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JCGG Symposia

The 9th Symposium of Japanese Consortium for Glycobiology and Glycotechnology

Establishing a Cuttingedge International Research Center Aiming for the Integrated Development of Glycoscience
November 24 - 25, 2011 Toyota auditorium (Nagoya University)
Chair’s message

JCGG Steering Committee Chair
Toshisuke Kawasaki

I am Toshisuke Kawasaki and have served as the chair of the JCGG steering committee since last year. I would like to welcome the participants on the occasion of this annual symposium.
We are happy to announce that we will be hosting the 9th Annual Symposium, the biggest event for JCGG, for the first this year in Nagoya. I wish to express my sincere appreciation to Dr. Koichi Furukawa, Chair of the organizing committee of this symposium, as well as other committee members for their efforts. We also would like to express our deep respect to the people in the Tohoku/Kanto area who, having gone through the devastating disaster of 3.11, the giant east Japan earthquake/tsunami, have resumed their research activities.
This year there was more activities than ever in the Glycoscience field, as a series of meetings/symposiums were held in Japan during summer/autumn. These include: The 31st Naito Conference on Glycan Expression and the Regulation [II]] Metabolites, Stress Response, Microdomains, and Beyond at Chateraise Gateaux Kingdom, Sapporo, (Sept. 13-16; Chair: Akemi Suzuki); the 3rd Glycobiology Japan-Netherlands Joint Seminar 2011 at Nagoya University School of Medicine, Nagoya (October 8-11; Chair: Koichi Furukawa); The 71st Okazaki Conference on “New perspectives on the molecular science of glycoconjugates” at NINS, Okazaki (Chair: Soichi Wakatsuki; Koichi Kato). These were all Gordon Research Conference-style international meetings that are limited to around 100 participants, and an impressive line-up of speakers brought about a series of exciting scientific discussions.
In terms of grant applications, I am happy to announce that the project “Deciphering sugar chain-based signals regulating integrative neuronal functions (neuro glycobiology)” was approved as a Grant-in-Aid for Scientific Research on Innovative Areas, MEXT (2011-2015: Project Leader, Kenji Kadomatsu). This success deserves special mention, since this is the first project granted from the Glycoscience field since this new grant system was launched in place of Grant-in-Aid for Specially Promoted Research, and I do hope that there will be more to follow next year. This year, a grant to support the “Development of integrated database/research support devices for glycosciences” (2011-2013: Project Leader, Hisashi Narimatsu) was also approved from the JST Project for Life Science Database Integration Project. I expect that this success will lead to further development of the JCGG database (JCGGDB). We also had a meeting focusing on a “Master-plan for the future of Life Science” organized by the Science Council of Japan (Chair: Naoyuki Taniguchi) and we proposed the formation of “a network of cutting-edge international centers with the aim of integrating the development of glycoscience”. This Master-plan project was by far the largest grant approved, both period- and budget-wise, and I hope we will be able to accomplish hour goals by literally gathering our “All-Japan” efforts from our community. The biggest news of all for the Glycoscience community this year was that Dr. Naoyuki Taniguchi was the recipient of the Japan Academy Award last spring. I express my sincere congratulations to Dr. Taniguchi and wish him all the best for his further success in the future.
As we all know, the field of Glycoscience has gone through enormous progress in the recent past. On the other hand, we sometimes feel that we face some limitations in Glycobiology, in that the epoch-making developments were based largely on the discipline of molecular biology. To put it in an extreme way, what can be done if KO-mice do not provide us with any concrete answers? This will be a long-term question for all of us. I hope, however, that the appreciation of the concept of “multidisciplinarity”, one of the important characteristics of Glycoscience, will lead us to innovative approaches that will permit us to address this problem. Fig. 1 shows a schematic view of multiple disciplines in Glycosciences (modified from the figure used for a symposium held about 10 years ago). I do hope that, together with creative ideas from young scientists, such versatile approaches will lead to significant break-throughs in our field. The success of “neuro-glycobiology”, naturally an interdisciplinary field of research between neuro science and glycobiology, in the Grant-in-Aid for Scientific Research on Innovative Areas or the promising impressions of the Okazaki Conference which highlights multidisciplinarity in glycosciences may all suggest that we start to follow this path of multidiciplinarity.
Lastly, we wish to introduce our activities for the research ethics committee. Our former JCGG planning commitee held discussions on the topic of research ethics, and upon reforming the organization, we organized an ethics committee as part of our steering committee. After setting up this committee, the steering committee received a petition from several young JCGG members dealing with the wrong-doings of a certain researcher’s research presentations. Our ethics committee decided to form an external investigation committee and closely examined the content of this petition. As a result, the investigation committee came to the conclusion that the presentations of the aforementioned researcher included content that may have misled the public. Based on the outcome of this investigation, the ethics committee sent a cautionary letter to this researcher. The demands for clear explanations of scientists’ outreach activities such as educational activity, not to mention the direct accountability to the public regarding the use of government funding, are increasing. I must agree that this is an important issue for all of us. The most important issue, however, will be to accurately tell the “scientific truth” to the public, either directly or indirectly through media. Otherwise science will lose it credibility and lose public support for not only one scientist, but for the entire research community if one exaggerates the usefulness of his research and it eventually turns out not to be true. JCGG therefore will continue to monitor such ethical issues. At the same time, I would like to ask all the JCGG members to maintain the highest level of ethics and social responsibility.

