Updated on 2022/07/02

写真a

 
Miyazaki Haruko
 
Organization
Faculty of Medicine, Dentistry and Pharmaceutical Sciences Assistant Professor
Position
Assistant Professor
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Degree

  • 博士(医学) ( 筑波大学 )

Research Interests

  • 無髄神経

  • 有髄神経

  • ミエリン

  • ハンチントン病

  • 大脳基底核

  • 神経変性疾患

  • ナトリウムチャネル

  • 神経科学

Research Areas

  • Life Science / Pathophysiologic neuroscience  / ナトリウムチャネル

  • Life Science / Pathophysiologic neuroscience  / 髄鞘

  • Life Science / Neurology  / 大脳基底核

  • Life Science / Pathophysiologic neuroscience  / ハンチントン病

  • Life Science / Neurology  / ハンチントン病

Education

  • University of Tsukuba     博士課程人間総合科学研究科病態制御医学専攻

    2005.4 - 2008.3

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  • University of Tsukuba     修士課程医科学研究科医科学専攻

    2003.4 - 2005.3

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  • Azabu University   獣医学部   動物応用科学科

    1995.4 - 1999.3

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Research History

  • Okayama University   Graduate School of Medicine , Dentistry and Pharmaceutical Sciences   Assistant Professor

    2021.5

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    Country:Japan

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  • Doshisha University   脳科学研究科 認知記憶加齢部門   Assistant Professor

    2014.4 - 2021.4

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  • 理化学研究所   研究員

    2008.4 - 2013.3

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  • 理化学研究所   ジュニアリサーチアソシエイト

    2005.4 - 2008.3

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  • 理化学研究所   テクニカルスタッフ

    2001.1 - 2005.3

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  • 理化学研究所   テクニカルスタッフ

    1999.4 - 2000.12

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Papers

  • FACS-array-based cell purification yields a specific transcriptome of striatal medium spiny neurons in a murine Huntington disease model. Reviewed International journal

    Haruko Miyazaki, Tomoyuki Yamanaka, Fumitaka Oyama, Yoshihiro Kino, Masaru Kurosawa, Mizuki Yamada-Kurosawa, Risa Yamano, Tomomi Shimogori, Nobutaka Hattori, Nobuyuki Nukina

    The Journal of biological chemistry   295 ( 29 )   9768 - 9785   2020.7

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    Huntington disease (HD) is a neurodegenerative disorder caused by expanded CAG repeats in the Huntingtin gene. Results from previous studies have suggested that transcriptional dysregulation is one of the key mechanisms underlying striatal medium spiny neuron (MSN) degeneration in HD. However, some of the critical genes involved in HD etiology or pathology could be masked in a common expression profiling assay because of contamination with non-MSN cells. To gain insight into the MSN-specific gene expression changes in presymptomatic R6/2 mice, a common HD mouse model, here we used a transgenic fluorescent protein marker of MSNs for purification via FACS before profiling gene expression with gene microarrays and compared the results of this "FACS-array" with those obtained with homogenized striatal samples (STR-array). We identified hundreds of differentially expressed genes (DEGs) and enhanced detection of MSN-specific DEGs by comparing the results of the FACS-array with those of the STR-array. The gene sets obtained included genes ubiquitously expressed in both MSNs and non-MSN cells of the brain and associated with transcriptional regulation and DNA damage responses. We proposed that the comparative gene expression approach using the FACS-array may be useful for uncovering the gene cascades affected in MSNs during HD pathogenesis.

    DOI: 10.1074/jbc.RA120.012983

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  • Singular localization of sodium channel beta 4 subunit in unmyelinated fibres and its role in the striatum Reviewed

    Haruko Miyazaki, Fumitaka Oyama, Ritsuko Inoue, Toshihiko Aosaki, Takaya Abe, Hiroshi Kiyonari, Yoshihiro Kino, Masaru Kurosawa, Jun Shimizu, Ikuo Ogiwara, Kazuhiro Yamakawa, Yoshinori Koshimizu, Fumino Fujiyama, Takeshi Kaneko, Hideaki Shimizu, Katsuhiro Nagatomo, Katsuya Yamada, Tomomi Shimogori, Nobutaka Hattori, Masami Miura, Nobuyuki Nukina

    NATURE COMMUNICATIONS   5   5525   2014.11

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:NATURE PUBLISHING GROUP  

    Voltage-gated Na+ channel beta-subunits are multifunctional molecules that modulate Na+ channel activity and regulate cell adhesion, migration and neurite outgrowth. beta-subunits including beta 4 are known to be highly concentrated in the nodes of Ranvier and axon initial segments in myelinated axons. Here we show diffuse beta 4 localization in striatal projection fibres using transgenic mice that express fluorescent protein in those fibres. These axons are unmyelinated, forming large, inhibitory fibre bundles. Furthermore, we report beta 4 dimer expression in the mouse brain, with high levels of beta 4 dimers in the striatal projection fascicles, suggesting a specific role of beta 4 in those fibres. Scn4b-deficient mice show a resurgent Na+ current reduction, decreased repetitive firing frequency in medium spiny neurons and increased failure rates of inhibitory postsynaptic currents evoked with repetitive stimulation, indicating an in vivo channel regulatory role of beta 4 in the striatum.

    DOI: 10.1038/ncomms6525

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  • Establishment of Feeder-Independent Cloned Caprine Trophoblast Cell Line Which Expresses Placental Lactogen and Interferon Tau Reviewed

    H. Miyazaki, M. Imai, T. Hirayama, S. Saburi, M. Tanaka, M. Maruyama, C. Matsuo, H. Meguro, K. Nishibashi, F. Inoue, J. Djiane, A. Gertler, S. Tachi, K. Imakawa, C. Tachi

    Placenta   23 ( 8-9 )   613 - 630   2002.9

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

    DOI: 10.1053/plac.2002.0846

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  • Amyloids facilitate DNA transfection in vivo. International journal

    Yukio Imamura, Akiko Hiyama, Haruko Miyazaki, Tomoyuki Yamanaka, Nobuyuki Nukina

    Neuroscience research   2022.3

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    Amyloid fibril deposits are a main source of pathology in neurodegenerative diseases. Normal proteins such as tau, alpha-synuclein, TDP-43 and others could form specific conformational fibrils called amyloid, which deposited in the brains of neurodegenerative diseases. Although the pathological roles of amyloids in cell death have been discussed a lot, their other functions have not been investigated well. Here, we studied the effect of amyloids on DNA transfection in vivo. We injected quantum dot labeled or non-labeled amyloid-preformed fibrils (PFFs) and a green fluorescent protein (EGFP) expression vector into organs including brain, testis, liver and calf muscle. GFP expression patterns were examined by immunohistochemistry and western blotting. At 24 h after injection, EGFP was predominantly expressed in the neurons in the cortex and the striatum, Leydig cells in testis, hepatocytes in the liver and muscle cells. EGFP expression was inhibited by an endocytosis inhibitor, sertraline in the brain and testis. The amyloid-PFFs potentiated Ca2+ transients shown by calcium imaging and EGFP expression in the brain was blocked by Ca blocker, cilnidipine. Our results show that amyloid-PFFs facilitate DNA transfection and can be used for a new gene delivery system in vivo.

    DOI: 10.1016/j.neures.2022.03.003

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  • The diffuse distribution of Nav1.2 on mid-axonal regions is a marker for unmyelinated fibers in the central nervous system. International journal

    Risa Yamano, Haruko Miyazaki, Nobuyuki Nukina

    Neuroscience research   2021.11

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    Unmyelinated fibers in the central nervous system are known to exist in hippocampal mossy fibers, cerebellar parallel fibers and striatal projection fibers. Previously, we and others reported diffuse distribution of Nav1.2, a voltage-gated sodium channel α-subunit encoded by the SCN2A gene, on unmyelinated striatal projection fibers. Mutations in the SCN2A gene are associated with epilepsies and autism. In this study, we investigated the distribution of Nav1.2 on the unmyelinated fibers in the corpus callosum and stria terminalis by immunohistochemistry and immunoelectron microscopy analysis, suggesting that diffuse localization of Nav1.2 on mid-axonal regions can be a useful marker for unmyelinated fibers.

    DOI: 10.1016/j.neures.2021.11.005

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  • Assessment of Possible Contributions of Hyaluronan and Proteoglycan Binding Link Protein 4 to Differential Perineuronal Net Formation at the Calyx of Held. International journal

    Kojiro Nojima, Haruko Miyazaki, Tetsuya Hori, Lydia Vargova, Toshitaka Oohashi

    Frontiers in cell and developmental biology   9   730550 - 730550   2021

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    The calyx of Held is a giant nerve terminal mediating high-frequency excitatory input to principal cells of the medial nucleus of the trapezoid body (MNTB). MNTB principal neurons are enwrapped by densely organized extracellular matrix structures, known as perineuronal nets (PNNs). Emerging evidence indicates the importance of PNNs in synaptic transmission at the calyx of Held. Previously, a unique differential expression of aggrecan and brevican has been reported at this calyceal synapse. However, the role of hyaluronan and proteoglycan binding link proteins (HAPLNs) in PNN formation and synaptic transmission at this synapse remains elusive. This study aimed to assess immunohistochemical evidence for the effect of HAPLN4 on differential PNN formation at the calyx of Held. Genetic deletion of Hapln4 exhibited a clear ectopic shift of brevican localization from the perisynaptic space between the calyx of Held terminals and principal neurons to the neuropil surrounding the whole calyx of Held terminals. In contrast, aggrecan expression showed a consistent localization at the surrounding neuropil, together with HAPLN1 and tenascin-R, in both gene knockout (KO) and wild-type (WT) mice. An in situ proximity ligation assay demonstrated the molecular association of brevican with HAPLN4 in WT and HAPLN1 in gene KO mice. Further elucidation of the roles of HAPLN4 may highlight the developmental and physiological importance of PNN formation in the calyx of Held.

