Papers - KINOSHITA, Makoto

Septin 3 regulates memory and L-LTP-dependent extension of endoplasmic reticulum into spines Reviewed Open Access

Ageta-Ishihara N, Fukazawa Y, Arima-Yoshida F, Okuno H, Ishii Y, Takao K, Konno K, Fujishima K, Ageta H, Hioki H, Tsuchida K, Sato Y, Kengaku M, Watanabe M, Watabe AM, Manabe T, Miyakawa T, Inokuchi K, Bito H, and Kinoshita M

Cell Reports   Vol. 44 ( 3 )   2025.2

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Authorship：Last author,　Corresponding author   Language：English  

DOI： 10.1016/j.celrep.2025.115352

Open Access

Expression analyses of Rich2/Arhgap44, a Rho family GTPase-activating protein, during mouse brain development. Reviewed

Goto N, Nishikawa M, Ito H, Noda M, Hamada N, Tabata H, Kinoshita M, Nagata K

Developmental Neuroscience   Vol. 45   page： 19 - 26   2023.1

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1159/000529051

Chr21 protein-protein interactions: enrichment in products involved in intellectual disabilities, autism and Late Onset Alzheimer Disease Reviewed International coauthorship

Julia Viard, Yann Loe-Mie, Rachel Daudin, Malik Khelfaoui, Christine Plancon, Anne Boland, Francisco Tejedor, Richard L. Huganir, Eunjoon Kim, Makoto Kinoshita, Guofa Liu, Volker Haucke, Thomas Moncion, Eugene Yu, Valérie Hindie, Henri Bléhaut, Clotilde Mircher, Yann Herault, Jean-François Deleuze, Jean-Christophe Rain, Michel Simonneau, Aude-Marie Lepagnol-Bestel

Life Science Alliance   Vol. 5 ( 12 ) page： e202101205   2022.8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Cold Spring Harbor Laboratory  

<title>ABSTRACT</title>Intellectual disability (ID) found in Down syndrome (DS), which is characterized by an extra copy of 234 genes on Chr21 is poorly understood. We first used two DS mouse models that either display an extra copy of the <italic>Dyrk1A</italic> gene or of the mouse Chr16 syntenic region. Exome sequencing of transcripts deregulated in embryonic hippocampus uncovers enrichment in genes involved in chromatin and synapse respectively. Using large-scale yeast two-hybrid screen (154 distinct screens) of human brain library containing at least 10⁷ independent fragments, we identified 3,636 novel protein-protein interactions with an enrichment of direct interactors of both Chromosome 21(Hsa21) baits and rebounds in ID-related genes. Using proximity ligation assays, we identified that Hsa21-encoded proteins are located at the dendritic spine postsynaptic density in a protein network located at the dendritic spine post synapse. Hsa21 DYRK1A and DSCAM that confers a ~ 20-fold increase in Autism Spectrum Disorders (ASDs) are part of this dendritic spine postsynaptic network. We found that a DSCAM intracellular domain binds either DYRK1A or DLGs that are multimeric scaffolds for the clustering of receptors, ion channels, and associated signaling proteins. The DYRK1A-DSCAM interaction is conserved from drosophila to humans. The identified postsynaptic.network is enriched in ARC-related synaptic plasticity, ASDs and Late-Onset Alzheimer Disease. Altogether, these results emphasize links between DS and brain diseases with complex genetics.

DOI： 10.26508/lsa.202101205

Mice with cleavage-resistant N-cadherin exhibit synapse anomaly in the hippocampus and outperformance in spatial learning tasks Reviewed

Asada-Utsugi M., Uemura K., Kubota M., Noda Y., Tashiro Y., Uemura T. M., Yamakado H., Urushitani M., Takahashi R., Hattori S., Miyakawa T., Ageta-Ishihara N., Kobayashi K., Kinoshita M., Kinoshita A.

  Vol. 14 ( 1 )   2021.1

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1186/s13041-021-00738-1

Web of Science

Developmental and postdevelopmental roles of septins in the brain. Invited Reviewed International journal

Natsumi Ageta-Ishihara, Makoto Kinoshita

Neuroscience research     2020.11

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Morphogenetic processes during brain development and postdevelopmental remodeling of neural architecture depend on the exquisite interplay between the microtubule- and actin-based cytoskeletal systems. Accumulation of evidence indicates cooperative roles of another cytoskeletal system composed of the septin family. Here we overview experimental findings on mammalian septins and their hypothetical roles in the proliferation of neural progenitor cells, neurite development, synapse formation and regulations. The diverse, mostly unexpected phenotypes obtained from gain- and loss-of-function mutants point to unknown molecular network to be elucidated, which may underlie pathogenetic processes of infectious diseases and neuropsychiatric disorders in humans.

DOI： 10.1016/j.neures.2020.08.006

PubMed

PCP-dependent transcellular regulation of actomyosin oscillation facilitates convergent extension of vertebrate tissue Reviewed International coauthorship

Asako Shindo, Yasuhiro Inoue, Makoto Kinoshita, John B. Wallingford

Developmental Biology   Vol. 446 ( 2 ) page： 159 - 167   2019.2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.ydbio.2018.12.017

Web of Science

UBL3 modification influences protein sorting to small extracellular vesicles Reviewed

Hiroshi Ageta, Natsumi Ageta-Ishihara, Keisuke Hitachi, Ozge Karayel, Takanori Onouchi, Hisateru Yamaguchi, Tomoaki Kahyo, Ken Hatanaka, Koji Ikegami, Yusuke Yoshioka, Kenji Nakamura, Nobuyoshi Kosaka, Masashi Nakatani, Akiyoshi Uezumi, Tomihiko Ide, Yutaka Tsutsumi, Haruhiko Sugimura, Makoto Kinoshita, Takahiro Ochiya, Matthias Mann, Mitsutoshi Setou, Kunihiro Tsuchida

Nature Communications   Vol. 9 ( 1 )   2018.12

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1038/s41467-018-06197-y

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PubMed

Other Link： http://www.nature.com/articles/s41467-018-06197-y

CDC42EP4, a perisynaptic scaffold protein in Bergmann glia, is required for glutamatergic tripartite synapse configuration. Invited Reviewed International journal

Natsumi Ageta-Ishihara, Kohtarou Konno, Maya Yamazaki, Manabu Abe, Kenji Sakimura, Masahiko Watanabe, Makoto Kinoshita

Neurochemistry International   Vol. 119   page： 190 - 198   2018.10

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

Configuration of tripartite synapses, comprising the pre-, post-, and peri-synaptic components (axon terminal or bouton, dendritic spine, and astroglial terminal process), is a critical determinant of neurotransmitter kinetics and hence synaptic transmission. However, little is known about molecular basis for the regulation of tripartite synapse morphology. Previous studies showed that CDC42EP4, an effector protein of a cell morphogenesis regulator CDC42, is expressed exclusively in Bergmann glia in the cerebellar cortex, that it forms tight complex with the septin heterooligomer, and that it interacts indirectly with the glutamate transporter GLAST and MYH10/nonmuscle myosin ΙΙB. Scrutiny of Cdc42ep4-/- mice had revealed that the CDC42EP4-septins-GLAST interaction facilitates glutamate clearance, while the role for CDC42EP4-septins-MYH10 interaction has remained unsolved. Here, we find anomalous configuration of the tripartite synapses comprising the parallel fiber boutons, dendritic spines of Purkinje cells, and Bergmann glial processes in Cdc42ep4-/- mice. The complex anomalies include 1) recession of Bergmann glial membranes from the nearest active zones, and 2) extension of nonactive synaptic contact around active zone. In line with the recession of Bergmann glial membranes by the loss of CDC42EP4, overexpression of CDC42EP4 in heterologous cells promotes cell spreading and partitioning of MYH10 to insoluble (i.e., active) fraction. Paradoxically, however, Cdc42ep4-/- cerebellum contained significantly more MYH10 and N-cadherin, which is attributed to secondary neuronal response mainly in Purkinje cells. Given cooperative actions of N-cadherin and MYH10 for adhesion between neurons, we speculate that their augmentation may reflect the extension of nonactive synaptic contacts in Cdc42ep4-/- cerebellum. Transcellular mechanism that links the absence of CDC42EP4 in Bergmann glia to the augmentation of N-cadherin and MYH10 in neurons is currently unknown, but the phenotypic similarity to GLAST-null mice indicates involvement of the glutamate intolerance. Together, the unique phenotype of Cdc42ep4-/- mice provides a clue to novel molecular network underlying tripartite synapse configuration.

DOI： 10.1016/j.neuint.2018.01.003

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PubMed

Septin-dependent remodeling of cortical microtubule drives cell reshaping during epithelial wound healing. Reviewed International coauthorship International journal

Asako Shindo, Anastasia Audrey, Maki Takagishi, Masahide Takahashi, John B Wallingford, Makoto Kinoshita

Journal of Cell Science   Vol. 131 ( 12 ) page： jcs212647 - jcs212647   2018.6

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Company of Biologists  

Wounds in embryos heal rapidly through contraction of the wound edges. Despite well-recognized significance of the actomyosin purse string for wound closure, roles for other cytoskeletal components are largely unknown. Here, we report that the septin cytoskeleton cooperates with actomyosin and microtubules to coordinate circumferential contraction of the wound margin and concentric elongation of wound-proximal cells in Xenopus laevis embryos. Microtubules reoriented radially, forming bundles along lateral cell cortices in elongating wound-proximal cells. Depletion of septin 7 (Sept7) slowed wound closure by attenuating the wound edge contraction and cell elongation. ROCK/Rho-kinase inhibitor-mediated suppression of actomyosin contractility enhanced the Sept7 phenotype, whereas the Sept7 depletion did not affect the accumulation of actomyosin at the wound edge. The cortical microtubule bundles were reduced in wound-proximal cells in Sept7 knockdown (Sept7-KD) embryos, but forced bundling of microtubules mediated by the microtubule-stabilizing protein Map7 did not rescue the Sept7-KD phenotype. Nocodazole-mediated microtubule depolymerization enhanced the Sept7-KD phenotype, suggesting that Sept7 is required for microtubule reorganization during cell elongation. Our findings indicate that septins are required for the rapid wound closure by facilitating cortical microtubule reorganization and the concentric elongation of surrounding cells.

DOI： 10.1242/jcs.212647

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PubMed

神経ネットワークの形と機能を制御する細胞骨格系分子群 Invited

木下　専

DOJIN BIOSCIENCE SERIES 28脳神経化学　脳はいま化学の言葉でどこまで語れるか     2018.4

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Authorship：Lead author,　Last author,　Corresponding author   Language：Japanese   Publishing type：Part of collection (book)  

グルタミン酸輸送体の足場となり、クリアランスを促進する シナプス周囲グリア突起内の蛋白質複合体 Invited

木下　専

日本生物学的精神医学会誌 （特集　精神神経疾患と興奮性アミノ酸受容体）   Vol. 28 ( 2 ) page： 84 - 86   2017.4

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Authorship：Lead author,　Last author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.11249/jsbpjjpp.28.2_84

Septin Interferes with the Temperature-Dependent Domain Formation and Disappearance of Lipid Bilayer Membranes. Reviewed International journal

Shunsuke Yamada, Takumi Isogai, Ryugo Tero, Yohko Tanaka-Takiguchi, Toru Ujihara, Makoto Kinoshita, Kingo Takiguchi

Langmuir : the ACS journal of surfaces and colloids   Vol. 32 ( 48 ) page： 12823 - 12832   2016.12

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

Domain formation or compartmentalization in a lipid bilayer membrane has been thought to take place dynamically in cell membranes and play important roles in the spatiotemporal regulation of their physiological functions. In addition, the membrane skeleton, which is a protein assembly beneath the cell membrane, also regulates the properties as well as the morphology of membranes because of its role as a diffusion barrier against constitutive molecules of the membrane or as a scaffold for physiological reactions. Therefore, it is important to study the relationship between lipid bilayer membranes and proteins that form the membrane skeleton. Among cytoskeletal systems, septin is unique because it forms arrays on liposomes that contain phosphoinositides, and this property is thought to contribute to the formation of the annulus in sperm flagellum. In this study, a supported lipid bilayer (SLB) was used to investigate the effect of septin on lipid bilayers because SLBs rather than liposomes are suitable for observation of the membrane domains formed. We found that SLBs containing phosphatidylinositol (PI) reversibly form domains by decreasing the temperature and that septin affects both the formation and the disappearance of the cooling-induced domain. Septin inhibits the growth of cooling-induced domains during decreases in temperature and inhibits the dispersion and the disappearance of those domains during increases in temperature. These results indicate that septin complexes, i.e., filaments or oligomers assembling on the surface of lipid bilayer membranes, can regulate the dynamics of domain formation via their behavior as an anchor for PI molecules.

