2014年08月, 日本細胞外小胞学会, 奨励賞　　　　　　　　　　　　　　　
神経細胞由来エクソソームのアミロイドß蛋白質除去効果

2013年04月, International Society for Extracellular Vesicles (ISEV), Young Investigator Award　　　　　　　　　　　　　　　

2012年10月, 第５回セラミド研究会学術集会, Young Investigator Award　　　　　　　　　　　　　　　

Odor identification score as an alternative method for early identification of amyloidogenesis in Alzheimer's disease.
Yukifusa Igeta, Isao Hemmi, Kohei Yuyama, Yasuyoshi Ouchi
Scientific reports, 14, 1, 4658, 4658, 2024年02月26日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A simple screening test to identify the early stages of Alzheimer's disease (AD) is urgently needed. We investigated whether odor identification impairment can be used to differentiate between stages of the A/T/N classification (amyloid,  tau, neurodegeneration) in individuals with amnestic mild cognitive impairment or AD and in healthy controls. We collected data from 132 Japanese participants visiting the Toranomon Hospital dementia outpatient clinic. The odor identification scores correlated significantly with major neuropsychological scores, regardless of apolipoprotein E4 status, and with effective cerebrospinal fluid (CSF) biomarkers [amyloid β 42 (Aβ42) and the Aβ42/40 and phosphorylated Tau (p-Tau)/Aβ42 ratios] but not with ineffective biomarkers [Aβ40 and the p-Tau/total Tau ratio]. A weak positive correlation was observed between the corrected odor identification score (adjusted for age, sex, ApoE4 and MMSE), CSF Aβ42, and the Aβ42/40 ratio. The odor identification score demonstrated excellent discriminative power for the amyloidogenesis stage , according to the A/T/N classification, but was unsuitable for differentiating between the p-Tau accumulation and the neurodegeneration stages. After twelve odor species were analyzed, a version of the score comprising only four odors-India ink, wood, curry, and sweaty socks-proved highly effective in identifying AD amyloidogenesis, showing promise for the screening of preclinical AD.

Extracellular vesicle proteome unveils cathepsin B connection to Alzheimer’s disease pathogenesis
Kohei Yuyama, Hui Sun, Risa Fujii, Isao Hemmi, Koji Ueda, Yukifusa Igeta
Brain, Oxford University Press (OUP), 2023年12月10日, ［査読有り］, ［筆頭著者］
研究論文（学術雑誌）, Abstract

Extracellular vesicles (EVs) are membrane vesicles that are released extracellularly and considered to be implicated in the pathogenesis of neurodegenerative diseases including Alzheimer’s disease. Here, CSF EVs of 16 ATN-classified cases were subjected to quantitative proteome analysis. In these CSF EVs, levels of 11 proteins were significantly altered during the ATN stage transitions (P < 0.05 and fold-change > 2.0). These proteins were thought to be associated with Alzheimer’s disease pathogenesis and represent candidate biomarkers for pathogenic stage classification. Enzyme-linked immunosorbent assay analysis of CSF and plasma EVs revealed altered levels of cathepsin B (CatB) during the ATN transition (seven ATN groups in validation set, n = 136). The CSF and plasma EV CatB levels showed a negative correlation with CSF amyloid-β42 concentrations. This proteomic landscape of CSF EVs in ATN classifications can depict the molecular framework of Alzheimer’s disease progression, and CatB may be considered a promising candidate biomarker and therapeutic target in Alzheimer’s disease amyloid pathology.

Stereochemistry‐activity relationship of ceramide‐induced exosome production to clear amyloid‐β in Alzheimer's disease
Mariam Abdelrasoul, Kohei Yuyama, Mahadeva M. M. Swamy, Yuta Murai, Kenji Monde
Chirality, 35, 9, 577, 585, Wiley, 2023年04月13日, ［査読有り］
研究論文（学術雑誌）, Abstract

Stereochemistry has a substantial impact on the biological activity of various drugs. We investigated the role of stereochemistry of ceramides in inducing the production of exosomes, a type of extracellular vesicle, from neuronal cells, with a potential benefit in improving the clearance of amyloid‐β (Aβ), a causal agent of Alzheimer's disease. A stereochemical library of diverse ceramides with different tail lengths was synthesized with the purpose of varying stereochemistry (D‐erythro: DE, D‐threo: DT, L‐erythro: LE, L‐threo: LT) and hydrophobic tail length (C6, C16, C18, C24). The exosome levels were quantified using TIM4‐based exosome enzyme‐linked immunosorbent assay after concentrating the conditioned medium using centrifugal filter devices. The results revealed a pivotal role of stereochemistry in determining the biological activity of ceramide stereoisomers, with the superiority of those based on DE and DT stereochemistry with C16 and C18 tails, which demonstrated significantly higher exosome production, without a significant change in the particle size of the released exosomes. In transwell experiments with Aβ‐expressed neuronal and microglial cells, DE‐ and DT‐ceramides with C16 and C18 tails significantly decreased extracellular Aβ levels. The results reported here are promising in the design of non‐classic therapies for the treatment of Alzheimer's disease.

Immuno-digital invasive cleavage assay for analyzing Alzheimer's amyloid ß-bound extracellular vesicles.
Kohei Yuyama, Hui Sun, Yasuyuki Igarashi, Kenji Monde, Takumi Hirase, Masato Nakayama, Yoichi Makino
Alzheimer's Research & Therapy, 14, 1, 140, 140, 2022年10月03日, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: The protracted preclinical stage of Alzheimer's disease (AD) provides the opportunity for early intervention to prevent the disease; however, the lack of minimally invasive and easily detectable biomarkers and their measurement technologies remain unresolved. Extracellular vesicles (EVs) are nanosized membrane vesicles released from a variety of cells and play important roles in cell-cell communication. Neuron-derived and ganglioside-enriched EVs capture amyloid-ß protein, a major AD agent, and transport it into glial cells for degradation; this suggests that EVs influence Aß accumulation in the brain. EV heterogeneity, however, requires the use of a highly sensitive technique for measuring specific EVs in biofluid. In this study, immuno-digital invasive cleavage assay (idICA) was developed for quantitating target-intact EVs. METHODS: EVs were captured onto ganglioside GM1-specific cholera toxin B subunit (CTB)-conjugated magnetic beads and detected with a DNA oligonucleotide-labeled Aß antibody. Fluorescence signals for individual EVs were then counted using an invasive cleavage assay (ICA). This idICA examines the Aß-bound and GM1-containing EVs isolated from the culture supernatant of human APP-overexpressing N2a (APP-N2a) cells and APP transgenic mice sera. RESULTS: The idICA quantitatively detected Aß-bound and GM1-containing EVs isolated from culture supernatants of APP-N2a cells and sera of AD model mice. The idICA levels of Aß-associated EVs in blood gradually increased from 3- to 12-month-old mice, corresponding to the progression of Aß accumulations in the brain of AD model mice. CONCLUSIONS: The present findings suggest that peripheral EVs harboring Aß and GM1 reflect Aß burden in mice. The idICA is a valuable tool for easy quantitative detection of EVs as an accessible biomarker for preclinical AD diagnosis.

