研究者業績

八幡 直樹

ヤハタ ナオキ  (Naoki Yahata)

基本情報

所属
藤田医科大学 医学部 解剖学I 講師
学位
博士(理学)(大阪大学)

研究者番号
60450607
J-GLOBAL ID
200901060668151915
researchmap会員ID
5000050395

受賞

 1

論文

 14
  • Naoki Yahata, Hiroko Boda, Ryuji Hata
    Molecular therapy. Methods & clinical development 20 54-68 2021年3月12日  
    Various mitochondrial diseases, including mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), are associated with heteroplasmic mutations in mitochondrial DNA (mtDNA). Herein, we refined a previously generated G13513A mtDNA-targeted platinum transcription activator-like effector nuclease (G13513A-mpTALEN) to more efficiently manipulate mtDNA heteroplasmy in MELAS-induced pluripotent stem cells (iPSCs). Introduction of a nonconventional TALE array at position 6 in the mpTALEN monomer, which recognizes the sequence around the m.13513G>A position, improved the mpTALEN effect on the heteroplasmic shift. Furthermore, the reduced expression of the new Lv-mpTALEN(PKLB)/R-mpTALEN(PKR6C) pair by modifying codons in their expression vectors could suppress the reduction in the mtDNA copy number, which contributed to the rapid recovery of mtDNA in mpTALEN-applied iPSCs during subsequent culturing. Moreover, MELAS-iPSCs with a high proportion of G13513A mutant mtDNA showed unusual properties of spontaneous, embryoid body-mediated differentiation in vitro, which was relieved by decreasing the heteroplasmy level with G13513A-mpTALEN. Additionally, drug-inducible, myogenic differentiation 1 (MYOD)-transfected MELAS-iPSCs (MyoD-iPSCs) efficiently differentiated into myosin heavy chain-positive myocytes, with or without mutant mtDNA. Hence, heteroplasmic MyoD-iPSCs controlled by fine-tuned mpTALENs may contribute to a detailed analysis of the relationship between mutation load and cellular phenotypes in disease modeling.
  • Tsuneyoshi Seki, Motoi Kanagawa, Kazuhiro Kobayashi, Hisatomo Kowa, Naoki Yahata, Kei Maruyama, Nobuhisa Iwata, Haruhisa Inoue, Tatsushi Toda
    The Journal of biological chemistry 295(11) 3678-3691 2020年3月13日  査読有り
    Alzheimer's disease (AD) is the most common type of dementia, and its pathogenesis is associated with accumulation of β-amyloid (Aβ) peptides. Aβ is produced from amyloid precursor protein (APP) that is sequentially cleaved by β- and γ-secretases. Therefore, APP processing has been a target in therapeutic strategies for managing AD; however, no effective treatment of AD patients is currently available. Here, to identify endogenous factors that modulate Aβ production, we performed a gene microarray-based transcriptome analysis of neuronal cells derived from human induced pluripotent stem cells, because Aβ production in these cells changes during neuronal differentiation. We found that expression of the glycophosphatidylinositol-specific phospholipase D1 (GPLD1) gene is associated with these changes in Aβ production. GPLD1 overexpression in HEK293 cells increased the secretion of galectin 3-binding protein (GAL3BP), which suppressed Aβ production in an AD model, neuroglioma H4 cells. Mechanistically, GAL3BP suppressed Aβ production by directly interacting with APP and thereby inhibiting APP processing by β-secretase. Furthermore, we show that cells take up extracellularly added GAL3BP via endocytosis and that GAL3BP is localized in close proximity to APP in endosomes where amyloidogenic APP processing takes place. Taken together, our results indicate that GAL3BP may be a suitable target of AD-modifying drugs in future therapeutic strategies for managing AD.
  • 関 恒慶, 金川 基, 小林 千浩, 古和 久朋, 八幡 直樹, 丸山 敬, 岩田 修永, 井上 治久, 戸田 達史
    Dementia Japan 33(4) 516-516 2019年10月  査読有り
