Faculty Profiles - KINOSHITA, Makoto

Papers - KINOSHITA, Makoto

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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

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

Web of Science

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

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

Web of Science

PubMed

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

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

Web of Science

PubMed

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

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Science $\mathplus$ Business Media

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

Web of Science

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

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

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

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：BIOMED CENTRAL LTD

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

Web of Science

Scopus

PubMed

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

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

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

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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PubMed

[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

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

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

上谷　大介、木下　専

日本細胞生物学会　細胞生物学用語集 2012

セプチン Invited Reviewed

上田（石原）奈津実、木下　専

脳科学辞典 2012

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

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

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

Web of Science

PubMed

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

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

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

Web of Science

PubMed

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

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

Septin-dependent microtubule control during mammalian neurite outgrowth Invited Reviewed

Molecular Biology of the Cell Vol. 22 2011

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

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

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

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

Web of Science

PubMed

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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Papers - KINOSHITA, Makoto