 

Summary
4. On “Post-doctoral fellows and non-tenure track assistant professors in the fi eld of life science: Current status and future prospects”, a recent proposition issued by the Science Council of Japan.

Takashi Angata

The Science Council of Japan has recently issued a proposition titled “Post-doctoral fellows and non-tenuretrack assistant professors in the field of life science: Current status and future prospects”. The proposition is based on the online survey conducted by the Council earlier this year, which targeted post-doctoral fellows and non-tenure track assistant professors belonging to one or more of 25 academic societies that comprise the Association of Biological Science Societies of Japan. The survey successfully collected answers from 1,147 postdocs and non-tenure track assistant professors, and extracted valuable pieces of information that illustrates current status of this precious yet precarious population. The document goes further to propose possible solutions to the problem known as “post-doc problem”, i.e., shortage of positions appropriate for these post-docs to occupy aft er they fi nish their trainings.
In this talk, I will briefly summarize the major findings of the survey and the recommendations by the Council, and time permitting, compare the survey result with those conducted elsewhere.

 

6. Isolation of useful human antibodies through construction of phage antibody libraries

Yoshikazu Kurosawa

Antibody (Ab) is only the type of a molecule that can specifically and tightly bind to biological macromolecules such as proteins and polysaccharides as well as small molecules like hormones. Since Keller and Milstein developed the hybridoma technology to obtain monoclonal Abs in 1975, it has been widely utilized in various research fields. On the other hand, the phage Ab technology has not become popular among researchers even after twenty years have already passed since its development. There seem to be several misunderstandings about this technology. 1) Ab libraries constructed by use of mRNA from human B cells cannot contain Abs specific against human proteins. 2) An Ab consists of a set of H and L chains. Since the Ab library is constructed by mixing of VH library and VL library both of which are separately constructed, the repertoire of Ab in the constructed library cannot reflect the in vivo repertoire. 3) Since the Abs in the library should be considered to be naive to antigens (Ags), they cannot show strong binding activities to the Ags. We have worked on improvement of phage Ab technology for longer than 16 years and realized that this technology is revolutionary and can be applied to various new fields. I am going to present such examples.

 

7. Transgenic Chickens Expressing Galactosyltransferase Produce Galactosylated Pharmaceutical Proteins into Egg White

Shinji Iijima

We previously reported production of erythropoietin, TNF receptor fused to Fc fragment, and single chain immunoglobulin in eggs laid by genetically manipulated chicken. In egg white, however, incomplete addition of terminal sugar such as sialic acid and galactose was found on N-linked glycans of both exogenous and endogenous proteins. The loss of these terminal sugars deteriorates the physiological and/or functional stability of recombinant proteins purified from chicken egg white as for pharmaceutical usage.
To overcome this problem, we first investigated galactosyltransferase expression and its enzymatic activity in magnum where the majority of egg white proteins are produced. In magnum, despite its high secretion capacity of proteins, lower β1,4-galactosyltransferase I (GalT1) expression and poor galactose-transfer activity was observed. Thus, we generated transgenic chickens expressing GalT1. By this, we could attain drastic improvement of galactosylation to native egg white protein as well as exogenous single chain immunoglobulin and human erythropoietin.
As a bio-factory, chicken has been proven its convenience with benefits of high productivity of eggs and low breeding cost comparing to other animals. We previously suceeded in production of erythropoietin, TNF receptor fused to Fc fragment, and single chain immunoglobulin in eggs laid by genetically manipulated chicken. In egg white, however, incomplete addition of terminal sugar such as sialic acid and galactose was found on N-linked glycans of both exogenous and endogenous proteins. This could be a considerable since the loss of these terminal sugars deteriorates the physiological and/or functional stability of recombinant proteins purified from chicken egg white as for pharmaceutical usage. To overcome this problem, we first investigated galactosyltransferase expression and enzymatic activity in magnum where the majority of egg white proteins are produced. In magnum, despite its high secretion capacity, lower β1,4-galactosyltransferase I (GalT1) expression and poor galactose-transfer activity was observed. Thus, we generated transgenic chickens expressing GalT1. In golgi fraction prepared from magnum of the transgenic chickens, significant galactosyltransferase activity was detected compare to wild type chicken. The series of analyses revealed the drastic improvement of galactosylation to native egg white protein as well as exogenous single chain immunoglobulin.
Acknowledgment. This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).