Evaluation of Plant Ceramide Species-Induced Exosome Release from Neuronal Cells and Exosome Loading Using Deuterium Chemistry
Yuta Murai, Takumi Honda, Kohei Yuyama, Daisuke Mikami, Koichi Eguchi, Yuichi Ukawa, Seigo Usuki, Yasuyuki Igarashi, Kenji Monde
International Journal of Molecular Sciences, 23, 18, 10751, 10751, MDPI AG, 2022年09月15日, ［査読有り］, ［責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, The extracellular accumulation of aggregated amyloid-β (Aβ) in the brain leads to the early pathology of Alzheimer’s disease (AD). The administration of exogenous plant-type ceramides into AD model mice can promote the release of neuronal exosomes, a subtype of extracellular vesicles, that can mediate Aβ clearance. In vitro studies showed that the length of fatty acids in mammalian-type ceramides is crucial for promoting neuronal exosome release. Therefore, investigating the structures of plant ceramides is important for evaluating the potential in releasing exosomes to remove Aβ. In this study, we assessed plant ceramide species with D-erythro-(4E,8Z)-sphingadienine and D-erythro-(8Z)-phytosphingenine as sphingoid bases that differ from mammalian-type species. Some plant ceramides were more effective than mammalian ceramides at stimulating exosome release. In addition, using deuterium chemistry-based lipidomics, most exogenous plant ceramides were confirmed to be derived from exosomes. These results suggest that the ceramide-dependent upregulation of exosome release may promote the release of exogenous ceramides from cells, and plant ceramides with long-chain fatty acids can effectively release neuronal exosomes and prevent AD pathology.

Ceramide Metabolism Regulated by Sphingomyelin Synthase 2 Is Associated with Acquisition of Chemoresistance via Exosomes in Human Leukemia Cells.
Makoto Taniguchi, Shingo Nagaya, Kohei Yuyama, Ai Kotani, Yasuyuki Igarashi, Toshiro Okazaki
International journal of molecular sciences, 23, 18, 2022年09月13日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Ceramide levels controlled by the sphingomyelin (SM) cycle have essential roles in cancer cell fate through the regulation of cell proliferation, death, metastasis, and drug resistance. Recent studies suggest that exosomes confer cancer malignancy. However, the relationship between ceramide metabolism and exosome-mediated cancer malignancy is unclear. In this study, we elucidated the role of ceramide metabolism via the SM cycle in exosomes and drug resistance in human leukemia HL-60 and adriamycin-resistant HL-60/ADR cells. HL-60/ADR cells showed significantly increased exosome production and release compared with parental chemosensitive HL-60 cells. In HL-60/ADR cells, increased SM synthase (SMS) activity reduced ceramide levels, although released exosomes exhibited a high ceramide ratio in both HL-60- and HL-60/ADR-derived exosomes. Overexpression of SMS2 but not SMS1 suppressed intracellular ceramide levels and accelerated exosome production and release in HL-60 cells. Notably, HL-60/ADR exosomes conferred cell proliferation and doxorubicin resistance properties to HL-60 cells. Finally, microRNA analysis in HL-60 and HL-60/ADR cells and exosomes showed that miR-484 elevation in HL-60/ADR cells and exosomes was associated with exosome-mediated cell proliferation. This suggests that intracellular ceramide metabolism by SMS2 regulates exosome production and release, leading to acquisition of drug resistance and enhanced cell proliferation in leukemia cells.

Penta-deuterium-labeled 4E, 8Z-sphingadienine for rapid analysis in sphingolipidomics study.
Yuta Murai, Kohei Yuyama, Daisuke Mikami, Yasuyuki Igarashi, Kenji Monde
Chemistry and physics of lipids, 245, 105202, 105202, 2022年03月22日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The use of deuterium-incorporated bioactive compounds is an efficient method for tracing their metabolic fate and for quantitative analysis by mass spectrometry without complicated HPLC separation even if their amounts are extremely small. Plant sphingolipids and their metabolites, which have C4, 8-olefins on a common backbone as a sphingoid base, show unique and fascinating bioactivities compared to those of sphingolipids in mammals. However, the functional and metabolic mechanisms of exogenous plant sphingolipids have not been elucidated due to the difficulty in distinguishing exogenous sphingolipids from endogenous sphingolipids having the same polarity and same molecular weight by mass spectrometric analysis. Their roles might be elucidated by the use of deuterated probes with original biological and physicochemical properties. In this study, we designed (2S,3R,4E,8Z)-2-aminooctadeca-4,8-diene-17,17,18,18,18-d5-1,3-diol (penta-deuterium-labeled 4E, 8Z-sphingadienine) as a tracer for exogenous metabolic studies. In addition, the sphingadienine was confirmed to be metabolized in HEK293 cells and showed distinct peaks in mass spectrometric analysis.

Konjac Ceramide (kCer)-Mediated Signal Transduction of the Sema3A Pathway Promotes HaCaT Keratinocyte Differentiation
Seigo Usuki, Noriko Tamura, Tomohiro Tamura, Kohei Yuyama, Daisuke Mikami, Katsuyuki Mukai, Yasuyuki Igarashi
Biology, 11, 1, 121, 121, MDPI AG, 2022年01月12日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Histamines suppress epidermal keratinocyte differentiation. Previously, we reported that konjac ceramide (kCer) suppresses histamine-stimulated cell migration of HaCaT keratinocytes. kCer specifically binds to Nrp1 and does not interact with histamine receptors. The signaling mechanism of kCer in HaCaT cells is also controlled by an intracellular signaling cascade activated by the Sema3A-Nrp1 pathway. In the present study, we demonstrated that kCer treatment induced HaCaT keratinocyte differentiation after migration of immature cells. kCer-induced HaCaT cell differentiation was accompanied by some features of keratinocyte differentiation markers. kCer induced activating phosphorylation of p38MAPK and c-Fos, which increased the protein levels of involucrin that was the latter differentiation marker. In addition, we demonstrated that the effects of both kCer and histamines are regulated by an intracellular mechanism of Rac1 activation/RhoA inhibition downstream of the Sema3A/Nrp1 receptor and histamine/GPCR pathways. In summary, the effects of kCer on cell migration and cell differentiation are regulated by cascade crosstalk between downstream Nrp1 and histamine-GPCR pathways in HaCaT cells.

