging analysis of insulin exocytosis
Mica Ohara-Imaizumi
Department of Biochemistry, Kyorin University School of Medicine
mimaizu@ks.kyorin-u.ac.jp

Abstract
A variant of the CDKAL1 gene was reported to be associated with type 2 diabetes. To determine
the role of CDKAL1 in β-cells, we studied insulin release profiles in CDKAL1 gene knockout(KO) mice using 4D-total internal reflection fluorescence (TIRF) microscopy. 4D-TIRF imagingof CDKAL1 KO β-cells showed that the number of fusion events during first-phase insulin release
was reduced with delayed and slow cytosolic Ca2+ increase. The responsiveness of K ATP channels
to glucose was blunted in KO cells. In addition, glucose-induced ATP generation was impaired.
Although CDKAL1 is homologous to CDK5RAP1, there was no difference in CDK5 activity
between WT and KO islets. Thus, CDKAL1 controls first-phase insulin exocytosis in β-cells byfacilitating ATP generation, K ATP channel responsiveness and subsequent Ca 2+ channels activity
through pathways other than CDK5-mediated regulation.
Keywords: TIRF microscopy, CDKAL1, insulin, pancreatic β cells, exocytosis, type 2 diabetes

Introduction
Recent genome-wide association studies (GWAS) have identified CDKAL1 (cyclin-dependent
kinase 5 regulatory subunit associated protein 1-like 1) as a susceptibility gene for type 2 diabetes,
which has been replicated in several populations (Zeggini et al. 2007; Steinthorsdottir et al. 2007;
Saxena et al. 2007; Scott et al. 2007). The CDKAL1 gene is located on chromosome 6p22.3 andencodes a 65-kDa protein (CDKAL1). Although the function of CDKAL1 is still unclear, CDKAL1
is similar to cyclin-dependent kinase 5 regulatory subunit-associated protein 1 (CDK5RAP1) which
inhibits cyclin-dependent kinase 5 (CDK5) activity by binding to the CDK5 activator p35 (Ching etal. 2002).
Recently, Groenewoud et al. (2008) and Stancakova et al. (2008) reported that a CDKAL1 variant(rs7754840) decreased first-phase insulin secretion but not second-phase insulin secretion duringhyperglycaemic clamps and intravenous glucose tolerance tests, respectively. Furthermore, the genevariants were not associated with the insulin sensitivity index, suggesting that CDKAL1 variants
influence the risk of type 2 diabetes by impairing first-phase insulin secretion. However, themolecular mechanisms through which CDKAL1 modulates insulin release in pancreatic β cells are
unknown. To understand the role of CDKAL1 in β cells, we studied the phenotype of mice deficientin CDKAL1 expression established using a gene-trap technique (CDKAL1 KO mice).

- 34 -

Results
4D-TIRF imaging of CDKAL1 KO β cells showed that the number of fusion events during firstphase insulin release was reduced. However, there was no significant difference in the number offusion events during second-phase release or high K+-induced release between WT and KO cells.
CDKAL1 deletion resulted in a delayed and slow increase in cytosolic free Ca 2+ concentration
during high glucose stimulation. Patch-clamp experiments revealed that the responsiveness of ATPsensitive K+ (K ATP) channels to glucose was blunted in KO cells. In addition, glucose-induced ATP
generation was impaired. Although CDKAL1 is homologous to cyclin-dependent kinase 5 (CDK5)
regulatory subunit-associated protein 1, there was no difference in the kinase activity of CDK5
between WT and CDKAL1 KO islets.

Discussion & Conclusion
In this study, we found that pancreatic β cells from mice null for CDKAL1, which was identified
as a susceptibility gene for type 2 diabetes in humans, have a reduced first-phase insulin releasein vitro. Our results indicate that CDKAL1 controls first-phase insulin exocytosis in β cells byfacilitating ATP generation, K ATP channel responsiveness and the subsequent activity of Ca 2+channels through pathways other than CDK5-mediated regulation. Defects in the process bydecreased CDKAL1 expression levels may confer an increased risk of diabetes.

References
1) Ohara-Imaizumi M. , Yoshida M . Aoyagi K, Saito T , Okamura T , Takenaka H , Akimoto Y ,Nakamichi Y, Takanashi-Yanobu R , Nishiwaki C, Kawakami H , Kato N , Hisanaga S , Kakei M, and Nagamatsu S (2010) Deletion of CDKAL1 Affects First-phase Glucose-stimulated Insulin
Exocytosis PLoS ONE 5, e15553
2) Aoyagi K, Ohara-Imaizumi M, Nishiwaki C, Nakamichi Y, Nagamatsu S. (2010) Insulin/
phosphatidylinositol 3-kinase pathway accelerates the glucose-induced first phase insulin
secretion through TrpV2 recruitment in pancreatic beta-cells. Biochem J. 432, 375-386- 35 -

Mechanism for development and homeostatic maintenance ofhuman dendritic cell subsets
Nobuyuki Onai
Department of Biodefense Research, Medical Research Institute,Tokyo Medical and Dental University
onai.bre@mri.tmd.ac.jp

Abstract
In the mice, we isolated mouse common dendritic cell progenitor (CDP), which gave rise to only
DC subsets but not other lineage in vivo. Thus, mouse DC subsets were derived from CDP in thebone marrow. While the origin and developmental pathway of human DC subsets are completely
unknown. In this study, we try to isolate human CDP counterpart using the humanized mice. After
reconstitution of human hematopoiesis in the mouse, we found lin-CD34+CD34+Flt3+M-CSFR+ cells,
which are quite similar cell surface maker expression with mouse CDP. Interestingly, these cells
gave rise to only conventional DC in vitro. Thus, humanized mice system is very useful to identified
human DC progenitors.
Keywords: Humanized mice, progenitor, dendritic cell, Flt3, M-CSF receptor

Introduction
In the mouse lymphoid organ, resident dendritic cell (DC) sub-divided into two subsets such asplasmacytoid DC (pDC) and conventional DC (cDC)1). It has not been clear whether these two DC
subsets are derived from common progenitor or different progenitors. We have identified mousecommon DC progenitor (CDP) from bone marrow based on the expression of Flt3 and M-CSF
receptor, which are important cytokine receptor for DC development. CDP gave rise to pDC andcDC but not other lineage cells, both in vitro as single cell level and in vivo as population level2).
Quantitative RT-PCR analysis revealed that CDP highly expressed DC development affiliated genes,
which have been shown by us previously3). Thus, based on these results, we conclude that mouse
pDC and cDC are mainly derived from CDP in the bone marrow2). While the origin, DC progenitors,
and developmental pathway of human DC subset are not clear. Because it is very difficult to collecthuman sample for using experiments. Most of studies about human DC subsets have been doneby using pDC and cDC directly isolated from peripheral blood, or ex vivo culture system using
CD34+ cells. To solve this problem, we used humanized mice system, which are reconstituted
human hematopoiesis by transplanting CD34+ human stem cell fraction into sub-lethally irradiatedimmunodeficiency mice (NOG mice). Using these humanized mice, we try to identified the mouse
CDP counterpart in the humanized mice, and clarify human DC developmental pathway.

- 36 -

Results
1) For generating humanized mice, human CD34+ cells are isolated from cord blood derived fromhealthy. The lineage negative cell fractions are purified from cord blood using combination of
PE-Cy5-lebaed lineage antibodies staining and Cy5 micro-beads, and AutoMACS. After preenrichment of lineage negative cells, CD34+CD3-lin- cells are highly purified using cell sorter.
2) 1x105 cells of CD34+ CD3 -lin- cells are transplanted into sub-lethally irradiated NOG miceintravenously. These transplanted mice are maintained SPF conditions.
3) 8 weeks after transplantation, 20~25% of human CD45+ cells were detected in peripheral blood oftransplanted mice (data not shown). These human CD45+ cells are include CD19+ B cells, CD33+
myeloid cells, gp-41+ erythroid cells, and some CD3+ T cells (data not shown).
4) The human pDC (CD123+ CD11c -) and cDC (CD123+ CD11c -) subsets are detected in PB of
humanized mice.
5) In the bone marrow of humanized mice, over 75% of cells expressed human CD45, suggest thatvery nice reconstitution is established in humanized mice.
In lineage negative fraction, CD34 + CD38int and CD34+ CD38+ cells, which are long-term
hematopoietic stem cells, and multi-potent progenitor cells, respectively. In the MPP cell fraction,
we found Flt3+M-CSFR+ cells which are quite similar cell surface maker expression with murine
CDP(Fig.A)

Figure Identification of human DC progenitor in bone marrow of humanized mice.
A) Lin- cells from CD45+ human cells, are divided by CD34 and CD38.
Lin-CD34+CD38+ cells faction contained Flt3+M-CSFR+, and Flt3+M-CSFR- cells factions.
B) Sorted Flt3+M-CSFR+ cells are co-cultured with mouse stromal cells Ac-6 + human Flt3-ligand for 12 days.
These cells differentiate into CD11c+CD123- cDCs.
C) CD11chightCD123- cDCs and CD11cintCD123- cDCs are expressed cDC makers, HLA-DR and GM-CSFRa.

- 37 -

6) lin-CD34+CD38+Flt3+M-CSFR+ cells are sort out, and cultured in mouse stromal cells :Flt3-ligand
for 12 days. These cells gave rise to only cDC (CD123+CD11c-) but not pDC (CD123+CD11c-).
In cDC cells derived from these cell, included two cDC fraction, CD11chigh and CD11cint (Fig.
B). CD11chigh cells are mature DC, since these cells showed higher expression of cDC makersuch as HLA-DR and GM-CSFRa than those in CD11cint cells (Fig.C). Thus, human linCD34+CD38+Flt3+M-CSFR+ cells gave rise to cDC, but not pDC in vitro.

Discussion & Conclusion
In this study, we established humanized mice which are transplanted human CD34+ stem
cell fraction from cord blood into immunodeficiency NOG mice, and try to identify humanDC progenitor cells in the bone marrow. 8 weeks after transplantation, we detected very nicereconstitution of human hematopoiesis in PB and bone marrow. In the PB, CD19+ B cells, CD33+
myeloid cells, gp-41+ erythroid cells, pDC (CD123+ CD11c -), cDC (CD123+ CD11c -) subsets andsome CD3+ T cells are detected in the mice. These results indicated that human hematopoiesis isreconstituted in the NOG mice, and humanized mice are optimal system for human hematopoiesis
in vivo. Furthermore, we detected lin- CD34+ CD38+Flt3+M-CSFR+ cells, which are similar, cellsurface maker expression with murine CDP. These cells gave rise to only cDC but not pDC in vitro.
These results are different with murine CDP, since murine CDP gave rise to both pDC and cDC in
vitro as well as in vivo. To identify human common DC progenitor , it would be better combined
staining other cell surface maker such as CD123 (IL3 receptor) and CD116 (GM-CSFRa), sinceboth cytokines are important human DC development. Now we are preparing the manuscript ofthese results4).

References
1) Shortman, K., and S.H. Naik. Steady-state and inflammation dendritic cell development. Nat.
Rev. Immunol.7:19-30, 2007.
2) Onai, N., Obata-Onai, A., Schmid, M.A., Ohteki, T., Jarrossay, D., and Manz, M.G. Identificationof clonogenic common Flt3+M-CSFR+ plasmacytoid and conventional dendritic cell progenitors
in mouse bone marrow. Nat. Immunol., 8, 1207-1216, 2007.
3) Onai, N., Obata-Onai, A., Tussiwand, R., Lanzavecchia, A., and Manz, M.G. Activation of the
Flt3 signal transduction cascade rescues and enhances type I interferon-producing and dendritic
cell development. J. Exp. Med., 203, 227-238, 2006.
4) Onai, N., Kurabayashi, K., Hosoi, M., and Ohteki, T. Perspective isolation of human DCprogenitor using humanized mice. (in preparation).

- 38 -

Research of tumorigenesis and invasion/metastasis mediated bysmall GTPase Ral
Hisanori Horiuchi
Institute of Development, Aging and Cancer, Tohoku University
horiuchi@idac.tohoku.ac.jp

Abstract
Reduced expression of RalGAPα2 promoted the invasion and metastasis of urothelial cancer.
A gelsolin family protein, Fli-I promoted formin-induced actin assembly in collaboration with
RhoA.
Keywords: Bladder cancer, Ral, GAP, actin, rho, mDia1, Fli-I

Introduction
Ral small GTPase plays a critical role in the invasion and metastasis of cancer. Pal is activated by
Ral guanine nucleotide exchange factors (GEFs) and inactivated by Ral GTPase-activating proteins(GAPs), heterodimers of a catalytic α1 or α2 subunit and a common β subunit.
Actin reorganization is important tumor invasion and metastasis, where small GTPase Rho and its
effector formin, such as mDia1 and DAAM1, play critical roles.

Results
We found that Ral is highly activated in invasive urothelial cancer cells due to reduced expressionof the RalGAPα2 subunit. We are extensively investigating the role of RalGAP as a tumor
progression suppressor.
We identified a gelsolin family protein, Fli-I as a binding partner of Daam1 and showed its
function in the formin-induced actin assembly regulated by RhoA.

Discussion & Conclusion
RalGAPα2 might play an important role in the invasion and metastasis of urothelial cancer.
A gelsolin family protein, Fli-I plays a critical role in formin-induced actin assembly.

References
T. Higashi, T. Ikeda, T. Murakami, R. Shirakawa, M. Kawato, K. Okawa, M. Furuse, T. Kimura, T.
Kita, H. Horiuchi* (2010) Flightless-I (Fli-I) regulates the actin assembly activity of Diaphanousrelated formins (DRFs), Daam1 and mDia1, in cooperation with active Rho GTPase. J Biol Chem.
285, 16231-16238- 39 -

Hepatic fatty acid profile relates to inflammation and fibrosis
in nonalcoholic steatohepatitis
Tsuguhito Ota
Frontier Science Organization, Kanazawa University
tota@staff.kanazawa-u.ac.jp

Abstract
To delineate the hepatic fatty acid (FA) compositions which are specific to NASH and simple
steatosis, we examined the profile of long chain FAs in the liver of NAFLD in mice by gas
chromatography. We found that normal liver, simple steatosis and NASH showed significant
differences in FA content and composition in the liver of mice. Importantly, these changes inhepatic FA profiles are associated with the progression of NAFLD/NASH and whole-body insulin
resistance.
Keywords: NASH, NAFLD, fatty acid, insulin resistanceIntroduction
Insulin resistance is crucially involved in the development of nonalcoholic steatohepatitis (NASH).
We have demonstrated that insulin resistance accelerates the pathology of NASH in geneticallyand diet-induced obese rodents (Gastroenterology 132:282, 2007; Hepatology 46:1392,2007). Thesestudies clearly showed that fatty acid (FA)-induced insulin resistance leads to the progression ofhepatic inflammation and fibrosis. In the present study, we clarified the profile of long chain FAs in
the liver of nonalcoholic fatty liver disease (NAFLD) and examined the association between insulin
sensitivity, histological features, and FA profiles in the liver of NAFLD mice.

Results
NAFLD was divided into simple steatosis (SS) and steatohepatitis (SH). We used the liver ofgenetically (ob/ob) and high fat (HF) diet-induced obese (DIO) mice as an SS model. As an SH
model, the liver from methionine choline deficient and HF (MCF) diet-induced NASH, andcholesterol and HF (CL+HF) diet-induced NASH mice were studied. Normal liver (NL) fromC57Bl/6 wild type mice used as controls. FAs were extracted from liver tissue by a modificationof the method of Folch, and their content per mg of wet liver tissue was determined by gas
chromatography.
The content of most fatty acids were significantly higher in the SS and SH than in the NL mice.
Because the content of C16:0 was highest in the liver of all the groups, we compared the ratio of each
FA to C16:0 between NL, SS, and SH. The ratio of C18:0/C16:0 was higher in the order of SH, SS,
and NL groups (P<0.05, n=8) and this ratio was associated with increased insulin resistance assessedby HOMA-R. In addition, C18:2/C16:0 was markedly higher in the liver of SH than that of SS- 40 -

(Fig.1). In contrast, C18:1/C16:0 was decreased in theorder of SH, SS, and NL groups. The ratio of C18:1/
C16:0 was decreased in SH group with hepatocellularballooning and advanced inflammation and fibrosis.
These histological changes were associated withdecreased mRNA expression of fibrogenesis (TGF-b,
type 1 collagen) and TNF-a. The C16:1/C16:0 was
similar between NL, SS, and SH groups.
Fig.1

Discussion & Conclusion
The NL, SS, and NASH had markedly different FA content and composition in the liver of mice.
These changes in hepatic FA profiles were associated with the progression of NAFLD/NASH andwhole-body insulin resistance. We plan clarify the hepatic FA profile in the patients with liverbiopsy-proven 80 NAFLD including 50 with SS and 30 with SH. By comparing the results between
human and animal studies, we will delineate the hepatic FA compositions specific to NASH and SS.

References
1) Ota T, Takamura T, Kurita S, Matsuzawa N, Kita Y, Uno M, Akahori H, Misu H, Sakurai M,
Zen Y, Nakanuma Y, Kaneko S: Insulin resistance accelerates a dietary rat model of nonalcoholicsteatohepatitis. Gastroenterology 132:282-293, 2007
2) Matsuzawa N, Takamura T, Kurita S, Misu H, Ota T, Ando H, Yokoyama M, Honda M, Zen Y,
Nakanuma Y, Miyamoto K, Kaneko S: Lipid-induced oxidative stress causes steatohepatitis inmice fed an atherogenic diet. Hepatology 46:1392-1403, 2007

- 41 -

Analysis of the role of CD317/tetherin, inhibitor ofvirus budding, in the whole body
Shoichiro Miyatake
Tokyo Metropolitan Institute of Medical Science
Miyatake-si@igakuken.or.jp

Abstract
CD317/tetherin deficient mouse was established. pDCs derived from CD317/tetherin deficient mice
produced elevated level of typeI IFN upon stimulation, suggesting that CD317/tetherin transmits
suppressive signal on typeI IFN induction. CD317/tetherin is involved in the antigen presentation
activity of both MHC I and MHC II restricted antigens.
Keywords: CD317, tetherin, cytokine, dendritic cell

Introduction
CD317/tetherin is specifically expressed on mouse plasmacytoid dendritic cells (pDC) but not
on other hematopoietic cells during the homeostatic state, therefore CD317/tetherin is used as a
specific marker for mouse pDC. In addition CD317/tetherin is induced on various cell types by
typeI or typeII IFN. Ab against CD317/tetherin suppressed typeI IFN production of the activated
pDC, suggesting the presence of ligands for CD317/tetherin and their role in typeI IFN production.
CD317/tetherin inhibits virus release from cells by tethering viruses such as HIV or Ebola virus
on the plasma membrane when the viruses bud from cells. Most of its effects on viruses wererevealed by the experiments utilizing human cell lines. To elucidate the role of this molecule in thewhole body and in immune responses
against pathogens or cancer cells, amouse carrying the CD317/tetherin genef lanked by loxP recognition elements
for Cre recombinase (floxed allele) was
created.

Results
Germline disruption of CD317/tetherin
gene did not affect mouse development.
CD317/tetherin def icient mice (-/-)
appeared normal at birth, developed aswell as their control littermates and werefertile.
The number of pDC isolated from -/-FigureCD317/tetherin was not detected on various cells derived from-/- mice. Spleen cells prepared from litter control(+/+), +/- or -/mouse were stimulated with 0.5 M CpG-A for 18h. CD317/tetherin
expression on B cells, T cells, cDC and pDC were analyzed
by flow cytometry. CD317/tetherin was not detected on cells
derived from -/- mouse. The CD317/tetherin expression (Mean
Fluorescence Intensity) of cells derived from +/- mouse was lessthan half of that of litter control.