 

8. Roles of glycans in interaction between IgG and Fcγ receptor III

Koichi Kato

The effector functions of immunoglobulin G (IgG) critically depend on N-glycosylation of its Fc region. It is well known that removal of the fucose residue from the N-glycans of the Fc portion of IgG results in a dramatic enhancement of antibody-dependent cellular cytotoxicity (ADCC) through improved affinity for Fcγ receptor IIIa (FcγRIIIa). Recently, we determined the 2.2-Å crystal structure of the complex formed between nonfucosylated IgG1-Fc and a soluble form of FcγRIIIa (sFcγRIIIa) with two N-glycosylation sites. The crystal structure demonstrates that one of the two N-glycans of sFcγRIIIa mediates the interaction with the N-glycan of non-fucosylated Fc, thereby stabilizing the complex.
However, fucosylation of the Fc N-glycans impairs this interaction because of steric hindrance. On the other hand, our NMR data demonstrated that Tyr296 of the non-fucosylated Fc glycoform exhibits conformational multiplicity in its uncomplexed state, suggesting that conformational selection is governed by the presence or absence of the fucose residue of the Fc N-glycan. Fucose depletion increases the incidence of the active conformation of Tyr296, and thereby accelerates the formation of the high-affinity complex. These findings offer a structural basis for improvement in ADCC of therapeutic antibodies by defucosylation.

 

9. An antibody cancer therapy targeting CCR4 from Japan

Takashi Ishida

Peripheral T-cell lymphomas (PTCL) including adult T-cell leukemia/lymphoma (ATL) have very poor prognoses, and no standard treatment strategies for these diseases have been developed. Because we previously found that CCR4 is expressed on tumor cells from most ATL patients, as well as on tumor cells from a subgroup of PTCL which has an unfavorable prognosis. Accordingly, the therapeutic humanized anti-CCR4 monoclonal antibody (mAb), KW-0761 was developed. The Fc region of the therapeutic anti-CCR4 mAb is defucosylated, to enhance ADCC. We demonstrated that robust ADCC of the defucosylated anti-CCR4 mAb against primary tumor cells from ATL and PTCL patients mediated by autologous effector cells were triggered both in vitro and in humanized mouse in vivo. Next, we conducted a phase I clinical trial of KW-0761 for patients with relapsed CCR4-positive PTCL including ATL. In this phase I trial, clinical responses were observed even at 0.01 mg/kg, which is approximately 1/1000 of the well-known therapeutic antibody rituximab dose, and it would be consistent with the concept of using defucosylation of therapeutic mAb to enhance ADCC. The phase I trial resulted in a recommended dose of 1.0 mg/kg for future clinical trials, and we subsequently completed a phase II clinical trial of KW-0761, and demonstrated that KW-0761 treatment showed potent antitumor activity in patients with relapsed ATL, with an acceptable toxicity profile. The promising result of this phase II study prompted us to proceed with further clinical development of this antibody, and we are now conducting a phase II trial of KW-0761 with chemotherapy for patients with previously untreated ATL and that of KW-0761 monotherapy for patients with relapsed PTCL other than ATL, both in Japan.