    DOI: 10.3389/fcell.2021.730550

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  • Proteomic analysis of subcellular compartments containing disseminated alpha-synuclein seeds. International journal

    Junya Kasahara, Yukio Imamura, Akiko Hiyama, Tomoyuki Yamanaka, Haruko Miyazaki, Nobuyuki Nukina

    Neuroscience research   2020.12

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    The pathological form of a-synuclein (a-syn) is transmitted through neural circuits in the brains of Parkinson disease (PD) patients and amplifies misfolded a-syn, further forming intracellular deposits. However, the details of a-syn pre-formed fibrils (PFFs) transmission in vivo have not been fully elucidated. By inoculating Quantum dots (QD)-labeled a-syn PFFs (QD-a-syn PFFs) into the unilateral striatum, we detected QD-a-syn PFFs in brain homogenates obtained from the ipsilateral and contralateral sides of the inoculated site and further obtained QD-a-syn PFFs enriched-particles with fluorescence-activated organelle sorting. Proteomic analysis suggested that QD-a-syn PFFs-enriched particles in the contralateral side were associated with component proteins of synapse. In contrast, QD-a-syn PFFs-enriched particles in the ipsilateral side were associated with proteins belonging to ER components. Immunostaining of brain sections confirmed that QD-a-syn PFFs in the contralateral side were co-localized with synaptic vesicle marker proteins in the cortex and striatum. Additionally, QD-a-syn PFFs in the ipsilateral side were more co-localized with ER marker proteins compared to the contralateral side. These results correspond to proteomic analysis. This study provides potential candidates for the subcellular localization of a-syn PFFs in vivo during the dissemination phase of seeds. These subcellular compartments could be involved in the transmission of seeds.

    DOI: 10.1016/j.neures.2020.11.009

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  • Gene expression profiling in neuronal cells identifies a different type of transcriptome modulated by NF-Y. International journal

    Tomoyuki Yamanaka, Haruko Miyazaki, Asako Tosaki, Sankar N Maity, Tomomi Shimogori, Nobutaka Hattori, Nobuyuki Nukina

    Scientific reports   10 ( 1 )   21714 - 21714   2020.12

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    A heterotrimeric transcription factor NF-Y is crucial for cell-cycle progression in various types of cells. In contrast, studies using NF-YA knockout mice have unveiled its essential role in endoplasmic reticulum (ER) homeostasis in neuronal cells. However, whether NF-Y modulates a different transcriptome to mediate distinct cellular functions remains obscure. Here, we knocked down NF-Y in two types of neuronal cells, neuro2a neuroblastoma cells and mouse brain striatal cells, and performed gene expression profiling. We found that down-regulated genes preferentially contained NF-Y-binding motifs in their proximal promoters, and notably enriched genes related to ER functions rather than those for cell cycle. This contrasts with the profiling data of HeLa and embryonic stem cells in which distinct down-regulation of cell cycle-related genes was observed. Clustering analysis further identified several functional clusters where populations of the down-regulated genes were highly distinct. Further analyses using chromatin immunoprecipitation and RNA-seq data revealed that the transcriptomic difference was not correlated with DNA binding of NF-Y but with splicing of NF-YA. These data suggest that neuronal cells have a different type of transcriptome in which ER-related genes are dominantly modulated by NF-Y, and imply that NF-YA splicing alteration could be involved in this cell type-specific gene modulation.

    DOI: 10.1038/s41598-020-78682-8

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  • Preserved proteinase K-resistant core after amplification of alpha-synuclein aggregates: Implication to disease-related structural study. Reviewed International journal

    Saki Yoshinaga, Tomoyuki Yamanaka, Haruko Miyazaki, Ayami Okuzumi, Akiko Hiyama, Shigeo Murayama, Nobuyuki Nukina

    Biochemical and biophysical research communications   522 ( 3 )   655 - 661   2020.2

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    Many pathological proteins related to neurodegenerative diseases are misfolded, aggregating to form amyloid fibrils during pathogenesis. One of the pathological proteins, alpha-synuclein (α-syn), accumulates in the brains of Parkinson disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), which are designated as synucleinopathies. Recently, structural properties of abnormal accumulated proteins are suggested to determine the disease phenotype. However, the biochemical and structural characteristics of those accumulated proteins are still poorly understood. We previously reported the sequence and seed-structure-dependent polymorphic fibrils of α-syn and the polymorphism was identified by proteinase K-resistant cores determined by mass spectrometry (MS) analysis. In this study, we applied this method to analyze α-syn aggregates of MSA and DLB. To perform MS analysis on proteinase K-resistant cores, we first performed amplification of α-syn aggregates by seeding reaction and protein misfolding cyclic amplification (PMCA) to obtain a sufficient amount of aggregates. Using SDS insoluble fraction of the disease brain, we successfully amplified enough α-syn aggregates for MS analysis. We differentiated between mouse and human α-syn aggregates by MS analysis on proteinase K-resistant cores of the aggregates before and after amplification. The results suggest that structural properties of amplified α-syn fibrils are preserved after PMCA and these methods can be applicable in the study of pathological proteins of the neurodegenerative disorders.

    DOI: 10.1016/j.bbrc.2019.11.142

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  • Non-coding RNA Neat1 and Abhd11os expressions are dysregulated in medium spiny neurons of Huntington disease model mice. Reviewed International journal

    Hongsun Park, Haruko Miyazaki, Tomoyuki Yamanaka, Nobuyuki Nukina

    Neuroscience research   147   58 - 63   2019.10

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    Huntington Disease (HD) is a neurodegenerative disorder caused by expanded CAG repeats in the exon1 of huntingtin gene (HTT). The mutant HTT affects the transcriptional profile of neurons by disrupting the activities of transcriptional machinery and alters expression of many genes. In this study, we identified dysregulated non-coding RNAs (ncRNAs) in medium spiny neurons of 4-week-old HD model mouse. Also, we observed the intracellular localizations of Abhd11os and Neat1 ncRNAs by ViewRNA in situ hybridization, which could provide more precise detection, suggesting that it is a useful method to investigate the expression changes of genes with low expression levels.

    DOI: 10.1016/j.neures.2018.10.013

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  • Rapid dissemination of alpha-synuclein seeds through neural circuits in an in-vivo prion-like seeding experiment. Reviewed International journal

    Ayami Okuzumi, Masaru Kurosawa, Taku Hatano, Masashi Takanashi, Shuuko Nojiri, Takeshi Fukuhara, Tomoyuki Yamanaka, Haruko Miyazaki, Saki Yoshinaga, Yoshiaki Furukawa, Tomomi Shimogori, Nobutaka Hattori, Nobuyuki Nukina

    Acta neuropathologica communications   6 ( 1 )   96 - 96   2018.9

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    Accumulating evidence suggests that the lesions of Parkinson's disease (PD) expand due to transneuronal spreading of fibrils composed of misfolded alpha-synuclein (a-syn), over the course of 5-10 years. However, the precise mechanisms and the processes underlying the spread of these fibril seeds have not been clarified in vivo. Here, we investigated the speed of a-syn transmission, which has not been a focus of previous a-syn transmission experiments, and whether a-syn pathologies spread in a neural circuit-dependent manner in the mouse brain. We injected a-syn preformed fibrils (PFFs), which are seeds for the propagation of a-syn deposits, either before or after callosotomy, to disconnect bilateral hemispheric connections. In mice that underwent callosotomy before the injection, the propagation of a-syn pathology to the contralateral hemisphere was clearly reduced. In contrast, mice that underwent callosotomy 24 h after a-syn PFFs injection showed a-syn pathology similar to that seen in mice without callosotomy. These results suggest that a-syn seeds are rapidly disseminated through neuronal circuits immediately after seed injection, in a prion-like seeding experiment in vivo, although it is believed that clinical a-syn pathologies take years to spread throughout the brain. In addition, we found that botulinum toxin B blocked the transsynaptic transmission of a-syn seeds by specifically inactivating the synaptic vesicle fusion machinery. This study offers a novel concept regarding a-syn propagation, based on the Braak hypothesis, and also cautions that experimental transmission systems may be examining a unique type of transmission, which differs from the clinical disease state.