- 42 -

mice was comparable to that of the litter control. CD317/tetherin was not detected on the pDC of-/- mouse identified by the expression of B220 and CD11c (Figure). The expression level of CD317/
tetherin on pDC of heterozygous (+/-) mice was less than half of that of the litter control.
pDCs derived from -/- or litter control mice were stimulated with TLR9 ligand CpG or H1N1
influenza virus (PR8). The level of typeI IFN produced by pDC derived from -/- mice was moreelevated than that from the litter control. As expected, when pDCs were stimulated by CpG or
influenza virus in the presence of anti-CD317/tetherin Ab, typeI IFN production by pDC of the littercontrol but not -/- mice was suppressed.
Conventional DC (cDC) was prepared from -/- or litter control mice as Ag presenting cells. CD4+
T cells from OT-2 mice or CD8+ T cells from OT-1 mice were prepared for responder T cells. As
antigen OVA protein or antigenic peptide derived from OVA was used. The Ag presentation activity
to either OT-1 or OT-2 T cells was reduced when cDC was derived from -/- mice.

Discussion & Conclusion
Since CD317/tetherin disrupted mice were born without defects, grow healthy and fertile, CD317/
tetherin is not required for normal development and reproduction. CD317/tetherin deficient pDC
produced elevated level of typeI IFN compared to wild type pDC, suggesting that CD317/tetherindelivers some signal that suppresses typeI IFN production of pDC. pDCs used in this experimentwere enriched but not nearly 100% pure, thus the possibility of the requirement of any ligand
molecules expressed on other cell types for this suppressive effect was not eliminated. There are no
putative signaling motifs in the cytoplasmic region of CD317/tetherin, and the signaling pathways
that CD317/tetherin may utilize are totally unknown.
CD317/tetherin contributes to the Ag presentation activity of cDC. Both MHC II restricted Ag
presentation and MHC I restricted cross presentation activity were affected by the absence ofCD317/tetherin. CD317/tetherin is present not only on the cell surface but also in the endosome. One
possible site of its action is Ag uptake and processing in lysosomes. Since crosspresentation wasaffected, critical machinery for the crosspresentation, retrotranslocon might be affected by CD317/
tetherin.

- 43 -

Driving mechanisms for axon guidance: studies on
asymmetric membrane dynamics in the growth cone
Hiroyuki Kamiguchi
RIKEN Brain Science Institute
Kamiguchi@brain.riken.jp

Abstract
An extending axon in the developing nervous system is tipped by a growth cone, a highly motile
structure that can translate extracellular guidance information into directed migration along thecorrect path. In this project, I found that guidance cues repel growth cones via clathrin-mediatedendocytosis. Also, an intracellular signal transduction cascade has been identified that links
guidance cues to clathrin-mediated endocytosis: influx of calcium ions from the extracellular space
followed by calcineurin activation. These results demonstrate for the first time that repulsive axon
guidance is driven by polarized membrane trafficking in the growth cone.
Keywords: Axon; Growth cone; Guidance; Calcium; Calcineurin; Clathrin; Endocytosis

Introduction
The formation of functional neuronal circuits relies on the spatial accuracy of axon extension.
The tip of each extending axon, a growth cone, recognizes molecular “guidance cues” in its
microenvironment and navigates the axon along the correct path. When a growth cone migrates
in a guidance cue gradient, the cue binds its receptor preferentially on the side of the growth cone
facing higher concentrations of the cue. This asymmetric receptor occupancy polarizes the growthcone for turning either toward increasing concentrations of the cue (attraction) or away from the cue(repulsion), via intracellular generation of Ca2+ signals. Ca2+ release from the endoplasmic reticulum
mediates growth cone turning toward the side with elevated Ca2+, whereas Ca2+ influx across theplasma membrane mediates repulsive turning if unaccompanied by secondary Ca2+ release fromthe endoplasmic reticulum (Ooashi et al., 2005; Tojima et al., 2009; Akiyama et al., 2009; Tojima
et al., 2011). We have previously reported that attractive Ca2+ signals cause growth cone turning byfacilitating centrifugal transport of membrane vesicles and exocytosis ensuing in the growth cone
periphery (Tojima et al., 2007; Akiyama and Kamiguchi 2010). However, signaling and driving
mechanisms underlying repulsive axon guidance have remained unclear. Here I report how repulsive
Ca2+ signals mediate growth cone turning.

Results
Dissociated cultures of dorsal root ganglion neurons derived from chicken embryos were usedin this study. Clathrin-mediated endocytosis in live growth cones was visualized by total internal
ref lection f luorescence imaging of clathrin and dynamin. When repulsive Ca 2+ signals were

- 44 -

generated by UV-induced photolysis of caged Ca2+ on one side of the growth cone, the formationof clathrin-coated pits was facilitated only on the side with elevated Ca 2+. Similarly, directional
application of repulsive guidance cues such as Semaphorin 3A caused asymmetric facilitation ofclathrin-mediated endocytosis. Such Ca 2+-elicited facilitation of clathrin-mediated endocytosis
required the activity of calcineurin that can dephosphorylate and activate endocytic-adaptor
proteins. In contrast, attractive Ca2+ signals did not influence the frequency of clathrin-mediatedendocytosis. Pharmacological or genetic Inhibition of clathrin-mediated endocytosis abolishedgrowth cone repulsion, but not attraction, induced by Ca 2+ or extracellular physiological cues.
Intriguingly, growth cone repulsion by Semaphorin 3A or myelin-associated glycoprotein wasconverted to attraction after inhibition of clathrin-mediated endocytosis, and this attraction was
blocked by tetanus neurotoxin that blocks vesicle-associated membrane protein 2 (VAMP2)dependent exocytosis. This result suggests that guidance cues can evoke both endocytosis andexocytosis, the balance of which determines the turning direction. Furthermore, we showed thatasymmetric perturbation of the balance of endocytosis and exocytosis in the growth cone wassufficient to initiate its turning toward the side with less endocytosis or more exocytosis.
To gain insight into mechanisms of how asymmetric endocytosis drives growth cone turning, weestimated the area of plasma membrane that was removed asymmetrically from the growth conesurface in response to Semaphorin 3A gradients. The number of newly formed clathrin-coated pits
in these growth cones after Semaphorin 3A application was 0.78 ± 0.11/µm 2/min on near sides and0.38 ± 0.06 /µm 2/min on far sides (mean ± SEM; n = 7 growth cones). Assuming the diameter ofclathrin-coated pits to be 120 nm, the difference in the area of membrane removal between the nearand far sides would be 0.018 µm 2 per 1-µm 2 growth cone area (1.8%) every minute, therefore thecumulative difference during the course of growth cone turning (40 min) would correspond to 72%.
This estimation suggests that clathrin-mediated endocytosis can generate a substantial asymmetry
in membrane removal across the growth cone during repulsive guidance.

Discussion & Conclusion
In conclusion, the present and previous (Tojima et al., 2007) studies demonstrate that growth cone
repulsion and attraction involve preferential removal and addition of plasma membrane componentsof the growth cone on the side with elevated Ca 2+, respectively, and that the balance between
these counteractive events may determine the directional polarity of axon guidance (Itofusa andKamiguchi 2011).

References
1) Akiyama H, Kamiguchi H: J Biol Chem 285: 41740-41748, 2010
2) Akiyama H, Matsu-ura T, Mikoshiba K, Kamiguchi H: Sci Signal 2: ra34, 20093) Itofusa R, Kamiguchi H: Mol Cell Neurosci (in press)
4) Ooashi N, Futatsugi A, Yoshihara F, Mikoshiba K, Kamiguchi H: J Cell Biol 170: 1159-1167, 2005

- 45 -

5) Tojima T, Itofusa R, Kamiguchi H: Neuron 66: 370-377, 2010
6) Tojima T, Itofusa R, Kamiguchi H: J Neurosci 29: 7886-7897, 20097) Tojima T, Hines JH, Henley JR, Kamiguchi H: Nat Rev Neurosci 12: 191-203, 2011

- 46 -

Mechanism of pathogenesis byhuman T-cell leukemia virus type 1
Masao Matsuoka
Institute for Virus Research, Kyoto University
mmatsuok@virus.kyoto-u.ac.jp

Abstract
Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL) and HTLV1 associated myelopathy (HAM). The HTLV-1 bZIP factor (HBZ) gene, which is encoded by theminus strand of the provirus, is expressed in all ATL cases and promotes proliferation of ATL
cells. Recently, we have reported that HBZ expression causes T-cell lymphomas and inflammatory
diseases, which are very similar to HTLV-1 infection. In HBZ expressing transgenic mice, and
HTLV-1 carriers, regulatory T cells increase. As a mechanism, we found that HBZ enhanced TGF-b
signaling via interaction with Smad2/3 and p300. This activity of HBZ enhanced transcription of
Foxp3 gene, which accounts for increased regulatory T cells in HBZ expressing cells. ATL patientsand HTLV-1 infected individuals succumb to opportunistic infections due to impaired cell-mediatedimmunity. However, the mechanism of immunodeficiency remains to be solved. We found that
HBZ-transgenic mice were susceptible to herpes simplex type 2 (HSV-2) and Listeria infection.
Production of interferon-g (IFN-g) was hindered in CD4+ T cells. HBZ interacted with NFAT andc-Jun, and inhibited DNA binding of these transcription factors to the promoter region of the IFNg gene. HBZ mediated inhibition of IFN-g is a possible mechanism of immunodeficiency caused by
HTLV-1 infection. Thus, HBZ plays an important role in pathogenesis by HTLV-1.
Keywords: HTLV-1, ATL, HBZ, Foxp3, regulatory T cell

Introduction
Human T-cell leukemia virus type 1 (HTLV-1) is a causal agent of adult T-cell leukemia (ATL)
and inflammatory diseases including HTLV-1 associated myelopathy (HAM).1 HTLV-1 causes ATL
after a long latent period. HTLV-1 has regulatory genes (tax and rex) and accessory genes (p12,p13, p30, and HBZ). After discovery of HTLV-1, many studies suggest that Tax plays critical roles
in pathogenesis of HTLV-1. However, we found that Tax expression was not detected in about 60%
of ATL cases.2 We identified three mechanisms to inactivate Tax expression in vivo: 1) nonsense
mutation, deletion and insertion of tax gene, 2) DNA methylation of 5’ long terminal repeat (LTR)
and 3) deletion of 5’LTR. However, HTLV-1 bZIP factor (HBZ) gene remains intact in all ATL
cases, indicating the importance of HBZ gene in ATL cells. We have reported that the HBZ geneis expressed in all ATL cases, and HBZ promotes proliferation of ATL cells.3 Recently, we havereported that transgenic expression of HBZ causes T-cell lymphomas and inflammatory diseases
in the mice.4 These data show that HBZ is a responsible gene for pathogenesis by HTLV-1. In HBZ-

- 47 -

transgenic (HBZ-Tg) mice, the number of regulatory T cells increases. We found that HBZ enhanced
transcription of Foxp3 gene in T cells, resulting in increased regulatory T cells. However, precise
mechanism remains unsolved.
ATL and HTLV-1 carriers frequently succumb to opportunistic infections caused by fungus,
virus and bacteria. In these patients, cell mediated immunity is impaired. However, its mechanism
remains unknown. HBZ is expressed in all ATL cases and HTLV-1 carriers, indicating theassociation with immunodeficiency caused by HTLV-1. We used HBZ-Tg mice that express sHBZ
in CD4 T cells and studied well-established infection models of two pathogens. The first model
involves intravaginal viral infection with herpes simplex virus type-2 (HSV-2). Interferon-g(IFN-g
production by CD4 T cells is critical for the exclusion of HSV-2 from the host. The other model
involves infection with the Gram-positive intracellular bacterium, Listeria monocytogenes (LM),which is known as an opportunistic pathogen. In LM infection, CD4 T cells play pivotal roles in theacquired immune response by producing IFN-g and inducing the activation of macrophages, which
eliminate LM by phagocytosis and subsequent bactericidal activity.

Results
1) Effect of HBZ on TGF-b signaling
Vector expressing HBZ was transfected into HepG2 cells and then transfected cells were treatedwith TGF-b. HBZ enhanced TGF-b signaling (Figure 1). Physiological level of HBZ could rescue
the repressed TGF-b responses by Tax.
2) Mechanism how HBZ enhances TGF-b signaling.
We checked binding of HBZ with Smad proteins using immunoprecipitation assay, and foundthat HBZ physically interacted with Smad2/3 (Figure 2), and colocalized with Smad3 protein in
the nucleus.
3) Detailed interaction between Smad and p300
HBZ enhanced TGF-b signaling through its N-terminal LXXLL motif, while C-terminal
MH2 domain of Smad3 was essential for interacting with HBZ. HBZ protein enhances TGF-b
signaling in a p300 dependent manner. HBZ formed a ternary complex with Smad3 and p300
(Figure 3), and enhanced Smad3-p300 interaction.
4) Effects of HBZ enhanced TGF-b signaling
By expression HBZ in naïve T cells, HBZ enhanced the transcription of PDGFB, SOX4, CTGF,
FOXP3, RUNX1, TSC22D1 genes and suppressed the ID2 gene expression; such effects weresimilar to those by TGF-b. HBZ enhanced Foxp3 expression in mouse and human naïve T cells(Figure 4). HBZ activated Foxp3 enhancer activity through its Smad binding site. HBZ was
recruited to the Foxp3 enhancer through forming a complex with Smad3 and p300.
5) Susceptibility of HBZ-Tg mice to HSV-2
In the primary infection assay, we found no significant difference in the viral titers between nonTg and HBZ-Tg mice at day 2 after inoculation, when innate immunity is responsible for the host- 48 -

defense. In contrast, at day 6 post-infection, when acquired immunity becomes important, HBZTg mice showed significantly higher viral titers of HSV-2 than non-Tg mice. In CD4+ T cells of
HBZ-Tg mice, productions of IFN-g, TNF- a, and IL-2 were suppressed.
6) Susceptibility of HBZ-Tg to Listreria infection
The number of LM recovered from HBZ-Tg spleen on day 2 was comparable to that from nonTg mice, yet the bacterial burden in HBZ-Tg mice at day 5 was higher than that in non-Tg mice,suggesting a reduced protection in HBZ-Tg mice against LM, especially when acquired immunity
is being established.
7) Mechanism of HBZ mediated suppression on IFN-g production
We found that HBZ suppressed transcription from the promoter of IFN-g. Deletion andmutation analyses of the promoter showed that NFAT and AP-1 binding sites were responsible
for suppressive effects of HBZ. HBZ can interact with NFAT and c-Jun. Chromatin
immunoprecipitation assay revealed that HBZ inhibited DNA binding of NFAT and c-Jun to thepromoter of IFN-g gene.

Discussion & Conclusion
FoxP3 was detected in two thirds of ATL cases, and FoxP3+ regulatory T cells were increased in
HTLV-1 infected individuals. These reports suggest that ATL is a leukemia of regulatory T cells,and HTLV-1 infects regulatory T cells. We have reported that HBZ expression induces transcriptionof Foxp3 gene, indicating that HBZ plays a central role in phenotype of regulatory T cells in ATL- 49 -

cells. This study revealed the detailed mechanism how HBZ induces transcription of Foxp3 gene.
HBZ can bind to Smad2/3 and p300, resulting in activation of TGF-b signaling. Transcriptions ofother cellular genes activated by TGF-b were also enhanced by HBZ expression.
Previous studies have reported that Tax suppresses TGF-b signaling. In contrast, this study showedthat HBZ enhanced TGF- b signaling. This study showed that enhancing activity by HBZ was
predominant to suppressive effects by Tax.
It is well known that ATL patients are complicated with opportunistic infections like AIDS
patients. One possible reason is increased regulatory T cells. This study first reported that HBZ was
implicated in immunodeficiency caused by HTLV-1. HBZ directly interacts with NFAT and c-Jun,
and hinders DNA binding of these transcription factors.
As shown in this study, HBZ has pleiotropic functions for pathogenesis by HTLV-1. Further
studies will disclose detailed mechanism of HBZ mediated pathogenesis.

References
1) Matsuoka M, Jeang KT. Human T-cell leukaemia virus type 1 (HTLV-1) infectivity and cellulartransformation. Nat Rev Cancer. 2007;7(4):270-280.
2) Takeda S, Maeda M, Morikawa S, et al. Genetic and epigenetic inactivation of tax gene in adult
T-cell leukemia cells. Int J Cancer. 2004;109(4):559-567.
3) Satou Y, Yasunaga J, Yoshida M, Matsuoka M. HTLV-I basic leucine zipper factor gene mRNA
supports proliferation of adult T cell leukemia cells. Proc Natl Acad Sci U S A. 2006;103(3):720725.
4) Satou Y, Yasunaga J, Zhao T, et al. HTLV-1 bZIP Factor Induces T-Cell Lymphoma and Systemic
Inflammation In Vivo. PLoS Pathog. 2011;7(2):e1001274.

- 50 -

Development of chemical fragmentation method for glycan sequencing
Shunji Natsuka
Niigata University
natsuka@bio.sc.niigata-u.ac.jp

Abstract
Micro-heterogeneity is a fundamental character of glycans. The structural variation mainly bears
on non-reducing ends of the glycans. In vertebrates, N-acetyllactosamine structure and its relatives
with various modifications are responsible to the variation of the non-reducing ends. We developed
analytical method for the non-reducing end units. Acetyl residue on N-acetylglucosamine wasremoved under alkaline condition, and then non-reducing end units were torn by deamination
reaction with nitrite. The newly appeared reducing end, which was 2,5-anhydromannose converted
from N-acetylglucosamine, was tagged with 2-aminopyridine to become f luorescent. The
pyridylamino (PA-) glycans derived from non-reducing ends were separated and analyzed with sizefractionation and reversed phase HPLCs. We optimized and fixed the reaction conditions for the
preparation steps of the PA-non-reducing end units.
Keywords: Carbohydrate sequence, Nitrous deamination, HPLC mapping

Introduction
Development of the sequencing techniques(i.e. the Edman method for protein and theSanger method for DNA) has expanded theresearch of these biomolecules. Although
the importance of the glycan molecules
is pointed out, a general spread of theglycan research is relatively small ascompared with that of protein or DNA.
The major cause of that is in the absenceof a sequencing technique of the glycans.
In this research, I built a new glycananalytical method which combined glycanfragmentation and sensitive separation.
This method can expect to develop into thesequencing technique of the glycans.

- 51 -

Results
The acetyl groups on pyridylaminated glycans were removed by heating under strong basic
condition, and then the glycans were cleaved at the reducting end of amino sugars by nitrite
deamination. As a strong base, sodium hydroxide, potassium hydroxide, and hydrazine wereexa m i ned u nde r seve r al cond it ion s.
The condition of deamination reaction
was examined with various times andtemperat u res. From results of thoseexperiments, optimum condition was fixed
as followed: de-acetylation with1M NaOH
at 90ºC for 15 h, and deamination with 0.2
M NaNO2 on ice for 60 min. The amino
sugars at the cleaving site were converted
into 2,5-anhydro sugars. Pyridylamino
residue was tagged to the 2,5-anhydros u g a r fo r f l u o r e s c e n c e -l a b el i n g ofgenerated fragments. An aldehyde of
2,5-anhydro sugar is more reactive than the aldehyde of common sugars, which is hemiacetal. Thus,
authentic condition (1) for pyridylamination was not suitable for this case, and I re-examined thepyridylamination condition. As a result, optimum condition was fixed as followed: tagging reaction
with PA-reagent at room temperature for 30 min, and reduction reaction at 60 ºC for 30 min.
The labeled fragments were separated by two modes of HPLCs, and analyzed by two dimensional
HPLC map method. The conditions of the HPLCs were fixed, and two dimensional HPLC map of
the fragments was made.