 

10. An innovative therapeutic antibody, tocilizumab (a humanized anti-IL-6 receptor antibody), for the treatment of rheumatoid arthritis

Yoshiyuki Ohsugi

Tocilizumab (Actemra®) is a humanized anti-human IL-6 receptor antibody. It is a genetic recombinant antibody produced using Chinese hamster ovary (CHO) cells. It was the first antibody drug to be developed in Japan, and the first IL-6 inhibitor to be developed anywhere in the world. Tocilizumab was launched as a treatment for Castleman’s disease in June 2005, and for rheumatoid arthritis (RA) and systemic juvenile idiopathic arthritis in April 2008. Tocilizumab is the fruit of joint research and grew steadily for 22 years from the combined “seeds” of many years of basic research into autoimmune disease at Chugai Pharmaceutical and the basic immunological research conducted by T. Kishimoto et al. at Osaka University.
This paper is to tell the story of the long road that led to the birth of tocilizumab. In particular, I wish to explain why we focused on IL-6 as a molecular target for the treatment of autoimmune diseases, why we attempted to develop an antibody drug. The other objective of this paper is to introduce recent topics related to the mechanism of RA that have come to light as a result of the treatment of RA with tocilizumab.

 

11. Development of the molecular probe that discriminates a specific structure of glycosaminoglycans

Tomio Yabe

Numerous studies on the critical role of heparin and heparan sulfate (HS) in biological events on the cell surface through changes in sulfation pattern have stimulated interest to elucidate the detailed structure of the polysaccharide chains. The in vivo modification of sugar chains with sulfates, however, is complicated, and the discrimination of different sulfation patterns is difficult.
Screening of heparin-associating peptides using phage display and antithrombin (AT)-bound affinity chromatography identified a peptide, heparin-associating peptide Y (HappY), that acts as a target of immobilized heparin. HappY peptide consists of 12 amino acid residues with characteristic three arginines and exclusively binds to heparin and HS but does not associate with other glycosaminoglycans. Furthermore, HappY peptide caused a dose-dependent increase in thrombin activity by hampering the formation of the heparin-AT complex. In addition, the neurite outgrowth in PC-12 cells by FGF-2 is significantly inhibited by HappY peptide. Thus, HappY peptide would play a role in several biological functions through the interaction with heparin and HS.

 

12. Synthesis and Biological Activities of Sialidase-resistant Ganglioside GM3 Analogues

Go Hirai

Gangliosides are glycosphingolipids that are ubiquitous components of mammalian cell membranes. GM3 was known to inhibit cell proliferation and auto-phosphorylation of EGFR stimulated by EGF. On the other hand, plasma membrane-associated human sialidase NEU3 hydrolyzes the glycosidic linkage of sialic acid in GM3 to produce lactosylceramide, and is up-regulated in various cancer cells and tissues. Recent reports suggested that enzymatic metabolism of GM3 by NEU3 may be involved in cancer malignancy. To clarify the relationship between GM3 metabolism and cancer malignancy by NEU3, we designed sialidase-resistant GM3 analogues with CF2- and CH2-sialoside linkages, in which the oxygen atom of the O-sialoside linkage is replaced by the CF2 or CH2 group respectively, as ganglioside probes. This time, we succeeded in the synthesis of both CF2- and CH2-linked GM3 analogues. During the course of the synthesis, we developed novel methodologies for the construction of the key C-sialoside linkages utilizing the Ireland-Claisen rearrangement reaction.
Moreover, extensive investigations for the glycosylation reaction of C-linked sialylgalactose donors revealed the unexpected reactivity of glycosyl donors and acceptors for the synthesis of GM3 analogues. Details of the synthesis and results of the biological activity of GM3 analogues will be presented.

 

13. DDS of triple-helical complex composed of glycan and nucleic acid

Kazuo Sakurai

Schizophyllan is a β-(1→3)-glucan and can form a macromolecular complex with DNA. The complex can be recognized by a receptor called dectin-1 expressed on antigen presenting cells and eventually ingested. This cellular specific uptake can be used to deliver CpG DNA and antisense DNA to macrophages.

 

14. Proteoliposome engineering for drug delivery system

Kazunari Akiyoshi

Membrane proteins play important roles in all living cells, such as signal transduction and cellular communication. In contrast to water-soluble proteins, the structural and functional studies of membrane proteins are not well established because they require a lipid bilayer for correct folding. Molecular chaperone technology assisting reconstruction of membrane proteins to liposome is important in membrane protein science. We developed three methods for preparations of proteoliposomes. I) Sugar-surfactant micelle-vesicle transition system caused by enzymatic polymerization was effective for the reconstitution and refolding of membrane proteins. II) The expressed membrane protein such as connexin 43 was incorporated directly into the liposome membrane upon in vitro synthesis, leading to pure membrane protein-containing liposomes. The chaperone-like function of liposomes prevents the aggregation of the water-insoluble membrane protein and aids the oligomerization of the membrane protein Cx43 in the membrane. III) Liposomes were fused by membrane protein recombinant baculovirus budded from insect cell. The original orientation of the membrane proteins such as connexin 43 and N-cadherin are maintained by the fusion method. Functional proteoliposomes would enable innovative breakthroughs in nanocarrier technologies for use in DDS and also in a new platform for membrane protein chips in bioanalysis and biodiagnostics.