DOI： 10.1021/acs.langmuir.6b03452

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Septin7 regulates inner ear formation at an early developmental stage. Reviewed International journal

Hiroko Torii, Atsuhiro Yoshida, Tatsuya Katsuno, Takayuki Nakagawa, Juichi Ito, Koichi Omori, Makoto Kinoshita, Norio Yamamoto

Developmental biology   Vol. 419 ( 2 ) page： 217 - 228   2016.11

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Septins are guanosine triphosphate-binding proteins that are evolutionally conserved in all eukaryotes other than plants. They function as multimeric complexes that interact with membrane lipids, actomyosin, and microtubules. Based on these interactions, septins play essential roles in the morphogenesis and physiological functions of many mammalian cell types including the regulation of microtubule stability, vesicle trafficking, cortical rigidity, planar cell polarity, and apoptosis. The inner ear, which perceives auditory and equilibrium sensation with highly differentiated hair cells, has a complicated gross morphology. Furthermore, its development including morphogenesis is dependent on various molecular mechanisms, such as apoptosis, convergent extension, and cell fate determination. To determine the roles of septins in the development of the inner ear, we specifically deleted Septin7 (Sept7), the non-redundant subunit in the canonical septin complex, in the inner ear at different times during development. Foxg1Cre-mediated deletion of Sept7, which achieved the complete knockout of Sept7 within the inner ear at E9.5, caused cystic malformation of inner ears and a reduced numbers of sensory epithelial cells despite the existence of mature hair cells. Excessive apoptosis was observed at E10.5,E11.5 and E12.5 in all inner ear epithelial cells and at E10.5 and E11.5 in prosensory epithelial cells of the inner ears of Foxg1Cre;Septin7floxed/floxed mice. In contrast with apoptosis, cell proliferation in the inner ear did not significantly change between control and mutant mice. Deletion of Sept7 within the cochlea at a later stage (around E15.5) with Emx2Cre did not result in any apparent morphological anomalies observed in Foxg1Cre;Septin7floxed/floxed mice. These results suggest that SEPT7 regulates gross morphogenesis of the inner ear and maintains the size of the inner ear sensory epithelial area and exerts its effects at an early developmental stage of the inner ear.

DOI： 10.1016/j.ydbio.2016.09.012

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PubMed

A septin requirement differentiates autonomous and contact-facilitated T cell proliferation. Reviewed International coauthorship International journal

Adriana M Mujal, Julia K Gilden, Audrey Gérard, Makoto Kinoshita, Matthew F Krummel

Nature Immunology   Vol. 17 ( 3 ) page： 315 - 22   2016.3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Nature  

T cell proliferation is initiated by T cell antigen receptor (TCR) triggering, soluble growth factors or both. In characterizing T cells lacking the septin cytoskeleton, we found that successful cell division has discrete septin-dependent and septin-independent pathways. Septin-deficient T cells failed to complete cytokinesis when prompted by pharmacological activation or cytokines. In contrast, cell division was not dependent on septins when cell-cell contacts, such as those with antigen-presenting cells, provided a niche. This septin-independent pathway was mediated by phosphatidylinositol-3-OH kinase activation through a combination of integrins and costimulatory signals. We were able to differentiate between cytokine- and antigen-driven expansion in vivo and thus show that targeting septins has strong potential to moderate detrimental bystander or homeostatic cytokine-driven proliferation without influencing expansion driven by conventional antigen-presentation.

DOI： 10.1038/ni.3330

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Facilitation of axon outgrowth via a Wnt5a-CaMKK-CaMKIα pathway during neuronal polarization Reviewed

MAKOTO KINOSHITA

Molecular Brain   Vol. 9 ( 8 )   2016.1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED CENTRAL LTD  

Background: Wnt5a, originally identified as a guidance cue for commissural axons, activates a non-canonical pathway critical for cortical axonal morphogenesis. The molecular signaling cascade underlying this event remains obscure.
Results: Through Ca2+ imaging in acute embryonic cortical slices, we tested if radially migrating cortical excitatory neurons that already bore primitive axons were sensitive to Wnt5a. While Wnt5a only evoked brief Ca2+ transients in immature neurons present in the intermediate zone (IZ), Wnt5a-induced Ca2+ oscillations were sustained in neurons that migrated out to the cortical plate (CP). We wondered whether this early Wnt5a-Ca2+ signaling during neuronal polarization has a morphogenetic consequence. During transition from round to polarized shape, Wnt5a administration to immature cultured cortical neurons specifically promoted axonal, but not dendritic, outgrowth. Pharmacological and genetic inhibition of the CaMKK-CaMKI alpha pathway abolished Wnt5a-induced axonal elongation, and rescue of CaMKI alpha in CaMKI alpha-knockdown neurons restored Wnt5a-mediated axon outgrowth.
Conclusions: This study suggests that Wnt5a activates Ca2+ signaling during a neuronal morphogenetic time window when axon outgrowth is critically facilitated. Furthermore, the CaMKK-CaMKI alpha cascade is required for the axonal growth effect of Wnt5a during neuronal polarization.

DOI： 10.1186/s13041-016-0189-3

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Methods for immunoblot detection and electron microscopic localization of septin subunits in mammalian nervous systems Invited

L. K. Parajuli, N. Ageta-Ishihara, H. Ageta, Y. Fukazawa, M. Kinoshita

Methods in Cell Biology   Vol. 136   page： 285 - 294   2016

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Part of collection (book)   Publisher：ELSEVIER ACADEMIC PRESS INC  

The minimal functional units of the mammalian septin system are diverse heterooligomers of SEPT1 1-14 subunits, which are most abundantly and differentially expressed in post mitotic neurons and glia. The subunit compositions of such heterooligomers are thought to differentiate their affinity for other proteins and lipids, and subcellular localization. Thus, high-precision quantification and mapping of each subunit is necessary to understand their subcellular functions and physiological roles. However, systematic information on the localization of individual septin subunits in the mammalian nervous system is limited. Here, we present our experimental workflows for the study of septin expression and localization in the rodent brain by immunoblot and serial section immunoelectron microscopy. Our protocols, based on standard methods, have been rigorously optimized and simplified for universality and reproducibility to aid non-experts in the field.

DOI： 10.1016/bs.mcb.2016.04.021

Web of Science

CDC42EP4/BORG4-septin complex beneath perisynaptic membrane domains of Bergmann glial processes facilitates GLAST-mediated glutamate clearance and motor learning.

Ageta-Ishihara N., Kinoshita M.

Molecular Biology of the Cell   Vol. 27   2016

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)  

Web of Science

A CDC42EP4/septin-based perisynaptic glial scaffold facilitates glutamate clearance. Reviewed International coauthorship International journal

Natsumi Ageta-Ishihara, Maya Yamazaki, Kohtarou Konno, Hisako Nakayama, Manabu Abe, Kenji Hashimoto, Tomoki Nishioka, Kozo Kaibuchi, Satoko Hattori, Tsuyoshi Miyakawa, Kohichi Tanaka, Fathul Huda, Hirokazu Hirai, Kouichi Hashimoto, Masahiko Watanabe, Kenji Sakimura, Makoto Kinoshita

Nature Communications   Vol. 6 ( 10090 ) page： 10090 - 10090   2015.12

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Nature  

The small GTPase-effector proteins CDC42EP1-5/BORG1-5 interact reciprocally with CDC42 or the septin cytoskeleton. Here we show that, in the cerebellum, CDC42EP4 is exclusively expressed in Bergmann glia and localizes beneath specific membrane domains enwrapping dendritic spines of Purkinje cells. CDC42EP4 forms complexes with septin hetero-oligomers, which interact with a subset of glutamate transporter GLAST/EAAT1. In Cdc42ep4(-/-) mice, GLAST is dissociated from septins and is delocalized away from the parallel fibre-Purkinje cell synapses. The excitatory postsynaptic current exhibits a protracted decay time constant, reduced sensitivity to a competitive inhibitor of the AMPA-type glutamate receptors (γDGG) and excessive baseline inward current in response to a subthreshold dose of a nonselective inhibitor of the glutamate transporters/EAAT1-5 (DL-TBOA). Insufficient glutamate-buffering/clearance capacity in these mice manifests as motor coordination/learning defects, which are aggravated with subthreshold DL-TBOA. We propose that the CDC42EP4/septin-based glial scaffold facilitates perisynaptic localization of GLAST and optimizes the efficiency of glutamate-buffering and clearance.

DOI： 10.1038/ncomms10090

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Transgenic supplementation of SIRT1 fails to alleviate acute loss of nigrostriatal dopamine neurons and gliosis in a mouse model of MPTP-induced parkinsonism [v1; ref status: awaiting peer review, http://f1000r.es/5a9] Reviewed

Yasuko Kitao, Natsumi Ageta-Ishihara, Ryosuke Takahashi, Makoto Kinoshita, Osamu Hori

F1000Research   Vol. 4 ( 130 ) page： -   2015.5

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Faculty of 1000 Ltd  

Background Dopamine (DA) neuron-selective uptake and toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes parkinsonism in humans. Loss of DA neurons via mitochondrial damage and oxidative stress is reproduced by systemic injection of MPTP in animals, which serves as models of parkinsonism and Parkinson's disease (PD). This study aimed to test whether pan-neural supplementation of the longevity-related, pleiotropic deacetylase SIRT1, which confers partial tolerance to at least three models of stroke and neurodegeneration, could also alleviate MPTP-induced acute pathological changes in nigrostriatal DA neurons and neighboring glia. Results We employed a line of prion promoter-driven Sirt1-transgenic (Sirt1Tg) mice that chronically overexpress murine SIRT1 in the brain and spinal cord. Sirt1Tg and wild-type (WT) male littermates (3-4 months old) were subjected to intraperitoneal injection of MPTP. Acute histopathological changes in the midbrain and striatum (caudoputamen) were assessed with serial coronal sections triply labeled for tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP), and nuclear DNA. In the substantia nigra pars compacta (SNpc) of the midbrain, the number of TH-positive neurons and the reactive gliosis were comparable between the Sirt1Tg and WT littermates. In the striatum, the relative fluorescence intensity of TH-positive nerve terminals and the level of gliosis did not differ by the genotypes. Conclusions Sirt1Tg and WT littermate mice exhibited comparable acute histopathological reactions to the systemic injection of MPTP, loss of TH-positive neurons and reactive gliosis. Thus, the genetic supplementation of SIRT1 does not confer histologically recognizable protection on nigrostriatal DA neurons against acute toxicity of MPTP.

DOI： 10.12688/f1000research.6386.1

Scopus

SIRT1 attenuates severe ischemic damage by preserving cerebral blood flow Reviewed International coauthorship International journal

Yorito Hattori, Yoko Okamoto, Kazuyuki Nagatsuka, Ryosuke Takahashi, Rajesh N. Kalaria, Makoto Kinoshita, Masafumi Ihara

NeuroReport   Vol. 26 ( 3 ) page： 113 - 117   2015.2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Ovid Technologies (Wolters Kluwer Health)  

Silent information regulator 2 homolog 1 (SIRT1) is a protein deacetylase that has been reported to suppress neurodegenerative and cardiovascular pathologies in model organisms. We have recently reported that SIRT1 overexpression preserves cerebral blood flow (CBF) after bilateral common carotid artery stenosis (∼50% stenosis) by the deacetylation of endothelial nitric oxide synthase. This study was designed to determine whether cerebral SIRT1 expression would be effective in a more severe model of cerebral ischemia caused by bilateral common carotid artery occlusion (BCAO) in vivo. Sirt1-overexpressing (Sirt1-Tg) mice (n=13) and their wild-type littermates (n=17) were subjected to BCAO for 10 min using microaneurysm clips. Temporal CBF changes were measured by laser speckle flowmetry before and 5, 10 min, and 2 h after BCAO. Histological evaluation of hippocampal changes was performed 7 days after BCAO. Histological findings were significantly less severe in Sirt1-Tg mice than in wild-type mice; wild-type mice showed strokes in the hippocampus, whereas Sirt1-Tg mice had minimal hippocampal damage 7 days after BCAO. Consistent with this observation, wild-type mice showed a severe reduction in CBF to ∼20-25% of the baseline level during BCAO, whereas Sirt1-Tg littermates showed significantly preserved CBF up to 45-50% of the baseline level. Our study provides evidence for the promising role of SIRT1 in protecting against cerebral global ischemia by preserving CBF and restoring the cerebrovascular reserve.