Discussion & Conclusion
The conditions of de-N-acetylation and nitrite deamination were set up for glycan fragmentation.
The optimization of the reaction for fluorescence tagging of the fragment was carried out. From
those experiments, the conditions for the fragmentation of glycans and the fluorescence labelingwere fixed. Moreover, the system for analysis of the prepared fragments with two-dimensional
HPLC mapping was established. From now on, the development to a glycan sequencer is aimed byusing this technique. On the other hand, this technique has a possibility of becoming a new methodof screening glycan biomarkesr.

References
1) Shunji Natsuka, Sumihiro Hase. Analysis of N- and O-glycans by pyridylamination. In Method
in Molecular Biology, Vol. 76: Glycoanalysis Protocols (Hounsell,E.F. ed) Humana Press Inc.
pp101-113 (1998).
2) Sumihiro Hase, Shunji Natsuka, Hisashi Oku, Tokuji Ikenaka. Identification method for twelve
oligomannose-type sugar chains thought to be processing intermediates of glycoproteins.
Analytical Biochemistry, 167, (2) 321-326 (1987).

- 52 -

Development of the direct arylation of C-H bonds innitrogen heteroaromatics
Mamoru Tobisu
Graduate School of Engineering, Osaka University
tobisu@chem.eng.osaka-u.ac.jp

Abstract
We previously repor ted nickel-catalyzed direct C-2 ar ylation of pyridine and related
N-heteroaromatics using arylzinc reagents. Based on this reaction, C-4 and C-9 arylation of acridine
ring system is developed using rhodium and nickel catalysts, respectively.
Keywords: Acridine, Arylation, Zinc, Rhodium, Nickel

Introduction
Electron-deficient heteroaromatics, such as pyridines and quinolines, are privileged scaffolds
that occupy a central role in many bioactive or medicinally relevant compounds. Therefore,
brilliant methods for their expeditious functionalization are strongly demanded. In this context,
arylation reactions, which can directly transform the C-H bonds in pyridines to C-C bonds should
represent an efficient and environmentally-friendly method. However, such reactions have notbeen accomplished, although success has been met for both electron-rich heteroaromatics, such as
pyrroles and thiophenes and ones with relatively acidic C-H bonds, such as azoles.

Results
We have previously established a powerful C-H arylation methodology by employing the
combination of a nickel-based catalyst and arylzinc reagents (eq 1). This is the first general catalytic
method for the regioselective C-Harylation of electron-def icient

N

H

ArZnR

cat. Ni(0)NAr

up to 99 %(1)

Tobisu, M et. al. JACS, 2009, 131, 12070.

heteroaromatics. A variety of arylPhgroups can be introduced into the
2-position of pyridine derivatives.

N

In this study, we applied thisacridine

Ph2 Zncat. [RhCl(cod)]2 / PCy 3
toluene130 °C, 72 h
C-9 Aryl ati on

(2)

N
75%

catalytic arylation to acridine ringsystem, in which no hydrogenis present at the 2-position, thus
alternation of the regioselectivity
is exp e ct e d . A f t e r ext e n sive
screening of the cat alyst andconditions, we found that arylationi

Pr 2ZnNno catalyst[O]toluene
70 °C, 12 h
C-9 Alkyl ati on

Ar
Ph2 ZnN
Ar = 4-NMe2 C6 H4- 53 -

cat. Ni(cod)2 / PCy 3
toluene160 °C, 20 h
C-4 Aryl ati on(3)

Nquant.
Ar
N
40%

(4)
Ph

took place at the 9 position exclusively when [RhCl(cod)]2 was used as a catalyst precursor and PCy3
as a supporting ligand (eq 2). Cu(OTf)2, FeCl3, and InCl3 also exhibited catalytic activity for this C-9arylation of acridine, although the yields were decreased compared to the rhodium-based catalyst.
The quality of the zinc reagents proved to affect the efficiency significantly. When diphenyl zinc
prepared from PhMgBr and ZnCl2 was used directly, the yield was dramatically decreased. The useof Mg salt-free zinc reagent is essential.
We next examined the possibility of installing an alkyl substituent onto acridine by using
dialkylzinc reagents. Among the zinc reagents examined (Me2Zn, Et 2Zn, iPr2Zn), only iPr2Znafforded the expected alkylated product. Interestingly, the addition of iPr2Zn proceeded in theabsence of any transition-metal catalysts to initially form dearomatized dihydroacridine, which wasthen converted into an aromatized product by the treatment with suitable oxidizing agents (eq 3).
On the other hand, when acridine was treated with diphenylzinc in the presence of a nickel
catalyst, a mixture of C-4 and C-9 arylation product was obtained. By using 9-substituted acridine,C-4 arylation product was selectively synthesized through the nickel-catalyzed reaction (eq 4).
Although the yield was modest, the reaction was quite clean and the corresponding amount of thestarting material was recovered. Further study is on going to improve the yield of this intriguingC-4 arylation of acridine.

Discussion & Conclusion
Three methods for the regioselective functionalization of acridine have been developed. 1) C-9
arylation by rhodium catalysis, 2) C-9 alkylation without transition metal catalysts, and 3) C-4
arylation by nickel catalysis. By properly choosing the catalyst and conditions, we can directly
functionalize the C-H bonds in acridine in a regioselective manner. Most of the reported methods
for the direct arylation of heteroaromatics depends on either nucleophilic or acidic reactivity, which
limits the applicable substrates to five-membered heterocycles. By contrast, our protocol utilizes theelectrophilic reactivity of heterocycles, thus 6-membered substrates, such as pyridines and related
heteroarenes are applicable. In the first two reactions (rhodium and no catalyst), the reaction occurs
at the most electrophilic C-9 position, exemplifying our reaction design concept. On the other hand,
the last reaction catalyzed by nickel delivered C-4 arylated products. This type of selectivity cannot
be explained by nucleophilic or electrophilic reactivity. Further study toward the elucidation of themechanism of this C4-selectivity and synthetic application is currently underway.

References
Nickel-Catalyzed Reaction of Arylzinc Reagents with N-Aromatic Heterocycles: A Straightforward
Approach to C-H Bond Arylation of Electron-Deficient Heteroaromatic Compounds
M. Tobisu, I.Hyodo, N. Chatani
J. Am. Chem. Soc. 131, 12070-12071 (2009).

- 54 -

Self-Defence Mechanism for Natural Toxin-Containing Species
Keiichi Konoki
Graduate School of Agricultural Science, Tohoku University
konoki@biochem.tohoku.ac.jp

Abstract
OABP2 might play a role in self-defense of the sponge Halichondria okadai.
Keywords: okadaic acid, secondary metabolite, binding protein, self-defense

Introduction
Physiological roles of secondary metabolites in secondary metabolite-producing or -accumulating
species have not been investigated in detail. Okadaic acid (OA)-binding proteins OABP1 andOABP2 were isolated from the sponge Halichondria okadai where OA was originally isolated. Since
OABP2 is not homologous to protein phosphatase 2A, the physiological target for OA in mammals,
it might be an important substance that helps us understand self-defense of the sponge.

Results
1. Cloning and expression of OABP1
Prior to this study, the cDNA cloning of OABP1 was performed in spite of being incomplete.
In this study, we determined the complete cDNA sequence. We subcloned it into bacterial
expression vector and expressed as a fusion protein. The recombinant protein was subjected tobinding assay and the dissociation constant Kd for OA was determined.
2. Immunocytochemistry of the sponge Halichondria okadai
The sponge Halichondria okadai was thought to possess OABP2 for reducing self-intoxication.
We attempted to elucidate localization of OABP2 in the sponge, though we faced a technicalproblem to perform.
3. Sponge cell culture
The fresh sponge specimen was cut into small pieces and suspended in sterilized seawater. The
sponge cells were dissociated and successfully kept for a long period. The sponge DNA wasextracted and OABP2 DNA was amplified by PCR. We thus concluded that OABP2 is the sponge
protein.
4. Variation of OA and OABP2 contents in the sponge
In order to obtain a clue as for the function of OABP2, the sponge specimen was collected inseveral places from Japan. OA content was measured with LC-MS/MS (MRM) and OABP2
content was measured with Western blotting or ELISA using antibody.

- 55 -

Discussion & Conclusion
1. The physical distance between OABP1/OABP2 and OA in the sponge tissue might be importantfor self-defense of the sponge Halichondria okadai. Further studies should be continued toelucidate localization of OA, OABP1 and OABP2.
2. Molecular biological experiments let us conclude that OABP2 would be the sponge protein.
3. Statistical analysis would be required, though all these results indicated that OABP2 plays a rolein self-defense of the sponge.

References
Keiichi Konoki, Kaori Saito, Hiroki Matsuura, Naoyuki Sugiyama, Yuko Cho, Mari YotsuYamashita, and Kazuo Tachibana. Binding of diarrheic shellfish poisoning toxins to okadaic acidbinding proteins purified from the sponge Halichondria okadai. Bioorg. Med. Chem. 2010, 18, 76077610.

- 56 -

Palladium-Catalyzed Enantioselective Synthesis ofHighly Substituted Epoxides
Hideki Yorimitsu
Kyoto University
yori@kuchem.kyoto-u.ac.jp

Abstract
Epoxides and aziridines are among the most synthetically useful intermediates and are foundin many biologically intriguing compounds. We found a conceptually new approach to thesestrained heterocycles. Treatment of tertiary allylic alcohols with aryl halides under palladiumcatalysis resulted in arylative cyclization to form the corresponding epoxides. When we employed
enantiomerically enriched allylic alcohol bearing a bulky t-butyl group and a small methyl groupat the oxygenated carbon, the reaction proceeded with exclusive diastereoselectivity as well as
perfect chirality transfer. Similar transformations were applicable to the synthesis of aziridines andmethylenecyclopropanes, starting from allylic amines and propargyl-substituted malonate esters,
respectively.
Keywords: Epoxides, Aziridines, Methylenecyclopropanes

Introduction
Epoxides are synthetically useful intermediates and are found in many natural products andbiologically active compounds. A number of methods for the preparation of epoxides have beenreported so far, such as epoxidation of olefins, reaction of carbonyl compounds with sulfur ylides,
and ring closure of vic-halohydrins.1) However, novel synthetic methods for the construction of
epoxides have been still required because of their synthetic importance in organic chemistry.
Recently, Wolfe reported that palladium-catalyzed carboetherification/arylation reactions ofalkenes with aryl bromides have provided a very efficient way to construct multisubstituted fivemembered heterocycles.2) However, his method is limited to the synthesis of unstrained mediumsized rings. We serendipitously found that Wolfe’s carboetherification is applicable to more rigid
three-membered ring construction, which is a new synthetic strategy for the preparation of epoxides.
Here we report palladium-catalyzed reactions of readily accessible tertiary allyl alcohols with aryl
halides. We also disclose herein that similar transformations were applicable to the synthesis ofaziridines and methylenecyclopropanes, starting from allylic amines and propargyl-substituted
malonate esters, respectively.

- 57 -

Results
1) Synthesis of Epoxides by Palladium-Catalyzed Reactions of Tertiary Allyl Alcohols with
Aryl Halides3)
Treatment of enantiomerically enriched tertiary allyl alcohol bearing a t-butyl and a methylgroup at the oxygenated carbon with 1-bromonaphthalene in the presence of sodium t-butoxide
under palladium/SPhos catalysis resulted in diastereoselective arylative cyclization to provide thecorresponding chiral epoxide (Figure 1). Based on our results and Wolfe’s report,2) we assume
the reaction mechanism as follows. Oxidative addition and subsequent ligand exchange between
the palladium intermediate and allyl alcohol in the presence of the base provide arylpalladium
alkoxide. Intramolecular oxypalladation then takes place to generate (2,3-epoxyalkyl)palladium
intermediate. Finally, reductive elimination gives the desired product and regenerates Pd(0).
The diastereoselectivity would be determined in the oxypalladation step. The intermediate in
the catalytic cycle is more favorable than the intermediate off the catalytic cycle because, in theformer, the larger t-butyl group is located at the pseudoequatorial position, thus minimizing thesteric repulsion from the vinylic proton or the ligand L (SPhos) that coordinates to the palladium
center.

Figure 1. Synthesis of Optically Enriched Epoxide and Plausible Mechanism.

2) Synthesis of Aziridines by Palladium-Catalyzed Reactions of Allylamines with Aryl and
Alkenyl Halides4)
The new epoxidation reaction could be extended to carboamination for the synthesis of aziridines,
especially when the aziridination employed N-allylanilines as substrates (Figure 2). The arylative
aziridination of N- allylanilines that have a phenyl and a methyl group at the allylic position
proceeded in a diastereoselective fashion. Interestingly, the sense of the diastereoselectivity is
opposite to that of the epoxidation reaction. The steric repulsion between the phenyl group on thenitrogen and the pseudoequatorial substituent can account for the stereochemical reversal.

- 58 -

Figure 2. Arylative Aziridination.

3) Palladium-Catalyzed Arylative Cyclization of N-Allylacetamides with Aryl Halides
Yielding Benzyl-Substituted Oxazolines5)
The reactions of N-allylacetamides, instead of N-arylallylamines, with aryl halides underconditions similar to the aziridination reaction resulted in the formation of five-memberedoxazolines without providing the corresponding aziridines (Figure 3). The nucleophilicity of thenitrogen atom decreases, leading to the facile formation of the unstrained 5-membered ring.

Figure 3. Synthesis of Oxazolines.

4) Synthesis of Methylenecyclopropanes by Palladium-Catalyzed Reaction of PropargylSubstituted Malonate Esters with Aryl Halides6)
Palladium-catalyzed arylative cyclization of propargyl-substituted malonate esters with aryl
halides proved to offer a stereoselective approach to methylenecyclopropanes. The reaction
proceeds by an anti-carbopalladation pathway, which guarantees the exclusive stereocontrol of
the resulting double bond (Figure 4). The highly strained as well as densely substituted skeletons

Figure 4. Cyclopropanative Arylation for Synthesis of Methylenecyclopropanes.

- 59 -

of the products facilitate further versatile transformations with the aid of Lewis acids. For
instance, Lewis acid-mediated ring-opening etherification proceeded with perfect retention of
the configuration of the double bond, thereby being useful as a new stereo- and regioselective
approach to tetrasubstituted alkenes. (Figure 5).

Figure 5. Ring-opening Reaction of Methylenecyclopropane.

5) NHC-Cu-Catalyzed Allenyl Transfer Reactions from 2,3-Alkadien-1-ols to Imines via
Cleavage of C–C Bonds: Application to Synthesis of 3-Pyrrolines7)
Serendipitously, we found that treatment of a 2,3-butadien-1-ol derivative with imine in thepresence of a catalytic amount of a copper-NHC complex and sodium t-butoxide resulted inallenyl group transfer and that, after the product was treated in hot aqueous ethanol, cyclization
took place to yield 3-pyrrolines (Figure 6).

Figure 6. Synthesis of 3-Pyrrolines by Allenyl Transfer/Cyclization

Discussion & Conclusion
Epoxides, aziridines, and methylenecyclopropanes are extremely important classes of cyclic
compounds, especially as versatile synthetic intermediates, in organic synthesis. Although numerous
methods for the preparation of these strained rings have been reported so far, novel approaches havebeen still urgently required.
Our approach to the strained rings is uniquely characterized by the followings.
(1) Tertiary allyl alcohols and N-allylanilines are readily available, and their enantiomerically pureforms are also available by the conventional asymmetric synthesis if required. The arylative
cyclization rapidly increases molecular complexity from readily available compounds, providing
a diverse range of substituted three-membered heterocycles in short steps. The reactions canprovide strained heterocycles that are otherwise difficult to synthesize, which contributes tofurther sophistication of synthetic organic chemistry.
(2) For the last f ive years, palladium-catalyzed intramolecular carboetherif ication andcarboamination reactions have been rapidly emerging as powerful tools for the construction of
five-membered heterocycles. On the other hand, one had thought it impossible to synthesize- 60 -

epoxides and aziridines by palladium-catalyzed cyclization of allyl alcohols and amines becauseof the high strain energy arising. We found that the combination of sodium t-butoxide and thebulky biphenyl-based phosphine ligand is uniquely effective for the synthesis of epoxides andaziridines. The reactions have made a quantum leap in the field of small-ring construction.
(3) Methylenecyclopropanes (MCPs) are very useful building blocks and also found in many
bioactive compounds. However, the conventional syntheses of multisubstituted MCPs are usuallyachieved by multi-step preparations, which require preformation of cyclopropane derivatives
or allenes. These methods often encountered difficulty of controlling the stereochemistry of
the carbon-carbon double bond of MCPs. Our protocol to MCPs is highly selective and thus
unique and efficient. The highly strained as well as densely substituted skeletons of the products
facilitate further versatile transformations, which underscores the importance of the products assynthetic intermediates.
(4) 3-Pyrrolines are prominent structural motifs in natural products, and straightforward methods
for their preparation are to be explored. Our copper-catalyzed protocol is not only useful but
also mechanistically interesting because development of efficient methods for transition-metalcatalyzed selective cleavage of C—C bonds and their application have been a challenging subjectof modern organic synthesis.

References
1) (a) Sello, G.; Fumagalli, T.; Orsini, F. Curr. Org. Synth. 2006, 3, 457–476. (b) Wong, O. A.; Shi, Y.
Chem. Rev. 2008, 108, 3958–3987. (c) Yang, D. Acc. Chem. Res. 2004, 37, 497–505. (d) Aggarwal,
V. K.; Winn, C. L. Acc. Chem. Res. 2004, 37, 611–620.
2) Wolfe, J. P. Synlett 2008, 2913–2937.
3) Hayashi, S.; Yorimitsu, H.; Oshima, K. J. Am. Chem. Soc. 2009, 131, 2052–2053.
4) Hayashi, S.; Yorimitsu, H.; Oshima, K. Angew. Chem. Int. Ed. 2009, 48, 7224–7226.
5) Fujino, D.; Hayashi, S.; Yorimitsu, H.; Oshima, K. Chem. Commun. 2009, 5754–5756.
6) Fujino, D.; Yorimitsu, H.; Oshima, K. Submitted.
7) Sai, M.; Yorimitsu, H.; Oshima, K. Angew. Chem. Int. Ed. 2011, 50, 3294–3298.