 

15. A day in cyanobacteria timed by clock protein KaiC

Takao Kondo

We reconstituted the self-sustained circadian oscillation in phosphorylation state of the cyanobacterial clock protein KaiC by incubating it with KaiA protein, KaiB protein, and ATP. This in vitro oscillation persisted robustly and the period was compensated against temperature changes. Period lengths observed in vivo in various kaiC mutants were consistent with those measured using in vitro mixtures containing the respective mutant KaiC proteins. These results indicate that the oscillation of KaiC phosphorylation is the primary pacemaker of the cyanobacterial circadian clock.We then found that the interactions between KaiA or KaiB with KaiC and mutual regulations of two neighboring phosphorylation sites of KaiC facilitated the phosphorylation cycle. Moreover, we showed that KaiC possesses extremely weak but temperature-compensated ATPase activity (10-15 ATPs/day/KaiC) and that activities of wild-type KaiC and five period-mutant proteins are directly proportional to their in vivo circadian frequencies, indicating that the ATPase activity defines the circadian period. Based on these observations, we propose the KaiC ATPase activity is the most fundamental reaction underlying circadian periodicity of cyanobacteria.

 

16. Allorecognition and its cellular response in ascidian fertilization

Hitoshi Sawada

Ascidians (primitive chordate) are hermaphroditic animals that release sperm and eggs nearly simultaneously. However, many species, including Halocynthia roretzi and Ciona intestinalis, show strict self-sterility. In H. roretzi, we have previously reported that a 70-kDa vitelline-coat (VC) protein consisting of 12 EGF-like repeats (HrVC70) is a promising candidate protein for allorecognition during gamete interaction. HrUrabin, a GPI-anchored CRISP family protein, and HrTTSP1, a type-II transmembrane serine protease, appear to be the binding partner for HrVC70.
In C. intestinalis, we revealed that the fibrinogen-like proteins (v-Themis-A/-B) and the PKD1-like proteins (s-Themis-A/-B) are female-side and male-side candidate allorecognition proteins, respectively. v-Themis-A/B are highly polymorphic proteins and s-Themis-A/B have a hyper-variable region at their N-terminal portions. Genetic analyses showed that sperm recognizes the VC as self, only if both s-Themis-A and s-Themis-B alleles recognize the same haplotypic v-Themis-A and v-Themis-B, respectively. This allorecognition or selfincompatibility system is very similar to those of flowering plants.
By live-imaging technique using a fluorescent Ca2+ indicator, we recently found that sperm intracellular Ca2+ concentration is dramatically increased when sperm attach to the vitelline coat of self-eggs but not of nonselfeggs. These results indicate that sperm intracellular Ca2+ plays a key role in the self-incompatibility system during ascidian fertilization.

 

17. How animals sense the spring: Glycosylation of springtime hormone

Takashi Yoshimura

Animals living outside the tropics use changes in day length to adapt to seasonal changes in environment, but the molecular and endocrine mechanisms underlying photoperiodic time measurement are not fully understood. The Japanese quail is a robust model for the study of these mechanisms because of its rapid and dramatic response to changes in photoperiod. In the previous study, we have demonstrated that local thyroid hormone catabolism within the mediobasal hypothalamus (MBH) by thyroid hormone-activating enzyme (type 2 deiodinase: DIO2) regulates photoperiodism. Recent systems biology analysis in quail demonstrated that long days induce thyrotropin (TSH) production in the pars tuberalis (PT) of the pituitary gland, which triggers DIO2 expression in the ependymal cells (EC) of the MBH. In mammals, nocturnal melatonin secretion provides an endocrine signal of the photoperiod to the PT that contains melatonin receptors in high density. We have also demonstrated the involvement of TSH signalling pathway in mammals by using the TSH receptor null mice. Since TSH is a glycoprotein, we are now studying the glycosylation of TSH.