DOI： 10.1097/WNR.0000000000000308

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Silent Information Regulator 2 Homolog 1 Counters Cerebral Hypoperfusion Injury by Deacetylating Endothelial Nitric Oxide Synthase Reviewed International coauthorship

Yorito Hattori, Yoko Okamoto, Takakuni Maki, Yumi Yamamoto, Naoya Oishi, Kenichi Yamahara, Kazuyuki Nagatsuka, Ryosuke Takahashi, Raj N. Kalaria, Hidenao Fukuyama, Makoto Kinoshita, Masafumi Ihara

STROKE   Vol. 45 ( 11 ) page： 3403 - 3411   2014.11

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

Background and Purpose Silent information regulator 2 homolog 1 (SIRT1) is a protein deacetylase that has been reported to suppress neurodegenerative and cardiovascular diseases in model organisms. We hypothesized that neurovascular protection is one of the diverse actions of SIRT1. This study was designed to determine whether SIRT1 protects against the consequences of cerebral hypoperfusion in vivo.
MethodsSirt1-overexpressing (Sirt1-Tg) mice driven by a prion promoter and their wild-type littermates were subjected to bilateral common carotid artery stenosis using external microcoils. Using Sirt1-Tg mice, we assessed the effect of SIRT1 on cerebral blood flow, cerebral angioarchitecture, histological and ultrastructural changes, and spatial working memory at several time points. We also evaluated the effects of preadministration of SIRT1 inhibitors or endothelial nitric oxide synthase inhibitors on cerebral blood flow after bilateral common carotid artery stenosis in Sirt1-Tg mice. Levels of acetylated and nonacetylated endothelial nitric oxide synthase were measured semiquantitatively with immunoblotting.
Results Cerebral hypoperfusion induced by bilateral common carotid artery stenosis caused memory impairment and histological changes in wild-type littermates. However, these phenotypes were rescued in Sirt1-Tg mice, where cerebral blood flow was maintained even poststenosis. Electron microscopic analyses showed irregularities in the vascular endothelia, such as tight junction openings in wild-type mice, which were absent in Sirt1-Tg littermates. Brain endothelial nitric oxide synthase was acetylated after cerebral hypoperfusion in wild-type littermates but remained unacetylated in Sirt1-Tg mice. Moreover, treatment with SIRT1 inhibitors and endothelial nitric oxide synthase inhibitors abolished the vasculoprotective effects of SIRT1.
Conclusions Our results indicate that neurovascular endothelial SIRT1 potentiation upregulates the nitric oxide system and counters cerebral hypoperfusion injury. This novel cerebral blood flow-preserving mechanism offers potential molecular targets for future therapeutic intervention.

DOI： 10.1161/STROKEAHA.114.006265

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Genetic deletion of SEPT7 reveals a cell type-specific role of septins in microtubule destabilization for the completion of cytokinesis. Reviewed International coauthorship International journal

Manoj B Menon, Akihiro Sawada, Anuhar Chaturvedi, Pooja Mishra, Karin Schuster-Gossler, Melanie Galla, Axel Schambach, Achim Gossler, Reinhold Förster, Michael Heuser, Alexey Kotlyarov, Makoto Kinoshita, Matthias Gaestel

PLoS Genetics   Vol. 10 ( 8 ) page： e1004558   2014.8

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Public Library of Science ({PLoS})  

Cytokinesis terminates mitosis, resulting in separation of the two sister cells. Septins, a conserved family of GTP-binding cytoskeletal proteins, are an absolute requirement for cytokinesis in budding yeast. We demonstrate that septin-dependence of mammalian cytokinesis differs greatly between cell types: genetic loss of the pivotal septin subunit SEPT7 in vivo reveals that septins are indispensable for cytokinesis in fibroblasts, but expendable in cells of the hematopoietic system. SEPT7-deficient mouse embryos fail to gastrulate, and septin-deficient fibroblasts exhibit pleiotropic defects in the major cytokinetic machinery, including hyperacetylation/stabilization of microtubules and stalled midbody abscission, leading to constitutive multinucleation. We identified the microtubule depolymerizing protein stathmin as a key molecule aiding in septin-independent cytokinesis, demonstrated that stathmin supplementation is sufficient to override cytokinesis failure in SEPT7-null fibroblasts, and that knockdown of stathmin makes proliferation of a hematopoietic cell line sensitive to the septin inhibitor forchlorfenuron. Identification of septin-independent cytokinesis in the hematopoietic system could serve as a key to identify solid tumor-specific molecular targets for inhibition of cell proliferation.

DOI： 10.1371/journal.pgen.1004558

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Phosphatidic Acid ( PA)-preferring Phospholipase A1 Regulates Mitochondrial Dynamics Reviewed International coauthorship

Takashi Baba, Yuriko Kashiwagi, Nagisa Arimitsu, Takeshi Kogure, Ayumi Edo, Tomohiro Maruyama, Kazuki Nakao, Hiroki Nakanishi, Makoto Kinoshita, Michael A. Frohman, Akitsugu Yamamoto, Katsuko Tani

JOURNAL OF BIOLOGICAL CHEMISTRY   Vol. 289 ( 16 ) page： 11497 - 11511   2014.4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Background: Phosphatidic acid (PA) is involved in membrane dynamics. Results: PA-preferring phospholipase A(1) (PA-PLA(1)) affects mitochondrial morphology in an activity-dependent manner. Gene disruption of PA-PLA(1) in mice causes sperm malformation due to mitochondrial organization defects. Conclusion: PA-PLA(1) regulates mitochondrial dynamics. Significance: We demonstrate an in vivo function of PA-PLA(1) and suggest a possible mechanism of PA regulation of the mitochondrial membrane.
Recent studies have suggested that phosphatidic acid (PA), a cone-shaped phospholipid that can generate negative curvature of lipid membranes, participates in mitochondrial fusion. However, precise mechanisms underling the production and consumption of PA on the mitochondrial surface are not fully understood. Phosphatidic acid-preferring phospholipase A(1) (PA-PLA(1))/DDHD1 is the first identified intracellular phospholipase A(1) and preferentially hydrolyzes PA in vitro. Its cellular and physiological functions have not been elucidated. In this study, we show that PA-PLA(1) regulates mitochondrial dynamics. PA-PLA(1), when ectopically expressed in HeLa cells, induced mitochondrial fragmentation, whereas its depletion caused mitochondrial elongation. The effects of PA-PLA(1) on mitochondrial morphology appear to counteract those of MitoPLD, a mitochondrion-localized phospholipase D that produces PA from cardiolipin. Consistent with high levels of expression of PA-PLA(1) in testis, PA-PLA(1) knock-out mice have a defect in sperm formation. In PA-PLA(1)-deficient sperm, the mitochondrial structure is disorganized, and an abnormal gap structure exists between the middle and principal pieces. A flagellum is bent at that position, leading to a loss of motility. Our results suggest a possible mechanism of PA regulation of the mitochondrial membrane and demonstrate an in vivo function of PA-PLA(1) in the organization of mitochondria during spermiogenesis.

DOI： 10.1074/jbc.M113.531921

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SIRT1 overexpression ameliorates a mouse model of SOD1-linked amyotrophic lateral sclerosis via HSF1/HSP70i chaperone system Reviewed

Seiji Watanabe, Natsumi Ageta-Ishihara, Shinji Nagatsu, Keizo Takao, Okiru Komine, Fumito Endo, Tsuyoshi Miyakawa, Hidemi Misawa, Ryosuke Takahashi, Makoto Kinoshita, Koji Yamanaka

Molecular Brain   Vol. 7 ( 1 ) page： 62 - 62   2014

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Background: Dominant mutations in superoxide dismutase 1 (SOD1) cause degeneration of motor neurons in a subset of inherited amyotrophic lateral sclerosis (ALS). The pathogenetic process mediated by misfolded and/or aggregated mutant SOD1 polypeptides is hypothesized to be suppressed by protein refolding. This genetic study is aimed to test whether mutant SOD1-mediated ALS pathology recapitulated in mice could be alleviated by overexpressing a longevity-related deacetylase SIRT1 whose substrates include a transcription factor heat shock factor 1 (HSF1), the master regulator of the chaperone system.
Results: We established a line of transgenic mice that chronically overexpress SIRT1 in the brain and spinal cord. While inducible HSP70 (HSP70i) was upregulated in the spinal cord of SIRT1 transgenic mice (PrP-Sirt1), no neurological and behavioral alterations were detected. To test hypothetical benefits of SIRT1 overexpression, we crossbred PrP-Sirt1 mice with two lines of ALS model mice: A high expression line that exhibits a severe phenotype (SOD1(G93A)-H) or a low expression line with a milder phenotype (SOD1(G93A)-L). The Sirt1 transgene conferred longer lifespan without altering the time of symptomatic onset in SOD1(G93A)-L. Biochemical analysis of the spinal cord revealed that SIRT1 induced HSP70i expression through deacetylation of HSF1 and that SOD1(G93A)-L/PrP-Sirt1 double transgenic mice contained less insoluble SOD1 than SOD1(G93A)-L mice. Parallel experiments showed that Sirt1 transgene could not rescue a more severe phenotype of SOD1(G93A)-H transgenic mice partly because their HSP70i level had peaked out.
Conclusions: The genetic supplementation of SIRT1 can ameliorate a mutant SOD1-linked ALS mouse model partly through the activation of the HSF1/HSP70i chaperone system. Future studies shall include testing potential benefits of pharmacological enhancement of the deacetylation activity of SIRT1 after the onset of the symptom.

DOI： 10.1186/s13041-014-0062-1

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Septins promote dendrite and axon development by negatively regulating microtubule stability via HDAC6-mediated deacetylation. Reviewed International coauthorship

Ageta-Ishihara N, Miyata T, Ohshima C, Watanabe M, Sato Y, Hamamura Y, Higashiyama T, Mazitschek R, Bito H, Kinoshita M.

Nature Communications   Vol. 4 ( 2532 )   2013.10

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Neurite growth requires two guanine nucleotide-binding protein polymers of tubulins and septins. However, whether and how those cytoskeletal systems are coordinated was unknown. Here we show that the acute knockdown or knockout of the pivotal septin subunit SEPT7 from cerebrocortical neurons impairs their interhemispheric and cerebrospinal axon projections and dendritogenesis in perinatal mice, when the microtubules are severely hyperacetylated. The resulting hyperstabilization and growth retardation of microtubules are demonstrated in vitro. The phenotypic similarity between SEPT7 depletion and the pharmacological inhibition of α-tubulin deacetylase HDAC6 reveals that HDAC6 requires SEPT7 not for its enzymatic activity, but to associate with acetylated α-tubulin. These and other findings indicate that septins provide a physical scaffold for HDAC6 to achieve efficient microtubule deacetylation, thereby negatively regulating microtubule stability to an optimal level for neuritogenesis. Our findings shed light on the mechanisms underlying the HDAC6-mediated coupling of the two ubiquitous cytoskeletal systems during neural development.

DOI： 10.1038/ncomms3532

Chronic overload of SEPT4, a parkin substrate that aggregates in Parkinson's disease, causes behavioral alterations but not neurodegeneration in mice. Reviewed International journal

Natsumi Ageta-Ishihara, Hodaka Yamakado, Takao Morita, Satoko Hattori, Keizo Takao, Tsuyoshi Miyakawa, Ryosuke Takahashi, Makoto Kinoshita

Molecular Brain   Vol. 6 ( 1 ) page： 35 - 35   2013.8

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BACKGROUND: In autosomal recessive early-onset Parkinsonism (PARK2), the pathogenetic process from the loss of function of a ubiquitin ligase parkin to the death of dopamine neurons remains unclear. A dominant hypothesis attributes the neurotoxicity to accumulated substrates that are exempt from parkin-mediated degradation. Parkin substrates include two septins; SEPT4/CDCrel-2 which coaggregates with α-synuclein as Lewy bodies in Parkinson's disease, and its closest homolog SEPT5/CDCrel-1/PNUTL1 whose overload with viral vector can rapidly eliminate dopamine neurons in rats. However, chronic effects of pan-neural overload of septins have never been examined in mammals. To address this, we established a line of transgenic mice that express the largest gene product SEPT4(54kDa) via the prion promoter in the entire brain. RESULTS: Histological examination and biochemical quantification of SEPT4-associated proteins including α-synuclein and the dopamine transporter in the nigrostriatal dopamine neurons found no significant difference between Sept4(Tg/+) and wild-type littermates. Thus, the hypothetical pathogenicity by the chronic overload of SEPT4 alone, if any, is insufficient to trigger neurodegenerative process in the mouse brain. Intriguingly, however, a systematic battery of behavioral tests revealed unexpected abnormalities in Sept4(Tg/+) mice that include consistent attenuation of voluntary activities in distinct behavioral paradigms and altered social behaviors. CONCLUSIONS: Together, these data indicate that septin dysregulations commonly found in postmortem human brains with Parkinson's disease, schizophrenia and bipolar disorders may be responsible for a subset of behavioral abnormalities in the patients.