    DOI: 10.1186/s40478-018-0587-0

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  • Parallel homodimer structures of the extracellular domains of the voltage-gated sodium channel beta 4 subunit explain its role in cell-cell adhesion Reviewed

    Hideaki Shimizu, Asako Tosaki, Noboru Ohsawa, Yoshiko Ishizuka-Katsura, Shisako Shoji, Haruko Miyazaki, Fumitaka Oyama, Takaho Terada, Mikako Shirouzu, Shun-ichi Sekine, Nobuyuki Nukina, Shigeyuki Yokoyama

    JOURNAL OF BIOLOGICAL CHEMISTRY   292 ( 32 )   13428 - 13440   2017.8

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

    Voltage-gated sodium channels (VGSCs) are transmembrane proteins required for the generation of action potentials in excitable cells and essential for propagating electrical impulses along nerve cells. VGSCs are complexes of a pore-forming alpha subunit and auxiliary beta subunits, designated as beta 1/beta 1B-beta 4 (encoded by SCN1B-4B, respectively), which also function in cell-cell adhesion. We previously reported the structural basis for the trans homophilic interaction of the beta 4 subunit, which contributes to its adhesive function. Here, using crystallo-graphic and biochemical analyses, we show that the beta 4 extracellular domains directly interact with each other in a parallel manner that involves an intermolecular disulfide bond between the unpaired Cys residues (Cys(58)) in the loop connecting strands B and C and intermolecular hydrophobic and hydrogen-bonding interactions of the N-terminal segments (Ser(30)-Val(35)). Under reducing conditions, an N-terminally deleted beta 4 mutant exhibited decreased cell adhesion compared with the wild type, indicating that the beta 4 cis dimer contributes to the trans homophilic interaction of beta 4 in cell- cell adhesion. Furthermore, this mutant exhibited increased association with the alpha subunit, indicating that the cis dimerization of beta 4 affects alpha-beta 4 complex formation. These observations provide the structural basis for the parallel dimer formation of beta 4 in VGSCs and reveal its mechanism in cell-cell adhesion.

    DOI: 10.1074/jbc.M117.786509

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  • Acidic mammalian chitinase is a proteases-resistant glycosidase in mouse digestive system Reviewed

    Misa Ohno, Masahiro Kimura, Haruko Miyazaki, Kazuaki Okawa, Riho Onuki, Chiyuki Nemoto, Eri Tabata, Satoshi Wakita, Akinori Kashimura, Masayoshi Sakaguchi, Yasusato Sugahara, Nobuyuki Nukina, Peter O. Bauer, Fumitaka Oyama

    SCIENTIFIC REPORTS   6   37756   2016.11

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    Chitinases are enzymes that hydrolyze chitin, a polymer of beta-1, 4-linked N-acetyl-D-glucosamine (GlcNAc). Chitin has long been considered as a source of dietary fiber that is not digested in the mammalian digestive system. Here, we provide evidence that acidic mammalian chitinase (AMCase) can function as a major digestive enzyme that constitutively degrades chitin substrates and produces (GlcNAc) 2 fragments in the mouse gastrointestinal environment. AMCase was resistant to endogenous pepsin C digestion and remained active in the mouse stomach extract at pH 2.0. The AMCase mRNA levels were much higher than those of four major gastric proteins and two housekeeping genes and comparable to the level of pepsinogen C in the mouse stomach tissues. Furthermore, AMCase was expressed in the gastric pepsinogen-synthesizing chief cells. The enzyme was also stable and active in the presence of trypsin and chymotrypsin at pH 7.6, where pepsin C was completely degraded. Mouse AMCase degraded polymeric colloidal and crystalline chitin substrates in the gastrointestinal environments in presence of the proteolytic enzymes. Thus, AMCase can function as a protease-resistant major glycosidase under the conditions of stomach and intestine and degrade chitin substrates to produce (GlcNAc) 2, a source of carbon, nitrogen and energy.

    DOI: 10.1038/srep37756

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  • Differential roles of NF-Y transcription factor in ER chaperone expression and neuronal maintenance in the CNS Reviewed

    Tomoyuki Yamanaka, Asako Tosaki, Haruko Miyazaki, Masaru Kurosawa, Masato Koike, Yasuo Uchiyama, Sankar N. Maity, Hidemi Misawa, Ryosuke Takahashi, Tomomi Shimogori, Nobutaka Hattori, Nobuyuki Nukina

    SCIENTIFIC REPORTS   6   34575   2016.9

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    The mammalian central nervous system (CNS) contains various types of neurons with different neuronal functions. In contrast to established roles of cell type-specific transcription factors on neuronal specification and maintenance, whether ubiquitous transcription factors have conserved or differential neuronal function remains uncertain. Here, we revealed that inactivation of a ubiquitous factor NF-Y in different sets of neurons resulted in cell type-specific neuropathologies and gene downregulation in mouse CNS. In striatal and cerebellar neurons, NF-Y inactivation led to ubiquitin/p62 pathologies with downregulation of an endoplasmic reticulum (ER) chaperone Grp94, as we previously observed by NF-Y deletion in cortical neurons. In contrast, NF-Y inactivation in motor neurons induced neuronal loss without obvious protein deposition. Detailed analysis clarified downregulation of another ER chaperone Grp78 in addition to Grp94 in motor neurons, and knockdown of both ER chaperones in motor neurons recapitulated the pathology observed after NF-Y inactivation. Finally, additional downregulation of Grp78 in striatal neurons suppressed ubiquitin accumulation induced by NF-Y inactivation, implying that selective ER chaperone downregulation mediates different neuropathologies. Our data suggest distinct roles of NF-Y in protein homeostasis and neuronal maintenance in the CNS by differential regulation of ER chaperone expression.

    DOI: 10.1038/srep34575

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  • Structure-based site-directed photo-crosslinking analyses of multimeric cell-adhesive interactions of voltage-gated sodium channel beta subunits Reviewed

    Hideaki Shimizu, Haruko Miyazaki, Noboru Ohsawa, Shisako Shoji, Yoshiko Ishizuka-Katsura, Asako Tosaki, Fumitaka Oyama, Takaho Terada, Kensaku Sakamoto, Mikako Shirouzu, Shun-ichi Sekine, Nobuyuki Nukina, Shigeyuki Yokoyama

    SCIENTIFIC REPORTS   6   26618   2016.5

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    The beta 1, beta 2, and beta 4 subunits of voltage-gated sodium channels reportedly function as cell adhesion molecules. The present crystallographic analysis of the beta 4 extracellular domain revealed an antiparallel arrangement of the beta 4 molecules in the crystal lattice. The interface between the two antiparallel beta 4 molecules is asymmetric, and results in a multimeric assembly. Structure-based mutagenesis and sitedirected photo-crosslinking analyses of the beta 4-mediated cell-cell adhesion revealed that the interface between the antiparallel beta 4 molecules corresponds to that in the trans homophilic interaction for the multimeric assembly of beta 4 in cell-cell adhesion. This trans interaction mode is also employed in the beta 1-mediated cell-cell adhesion. Moreover, the beta 1 gene mutations associated with generalized epilepsy with febrile seizures plus (GEFS+) impaired the beta 1-mediated cell-cell adhesion, which should underlie the GEFS+ pathogenesis. Thus, the structural basis for the beta-subunit-mediated cell-cell adhesion has been established.

    DOI: 10.1038/srep26618

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  • Proteomic analysis of native cerebellar iFGF14 complexes Reviewed

    Marie K. Bosch, Jeanne M. Nerbonne, R. Reid Townsend, Haruko Miyazaki, Nobuyuki Nukina, David M. Ornitz, Celine Marionneau

    CHANNELS   10 ( 4 )   297 - 312   2016

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:TAYLOR & FRANCIS INC  

    Intracellular Fibroblast Growth Factor 14 (iFGF14) and the other intracellular FGFs (iFGF11-13) regulate the properties and densities of voltage-gated neuronal and cardiac Na+ (Nav) channels. Recent studies have demonstrated that the iFGFs can also regulate native voltage-gated Ca2+ (Cav) channels. In the present study, a mass spectrometry (MS)-based proteomic approach was used to identify the components of native cerebellar iFGF14 complexes. Using an anti-iFGF14 antibody, native iFGF14 complexes were immunoprecipitated from wild type adult mouse cerebellum. Parallel control experiments were performed on cerebellar proteins isolated from mice (Fgf14(-/-)) harboring a targeted disruption of the Fgf14 locus. MS analyses of immunoprecipitated proteins demonstrated that the vast majority of proteins identified in native cerebellar iFGF14 complexes are Nav channel pore-forming () subunits or proteins previously reported to interact with Nav subunits. In contrast, no Cav channel or accessory subunits were revealed in cerebellar iFGF14 immunoprecipitates. Additional experiments were completed using an anti-PanNav antibody to immunoprecipitate Nav channel complexes from wild type and Fgf14(-/-) mouse cerebellum. Western blot and MS analyses revealed that the loss of iFGF14 does not measurably affect the protein composition or the relative abundance of Nav channel interacting proteins in native adult mouse cerebellar Nav channel complexes.