Intracerebral Transplantation of Mesenchymal Stromal Cell Compounded with Recombinant Peptide Scaffold against Chronic Intracerebral Hemorrhage Model.
Soichiro Takamiya, Masahito Kawabori, Tsukasa Kitahashi, Kentaro Nakamura, Yuki Mizuno, Hironobu Yasui, Yuji Kuge, Aki Tanimori, Yasuyuki Takamatsu, Kohei Yuyama, Hideo Shichinohe, Miki Fujimura
Stem cells international, 2022, 8521922, 8521922, 2022年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Background: Due to the lack of effective therapies, stem cell transplantation is an anticipated treatment for chronic intracerebral hemorrhage (ICH), and higher cell survival and engraftment are considered to be the key for recovery. Mesenchymal stromal cells (MSCs) compounded with recombinant human collagen type I scaffolds (CellSaics) have a higher potential for cell survival and engraftment compared with solo-MSCs, and we investigated the validity of intracerebral transplantation of CellSaic in a chronic ICH model. Methods: Rat CellSaics (rCellSaics) were produced by rat bone marrow-derived MSC (rBMSCs). The secretion potential of neurotrophic factors and the cell proliferation rate were compared under oxygen-glucose deprivation (OGD) conditions. rCellSaics, rBMSCs, or saline were transplanted into the hollow cavity of a rat chronic ICH model. Functional and histological analyses were evaluated, and single-photon emission computed tomography for benzodiazepine receptors was performed to monitor sequential changes in neuronal integrity. Furthermore, human CellSaics (hCellSaics) were transplanted into a chronic ICH model in immunodeficient rats. Antibodies neutralizing brain-derived neurotrophic factor (BDNF) were used to elucidate its mode of action. Results: rCellSaics demonstrated a higher secretion potential of trophic factors and showed better cell proliferation in the OGD condition. Animals receiving rCellSaics displayed better neurological recovery, higher intracerebral BDNF, and better cell engraftment; they also showed a tendency for less brain atrophy and higher benzodiazepine receptor preservation. hCellSaics also promoted significant functional recovery, which was reversed by BDNF neutralization. Conclusion: Intracerebral transplantation of CellSaics enabled neurological recovery in a chronic ICH model and may be a good option for clinical application.

こんにゃく由来グルコシルセラミド摂取による脳内アミロイドβ蓄積に対する抑制効果の探索的試験 —プラセボ対照ランダム化二重盲検並行群間比較試験—　　　　　　　　　　　　　　　
江口晃一, 向井克之, 湯山耕平, 臼杵靖剛, 栗本成敬, 田中藍子, 勝山(鏡)豊代, 西平 順, 門出健次, 五十嵐 靖之
薬理と治療, 49, 8, 1225, 1239, 2021年08月, ［査読有り］
日本語, 研究論文（学術雑誌）

Comprehensive Glycomic Approach Reveals Novel Low-Molecular-Weight Blood Group-Specific Glycans in Serum and Cerebrospinal Fluid.
Jun-Ichi Furukawa, Hisatoshi Hanamatsu, Ikuko Yokota, Megumi Hirayama, Tomohiro Ando, Hiroyuki Kobayashi, Shunsuke Ohnishi, Nobuaki Miura, Kazue Okada, Shota Sakai, Kohei Yuyama, Yasuyuki Igarashi, Makoto Ito, Yasuro Shinohara, Naoya Sakamoto
Journal of proteome research, 20, 5, 2812, 2822, 2021年03月10日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, ABO blood antigens on the human red blood cell membrane as well as different cells in various human tissues have been thoroughly studied. Anti-A and -B antibodies of IgM are present in serum/plasma, but blood group-specific glyco-antigens have not been extensively described. In this study, we performed comprehensive and quantitative serum glycomic analyses of various glycoconjugates and free oligosaccharides in all blood groups. Our comprehensive glycomic approach revealed that blood group-specific antigens in serum/plasma are predominantly present on glycosphingolipids on lipoproteins rather than glycoproteins. Expression of the ABO antigens on glycosphingolipids depends not only on blood type but also on secretor status. Blood group-specific glycans in serum/plasma were classified as type I, whereas those on RBCs had different structures including hexose and hexosamine residues. Analysis of free oligosaccharides revealed that low-molecular-weight blood group-specific glycans, commonly containing lacto-N-difucotetraose, were expressed in serum/plasma according to blood group. Furthermore, comprehensive glycomic analysis in human cerebrospinal fluid showed that many kinds of free oligosaccharides were highly expressed, and low-molecular-weight blood group-specific glycans, which existed in plasma from the same individuals, were present. Our findings provide the first evidence for low-molecular-weight blood group-specific glycans in both serum/plasma and cerebrospinal fluid.

Structure-dependent absorption of atypical sphingoid long-chain bases from digestive tract into lymph
Daisuke Mikami, Shota Sakai, Megumi Nishimukai, Kohei Yuyama, Katsuyuki Mukai, Yasuyuki Igarashi
Lipids in Health and Disease, 20, 1, Springer Science and Business Media LLC, 2021年03月01日, ［査読有り］
英語, 研究論文（学術雑誌）,

Dietary sphingolipids have various biofunctions, including skin barrier improvement and anti-inflammatory and anti-carcinoma properties. Long-chain bases (LCBs), the essential backbones of sphingolipids, are expected to be important for these bioactivities, and they vary structurally between species. Given these findings, however, the absorption dynamics of each LCB remain unclear.




In this study, five structurally different LCBs were prepared from glucosylceramides (GlcCers) with LCB 18:2(4E,8Z);2OH and LCB 18:2(4E,8E);2OH moieties derived from konjac tuber (Amorphophallus konjac), from GlcCers with an LCB 18(9Me):2(4E,8E);2OH moiety derived from Tamogi mushroom (Pleurotus cornucopiae var. citrinopileatus), and from ceramide 2-aminoethyphosphonate with LCB 18:3(4E,8E,10E);2OH moiety and LCB 18(9Me):3(4E,8E,10E);2OH moiety derived from giant scallop (Mizuhopecten yessoensis), and their absorption percentages and metabolite levels were analyzed using a lymph-duct-cannulated rat model via liquid chromatography tandem mass spectrometry (LC/MS/MS) with a multistage fragmentation method.




The five orally administered LCBs were absorbed and detected in chyle (lipid-containing lymph) as LCBs and several metabolites including ceramides, hexosylceramides, and sphingomyelins. The absorption percentages of LCBs were 0.10–1.17%, depending on their structure. The absorption percentage of LCB 18:2(4E,8Z);2OH was the highest (1.17%), whereas that of LCB 18:3(4E,8E,10E);2OH was the lowest (0.10%). The amount of sphingomyelin with an LCB 18:2(4E,8Z);2OH moiety in chyle was particularly higher than sphingomyelins with other LCB moieties.




Structural differences among LCBs, particularly geometric isomerism at the C8–C9 position, significantly affected the absorption percentages and ratio of metabolites. This is the first report to elucidate that the absorption and metabolism of sphingolipids are dependent on their LCB structure. These results could be used to develop functional foods that are more readily absorbed.

Mevalonate pathway-mediated ER homeostasis is required for haploid stability in human somatic cells.
Kan Yaguchi, Kimino Sato, Koya Yoshizawa, Daisuke Mikami, Kohei Yuyama, Yasuyuki Igarashi, Gabor Banhegyi, Eva Margittai, Ryota Uehara
Cell structure and function, 2020年12月22日, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, The somatic haploidy is unstable in diplontic animals, but cellular processes determining haploid stability remain elusive. Here, we found that inhibition of mevalonate pathway by pitavastatin, a widely used cholesterol-lowering drug, drastically destabilized the haploid state in HAP1 cells. Interestingly, cholesterol supplementation did not restore haploid stability in pitavastatin-treated cells, and cholesterol inhibitor U18666A did not phenocopy haploid destabilization. These results ruled out the involvement of cholesterol in haploid stability. Besides cholesterol perturbation, pitavastatin induced endoplasmic reticulum (ER) stress, the suppression of which by a chemical chaperon significantly restored haploid stability in pitavastatin-treated cells. Our data demonstrate the involvement of the mevalonate pathway in the stability of the haploid state in human somatic cells through managing ER stress, highlighting a novel link between ploidy and ER homeostatic control. Key words: Haploid, ER stress, Mevalonate, pathway.