DOI： 10.1186/1756-6606-6-35

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セプチンによるドーパミン神経伝達機構の制御 Invited

木下　専、田（石原）奈津実

脳21 特集 トランスポートソーム：トランスポーター・チャネル群の相互作用に基づく神経系の機能構築   Vol. 16 ( 3 ) page： 40 - 45   2013.7

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運動による脳の活性化のしくみ Invited Reviewed

Ageta-Ishihara N, Morita T, Yamauchi Y, Kinoshita M

健康医科学   Vol. 28   page： 44 - 51   2013.3

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Localization of septin proteins in the mouse cochlea. Reviewed International coauthorship International journal

Atsuhiro Yoshida, Norio Yamamoto, Makoto Kinoshita, Noboru Hiroi, Takeshi Hiramoto, Gina Kang, William S Trimble, Kenji Tanigaki, Takayuki Nakagawa, Juichi Ito

Hearing Research   Vol. 289 ( 1-2 ) page： 40 - 51   2012.7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

Septins are a family of GTP binding proteins that are well conserved in eukaryotic species except plants. Septins contribute to the lateral compartmentalization of membranes, cortical rigidity, and the regulation of membrane trafficking by associating with membrane lipids, actin, and microtubules. The organ of Corti in the cochlea has pivotal roles in auditory perception and includes two kinds of highly polarized cells, hair and supporting cells, both of which are rich in actin and microtubules. To identify the roles of septins in the cochlea, we analyzed the localization of three septin proteins, septin 4 (SEPT4), septin 5 (SEPT5), and septin 7 (SEPT7) that are abundantly expressed in brain tissues, and also examined auditory functions of Sept4 and Sept5 null mice. SEPT4, SEPT5, and SEPT7 were expressed in inner and outer pillar cells and Deiters' cells but the distribution patterns of each protein in Deiters' cells were different. SEPT4 and SEPT7 were expressed in the phalangeal process where SEPT5 was not detected. In addition to these cells SEPT5 and SEPT7 were co-localized with presynaptic vesicles of efferent nerve terminals. Only SEPT7 was expressed in the cochlea at embryonic stages. Although expression patterns of septin proteins suggested their important roles in the function of the cochlea, both Sept4 and Sept5 null mice had similar auditory functions to their wild type littermates. Immunohistochemical analysis of Sept4 null mice showed that compensatory expression of SEPT5 in the phalangeal process of Deiters' cells may have caused functional compensation of hearing ability in Sept4 null mice.

DOI： 10.1016/j.heares.2012.04.015

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[Role of septin cytoskeleton in dopaminergic neurotransmission and neurodegeneration]. Invited

Makoto Kinoshita

Nihon shinkei seishin yakurigaku zasshi = Japanese journal of psychopharmacology   Vol. 32 ( 1 ) page： 25 - 9   2012.2

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Cytoskeletal polymers play pleiotropic roles in neuroglial morphogenesis, intracellular transport, organization of pre- and post-synaptic scaffolds, etc. Thus, neuroglial dysfunction and degeneration are often accompanied by abnormalities in microtubules, actin and/or intermediate filament systems. Although our understanding of an unconventional cytoskeletal system composed of the septin family of GTP-binding proteins is far behind, recent studies have been revealing that qualitative and/or quantitative abnormalities of septins are also associated with neurodegenerative disorders including hereditary neuralgic amyotrophy, Parkinson disease, schizophrenia and bipolar disorder. A better understanding of the physiological and pathophysiological roles of the septin system should help develop useful biomarkers and therapeutic strategies for these diseases.

PubMed

セプチン Invited Reviewed

上田（石原）奈津実、木下　専

脳科学辞典     2012

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DOI： 10.14931/bsd.484

セプチン Invited

上谷　大介、木下　専

日本細胞生物学会　細胞生物学用語集     2012

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Downregulation of the Wnt antagonist Dkk2 links the loss of Sept4 and myofibroblastic transformation of hepatic stellate cells. Reviewed International journal

Atsuko Yanagida, Keiko Iwaisako, Etsuro Hatano, Kojiro Taura, Fumiaki Sato, Masato Narita, Hiromitsu Nagata, Hiroyuki Asechi, Shinji Uemoto, Makoto Kinoshita

Biochimica et Biophysica Acta - Molecular Basis of Disease   Vol. 1812 ( 11 ) page： 1403 - 1411   2011.11

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BACKGROUND/AIMS: Sept4, a subunit of the septin cytoskeleton specifically expressed in quiescent hepatic stellate cells (HSCs), is downregulated through transdifferentiation to fibrogenic and contractile myofibroblastic cells. Since Sept4(-/-)mice are prone to liver fibrosis, we aimed to identify the unknown molecular network underlying liver fibrosis by probing the association between loss of Sept4 and accelerated transdifferentiation of HSCs. METHODS: We compared the transcriptomes of Sept4(+/+) and Sept4(-/-) HSCs undergoing transdifferentiation by DNA microarray and quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. Because Dickkopf2 (Dkk2) gene expression is reduced in Sept4(-/-) HSCs, we tested whether supplementing Dkk2 could suppress myofibroblastic transformation of Sept4(-/-) HSCs. We tested the involvement of the canonical Wnt pathway in this process by using a lymphoid enhancer-binding factor/transcription factor-luciferase reporter assay. RESULTS: We observed consistent upregulation of Dkk2 in primary cultured HSCs and in a carbon tetrachloride liver fibrosis in mice, which was decreased in the absence of Sept4. Supplementation with Dkk2 suppressed the induction of pro-fibrotic genes (α-smooth muscle actin and 2 collagen genes) and induced an anti-fibrotic gene (Smad7) in Sept4(-/-) HSCs. In human liver specimens with inflammation and fibrosis, Dkk2 immunoreactivity appeared to be positively correlated with the degree of fibrotic changes. CONCLUSIONS: Pro-fibrotic transformation of HSCs through the loss of Sept4 is, in part, due to reduced expression of Dkk2 and its homologues, and the resulting disinhibition of the canonical Wnt pathway.

DOI： 10.1016/j.bbadis.2011.06.015

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Submembranous septins as relatively stable components of actin-based membrane skeleton. Reviewed International journal

Akari Hagiwara, Yasuhiro Tanaka, Rie Hikawa, Nobuhiro Morone, Akihiro Kusumi, Hiroshi Kimura, Makoto Kinoshita

Cytoskeleton (Hoboken, N.J.)   Vol. 68 ( 9 ) page： 512 - 525   2011.9

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The cell cortex is organized by the dynamic interplay between the plasma membrane, membrane proteins, and the cytoskeleton. Despite the cortical localization of septin heteropolymers in vivo and their direct interaction with phospholipid membranes in vitro, their behavior and roles remain elusive. This study characterizes the major cortical septin assembly found in mammalian tissue culture cells by fluorescence recovery after photobleaching analysis. GFP-tagged septin subunits, which colocalized with cortical actin, exhibited slower turnover than some other cortical proteins that were analyzed (e.g., actin, syntaxin-1A and a glutamate aspartate transporter [GLAST]). Perturbation of actin turnover by cytochalasin D or jasplakinolide retarded the cortical septin turnover, while septin depletion by RNAi did not recognizably affect cortical actin turnover. These phenomena are compatibly interpreted by septins' selective association with a subset of actin-based membrane skeleton, as revealed by rapid-freeze deep-etch immuno-replica electron microscopy. We applied the assay system to test septins' presumptive scaffold function on their physiological binding partners. Septin filament destabilization by RNAi-mediated subunit depletion facilitated the turnover of GLAST, depending on the carboxyl-terminal 29 residues, while a septin filament-stabilizing drug forchlorfenuron restrained more GLAST in the unexchangeable fraction. These data indicate that cortical septin heteropolymers are components of the actin-based membrane skeleton providing scaffolds for their interacting partners probably by impeding their lateral diffusion. We predict that diverse submembranous septin clusters found in vivo may serve as scaffolds or reserve pools for specific membrane-bound proteins.

DOI： 10.1002/cm.20528

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Ultrastructural localization analysis of septins in mammalian nervous system

Hiroyuki Kurita, Yugo Fukazawa, Natsumi Ageta-Ishihara, Ryuichi Shigemoto, Makoto Kinoshita

Neuroscience Research   Vol. 71   2011.9

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DOI： 10.1016/j.neures.2011.07.505

トランスポートソームの組織化における細胞骨格系の役割 Reviewed

藤原敬広、木下　専

トランスポートソームの世界-膜輸送研究の源流から未来へ     page： 380 - 386   2011.4

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Septin-dependent microtubule control during mammalian neurite outgrowth Invited Reviewed

Molecular Biology of the Cell   Vol. 22   2011

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Role of the Septin Cytoskeleton for the Functional Organization of the Plasma Membrane Invited

Makoto Kinoshita

WATER: THE FORGOTTEN BIOLOGICAL MOLECULE     page： 259 - 267   2011

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Entrapment of intracytosolic bacteria by septin cage-like structures. Reviewed International coauthorship International journal

Serge Mostowy, Matteo Bonazzi, Mélanie Anne Hamon, To Nam Tham, Adeline Mallet, Mickaël Lelek, Edith Gouin, Caroline Demangel, Roland Brosch, Christophe Zimmer, Anna Sartori, Makoto Kinoshita, Marc Lecuit, Pascale Cossart

Cell host & microbe   Vol. 8 ( 5 ) page： 433 - 44   2010.11

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Actin-based motility is used by various pathogens for dissemination within and between cells. Yet host factors restricting this process have not been identified. Septins are GTP-binding proteins that assemble as filaments and are essential for cell division. However, their role during interphase has remained elusive. Here, we report that septin assemblies are recruited to different bacteria that polymerize actin. We observed that intracytosolic Shigella either become compartmentalized in septin cage-like structures or form actin tails. Inactivation of septin caging increases the number of Shigella with actin tails and enhances cell-to-cell spread. TNF-α, a host cytokine produced upon Shigella infection, stimulates septin caging and restricts actin tail formation and cell-to-cell spread. Finally, we show that septin cages entrap bacteria targeted to autophagy. Together, these results reveal an unsuspected mechanism of host defense that restricts dissemination of invasive pathogens.

DOI： 10.1016/j.chom.2010.10.009

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Rho and Anillin-dependent Control of mDia2 Localization and Function in Cytokinesis Reviewed

Molecular Biology of the Cell   Vol. 21 ( 18 ) page： 3193 - 3204   2010.9

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DOI： 10.1091/mbc.E10-04-0324

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[Diverse physiological functions of the septin system: the protean cytoskeleton]. Invited Reviewed

Makoto Kinoshita

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme   Vol. 54 ( 9 ) page： 1150 - 8   2009.7

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パーキンソン病関連遺伝子Sept4 Invited Reviewed

猪原匡史、木下　専

「パーキンソン病―基礎・臨床研究のアップデート」日本臨牀 76巻増刊号4（通巻1142号）   Vol. 76 ( 4 ) page： 75 - 78   2009.6

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[Functions of the septin cytoskeleton and its roles in dopaminergic neurotransmission]. Invited Reviewed

Masafumi Ihara, Makoto Kinoshita

Brain and nerve = Shinkei kenkyu no shinpo   Vol. 61 ( 4 ) page： 419 - 28   2009.4

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Cytoskeletal polymers are component of cellular infrastructure that are required for fundamental biological processes ranging from cell division to brain functions. Unlike the knowledge available for tubulin and actin, our understanding of unconventional cytoskeletal structures composed of GTP-binding proteins belonging to the septin family is limited, despite their ubiquity and implications in human diseases. Recent studies have revealed that septin plays unique modulatory roles as an accessory component of microtubules and the actin cytoskeleton. Morphological analyses of the mammalian brain and neural cells have revealed that septins preferentially cluster beneath the extra-synaptic membrane domains in dendritic shafts and spine necks, presynaptic terminals of major neurons, and astroglial processes. Live imaging analysis revealed that septin polymers are remarkably stable in these clusters, which may serve as local cytoskeleton and/or scaffold for the organization of specialized cortical domains in neurons and glia. This hypothesis has been supported by the hypo-dopaminergic phenotype of mice that lack the Sept4 subunit and the hyper-dopaminergic phenotype of those with excess Sept4. In these cases, the septin scaffold in the dopamine neurons is considered as a determinant of the quantity of a subset of presynaptic molecules, including tSNAREs (membrane-fusion machinery) and the dopamine transporters. This finding in mouse models is in agreement with the recent findings that qualitative and/or quantitative dysregulation of septins is involved in neurodegenerative disorders such as Parkinson disease and psychological disorders such as schizophrenia and bipolar disorder. Studies on tubulin/actin indicate that a better understanding of the septin family of proteins will improve our insight into neuropathological phenomena in neurodegenerative and psychological disorders, which may help develop diagnostic markers and therapeutic strategies for such diseases.

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Other Link： http://orcid.org/0000-0002-7102-4048

Spatiotemporal association of DNAJB13 with the annulus during mouse sperm flagellum development. Reviewed International coauthorship International journal

Jikui Guan, Makoto Kinoshita, Li Yuan

BMC developmental biology   Vol. 9 ( 1 ) page： 23 - 23   2009.3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science $\mathplus$ Business Media  

BACKGROUND: The sperm annulus is a septin-based fibrous ring structure connecting the midpiece and the principal piece of the mammalian sperm flagellum. Although ultrastructural abnormalities and functional importance of the annulus have been addressed in Sept4-null mutant mice and a subset of human patients with asthenospermia syndrome, little is known about how the structure is assembled and positioned to the midpiece-principal piece junction during mammalian sperm flagellum development. RESULTS: By performing immunofluorescence and biochemical approaches with antibodies against DNAJB13 and an annulus constituent SEPT4, we report here a spatiotemporal association of DNAJB13 with sperm annulus during mouse sperm flagellum development. DNAJB13 co-localized with SEPT4 to the annulus, and both were first able to be detected in step 9 spermatids. As spermiogenesis proceeded, the annular DNAJB13 immunosignal increased until the annulus reached the midpiece-principal piece junction, and then gradually disappeared from it in late spermiogenesis. In contrast, the SEPT4 immunosignal was relatively unaltered, and still present on annulus of mature spermatozoa. In Sept4-null mouse spermatids lacking the annulus structure, the annulus-like DNAJB13 immunosignal was still able to be detected, albeit weaker, at the neck region of the flagella. In vitro DNAJB13 was co-localized and interacted with SEPT4 directly. CONCLUSION: The direct interaction of DNAJB13 with SEPT4 in vitro and its spatiotemporal association with the annulus during sperm flagellum development, and even its annulus-like appearance in the annulus-deficient spermatids, suggest that DNAJB13 may be involved in assembling the annulus structure and positioning it towards the midpiece-principal piece junction.