  • 関 恒慶, 金川 基, 小林 千浩, 古和 久朋, 八幡 直樹, 丸山 敬, 岩田 修永, 井上 治久, 戸田 達史
    日本生化学会大会プログラム・講演要旨集 92回 [2T17m-02] 2019年9月  査読有り
  • Naoki Yahata, Yuji Matsumoto, Minoru Omi, Naoki Yamamoto, Ryuji Hata
    SCIENTIFIC REPORTS 7 15557 2017年11月  査読有り
    Induced pluripotent stem cells (iPSCs) are suitable for studying mitochondrial diseases caused by mitochondrial DNA (mtDNA) mutations. Here, we generated iPSCs from a patient with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) with the m.13513G>A mutation. The patient's dermal fibroblasts were reprogrammed, and we established two iPSC clones with and without mutant mtDNA. Furthermore, we tried to decrease mutant mtDNA level in iPSCs using transcription activator-like effector nucleases (TALENs). We originally engineered platinum TALENs, which were transported into mitochondria, recognized the mtDNA sequence including the m.13513 position, and preferentially cleaved G13513A mutant mtDNA (G13513A-mpTALEN). The m.13513G>A heteroplasmy level in MELAS-iPSCs was decreased in the short term by transduction of G13513A-mpTALEN. Our data demonstrate that this mtDNA-targeted nuclease would be a powerful tool for changing the heteroplasmy level in heteroplasmic iPSCs, which could contribute to elucidation of the pathological mechanisms of mitochondrial diseases caused by mtDNA mutations.
  • Takayuki Kondo, Masashi Asai, Kayoko Tsukita, Yumiko Kutoku, Yutaka Ohsawa, Yoshihide Sunada, Keiko Imamura, Naohiro Egawa, Naoki Yahata, Keisuke Okita, Kazutoshi Takahashi, Isao Asaka, Takashi Aoi, Akira Watanabe, Kaori Watanabe, Chie Kadoya, Rie Nakano, Dai Watanabe, Kei Maruyama, Osamu Hori, Satoshi Hibino, Tominari Choshi, Tatsutoshi Nakahata, Hiroyuki Hioki, Takeshi Kaneko, Motoko Naitoh, Katsuhiro Yoshikawa, Satoko Yamawaki, Shigehiko Suzuki, Ryuji Hata, Shu-ichi Ueno, Tsuneyoshi Seki, Kazuhiro Kobayashi, Tatsushi Toda, Kazuma Murakami, Kazuhiro Irie, William L. Klein, Hiroshi Mori, Takashi Asada, Ryosuke Takahashi, Nobuhisa Iwata, Shinya Yamanaka, Haruhisa Inoue
    CELL STEM CELL 12(4) 487-496 2013年4月  査読有り
    Oligomeric forms of amyloid-beta peptide (A beta) are thought to play a pivotal role in the pathogenesis of Alzheimer's disease (AD), but the mechanism involved is still unclear. Here, we generated induced pluripotent stem cells (iPSCs) from familial and sporadic AD patients and differentiated them into neural cells. A beta oligomers accumulated in iPSC-derived neurons and astrocytes in cells from patients with a familial amyloid precursor protein (APP)-E693 Delta mutation and sporadic AD, leading to endoplasmic reticulum (ER) and oxidative stress. The accumulated A beta oligomers were not proteolytically resistant, and docosahexaenoic acid (DHA) treatment alleviated the stress responses in the AD neural cells. Differential manifestation of ER stress and DHA responsiveness may help explain variable clinical results obtained with the use of DHA treatment and suggests that DHA may in fact be effective for a subset of patients. It also illustrates how patient-specific iPSCs can be useful for analyzing AD pathogenesis and evaluating drugs.
  • Yahata N, Asai M, Kitaoka S, Takahashi K, Asaka I, Hioki H, Kaneko T, Maruyama K, Saido TC, Nakahata T, Asada T, Yamanaka S, Iwata N, Inoue H
    PloS one 6(9) e25788 2011年9月  査読有り
  • Yahata N, Inoue H
    Nihon rinsho. 69(8) 282-285 2011年  査読有り
  • Yahata N, Asai M, Kitaoka S, Takahashi K, Asaka I, Hioki H, Kaneko T, Maruyama K, Saido TC, Nakahata T, Asada T, Yamanaka S, Iwata N, Inoue H
    PLoS ONE 6(9) 2011年  査読有り