Lysosomal-associated transmembrane protein 4B regulates ceramide-induced exosome release.
Kohei Yuyama, Hui Sun, Daisuke Mikami, Tetsuo Mioka, Katsuyuki Mukai, Yasuyuki Igarashi
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2020年10月08日, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Exosomes are extracellular vesicles that mediate the transport of intracellular molecules, including neurodegenerative agents. Exogenously administrated ceramides have been implicated in the acceleration of exosome production by neurons; however, the molecular machinery involved in this process is unknown. Here, we found that ceramides, especially those consisting of long fatty acids, were internalized into the endocytic pathway in neuroblastoma SH-SY5Y cells to induce exosome secretion through lysosome-associated protein transmembrane 4B (LAPTM4B). Knockdown of LAPTM4B inhibited the ceramide-mediated increase in exosome release completely. Fluorescence microscopy observations indicated that exogenous ceramides promote the transport of multivesicular bodies to the plasma membranes in a LAPTM4B-dependent manner. Similarly, inhibition of acid ceramidase, which tends to induce intracellular ceramide accumulation, increased exosome production by SH-SY5Y cells in a LAPTM4B-dependent manner. Furthermore, the level of amyloid-ß protein (Aß) was decreased in neuronal cells following treatment with exogenous ceramide or inhibition of acid ceramidase, and this effect was attributed to the LAPTM4B-dependent efflux of Aß-containing exosomes. Overall, these findings reveal the novel machinery involved in exosome secretion regulated by ceramides and LAPTM4B, and may contribute to efforts to ameliorate the cellular accumulation of neurodegenerative agents such as Aß.

Glucocerebrosidases catalyze a transgalactosylation reaction that yields a newly-identified brain sterol metabolite, galactosylated cholesterol.
Hisako Akiyama, Mitsuko Ide, Yasuko Nagatsuka, Tomoko Sayano, Etsuro Nakanishi, Norihito Uemura, Kohei Yuyama, Yoshiki Yamaguchi, Hiroyuki Kamiguchi, Ryosuke Takahashi, Johannes M F G Aerts, Peter Greimel, Yoshio Hirabayashi
The Journal of biological chemistry, 295, 16, 5257, 5277, 2020年04月17日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, β-Glucocerebrosidase (GBA) hydrolyzes glucosylceramide (GlcCer) to generate ceramide. Previously, we demonstrated that lysosomal GBA1 and nonlysosomal GBA2 possess not only GlcCer hydrolase activity, but also transglucosylation activity to transfer the glucose residue from GlcCer to cholesterol to form β-cholesterylglucoside (β-GlcChol) in vitro β-GlcChol is a member of sterylglycosides present in diverse species. How GBA1 and GBA2 mediate β-GlcChol metabolism in the brain is unknown. Here, we purified and characterized sterylglycosides from rodent and fish brains. Although glucose is thought to be the sole carbohydrate component of sterylglycosides in vertebrates, structural analysis of rat brain sterylglycosides revealed the presence of galactosylated cholesterol (β-GalChol), in addition to β-GlcChol. Analyses of brain tissues from GBA2-deficient mice and GBA1- and/or GBA2-deficient Japanese rice fish (Oryzias latipes) revealed that GBA1 and GBA2 are responsible for β-GlcChol degradation and formation, respectively, and that both GBA1 and GBA2 are responsible for β-GalChol formation. Liquid chromatography-tandem MS revealed that β-GlcChol and β-GalChol are present throughout development from embryo to adult in the mouse brain. We found that β-GalChol expression depends on galactosylceramide (GalCer), and developmental onset of β-GalChol biosynthesis appeared to be during myelination. We also found that β-GlcChol and β-GalChol are secreted from neurons and glial cells in association with exosomes. In vitro enzyme assays confirmed that GBA1 and GBA2 have transgalactosylation activity to transfer the galactose residue from GalCer to cholesterol to form β-GalChol. This is the first report of the existence of β-GalChol in vertebrates and how β-GlcChol and β-GalChol are formed in the brain.

Nrp1 is Activated by Konjac Ceramide Binding-Induced Structural Rigidification of the a1a2 Domain.
Usuki S, Yasutake Y, Tamura N, Tamura T, Tanji K, Saitoh T, Murai Y, Mikami D, Yuyama K, Monde K, Mukai K, Igarashi Y.
Cells, 9, 2, 2020年02月24日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Konjac ceramide (kCer) is a plant-type ceramide composed of various long-chain bases and a-hydroxyl fatty acids. The presence of d4t,8t-sphingadienine is essential for semaphorin 3A (Sema3A)-like activity. Herein, we examined the three neuropilin 1 (Nrp1) domains (a1a2, b1b2, or c), and found that a1a2 binds to d4t,8t-kCer and possesses Sema3A-like activity. kCer binds to Nrp1 with a weak affinity of mM dissociation constant (Kd). We wondered whether bovine serum albumin could influence the ligand-receptor interaction that a1a2 has with a single high affinity binding site for kCer (Kd in nM range). In the present study we demonstrated the influence of bovine serum albumin. Thermal denaturation indicates that the a1a2 domain may include intrinsically disordered region (IDR)-like flexibility. A potential interaction site on the a1 module was explored by molecular docking, which revealed a possible Nrp1 activation mechanism, in which kCer binds to Site A close to the Sema3A-binding region of the a1a2 domain. The a1 module then accesses a2 as the IDR-like flexibility becomes ordered via kCer-induced protein rigidity of a1a2. This induces intramolecular interaction between a1 and a2 through a slight change in protein secondary structure.

Blood-brain barrier permeability analysis of plant ceramides.
Koichi Eguchi, Daisuke Mikami, Hui Sun, Takuya Tsumita, Kaori Takahashi, Katsuyuki Mukai, Kohei Yuyama, Yasuyuki Igarashi
PloS one, 15, 11, e0241640, 2020年, ［査読有り］, ［責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Ceramides, a type of sphingolipid, are cell membrane components and lipid mediators that modulate a variety of cell functions. In plants, ceramides are mostly present in a glucosylated glucosylceramide (GlcCer) form. We previously showed that oral administration of konjac-derived GlcCer to a mouse model of Alzheimer's disease reduced brain amyloid-β and amyloid plaques. Dietary plant GlcCer compounds are absorbed as ceramides, but it is unclear whether they can cross the blood-brain barrier (BBB). Herein, we evaluated the BBB permeability of synthetic plant-type ceramides (4, 8-sphingadienine, d18:2) using mouse and BBB cell culture models, and found that they could permeate the BBB both in vivo and in vitro. In addition, administrated ceramides were partially metabolized to other sphingolipid species, namely sphingomyelin (SM) and GlcCer, while crossing the BBB. Thus, plant ceramides can cross the BBB, suggesting that ceramides and their metabolites might affect brain functions.