DOI： 10.1186/1471-213X-9-23

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Septin-Mediated Uniform Bracing of Phospholipid Membranes Reviewed

Yohko Tanaka-Takiguchi, Makato Kinoshita, Kingo Takiguchi

Current Biology   Vol. 19 ( 2 ) page： 140 - 145   2009.1

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Cell shape is determined by the interplay between the lipid bilayer and the underlying network of protein polymers [1]. We explored unknown determinants involved in cell morphogenesis as factors that transform phospholipid-based liposomes (diameter 5-20 mu m). Unlabeled giant liposomes, observed through dark-field optics, were metastable in an aqueous suspension. In contrast, liposomes robustly protruded uniform tubules immediately after the addition of a brain extract to the suspension. The tubulation reaction was greatly facilitated when the liposomes contained PIP or PIP2. Biochemical analysis of the brain extract revealed that heteromeric complexes of septins, a family of polymerizing GTP/GDP-binding proteins, are responsible for the membrane transformation. Ultrastructural analysis established that each membrane tubule (diameter 0.43 +/- 0.079 mu m) is braced by a circumferential array of septin filaments. Although submembranous septin assemblies are associated with diverse cortical morphogenesis from yeast to mammals [2-5], the biophysical basis for the septin-membrane interplay remains largely unknown. Further, there is a biochemical discrepancy between the fast septin remodeling in cells and their slow self-assembly in vitro [6, 7]. This membrane-facilitated fast septin assembly demonstrated for the first time by our unique experimental system should provide important clues to characterize these processes.

DOI： 10.1016/j.cub.2008.12.030

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Amoeboid T lymphocytes require the septin cytoskeleton for cortical integrity and persistent motility. Reviewed International coauthorship International journal

Aaron J Tooley, Julia Gilden, Jordan Jacobelli, Peter Beemiller, William S Trimble, Makoto Kinoshita, Matthew F Krummel

Nature Cell Biology   Vol. 11 ( 1 ) page： 17 - 26   2009.1

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The systems that refine actomyosin forces during motility remain poorly understood. Septins assemble on the T-cell cortex and are enriched at the mid-zone in filaments. Septin knockdown causes membrane blebbing, excess leading-edge protrusions and lengthening of the trailing-edge uropod. The associated loss of rigidity permits motility, but cells become uncoordinated and poorly persistent. This also relieves a previously unrecognized restriction to migration through small pores. Pharmacologically rigidifying cells counteracts this effect, and relieving cytoskeletal rigidity synergizes with septin depletion. These data suggest that septins tune actomyosin forces during motility and probably regulate lymphocyte trafficking in confined tissues.

DOI： 10.1038/ncb1808

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Sept4 Invited

祝迫惠子、木下　専

分子細胞治療 (先端医学社)   Vol. 8 ( 2 ) page： 68 - 69   2009

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Language：Japanese  

物質輸送システムの支持機構としてのセプチン系とその破綻 Invited Reviewed

木下　専

創薬研究者必見!最新トランスポーター研究2009 (遺伝子医学MOOK)     page： 95 - 101   2009

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Septins as diagnostic markers for a subset of human asthenozoospermia. Reviewed International journal

Yoshio Sugino, Kentaro Ichioka, Takeshi Soda, Masafumi Ihara, Makoto Kinoshita, Osamu Ogawa, Hiroyuki Nishiyama

The Journal of Urology   Vol. 180 ( 6 ) page： 2706 - 2709   2008.12

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PURPOSE: Septins are the major constituents of the annulus, a submembranous ring that separates the middle and principal pieces of spermatozoa. We previously reported its essential role in spermiogenesis and reproduction in mice. In the current study we investigated septin abnormality in infertile men. MATERIALS AND METHODS: Semen samples from 108 infertile patients and 21 healthy volunteers were analyzed for sperm concentration and motility. Spermatozoa were immunostained for the 2 representative septin subunits SEPT4 and SEPT7. Peripheral blood DNA from 8 patients with asthenozoospermia who had defective SEPT4 and/or SEPT7 labeling in the annuli was analyzed by direct sequencing. Clinical information and a followup review of pregnancy were obtained retrospectively from medical records. RESULTS: Specific antibodies for SEPT4 and SEPT7 consistently labeled the annuli in spermatozoa from the 21 healthy volunteers, while 14 of 108 samples (13%) from infertile patients showed defective labeling. In 33 patients with asthenozoospermia 10 samples (30%) demonstrated defective labeling for SEPT4 and/or SEPT7. We could not detect exon mutations in the SEPT4 gene by sequencing peripheral blood DNA from 8 patients with asthenozoospermia who had defective SEPT4 and/or SEPT7 labeling. During followup 8 of 14 patients (57%) with SEPT4 and/or SEPT7 labeling defects achieved successful pregnancies. CONCLUSIONS: Annulus defects were found exclusively in infertile patients. Although their prognoses do not differ from those without annulus defects, annulus labeling by septin antibodies may serve as an index for classifying a subset of spermatogenesis defects and monitoring sperm quality.

DOI： 10.1016/j.juro.2008.08.005

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Loss of Sept4 exacerbates liver fibrosis through the dysregulation of hepatic stellate cells Reviewed

Keiko Iwaisako, Etsuro Hatano, Kojiro Taura, Akio Nakajima, Masaharu Tada, Satoru Seo, Nobuyuki Tamaki, Fumiaki Sato, Iwao Ikai, Shinji Uemoto, Makoto Kinoshita

Journal of Hepatology   Vol. 49 ( 5 ) page： 768 - 778   2008.11

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Background/Aims: Septins are ubiquitous and multifunctional scaffold proteins involved in cytoskeletal organization, exocytosis and other cellular processes. We disclose the quiescent hepatic stellate cells (HSCs)-specific expression of a septin subunit Sept4 in the liver, and explore the significance of the septin system in liver fibrosis.
Methods: We analyzed the expression of alpha-smooth muscle actin (alpha-SMA), collagens and other markers in primary cultured HSCs derived from wild-type and Sept4(-/-) mice. We compared susceptibility of these mice to liver fibrosis induced by either carbon tetrachloride treatment, bile duct ligation or methionine/choline-deficient diet. Collagen deposition, the principal parameter of liver fibrosis, was quantified both histochemically (Masson's trichrome stain) and biochemically (hydroxyproline content).
Results: In vitro, Sept4 mRNA/protein was remarkably downregulated in HSCs through myofibroblastic transformation. Sept4(-/-) HSCs showed normal morphology and proliferation, while myofibroblastic transformation as monitored by the upregulation of alpha-SMA and collagen was accelerated compared to wild-type HSCs. In vivo, liver fibrosis was consistently more severe in Sept4(-/-) mice than in wild-type littermates in all of the three paradigms of hepatitis/liver fibrosis.
Conclusions: These data concordantly indicate that the HSC-specific septin subunit Sept4 and perhaps the septin system are involved in the suppressive modulation of myofibroblastic transformation and fibrogenesis associated with liver diseases. (C) 2008 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jhep.2008.05.026

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Structural insights shed light onto septin assemblies and function Invited Reviewed International coauthorship

Yves Barral, Makoto Kinoshita

Current Opinion in Cell Biology   Vol. 20 ( 1 ) page： 12 - 18   2008.2

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While the original septin mutants were identified more than 30 years ago for their role in cytokinesis [Hartwell, LH: Genetic control of the cell division cycle in yeast. IV. Genes controlling bud emergence and cytokinesis. Exp Cell Res 1971, 69: 265-276], the architecture of septin complexes and higher order structures has remained a mystery up until very recently. Over the last few months a number of converging approaches have suddenly provided a wealth of structural information about the different levels of septin organization. Here, we review these advancements and highlight their functional consequences.

DOI： 10.1016/j.ceb.2007.12.001

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Morphogenesis of polarized epithelia requires Sept2-mediated coupling of vesicle transport to polyglutamylated microtubules. Reviewed International coauthorship

Spiliotis ET, Hunt S, Hu Q, Kinoshita M, Nelson WJ

Journal of Cell Biology   Vol. 180   page： 295-303   2008

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Parkin deficient mice display motor disfunction and learning and memory impairment Reviewed

Neuroscience Research   Vol. 61   page： S136 - S136   2008

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Role of Septin Cytoskeleton in Spine Morphogenesis and Dendrite Development in Neurons Reviewed International coauthorship

Tomoko Tada, Alyson Simonetta, Matthew Batterton, Makoto Kinoshita, Dieter Edbauer, Morgan Sheng

Current Biology   Vol. 17 ( 20 ) page： 1752 - 1758   2007.10

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Septins are GTP-binding proteins that polymerize into heteromeric filaments and form microscopic bundles or ring structures in vitro and in vivo. Because of these properties and their ability to associate with membrane, F-actin, and microtubules, septins have been generally regarded as cytoskeletal components [1, 2]. Septins are known to play roles in cytokinesis, in membrane trafficking, and as structural scaffolds; however, their function in neurons is poorly understood. Many members of the septin family, including Septin 7 (Sept7), were found by mass-spectrometry analysis of postsynaptic density (PSD) fractions of the brain [3, 4], suggesting a possible postsynaptic function of septins in neurons. We report that Sept7 is localized at the base of dendritic protrusions and at dendritic branch points in cultured hippocampal neurons-a distribution reminiscent of septin localization in the bud neck of budding yeast. Overexpression of Sept7 increased dendrite branching and the density of dendritic protrusions, whereas RNA interference (RNAi)-mediated knockdown of Sept7 led to reduced dendrite arborization and a greater proportion of immature protrusions. These data suggest that Sept7 is critical for spine morphogenesis and dendrite development during neuronal maturation.

DOI： 10.1016/j.cub.2007.09.039

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Connecting the dots between septins and the DNA damage checkpoint Invited Reviewed

Makoto Kinoshita, Shunichi Takeda

Cell   Vol. 130 ( 5 ) page： 777 - 779   2007.9

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In budding yeast, septins are involved in the morphogenesis checkpoint and the DNA damage checkpoint, both of which regulate cell-cycle progression. In this issue of Cell, Kremer et al. (2007) link septins to DNA damage in mammalian cells by identifying a new signaling pathway that includes the adaptors SOCS7 and NCK. As NCK controls actin dynamics, this pathway may connect DNA damage responses and cellular morphology in metazoans.

DOI： 10.1016/j.cell.2007.08.022

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パーキンソン病の病態に迫る Invited

猪原匡史、木下　専

化学と生物 (日本農芸化学会誌)   Vol. 45 ( 9 ) page： 598 - 599   2007.9

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DOI： 10.1271/kagakutoseibutsu1962.45.596

Phosphorylation of adult type Sept5 (CDCrel-1) by cyclin-dependent kinase 5 inhibits interaction with syntaxin-1 Reviewed

Makoto Taniguchi, Masato Taoka, Makoto Itakura, Akiko Asada, Taro Saito, Makoto Kinoshita, Masami Takahashi, Toshiaki Isobe, Shin-ichi Hisanaga

Journal of Biological Chemistry   Vol. 282 ( 11 ) page： 7869 - 7876   2007.3

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Increasing evidence implicates cyclin-dependent kinase 5 (Cdk5) in neuronal synaptic function. We searched for Cdk5 substrates in synaptosomal fractions prepared from mouse brains. Mass spectrometric analysis after two-dimensional SDS-PAGE identified several synaptic proteins phosphorylated by Cdk5-p35; one protein identified was Sept5 (CDCrel-1). Although septins were isolated originally as cell division-related proteins in yeast, Sept5 is expressed predominantly in neurons and is implicated in exocytosis. We confirmed that Sept5 is phosphorylated by Cdk5p35 in vitro and identified Ser(17) of adult type Sept5 (Sept5_v1) as a major phosphorylation site. We found that Ser17 of Sept5_v1 is phosphorylated in mouse brains. Coimmunoprecipitation from synaptosomal fractions and glutathione S-transferase-syntaxin-1A pulldown assays of Sept5_v1 expressed in COS-7 cells showed that phosphorylation of Sept5_v1 by Cdk5-p35 decreases the binding to syntaxin-1. These results indicate that the interaction of Sept5 with syntaxin-1 is regulated by the phosphorylation of Sept_v1 at Ser(17) by Cdk5-p35.