    DOI: 10.1080/19336950.2016.1153203

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  • Sodium Channel beta 1 Subunit Localizes to Axon Initial Segments of Excitatory and Inhibitory Neurons and Shows Regional Heterogeneity in Mouse Brain Reviewed

    Verena C. Wimmer, Rosemary C. Harty, Kay L. Richards, A. Marie Phillips, Haruko Miyazaki, Nobuyuki Nukina, Steven Petrou

    JOURNAL OF COMPARATIVE NEUROLOGY   523 ( 5 )   814 - 830   2015.4

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-BLACKWELL  

    The 1 subunit of voltage-gated sodium channels, Na(v)1, plays multiple roles in neurons spanning electrophysiological modulation of sodium channel subunits to cell adhesion and neurite outgrowth. This study used immunohistochemistry to investigate Na(v)1 subneuronal and regional expression. Na(v)1 was enriched at axon initial segments (AIS) and nodes of Ranvier. Na(v)1 expression at the AIS was detected throughout the brain, predominantly in the hippocampus, cortex, and cerebellum. Despite expression of Na(v)1 in both excitatory and inhibitory AIS, it displayed a marked and fine-grained heterogeneity of expression. Such heterogeneity could have important implications for the tuning of single neuronal and regional excitability, especially in view of the fact that Na(v)1 coexpressed with Na(v)1.1, Na(v)1.2, and Na(v)1.6 subunits. The disruption of Na(v)1 AIS expression by a human epilepsy-causing C121W genetic mutation in Na(v)1 was also investigated using a mouse model. AIS expression of Na(v)1 was reduced by approximately 50% in mice heterozygous for the C121W mutation and was abolished in homozygotes, suggesting that loss of Na-v subunit modulation by Na(v)1 contributes to the mechanism of epileptogenesis in these animals as well as in patients. J. Comp. Neurol. 523:814-830, 2015. (c) 2015 Wiley Periodicals, Inc.

    DOI: 10.1002/cne.23715

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  • FUS/TLS deficiency causes behavioral and pathological abnormalities distinct from amyotrophic lateral sclerosis Reviewed

    Yoshihiro Kino, Chika Washizu, Masaru Kurosawa, Mizuki Yamada, Haruko Miyazaki, Takumi Akagi, Tsutomu Hashikawa, Hiroshi Doi, Toru Takumi, Geoffrey G. Hicks, Nobutaka Hattori, Tomomi Shimogori, Nobuyuki Nukina

    ACTA NEUROPATHOLOGICA COMMUNICATIONS   3   24   2015.4

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

    Introduction: FUS/TLS is an RNA-binding protein whose genetic mutations or pathological inclusions are associated with neurological diseases including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration, and essential tremor (ET). It is unclear whether their pathogenesis is mediated by gain or loss of function of FUS/TLS.
    Results: Here, we established outbred FUS/TLS knockout mice to clarify the effects of FUS/TLS dysfunction in vivo. We obtained homozygous knockout mice that grew into adulthood. Importantly, they did not manifest ALS-or ET-like phenotypes until nearly two years. Instead, they showed distinct histological and behavioral alterations including vacuolation in hippocampus, hyperactivity, and reduction in anxiety-like behavior. Knockout mice showed transcriptome alterations including upregulation of Taf15 and Hnrnpa1, while they have normal morphology of RNA-related granules such as Gems.
    Conclusions: Collectively, FUS/TLS depletion causes phenotypes possibly related to neuropsychiatric and neurodegenerative conditions, but distinct from ALS and ET, together with specific alterations in RNA metabolisms.

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  • Modulation of voltage-gated K+ channels by the sodium channel beta 1 subunit Reviewed

    Hai M. Nguyen, Haruko Miyazaki, Naoto Hoshi, Brian J. Smith, Nobuyuki Nukina, Alan L. Goldin, K. George Chandy

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   109 ( 45 )   18577 - 18582   2012.11

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    Voltage-gated sodium (Na-V) and potassium(K-V) channels are critical components of neuronal action potential generation and propagation. Here, we report that Na-V beta 1 encoded by SCN1b, an integral subunit of Na-V channels, coassembles with and modulates the biophysical properties of K(V)1 and K(V)7 channels, but not K(V)3 channels, in an isoform-specific manner. Distinct domains of Na-V beta 1 are involved in modulation of the different K-V channels. Studies with channel chimeras demonstrate that Na-V beta 1-mediated changes in activation kinetics and voltage dependence of activation require interaction of Na-V beta 1 with the channel's voltage-sensing domain, whereas changes in inactivation and deactivation require interaction with the channel's pore domain. A molecular model based on docking studies shows Na-V beta 1 lying in the crevice between the voltage-sensing and pore domains of K-V channels, making significant contacts with the S1 and S5 segments. Cross-modulation of Na-V and K-V channels by Na-V beta 1 may promote diversity and flexibility in the overall control of cellular excitability and signaling.

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  • Mutant huntingtin fragment selectively suppresses Brn-2 POU domain transcription factor to mediate hypothalamic cell dysfunction Reviewed

    Tomoyuki Yamanaka, Asako Tosaki, Haruko Miyazaki, Masaru Kurosawa, Yoshiaki Furukawa, Mizuki Yamada, Nobuyuki Nukina

    HUMAN MOLECULAR GENETICS   19 ( 11 )   2099 - 2112   2010.6

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    In polyglutamine diseases including Huntington's disease (HD), mutant proteins containing expanded polyglutamine stretches form nuclear aggregates in neurons. Although analysis of their disease models suggested a significance of transcriptional dysregulation in these diseases, how it mediates the specific neuronal cell dysfunction remains obscure. Here we performed a comprehensive analysis of altered DNA binding of multiple transcription factors using R6/2 HD model mice brains that express an N-terminal fragment of mutant huntingtin (mutant Nhtt). We found a reduction of DNA binding of Brn-2, a POU domain transcription factor involved in differentiation and function of hypothalamic neurosecretory neurons. We provide evidence supporting that Brn-2 loses its function through two pathways, its sequestration by mutant Nhtt and its reduced transcription, leading to reduced expression of hypothalamic neuropeptides. In contrast to Brn-2, its functionally related protein, Brn-1, was not sequestered by mutant Nhtt but was upregulated in R6/2 brain, except in hypothalamus. Our data indicate that functional suppression of Brn-2 together with a region-specific lack of compensation by Brn-1 mediates hypothalamic cell dysfunction by mutant Nhtt.

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  • Harnessing chaperone-mediated autophagy for the selective degradation of mutant huntingtin protein Reviewed

    Peter O. Bauer, Anand Goswami, Hon Kit Wong, Misako Okuno, Masaru Kurosawa, Mizuki Yamada, Haruko Miyazaki, Gen Matsumoto, Yoshihiro Kino, Yoshitaka Nagai, Nobuyuki Nukina

    NATURE BIOTECHNOLOGY   28 ( 3 )   256 - U111   2010.3

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    Huntington's Disease (HD) is a dominantly inherited pathology caused by the accumulation of mutant huntingtin protein (HTT) containing an expanded polyglutamine (polyQ) tract. As the polyglutamine binding peptide 1 (QBP1) is known to bind an expanded polyQ tract but not the polyQ motif found in normal HTT, we selectively targeted mutant HTT for degradation by expressing a fusion molecule comprising two copies of QBP1 and copies of two different heat shock cognate protein 70 (HSC70)-binding motifs in cellular and mouse models of HD. Chaperone-mediated autophagy contributed to the specific degradation of mutant HTT in cultured cells expressing the construct. Intrastriatal delivery of a virus expressing the fusion molecule ameliorated the disease phenotype in the R6/2 mouse model of HD. Similar adaptor molecules comprising HSC70-binding motifs fused to an appropriate structure-specific binding agent(s) may have therapeutic potential for treating diseases caused by misfolded proteins other than those with expanded polyQ tracts.

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  • Functional reciprocity between Na+ channel Na(v)1.6 and beta 1 subunits in the coordinated regulation of excitability and neurite outgrowth Reviewed

    William J. Brackenbury, Jeffrey D. Calhoun, Chunling Chen, Haruko Miyazaki, Nobuyuki Nukina, Fumitaka Oyama, Barbara Ranscht, Lori L. Isom

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   107 ( 5 )   2283 - 2288   2010.2

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    Voltage-gated Na+ channel (VGSC) beta 1 subunits regulate cell-cell adhesion and channel activity in vitro. We previously showed that beta 1 promotes neurite outgrowth in cerebellar granule neurons (CGNs) via homophilic cell adhesion, fyn kinase, and contactin. Here we demonstrate that beta 1-mediated neurite outgrowth requires Na+ current (I-Na) mediated by Na(v)1.6. In addition, beta 1 is required for high-frequency action potential firing. Transient INa is unchanged in Scn1b (beta 1) null CGNs; however, the resurgent I-Na, thought to underlie high-frequency firing in Na(v)1.6-expressing cerebellar neurons, is reduced. The proportion of axon initial segments (AIS) expressing Nav1.6 is reduced in Scn1b null cerebellar neurons. In place of Na(v)1.6 at the AIS, we observed an increase in Na(v)1.1, whereas Na(v)1.2 was unchanged. This indicates that beta 1 is required for normal localization of Nav1.6 at the AIS during the postnatal developmental switch to Na(v)1.6-mediated high-frequency firing. In agreement with this, beta 1 is normally expressed with a subunits at the AIS of P14 CGNs. We propose reciprocity of function between beta 1 and Na(v)1.6 such that beta 1-mediated neurite outgrowth requires Na(v)1.6-mediated I-Na, and Na(v)1.6 localization and consequent high-frequency firing require beta 1. We conclude that VGSC subunits function in macromolecular signaling complexes regulating both neuronal excitability and migration during cerebellar development.