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Generation of a new common marmoset glioma model
Tomotoshi Marumoto
Division of Molecular and Clinical Genetics, Medical Institute of Bioregulation,
Kyushu University
marumoto@sentan.med.kyushu-u.ac.jp

Abstract
Glioblastoma (GBM) is the most malignant form of glioma in adults. Patients with GBM die within
a year from the time of diagnosis. Therefore it is important to find a new strategy of treatment forthis severe disease. To develop a novel strategy of treatment, appropriate animal models are needed
to test the efficacy of new approaches. The Common Marmoset (CM, callithrix jacchus), a New
World primate, is considered one of the most useful experimental animals for medical researchbecause CM is similar to human especially in immune systems. In this study we have attempted togenerate a new CM glioma model using Cre controlled lentiviral vectors.
Keywords: Cancer, Glioma, Animal model, Lentiviral Vector

Introduction
Gliomas are the most common central nervous system tumor in adults. They are histologicallyclassified as astrocytomas, oligodendrogliomas and oligoastrocytomas. According to their degreeof malignancey, they are graded on the World Health Organization (WHO) grading system (Furnari
FB et al., , et al. Genes Dev 21, 2683-2710, 2007). The most malignant GradeIV gliomas are called
glioblastoma (GBM) which exhibits advanced features of malignancy, including necrosis, vascularproliferation and pleomorphism. Patients with GBMs generally die within a year from the time ofdiagnosis due to their strong resistance to conventional therapies including surgery, chemotherapy
and irradiation. Although, until recently, chemotherapy for GBMs did not substantially improveoutcomes when added to the other treatment methods, a randomized clinical trial showed thatcombined therapy of chemotherapy using temozolomide and radiation was superior to radiation
alone (Stupp R et al. N England J of Med 352, 987-996, 2005). The median survivial of the patients
who received this combination treatment was 14.6 months. This is 2.5 months superior compared
to a median survival time of the patients who received radiation alone. Combination therapy using
temozolomide and radiation is now widely used all over the world, however patients and physicians
certainly need more effective drug for GBMs. To find the new strategy of treatment for GBMs,
understanding of molecular mechanisms related to the initiation and progression of GBMs and thepresence of animal models being able to test the new strategy of treatment are still of importance.
In this study we have tried to generate a novel Common Marmoset (CM, callithrix jacchus) glioma
model using Cre controlled lentiviral vectors.

- 62 -

Results
Recently we have generated a novel mouse glioma model using Cre controlled lentiviral vectors(Marumoto et al. Nat Med 15, 110-116. 2009). This model can induce oncogenic events in a cell typeand region specific manner in adult GFAP-Cre mice. The induction of H-Ras in GFAP+ cells in the
hippocampus or subventricular zone in GFAP+/p53+/- mouse brains resulted in the generation of
GBM like tumors.
To establish the technology to induce oncogenic events in a cell type and region specific mannerin adult CM brains, we constructed pToDi lentiviral vectors (pToDi LV, Fig.1) and Cre recombinase
expressing lentiviral vectors under the control of GFAP (GFAP Cre-LV), and planned to inject both
vectors into the hippocampus and subventricular zone of adult CM. As shown in Fig.1, pToDi LVs
are capable of expressing shRNAs targeting tumor suppressors and oncogenes in the presence ofCre recombinase.

Fig.1 Cre controlled lentiviral vector, pToDi: In the presence of Cre, the stuffer
sequence, RFP, would be cut out and the oncogene would be expressed. ShRNAtargeting for tumor suppressorswould also be expressed under the control of H1
promoter.

We found that all the GFAP-Cre mice injected with pToDi LVs into the hippocampus showed theformation of GBM-like tumors (data not shown).
Next we are planning to generate a new CM GBM model by injecting both pToDi LVs and GFAPCre LVs in adult CM.

Discussion &Conclusion
To generate CM GBM modelby injecting both pToDi LVs
and GFAP-Cre LVs into thebrains of adult CM, we plan toexpress H-RasV12 which is anactivated form of H-Ras andinhibit p53 tumor suppressor
by shRNA (Fig. 2). Injectionof these vectors will probably
induce GBM formation in adult
CM. If this approach could not

Fig.2 Novel common marmoset GBM model: Injection of both Cre
controlled lentiviral vectors expressing oncogenes and Cre expressing
lentiviral vectors under the control of GFAP promoter into the brain of adult
CM can induce oncogenic events in a cell type and region specific manner.

- 63 -

induce GBM formation, we would test other combinations. For example, activation of EGFR andinactivation of p53 or p16.
If the tumor arose, we would perform pathological analyses and try to generate CM CBM cell line
in culture to characterize them.

References
1) Furnari, F.B., Fenton, T., Bachoo, R.M., Mukasa, A., Stommel, J.M., Stegh, A., Hahn, W.C.,
Ligon, K.L., Louis, D.N., Brennan, C., et al. Malignant astrocytic glioma: genetics, biology, andpaths to treatment. Genes Dev 21: 2683-2710, 2007
2) Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, Belanger K, Brandes
AA, Marosi C, Bogdahn U, Curschmann J, Janzer RC, Ludwin SK, Gorlia T, Allgeier A,
Lacombe D, Cairncross JG, Eisenhauer E, Mirimanoff RO; European Organisation for Research
and Treatment of Cancer Brain Tumor and Radiotherapy Groups; National Cancer Institute ofCanada Clinical Trials Group. Radiotherapy plus concomitant and adjuvant temozolomide forglioblastoma. N Engl J Med. 10;352(10):987-96, 2005
3) Marumoto T, Tashiro A, Friedmann-Morvinski D, Scadeng M, Soda Y, Gage FH, VermaIM. Development of a novel mouse glioma model using lentiviral vectors. Nat Med. 2009
Jan;15(1):110-6.

- 64 -

Revealing olfactory processing algorithm by the study of insects
Makoto Mizunami
Hokkaido University
mizunami@sci.hokudai.ac.jp

Abstract
Representation of odors in the primary olfactory center was studied by using calcium-imaging
techniques in the American cockroach. Presentations of each of 20 different kinds of odors toan antenna induced each specific pattern of activities in a group of 45 glomeruli in the antennal
lobe. We found no evidence of “odotopic map”, i.e., no specific relationship between the chemical
properties of odorant molecules and the location of responded glomeruli. Response to a mixture ofsome of these odors often deviated from linear summation of responses to components, indicating
non-linear interaction among glomeruli. The results provide solid basis for further examining
olfactory processing algorithm in the primary olfactory center.
Keywords: Calcium imaging, odor representation, primary olfactory center, non-linear summation,
cockroach

Introduction
Cockroaches have been used as model animals to study neural mechanisms of olfaction andolfactory learning (1,2,3). We have recently established neuroanatomical map of glomeruli in theantennal lobe, primary olfactory center, of the cockroach, Periplaneta Americana (1): Cockroaches
possess about 206 glomeruli in the antennal lobe, which could be divided into ten clusters. In eachglomerulus, axon terminals of a specific type of olfactory receptor neurons on the antennae makesynaptic connections with dendrites of a few interneurons. In this study, I examined responses ofabout 45 glomeruli in the frontal surface of the antennal lobe to 20 kinds of odors by using calciumimaging techniques.

Results
By loading the antennal lobe with a calcium-sensitive dye (calcium green AM) and by using optical
recording techniques, we measured responses of neurons in the antennal lobe to 20 different kindsof odors presented to an antenna: Responses of neurons to an odor could be optically measuredby accompanying changes of calcium concentration in the neurons. Several lines of evidence
suggested that the recorded optical signals reflect, in large part, activities of axon terminals ofreceptor neurons. Odors were selected on the basis of the results of previous studies on olfactory
receptor neurons (3). These included 1-butanol, 1-pentanol, 1-hexanol, 1-octanol, trans-2-hexen-1ol, 2-pentanol, 2-hexanol, 2-octanol, hexanon, cineol, menthol, linalool, geraniol, citral and eugenol.
After recording, the antennal lobe was stained with a dye (neutral red) to facilitate matching of the- 65 -

neural activity with neuroanatomical
map of glomeruli (Fig. 1A).
We observed that presentation of anodor induced responses in a specific
set of glomeruli, and each glomerulus
responded to a specific set of odors,
indicating overlapping, combinatorycode of odors in an array of glomeruli(Fig. 1B). We obser ved no specif ic
relationship between chemical structuresof odorant molecules and locations ofresponded glomeruli in the antennal
lobe, indicating no “odotopic map” inthe antennal lobe of the cockroach.
We also compared the response to a
mixture of odors and the responses to its
components. In some cases, responses
to a mixture of odors roughly matched
to a linear summation of responses
to components. In many other cases,
however, response to a mixt u re ofodors markedly differed from a linear

Figure 1.
A: Morphologically identified 45 glomeruli at the frontal surfaceof the antennal lobe of the cockroach.
B: Representation of odors in an array of 45 glomeruli in theantennal lobe of the cockroach. The intensity of response to anodor is shown with a color code. There is a highly overlapping
representation of odors in an array of glomeruli.

summation of responses to components, indicating non-linear, inhibitory interactions among
glomeruli in the antennal lobe.

Discussion & Conclusion
We observed a highly overlapping, combinatory code of odors in an array of glomeruli in the
primary olfactory center of cockroaches. Similar observations have been made in the olfactory
bulb in mammals and the antennal lobe of honey bees and fruit-f lies, suggesting a common
feature among olfactory systems in vertebrates and invertebrates. A characteristic feature ofodor representation in the cockroach antennal lobe is that there is no obvious spatial arrangement
regarding the chemical features of the odorant molecules. This is in contrast to that some of previous
studies in mammalian olfactory bulb and insect antennal lobe emphasized the presence of “odotopic
map” (4). Our findings provide a solid basis for further examining the odor processing in theantennal lobe in the cockroach.
We found nonlinear summation effect in the response to a mixture of odors, indicating non-linear,inhibitory interaction among glomeruli. Such interaction is likely to be mediated by GABA-ergic
local interneurons that connect among glomeruli (3). Future study using a specific antagonist of
GABA receptors will help to clarify neural mechanisms of odor processing in the antennal lobe of
the cockroach.

- 66 -

References
1) Watanabe H, Nishino H, Nishikawa M, Mizunami M, Yokohari F. (2010) Complete mappingof glomeruli based on sensory nerve branching pattern in the primary olfactory center of the
cockroach Periplaneta americana. J. Comp. Neurol. 518: 3907-3930.
2) Sakura M, Okada R, Mizunami M. (2002) Olfactory discrimination of structurally similaralcohols by cockroaches. J. Comp. Physiol. A. 188: 787-797.
3) Boeckh J, Ernst K-D. (1987) Contribution of single unit analysis in insects to an understanding ofolfactory function. J. Comp. Physiol., 161: 549-565.
4) Christensen TA, White J. (2000) Representation of Olfactory Information in the Brain, eds.
Finger TE, Silver WL, Restrepo D. (Wiley-Liss), pp. 202-232.

- 67 -

Human RNA dependent RNA polymerase and cancer stem cell
Kenkichi Masutomi
National Cancer Center Research Institute, Division of Cancer Stem Cell
kmasutom@ncc.go.jp

Abstract
A complex of hTERT, BRG1 and the nucleolar GTP-binding proteins GNL3L and nucleostemin
regulates tumor initiating cell behavior.
Keywords: Telomerase, cancer stem cell, RMRP, RdRP

Introduction
Accumulating evidence indicates that the human telomerase catalytic subunit (hTERT) contributes
to stem cell turnover and the transformation of human cells through both telomere-dependent andtelomere-independent mechanisms. Constitutive expression of hTERT not only stabilizes telomere
length and facilitates cell immortalization but also increases tumor susceptibility and alters stem
cell cycling in vivo even when telomere lengths are not limiting.

Results
We show that hTERT forms a protein complex with the SWI/SNF component BRG1 and thenucleolar GTP-binding proteins, nucleostemin (NS) or GNL3L. Although overexpression or
suppression of GNL3L or NS did not affect telomerase activity or telomere length, cells that
constitutively express NS/GNL3L exhibit increased hTERT expression. Moreover, cells that
constitutively express elevated levels of hTERT, BRG1 and NS/GNL3L exhibit increased CD133
and CD44 expression and enhanced tumorigenicity at limiting cell numbers. These phenotypes
were dependent on the expression of hTERT and BRG1 and NS immunoprecipitated RMRP. Theseobservations indicate that a complex composed of hTERT, BRG1 and NS or GNL3L participates in
the regulation of tumor initiating cell phenotypes and imply potential link between cancer stem celland RNA dependent RNA polymerase (RdRP).

- 68 -

Discussion & Conclusion
These observations demonstrate that a complex composed of hTERT, BRG1 and the nucleolar
GTP binding proteins NS and GNL3L regulates pathways implicated in the regulation of tumor
initiating cells. Overexpression of NS and GNL3L leads to altered tumor initiating cell phenotypes
in a manner that is dependent on hTERT but does not depend on either of the two non-coding RNAs
associated with hTERT, hTERC and RMRP. These observations indicate that hTERT exists at least
three complexes: one composed of hTERT and hTERC that is involved in telomere maintenance, aRNA-dependent RNA polymerase composed of hTERT and RMRP, and a third complex composedof BRG1 and NS/GNL3L that regulates Wnt/β-catenin signaling in both normal stem cells andglioblastoma derived tumor initiating cells.

References
Maida et al., “An RNA-dependent RNA polymerase formed by TERT and the RMRP RNA”; Nature
2009
Okamoto et al., “Induction of tumor initiating cells of defined genetic composition by a complex
composed of nucleostemin, hTERT and BRG1” ; revised manuscript under peer review- 69 -

Pattern formation played by growth factors signaling in the
mammalian embryo
Isao Matsuo
Osaka Medical Center and Research Institute for Maternal and Child HealthOsaka Prefectural Hospital Organization
imatsuo@mch.pref.osaka.jp

Abstract
Heparan sulfate (HS) proteoglycans modulate the activity of multiple growth factors on thecell surface and extracellular matrix. However, it remains unclear how the HS chains control
the movement and reception of growth factors into targeted receiving cells during mammalianmorphogenetic processes. In order to clarify the short-range spreading mechanism of FGF-signaling
activation by HS chains during mouse extraembryonic ectoderm (ExE) development, several
chemical reagents, which were previously proposed to specifically inhibit the movement processesof HS chains; planar transcytosis, cyotoneme (filopodia), proteolytic and heparanase cleavages were
exploited. FGF-signaling activity was affected by inhibitors for serine protease but neither for actin
filaments, heparanase, metalloproteinase, nor other proteases in the ExE. These findings suggestthat FGF-signaling activation is likely to be mediated by serine protease-dependent processes. To
verify the specificity of roles of serine protease inhibitors on activation of FGF signaling, expressionof other FGF targets and non-FGF targets were also analyzed. These above studies together suggestthat the proteolytic cleavage of HS proteoglycans through the secreted serine protease can contributeto proper FGF-signaling activation, including the spread of FGF signaling during mammalian ExE
development.
Keywords: Ext2, Heparan sulfate, FGF signaling, Protease inhibitors

Introduction
The stability, movement and reception of growth factors have been suggested to be controlled by
HS proteoglycans (Haecker et al., 2005; Bishop et al., 2007; Yan and Lin, 2009). In other words, HS
proteoglycans modulate the activity of signaling factors by controlling extracellular stabilization,
movement and retention both on the cell surface and on the ECM during development and growth.
HS proteoglycans are formed by HS-chains and core proteins via a multi-step process in the Golgi
apparatus (Haecker et al., 2005; Yan and Lin, 2009). However, it remains poorly understood how
HS chains control the movement and reception of growth factors into targeted receiving cells during
mammalian morphogenetic processes. In order to clarify the short-range spreading mechanismof FGF-signaling activation by HS chains, we have exploited several chemical reagents whichwere previously proposed to specifically inhibit the following movement processes of HS chains(Haecker et al., 2005; Yan and Lin, 2009): planar transcytosis, cyotoneme (filopodia), proteolytic
and heparanase cleavages (Fig.1).

- 70 -

Results
Since the above mechanismsof planar t ranscy tosis andcy toneme are required forthe actin cytoskeleton (Fig.1;
mo del I ), w i ld -t y p e E 6.5
embryos were treated withcytochalasin D, and then dpErk expression was examinedt o d e t e r m i n e w h e t h e r i t Fig.1: Schematic models illustrate the role of HS chains in the
movement or transport of FGF ligands.
is actin-dependent or not.
Although F-actin staining clearly showed that cytochalasin D abolished the actin cytoskeleton,
under this condition dp-Erk expression of the ExE was unchanged. These findings indicate that
FGF-signaling activity was not affected by actin filaments, suggesting that FGF-signaling activation
is likely to be mediated by actin-independent processes. Next, to determine whether proteolytic
or heparanase cleavages can mediate activation of FGF signaling (Fig.1; model II), embryos wereincubated with two serine proteinase inhibitors benzamide and phenylmethanesulfonyl fluoride(PMSF) (Fig.1; model II-1), and a heparanase inhibitor, OGT2115 (Fig.1; model II-2). The results
showed that dp-Erk expression was completely lost in the ExE of the embryos treated with serine
proteinase inhibitors. However, dp-Erk expression was unaffected by the heparanase inhibitor.
These findings suggest that the proteolytic cleavage of HS proteoglycans through the secreted
serine protease but not heparanase is involved in FGF-signaling activation via HS chains in the ExE.
Additionally, the matrix metalloproteinase inhibitor, G6001, which broadly inhibits ECM-localized
proteinase activity, did not reduce dp-Erk expression. Moreover, other four protease inhibitors, i.e.
E-64 and Chymostatin, which are specific for a cysteine protease, Pepstatin A, which is specific foran aspartic protease, Calpain inhibitor I, which is specific for Calpain and Cathepsin, did not reducethe dp-Erk expression at all. These results confirm that proteolytic release of ECM-associatedproteoglycans and other proteases with the exception of a serine protease may not primarily
participate in activation of FGF signaling via HS chains.
To further verify the specificity of roles of serine protease inhibitors on activation of FGF
signaling, we have examined the expression of other FGF targets, Cdx2 and Dusp6/Mkp3, andexpression of non-FGF targets, phosphorylation of Smad2 and Smad1/5/8 for Nodal and BMP
signaling, respectively. Consequently, after incubation with the serine protease inhibitor, both Cdx2
and Dusp6/Mkp3 expression were significantly reduced while the expressions of non-FGF targets,
pSmad2 and pSmad1/5/8, were unaffected. These findings together suggest that the proteolytic
cleavage of HS proteoglycans through the secreted serine protease can contribute to proper FGFsignaling activation, including the spread of short-range FGF signaling during ExE development.

- 71 -

Discussion & Conclusion
The present experiments are in good agreement with previous models in which the following
proposals were made to explain the modulation of the activity of multiple growth factors by HS
chains (Haecker et al., 2005; Bishop et al., 2007; Yan and Lin, 2009): HS chains mediate themovement of growth factors through diffusion or transport, via transcellular transport of vesicles,
filopodial processes (cytoneme), sheddases (proteolytic release), endoglycosidase (heparanase) andso on. Our expression analyses of FGF targets with the serine proteinase inhibitors suggest thatcell surface-tethered HS chains can spread out FGF ligands to adjacent HS-deficient cells withina short distance partly through proteolytic cleavage. It was previously thought that the complex of
HS chains and FGF ligands could be transported across longer-range distances after having beencleaved by sheddases or endoglycosidase (Yan and Lin, 2009). However, it is noteworthy that the
spreading of FGF signaling is likely to be limited to much shorter distances in the early mouseembryo than previously proposed (Hou et al., 2007).

References
1) Bishop, J.R., Schuksz, M., Esko, J.D. (2007). Heparan sulphate proteoglycans fine-tune
mammalian physiology. Nature 446, 1030-1037.
2) Haecker, U., Nybakken, K., and Perrimon, N. (2005). Heparan sulphate proteoglycans: the sweet
side of development. Nature Rev. Mol. Cell. Biol. 6, 530-541.
3) Hou, S., Maccarana, M., Min, T.H., Strate, I., and Pera, E.M. (2007). The secreted serine protease
xHtrA1 stimulates long-range FGF signaling in the early Xenopus embryo. Dev. Cell 13, 226-241.
4) Yan, D. and Lin, X. (2009). Shaping morphogen gradients by proteoglycans. Cold Spring Harb.
Perspect. Biol. 1, a002493.