 

18. Hydroxyproline arabinosylation in plant peptide hormones

Yoshikatsu Matsubayashi

Posttranslational modification often plays a crucial role in peptide hormone signaling. Here, we show, by nano-LC-MS/MS analysis of apoplastic peptides of Arabidopsis plants, that CLV3 peptide, a regulator of the stem-cell population in the shoot apical meristem, is present in the apoplast as a 13-amino-acid arabinosylated glycopeptide. We also determined that CLE2, one of the CLV3 homolog is a 12-amino-acid arabinosylated glycopeptide. Given these and the ability of purified CLV3 and CLE2 glycopeptide to complement clv3 mutant through high-affinity binding to CLV1 receptor, we propose CLV3 and CLE2 as arabinosylated glycopeptide ligands for CLV1.

 

19. AMOR, a novel signaling molecule of flowering plants required for capcitation of the pollen tube, and its relation with sugar chains

Tetsuya Hihgashiyama

During the fertilization of flowering plants, the pollen tube grows directionally inside the pistil and delivers two immotile male gametes to the embryo sac (haploid female gametophyte). Due to difficulties associated with inaccessibility to the embryo sac embedded in the maternal tissue, little is known about the molecular mechanism of plant fertilization. The role of the pistil for pollen tube guidance has been thought to simply provide directional signals and growth path. By using a unique plant Torenia fournieri, which has a protruding embryo sac, we developed an in vitro system whereby pollen tubes growing through a cut style were precisely attracted to the embryo sac. This experiment suggested that female tissues have an ability to make pollen tubes competent to the attraction signal. The attraction signal was identified as defensin-line LURE peptides derived from the synergid cell on the side of the egg cell. Moreover, pollen tubes were found to be capacitated by twostep control; the first step was growing through a cut style and the second step was to receive an ovular factor named AMOR (Activation Molecule for Responsecapability). In this symposium, I will talk about the characterization and purification of AMOR and its relation with sugar chains.

 

20. Structural basis for gibberellin recognition by its receptor, GID1

Makoto Matsuoka

Gibberellins (GAs) are phytohormones essential for many developmental processes in plants. A nuclear GA receptor, GIBBERELLIN INSENSITIVE DWARF1 (GID1), has a primary structure similar to that of the hormone-sensitive lipases (HSLs). The structure analysis of rice GID1 interacting with GA4 revealed that GID1 has an α/β-hydrolase fold similar to that of HSLs. On the basis of the GID1 structure, we mutagenized important residues for GA binding and examined their binding activities. Almost all of them showed very little or no activity, confirming that the residues revealed by structural analysis are important for GA binding. The replacement of Ile 133 with Leu or Val-residues corresponding to those of the lycophyte Selaginella moellendorffii GID1s-caused an increase in the binding affinity for GA34, a 2β-hydroxylated GA4. These observations indicate that GID1 originated from HSL and was further modified to have higher affinity and more strict selectivity for bioactive GAs by adapting the amino acids involved in GA binding in the course of plant evolution.

 

21. Molecular mechanism underlying axon formation

Shinichi Nakamuta

Neurons are highly polarized cells that possess structurally distinct processes--the axons and dendrites--that differentiate from common immature neurites. In cultured hippocampal neurons, one of these immature neurites stochastically initiates rapid extension and becomes an axon, whereas the others become dendrites. Various extracellular and intracellular signals have been identified as contributing to axon specification. However, the specific intracellular pathways whereby particular extracellular stimuli lead to axon specification remain to be delineated. Here, we found that the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), were required for axon specification in an autocrine or a paracrine fashion. Using local application with a micropipette to selectively stimulate individual neurites, we found that stimulation of a selected neurite by BDNF and NT-3 induced neurite outgrowth and subsequent axon formation. NT-3 induced a rapid increase in Ca2+ in an inositol 1, 4, 5-trisphosphate (IP3)-dependent fashion as well as local activation of the Ca2+ effector calmodulin-dependent protein kinase kinase (CaMKK) in the growth cone. Inhibition of neurotrophin receptors or CaMKK attenuated NT-3-induced axon specification in cultured neurons and axon formation in cortical neurons in vivo. These results identify a role for IP3- Ca2+-CaMKK signaling in axon specification.