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  • A Functional Null Mutation of SCN1B in a Patient with Dravet Syndrome Reviewed

    Gustavo A. Patino, Lieve R. F. Claes, Luis F. Lopez-Santiago, Emily A. Slat, Raja S. R. Dondeti, Chunling Chen, Heather A. O'Malley, Charles B. B. Gray, Haruko Miyazaki, Nobuyuki Nukina, Fumitaka Oyama, Peter De Jonghe, Lori L. Isom

    JOURNAL OF NEUROSCIENCE   29 ( 34 )   10764 - 10778   2009.8

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    Dravet syndrome (also called severe myoclonic epilepsy of infancy) is one of the most severe forms of childhood epilepsy. Most patients have heterozygous mutations in SCN1A, encoding voltage-gated sodium channel Na(v)1.1 alpha subunits. Sodium channels are modulated by beta 1 subunits, encoded by SCN1B, a gene also linked to epilepsy. Here we report the first patient with Dravet syndrome associated with a recessive mutation in SCN1B (p.R125C). Biochemical characterization of p.R125C in a heterologous system demonstrated little to no cell surface expression despite normal total cellular expression. This occurred regardless of coexpression of Na(v)1.1 alpha subunits. Because the patient was homozygous for the mutation, these data suggest a functional SCN1B null phenotype. To understand the consequences of the lack of beta 1 cell surface expression in vivo, hippocampal slice recordings were performed in Scn1b(-/-) versus Scn1b(+/+) mice. Scn1b(-/-) CA3 neurons fired evoked action potentials with a significantly higher peak voltage and significantly greater amplitude compared with wild type. However, in contrast to the Scn1a(+/-) model of Dravet syndrome, we found no measurable differences in sodium current density in acutely dissociated CA3 hippocampal neurons. Whereas Scn1b(-/-) mice seize spontaneously, the seizure susceptibility of Scn1b(+/-) mice was similar to wild type, suggesting that, like the parents of this patient, one functional SCN1B allele is sufficient for normal control of electrical excitability. We conclude that SCN1B p.R125C is an autosomal recessive cause of Dravet syndrome through functional gene inactivation.

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  • Genetically Determined Differences in Sodium Current Characteristics Modulate Conduction Disease Severity in Mice With Cardiac Sodium Channelopathy Reviewed

    Carol Ann Remme, Brendon P. Scicluna, Arie O. Verkerk, Ahmad S. Amin, Sandra van Brunschot, Leander Beekman, Vera H. M. Deneer, Catherine Chevalier, Fumitaka Oyama, Haruko Miyazaki, Nobuyuki Nukina, Ronald Wilders, Denis Escande, Remi Houlgatte, Arthur A. M. Wilde, Hanno L. Tan, Marieke W. Veldkamp, Jacques M. T. de Bakker, Connie R. Bezzina

    CIRCULATION RESEARCH   104 ( 11 )   1283 - U112   2009.6

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    Conduction slowing of the electric impulse that drives the heartbeat may evoke lethal cardiac arrhythmias. Mutations in SCN5A, which encodes the pore-forming cardiac sodium channel alpha subunit, are associated with familial arrhythmia syndromes based on conduction slowing. However, disease severity among mutation carriers is highly variable. We hypothesized that genetic modifiers underlie the variability in conduction slowing and disease severity. With the aim of identifying such modifiers, we studied the Scn5a(1798insD/+) mutation in 2 distinct mouse strains, FVB/N and 129P2. In 129P2 mice, the mutation resulted in more severe conduction slowing particularly in the right ventricle (RV) compared to FVB/N. Pan-genomic mRNA expression profiling in the 2 mouse strains uncovered a drastic reduction in mRNA encoding the sodium channel auxiliary subunit beta 4 (Scn4b) in 129P2 mice compared to FVB/N. This corresponded to low to undetectable beta 4 protein levels in 129P2 ventricular tissue, whereas abundant beta 4 protein was detected in FVB/N. Sodium current measurements in isolated myocytes from the 2 mouse strains indicated that sodium channel activation in myocytes from 129P2 mice occurred at more positive potentials compared to FVB/N. Using computer simulations, this difference in activation kinetics was predicted to explain the observed differences in conduction disease severity between the 2 strains. In conclusion, genetically determined differences in sodium current characteristics on the myocyte level modulate disease severity in cardiac sodium channelopathies. In particular, the sodium channel subunit beta 4 (SCN4B) may constitute a potential genetic modifier of conduction and cardiac sodium channel disease. (Circ Res. 2009; 104: 1283-1292.)

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  • Inhibition of Rho Kinases Enhances the Degradation of Mutant Huntingtin Reviewed

    Peter O. Bauer, Hon Kit Wong, Fumitaka Oyama, Anand Goswami, Misako Okuno, Yoshihiro Kino, Haruko Miyazaki, Nobuyuki Nukina

    JOURNAL OF BIOLOGICAL CHEMISTRY   284 ( 19 )   13153 - 13164   2009.5

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    Huntington disease (HD) is a fatal hereditary neurodegenerative disease caused by an expansion of the polyglutamine (polyQ) stretch in huntingtin (htt). Whereas the pathological significance of the expanded polyQ has been clearly established and a tremendous effort to develop therapeutic tools for HD has been exerted, there is yet no effective cure. Whereas many molecules able to reduce the polyQ accumulation and aggregation have been identified, including several Rho kinase (ROCK) inhibitors, it remains very important to determine the mechanism of action of the potential drugs. ROCK inhibitors, including Y-27632 were reported to decrease aggregation of htt and androgen receptor (AR) through ROCK1 and protein kinase C-related protein kinase-2 (PRK-2). A downstream effector of ROCK1, actin-binding factor profilin, was shown to inhibit the mutant htt aggregation but not AR by direct interaction. We found that the anti-aggregation effect of ROCK inhibitors was not limited to the mutant htt and AR and that Y-27632 was also able to reduce the aggregation of ataxin-3 and atrophin-1 with expanded polyQ. These results suggested that in addition to the mechanism reported for htt and AR, there might also be other common mediators involved in the reduced aggregation of different polyQ proteins. In this study, we show that Y-27632 not only reduced the mutant htt aggregation by enhancing its degradation, but surprisingly was able to activate the main cellular degradation pathways, proteasome, and macroautophagy. We also show that this unique effect was mediated by ROCK1 and ROCK2.

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  • BIG-2 mediates olfactory axon convergence to target glomeruli. Reviewed International journal

    Tomomi Kaneko-Goto, Sei-Ichi Yoshihara, Haruko Miyazaki, Yoshihiro Yoshihara

    Neuron   57 ( 6 )   834 - 46   2008.3

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    Olfactory sensory neurons expressing a given odorant receptor converge axons onto a few topographically fixed glomeruli in the olfactory bulb, leading to establishment of the odor map. Here, we report that BIG-2/contactin-4, an axonal glycoprotein belonging to the immunoglobulin superfamily, is expressed in a subpopulation of mouse olfactory sensory neurons. A mosaic pattern of glomerular arrangement is observed with strongly BIG-2-positive, weakly positive, and negative axon terminals in the olfactory bulb, which is overlapping but not identical with those of Kirrel2 and ephrin-A5. There is a close correlation between the BIG-2 expression level and the odorant receptor choice in individual sensory neurons. In BIG-2-deficient mice, olfactory sensory neurons expressing a given odorant receptor frequently innervate multiple glomeruli at ectopic locations. These results suggest that BIG-2 is one of the axon guidance molecules crucial for the formation and maintenance of functional odor map in the olfactory bulb.

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  • Mutant Huntingtin reduces HSP70 expression through the sequestration of NF-Y transcription factor Reviewed

    Tomoyuki Yamanaka, Haruko Miyazaki, Fumitaka Oyama, Masaru Kurosawa, Chika Washizu, Hiroshi Doi, Nobuyuki Nukina

    EMBO JOURNAL   27 ( 6 )   827 - 839   2008.3

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    In Huntington's disease (HD), mutant Huntingtin, which contains expanded polyglutamine stretches, forms nuclear aggregates in neurons. The interactions of several transcriptional factors with mutant Huntingtin, as well as altered expression of many genes in HD models, imply the involvement of transcriptional dysregulation in the HD pathological process. The precise mechanism remains obscure, however. Here, we show that mutant Huntingtin aggregates interact with the components of the NF-Y transcriptional factor in vitro and in HD model mouse brain. An electrophoretic mobility shift assay using HD model mouse brain lysates showed reduction in NF-Y binding to the promoter region of HSP70, one of the NF-Y targets. RT-PCR analysis revealed reduced HSP70 expression in these brains. We further clarified the importance of NF-Y for HSP70 transcription in cultured neurons. These data indicate that mutant Huntingtin sequesters NF-Y, leading to the reduction of HSP70 gene expression in HD model mice brain. Because suppressive roles of HSP70 on the HD pathological process have been shown in several HD models, NF-Y could be an important target of mutant Huntingtin.