  • Naoki Yahata, Harushisa Inoue, Shiho Kitaoka, Kayoko Tsukita, Takayuki Kondo, Naohiro Egawa, Isao Asaka, Kazutoshi Takahashi, Tatsutoshi Nakahata, Shinobu Kawakatsu, Ryosuke Takahashi, Takashi Asada, Shinya Yamanaka
    NEUROSCIENCE RESEARCH 68 E305-E305 2010年  査読有り
  • Naoki Yahata, Shigeki Yuasa, Toshiyuki Araki
    JOURNAL OF NEUROSCIENCE 29(19) 6276-6284 2009年5月  査読有り
    Studies of naturally occurring mutant mice, wld(s), showing delayed Wallerian degeneration phenotype, suggest that axonal degeneration is an active process. We previously showed that increased nicotinamide adenine dinucleotide (NAD)-synthesizing activity by overexpression of nicotinamide mononucleotide adenylyltransferase (NMNAT) is the essential component of the Wld(s) protein, the expression of which is responsible for the delayed Wallerian degeneration phenotype in wld(s) mice. Indeed, NMNAT overexpression in cultured neurons provides robust protection to neurites, as well. To examine the effect of NMNAT overexpression in vivo and to analyze the mechanism that causes axonal protection, we generated transgenic mice (Tg) overexpressing NMNAT1 (nuclear isoform), NMNAT3 (mitochondrial isoform), or the Wld(s) protein bearing a W258A mutation, which disrupts NAD-synthesizing activity of the Wld(s) protein. Wallerian degeneration delay in NMNAT3-Tg was similar to that in wld(s) mice, whereas axonal protection in NMNAT1-Tg or Wld(s)(W258A)-Tg was not detectable. Detailed analysis of subcellular localization of the overexpressed proteins revealed that the axonal protection phenotype was correlated with localization of NMNAT enzymatic activity to mitochondrial matrix. Furthermore, we found that isolated mitochondria from mice showing axonal protection expressed unchanged levels of respiratory chain components, but were capable of increased ATP production. These results suggest that axonal protection by NMNAT expression in neurons is provided by modifying mitochondrial function. Alteration of mitochondrial function may constitute a novel tool for axonal protection, as well as a possible treatment of diseases involving axonopathy.
  • Y Takayama, Y Kobayashi, N Yahata, T Saitoh, H Hori, T Ikegami, H Akutsu
    BIOCHEMISTRY 45(10) 3163-3169 2006年3月  査読有り
    Carbon monoxide (CO) has been identified as another bioactive molecule like NO. Binding of CO to a tetraheme cytochiome c(3) (cyt c(3)) was investigated using visible absorption spectroscopy, circular dichroism (CD), and NMR. CO was found to bind to the four hemes in different manners. CD spectra, however, indicated that only single-site CO binding can keep the protein intact. The K-d for the single-site binding was 8.0 mu M, which is a typical value for a CO sensor protein. Furthermore, NMR spectra of uniformly N-15-labeled and specifically [N-15]His-labeled proteins have provided evidence that CO specifically binds to the sixth coordination site of heme 2 via single-site binding. The CO-bound cyt c(3) could conduct redox reactions. In light of triheme cytochrome c(7), the CO-bound cyt c(3) may work as an electron transporter. It was reported for sulfate-reducing bacteria that CO can be used as an energy source and CO cycling is operating like H-2 cycling. Therefore, the CO-bound cyt c(3) may play a role in maintaining electron transport pathways on accumulation of toxic CO for its utilization.
  • N Yahata, T Saitoh, Y Takayama, K Ozawa, H Ogata, Y Higuchi, H Akutsu
    BIOCHEMISTRY 45(6) 1653-1662 2006年2月  査読有り