 

22. Molecular mechanisms of neuronal growth cone navigation by nerve growth factor

Hiroki Akiyama

During development, neurons extend axons to create neuronal networks. To guide extending axons, environmental cues polarize growth cones at the tip of the axons via asymmetric generation of Ca2+ signals and subsequent intracellular mechanical events, including membrane trafficking and cytoskeletal reorganization. However, the precise mechanisms of growth cone navigation including how to generate asymmetric Ca2+ signals are unclear. Here, using nerve growth factor (NGF) as a chemoattractant, we determined the crucial roles of localized IP3 production in generation of asymmetric Ca2+ signals, and of phosphatydilinositol-3 kinase (PI3K) activity in asymmetric membrane trafficking required for growth cone chemoattraction. We found that IP3 and ensuing Ca2+ signals were produced asymmetrically across growth cones exposed to an extracellular NGF gradient and mediated growth cone attraction. Moreover, photolysis-induced production of IP3 on one side of a growth cone was sufficient to initiate growth cone turning toward the side with IP3 production. We also found that PI3K facilitated microtubule advance into the growth cone periphery thereby contributes to asymmetric membrane trafficking downstream of Ca2+ signals. By identifying the signaling cascade underlying NGF-mediated attraction, we show that locally produced IP3 encodes spatial information and PI3K facilitates membrane trafficking, which polarize the growth cone for guided migration.

 

23. Neural stem cell stemness is regulated by a unique LewisX-synthesizing α1,3-fucosyltransferase

Seiji Hitoshi

LewisX (LeX), or stage specific embryonic antigen-1 (SSEA-1), carbohydrate antigen is a good marker for undifferentiated state of stem cell populations. For example, LeX is rapidly downregulated during a differentiating process of embryonic stem cells. However, it is currently unknown whether LeX that consists of only three sugars exhibits full function for maintaining cells undifferentiated or whether LeX expressed on specific structures of N-glycans or even specific glycoproteins plays significant roles. To clarify those possibilities, we utilized a 3-dimensional HPLC analysis system and characterized whole N-glycan profiles of murine neural stem cells as well as embryonic stem cells. We found several novel LeX-containing N-glycans, which are downregulated after differentiation. Interestingly, some of those LeX-containing N-glycans were present in neural precursor cells derived from mice deficient for Fut9, which is known to be responsible for the synthesis of LeX in the brain. We also isolated a unique α1,3-fucosyltransferase gene (Fut10) that specifically synthesizes one of those novel LeX-containing N-glycans. We are characterizing roles of Fut10 played in the neural development and in the maintenance and differentiation of neural stem cells in the mouse brain.

 

24. The enhancement of protein degradation systems exerts therapeutic effects in the polyglutamine-mediated motor neuron disease

Hiroaki Adachi

A common characteristic of neurodegenerative diseases is the accumulation of abnormal proteins. This reflects a severe disturbance of cellular homeostasis. Here, we report the activation of the two major proteolytic machineries, the ubiquitin proteasome system (UPS) and the autophagy/lysosomal system, exerted the therapeutic effects in the models of spinal and bulbar muscular atrophy (SBMA). These proteolytic systems cooperate to clean up the pathogenic proteins and maintain the proteostasis. p62 is well known as the adaptor protein that binds to ubiquitin and LC-3 in autophagic degradation pathway. Deletion of the p62 significantly exacerbated numerous behavioral and neuropathological outcome measures in the SBMA mouse model. Deletion of p62 significantly increased in monomeric mutant AR and mutant AR protein complexes in the SBMA models. A peony extract (PE) is the principal active ingredient extracted from the root of Paeoniae alba, a Chinese herb commonly used to treat neurodegenerative disorders. It was found that PE significantly up regulated the expression of molecules related to proteolytic machineries. Moreover, PE was highly efficacious in alleviating SBMA pathogenesis. These findings demonstrated for the first time that PE could enhance the broad quality control system by regulating the expression of NF-Y and thus produced protective effects against SBMA.

 

25. The role of keratan sulfate in amyotrophic lateral sclerosis

Tomohiro Ohgomori

Amyotrophic lateral sclerosis (ALS), an adult-onset neurodegenerative disease, is characterized by motor neuron loss. Although most ALS cases are sporadic, approximately 10% are familial and they have the dominant mutants in the gene encoding for superoxide dismutase 1 (SOD1). Transgenic mice carrying mutant human SOD1 genes broadly recapitulate the human disease. Proteoglycans are glycoproteins which have glycosaminoglycan on the core proteins. Recently, we found that keratan sulfate proteoglycans (KSPGs) were critical inhibitors of axonal regeneration after spinal cord injury, using KS synthase N-acethylglucosamine 6-O-sulfotransferase-1 knock out mice and keratanase. It is suggested that proteoglycans are important for the progression of neurodegenerative disease. KS was upregulated after the onset of ALS. In ALS spinal cord, the number of microglia was increased and activated at the end of ALS. We reported that GlcNAc6ST-1 is essential for the synthesis of KS sugar chains. ALS model mice were mated with GlcNAc6ST-1 KO mice. Interestingly, the onset of ALS in KS-deficit ALS mice were earlier than that in KS-bearing ALS mice. We suggest that microglial KS have neuroprotective function in ALS.