DOI： 10.1074/jbc.M609457200

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Sept4, a Component of Presynaptic Scaffold and Lewy Bodies, Is Required for the Suppression of α-Synuclein Neurotoxicity Reviewed

Masafumi Ihara, Nobuyuki Yamasaki, Akari Hagiwara, Ai Tanigaki, Ayumi Kitano, Rie Hikawa, Hidekazu Tomimoto, Makoto Noda, Masashi Takanashi, Hideo Mori, Nobutaka Hattori, Tsuyoshi Miyakawa, Makoto Kinoshita

Neuron   Vol. 53 ( 4 ) page： 519 - 533   2007.2

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In Parkinson disease (PID), alpha-synuclein aggregates called Lewy bodies often involve and sequester Septin4 (Sept4), a polymerizing scaffold protein. However, the pathophysiological significance of this phenomenon is unclear. Here, we show the physiological association of Sept4 with alpha-synuclein, the dopamine transporter, and other presynaptic proteins in dopaminergic neurons; mice lacking Sept4 exhibit diminished dopaminergic neurotransmission due to scarcity of these presynaptic proteins. These data demonstrate an important role for septin scaffolds in the brain. In transgenic mice that express human alpha-synuclein(A53T) (a mutant protein responsible for familial PD), loss of Sept4 significantly enhances neuropathology and locomotor deterioration. In this PID model, insoluble deposits of Ser(129)-phosphorylated alpha-synuclein(A53T) are negatively correlated with the dosage of Sept4. In vitro, direct association with Sept4 protects alpha-synuclein against self-aggregation and Ser(129) phosphorylation. Taken together, these data show that Sept4 may be involved in PID as a dual susceptibility factor, as its insufficiency can diminish dopaminergic neurotransmission and enhance alpha-synuclein neurotoxicity.

DOI： 10.1016/j.neuron.2007.01.019

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蛍光偏光顕微鏡が解いたセプチン線維配向の謎 Invited Reviewed

木下　専

蛋白質核酸酵素   Vol. 52 ( 1 )   2007.1

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Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen Invited

Kinoshita, M., Watanabe, N.

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme   Vol. 52 ( 13 Suppl ) page： 1796 - 1799   2007

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表現型スクリーニングで同定された紡錘体双極性の阻害剤 Invited

木下　専、渡邊直樹

蛋白質核酸酵素　融合発展する構造生物学とケミカルバイオロジーの最前線   Vol. 53 ( 13 ) page： 1796 - 1799   2007

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細胞分裂と細胞形態形成におけるGTP結合蛋白質セプチンの役割 Invited

木下　専

生化学(日本生化学会誌)   Vol. 78 ( 8 ) page： 755 - 759   2006.8

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DOI： 10.14952/SEIKAGAKU.2021.930001

Diversity of septin scaffolds Invited Reviewed

Makoto Kinoshita

Current Opinion in Cell Biology   Vol. 18 ( 1 ) page： 54 - 60   2006.2

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Septins are ubiquitous GTP-binding proteins generally regarded as cytoskeletal components. Higher-order septin assemblies represented by the yeast septin collar function as cytoskeleton, providing structural support and scaffolds for many cellular factors. In metazoans, however, typical higher-order septin assemblies are often less predominant than dispersed 'low-order' septin populations. Recent studies revealed that septin populations with no obvious structure that had previously escaped our attention serve as scaffolds for kinetochore motor proteins and as sequestering depots for microtubule regulators. Unlike classic cytoskeletal polymers, which form uniform, continuous networks, septin polymers, being diverse, discontinuous and relatively static, seem suited to form discrete scaffolds. Thus, the septin system might be redefined as discrete scaffolds that are conditionally united to behave like cytoskeleton.

DOI： 10.1016/j.ceb.2005.12.005

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再構成系と遺伝子破壊マウスによるセプチン系の解析 Invited

木下　専

細胞工学   Vol. 25 ( 7 ) page： 785 - 789   2006

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Disruption of Sept6, a fusion partner gene of MLL, does not affect ontogeny, leukemogenesis induced by MLL-SEPT6, or phenotype induced by the loss of Sept4 Reviewed

R Ono, M Ihara, H Nakajima, K Ozaki, Y Kataoka-Fujiwara, T Taki, K Nagata, M Inagaki, N Yoshida, T Kitamura, Y Hayashi, M Kinoshita, T Nosaka

Molecular and Cellular Biology   Vol. 25 ( 24 ) page： 10965 - 10978   2005.12

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Septins are evolutionarily conserved GTP-binding proteins that can heteropolymerize into filaments. Recent studies have revealed that septins are involved in not only diverse normal cellular processes but also the pathogenesis of various diseases, including cancer. SEPT6 is ubiquitously expressed in tissues and one of the fusion partner genes of MLL in the 11q23 translocations implicated in acute leukemia. However, the roles of this septin in vivo remain elusive. We have developed Sept6-deficient mice that exhibited neither gross abnormalities, changes in cytokinesis, nor spontaneous malignancy. Sept6 deficiency did not cause any quantitative changes in any of the septins evaluated in this study, nor did it cause any additional changes in the Sept4-deficient mice. Even the depletion of Sept11, a close homolog of Sept6, did not affect the Sept6-null cells in vitro, thus implying a high degree of redundancy in the septin system. Furthermore, a loss of Sept6 did not alter the phenotype of myeloproliferative disease induced by MLL-SEPT6, thus suggesting that Sept6 does not function as a tumor suppressor. To our knowledge, this is the first report demonstrating that a disruption of the translocation partner gene of MLL in 11q23 translocation does not contribute to leukemogenesis by the MLL fusion gene.

DOI： 10.1128/MCB.25.24.10965-10978.2005

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セプチン系細胞骨格と精子無力症 Invited

西山博之、木下　専

医学のあゆみ   Vol. 214 ( 3 ) page： 221 - 222   2005.7

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A mitotic septin scaffold required for mammalian chromosome congression and segregation Reviewed International coauthorship International journal

  Vol. 307   page： 1781 - 1785   2005.3

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DOI： 10.1126/science.1106823

Cortical Organization by the Septin Cytoskeleton Is Essential for Structural and Mechanical Integrity of Mammalian Spermatozoa Reviewed

Masafumi Ihara, Ayae Kinoshita, Shuichi Yamada, Hiromitsu Tanaka, Ai Tanigaki, Ayumi Kitano, Motohito Goto, Kazutoshi Okubo, Hiroyuki Nishiyama, Osamu Ogawa, Chiaki Takahashi, Shigeyoshi Itohara, Yoshitake Nishimune, Makoto Noda, Makoto Kinoshita

Developmental Cell   Vol. 8 ( 3 ) page： 343 - 352   2005.3

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Septins are polymerizing GTP binding proteins required for cortical organization during cytokinesis and other cellular processes. A mammalian septin gene Sept4 is expressed mainly in postmitotic neural cells and postmeiotic male germ cells. In mouse and human spermatozoa, SEPT4 and other septins are found in the annulus, a cortical ring which separates the middle and principal pieces. Sept4(-/-) male mice are sterile due to defective morphology and motility of the sperm flagellum. In Sept4 null spermatozoa, the annulus is replaced by a fragile segment lacking cortical material, beneath which kinesin-mediated intraflagellar transport stalls. The sterility is rescued by injection of sperm into oocytes, demonstrating that each Sept4 null spermatozoon carries an intact haploid genome. The annulus/septin ring is also disorganized in spermatozoa from a subset of human patients with asthenospermia syndrome. Thus, cortical organization based on circular assembly of the septin cytoskeleton is essential for the structural and mechanical integrity of mammalian spermatozoa.

DOI： 10.1016/j.devcel.2004.12.005

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Septins and Cytokinesis Invited Reviewed International coauthorship

Encyclopedia of Biological Chemistry   Vol. 4   page： 22 - 26   2004.11

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DOI： 10.1016/B0-12-443710-9/00618-9

Endomucin, a sialomucin expressed in high endothelial venules, supports L-selectin-mediated rolling Reviewed

H Kanda, T Tanaka, M Matsumoto, E Umemoto, Y Ebisuno, M Kinoshita, M Noda, R Kannagi, T Hirata, T Murai, M Fukuda, M Miyasaka

International Immunology   Vol. 16 ( 9 ) page： 1265 - 1274   2004.9

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Lymphocyte homing to lymph nodes is regulated by transient but specific interactions between lymphocytes and high endothelial venules (HEVs), the initial phase of which is mainly governed by the leukocyte adhesion molecule L-selectin, which recognizes sulfated and sialylated O-linked oligosaccharides displayed on sialomucin core proteins. One of the sialomucin proteins, endomucin, is predominantly expressed in vascular endothelial cells of a variety of tissues including the HEVs of lymph nodes; however, whether it functions as a ligand for L-selectin remains to be formally proven. Here we show that the endomucin splice isoform a is predominantly expressed in Ill HEVs and MAdCAM-1(+) HEVs, as seen in non-HEV-type vascular endothelial cells. Using affinity purification with soluble L-selectin, we found that HEV endomucin is specifically modified with L-selectin-reactive oligosaccharides and can bind L-selectin as well as an HEV-specific mAb, MECA-79. Our results also indicated that a 90-100 kDa endomucin species is preferentially decorated with L-selectin-reactive sugar chains, whereas an 80 kDa species represents conventional forms expressed in non-HEV-type vascular endothelial cells in lymph nodes. Furthermore, a CHO cell line expressing endomucin together with a specific combination of carbohydrate-modifying enzymes [core-2 beta-1,6-N-acetylglucosaminyltransferase (C2GnT), alpha-1,3-fucosyltransferase VII (FucTVII) and L-selectin ligand sulfotransferase (LSST)] showed L-selectin-dependent rolling under flow conditions in vitro. These results suggest that when endomucin is appropriately modified by a specific set of glycosyltransferases and a sulfotransferase, it can function as a ligand for L-selectin, and that the endomucin expressed in HEVs may represent another sialomucin ligand for L-selectin.

DOI： 10.1093/intimm/dxh128

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Chronic cerebral hypoperfusion induces white matter lesions and loss of oligodendroglia with DNA fragmentation in the rat Reviewed

H. Tomimoto, M. Ihara, H. Wakita, R. Ohtani, J. X. Lin, I. Akiguchi, M. Kinoshita, H. Shibasaki

Acta Neuropathologica   Vol. 106 ( 6 ) page： 527 - 534   2003.12

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Cerebrovascular white matter lesions represent an age-related neurodegenerative condition that appears as a hyperintense signal on magnetic resonance images. These lesions are frequently observed in aging, hypertension and cerebrovascular disease, and are responsible for cognitive decline and gait disorders in the elderly population. In humans, cerebrovascular white matter lesions are accompanied by apoptosis of oligodendroglia, and have been thought to be caused by chronic cerebral ischemia. In the present study, we tested whether chronic cerebral hypoperfusion induces white matter lesions and apoptosis of oligodendroglia in the rat. Doppler flow meter analysis revealed an immediate reduction of cerebral blood flow ranging from 30% to 40% of that before operation; this remained at 52-64% between 7 and 30 days after operation. Transferrin-immunoreactive oligodendroglia decreased in number and the myelin became degenerated in the medial corpus callosum at 7 days and thereafter. Using the TUNEL method, the number of cells showing DNA fragmentation increased three- to eightfold between 3 and 30 days post-surgery compared to sham-operated animals. Double labeling with TUNEL and immunohistochemistry for markers of either astroglia or oligodendroglia showed that DNA fragmentation occurred in both of these glia. Messenger RNA for caspase-3 increased approximately twofold versus the sham-operated rats between 1 and 30 days post-surgery. Immunohistochemistry revealed up-regulation of caspase-3 in the oligodendroglia of the white matter, and also in the astroglia and neurons of the gray matter. Molecules involved in apoptotic signaling such as TNF-α and Bax were also up-regulated in glial cells. These results indicate that chronic cerebral hypoperfusion induces white matter degeneration in association with DNA fragmentation in oligodendroglia.

DOI： 10.1007/s00401-003-0749-3

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The Septins (Protein Family Review) Invited Reviewed

Makoto Kinoshita

Genome Biology   Vol. 4 ( 236 ) page： 1 - 9   2003.10

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The septins make up a family of guanine-nucleotide binding proteins, most of which polymerize to form filaments. Septin genes have been found in fungi and animals but not in protozoa or plants
yeasts have seven septin genes and humans have twelve, but Caenorhabditis elegans has only two. Some septin genes generate multiple polypeptides by alternative splicing or alternative translation start sites. Of the five conserved motifs found in other members of the GTPase superfamily, three are highly conserved in septins. Septin filaments are thought to form a cytoskeletal system that organizes higher-order structures by self-assembly and templated assembly. These multifunctional proteins are best known for their role in cytokinesis, but other functions in dividing and non-dividing cells have evolved in different lineages: budding yeast has septins specific for sporulation
nematode septins are implicated in postembryonic morphogenesis of multiple cell lineages
fly septins are associated with the development of germ cells, photoreceptor cells and nervous system
and mammalian septins are implicated in exocytosis, tumorigenesis, apoptosis, synaptogenesis and neurodegeneration.