Plant sphingolipids promote extracellular vesicle release and alleviate amyloid-β pathologies in a mouse model of Alzheimer's disease.
Yuyama K, Takahashi K, Usuki S, Mikami D, Sun H, Hanamatsu H, Furukawa J, Mukai K, Igarashi Y
Scientific reports, 9, 1, 16827, 16827, 2019年11月14日, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, The accumulation of amyloid-β protein (Aβ) in brain is linked to the early pathogenesis of Alzheimer's disease (AD). We previously reported that neuron-derived exosomes promote Aβ clearance in the brains of amyloid precursor protein transgenic mice and that exosome production is modulated by ceramide metabolism. Here, we demonstrate that plant ceramides derived from Amorphophallus konjac, as well as animal-derived ceramides, enhanced production of extracellular vesicles (EVs) in neuronal cultures. Oral administration of plant glucosylceramide (GlcCer) to APP overexpressing mice markedly reduced Aβ levels and plaque burdens and improved cognition in a Y-maze learning task. Moreover, there were substantial increases in the neuronal marker NCAM-1, L1CAM, and Aβ in EVs isolated from serum and brain tissues of the GlcCer-treated AD model mice. Our data showing that plant ceramides prevent Aβ accumulation by promoting EVs-dependent Aβ clearance in vitro and in vivo provide evidence for a protective role of plant ceramides in AD. Plant ceramides might thus be used as functional food materials to ameliorate AD pathology.

Malabaricone C as Natural Sphingomyelin Synthase Inhibitor against Diet-Induced Obesity and Its Lipid Metabolism in Mice.
Muhamad Aqmal Othman, Kohei Yuyama, Yuta Murai, Yasuyuki Igarashi, Daisuke Mikami, Yasodha Sivasothy, Khalijah Awang, Kenji Monde
ACS medicinal chemistry letters, 10, 8, 1154, 1158, 2019年08月08日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The interaction between natural occurring inhibitors and targeted membrane proteins could be an alternative medicinal strategy for the treatment of metabolic syndrome, notably, obesity. In this study, we identified malabaricones A-C and E (1-4) isolated from the fruits of Myristica cinnamomea King as natural inhibitors for sphingomyelin synthase (SMS), a membrane protein responsible for sphingolipid biosynthesis. Having the most promising inhibition, oral administration of compound 3 exhibited multiple efficacies in reducing weight gain, improving glucose tolerance, and reducing hepatic steatosis in high fat diet-induced obesity mice models. Liver lipid analysis revealed a crucial link between the SMS activities of compound 3 and its lipid metabolism in vitro and in vivo. The nontoxic nature of compound 3 makes it a suitable candidate in search of drugs which can be employed in the treatment and prevention of obesity.

Isolation of Sphingoid Bases from Starfish Asterias amurensis Glucosylceramides and Their Effects on Sphingolipid Production in Cultured Keratinocytes　　　　　　　　　　　　　　　
Daisuke Mikami, Shota Sakai, Kohei Yuyama, Yasuyuki Igarashi
Journal of Oleo Science, 68, 5, 427, 441, 2019年, ［査読有り］
英語, 研究論文（学術雑誌）

Extracellular vesicles from amnion-derived mesenchymal stem cells ameliorate hepatic inflammation and fibrosis in rats
Ohara M, Ohnishi S, Hosono H, Yamamoto K, Yuyama K, Nakamura H, Fu Q, Maehara O, Suda G, Sakamoto N.
Stem Cells International, 2018, 3212643, 3212643, 2018年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Background: There are no approved drug treatments for liver fibrosis and nonalcoholic steatohepatitis (NASH), an advanced stage of fibrosis which has rapidly become a major cause of cirrhosis. Therefore, development of anti-inflammatory and antifibrotic therapies is desired. Mesenchymal stem cell- (MSC-) based therapy, which has been extensively investigated in regenerative medicine for various organs, can reportedly achieve therapeutic effect in NASH via paracrine action. Extracellular vesicles (EVs) encompass a variety of vesicles released by cells that fulfill functions similar to those of MSCs. We herein investigated the therapeutic effects of EVs from amnion-derived MSCs (AMSCs) in rats with NASH and liver fibrosis. Methods: NASH was induced by a 4-week high-fat diet (HFD), and liver fibrosis was induced by intraperitoneal injection of 2 mL/kg 50% carbon tetrachloride (CCl4) twice a week for six weeks. AMSC-EVs were intravenously injected at weeks 3 and 4 in rats with NASH (15 μg/kg) and at week 3 in rats with liver fibrosis (20 μg/kg). The extent of inflammation and fibrosis was evaluated with quantitative reverse transcription polymerase chain reaction and immunohistochemistry. The effect of AMSC-EVs on inflammatory and fibrogenic response was investigated in vitro. Results: AMSC-EVs significantly decreased the number of Kupffer cells (KCs) in the liver of rats with NASH and the mRNA expression levels of inflammatory cytokines such as tumor necrosis factor- (Tnf-) α, interleukin- (Il-) 1β and Il-6, and transforming growth factor- (Tgf-) β. Furthermore, AMSC-EVs significantly decreased fiber accumulation, KC number, and hepatic stellate cell (HSC) activation in rats with liver fibrosis. In vitro, AMSC-EVs significantly inhibited KC and HSC activation and suppressed the lipopolysaccharide (LPS)/toll-like receptor 4 (TLR4) signaling pathway. Conclusions: AMSC-EVs ameliorated inflammation and fibrogenesis in a rat model of NASH and liver fibrosis, potentially by attenuating HSC and KC activation. AMSC-EV administration should be considered as a new therapeutic strategy for chronic liver disease.

Multiple roles of Sms2 in white and brown adipose tissues from diet-induced obese mice　　　　　　　　　　　　　　　
Hisatoshi Hanamatsu, Susumu Mitsutake, Shota Sakai, Toshiro Okazaki, Ken Watanabe, Yasuyuki Igarashi, Kohei Yuyama
J Metabolic Syndrome, 2018年, ［査読有り］, ［最終著者, 責任著者］
英語, 研究論文（学術雑誌）

Konjac ceramide (kCer) regulates NGF-induced neurite outgrowth via the sema3A signaling pathway
Seigo Usuki, Noriko Tamura, Kohei Yuyama, Tomohiro Tamura, Katsuyuki Mukai, Yasuyuki Igarashi
Journal of Oleo Science, 67, 1, 77, 86, Japan Oil Chemists Society, 2018年, ［査読有り］
英語, 研究論文（学術雑誌）, The tuber of the konjac plant is a source enriched with GlcCer (kGlcCer), and has been used as a dietary supplement to improve the dry skin and itching that are caused by a deficiency of epidermal ceramide. Previously, we showed chemoenzymatically prepared konjac ceramide has a neurite-outgrowth inhibitory effect that is very similar to that of Sema3A and is not seen with animal-type ceramides. While, it has been unclear whether kCer may act on Sema3A or TrkA signaling pathway. In the present study, we showed kCer induces phosphorylation of CRMP2 and microtubules depolymerization via Sema3A signaling pathway not TrkA. It is concluded that kCer may be a potential Sema3A-like agonist that activates Sema3A signaling pathway directly.