- 72 -

Inhibition of USAG-1, a BMP antagonist, ameliorates hereditarykidney disease of glomerular basement membrane
Motoko Yanagita
Hakubi Project, Kyoto University Graduate School of Medicine
motoy@kuhp.kyoto-u.ac.jp

Abstract
The glomerular basement membrane (GBM) is a key component of the filtering unit in the kidney.
Mutations involving COL4A3/4/5 affect the assembly of GBM, and cause Alport syndrome, aprogressive hereditary kidney disease with no definitive therapy.
Previously, we demonstrated that the BMP antagonist USAG-1 negatively regulates the renoprotective action of bone morphogenetic protein7 (BMP7) in tubular injury during acute renal
failure. This study demonstrates that genetic ablation of USAG-1 led to significant attenuation of
disease progression and to preservation of renal function in COL4A3-/- mice, a model for human
Alport syndrome. The pathogenetic role of USAG-1 might involve crosstalk between kidney tubules
and glomerulus, since USAG-1 and BMP7 colocalized in the macula densa in the distal tubules,
lying in direct contact with glomerular mesangial cells, where BMP7 attenuated and USAG-1
enhanced the expression of MMP-12 that degrade GBM. We propose that the inhibition of USAG-1
may be developed as a novel therapeutic approach for the treatment of this hereditary disease.
Keywords: Chronic Kidney Disease, USAG-1, BMP7, BMP antagonist, prognosis

Introduction
The renal glomerular basement membrane (GBM) contributes importantly to maintenance of thestructural integrity of the glomerular capillaries. Type IV collagen is the major component of the
GBM and its mutations have been linked to the genetic disorder Alport syndrome, a progressive
hereditary kidney disease associated with sensoryneural deafness. With a genetic frequency of
about 1 in 5000 people, it counts among the more prevalent of known genetic disorders. The
disease is caused by the mutations in any one of the genes encoding the α3, α4, and α5 chains oftype IV collagen (COL4A3, COL4A4 and COL4A5), and a mutation affecting one of these chainsforming the α3/α4/α5(IV) collagen network can alter or abolish the GBM expression not only of thecorresponding chain but also of the other two chains. The GBM in Alport syndrome instead retains
the fetal α1/α1/α2(IV) collagen network, which confers an increased susceptibility to proteolytic
enzyme, leading to progressive destruction of the GBM with subsequent hematuria and proteinuria,
glomerulosclerosis, and ultimately end-stage renal disease. The current therapy is limited to dialysis
and transplantation, with a higher risk of anti-glomerular basement membrane disease in thetransplanted organs due to immune reaction against the type IV collagen chains.
BMP7 is a promising candidate to treat Alport syndrome. BMP7 belongs to the transforming- 73 -

growth factor-β (TGF-β superfamily and the kidney is the major site of BMP7 expression during
both embryogenesis and postnatal developmen. Pharmacological doses of BMP7 can repair
damaged renal tubules and preserve renal function in several models of renal diseases, including the
Col4A3 knockout model of Alport syndrome. However, the exact role of endogenous BMP7 and its
mechanism of action remain unclear. In addition, the administration of recombinant BMP7, whosetarget cells are widely expressed throughout the body, might also produce some undesired extrarenal
effect.
The local activity of endogenous BMPs is controlled by certain classes of binding molecules thatact as positive or negative regulators of BMP signaling activity. BMP antagonists function throughdirect association with BMP, thus inhibiting the binding of BMP to its receptors, and defining theboundaries of BMP activity.
The product of uterine sensitization-associated gene-1 (USAG-1) acts as a kidney-specific BMP
antagonist, and USAG-1 binds to and inhibits the biological activity of BMP7. USAG-1 is expressedin distal tubules, and colocalizes with BMP7 in distal convoluted tubules and connecting tubules.
Furthermore, USAG-1-/- mice are resistant to tubular injury such as acute renal failure and interstitial
fibrosis, and USAG-1 is the central negative regulator of BMP function in the adult kidney. Because
in adults the expression of USAG-1 is confined to the kidneys, targeting the activity of this protein
might yield safer and more kidney-specific therapies than the administration of BMP7. For this, itwill be important to first elucidate the role of USAG-1 in the pathology of progressive glomerular
injury.

Results
COL4A3 - /- m ice, a mou se model of hu ma n A lpor t sy nd rome, develop prog re ssive
glomerulonephritis associated with tubulointerstitial fibrosis leading to renal failure. To test therole of USAG-1 in the progression of end-stage renal disease originated from glomerular injury,
mice deficient in both COL4A3 gene and USAG-1 gene were generated (USAG-1-/-:COL4A3-/- mice).
A histological examination of the kidneys from USAG-1+/+:COL4A3 -/- mice revealed segmental
sclerosis and intraglomerular hemorrhage at 6 weeks of age, while these changes were almost
completely absent in USAG-1-/-:COL4A3-/- mice. At 10 weeks of age, USAG-1+/+:COL4A3-/- micedemonstrated glomerulosclerosis associated with inflammatory cell infiltration, interstitial fibrosis, tubular
atrophy and cast formation, while these changes were significantly decreased in USAG-1-/-:COL4A3-/mice. In addition, USAG-1-/-:COL4A3-/- mice showed less albuminuria and preserved renal function
in comparison to that of USAG-1+/+:COL4A3-/- mice, consistent with the results of renal histology.
Furthermore, upon aging beyond 13 weeks, USAG-1-/-:COL4A3-/- mice showed less mortality than
USAG-1+/+:COL4A3-/- mice. Renal expression of inflammatory cytokines as well as MMPs, GBMdegrading enzyme which play key roles in progression of Alport syndrome, was significantly
reduced in USAG-1-/-:COL4A3-/- mice.
Deficiency of USAG-1 significantly attenuated glomerular pathology in the COL4A3-/- mouse- 74 -

model of Alport syndrome, in spite of the absence of USAG-1 expression in glomeruli. Instead,both BMP7 and USAG-1 is expressed in the part of the distal tubule which came in contact with
its own glomerulus, the macula densa. To investigate potential mechanisms that are responsible forthe beneficial effect of USAG-1 deficiency in Alport syndrome, the effect of BMP7 and USAG-1 incultured mesangial cells was examined. The administration of BMP7 suppressed TGF-β-induced
MMP-12 up-regulation in mesangial cells, and simultaneous administration of USAG-1 antagonized
the suppressive effect of BMP7. These results indicate that USAG-1 secreted from macula densa
might enhance MMP-12 expression in the glomeruli by suppressing the inhibitory effect of BMP7
and exacerbate glomerular disease progression in Alport syndrome (Figure).

Discussion & Conclusion
At present there is no definitive therapy to prevent or slow renal disease progression in Alport
syndrome. Several studies using a mouse model of Alport syndrome have provided potential
therapies, such as MMP inhibitor, angiotensin-converting enzyme inhibitor, statins, transplantationof bone-marrow derived stem cells, and total body irradiation. The results of the present study
support the notion that therapeutic trials to inhibit the function of USAG-1 may become a novel
therapeutic approach for Alport syndrome either alone or in combination with other approaches.
A therapeutic trial targeting USAG-1 is promising because it is expected to be effective in bothglomerular and tubular injuries, and is more kidney-specific and has fewer extra-renal effects,
because the expression of USAG-1 is confined to the kidney.

References
1) Mari Tanaka, Misako Asada, Atsuko Y Higashi, Jin Nakamura, Akiko Oguchi, Mayumi Tomita,
Sachiko Yamada, Nariaki Asada, Masayuki Takase, Tomohiko Okuda, Hiroshi Kawachi, Aris N.
Economides, Elizabeth Robertson, Satoru Takahashi, Takeshi Sakurai, Roel Goldschmeding, Eri
Muso, Atsushi Fukatsu, Toru Kita, and Motoko Yanagita
Loss of the BMP antagonist, USAG-1 ameliorates disease in a mouse model of progressive
hereditary kidney disease Alport syndrome
J Clin Invest. 120 (3):768-77, 2010

- 75 -

2) Motoko Yanagita
BMP antagonists in kidney diseases
Curr Opin Invest Drugs. 11(3):315-22, 2010.
3) Atsuko Y. Higashi, Tomokatsu Ikawa, Masamichi Muramatsu, Aris N. Economides, Akira Niwa,
Tomohiko Okuda, Andrew J. Murphy, Jose Rojas, Toshio Heike, Tatsutoshi Nakahata, Hiroshi
Kawamoto, Toru Kita, and Motoko Yanagita
Direct hematological toxicity and illegitimate chromosomal recombination caused by thesystemic activation of CreERT2Journal of Immunology 182 (9):5633-40, 2009.
4) New horizons at the caudal embryos: coordinated urogenital/reproductive organ formation by
growth factor signaling.
Kentaro Suzuki, Aris Economides, Motoko Yanagita, Daniel Graf, Gen Yamada.
Curr Opin Genet Dev. 19(5):491-6, 2009
5) Motoko Yanagita
BMP modulators regulate the function of BMP during body
patterning and disease progression
Biofactors 35(2):113-9, 2009.

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Molecular analysis of social behavior
using a humanoid model mouse of autism
Toru Takumi
Hiroshima University
takumi@hiroshima-u.ac.jp

Abstract
Autism spectrum disorders (ASDs) have garnered significant attention as an important groupingof developmental brain disorders. Recent genomic studies have revealed that inherited or de novo
copy number variations (CNVs) are significantly involved in the pathophysiology of ASDs. In aprevious report from our laboratory, we generated mice with CNVs as a model of ASDs, with aduplicated mouse chromosome 7C that is orthologous to human chromosome 15q11-13. Behavioral
analyses revealed paternally duplicated (patDp/+) mice displayed abnormal behaviors resembling
the symptoms of ASDs. In the present study, we extended these findings by performing various
behavioral tests with C57BL/6J patDp/+ mice, and comprehensively measuring brain monoaminelevels with ex vivo high performance liquid chromatography. Compared with wild-type controls,
patDp/+ mice exhibited decreased locomotor and exploratory activities in the open field test,
Y-maze test, and fear-conditioning test. Furthermore, their decreased activity levels overcame
increased appetite induced by 24 hours of food deprivation in the novelty suppressed feeding test.
Serotonin levels in several brain regions of adult patDp/+ mice were lower than those of wildtype control, with no concurrent changes in brain levels of dopamine or norepinephrine. Moreover,analysis of monoamines in postnatal developmental stages demonstrated reduced brain levelsof serotonin in young patDp/+ mice. These findings suggest that a disrupted brain serotonergic
system, especially during postnatal development, may generate the phenotypes of patDp/+ mice.
Keywords: Autism, mouse model, behavior, serotonin

Introduction
Autism is a widely accepted neurodevelopmental disorder characterized by several major criteria,
including impairments in social interaction, verbal and non-verbal communication difficulties,
repetitive or rigid behavior, and restricted interest. Autism spectrum disorders (ASDs) compriseautistic disorder, Rett’s disorder, Asperger’s disorder, and pervasive developmental disorder nototherwise specified (PDD-NOS) in DSM-IV. The prevalence of ASDs was recently estimated tohave increased to 1 in 166 births. According to previous studies of twins, these disorders are highly
heritable because concordance rates for monozygotic twins (70–90%) are several fold higher thanthe corresponding rates for dizygotic twins (0–10%). Although there are many candidate genes forpathophysiology in ASDs, recent high-resolution genetic techniques revealed that a de novo copy
number variation (CNV) is also a significant risk factor for ASDs, and is a more important risk

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factor than previously recognized.
In several candidate chromosomal regions with relevance for ASDs, duplication of humanchromosome 15q11-13 is the most frequent chromosome rearrangement, found in 1–4% of
ASDs patients. Moreover, this region is an imprinting region where deletions or methylation
abnormalities of paternal and maternal alleles leads to Prader-Willi syndrome and Angelman
syndrome, respectively. In chromosomal duplication, these imprinting effects can also affect theresulting phenotypes. Maternal duplication of human chromosome 15q11-13 is believed to cause
ASDs, whereas paternal duplication usually results in a normal behavioral phenotype. However,recent findings suggest that maternal interstitial duplications may be less prevalent than previously
assumed, and indeed paternal duplication of this region may lead to autistic-like behavior.
Various neurotransmitters may be involved in the pathophysiology of ASDs. Notably, serotonin (5HT) concentrations in platelets of patients with ASDs are higher and more widely distributed thanthose of controls. However, this abnormality may not result in abnormal brain 5-HT levels because
5-HT generally does not penetrate the blood brain barrier (BBB). Nevertheless, a recent report
showed that 5-HT could cross the BBB in rats. Thus, the relationship between peripheral and central
5-HT in ASDs remains unclear, and might be more complex than established mechanisms would
indicate. As further evidence for 5-HT abnormality in ASDs, administration of selective serotonin
reuptake inhibitors (SSRIs) improved repetitive behavior which is a core symptom in ASDs.

Results
During the 120 min test period of the open field test, patDp/+ mice exhibited less distancetraveled (Fig. 1B; F1,42=5.84; p=0.02), less time spent in the center area accompanied by thigmotaxis(Fig. 1A; averaged trace images of each genotype) (Fig. 1C; time spent in center area; F1,42=11.61;
p<0.01), and less vertical activity (Fig.
1D; F1,42=10.12; p<0.01) compared with
WT mice. The ratio of center time/
total distance for the first 30 min was
also calculated as “anxiet y index”.
patDp/+ mice significantly showed thehigher ratio than WT mice (Fig. 1E;t 42 =2.2, p<0.05, t-test). These results
suggest that patDp/+ mice exhibited
increased anxiety-like behavior and/or
decreased locomotor exploration in novel
environments.
We used the novelty suppressed feeding
test to deter mine whether patDp/+
mice exhibit anxiety-like behaviors or- 78 -

are the lowered motivation
caused by reduced activity.
In this test, patDp/+ mice
had longer latencies to feed
than WT mice (Fig. 2B).
Fu r ther more, most WT
mice ate chow within 5 min,whereas approximately 60%
of patDp/+ mice did not (Fig. 2A). Kaplan-Meier survival analysis and the Mantel-Cox log-rank test
revealed that this was a significant difference (p<0.01). The amount of food consumed in the home
cage after the test and body weight decreased (%) by 24 h food deprivation were not significantly
different between patDp/+ and WT mice (t25=1.57, p=0.13, t-test, for food consumed, t25=0.51,
p=0.61 for body weight, data not shown). These results indicate that patDp/+ mice have increased
anxiety-like behavior and/or decreased activity, even when food deprivation opposes these effects.
To establish the relationship between brain neurochemistry and behavioral disruptions in patDp/+
mice, we quantified levels of biogenic monoamines ex vivo, including 5-HT and its major metabolite
5-hydroxyindoleacetic acid (5-HIAA), using HPLC in several adult brain regions. We specificallyfocused on 5-HT because it is one of the most notable molecules for the pathophysiology of ASDs.
Levels of 5-HT and 5-HIAA were significantly decreased in several brain regions from patDp/+
mice (5-HIAA; OB, Ce, Mid, t18=2.72, 2.20, 2.99, p<0.05, 5-HT; OB, Mid, t18=2.23, 2.67, p<0.05). In
contrast, there was no change in levels of the other monoamines, dopamine (DA) and norepinephrine(NE), compared with WT mice in any brain region. These findings suggest that the neurochemicaldifferences between patDp/+ and WT mice are specific for the serotonergic system. However,
metabolic rates of 5-HT turnover, determined from the ratio of 5-HIAA to 5-HT, in patDp/+ mice
were not altered compared with WT mice, suggesting that monoamine oxidase (MAO) functions at
a similar rate in both WT and patDp/+ adult mice. Collectively, these results demonstrate alterationsin the serotonergic system of patDp/+ mice that may contribute to their behavioral phenotypes.
ASDs are thought to be developmental disorders, and there are several neurodevelopmental
disorders wherein subjects exhibit low brain levels of biogenic amines. Thus, we performed acomprehensive investigation of brain monoamine levels in several brain regions of mice frompostnatal weeks 1 to 3. Two-way ANOVA revealed that 5-HT concentrations in patDp/+ mice tended
to decrease in all brain regions tested over these developmental stages (genotype effect) (Fig. 3).
We also found that DA and its metabolites homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic
acid (DOPAC) tended to increase over these developmental stages in some brain regions of patDp/+
mice, such as the pons and medulla (Fig. 3). Conversely, brain levels of NE and its metabolite
3-methoxy-4-hydroxyphenylglycol (MHPG) did not consistently increase or decrease over thesedevelopmental stages in patDp/+ mice . These results suggest that 5-HT signaling in the brains of
patDp/+ mice is altered during these developmental stages.

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Discussion & Conclusion
Major autistic phenotypes such as impaired social interaction in the 3-chamber sociability test
and perseverative responding in reversal learning tests have been reported in patDp/+ micefrom either the C57BL/6J or 129S6 strains. In the present study, we report additional behavioral
abnormalities of C57BL/6J patDp/+ mice. When conducting daily breeding procedures with theseanimals, we observed decreased movements and behavior. In support of this observation, patDp/+
mice exhibited decreased exploratory activity in the open field test. Furthermore, this abnormal
phenotype was observed in other behavioral tests, such as the Y-maze test. Collectively, thesefindings strongly suggest that patDp/+ mice have decreased exploratory activity. In an evaluationof the underlying causes of decreased exploratory activity in patDp/+ mice, motor tests showedthat patDp/+ mice possess normal motor coordination. This excludes the possibility that decreased
exploratory activity in these animals resulted from dysfunctional motor coordination. Furthermore,
lower exploratory activity in patDp/+ mice overcame increased appetite induced by 24 hours offood deprivation. As another example, in the marble burying test, patDp/+ mice buried significantly
fewer marbles than WT mice, but no difference in locomotor activity between patDp/+ and WT
mice were observed. These results further suggest that decreased activity in patDp/+ mice may
be elicited by anxiety induced by novel environmental conditions. However, decreased activity
in patDp/+ mice was observed in their home cages and no significant differences were observed- 80 -

in the elevated plus maze test. Regarding these discrepancies, it is important to note that thesemeasures are not inherently correlated and may reflect unique aspects of anxiety-like behaviors.
However, reduced locomotor activity in the open field test was not reported in 129S6 mice. Thismay be due to lower levels of spontaneous locomotor activity in 129S6 mice compared with
C57BL/6J. Interestingly, longer latencies to approach food during either eight-arm radial maze or
T-maze tests have been observed in 129S6 mice. Taken together, these findings indicate that thepatDp/+ phenotype includes decreased exploratory activity, increased fear, or decreased motivation.
In addition, patients with paternally derived duplication of 15q11-13 display motor coordination
problems. The mouse cerebellum is involved in exploratory behavior and motivational processes,
and reduced exploration and stereotyped behavior in children with autism is linked with cerebellar
hypoplasia. Thus, the cerebellum of patDp/+ mice provides an intriguing target for further study.
The results of the present study demonstrate behavioral and neurochemical abnormalities in
patDp/+ mice backcrossed to the C57BL/6J strain. These animals displayed lower locomotor/
exploratory activity in most of the activity-related behavioral tests studied, and showed decreased
5-HT levels in several brain regions during development and at adulthood. We also found that tissuelevels of DA and its metabolites were increased during development. Based on these findings, we
propose that alterations in the serotonergic system at an early stage may contribute to abnormal
behavioral phenotypes in adult patDp/+ mice.