DOI： 10.1186/gb-2003-4-11-236

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Assembly of mammalian septins Invited

Makoto Kinoshita

Journal of Biochemistry   Vol. 134 ( 4 ) page： 491 - 496   2003.10

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Septins are a conserved family of polymerizing guanine nucleotide binding proteins associated with diverse processes in dividing and non-dividing cells. In humans, 12 septin genes generate dozens of polypeptides, many of which comprise heterooligomeric complexes. Native and recombinant mammalian septin complexes are purified as similar to8-nm-thick filaments of variable length. Ultrastructurally, a mammalian septin filament appears an irregular array of structural segments, whose polarity is obscure. The filaments have a potential to self-assemble into higher-order structures by lateral stacking and tandem annealing, eventually forming uniformly curved bundles, i.e., rings and coils. The septin filaments also undergo templated assembly along existing actin bundles containing an adapter protein, anillin. The resultant higher-order assembly of septin filaments may provide scaffolds to recruit other molecules and/or help organize the actin-based structures. The in vitro self-assembly is an irreversible process, which is not coupled with robust nucleotide exchange or hydrolysis. In contrast, septin-based structures rearrange and disassemble in cells, which might be controlled by diverse factors (e.g., the Cdc42-borg system, anillin, syntaxin, phospholipids) and covalent modifications (e.g., phosphorylation, ubiquitination, sumoylation). An immediate goal of septin biochemistry is to define the mechanisms of assembly and disassembly of this elusive cytoskeleton.

DOI： 10.1093/jb/mvg182

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Association of the cytoskeletal GTP-binding protein Sept4/H5 with cytoplasmic inclusions found in Parkinson's disease and other synucleinopathies Reviewed

M Ihara, H Tomimoto, H Kitayama, Y Morioka, Akiguchi, I, H Shibasaki, M Noda, M Kinoshita

Journal of Biological Chemistry   Vol. 278 ( 26 ) page： 24095 - 24102   2003.6

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alpha-Synuclein-positive cytoplasmic inclusions are a pathological hallmark of several neurodegenerative disorders including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Here we report that Sept4, a member of the septin protein family, is consistently found in these inclusions, whereas five other septins (Sept2, Sept5, Sept6, Sept7, and Sept8) are not found in these inclusions. Sept4 and alpha-synuclein can also be co-immunoprecipitated from normal human brain lysates. When co-expressed in cultured cells, FLAG-tagged Sept4 and Myc-tagged alpha-synuclein formed detergent-insoluble complex, and upon treatment with a proteasome inhibitor, they formed Lewy body-like cytoplasmic inclusions. The tagged Sept4 and alpha-synuclein synergistically accelerated cell death induced by the proteasome inhibitor, and this effect was further enhanced by expression of another Lewy body-associated protein, synphilin-1, tagged with the V5 epitope. Moreover, co-expression of the three proteins (tagged Sept4, alpha-synuclein, and synphilin-1) was sufficient to induce cell death. These data raise the possibility that Sept4 is involved in the formation of cytoplasmic inclusions as well as induction of cell death in alpha-synuclein-associated neurodegenerative disorders.

DOI： 10.1074/jbc.M301352200

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細胞生物学から「ケミカルジェネティクス」へ：ハーバード大学Mitchison研究室における研究の展開 Invited Reviewed

木下　専

現代化学 2003年2月号     page： 21 - 24   2003.1

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Self- and Actin-Templated Assembly of Mammalian Septins Reviewed International coauthorship

Makoto Kinoshita, Christine M. Field, Margaret L. Coughlin, Aaron F. Straight, Timothy J. Mitchison

Developmental Cell   Vol. 3 ( 6 ) page： 791 - 802   2002.12

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Septins are polymerizing GTPases required for cytokinesis and cortical organization. The principles by which they are targeted to, and assemble at, specific cell regions are unknown. We show that septins in mammalian cells switch between a linear organization along actin bundles and cytoplasmic rings, approximately 0.6 mum in diameter. A recombinant septin complex self-assembles into rings resembling those in cells. Linear organization along actin bundles was reconstituted by adding an adaptor protein, anillin. Perturbation of septin organization in cells by expression of a septin-interacting fragment of anillin or by septin depletion via siRNA causes loss of actin bundles. We conclude that septins alone self-assemble into rings, that adaptor proteins recruit septins to actin bundles, and that septins help organize these bundles.

DOI： 10.1016/s1534-5807(02)00366-0

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Mammalian septins nomenclature Reviewed International coauthorship

IG Macara, R Baldarelli, CM Field, M Glotzer, Y Hayashi, SC Hsu, MB Kennedy, M Kinoshita, M Longtine, C Low, LJ Maltais, L McKenzie, TJ Mitchison, T Nishikawa, M Noda, EM Petty, M Peifer, Pringle, JR, PJ Robinson, D Roth, SEH Russell, H Stuhlmann, M Tanaka, T Tanaka, WS Trimble, J Ware, NJ Zeleznik-Le, B Zieger

Molecular Biology of the Cell   Vol. 13 ( 12 ) page： 4111 - 4113   2002.12

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There are 10 known mammalian septin genes, some of which produce multiple splice variants. The current nomenclature for the genes and gene products is very confusing, with several different names having been given to the same gene product and distinct names given to splice variants of the same gene. Moreover, some names are based on those of yeast or Drosophila septins that are not the closest homologues. Therefore, we suggest that the mammalian septin field adopt a common nomenclature system, based on that adopted by the Mouse Genomic Nomenclature Committee and accepted by the Human Genome Organization Gene Nomenclature Committee. The human and mouse septin genes will be named SEPT1-SEPT10 and Sept1-Sept10, respectively. Splice variants will be designated by an underscore followed by a lowercase "v" and a number, e.g., SEYT4_v1.

DOI： 10.1091/mbc.E02-07-0438

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Roles of septins in the mammalian cytokinesis machinery Invited Reviewed

Kinoshita M, Noda M

Cell Structure and Function   Vol. 26   page： 647 - 650   2002.4

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DOI： doi.org/10.1247/csf.26.667

Borg proteins control septin organization and are negatively regulated by Cdc42 Reviewed International coauthorship

G Joberty, RR Perlungher, PJ Sheffield, M Kinoshita, M Noda, T Haystead, IG Macara

Nature Cell Biology   Vol. 3 ( 10 ) page： 861 - 866   2001.10

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MACMILLAN PUBLISHERS LTD  

The Cdc42 GTPase binds to numerous effector proteins that control cell polarity, cytoskeletal remodelling and vesicle transport. In many cases the signalling pathways downstream of these effectors are not known. Here we show that the Cdc42 effectors Borg1 to Borg3 bind to septin GTPases. Endogenous septin Cdc10 and Borg3 proteins can be immunoprecipitated together by an anti-Borg3 antibody. The ectopic expression of Borgs disrupts normal septin organization. Cdc42 negatively regulates this effect and inhibits the binding of Borg3 to septins. Borgs are therefore the first known regulators of mammalian septin organization and provide an unexpected link between the septin and Cdc42 GTPases.

DOI： 10.1038/ncb1001-861

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Identification of human endomucin-1 and -2 as membrane-bound O-sialoglycoproteins with anti-adhesive activity11The nucleotide sequences reported in this paper are available via GenBank accession numbers AB034694 (mouse endomucin-1), AB034695 (human endomucin-1), and AB034696 (human endomucin-2). Reviewed

Makoto Kinoshita, Tomoyuki Nakamura, Masafumi Ihara, Tokuko Haraguchi, Yasushi Hiraoka, Kei Tashiro, Makoto Noda

FEBS Letters   Vol. 499 ( 1-2 ) page： 121 - 126   2001.6

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DOI： 10.1016/s0014-5793(01)02520-0

PubMed

Chronic cerebral hypoperfusion induces MMP-2 but not MMP-9 expression in the microglia and vascular endothelium of white matter Reviewed

Ihara M, Tomimoto H, Kinoshita M, Oh J, Noda M, Wakita H, Akiguchi I, Shibasaki H

Journal of Cerebral Blood Flow and Metabolism   Vol. 21   page： 828-834   2001

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DOI： 10.1097/00004647-200107000-00008

Roles of Septins in the Mammalian Cytokinesis Machinery Reviewed

Makoto Kinoshita, Makoto Noda

Cell Structure and Function   Vol. 26 ( 6 ) page： 667 - 670   2001

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DOI： 10.1247/csf.26.667

PubMed

J-GLOBAL

Phosphorylation of a New Brain-specific Septin, G-septin, by cGMP-dependent Protein Kinase Reviewed International coauthorship

Jing Xue, Xin Wang, Chandra S. Malladi, Makoto Kinoshita, Peter J. Milburn, Imre Lengyel, John A. P. Rostas, Phillip J. Robinson

Journal of Biological Chemistry   Vol. 275 ( 14 ) page： 10047 - 10056   2000.3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Biochemistry {\&} Molecular Biology ({ASBMB})  

The septins are a family of GTPase enzymes, some of which are required for the cytokinesis stage of cell di vision and others of which are associated with exocytosis. me purified and cloned the cDNA for a 40-kDa protein from rat brain that is a substrate for type I cGMP-dependent protein kinase (PKG). The amino acid sequences of two tryptic peptides of P40 showed high homology to the septins. Molecular cloning revealed the 358-amino acid P40 to be a new member of the septin family. P40 was named G-septin, as it is phosphorylated in, vitro by PKG, but relatively poorly by the related cAMP dependent protein kinase and not by protein kinase C, Two splice variants of G-septin (alpha and beta) were found with distinct N and C termini, but a common GTPase domain. G-septin lacks the C-terminal coiled-coil domain characteristic of all other mammalian septins and uniquely has two predicted phosphorylation site motifs for type I PKG. Photoaffinity labeling with [alpha-P-32]GTP confirmed that G-septin is a GTP-binding protein. Northern blotting showed that G-septin mRNA (5.0 kilobases) is highly expressed in brain and undetectable in 12 other tissues, indicating that the G-septins are primarily neuronal proteins. Very low levels of 6.0-, 3.4-, and 2.6-kilobase transcripts were found in testis. Our results reveal a new class of brain-specific septins that may be regulated by PKG in neurons.

DOI： 10.1074/jbc.275.14.10047

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PubMed

Differential localization of septins in the mouse brain Reviewed

Ayae Kinoshita, Makoto Noda, Makoto Kinoshita

Journal of Comparative Neurology   Vol. 428 ( 2 ) page： 223 - 239   2000

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley-Blackwell  

We have carried out a comparative immunohistochemical study on four members of the septin family, CDCrel-1, Septin6, CDC10, and H5, which are abundantly expressed in the adult mouse brain. We found that each septin showed overlapping but distinct distribution at the levels of light and electron microscopy. CDCrel-1 was abundant in inhibitory presynaptic terminals and associated with GABAergic vesicles in the thalamus, globus pallidus, and cerebellar nuclei. Septin6 was associated with synaptic vesicles in various brain regions, including glomeruli of the olfactory bulb. CDC10 was diffusely expressed in the brain and was localized beneath presynaptic membrane and astroglial processes. H5 was localized in the astroglial processes in some specific brain regions. The differential expression and subcellular localization of these septins indicates that a given neuron or glial cell expresses a specific set of septin monomers and that the resulting septin complexes with distinct compositions may play distinct roles in the brain. (C) 2000 Wiley-Liss, Inc.

DOI： 10.1002/1096-9861(20001211)428:2<223::aid-cne3>3.0.co;2-m

Web of Science

PubMed

Identification of Septins in Neurofibrillary Tangles in Alzheimer's Disease Reviewed

Ayae Kinoshita, Makoto Kinoshita, Haruhiko Akiyama, Hidekazu Tomimoto, Ichiro Akiguchi, Sharad Kumar, Makoto Noda, Jun Kimura

The American Journal of Pathology   Vol. 153 ( 5 ) page： 1551 - 1560   1998.11

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Septins are evolutionarily conserved cytoskeletal GTPases that can form heteropolymer complexes involved hi cytokinesis and other cellular processes. We detected expression of the human septin genes Nedd5, H5, Diff6, and hCDC10 in postmortem brain tissues using the reverse transcription-coupled polymerase chain reaction and their products by immunoblot analysis, Four antibodies directed against three septins, Nedd5, H5, and Diff6, consistently labeled neurofibrillary tangles, neuropil threads, and dystrophic neurites in the senile plaques in brains affected by Alzheimer's disease but did not label obvious structures in young control brains. Immunoelectron microscopy revealed that Nedd5 localized to the paired helical filaments, Pre-tangles, the precursory granular deposits that accumulate in the neuronal cytoplasm, also were labeled with the antibodies. These findings suggest that at least the three septins are associated with tau-based paired helical filament core, and may contribute to the formation of neurofibrillary tangle as integral constitutents of paired helical filaments.