Direct Involvement of Arachidonic Acid in the Development of Ear Edema via TRPV3
Takao Sanaki, Erika Kasai-Yamamoto, Takeshi Yoshioka, Shota Sakai, Kohei Yuyama, Takuji Fujiwara, Yoshito Numata, Yasuyuki Igarashi
JOURNAL OF OLEO SCIENCE, 66, 6, 591, 599, JAPAN OIL CHEMISTS SOC, 2017年06月, ［査読有り］
英語, 研究論文（学術雑誌）, Arachidonic acid (AA) plays a pivotal role in the development of edema via its oxidized metabolites derived from cyclooxygenase (COX) and lipoxygenase (LOX), and is recently recognized as an activator of TRPV3. However, it is not clear whether AA plays some TRPV3-mediated pathological roles in the development of edema. Pharmacological and histological studies using ICRTRPV3+/+ and ICRTRPv3-/- mice indicated that higher ear edema responses to topical application of AA were observed in ICRTRPv3+/+ mice compared with ICRTRPv3-/- mice. However, there was no difference in the ear edema response to 12-O-tetradecanoylphorbol 13-acetate, skin histology, and skin barrier function between these mouse strains. Furthermore, oxidized fatty acids from the lesional site were analyzed to elucidate the TRPV3-mediated pathological roles of AA, and the results revealed that there were no differences in the level of COX or LOX metabolites derived from AA between both mouse strains. We concluded that AA plays a role in the development of TRPV3-mediated ear edema and that this result may contribute to better understanding of the pathophysiological mechanisms involved in the development of a certain type of edema.

Synthesis of Nontoxic Fluorous Sphingolipids as Molecular Probes of Exogenous Metabolic Studies for Rapid Enrichment by Fluorous Solid Phase Extraction
Shota Saito, Yuta Murai, Seigo Usuki, Masafumi Yoshida, Mostafa A. S. Hammam, Susumu Mitsutake, Kohei Yuyama, Yasuyuki Igarashi, Kenji Monde
EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, 2017, 6, 1045, 1051, WILEY-V C H VERLAG GMBH, 2017年02月, ［査読有り］
英語, 研究論文（学術雑誌）, Fluorous solid-phase extraction (FSPE) is a useful technique for efficient selective enrichment of fluorous compounds from nonfluorous molecules. Sphingolipids and their metabolites, which are ubiquitous building blocks of eukaryotic and prokaryotic cell membranes, play crucial roles, for example, as signaling molecules. However, details of the functions and metabolic mechanisms of exogenous sphingolipids have remained unknown compared with those of their endogenous analogs. To better understand these unknown roles, chemical probes with appropriate biological and physicochemical properties are needed. In this study, we designed and synthesized new fluorous sphingolipids to reveal these roles. Furthermore, we confirmed that they could be efficiently and rapidly separated from normal sphingolipids by FSPE, and that they hardly showed any cytotoxic activity, similarly to normal sphingolipids at the same dose. We also showed that these fluorinated ceramides could act as metabolic substrates for sphingomyelin synthase 2 (SMS2). This demonstrates their potential for further biological studies.

A potential function for neuronal exosomes: sequestering intracerebral amyloid-ß peptide
Yuyama K, Sun H, Usuki S, Sakai S, Hanamatsu H, Mioka T, Kimura N, Okada M, Tahara H, Furukawa J, Fujitani N, Shinohara Y, Igarashi Y
FEBS Lett., 589, 1, 84, 88, WILEY, 2015年01月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, Elevated amyloid-beta peptide (A beta) in brain contributes to Alzheimer's disease (AD) pathogenesis. We demonstrated the presence of exosome-associated A beta in the cerebrospinal fluid (CSF) of cynomolgus monkeys and APP transgenic mice. The levels of exosome-associated A beta notably decreased in the CSF of aging animals. We also determined that neuronal exosomes, but not glial exosomes, had abundant glycosphingolipids and could capture A beta. Infusion of neuronal exosomes into brains of APP transgenic mice decreased A beta and amyloid depositions, similarly to what reported previously on neuroblastoma-derived exosomes. These findings highlight the role of neuronal exosomes in A beta clearance, and suggest that their downregulation might relate to Ab accumulation and, ultimately, the development of AD pathology. (C) 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Altered levels of serum sphingomyelin and ceramide containing distinct acyl chains in young obese adults
H. Hanamatsu, S. Ohnishi, S. Sakai, K. Yuyama, S. Mitsutake, H. Takeda, S. Hashino, Y. Igarashi
NUTRITION & DIABETES, 4, e141, NATURE PUBLISHING GROUP, 2014年10月, ［査読有り］
英語, 研究論文（学術雑誌）, OBJECTIVE: Recent studies indicate that sphingolipids, sphingomyelin (SM) and ceramide (Cer) are associated with the development of metabolic syndrome. However, detailed profiles of serum sphingolipids in the pathogenesis of this syndrome are lacking. Here we have investigated the relationship between the molecular species of sphingolipids in serum and the clinical features of metabolic syndrome, such as obesity, insulin resistance, fatty liver disease and atherogenic dyslipidemia.
SUBJECTS: We collected serum from obese (body mass index, BMI >= 35, n = 12) and control (BMI = 20 - 22, n = 11) volunteers (18 - 27 years old), measured the levels of molecular species of SM and Cer in the serum by liquid chromatography-mass spectrometry and analyzed the parameters for insulin resistance, liver function and lipid metabolism by biochemical blood test.
RESULTS: The SM C18:0 and C24:0 levels were higher, and the C20:0 and C22:0 levels tended to be higher in the obese group than in the control group. SM C18:0, C20:0, C22:0 and C24:0 significantly correlated with the parameters for obesity, insulin resistance, liver function and lipid metabolism, respectively. In addition, some Cer species tended to correlate with these parameters. However, SM species containing unsaturated acyl chains and most of the Cer species were not associated with these parameters.
CONCLUSIONS: The present results demonstrate that the high levels of serum SM species with distinct saturated acyl chains (C18:0, C20:0, C22:0 and C24:0) closely correlate with the parameters of obesity, insulin resistance, liver function and lipid metabolism, suggesting that these SM species are associated with the development of metabolic syndrome and serve as novel biomarkers of metabolic syndrome and its associated diseases.

Decreased Amyloid-β Pathologies by Intracerebral Loading of Glycosphingolipid-enriched Exosomes in Alzheimer Model Mice
Yuyama K, Sun H, Sakai S, Mitsutake S, Okada M, Tahara H, Furukawa JI, Fujitani N, Shinohara Y, Igarashi Y
J. Biol. Chem, 289, 35, 24488, 24498, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2014年08月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, Background: Exosome, a type of extracellular vesicles, can associate with A in vitro. Results: Intracerebrally injected exosomes trapped A on surface glycosphingolipids and transported it into microglia in AD mouse brains, resulting in reductions in A pathology. Conclusion: Exogenous exosomes act as potent scavengers for A in mouse brains. Significance: The findings provide a novel therapeutic approach for AD.Elevated levels of amyloid- peptide (A) in the human brain are linked to the pathogenesis of Alzheimer disease. Recent in vitro studies have demonstrated that extracellular A can bind to exosomes, which are cell-secreted nanovesicles with lipid membranes that are known to transport their cargos intercellularly. Such findings suggest that the exosomes are involved in A metabolism in brain. Here, we found that neuroblastoma-derived exosomes exogenously injected into mouse brains trapped A and with the associated A were internalized into brain-resident phagocyte microglia. Accordingly, continuous intracerebral administration of the exosomes into amyloid- precursor protein transgenic mice resulted in marked reductions in A levels, amyloid depositions, and A-mediated synaptotoxicity in the hippocampus. In addition, we determined that glycosphingolipids (GSLs), a group of membrane glycolipids, are highly abundant in the exosomes, and the enriched glycans of the GSLs are essential for A binding and assembly on the exosomes both in vitro and in vivo. Our data demonstrate that intracerebrally administered exosomes can act as potent scavengers for A by carrying it on the exosome surface GSLs and suggest a role of exosomes in A clearance in the central nervous system. Improving A clearance by exosome administration would provide a novel therapeutic intervention for Alzheimer disease.