 

26. Gangliosdes are essential for regulation of inflammation and neurodegeneration

Yuhsuke Ohmi

Gangliosides are essential in the maintenance and repair of nervous tissues. However, the mechanisms of ganglioside for the regulation of neuronal function have been unknown. We examined gene expression profiles in double knockout (DKO) mice of GM2/GD2 synthase and GD3 synthase genes, showing that almost all genes of complement system and their receptors were upregulated in cerebellum of DKO mice. Inflammatory reaction was demonstrated with increased reactive astrocytes and microglia and up-regulated inflammatory cytokines, indicating the presence of complement activation and inflammation as reported in many neurodegenerative diseases. Immunoblotting of fractionated membrane extracts revealed that complement-regulatory molecules such as DAF and CD59 dispersed from glycolipid-enriched microdomain (GEM)/rafts in DKO cerebella. Furthermore, phospholipids and cholesterol also tended to decrease in GEM/rafts of DKO mine, although total amounts were almost equivalent. These results suggested that dysfunction of complement-regulatory molecules due to abnormal GEM/rafts triggered complement activation, inflammation, and subsequent neurodegeneration in DKO mice. Generation of TKO mice lacking complement activity in addition to the two glycosyltransferases revealed that complement activation is essentially involved in the inflammatory reactions and neurodegeneration caused by the ganglioside deficiency. These results are suggested that gangliosdes are essential for maintenance of architecture of lipid rafts, and of the integrity of the nervous tissues.

 

27. Neuron-glia-glycanic interactions for synaptic plasticity

Wataru Kakegawa

Synaptic plasticity such as long-term potentiation and depression (LTD) is thought to be a molecular basis of learning and memory. Recently, glial cell, which tightly enwraps synaptic structures, is considered to regulate synaptic plasticity by releasing glia-derived factors including gliotransmitters (e.g., D-Ser, Glu, ATP) or extracellular matrix molecules (e.g., proteoglycans). Delta2-type ionotropic glutamate receptors (GluD2s), selectively expressed at cerebellar parallel fiber (PF)-Purkinje cell synapses, are essential for cerebellar synaptic plasticity: mice lacking GluD2 (GluD2-null mice) impaired LTD at PF-Purkinje cell synapses and motor learning. Despite its importance, the mechanism by which GluD2 functions in vivo has remained unclear because any ligands to activate GluD2 have not been identified. We recently found that glial D-Ser serves as a ligand for GluD2 to regulate LTD at PF-Purkinje cell synapses in immature cerebellum. Endogenous DSer rapidly induced endocytosis of AMPA receptors and mutually occluded LTD in wild-type, but not in GluD2-null Purkinje cells. Furthermore, mice expressing mutant GluD2, in which the binding site for DSer was disrupted, exhibited impaired LTD and motor dyscoordination during development. These results indicated that D-Ser regulates synaptic plasticity and cerebellar functions during development by interacting with GluD2.

 

28. Sulfation patterns of chondroitin sulfate regulate neuronal plasticity

Shinji Miyata

Experience-dependent plasticity is most evident during a critical period in early life, but the mechanisms that restrict plasticity after the critical period are poorly understood. Chondroitin sulfate proteoglycans (CSPGs) consist of a core protein with covalently attached chondroitin sulfate (CS) glycosaminoglycan chains and are major components of the brain extracellular matrix. CSPGs condense around parvalbumin-expressing inhibitory interneurons (PV-cells) and form perineuronal nets (PNNs) during postnatal development. Digestion of CS chains with chondroitinase ABC disrupts PNNs and reactivates experience-dependent plasticity in adult animals after the closure of the critical period. Recent studies have proposed that functional information of CSPGs is encoded by specific sulfation patterns of CS chains. In this study, we showed that CS sulfation patterns are dynamic during brain development. Therefore, we hypothesized that CS sulfation patterns regulate the duration of critical periods for experience-dependent plasticity by limiting plasticity. Using a transgenic mouse model, in which CS sulfation patterns are altered, we found that developmental changes in CS sulfation patterns play an important role in condensation of CSPGs into PNNs, and led to termination of the critical period for experience-dependent plasticity.