DOI： 10.1016/s0002-9440(10)65743-4

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PubMed

Caspases in Cell Death Reviewed International coauthorship

Loretta Dorstyn, Makoto Kinoshita, Sharad Kumar

Results and Problems in Cell Differentiation   Vol. 24   page： 1 - 24   1998

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science $\mathplus$ Business Media  

DOI： 10.1007/978-3-540-69185-3_1

cDNA Cloning, Expression Analysis, and Mapping of the MouseNedd4Gene Reviewed International coauthorship

Sharad Kumar, Kieran F. Harvey, Makoto Kinoshita, Neal G. Copeland, Makoto Noda, Nancy A. Jenkins

Genomics   Vol. 44 ( 1 ) page： 156 - 156   1997.8

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DOI： 10.1006/geno.1997.4882

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A novel gene encoding a ferredoxin reductase-like protein expressed in the neuroectoderm in Xenopus neurula Reviewed

Seigo Hatada, Makoto Kinoshita, Hirofumi Sakumoto, Reina Nishihara, Makoto Noda, Makoto Asashima

Gene   Vol. 194 ( 2 ) page： 297 - 299   1997.7

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In an attempt to elucidate the molecular mechanisms of early neural development in Xenopus laevis, we identified, using a differential display method, several genes that are induced after Concanavalin A treatment in the animal caps prepared from stage 9 blastula. One such gene was found to encode a possible type IIIa membrane protein of 66.2 kDa sharing similarities with several prokaryotic and eukaryotic redox enzymes, hence the putative product was named Nfrl, neurula-specific ferredoxin reductase-like protein. Northern blot analysis confirmed that the expression of the Nfrl gene is up-regulated around the neurula stage, and is much lower in embryos of earlier stages and in adult tissues. The temporally limited expression of this gene implies neurula- and early larva-specific redox reactions of certain substrates, the nature of which remains to be elucidated. (C) 1997 Elsevier Science B.V.

DOI： 10.1016/s0378-1119(97)00208-4

Web of Science

Nedd5, a mammalian septin, is a novel cytoskeletal component interacting with actin-based structures Reviewed International coauthorship

M Kinoshita, S Kumar, A Mizoguchi, C Ide, A Kinoshita, T Haraguchi, Y Hiraoka, M Noda

Genes & Development   Vol. 11 ( 12 ) page： 1535 - 1547   1997.6

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The mouse Nedd5 gene encodes a 41.5-kD GTPase similar to the Saccharomyces and Drosophila septins essential for cytokinesis. Nedd5 accumulates near the contractile ring from anaphase through telophase, and finally condenses into the midbody. Microinjection of anti-Nedd5 antibody interferes with cytokinesis, giving rise to binucleated cells. In interphase and postmitotic cells, Nedd5 localizes to fibrous or granular structures depending on the growth state of the cell. The Nedd5-containing fibers are disrupted by microinjection of GTP gamma S and by Nedd5 mutants lacking GTP-binding activity, implying that GTP hydrolysis is required for its assembly. The Nedd5-containing fibers also appear to physically contact actin bundles and focal adhesion complexes and are disrupted by cytochalasin D, C3 exoenzyme, and serum starvation, suggesting a functional interaction with the actin-based cytoskeletal systems in interphase cells.

DOI： 10.1101/gad.11.12.1535

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PubMed

Up-Regulation of the Nedd2 Gene Encoding an ICE/Ced-3-Like Cysteine Protease in the Gerbil Brain After Transient Global Ischemia Reviewed International coauthorship

Makoto Kinoshita, Hidekazu Tomimoto, Ayae Kinoshita, Sharad Kumar, Makoto Noda

Journal of Cerebral Blood Flow & Metabolism   Vol. 17 ( 5 ) page： 507 - 514   1997.5

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We assessed the expression of several genes encoding pro-apoptotic cysteine proteases similar to interleukin-1 beta converting enzyme (ICE) and nematode Ced-3 in association with delayed neuronal death (DND) after transient forebrain ischemia in Mongolian gerbil. The levels of the two species of Nedd2 mRNA concomitantly increased about twofold in the whole forebrain at 3-6 h after 10-min ischemia and declined to the basal level by 24 h. In situ hybridization revealed that the Nedd2 gene was up-regulated in some neuronal populations in CA(1) and CA(3) regions of the hippocampus. In contrast, expression of ICE, CPP32/Yama/Apopain, and TX/ICErelII did not change within 48 h. These observations raise the possibility that up-regulation of Nedd2 in the vulnerable neurons may contribute to the proteolytic processes preceding the manifestation of apoptosis and/or necrosis after ischemic insult.

DOI： 10.1097/00004647-199705000-00004

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PubMed

Characterization of a mammalian cell death gene Nedd2 Reviewed International coauthorship

Kumar S, Kinoshita M, Noda M

Leukemia   Vol. 11 Suppl 3   page： 385 - 386   1997.4

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PubMed

cDNA Cloning, Expression Analysis, and Mapping of the Mouse Nedd4 Gene Reviewed International coauthorship

Sharad Kumar, Kieran F. Harvey, Makoto Kinoshita, Neal G. Copeland, Makoto Noda, Nancy A. Jenkins

Genomics   Vol. 40 ( 3 ) page： 435 - 443   1997.3

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The Nedd4 gene was initially identified by a subtraction cloning approach as a highly expressed transcript in the mouse embryonic brain. Cloning of the Nedd4 cDNA indicated that it can encode a protein of approximately 103 kDa, consisting of a Ca2+ and phospholipid binding domain, three putative protein-protein interaction domains (the WW domains), and a carboxyl-terminus region similar to the ubiquitin-protein ligase domain (hect domain). In mouse embryos, the expression of Nedd4 in the central nervous system is highest during neurogenesis and decreases as development progresses. In addition to the central nervous system, the expression of Nedd4 is detected in various embryonic tissues and persists in most adult tissues. Using an antibody raised against a fusion protein, we show that Nedd4 protein is localized to the cellular cytoplasm. We have mapped the mouse Nedd4 gene to chromosome 9 using an interspecific backcross panel. Nedd4 maps to a previously defined homologous region between human and mouse chromosomes and thus provides additional information regarding interspecies comparative mapping. (C) 1997 Academic Press.

DOI： 10.1006/geno.1996.4582

Web of Science

Origin, expression and possible functions of the two alternatively spliced forms of the mouse Nedd2 mRNA. Reviewed International coauthorship

Kumar S, Kinoshita M, Dorstyn L, Noda M

Cell Death and Differentiation   Vol. 4   page： 378-387   1997

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An interferon regulatory factor-related gene (xIRF-6) is expressed in the posterior mesoderm during the early development of Xenopus laevis Reviewed

Seigo Hatada, Makoto Kinoshita, Shuji Takahashi, Reina Nishihara, Hirofumi Sakumoto, Akimasa Fukui, Makoto Noda, Makoto Asashima

Gene   Vol. 203 ( 2 ) page： 183 - 188   1997

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

Out of a Xenopus neurula cDNA library, we isolated a clone which encodes a 52.4-kDa protein highly similar to the mouse interferon regulatory factor, IRF-6, whose function is unknown. The mRNA of this gene, named xIRF-6, seems to be maternally transmitted, but its amount rapidly decreases after the tailbud stage. Whole-mount in situ hybridization showed that xIRF-6 mRNA is expressed in the presumptive semitic mesoderm in the late gastrula, and then confined to a segment of posterior somite during the neurula through the tailbud stage. The temporally and spatially limited expression of the xIRF-6 gene product may contribute to the transcriptional regulation of specific genes which are necessary for the development of the posterior somites. (C) 1997 Elsevier Science B.V.

DOI： 10.1016/s0378-1119(97)00512-x

Web of Science

Origin, expression and possible functions of the two alternatively spliced forms of the mouse Nedd2 mRNA Reviewed

Sharad Kumar, Makoto Kinoshita, Loretta Dorstyn, Makoto Noda

Cell Death and Differentiation   Vol. 4 ( 5 ) page： 378 - 387   1997

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing Group  

The Nedd2 gene, a member of the ICE/ced-3 family, transcribes two mRNA species by differential splicing. One of these encodes a shorter truncated version of the protein termed Nedd2s/ICH-1s. While Nedd2/ICH-1 induces apoptosis, ICH-1s was proposed to be a negative regulator of cell death. In this communication we have analyzed various aspects of the Nedd2s/ICH-1s. The coding region of mouse Nedd2 consists of 10 exons and alternative splicing of the 7th exon results in the generation of two transcripts. Although Nedd2s expression conferred partial resistance to serum withdrawal-induced apoptosis in NIH-3T3 cells, it failed to prevent cell death induced by the overexpression of Nedd2 and was unable to suppress apoptosis in two other cell lines (N18 and FDC-P1) under factor deprived conditions. Our results indicate that Nedd2s/ICH-1s is not a general modulator of cell death.

DOI： 10.1038/sj.cdd.4400251

Web of Science

Caspase-2/Nedd2/Ich-1 Invited Reviewed

木下　専、野田　亮

アポトーシス (Bio Science用語ライブラリー)     page： 105 - 106   1996.12

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Identification of a Xenopus glutamine synthetase gene abundantly expressed in the embryonic nervous system but not in adult brain Reviewed

Seigo Hatada, Makoto Kinoshita, Makoto Noda, Makoto Asashima

FEBS Letters   Vol. 371 ( 3 ) page： 287 - 292   1995.9

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We used a PCR-based subtraction cloning procedure with concanavalin A-treated and -untreated animal caps from stage 9 Xenopus embryos to search for genes up-regulated during early neural development. One such gene was found to encode a protein homologous to several known glutamine synthetases, and we named it xGS, Molecular hybridization studies revealed that xGS mRNA is maternally transmitted and abundantly expressed in neuroectoderm-derived tissues during the gastrula and neurula stages, The expression of xGS mRNA in the nervous system continues until the larval stages, but declines thereafter and becomes undetectable in adult brain, Considering its metabolic activity and potential neuroprotective effect against the neurotoxic substances such as glutamate and ammonia, the glutamine synthetase may play an important role in the early stages of vertebrate neural development.

DOI： 10.1016/0014-5793(95)00913-t

Web of Science

HMG-X, a Xenopus gene encoding an HMG1 homolog, is abundantly expressed in the developing nervous system Reviewed

FEBS Letters   Vol. 352 ( 2 ) page： 191 - 196   1994.9

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DOI： 10.1016/0014-5793(94)00909-0

Web of Science

Induction of apoptosis by the mouse Nedd2 gene, which encodes a protein similar to the product of the Caenorhabditis elegans cell death gene ced-3 and the mammalian IL-1 beta-converting enzyme Reviewed International coauthorship

S Kumar, M Kinoshita, M Noda, N G Copeland, N A Jenkins

Genes & Development   Vol. 8 ( 14 ) page： 1613 - 1626   1994.7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COLD SPRING HARBOR LAB PRESS  

By subtraction cloning we previously identified a set of mouse genes (named Nedd1 through Nedd10) with developmentally down-regulated expression in brain. We now show that one such gene, Nedd2, encodes a protein similar to the mammalian interleukin-1 beta-converting enzyme (ICE) and the product of the Caenorhabditis elegans cell death gene ced-3 (CED-3). Both ICE and CED-3 are known to encode putative cysteine proteases and induce apoptosis when overexpressed in cultured cells. Overexpression of Nedd2 in cultured fibroblast and neuroblastoma cells also resulted in cell death by apoptosis, which was suppressed by the expression of the human bcl-2 gene, indicating that Nedd2 is functionally similar to the ced-3 gene in C. elegans. We also show that during embryonic development, Nedd2 is highly expressed in several types of mouse tissue undergoing high rates of programmed cell death such as central nervous system and kidney. Our data suggest that Nedd2 is an important component of the mammalian programmed cell death machinery.

DOI： 10.1101/gad.8.14.1613

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PubMed

Vector Analysis of Corneal Astigmatism after Scleral Buckling Surgery Reviewed

Makoto Kinoshita, Hidenobu Tanihara, Akira Negi, Shin-Ichiro Kawano, Hitoshi Ishigouoka, Yoshiki Veda, Satomi Suzuki-Yoshida, Yoshihito Honda

Ophthalmologica   Vol. 208 ( 5 ) page： 250 - 253   1994

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：S. Karger {AG}  

We conducted a short-term prospective study of 125 eyes with retinal detachment to examine changes of corneal astigmatism about 2 weeks after scleral buckling surgery, using a vector method on 2-fold-angle rectangular coordinates. Segmental buckles of one to less than two quadrants produced significantly greater changes in astigmatism (1.65+/-0.97 dptr) than those of less than one quadrant (0.88+/-0.75 dptr) and those spanning two quadrants or more (1.09+/-0.38 dptr) (p=0.0005, Kruskal-wallis test; n=73). The amplitude of differential vectors after explant buckling surgery (1.33+/-0.89 dptr, n=24) was significantly greater than after implant buckling surgery (0.65+/-0.45 dptr, n=15) (p=0.009, Mann-Whitney U-test) with buckles of one quadrant or less. Differential vectors tended to direct toward the buckles. There was no obvious directional tendency in the case of encircling procedures (n=52).

DOI： 10.1159/000310501

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癌遺伝子と神経系の発生・分化・機能 Invited Reviewed

木下　専、野田　亮

実験医学 増刊「脳神経系の発生・分化と可塑性」   Vol. 11 ( 10 ) page： 104 - 112   1993.6

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Expression of DRG during murine embryonic development Reviewed International coauthorship

Takashi Sazuka, Makoto Kinoshita, Yasuhiro Tomooka, Yoji Ikawa, Makoto Noda, Sharad Kumar

Biochemical and Biophysical Research Communications   Vol. 189 ( 1 ) page： 371 - 377   1992.11

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DOI： 10.1016/0006-291x(92)91568-b

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Central serous chorioretinopathy

    1991

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