Involvement of gangliosides in the process of Cbp/PAG phosphorylation by Lyn in developing cerebellar growth cones
Naoko Sekino-Suzuki, Kohei Yuyama, Toshiaki Miki, Mizuho Kaneda, Hidenori Suzuki, Naomasa Yamamoto, Tadashi Yamamoto, Chitose Oneyama, Masato Okada, Kohji Kasahara
JOURNAL OF NEUROCHEMISTRY, 124, 4, 514, 522, WILEY-BLACKWELL, 2013年02月, ［査読有り］
英語, 研究論文（学術雑誌）, The association of gangliosides with specific proteins in the central nervous system was examined by coimmunoprecipitation with an anti-ganglioside antibody. The monoclonal antibody to the ganglioside GD3 (R24) immunoprecipitated the Csk (C-terminal src kinase)-binding protein (Cbp). Sucrose density gradient analysis showed that Cbp of rat cerebellum was detected in detergent-resistant membrane (DRM) raft fractions. R24 treatment of the rat primary cerebellar cultures induced Lyn activation and tyrosine phosphorylation of Cbp. Treatment with anti-ganglioside GD1b antibody also induced tyrosine phosphorylation. Furthermore, over-expressions of Lyn and Cbp in Chinese hamster ovary (CHO) cells resulted in tyrosine 314 phosphorylation of Cbp, which indicates that Cbp is a substrate for Lyn. Immunoblotting analysis showed that the active form of Lyn and the Tyr314-phosphorylated form of Cbp were highly accumulated in the DRM raft fraction prepared from the developing cerebellum compared with the DRM raft fraction of the adult one. In addition, Lyn and the Tyr314-phosphorylated Cbp were highly concentrated in the growth cone fraction prepared from the developing cerebellum. Immunoelectron microscopy showed that Cbp and GAP-43, a growth cone marker, are localized in the same vesicles of the growth cone fraction. These results suggest that Cbp functionally associates with gangliosides on growth cone rafts in developing cerebella.

Cooperative Synthesis of Ultra Long-Chain Fatty Acid and Ceramide during Keratinocyte Differentiation.
Yukiko Mizutani, Hui Sun, Yusuke Ohno, Takayuki Sassa, Takeshi Wakashima, Mari Obara, Kohei Yuyama, Akio Kihara, Yasuyuki Igarashi
PloS one, 8, 6, e67317, 2013年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The lipid lamellae in the stratum corneum is important for the epidermal permeability barrier. The lipid lamellae component ceramide (CER), comprising an ultra long-chain (ULC) fatty acid (FA) of ≥26 carbons (ULC CER), plays an essential role in barrier formation. ULC acyl-CoAs, produced by the FA elongase ELOVL4, are converted to ULC CERs by the CER synthase CERS3. In the presented study, we observed that ELOVL4 and CERS3 mRNAs increased during keratinocyte differentiation in vivo and in vitro. We also determined that peroxisome proliferator-activated receptor β/δ is involved in the up-regulation of the mRNAs. Knockdown of CERS3 caused a reduction in the elongase activities toward ULC acyl-CoAs, suggesting that CERS3 positively regulates ULCFA. Thus, we reveal that the two key players in ULC CER production in epidermis, CERS3 and ELOVL4, are coordinately regulated at both the transcriptional and enzymatic levels.

Sphingolipid-modulated exosome secretion promotes clearance of amyloid-β by microglia.
Yuyama K, Sun H, Mitsutake S, Igarashi Y
The Journal of biological chemistry, 287, 14, 10977, 10989, 14, 2012年03月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, Background: Exosome is a membrane vesicle released from several types of cells, including neurons.
Results: Neuronal exosomes accelerate A beta fibril formation, and the exosome-associated A beta is taken into microglia to degrade it.
Conclusion: Exosomes promote A beta clearance.
Significance: These findings provide a new function of exosome in the brain and also suggest its involvement in the development of Alzheimer disease.

Ganglioside GD3 monoclonal antibody-induced paxillin tyrosine phosphorylation and filamentous actin assembly in cerebellar growth cones
Kohei Yuyama, Naoko Sekino-Suzuki, Naomasa Yamamoto, Kohji Kasahara
JOURNAL OF NEUROCHEMISTRY, 116, 5, 845, 850, WILEY-BLACKWELL, 2011年03月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, We have demonstrated that antibody to ganglioside GD3 (R24) immunoprecipitates src-family tyrosine kinase Lyn from primary cerebellar granule cells and R24 treatment of the intact cells induces Lyn activation and rapid tyrosine phosphorylation of several substrates, suggesting the functional association of ganglioside GD3 with Lyn. In this study, R24 treatment of primary cerebellar granule cells enhances phosphorylation of paxillin at tyrosine residue 118 and induces filamentous actin assembly and neurite outgrowth. R24 treatment of cerebellar growth cone membrane fraction induces prominent tyrosine phosphorylation of 68 kDa protein which comigrates with phosphopaxillin at tyrosine residue 118. Tyrosine phosphorylation of paxillin is known to regulate actin cytoskeleton-dependent changes in cell morphology. Signal transduction by ganglioside GD3 is involved in growth cone morphology via tyrosine phosphorylation of paxillin.