References
1) Tamada, K., Tomonaga, S., Hatanaka, F., Nakai, N., Takao, K., Miyakawa, T., Nakatani, J. and
Takumi, T. Decreased exploratory activity in a mouse model of 15q duplication syndrome;
implications for disturbance of serotonin signaling. PLoS ONE 15, e15126, 2010.
2) Takumi, T. A humanoid mouse model of autism. Brain Dev. 32, 753-758, 2010.
3) Nakatani, J., Tamada, K., Hatanaka, F., Ise, S., Ohta, H., Inoue, K., Tomonaga, S., Watanabe, Y.,
Chung, Y. J., Banerjee, R., Iwamoto, K., Kato, T., Okazawa, M., Yamauchi, K., Tanda, K., Takao,K., Miyakawa, T., Bradley, A. and Takumi, T. Abnormal behavior in a chromosome-engineeredmouse model for human 15q11-13 duplication seen in autism. Cell 137, 1235-1246, 2009. Cover
Picture.

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Novel signaling pathway from DNA damage repair to RNAi
Hitoshi Nakayashiki
Kobe University
hnakaya@kobe-u.ac.jp

Abstract
RNA silencing is thought to have evolved as a mechanism to defend the genome against invadingviruses and endogenous transposable elements. However, exactly how genome defense against
transposable elements is triggered remains largely unknown at the molecular level. Here we showthat the conserved catalytic DD(35)E motif in integrase was crucial for inducing RNA silencing
against the LTR-retrotransposn MAGGY in the fungus Magnaporthe oryzae.
Keywords: RNAi, RNA silencing, DNA damage, LTR-retrotransposon

Introduction
To date, the molecular mechanisms leading to transposon suppression by RNA silencing havebeen demonstrated in some cases. For example, fortuitous read-through transcription of dispersed
Tc1 copies can lead to dsRNA formation as a result of a ‘snap back’ of the terminal inverted repeats(TIR), which serve as a trigger for RNA silencing against the element (1). In the case of the MuDR
transposon in maize, a locus, called Mu killer (Muk), which resulted from the inverted duplicationof a partially deleted autonomous MuDR element, produces a hairpin transcript that can form
dsRNA, which is processed into small RNAs (2). It is not clear, however, how generally applicablethese mechanisms are for triggering RNA silencing against various types of transposable elements.
Relatively long TIRs, which could serve as a source for dsRNA triggers, are detectable only incertain groups of DNA transposons. A significantly large proportion of transposable elements,
especially retroelements, do not carry inverted repeats that are any longer than the length of siRNAs(21-26 nt). In addition, if spontaneous generation of inverted duplication of transposable elementsin the genome were a prerequisite for triggering RNA silencing, it would not be a potent genome
defense mechanism.
To address molecular mechanisms that lead to the initiation of RNA silencing against
retrotransposons, we made a series of deletion and site-directed mutants of the Ty3/Gypsy LTRretrotransposon, MAGGY, identified in the phytopathogenic fungus, Magnaporthe oryzae, as amodel. Previous studies showed that the MAGGY element was targeted for RNA silencing and
DNA methylation in the original host M. oryzae strains, as well as in MAGGY-free strains whenintroduced by transformation (3,4).

- 82 -

Results
We first constructed deletion and frameshift mutants of MAGGY in the gag (Δgag), reverse
transcriptase (ΔRT) and integrase (ΔINT) domains, and examined their ability to trigger RNA
silencing. In addition, a site-directed mutant (T5244G) of the chromodomain-like module in the
integrase domain was also examined.
The mutant and wild-type MAGGY constructs were introduced into the MAGGY-free M. oryzae
strain, Br48. All the MAGGY mutants were severely impaired in transposition activity (data notshown). Northern analysis revealed that the level of MAGGY siRNA accumulation, a hallmark
of RNA silencing, differed significantly among the mutants. No detectable siRNA accumulation
was found in the Δgag and ΔRT transformants,
except for a very faint signal in one of the ΔRTtransformants. Some siRNA accumulation was
detected i n t he Δ I N T t r a n sfor ma nt s wh ile,
sur prisingly, the siR NA accumulation in theT5244G transformants was almost the same as thatin the wild-type MAGGY transformants. The typeof mutant, rather than the copy number integrated
in the genome, appeared to account for the level ofsiRNA accumulation in the transformants (data notshown), suggesting that some biological activity of
MAGGY was crucial for the induction of siRNAaccumulation in M. oryzae.
Site-directed mutations were then generated in the
N-terminus conserved signature, Zinc finger andDD(35)E motifs, which are widely conserved among
integrases of retroviruses and retrotransposons.
Intriguingly, northern analysis revealed that thelevel of siRNA accumulation was significantly
reduced in the Zinc finger (HHcc) and DDE (Dde)Fig ure 1. The levels of siR NA accumulationi nduce d by t r a n sp osit ion- def icie nt M AG GY
integrase mutants were largely dependent on the typeof mutation rather than on the copy number in the
genome. Genomic Southern (a) and northern analysis(b) of Magnaporthe oryzae transformants withtransposition-deficient MAGGY mutants. MAGGY
siRNAs were detected in (b). Equal loading of total
RNA was estimated by EtBr staining of predominantRNAs. HHcc, zinc finger mutant; Dde, catalytic coremutants; T5244G, chromodomain mutant; WT, wildtype. kb. kilo-basepairs.

mutants regardless of their genomic copy numbers, indicating a crucial role of active integrase in the
induction of RNA silencing (Fig. 1a, b). We further examined which step of retrotransposition was
blocked in the DDE mutants. The cDNA intermediate of MAGGY was formed in the DDE mutants
as well as in wild-type MAGGY. Translocation of the cDNA intermediate to the nucleus was also
not impaired in the DDE mutants (data not shown). There results suggested that the integrasemediated reaction in the nucleus could be a key process for invoking RNA silencing against theelement.

- 83 -

Discussion & Conclusion
Due to environmental factors and normal metabolic processes, hydrolytic DNA damage isestimated to occur at a rate of several 104 lesions per cell per day (5). To immediately detect andrepair DNA damage on the huge molecular complex that is the chromosome, genome surveillancesystems for DNA damage should be efficient and exhaustive. It would be highly effective if such
surveillance systems participated in generating signals for RNA silencing against transposable
elements when DNA damage caused by the elements is detected and repaired. This study indicatedthat such a connection between RNA silencing and DNA damage could indeed exist. Detailed
molecular mechanisms generating signals from DNA damage to RNA silencing are intriguing
points to address in the future as an extension from this work.

References
1) Sijen, T. & Plasterk, R. H. Transposon silencing in the Caenorhabditis elegans germ line bynatural RNA. Nature 426, 310-314 (2003).
2) Slotkin, R. K., Freeling, M. & Lisch, D. Heritable transposon silencing initiated by a naturally
occurring transposon inverted duplication. Nat. Genet. 37, 641-644 (2005).
3) Murata, T. et al. siRNA-dependent and -independent post-transcriptional cosuppression of the
LTR-retrotransposon MAGGY in the phytopathogenic fungus Magnaporthe oryzae. Nucleic
Acids Res. 35, 5987-5994 (2007).
4) Nakayashiki, H., Ikeda, K., Hashimoto, Y., Tosa, Y. & Mayama, S. Methylation is not the main
force repressing the retrotransposon MAGGY in Magnaporthe grisea. Nucleic Acids Res. 29,
278-284 (2001).
5) Bernstein,C. & Bernstein,H. Aging, Sex and DNA Repair (Academic Press Inc., San Diego, 1991).

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Identification of autoimmune susceptible genes using murine models
of autoimmune diseases.
Taku Okazaki
Institute for Genome Research, The University of Tokushima
tokazaki@genome.tokushima-u.ac.jp

Abstract
Programmed cell death 1 (PD-1) belongs to the CD28/CTLA-4 co-receptor family and negatively
regulates immune responses. PD-1KO mice serve as a useful model to analyze the influence ofgenetic background on autoimmune diseases because PD-1KO mice develop different types of
autoimmune diseases when genetic background is replaced by backcrossing. To date, we havereported that PD-1KO mice develop autoimmune dilated cardiomyopathy and gastritis, myocarditis,
polyneuropathy and sialadenitis, and accelerated type I diabetes on BALB/c, MRL, NOD-H2b, andNOD backgrounds, respectively. Based on these findings, we are trying to identify strain-specificgenetic factors that determine the target organ or magnitude of the autoimmune response.
By genetic linkage analyses using PD-1KO mice, we have identified 2 to 7 loci each for type I
diabetes, myocarditis, polyneuropathy, gastritis, and sialadenitis. We have been trying to generatecongenic mice for these loci to analyze their effect and identify their causal genes. So far, we havesucceeded in the genetic reconstitution of type I diabetes in C57BL/6 mice by introducing 4 NODderived chromosomal regions and PD-1 deficiency.
These results indicate that these 4 loci and PD-1 deficiency are sufficient for the development oftype I diabetes in C57BL/6 mice. Further analyses are expected to identify their causal genes andunravel the complete molecular pathology of the disease.
Keywords: autoimmunity, linkage analysis, co-stimulation, type I diabetes

Introduction
Since most autoimmune diseases are polygenic, analysis of the synergistic involvement of various
immune-regulators is essential for the full understanding of the molecular pathology of these
diseases. Linkage analyses in animal models of autoimmune diseases have provided informative
results on the polygenic regulation of autoimmune diseases. Many autoimmune susceptible locihave been identified in each animal model and the immunological function of each locus has been
vigorously analyzed by generating various congenic animals. However, the mutual relationship ofthese loci has not been analyzed well because of the many possible combinations and rather limited
number of congenic mice, which can be established only by repeated backcrossing. Moreover, thelow resolution of the linkage analyses on polygenic diseases prevented identification of a single genefrom each susceptibility locus. Taken together, our knowledge about the polygenic regulation of
autoimmune diseases is very limited.

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Programmed cell death 1 (PD1), an immuno-receptor belonging
t o t he C D28/C T L A- 4 fa m i ly
provides negative co-stimulation to
antigen stimulation by recruiting
the protein tyrosine phosphatase,
SHP-2 (src homology 2-domain
containing tyrosine phosphatase2).1 PD -1 def iciency has beenshown to accelerate autoimmune
pre d isp osit ion a nd t o i nduce

Figure 1. Autoimmune diseases in PD-1KO mice. PD-1KO
autoi m mu ne diseases.1-3 PD -1 mice develop lupus-like glomerulonephritis and arthritis on
the C57BL/6 background, autoimmune dilated cardiomyopathy
knock out (PD-1KO) mice develop and gastritis on the BALB/c background, acute type I diabetes
lupus-like glomer uloneph r itis on the NOD background, and lethal myocarditis on the MRL
background.
a nd a r th r it is on t he C57BL/6
background, autoimmune dilated cardiomyopathy and gastritis on the BALB/c background,lethal myocarditis on the MRL background, acute type I diabetes on the NOD background, andpolyneuropathy, sialadenits, and gastritis on the NOD.H2b background (Figure 1). The development
of different forms of autoimmune diseases on different genetic backgrounds of mice indicates that
autoimmune phenotypes of PD-1KO mice are influenced by other genetic factors. Conversely, we
may be able to identify strain specific autoimmune susceptible genes more efficiently by using PD1KO mice.

Results
We have performed genetic linkage analyses of type I diabetes using F2 and BC1 progenies of
NOD-PD-1KO mice and C57BL/6-PD-1KO mice. Among more than 20 reported loci for type I
diabetes (Idd loci), only 2 of them (Idd1 and Idd17) showed significant association to type I diabetes
in the BC1 experiment. In the F2 experiment, we identified two new susceptible loci for type I
diabetes (Iddp1 and Iddp2), which functioned in a dominant fashion.4 Then, we started to generatecongenic mice in which these 4 loci are derived from NOD mice but all the other chromosomal
regions are derived from C57BL/6 mice (C57BL/6.Idd1/17/p1/p2 mice). As expected C57BL/6.
Idd1/17/p1/p2-PD-1KO mice developed type I diabetes, indicating that these 4 loci are sufficient forthe development of type I diabetes in C57BL/6-PD-1KO mice. We have planed to generate 10 linesof sub-congenic mice that harbor shorter NOD-derived chromosomal regions. To date, 3 of them
have been successfully generated and the other lines are under construction.
Because Idd1, which contains MHC genes showed very high association to type I diabetes in
the linkage analysis, we have introduced NOD congenic mice carrying C57BL/10-derived MHC
haplotype (NOD.H2b) from Jackson laboratory. NOD.H2b mice did not develop type I diabetes
even in the absence of PD-1 gene as expected. However, NOD.H2b-PD-1KO mice developed- 86 -

several autoimmune phenotypes including polyneuropathy, gastritis, and sialadenitis.2 By genetic
linkage analyses using BC1 progenies of NOD.H2b-PD-1KO mice and C57BL/6-PD-1KO mice,
we have identified 7 loci for polyneuropathy. In addition, 2, 4, and 7 loci for sialadenitis, gastritis,
and vasculitis, respectively were identified.5 Based on these findings, we have started to generatevarious kinds of congenic mice that may either develop or escape from each phenotypes. To date,the backcrossing has been repeated for 3 to 5 generations.
Next, we have performed genetic linkage analyses of myocarditis using F2 progenies of MRLPD-1KO mice and BALB/c-PD-1KO mice and identified 5 and 2 loci on MRL and BALB/c chromosomes, respectively. We have started to introduce 5 MRL loci into BALB/c mice bybackcrossing. To date, the backcrossing has been repeated for 5 generations and we have started theintercross to generate homozygous mice.

Discussion & Conclusion
In the current project, we have been trying to identify genetic factors that function together
with PD-1 deficiency to induce autoimmune diseases. It is noteworthy that type I diabetes was
genetically reconstituted in diabetes-resistant strain, C57BL/6 mice by introducing 4 NOD-derived
chromosomal regions and PD-1 deficiency. The successful reconstitution indicates that these 5
genetic factors are sufficient for the development of type I diabetes in C57BL/6 mice and that theclarification of these factors leads to the comprehensive understanding of the molecular mechanismsof type I diabetes.
The generation of congenic mice needs a long time and enormous efforts. For example, we aretrying to introduce 5 MRL-derived chromosomal regions into BALB/c mice. The probability of atransmission of a 50 cM chromosomal region from a heterozygous parent to its progeny without
internal recombination is 1 out of 4. Therefore, we have to generate more than 1,000 (45=1024) mice
to make one generation forward. In addition, it is still hard to make these regions homozygous. The
expectation value for the progeny that is homozygous for all 5 chromosomal regions (50 cM each) is
1 out of 1,048,576 (165). Although the actual frequencies are higher than these values because mostof the regions are shorter than 50 cM and we can make 5 regions homozygous in a stepwise manner,
the backcrossing took longer time than expected. Although we are aware that we have to improve
our system, the generation of congenic mice is the only method that allows us to examine thesufficiency of identified loci for the disease development and congenic mice would be irreplaceable
materials for further analyses when diseases are successfully reconstituted.
More and more practical studies are favored these days and grants for basic and time-consuming
studies like us are limited. Therefore, we really appreciate the generous support by the Novartis
Foundation (Japan) for the Promotion of Science.

- 87 -

References
1) Okazaki T and Honjo T. The PD-1-PD-L pathway and immunological tolerance. Trends.
Immunol. 27:195-201 (2006)
2) Yoshida T, Jiang F, Honjo T, and Okazaki T. PD-1 deficiency reveals various tissue-specificautoimmunity by H-2b and dose-dependent requirement of H-2g7 for diabetes on NOD
background. Proc. Natl. Acad. Sci. USA. 105:3533-3538 (2008)
3) Wang J, Okazaki IM, Yoshida T, Chikuma S, Kato Y, Nakaki F, Hiai H, Honjo T, Okazaki T.
PD-1 deficiency results in the development of fatal myocarditis in MRL mice. Int Immunol.
22:443-52 (2010)
4) Wang J, Yoshida T, Nakaki F, Hiai H, Okazaki T, and Honjo T. Establishment of NOD-Pdcd1–/–
mice as an efficient animal model of type I diabetes. Proc. Natl. Acad. Sci. USA. 102:11823-11828(2005)
5) Jiang F, Yoshida T, Nakaki F, Terawaki S, Chikuma S, Kato Y, Okazaki IM, Honjo T, andOkazaki T. Identification of QTLs that modify peripheral neuropathy in NOD.H2b –Pdcd1–/– mice.
Int. Immunol. 21:499-509 (2009)- 88 -

The Mechanisms How Myocardial Ischemia-reperfusion Initiates
Inflammatory Response in the Myocardium
Masafumi Takahashi
Division of Bioimaging Sciences,
Center for Molecular Medicine, Jichi Medical University
masafumi2@jichi.ac.jp

Abstract
Inflammation plays a key role in the pathophysiology of myocardial ischemia-reperfusion (I/R)
injury; however, the mechanism by which myocardial I/R induces inflammation remains unclear.
Recent evidence indicates that a sterile inflammatory response triggered by tissue damage ismediated through a multiple-protein complex called the inflammasome. In the present study,
we investigated the role of inflammasome in myocardial I/R injury. Here, we have shown that
inflammasomes are formed by I/R and that its subsequent activation of inflammasomes leads tointerleukin-1β (IL-1β) production, resulting in inflammatory responses such as inflammatory cell
infiltration and cytokine expression in the heart. In mice deficient for ASC (apoptosis-associatedspeck-like adaptor protein) and caspase-1, these inflammatory responses and subsequent injuries,
including infarct development and myocardial fibrosis and dysfunction, were markedly diminished.
Our results demonstrate the molecular basis for the initial inflammatory response after I/R andsuggest that the inflammasome is a potential novel therapeutic target for preventing myocardial I/R
injury.
Keywords: Cytokine, Heart, Inflammation, Interleukin, Leukocyte

Introduction
Increasing evidence indicates that inflammation is involved in the pathophysiology of myocardial
I/R injury [1]. One prominent and early mediator for inflammation in I/R injury is IL-1β [2].
In the generation of IL-1β, pro-IL-1β, an inactive precursor, undergoes proteolysis by theconverting enzyme caspase-1. Caspase-1 is activated within a cytosolic multiprotein complex—
the inflammasome. The inflammasome contains adaptor protein ASC which in turn recruits andactivates caspase-1 [3,4]. Therefore, we hypothesized that the inflammasome is an initial sensor
for danger signal(s) in myocardial I/R injury. In the present study, we investigated the role of theinflammasome and ASC in the pathophysiology of myocardial I/R injury.

Results
We first investigated whether ASC was expressed in myocardial tissues obtained from patients
who had died after an acute myocardial infarction and found that ASC was highly expressed in
the infiltrated inflammatory cells and weakly expressed in cardiomyocytes and the interstitial- 89 -

cells. Similarly, when experimental myocardial I/R was produced by occluding the left anterior
descending followed by reperfusion in mice (C57BL/6), ASC were clearly expressed at the site of
the injured myocardium. To determine whether ASC is critical for myocardial I/R injury, we used
mice deficient for ASC. ASC deficiency clearly reduced myocardial infarct areas and improved
function in a murine model of myocardial I/R injury. Furthermore, ASC deficiency attenuatedinflammatory responses, such as the infiltration of macrophages and neutrophils and the expressionof inflammatory cytokines, in the injured myocardium. However, the capillary density was not
influenced by ASC deficiency. Supporting this finding, flow cytometry analysis showed no effectof ASC deficiency on the percentage of peripheral CD34+/Flk-1+ cells (an ordinary endothelial
progenitor cell marker) after myocardial I/R injury.