    Cytochrome (C3) isolated from a sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki F, is a tetraheme protein. Its physiological partner, [NiFe] hydrogenase, catalyzes the reversible oxidoreduction of molecular hydrogen. To elucidate the mechanism of electron transfer between cytochrome C3 and [NiFe] hydrogenase, the transient complex formation by these proteins was investigated by means of NMR. All NH signals of uniformly N-15-labeled ferric cytochrome C3 except N-terminus, Pro, and Gly73 were assigned. H-1-N-15 HSQC spectra were recorded for 15N-labeled ferric and ferrous cytochrome C3, in the absence and presence of hydrogenase. Chemical shift perturbations were observed in the region around heme 4 in both oxidation states. Additionally, the region between hemes I and 3 in ferrous cytochrome C3 was affected in the presence of hydrogenase, suggesting that the mode of interaction is different in each redox state. Heme 3 is probably the electron gate for ferrous cytochrome C3. To investigate the transient complex of cytochrome C3 and hydrogenase in detail, modeling of the complex was performed for the oxidized proteins using a docking program, ZDOCK 2.3, and NMR data. Furthermore, the roles of lysine residues of cytochrome C3 in the interaction with hydrogenase were investigated by site-directed mutagenesis. When the lysine residues around heme 4 were replaced by an uncharged residue, methionine, one by one, the, K-m, of the electron-transfer kinetics increased. The results showed that the positive charges of Lys60, Lys72, Lys95, and Lys101 around heme 4 are important for formation of the transient complex with [NiFe] hydrogenase in the initial stage of the cytochrome C3 reduction. This finding is consistent with the most possible structure of the transient complex obtained by modeling.
  • Yahata, N, Ozawa, K, Tomimoto. Y, Morita, K, Komori, H, Ogata, H, Higuchi, Y, Akutsu, H
    BIOPHYSICS 2 45-56 2006年  査読有り
    Complicated pH-properties of the tetraheme cytochrome c3 (cyt c3) from Desulfovibrio vulgaris Miyazaki F (DvMF) were examined by the pH titrations of 1H-15N HSQC spectra in the ferric and ferrous states. The redox-linked pKa shift for the propionate group at C13 of heme 1 was observed as the changes of the NH signals around it. This pKa shift is consistent with the redox-linked conformational alteration responsible for the cooperative reduction between hemes 1 and 2. On the other hand, large chemical shift changes caused by the protonation/deprotonation of Glu41 and/or Asp42, and His67 were redox-independent. Nevertheless, these charged residues affect the redox properties of the four hemes. Furthermore, one of interesting charged residues, Glu41, was studied by site-directed mutagenesis. E41K mutation increased the microscopic redox potentials of heme 1 by 46 and 34 mV, and heme 2 by 35 and 30 mV at the first and last reduction steps, respectively. Although global folding in the crystal structure of E41K cyt c3 is similar to that of wild type, local change was observed in 1H NMR spectrum. Glu41 is important to keep the stable conformation in the region between hemes 1 and 2, controlling the redox properties of DvMF cyt c3. In contrast, the kinetic parameters for electron transfer from DvMF [NiFe] hydrogenase were not influenced by E41K mutation. This suggests that the region between hemes 1 and 2 is not involved in the interaction with [NiFe] hydrogenase, and it supports the idea that heme 4 is the exclusive entrance gate to accept the electron in the initial reduction stage.<br>

MISC

 21

書籍等出版物

 2

講演・口頭発表等

 34

担当経験のある科目(授業)

 4

共同研究・競争的資金等の研究課題

 9

産業財産権

 1

その他

 2
  • 特になし
  • 変異ミトコンドリアDNAを標的としたTALEN(変異ミトコンドリアDNAの割合を改変する部位特的変異ヌクレアーゼ。Yahata et al. Sci Rep. 7:15557, 2017; Mol Ther Methods Clin Dev. 20:54-68, 2021) *本研究シーズに関する産学共同研究の問い合わせは藤田医科大学産学連携推進センター(fuji-san@fujita-hu.ac.jp)まで