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  • RNA-binding protein TLS is a major nuclear aggregate-interacting protein in huntingtin exon 1 with expanded polyglutamine-expressing cells Reviewed

    Hiroshi Doi, Kazumasa Okamura, Peter O. Bauer, Yoshiaki Furukawa, Hideaki Shimizu, Masaru Kurosawa, Yoko Machida, Haruko Miyazaki, Kenichi Mitsui, Yoshiyuki Kuroiwa, Nobuyuki Nukina

    JOURNAL OF BIOLOGICAL CHEMISTRY   283 ( 10 )   6489 - 6500   2008.3

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    Formation of intracellular aggregates is the hallmark of polyglutamine (polyQ) diseases. We analyzed the components of purified nuclear polyQ aggregates by mass spectrometry. As a result, we found that the RNA-binding protein translocated in liposarcoma (TLS) was one of the major components of nuclear polyQ aggregate-interacting proteins in a Huntington disease cell model and was also associated with neuronal intranuclear inclusions of R6/2 mice. In vitro study revealed that TLS could directly bind to truncated N-terminal huntingtin (tNhtt) aggregates but could not bind to monomer GST-tNhtt with 18, 42, or 62Q, indicating that the tNhtt protein acquired the ability to sequester TLS after forming aggregates. Thioflavin T assay and electron microscopic study further supported the idea that TLS bound to tNhtt-42Q aggregates at the early stage of tNhtt-42Q amyloid formation. Immunohistochemistry showed that TLS was associated with neuronal intranuclear inclusions of Huntington disease human brain. Because TLS has a variety of functional roles, the sequestration of TLS to polyQ aggregates may play a role in diverse pathological changes in the brains of patients with polyQ diseases.

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  • Floxed allele for conditional inactivation of the voltage-gated sodium channel beta 1 subunit Scn1b Reviewed

    Chunling Chen, Travis L. Dickendesher, Fumitaka Oyama, Haruko Miyazaki, Nobuyuki Nukina, Lori L. Isom

    GENESIS   45 ( 9 )   547 - 553   2007.9

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    The voltage-gated sodium channel gene Scn1b encodes the auxiliary subunit beta 1, which is widely distributed in neurons and glia of the central and peripheral nervous systems, cardiac myocytes, skeletal muscle myocytes, and neuroendocrine cells. We showed previously that the Scn1b null mutation results in a complex and severe phenotype that includes retarded growth, seizures, ataxia, and death by postnatal day 21. We generated a floxed allele of Scn1b by inserting loxP sites surrounding the second coding exon. Ubiquitous deletion of the floxed exon by Cre recombinase using CMV-Cre-transgenic mice produced the Scn1b(del) allele. The null phenotype of Scn1b(del) homozygotes is indistinguishable from that of Scn1b nulls and confirms the in vivo inactivation of Scn1b. Conditional inactivation of the floxed allele will make it possible to circumvent the lethality that results from complete loss of this gene, such that the physiological role of Scn1b in specific cell types and/or specific developmental time points can be investigated.

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  • BACE1 modulates filopodia-like protrusions induced by sodium channel beta 4 subunit Reviewed

    Haruko Miyazaki, Fumitaka Oyama, Hon-Kit Wong, Kumi Kaneko, Takashi Sakurai, Akira Tamaoka, Nobuyuki Nukina

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   361 ( 1 )   43 - 48   2007.9

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    Processing of APP by BACE1 plays a crucial role in the pathogenesis of Alzheimer disease (AD). Recently, the voltage-gated sodium channel (Na-v) beta(4) subunit (beta(4)), an auxiliary subunit of Nav that is supposed to serve as a cell adhesion molecule, has been identified as a substrate for BACE1. However, the biological consequence of BACE1 processing of beta 4 remains illusive. Here, we report the biological effects of beta(4) processing by BACE1 Overexpression of beta 4 in Neuro2a cells promoted neurite extension and increased the number of F-actin rich filopodia-like protrusions. While coexpression of BACE1 together with beta(4) further accelerated neurite extension, the number of filopodia-like protrusions was reduced. Overexpression of C-terminal fragment of beta(4) that was generated by BACE1 (beta(4)-CTF) partially recapitulated the results obtained with BACE1 overexpression. These results suggest that the processing of beta(4) by BACE1 regulates neurite length and filopodia-like protrusion density in neurons. (c) 2007 Elsevier Inc. All rights reserved.

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  • Sodium channel beta 4 subunit: down-regulation and possible involvement in neuritic degeneration in Huntington's disease transgenic mice Reviewed

    F Oyama, H Miyazaki, N Sakamoto, C Becquet, Y Machida, K Kaneko, C Uchikawa, T Suzuki, M Kurosawa, T Ikeda, A Tamaoka, T Sakurai, N Nukina

    JOURNAL OF NEUROCHEMISTRY   98 ( 2 )   518 - 529   2006.7

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    Sodium channel beta 4 is a very recently identified auxiliary subunit of the voltage-gated sodium channels. To find the primarily affected gene in Huntington's disease (HD) pathogenesis, we profiled HD transgenic mice using a high-density oligonucleotide array and identified beta 4 as an expressed sequence tag (EST) that was significantly down-regulated in the striatum of HD model mice and patients. Reduction in beta 4 started at a presymptomatic stage in HD mice, whereas other voltage-gated ion channel subunits were decreased later. In contrast, spinal cord neurons, which generate only negligible levels of expanded polyglutamine aggregates, maintained normal levels of beta 4 expression even at the symptomatic stage. Overexpression of beta 4 induced neurite outgrowth in Neuro2a cells, and caused a thickening of dendrites and increased density of dendritic spines in hippocampal primary neurons, indicating that beta 4 modulates neurite outgrowth activities. These results suggest that down-regulation of beta 4 may lead to abnormalities of sodium channel and neurite degeneration in the striatum of HD transgenic mice and patients with HD.

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  • Expanded polyglutamines impair synaptic transmission and ubiquitin-proteasome system in Caenorhabditis elegans Reviewed

    LA Khan, PO Bauer, H Miyazaki, KS Lindenberg, BG Landwehrmeyer, N Nukina

    JOURNAL OF NEUROCHEMISTRY   98 ( 2 )   576 - 587   2006.7

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    Polyglutamine (polyQ) expansion in many proteins, including huntingtin and ataxin-3, is pathogenic and responsible for neuronal dysfunction and degeneration. Although at least nine neurodegenerative diseases are caused by expanded polyQ, the pathogenesis of these diseases is still not well understood. In the present study, we used Caenorhabditis elegans to study the molecular mechanism of polyQ-mediated toxicity. We expressed full-length and truncated ataxin-3 with different lengths of polyQ in the nervous system of C. elegans. We show that expanded polyQ interrupts synaptic transmission, and induces swelling and aberrant branching of neuronal processes. Using an ubiquitinated fluorescence reporter construct, we also showed that polyQ aggregates impair the ubiquitin-proteasome system in C. elegans. These results may provide information for further understanding the pathogenesis of polyQ diseases.

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  • beta subunits of voltage-gated sodium channels are novel substrates of beta-site amyloid precursor protein-cleaving enzyme (BACE1) and gamma-secretase Reviewed

    HK Wong, T Sakurai, F Oyama, K Kaneko, K Wada, H Miyazaki, M Kurosawa, B De Strooper, P Saftig, N Nukina

    JOURNAL OF BIOLOGICAL CHEMISTRY   280 ( 24 )   23009 - 23017   2005.6

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    Sequential processing of amyloid precursor protein (APP) by membrane-bound proteases, BACE1 and gamma-secretase, plays a crucial role in the pathogenesis of Alzheimer disease. Much has been discovered on the properties of these proteases; however, regulatory mechanisms of enzyme-substrate interaction in neurons and their involvement in pathological changes are still not fully understood. It is mainly because of the membrane-associated cleavage of these proteases and the lack of information on new substrates processed in a similar way to APP. Here, using RNA interference-mediated BACE1 knockdown, mouse embryonic fibroblasts that are deficient in either BACE1 or presenilins, and BACE1-deficient mouse brain, we show clear evidence that beta subunits of voltage-gated sodium channels are sequentially processed by BACE1 and gamma-secretase. These results may provide new insights into the underlying pathology of Alzheimer disease.