Discussion & Conclusion
There is increasing evidence that indicates the importance of inflammation in the pathophysiology
of myocardial I/R injury [1]. For instance, interventions targeted at leukocytes or inflammatory
mediators substantially reduce myocardial I/R injury. In particular, the neutralization of IL-1
reduces injury, suggesting that IL-1β is the key mediator in the pathophysiology of myocardial I/
R injury [2,5]. This finding is supported by the observed reduction in injury after myocardial
infarction in the absence of caspase-1 activity. However, recent evidence indicates that sterile
inflammation is triggered by tissue damage, and endogenous danger signals are mediated by the
inflammasome. Thus, we hypothesized that the inflammasome is an initial sensor in myocardial
I/R injury and showed that ASC deficiency considerably improves inf lammatory responses
and subsequent injury after myocardial I/R [6]. These findings highlight the importance of theinflammasome in the initial inflammatory response to myocardial I/R injury.
Several limitations in this study should be noted. First, although the present study demonstrated
that inhibition of initial inf lammation improved cardiac function and remodeling after I/R,
inflammation in myocardial I/R has been shown to be not only deleterious not also beneficial in theprocess of myocardial damage and remodeling. Second, although the inflammatory response after
myocardial infarction in mice shares many common characteristics with higher mammalian species
and is a excellent model for exploration of cellular and molecular mechanisms, we should understandthe potential limitations of extrapolating data from mice to humans. Thus, further investigations arenecessary to understand the precise role of inflammasome in myocardial I/R injury.
In summary, this study is the first to demonstrate the essential role of the inflammasome in thepathophysiology of myocardial I/R injury. Moreover, our results suggest that the inflammasome is a
novel therapeutic target for preventing myocardial I/R injury.

- 90 -

References
1) D.M. Yellon, D.J. Hausenloy, Myocardial reperfusion injury, N Engl J Med 357 (2007) 1121-1135.
2) A.A. Wanderer, Ischemic-reperfusion syndromes: biochemical and immunologic rationale forIL-1 targeted therapy, Clin Immunol 128 (2008) 127-132.
3) F. Martinon, A. Mayor, J. Tschopp, The inflammasomes: guardians of the body, Annu Rev
Immunol 27 (2009) 229-265.
4) N. Yajima, M. Takahashi, H. Morimoto, Y. Shiba, Y. Takahashi, J. Masumoto, H. Ise, J. Sagara,
J. Nakayama, S. Taniguchi, U. Ikeda, Critical role of bone marrow apoptosis-associated specklike protein, an inflammasome adaptor molecule, in neointimal formation after vascular injury in
mice, Circulation 117 (2008) 3079-3087.
5) M.W. Hwang, A. Matsumori, Y. Furukawa, K. Ono, M. Okada, A. Iwasaki, M. Hara, T.
Miyamoto, M. Touma, S. Sasayama, Neutralization of interleukin-1beta in the acute phase of
myocardial infarction promotes the progression of left ventricular remodeling, J Am Coll Cardiol
38 (2001) 1546-1553.
6) M. Kawaguchi, M. Takahashi, T. Hata, Y. Kashima, F. Usui, H. Morimoto, A. Izawa, Y.
Takahashi, J. Masumoto, J. Koyama, M. Hongo, T. Noda, J. Nakayama, J. Sagara, S. Taniguchi,
U. Ikeda, Inflammasome activation of cardiac fibroblasts is essential for myocardial ischemia/
reperfusion injury, Circulation 123 (2011) 594-604.

- 91 -

Novel strategy towards therapeutic approach for lissencephaly using
calpain inhibition.
Hirotsune Shinji
Osaka City University Graduate School of Medicine
shinjih@med.osaka-cu.ac.jp

Abstract
Lissencephaly is a devastating neurological disorder characterized by smooth cerebral surface,
thick cortex and dilated ventricules due to defective neuronal migration. LIS1 was identified as
the gene mutated in lissencephaly patients, and was found to be a regulator of cytoplasmic dynein.
Interestingly, more than half of LIS1 protein is degraded at the cell cortex after transport to the plusend of MTs via calpain-dependent proteolysis. We demonstrated that an inhibition or knockdown of
calpain protected LIS1 from proteolysis resulting in the augmentation of LIS1 levels in Lis1+/- mouseembryonic fibroblast (MEF) cells, which lead to rescue of the aberrant distribution of cytoplasmic
dynein and intracellular components including mitochondria. We also show that presence of
calpain inhibitors improves neuronal migration of Lis1+/- cerebellar granular neurons. Strikingly,
intra-peritoneal injection of ALLN to pregnant Lis1+/- dams rescued apoptotic neuronal cell
death, reduction of brain weight and neuronal migration defects in Lis1+/- offspring. Furthermore,
knockdown of calpain during neuronal migration by in utero injection of stRNA against calpain
rescued cortical layering in Lis1+/- mice. Thus, our findings suggest that the inhibition of calpain isa potential therapeutic intervention for the devastating human disorder lissencephaly.
Keywords: Lissencephaly, Calpain inhibitor, Haploinsufficiency

Introduction
Lissencephaly is a severe human neuronal migration defect characterized by a smooth cerebral
surface, mental retardation and seizures. LIS1 was the first gene cloned in any organism importantfor neuronal migration, as it was deleted or mutated in humans with lissencephaly in a heterozygous
fashion. LIS1 and its binding protein, NDEL1 regulate cytoplasmic dynein. We recently reported
that LIS1 and NDEL1 cooperatively regulate anterograde transport of cytoplasmic dynein in akinesin dependent fashion. In addition, NDEL1 also regulates microtubule organization with
Aurora-A, which is essential for neurite extension. In the series of experiments, we found that halfof LIS1 is degraded via calpain-dependent proteolysis, and that inhibition or knockdown of calpains
protects LIS1 from proteolysis, which suggests that calpain inhibitor will be applicable towards
therapeutic intervention for lissencephaly.

- 92 -

Results
1. We first examined the response of LIS1 protein levels to
the inhibition of calpain by ALLN using mouse embryonic

Fig. 1

fibroblast (MEF) cells established from Lis1+/+ or Lis1+/embryos. We explored time course of the response. LIS1
protein gradually increased in the Lis1+/- MEF cells, andreached to plateau two hours after the start of ALLN treatment(Fig. 1). After two hours of administration of ALLN, LIS1
protein was increased from 0.5 (where 1.0 is equivalent towild-types levels of LIS1) to 1.1. To address the effect of othercalpain inhibitors, we also examined the effect E64d andobtained similar results (Fig. 1).
2. We previously demonstrated that LIS1 is required for plus-end directed transport of cytoplasmic
dynein5. When Lis1 is mutated, the centrosomal accumulation of LIS1 is reduced, resulting in
the impairment of plus-end directed transportof cytoplasmic dynein. As a result, cytoplasmic

Fig. 2dynein is abnormally accumulated around thecentrosome. Therefore, we determined whether
preventing the degradation of LIS1 rescued the
distribution of LIS1 and cytoplasmic dynein
within the Lis1+/- MEF cells. Treatment of Lis1+/MEF cells by ALLN or E64d clearly improved
the reduction of centrosomal concentration of
LIS1 after 2 hours of the treatment.
In addition, the abnor mal accumulation ofcytoplasmic dynein around the centrosome was
rescued by ALLN or E64d treatment (Fig. 2).
3. To define the effect of calpain inhibitors on mammalian neuronal migration, we used mouse
cerebellar granule neurons in an in vitro
migration assay combined with ALLN or E64d
treatment. We previously showed that Lis1+/–
cerebellar granule neurons displayed defective
migration compared with Lis1+/+ cerebellar
granule neurons. We confirmed that Lis1+/–
cerebellar granule neurons displayed a leftward
shift of the bin dist ribution of migration
distance, and the mean distance decreased by
50% from the WT level. We next tested whether- 93 -

Fig. 3

ALLN or E64d treatment was sufficient to rescue the migration defect in Lis1+/– cerebellar
granule neurons. Lis1+/– cerebellar granule neurons in the presence of ALLN or E64d displayeda shift in migration distance bins back toward the right, with a similar distribution to that of WT
neurons (Fig. 3).
4. We next examine whether the administration of ALLN to pregnant Lis1+/- dams rescued
defective neuronal migration in vivo. In Lis1+/- mice, apoptotic cell death was clearly increased
as the previous report. In contrast, administration of ALLN suppressed apoptotic cell death in
Lis1+/- mice. We next examined the effect of ALLN on the cortical and hippocampal layeringof neurons. Lis1+/- mice exhibited laminar splitting and discontinuities of pyramidal cells
in the CA3 and CA2 region of the hippocampus. After administration of ALLN in utero,
Lis1+/- mice also displayed splitting and diffuse pyramidal cells of the hippocampus, but these
defects were markedly improved (Fig. 4). To examine cortical lamination, we analyzed Brn1 immunoreactivity, to label interneurons of layer 2 and 3. In Lis1+/- mice, Brn-1 positiveFig. 4cells (which migrate at a laterst ages) ex h ibited a broader
dist r ibution compared to
Lis1+/+ mice. Administration of
ALLN resulted in more packed
lamination in Lis1+/- mice.

Discussion & Conclusion
LIS1 levels correlate with in vivo phenotypes in mice and humans. Reductions of LIS1 protein
result in progressively more severe phenotypes in mice in a dose-dependent fashion. Recently, it
was shown that increased LIS1 expression affects human and mouse brain development. In ourcase, inhibition of calpain activity results in normalization close to the wild type levels rather thanaccumulation of LIS1 in excess. We believe that the restoration of more normal LIS1 levels was one
among others that calpain inhibition resulted in phenotypic improvement of Lis1+/- mice.
In spite of this encouraging first step in a preclinical model, several problems remain and must be
overcome if this promising avenue of therapy can be eventually tested in human lissencephaly. It
will probably be necessary to further refine the use of calpain inhibitors or select other drugs for theeffective inhibition of LIS1 degradation. In addition, the safe and effective delivery of such drugs
at early enough time points to achieve effective blood concentration will be essential for clinicallyeffective treatment of human lissencephaly. Although it will not treat lissencephaly, there may besome benefits to restore LIS1 to more normal levels even after birth, since this may improve theoften difficult to treat epilepsy displayed by patients with lissencephaly. To treat postnatal patients,
delivering therapeutic agents across blood-brain barrier (BBB) will be essential. In spite of thechallenges, our work provides a potential avenue to consider therapeutic strategies for early brain
developmental defects such as lissencephaly due to LIS1 mutations, or any other disorder that resultsfrom haploinsufficiency.

- 94 -

References
1) Torisawa, T., Nakayama, A., Furuta, K., Yamada, M., Hirotsune, S., Toyoshima, YY. (2010)
Functional dissection of LIS1 and NDEL1 towards understanding the molecular mechanism ofcytoplasmic dynein regulation. J Biol Chem (in press)
2) Pramparo, T., Youn, YH., Yingling, J., Hirotsune, S., Wynshaw-Boris A. (2010) Novel embryonicneuronal migration and proliferation defects in Dcx mutant mice are exacerbated by Lis1
reduction. J Neurosci 30: 3002-3012.
3) Yamada, M., Toba, S., Takitoh, T., Yoshida, Y., Mori, D., Nakamura, T., Iwane, A. H., Yanagida,T., Imai, H., Yu-Lee, L. Y., Schroer, T., Wynshaw-Boris, A., and Hirotsune, S. (2010) mNUDC
is required for plus-end-directed transport of cytoplasmic dynein and dynactins by kinesin-1,
EMBO J. 29: 517-531.
4) Youn, Y. H., Pramparo, T., Hirotsune, S., and Wynshaw-Boris, A. (2009) Distinct dose-dependent
cortical neuronal migration and neurite extension defects in Lis1 and Ndel1 mutant mice, J
Neurosci 29, 15520-15530.
5) Yamada, M., Yoshida, Y., Mori, D., Takitoh, T., Kengaku, M., Umeshima, H., Takao, K.,
Miyakawa, T., Sato, M., Sorimachi, H., Wynshaw-Boris, A., and Hirotsune, S. (2009) Inhibitionof calpain increases LIS1 expression and partially rescues in vivo phenotypes in a mouse modelof lissencephaly, Nat Med 15, 1202-1207.
6) Mori, D., Yamada, M., Mimori-Kiyosue, Y., Shirai, Y., Suzuki, A., Ohno, S., Saya, H., WynshawBoris, A., and Hirotsune, S. (2009) An essential role of the aPKC-Aurora A-NDEL1 pathway inneurite elongation by modulation of microtubule dynamics, Nat Cell Biol 11, 1057-1068.

- 95 -

III.
Reports from the Recipients of Grants
for International Meetings- 96 -

The 5th International Symposium on Filoviruses (Filo2010)
1. Representative
Yoshihiro Kawaoka, D.V.M., Ph.D.
Institute of Medical Science, The University of Tokyo
2. Opening period and Place
April 18 (Sun) - 21 (Wed), 2010
Sheraton Miyako Hotel Tokyo (Daigo, Saga, Ise, Magnolia)
3. Number of participants / Number of participating countries and areas
107 participants from 15 countries
4. Total cost
¥23,702,085 JPY
5. Main use of subsidy
Travel expenses for invited speakers
6. Result and Impression
Due to the volcanic eruption in Iceland, many of the registrants and speakers could not fly toTokyo. It caused program change and great decrease of the number of participants. Still meeting
itself was a great success and closed with standing ovation to the organizers from the audience.
7. Additional description
There was a great accomplishment in the filoviruses research, the organizers and theparticipants are now planning to publish the original work on filoviruses as a supplement to the
Journal of Infectious Diseases.

- 97 -

The International Symposium on Protein Community
1. Representative
Toshiya Endo (Nagoya University)
2. Opening period and Place
Sept. 13-16, 2010
3. Number of participants / Number of participating countries and areas
270/8（Japan, USA, UK, Germany, Netherland, Sweden, Korea, Taiwan）
4. Total cost
¥14,716,000 JPY
5. Main use of subsidy
Travel expenses for a foreign invited speaker
6. Result and Impression
The International Conference on Protein Community brought together nearly 300 scientists inJapan to exchange ideas on how proteins are expressed, folded, translocated, assembled, anddisassembled in health, and the events that can go awry leading to a myriad of protein
conformational diseases. The meeting, held in September, 2010, coincided with the 1,300thbirthday of Nara, Japan’s ancient capital, and provided a meditative setting for reflection on theimpact of this field in biology and medicine and the success of this program in Japan since thisgroup’s first meeting in Kyoto on the Stress Response (1989) and in Awaji Island on the Life ofProteins (2005).

The highlight was, without question, the social periods held after the long daysof talks and posters at which graduate students, postdocs, and all of the speakers were together
sitting on tatami-mats at low tables enjoying each others company in a relaxing and open forum.
(Reported by Rick Morimoto)7. Additional description
The meeting report “The life of proteins: the good, the mostly good, and the ugly” (written by
Richard I. Morimoto, Arnold J. M. Driessen, Ramanujan S. Hegde, and Thomas Langer) willappear in January 2011 issue of Nature Structural and Molecular Biology.

- 98 -

The 17th International RUNX Workshop
1. Representative
Ichiro Taniuchi,
President of the organizing committee for the 17th International RUNX Workshop , Team Leader,
Laboratory for Transcriptional Regulation, RIKEN, RCAI
2. Opening period and Place
From 11th July 2010 to 14th July 2010 at the Oriental Hotel Hiroshima, Hiroshima, JAPAN3. Number of participants / Number of participating countries and areas
102 participants including 61 participants from 13 oversea countries including Israel, France,
Germany, United Kingdom, Scotland, USA, India, Thailand, Singapore, China, Taiwan, andKorea.
4. Total cost
¥6,730,347 JPY
5. Main use of subsidy
The cost of the conference room (screen, projector, microphones, partitions, signs)
6. Result and Impression
The meeting was very successful with total 52 oral presentations in 6 sessions and 10 poster
presentations. Most participants had very nice impression on the organization of the meeting, inparticular on the very smooth and on-time regulation of scientific program. All participants also
enjoyed food and culture in Japan.
7. Additional description
The Runx family genes were identified at the same time in 1993 from several fields such as
virology, oncology and development. This history of discovery of Runx genes already represented
multiple biological functions of Runx family genes, stimulating the researchers to set up the joint
meeting to promote research of Runx genes. Then the 1st Runx workshop has started in 1994 justwith a few laboratories and has been followed up annually. Because of a rapid growth of thisresearch area and several epoch-making presentation with a meeting split of sharing unpublishedresults, the International RUNX Workshop became well recognized all over the world.
The International RUNX Workshop came back to Japan this year since Dr. Yoshiakai Ito, Kyoto
University at that time organized the 7th Runx workshop at Kyoto in 2000. Dr. Ichiro Taniuchi at
RIKEN, Research Center for Allergy and Immunology, who is a present of the 17th International
RUNX Workshop, selected Hiroshima as a meeting venue. Hiroshima had a disaster by Atomic
Bomb attack and peoples in Hiroshima have been suffering from diseases mainly leukemia.
Considering that one of the major topics in the Runx workshop is a leukemogensis by- 99 -

Runx1/AML1 dysfunction as well as an international movement for an abolition of nuclear
weapons, Dr. Taniuchi decided to have the meeting at Hiroshima. Indeed almost all participants
visited the Hiroshima Peace Memorial Museum during the meeting.
The 18th international RUNX Workshop will be held next at San Diego in USA.

- 100 -

2010 International Polyamine Conference: Progress in Medicine and Life Sciences
1. Representative
Senya Matsufuji, MD, PhD
Professor, The Jikei University School of Medicine
2. Opening period and Place
June 14 - 18, 2010
Gotemba Kogen Resort (Gotemba-shi, Shizuoka, Japan)
3. Number of participants / Number of participating countries and areas
Number of participants: 115 (26 from abroad and 86 domestic)
Number of participating countries 13 (Finland, Ireland, Israel, Italy, Portugal, South Africa, South
Korea, Spain, Sweden, UK, USA, Vietnam, and Japan)
4. Total cost
¥9,813,050
5. Main use of subsidy
Travel and accommodation costs for invited speakers from abroad.
6. Result and Impression
The conference consisted of 8 sessions with total of 33 oral presentations, including 18 invitedlectures. They covered function, regulation, molecular evolution and structure of polyamines,
polyamines in plants and microorganisms, polyamines in medicine, and polyamines as the targetof drug discovery.

In addition, 34 posters were presented and 4 of them were selected for shortoral presentations in an extra session. The presentations clearly showed the progress inmolecular-level understanding for the function and regulation of polyamines. Analyses ofgenetically altered model organisms revealed biological significance of the regulatory system ofpolyamines. Topics related to clinical medicine, such as diagnosis of early cancer with polyamine
derivatives in urine and large-scale clinical trials for polyamine-related drugs drew much attention.
In particular, lectures on drug discovery that targets polyamine function to control epigenetic gene
regulation were impressive. Overall, the conference was a good blend of basic, applied, andclinical sciences. The extra-academic settings such as the venue in a resort with hot spring, fine
food and drinks, and a view of Mt. Fuji also helped the success of the conference. The conference
has greatly contributed to an advance of the research field both in Japan and the world.
7. Additional description
The organizing committee greatly appreciates the NOVARTIS Foundation for the support.