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  • Decreased expression of hypothalamic neuropeptides in Huntington disease transgenic mice with expanded polyglutamine-EGFP fluorescent aggregates Reviewed

    S Kotliarova, NR Jana, N Sakamoto, M Kurosawa, H Miyazaki, M Nekooki, H Doi, Y Machida, HK Wong, T Suzuki, C Uchikawa, Y Kotliarov, K Uchida, Y Nagao, U Nagaoka, A Tamaoka, K Oyanagi, F Oyama, N Nukina

    JOURNAL OF NEUROCHEMISTRY   93 ( 3 )   641 - 653   2005.5

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    Huntington disease is caused by polyglutamine (polyQ) expansion in huntingtin. Selective and progressive neuronal loss is observed in the striatum and cerebral cortex in Huntington disease. We have addressed whether expanded polyQ aggregates appear in regions of the brain apart from the striatum and cortex and whether there is a correlation between expanded polyQ aggregate formation and dysregulated transcription. We generated transgenic mouse lines expressing mutant truncated N-terminal huntingtin (expanded polyQ) fused with enhanced green fluorescent protein (EGFP) and carried out a high-density oligonucleotide array analysis using mRNA extracted from the cerebrum, followed by TaqMan RT-PCR and in situ hybridization. The transgenic mice formed expanded polyQ-EGFP fluorescent aggregates and this system allowed us to directly visualize expanded polyQ aggregates in various regions of the brain without performing immunohistochemical studies. We show here that polyQ-EGFP aggregates were intense in the hypothalamus, where the expression of six hypothalamic neuropeptide mRNAs, such as oxytocin, vasopressin and cocaine-amphetamine-regulated transcript, was down-regulated in the transgenic mouse brain without observing a significant loss of hypothalamic neurons. These results indicate that the hypothalamus is susceptible to aggregate formation in these mice and this may result in the down-regulation of specific genes in this region of the brain.

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  • Gem GTPase and Tau - Morphological changes induced by Gem GTPase in Cho cells are antagonized by tau Reviewed

    F Oyama, S Kotliarova, A Harada, M Ito, H Miyazaki, Y Ueyama, N Hirokawa, N Nukina, Y Ihara

    JOURNAL OF BIOLOGICAL CHEMISTRY   279 ( 26 )   27272 - 27277   2004.6

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    A series of observations have indicated that tau, one of the major microtubule-associated proteins, is involved in neuronal cell morphogenesis and axonal maintenance. Tau is also the major component of paired helical filaments found in brains affected by Alzheimer's disease. To explore an as yet unidentified role of tau in vivo, similar to 11,000 mRNAs were profiled from tau-deficient mouse brains and compared with those from control brains at the same ages. The expression of Gem GTPase, a small GTP-binding protein of the ras superfamily, was significantly increased in the brains of tau-deficient mice at 8 weeks of age. Because Gem GTPase is a negative regulator of the Rho-Rho kinase pathway for cytoskeletal organization, this protein was transiently overexpressed in Chinese hamster ovary cells that do not express tau. Overexpression of Gem GTPase induced a marked elongation of Chinese hamster ovary cells, and simultaneous expression of tau eliminated this effect, although tau did not bind directly to Gem GTPase. This anti-elongation activity of tau was attributed to its microtubule-binding domain, and homologous domains of microtubule-associated proteins 2 and 4 exhibited similar antagonistic activities. Taken together, the present results indicate that the level of Gem GTPase and its cell elongation activity are modulated by tau and suggest that tau may be involved in a Gem GTPase-mediated signal transduction pathway.

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  • Pro-apoptotic protein kinase C delta is associated with intranuclear inclusions in a transgenic model of Huntington's disease Reviewed

    EA Zemskov, NR Jana, M Kurosawa, H Miyazaki, N Sakamoto, M Nekooki, N Nukina

    JOURNAL OF NEUROCHEMISTRY   87 ( 2 )   395 - 406   2003.10

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    In order to investigate any effect of truncated mutant huntingtin (tNhtt) aggregation on protein kinase C (PKC) signaling in Huntington's disease (HD), we studied a possible association of PKC isoforms with the aggregates using cellular and transgenic models of HD. In this report we describe an association of mutant tNhtt with at least three PKC isoforms (alpha, delta, zeta), as revealed by co-immunoprecipitation assays and immunocytochemistry in a cellular model of HD (Neuro2a cells expressing tNhtt-150Q-EGFP), as well as a specific association of PKCdelta with intranuclear aggregates in a transgenic model (R6/2 mice). Immunoblot analysis of isolated nuclear fractions shows an elevation of nuclear PKCdelta in transgenic brain tissue. The observed elevation has a strong similarity with the apoptotic translocation of PKCdelta detected in experiments with the mouse neuroblastoma Neuro2a cells. Using a Neuro2a cell line expressing tNhtt with the nuclear localization signal, we demonstrate the association of PKCdelta with intranuclear aggregates and present evidence that accumulation of PKCdelta in cell nuclei does not depend on mutant htt nuclear translocation. Our results suggest that the association of PKCdelta with intranuclear htt-aggregates may affect its apoptotic function in a transgenic model of HD.

    DOI: 10.1046/j.1471-4159.2003.02002.x

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MISC

  • マウス脳における脳梁離断を用いた線維状αsynucleinの伝播経路の検討

    奥住 文美, 波田野 琢, 黒澤 大, 山中 智行, 宮崎 晴子, 古川 良明, 服部 信孝, 貫名 信行

    パーキンソン病・運動障害疾患コングレスプログラム・抄録集   11回   68 - 68   2017.10

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    Language:Japanese   Publisher:Movement Disorder Society of Japan (MDSJ)  

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  • Structural and functional analysis of voltage-gated sodium channel β-subunit and epilepsy-causing mutations

    Hideaki Shimizu, Haruko Miyazaki, Shisako Shoji, Mikako Shirouzu, Nobuyuki Nukina, Shigeyuki Yokoyama

    The 35th Annual Meeting of the Japan Neuroscience Society   P4-j19   2012.9

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  • 電位依存性ナトリウムチャネルβサブユニットの分子間相互作用の解析

    清水英明, 宮崎晴子, 庄司志咲子, 白水美香子, 貫名信行, 横山茂之

    日本分子生物学会年会プログラム・要旨集(Web)   34th   4P-0276   2011.12

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    J-GLOBAL

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  • Correlation between nuclear accumulation and dysregulation of sodium channel beta 4 subunit in HD transgenic mice

    Haruko Miyazaki, Fumitaka Oyama, Masaru Kurosawa, Mizuki Yamada, Nobuyuki Nukina

    NEUROSCIENCE RESEARCH   65   S247 - S247   2009

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    Language:English   Publishing type:Research paper, summary (international conference)   Publisher:ELSEVIER IRELAND LTD  

    DOI: 10.1016/j.neures.2009.09.1400

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  • Scn4b Is A Genetic Modifier Of Cardiac Conduction Disease In Mice

    Brandon P. Scicluna, Carol A. Remme, Arie O. Verkerk, Ahmad S. Amin, Michael W. Tanck, Leander Beekman, Vera H. Deneer, Catherine Chevaller, Fumitaka Oyama, Haruko Miyazaki, Nobuyuki Nukina, Denis Escande, Remi Houlgatte, Jacques M. de Bakker, Marieke Veldkamp, Hanno L. Tan, Arthur A. Wilde, Connie R. Bezzina

    CIRCULATION   118 ( 18 )   S339 - S339   2008.10

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    Language:English   Publishing type:Research paper, summary (international conference)   Publisher:LIPPINCOTT WILLIAMS & WILKINS  

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  • Mutant Huntingtin reduces HSP70 expression through the sequestration of NF-Y transcription factor

    Tomoyuki Yamanaka, Haruko Miyazaki, Fumitaka Oyama, Masaru Kurosawa, Chika Washizu, Hiroshi Doi, Nobuyuki Nukina

    NEUROSCIENCE RESEARCH   61   S45 - S45   2008

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  • Downregulation of sodium channel beta 4 in Huntington disease transgenic mice

    Fumitaka Oyama, Haruko Miyazaki, Masaru Kurosavva, Mizuki Yamada, Nobuyuki Nukina

    NEUROSCIENCE RESEARCH   61   S204 - S204   2008

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  • beta subunits of voltage-gated sodium channels are novel substrates of beta-site amyloid precursor protein-cleaving enzyme (BACE1) and gamma-secretase

    T Sakurai, HK Wong, F Oyama, K Kaneko, K Wada, H Miyazaki, M Kurosawa, B De Strooper, P Saftig, N Nukina

    JOURNAL OF PHARMACOLOGICAL SCIENCES   100   244P - 244P   2006

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    Language:English   Publishing type:Research paper, summary (international conference)   Publisher:JAPANESE PHARMACOLOGICAL SOC  

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  • Dysregulation of sodium channel beta 4 subunit by expanded polyglutamine in Huntington disease transgenic mice

    Fumitaka Oyama, Haruko Miyazaki, Kazumasa Okamura, Yoko Machida, Kurosawa Masaru, Takashi Sakurai, Nobuyuki Nukina

    NEUROSCIENCE RESEARCH   55   S257 - S257   2006

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  • Region specific changes of gene expression in a transgenic mouse model for Huntington disease.

    F Oyama, H Miyazaki, N Sakamoto, NR Jana, SE Kotliarova, N Nukina

    AMERICAN JOURNAL OF HUMAN GENETICS   73 ( 5 )   544 - 544   2003.11

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    Language:English   Publishing type:Research paper, summary (international conference)   Publisher:UNIV CHICAGO PRESS  

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Presentations

  • Tracing and immunohistochemical study of the cortico-nigral projection in the mouse brain

    Haruko Miyazaki, Tetsuya Tatsukawa, Yasuharu Hirai, Fuyuki Karube, Tomohiro Miyasaka, Fumino Fujiyama, Kazuhiro Yamakaw, Nobuyuki Nukina

    The 44th Annual Meeting of the Japan Neuroscience Society  2021.7.31 

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    Event date: 2021.7.28 - 2021.7.31

    Language:English   Presentation type:Poster presentation  

    File: 4P-161_宮﨑.pdf

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  • A novel unmyelinated fiber projection to substantia nigra

    Haruko Miyazaki, Tetsuya Tatsukawa, Kazuhiro Yamakawa, Nobuyuki Nukina

    The 43rd Annual Meeting of the Japan Neuroscience Society  2020.7.31 

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    Event date: 2020.7.29 - 2020.8.1

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  • Mass spectrometry analysis for MBP (+) and MBP (-) membrane fraction from striatonigral fiber enriched region in mouse brain International conference

    Haruko Miyazaki, Nobuyuki Nukina

    NEURO2019  2019.7.25 

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  • Screening specific molecules of unmyelinated neurons in central nervous system International conference

    Haruko Miyazaki, Risa Yamano, Saki Nishioka, Masahito Ikawa, Nobuyuki Nukina

    NEURO2018  2018.7.26 

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  • Gene expression profiling of medium spiny neurons in Huntington’s disease model mouse. International conference

    Haruko Miyazaki, Fumitaka Oyama, Yoshihiro Kino, Masaru Kurosawa, Mizuki Kurosawa, Tomoyuki Yamanaka, Nobutaka Hattori, Tomomi Shimogori, Nobuyuki Nukina

    23th World Congress of Neurology  2017.9.16 

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  • 2. Gene expression profiling of medium spiny neurons in Huntington’s disease model mice International conference

    Haruko Miyazaki, Fumitaka Oyama, Yoshihiro Kino, Masaru Kurosawa, Mizuki Kurosawa, Kenji Ohtawa, Nobutaka Hattori, Tomomi Shimogori, Nobuyuki Nukina

    NEURO2016  2016.7.20 

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  • 1. 遺伝性神経疾患の病態研究から分かってきたこと

    宮崎 晴子

    麻布大学 特別講義  2016.5.19 

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    Language:Japanese   Presentation type:Oral presentation (invited, special)  

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  • 3. Singular localization of sodium channel β4 subunit in unmyelinated fibres in the striatum International conference

    Haruko Miyazaki

    NEURO2015  2015.7.28 

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  • 4. Transcriptional profiling of FACS-purified striatal medium spiny neurons in early pathological phase of Huntington disease model mice International conference

    Haruko Miyazaki

    NEURO2014  2014.9.11 

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  • 5. Correlation between nuclear accumulation and dysregulation of sodium channel beta4 subunit in HD transgenic mice. International conference

    Haruko Miyazaki

    39th Society for Neuroscience  2009.10.17 

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  • 6. BACE1 cleavage mediates neurite morphology induced by sodium channel ß4 subunit International conference

    Haruko Miyazaki

    NEURO2007  2007.9.10 

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  • 7. BACE1によるナトリウムチャネル ß4 サブユニットの神経突起伸長活性の制御

    宮崎 晴子

    第12回病態と治療におけるプロテアーゼとインヒビター学会  2007.8 

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  • 8. Sodium channel ß4 subunit: downregulation and possible involvement of neuritic degeneration in Huntington Disease transgenic mice International conference

    Haruko Miyazaki

    NEURO2005  2005.7.26 

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  • マウス嗅覚神経系発達過程における細胞接着分子BIG-1およびBIG-2の発現解析

    Haruko Miyazaki

    2000.9.4 

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Awards

  • 大藤内分泌医学賞

    2021.8   令和3年度 大藤内分泌医学賞   ハンチントン病モデルマウスにおける神経ペプチド遺伝子群発現低下メカニズムの解析

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  • 学術奨励賞

    2007.8   病態と治療におけるプロテアーゼとインヒビター学会  

    宮崎 晴子

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Research Projects

  • 自閉症関連蛋白質Nav1.2の翻訳制御機構の解明

    2021.12 - 2022.12

    公益財団法人岡山医学振興会  公益財団法人岡山医学振興会 第21回公募助成 

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  • Investigation of Nav1.2 distribution in the unmyelinated fibers of central nervous system.

    2020.10 - 2021.10

    The Ichiro Kanehara Foundation for the promotion of Medical Science and Medical Care  第35回基礎医学医療研究助成金 

    Haruko Miyazaki

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  • 中枢無髄神経特異的に分布する蛋白質の探索

    2018.10 - 2021.05

    公益財団法人 武田科学振興財団  ライフサイエンス研究助成 

    宮崎 晴子

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  • 髄鞘化における有髄・無髄の決定にかかわるメカニズムの解析

    2018.04 - 2019.03

    同志社大学赤ちゃん学研究センター  計画共同研究  計画共同研究

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  • 中枢神経系無髄神経の包括的分子機能解析

    Grant number:17H01564  2017.04 - 2022.03

    日本学術振興会  科学研究費助成事業 基盤研究(A)  基盤研究(A)

    貫名 信行, 山川 和弘, 宮崎 晴子

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    Grant amount:\41860000 ( Direct expense: \32200000 、 Indirect expense:\9660000 )

    中枢神経系の無髄神経の分布に関してNav1.2をマーカーにして検討を行っている。脳梁・分界条の線維に無髄が存在することを免疫電顕を含めて確認した。現在その起始細胞を同定しようと検討している。
    多系統萎縮症において線条体のNav1.2の染色性の脱落を見出した。この起始核である中型有棘細胞の脱落との関係を検討中である。無髄関連タンパク質と考えているGolliについてはconditional knock outマウス作成のためのFloxマウスの作製に成功した。現在その確認を行っている。Nav1.2のconditional knockoutマウスについては線条体、小脳顆粒細胞におけるknockoutを、作製中である。
    無髄神経関連タンパク質のプロテオーム解析は有髄線維の混入のため、難航している。線条体中型有棘細胞を蛍光ラベルしたマウスの使用なども考慮中である。ハンチントン病中型有棘細胞のtranscriptome解析を行い、データを投稿中である。この投射線維は無髄であり、プロテオーム解析と合わせて、解析を進められる可能性が出てきた。
    線条体の回路は皮質線条体とサーキットを作っているが、皮質線条体がStxbp1やScn2aのヘテロ欠損マウスにおけるてんかんの発症に関与していることを示した(Miyamoto et al Nat Commun2019,Tatsukawa et al Mol Autism, Ogiwara et al Commun Biol2018)。

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  • Distribution and characteristic of unmyelinated fibers in the central nervous system

    Grant number:16K07005  2016.04 - 2020.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    Haruko Miyazaki

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    Grant amount:\4940000 ( Direct expense: \3800000 、 Indirect expense:\1140000 )

    In the central nerve system, not all axons are myelinated. A large proportion of axons are unmyelinated but their distribution and function still unclear. Here, we identified parts of fibers in the corpus callosum and stria terminalis as novel unmyelinated fibers by immunostaining using anti-Nav1.2, which is a marker of unmyelinated fibers. To obtain the information relating to unmyelinated neurons/fibers, we generated gene expression profile of striatal medium spiny neurons, which are unmyelinated neurons. Next, we performed proteome analysis using MBP (+) and MBP (-) membrane fraction of striatal projection fibers and compared enriched proteins in each fraction. Gene ontology analysis of these data suggested that unmyelinated fibers may interact with glial cells on the surface of fibers and form synapses with other projection fibers. Indeed, we found the striatonigral fibers were surrounded by astrocyte processes by immunostaining using astrocyte markers.

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  • Long ncRNA expression abnormality in polyglutmine diseases

    Grant number:25253066  2013.04 - 2016.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)  Grant-in-Aid for Scientific Research (A)

    Nukina Nobuyuki, Miyazaki Haruko

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    Grant amount:\45110000 ( Direct expense: \34700000 、 Indirect expense:\10410000 )

    To reveal the pathological significance of the gene expression abnormality in Huntington disease(HD), we developed the method to identify the gene expression changes in specific neuronal fraction such as medium spiny neurons(MSN) in striatum. We isolated MSN using cell sorter from control and HD model mice with Venus expression in MSN, then compared their gene expressions with microarray. We identified many genes, which showed expression changes, those include previously reported and unreported ones.Those could be confirmed by RT-PCR if the expression levels are above the certain level. We also identified the expression abnormality of long non-coding RNA, however, many of them showed low expression level. Thus, new methods for confirmation is necessary.

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  • The development and analysis of selective neuronal pathology

    Grant number:22110004  2010.04 - 2015.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)  Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

    NUKINA Nobuyuki, MIYAZAKI Haruko, YAMANAKA Tomoyuki, MATSUMOTO Gen

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    Grant amount:\111670000 ( Direct expense: \85900000 、 Indirect expense:\25770000 )

    To elucidate the mechaism of selective neuronal degeneration of Huntington disease, we analyzed aggregate interacting proteins including FUS/TLS,NF-YA,and p62 using knockout mice and showed their in vivo functions.Investigating sodium channel beta4 subunit using knockout mouse, we demonstrated the resurgent current is regulated by beta4 in medium spiny neurons(MSNs) and during this study, we found the MSNs have unmyelinated fibers, which was first clearly demonstrated in this study.We established the methods to analyze the gene expressions of selected neurons by FACS and found new genes which changed in HD MSNs.

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