- 101 -

The 16th International Conference of the International Society of Differentiation
1. Representative
Naoto Ueno, Professor, National Institute for Basic Biology
2. Opening period and Place
Nov. 14 – 18, 2010
Nara-Ken New Public Hall, Kasugano-cho 101, Nara 630-8212, Japan
3. Number of participants / Number of participating countries and areas
250 / 22 countries (Australia, Canada, China, Czech, Denmark, Finland, France, Germany, Hong
Kong, Iran, Korea, Malaysia, Netherlands, Saudi Arabia, Singapore, Sweden, Switzerland, Taiwan,
UK, USA & Japan)
4. Total cost
¥18,836,443 JPY (Subsidy from The Novartis Foundation for the Promotion Sciences: 400,000 JPY)
5. Main use of subsidy
Invited speakers’ travel expenses (Airfare & Accommodation fee)
6. Result and Impression
The three and half day conference in which 250 attendants representing 22 countries got
together turned out to be very successful in terms of richness in science as well as of deepness ofinteraction among attendants and speakers. Seamless stream of excellent presentations by oral
speakers in addition to poster presentations from basic cell and developmental biology to stem celland cancer biology stimulated all the attendants.
7. Additional description
In this conference, we had students’session and offered platform presentation opportunity to 6
students from poster presentations. We also awarded three excellent students with Beverly
McKinnell Award to encourage them.

- 102 -

第 24 期（2010 年度）助成事業報告
当財団は、1987 年 9 月 4 日文部大臣の認可のもとに設立されて以来、寄附行為に定めた研究助成を中心とした公益事業を行ってきました。2010 年度は、下記に示した総額 4,000 万円の助成事業を行っております。
ノバルティス研究奨励金
研究集会助成38 件 （1 件
5 件 （1 件

100 万円）
3,800 万円
40 万円）
200 万円
総額 4,000 万円

2010 年度ノバルティス研究奨励金贈呈者
この事業は、生物・生命科学およびそれに関連する化学の領域において、我が国で行われる創造的な研究の助成を目的としています。
( 受付順、 敬称略、 所属 ・ 職位は申請時。 助成金額 ： 1 件 100 万円）
番号
1
2
3

4
5
6
7
8
9
10
11
12
13
14
15

氏

名

研究機関名
琉球大学大学院
齊藤 峰輝
医学研究科

役職

研究テーマ
HTLV-1 関連脊髄症発症における HBZ
准教授
遺伝子の作用機構の解明
光情報伝達における翻訳後修飾 SUMO
大 西 暁 士 大阪バイオサイエンス研究所
研究員
化の生理機能と作用機序の解明
創薬を目的とした有用天然物の生合成
渡 辺 賢 二 静岡県立大学薬学部
准教授 遺伝子発現によるシンセティックバイオロ
ジーの確立
レジスチンを標的とした 2 型糖尿病遺伝
愛媛大学大学院
大澤 春彦
教授 子 ・ 環境因子相互作用の分子機構解明
医学系研究科へのエピジェネティクスの応用
北海道大学
正常上皮細胞と癌細胞の相互作用－新藤田 恭之
教授
遺伝子病制御研究所
規な癌治療法の開発を目指して－愛知県心身障害者コロニー発達
統合失調症の神経発達障害仮説の実験
永田 浩一
部長
障害研究所
的検証
藤田保健衛生大学
ゲノムとマウスを用いた精神疾患感受性
岩田 仲生
教授
医学部
の同定
低酸素応答性転写因子による肝臓内代
早稲田大学
合田 亘人
教授 謝制御機構とその破綻に起因した代謝性
理工学術院
疾患の発症機構の解明
末梢 T 細胞の恒常性を制御する自己認
徳島大学疾患ゲノム
高田 健介
講師
識のメカニズム
研究センター
秋田大学
体外培養下でのヒト休眠原始卵胞の人為
講師
河村 和弘
医学部附属病院的活性化による成熟卵の作出
脳サイズ ・ 神経細胞数を決定する分子メ
小曽戸 陽一 川崎医科大学
准教授
カニズムの解明
東北大学大学院
海洋天然物の実用的合成を基盤とする
佐々木 誠
教授
生命科学研究科
革新的抗がん剤リード化合物の創製
自然科学研究機構岡崎統合
ガス分子により駆動される生体内シグナ
青野 重利
教授
バイオサイエンスセンター
ル伝達の分子機構解明
大阪大学
特任独立 染色体末端テロメアによる細胞分裂期制
加納 純子
蛋白質研究所准教授 御メカニズム
千葉大学大学院
南 野
徹
講師 次世代の血管新生治療の開発
医学研究院- 103 -

番号

研究機関名
東京大学大学院
16 河 崎 洋 志
医学系研究科
慶應義塾大学17 南 嶋 洋 司
医学部
徳島大学疾患酵素学
18 小 迫 英 尊
研究センター
19 秦

氏

名

猛

志

東京工業大学大学院
生命理工学研究科

自然科学研究機構
基礎生物学研究所
横浜市立大学大学院
21 來 生知
医学研究科
新潟大学大学院
22 日 比 野 浩
医歯学総合研究科
20 田

中

実

23 齋

藤

望

北海道大学大学院
薬学研究院

24 黒 河 博 文

東北大学大学院
医学系研究科25 北

名古屋市立大学
大学院医学研究科

村

浩

役職
研究テーマ
特任 感覚神経回路の時空間的な形成制御メ
准教授 カニズムの解明
特別研究 HIF の水酸化酵素 PHD2 をターゲットとし助教 た新たな虚血再還流障害治療法の可能性
細胞内情報伝達によるリン酸化を介した
准教授
核—細胞質間輸送装置の機能制御の解明
テトラヒドロピラン骨格を有する生物活性
助教 化合物の触媒的不斉合成及び構造活性相関研究
エネルギー代謝による幹細胞 ・ 性分化制
准教授
御機構の解析
がん再発に関わる CD11b+ 骨髄単球細講師
胞の腫瘍血管再形成における役割
内耳内リンパ液の特異環境の破綻に立教授
脚する難聴の病態生理の解明
アレンインの触媒的環化付加反応を機軸
准教授 とする Welwitindolinone A isonitrile 及びその類縁体の合成
Keap1-Nrf2 生体防御系による酸化ストレ講師
ス感知の構造基盤
新しいマクロファージ機能制御分子准教授 M-mod の肥満性糖尿病における病理学的役割の解析

理化学研究所
研究員
生命分子システム基盤研究領域
名古屋大学大学院
27 花 房
洋
助教
理学研究科
名古屋大学
28 松 尾 拓 哉
助教
遺伝子実験施設
26 西 田 元 彦

29 河

野

肇 帝京大学医学部

講師

名古屋大学大学院
生命農学研究科

助教

30 竹 本 大 吾

31 高 橋 智 聡 金沢大学がん研究所
財団法人癌研究会
癌研究所部長

忍 東京大学医科学研究所

助教

32 中 村 卓 郎
33 西

城

教授

34 小 室 一 成

大阪大学大学院
医学系研究科教授

35 江 本 憲 昭

神戸薬科大学
臨床薬学研究室教授

36 金 城 雄 樹

国立感染症研究所
生物活性物質部室長

37 脇 本 敏 幸

東京大学大学院
薬学系研究科講師

38 新 明 洋 平

熊本大学大学院
生命科学研究部

助教- 104 -

内向き整流性カリウムチャンネルの研究
ROCO ファミリーキナーゼ LRRK1 による
EGFR メンブレントラフィック制御
植物時計の進化の解明に向けた緑藻の生物時計の解析
動脈硬化における自然免疫炎症の関与とその制御
植物共生糸状菌の共生確立における活性酸素生成酵素複合体の役割の解明
Retinoblastoma がん抑制遺伝子不活性化による蛋白質ファルネシル化亢進の薬理学的 ・ 臨床的意義
Trib1 による白血病発症機構の解析
C 型レクチンによる免疫系制御機構の解明
p53 を標的とした新規心不全治療法の開発
腎糸球体における血管内皮 - 上皮細胞
間シグナルを介した慢性腎臓病の病態解明
播種性カンジダ症に対する免疫療法の開発
タンパク質脱リン酸化酵素阻害剤
Calyculin A に基づくサブタイプ選択的阻
害剤の開発
脳梁形成における軸索ガイダンス分子
draxin の役割

2010 年度研究集会助成金贈呈集会
この事業は、生物・生命化学およびそれに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。
（受付順、敬称略、所属・職位は申請時。助成金額：1 件 40 万円）
番号

研 究 集 会 名

開催期日
（開 催 地）助 成 先 代 表 者
所 属・職 位氏

名

1

第 5 回国際フィロウイルス
シンポジウム東京大学
2010.4.18 ～ 4.21
医科学研究所( 東京 )
教授

河岡 義裕

2

タンパク質社会に関する国際会議

名古屋大学
2010.9.12 ～ 9.16
大学院理学研究科( 奈良 )
教授

遠藤 斗志也3

第 17 回国際ＲＵＮＸワークショップ
2010独） 理化学研究所
2010.7.11 ～ 7.14 免疫 ・ アレルギー科学総合
谷内 一郎( 神奈川 )
研究センター
チームリーダー

42010 国際ポリアミン会議- 医学 ・ 生命科学への展開 -2010.6.14 ～ 6.18 東京慈恵会医科大学( 静岡 )
教授

松藤 千弥

5

第 16 回国際分化学会国際会議

自然科学研究機構
2010.11.14 ～ 11.18
基礎生物学研究所( 奈良 )
教授

上野 直人- 105 -

第 24 期（2010 年度）財務報告

貸借対照表

収支計算書

2011 年 3 月 31 日現在

2010 年 4 月 1 日から 2011 年 3 月 31 日まで（単位：円）（単位：円）

科目
Ⅰ資産の部
１． 流動資産
流動資産合計
２． 固定資産(1) 基本財産
投資有価証券 （指定）投資有価証券 （一般）
基本財産合計(2) 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
Ⅱ負債の部
１． 流動負債
流動負債合計
負債合計
Ⅲ正味財産の部
１． 指定正味財産
指定正味財産合計（うち基本財産への充当額）
２．一般正味財産（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

金

額

46,845,925

1,000,000,000
100,000,000
1,100,000,000
40,092,646
1,140,092,646
1,186,938,571

38,061,430
38,061,430

1,000,000,000(1,000,000,000)
148,877,141(100,000,000)
1,148,877,141
1,186,938,571- 106 -

科
目
Ⅰ事業活動収支の部
１． 事業活動収入
寄付金収入
事業活動収入計
２． 事業活動支出
事業費支出
助成金支出
管理費支出
事業活動支出計
事業活動収支差額
Ⅱ投資活動収支の部
１． 投資活動収入
投資活動収入計
２． 投資活動支出
投資活動支出計
投資活動収支差額
Ⅲ財務活動収支の部
１． 財務活動収入
２． 財務活動支出
財務活動収支差額
当期収支差額
前期繰越収支差額
次期繰越収支差額

決算額

40,038,000
68,622,359
50,720,917
40,000,000
4,522,946
55,243,863
13,378,496

200,000,000
200,000,000
000013,378,496△ 4,594,001
8,784,495

役員名簿（理事・評議員・選考委員）

理事会2011 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・非常勤理事長 金 子

章 道 畿央大学大学院健康科学研究科教授
慶應義塾大学名誉教授

2003 年 6 月 10 日

非常勤理

茂 隆 早稲田大学先端科学・健康医療融合研究機構長 東京大学名誉教授

1999 年 6 月 4 日

非常勤

石 川

裕 子 ノバルティス ファーマ株式会社
2004 年 6 月 7 日
常務取締役 人事・コミュニケーション本部長

非常勤

大 島

泰 郎 共和化工株式会社 環境微生物学研究所長
東京工業大学名誉教授

1997 年 6 月 8 日

非常勤1999 年 6 月 4 日

非常勤1999 年 6 月 4 日

非常勤1987 年 9 月 16 日

非常勤

事 浅 野

黒

川

眞 崎

清 政策研究大学院大学教授
東京大学名誉教授
知 生 筑波大学名誉教授
京都大学名誉教授

マックス ･ブルガー
眞 弓

忠 範 大阪大学名誉教授

2004 年 6 月 7 日

非常勤

三 谷

宏 幸 ノバルティス ファーマ株式会社
代表取締役社長

2007 年 9 月 5 日

非常勤

村 崎

光 邦 CNS薬理研究所長
北里大学名誉教授

2001 年 6 月 1 日

非常勤2005 年 6 月 13 日

非常勤2006 年 6 月 5 日

非常勤1998 年 2 月 10 日

非常勤

森
監

ノバルティス サイエンスボード議長
バーゼル大学教授

美 和 子 北海道医療大学客員教授
北海道大学名誉教授

事 中 嶋

徳 三 中嶋徳三公認会計士事務所
公認会計士

松本 秀三郎 元ノバルティス ファーマ株式会社
常勤監査役- 107 -

評 議 員 会
2011 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2002 年 2 月 7 日

非常勤

評 議 員 赤池 紀扶 熊本保健科学大学リハビリテーション学科教授 1999 年 6 月 4 日
銀杏学園理事 ・ 副学長
九州大学名誉教授

非常勤

評議員会 黒岩 常祥 立教大学極限生命情報センター長
議 長
東京大学名誉教授

赤沼 安夫 財団法人朝日生命成人病研究所名誉所長

2001 年 6 月 1 日

非常勤

浅島

1999 年 6 月 4 日

非常勤

遠藤 政夫 山形大学名誉教授

1997 年 6 月 8 日

非常勤

小川

聡 国際医療福祉大学三田病院長
慶應義塾大学名誉教授

2001 年 6 月 1 日

非常勤川嵜 敏祐 立命館大学糖鎖工学研究センター長
京都大学名誉教授

1999 年 6 月 4 日

非常勤

川島 博行 元新潟大学大学院医歯学総合研究科教授

2001 年 6 月 1 日

非常勤

北1999 年 6 月 4 日

非常勤

後藤 勝年 JST サテライト茨城センター長

2001 年 6 月 1 日

非常勤榊佳 之 豊橋技術科学大学学長
東京大学名誉教授

2001 年 6 月 1 日

非常勤

柴崎 正勝 財団法人微生物化学研究会
微生物化学研究所長

2005 年 6月13日

非常勤

健 昭和大学附属豊洲病院乳癌検診 ･ 治療センター 1998 年 6 月 5 日
元センター長 ・ 教授

非常勤

冨永

誠 東京大学大学院総合文化研究科客員教授
東京大学名誉教授

徹 神戸市立医療センター中央市民病院
病院長

中西 重忠 財団法人大阪バイオサイエンス研究所所長
京都大学名誉教授

1999 年 6 月 4 日

非常勤

長田 敏行 法政大学生命科学部学部長
東京大学名誉教授

2005 年 6月13日

非常勤

西川 武二 慶應義塾大学名誉教授
日本ワックスマン財団常務理事

2001 年 6 月 1 日

非常勤

西宗 義武 大阪大学微生物病研究所特任教授

1999 年 6 月 4 日

非常勤水野 美邦 順天堂大学医学部教授

1999 年 6 月 4 日

非常勤- 108 -

選考委員会
2011 年 10 月 1 日現在 ( 順不同、敬称略 )
職 名
選考
委員長

氏

名

現

職

村 田 道 雄 大阪大学大学院理学研究科教授

選考委員 一 條 秀 憲 東京大学大学院薬学系研究科教授

就任年月日

常勤・非常勤2008 年 6月20日

非常勤

2011年 6月17日

非常勤岡

慎 一 国立国際医療センター
エイズ治療 ・ 研究開発センター長

2011年 6月17日

非常勤岡芳 知 東北大学大学院医学系研究科教授

2008 年 6月20日

非常勤

貝 淵 弘 三 名古屋大学大学院医学研究科教授

2009 年 6月19日

非常勤

狩 野 方 伸 東京大学大学院医学系研究科教授

2009 年 6月19日

非常勤

岸 本 健 雄 東京工業大学大学院生命理工学研究科教授 2011年 6月17日

非常勤

笹 井 芳 樹 理化学研究所発生 ・ 再生総合研究センター 2008 年 6月20日
グループディレクター

非常勤

笹 井 宏 明 大阪大学産業科学研究所教授

2010 年 6月18日

非常勤

佐 田 政 隆 徳島大学大学院ヘルスバイオサイエンス研究部教授

2009 年 6月19日

非常勤

鹿 内 利 治 京都大学大学院理学研究科教授

2011年 6月17日

非常勤

高柳

広 東京医科歯科大学大学
大学院医歯学総合研究科教授

2011年 6月17日

非常勤

竹内

勤 慶應義塾大学医学部教授

2009 年 6月19日

非常勤

西 田 篤 司 千葉大学大学院薬学研究科教授

2009 年 6月19日

非常勤

長谷部 光泰 自然科学研究機構 基礎生物学研究所教授

2009 年 6月19日

非常勤

福 田 恵 一 慶応義塾大学医学部教授

2008 年 6月20日

非常勤

南

雅 文 北海道大学大学院薬学研究院教授

2011年 6月17日

非常勤

峯岸

敬 群馬大学大学院医学系研究科教授

2010 年 6月18日

非常勤

宮 園 浩 平 東京大学大学院医学系研究科教授

2009 年 6月19日

非常勤

室 原 豊 明 名古屋大学大学院医学系研究科教授

2011年 6月17日

非常勤- 109 -

事務局便りご寄附のお願い
当財団は、 生物 ・ 生命科学および関連する化学の領域において、 創造的な研究等を支援することにより、 学術の進展と福祉の向上に寄与することを目的にしております。
これらの公益事業は、 寄附金および基本財産の運用益にて行われており、 当財団は事業の趣旨にご賛同いただける方々からのご寄附を募っております。
なお、 当財団は特定公益増進法人の認定を受けております。
特定公益増進法人とは、 公益法人等のうち教育または科学の振興、 文化の向上、 社会福祉への貢献その他公益の増進に著しく寄与するものとして認定された法人をいいます。
これらの法人に対して、 個人または法人が寄附を行った場合は、 下記の税法上の優遇措置を受けることができます。
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個人 ： 支 出 し た 寄 附 金 （ そ の 年 の 総 所 得 額 の 40 ％ を 限 度 と す る ） のう ち、 5 千 円 を 超 え る 部 分 に つ い て 寄 附 金 控 除 が 認 め ら れ ま す。
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（電話 ： 03-5464-1460、 E メール ： novartisfound.japan@novartis.com）

事務局より
東日本大震災で被災された皆様に、 心よりお見舞いを申し上げます。
本年度も財団年報を発行できますことは、 助成を受けられた皆様および財団関係者のご尽力の賜物と厚く御礼申し上げます。
さて、 公益法人制度の改制に伴いまして、 当財団は引き続き税制の優遇措置が受けられる公益財団法人への移行を目指し、 本年度に申請を行いました。 現在審査が進行中ですが、 来年度には移行のご報告ができるものと考えております。
なお、 助成事業は財団の設立以来 1,413 件、 総額 17 億 2 千 7 百万円余りに達しました。 今後も事務局は、 学術の進展に寄与できる喜びを胸に助成事業に邁進する所存です。
今後ともご指導、 ご支援の程よろしくお願い申し上げます。
事務局長 松田光陽〒 106-0031 東京都港区西麻布 4-16-13
西麻布 28 森ビル 2F
Tel:03-5464-1460 Fax:03-5467-3055
E- メール ： novartisfound.japan@novartis.com
ホームページ :http://www.novartisfound.or.jp- 110 -