Sphingomyelin accumulation provides a favorable milieu for GM1 ganglioside-induced assembly of amyloid beta-protein
Kohei Yuyama, Katsuhiko Yanagisawa
NEUROSCIENCE LETTERS, 481, 3, 168, 172, ELSEVIER IRELAND LTD, 2010年09月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, The assembly of amyloid beta-protein into fibrils is an initial event of Alzheimer's disease (AD). Previous studies suggest that ganglioside-bound amyloid beta-protein (A beta), GA beta, is an endogenous seed for amyloid in Alzheimer's disease (AD) brain and that GA beta is generated in the membrane microdomains, comprising cholesterol, sphingomyelin (SM) and GM1 ganglioside. In this study, we showed that the GA beta-dependent amyloidogenesis was accelerated on the surface of PC12 cells that had been pretreated with a sphingomyelinase inhibitor. Conversely, the enhanced GAP-dependent amyloidogenesis under the endocytic dysfunction, which is one of the cell-pathological features of AD, was suppressed by pretreatment with a SM synthase inhibitor. These suggest that Sm is one of the key molecules for GA beta generation and further imply that the interaction of A beta with membrane lipids is critical in amyloid fibrillization in the brain. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Gangliosides determine the amyloid pathology of Alzheimer's disease
Naoto Oikawa, Haruyasu Yamaguchi, Koichi Ogino, Takao Taki, Kohei Yuyama, Naoki Yamamoto, Ryong-Woon Shin, Koichi Furukawa, Katsuhiko Yanagisawa
NEUROREPORT, 20, 12, 1043, 1046, LIPPINCOTT WILLIAMS & WILKINS, 2009年08月, ［査読有り］
英語, 研究論文（学術雑誌）, Gangliosides, GM3 and GM1, are suggested to accelerate the deposition of the amyloid beta-protein as amyloid angiopathy and senile plaques, respectively, in the Alzheimer brain. We investigated the profile of amyloid deposition in the brains of transgenic mice expressing a mutant amyloid precursor protein with a disrupted GM2 synthase gene, in which GM3 accumulates whereas GM1 is lacking. These mice showed a significantly increased level of deposited amyloid beta-protein in the vascular tissues. Furthermore, formation of severe dyshoric-form amyloid angiopathy, in which amyloid extended from the blood vessel walls deeply into the surrounding parenchyma was observed. Our results indicate that the expression of gangliosides is a critical determinant for the amyloid pathology in the Alzheimer brain. NeuroReport 20:1043-1046 (C) 2009 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

Late endocytic dysfunction as a putative cause of amyloid fibril formation in Alzheimer's disease
Kohei Yuyama, Katsuhiko Yanagisawa
JOURNAL OF NEUROCHEMISTRY, 109, 5, 1250, 1260, WILEY-BLACKWELL PUBLISHING, INC, 2009年06月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, The assembly of amyloid beta-protein to amyloid fibrils is a critical event in Alzheimer's disease. Evidence exists that endocytic pathway abnormalities, including the enlargement of early endosomes, precede the extraneuronal amyloid fibril deposition in the brain. We determined whether endocytic dysfunction potently promotes the assembly of amyloid beta-protein on the surface of cultured cells. Blocking the early endocytic pathway by clathrin suppression, inactivation of small GTPases, removal of membrane cholesterol, and Rab5 knockdown did not result in amyloid fibril formation on the cell surface from exogenously added soluble amyloid beta-protein. In contrast, blocking the late endocytic pathway by Rab7 suppression markedly induced the amyloid fibril formation in addition to the enlargement of early endosomes. Notably, a monoclonal antibody specific to GM1-ganglioside-bound amyloid beta-protein, an endogenous seed for Alzheimer amyloid, completely blocks the amyloid fibril formation. Our results suggest that late but not early endocytic dysfunction contributes to the amyloid fibril formation by facilitating the generation of amyloid seed in the Alzheimer's brain.

Accelerated release of exosome-associated GM1 ganglioside (GM1) by endocytic pathway abnormality: another putative pathway for GM1-induced amyloid fibril formation
Kohei Yuyama, Naoki Yamamoto, Katsuhiko Yanagisawa
JOURNAL OF NEUROCHEMISTRY, 105, 1, 217, 224, BLACKWELL PUBLISHING, 2008年04月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, Exosomes are extracellularly released small vesicles that are derived from multivesicular bodies formed via the endocytic pathway. We treated pheochromocytoma PC12 cells with chloroquine, an acidotropic agent, which potently perturbs membrane trafficking from endosomes to lysosomes. Chloroquine treatment increased the level of GM1 ganglioside in cell media only when the cells were exposed to KCl for depolarization, which is known to enhance exosome release from neurons. In the sucrose-density-gradient fractionation of cell media, GM1 ganglioside was exclusively recovered with Alix, a specific marker of exosomes, in the fractions with the density corrresponding to that of exosomes. Notably, amyloid-beta assembly was markedly accelerated when incubated with the exosome fraction prepared from the culture media of PC12 cells treated with chloroquine and KCl. Furthermore, amyloid-beta assembly was significantly suppressed by the co-incubation with an antibody specific to GM1-bound amyloid-beta, an endogenous seed for amyloid formation of Alzheimer's disease. Together with our previous finding that chloroquine treatment induces the accumulation of GM1 ganglioside in early endosomes, results of this study suggest that endocytic pathway abnormality accelerates the release of exosome-associated GM1 ganglioside following its accumulation in early endosomes. Furthermore, this study also suggests that extracellular amyloid fibril formation is induced by not only GM1 gangliosides accumulated on the surface of the cells but also those released in association with exosomes.

Translocation of activated heterotrimeric G protein G alpha(o) to ganglioside-enriched detergent-resistant membrane rafts in developing cerebellum
Kohei Yuyama, Naoko Sekino-Suzuki, Yutaka Sanai, Kohji Kasahara
JOURNAL OF BIOLOGICAL CHEMISTRY, 282, 36, 26392, 26400, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2007年09月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, The association of gangliosides with specific proteins in the central nervous system was examined by co-immunoprecipitation with an anti-ganglioside antibody. The monoclonal antibody to the ganglioside GD3 immunoprecipitated phosphoproteins of 40, 53, 56, and 80 kDa from the rat cerebellum. Of these proteins, the 40-kDa protein was identified as the alpha-subunit of a heterotrimeric G protein, Go ( G alpha(o)). Using sucrose density gradient analysis of cerebellar membranes, G alpha(o), but not G beta gamma, was observed in detergent-resistant membrane (DRM) raft fractions in which GD3 was abundant after the addition of guanosine 5 '-O-( thiotriphosphate) (GTP gamma S), which stabilizes Go in its active form. On the other hand, both G alpha(o) and G beta gamma were excluded from the DRM raft fractions in the presence of guanyl-5 '-yl thiophosphate, which stabilizes Go in its inactive form. Only G alpha(o) was observed in the DRM fractions from the cerebellum on postnatal day 7, but not from that in adult. After pertussis toxin treatment, G alpha(o) was not observed in the DRM fractions, even from the cerebellum on postnatal day 7. These results indicate the activation-dependent translocation of G alpha(o) into the DRM rafts. Furthermore, G alpha o was concentrated in the neuronal growth cones. Treatment with stromal cell-derived factor-1 alpha, a physiological ligand for the G protein-coupled receptor, stimulated [S-35] GTP gamma S binding to G alpha(o) and caused G alpha(o) translocation to the DRM fractions and RhoA translocation to the membrane fraction, leading to the growth cone collapse of cerebellar granule neurons. The collapse was partly prevented by pretreatment with the cholesterol-sequestering and raftdisrupting agent methyl- beta-cyclodextrin. These results demonstrate the involvement of signal-dependent G alpha(o) translocation to the DRM in the growth cone behavior of cerebellar granule neurons.

Permeability of water through a raft model membrane clarified by time-resolved SANS and SAXS
Mitsuhiro Hirai, Teruaki Onai, Masaharu Koizumi, Harutaka Hirai, Kohji Kasahara, Naoko Suzuki, Kohei Yuyama, Katsuaki Inoue
JOURNAL OF APPLIED CRYSTALLOGRAPHY, 40, S159, S164, WILEY-BLACKWELL PUBLISHING, INC, 2007年04月, ［査読有り］
英語, 研究論文（学術雑誌）, We have characterized the structures of ganglioside (G(D3)) micelle and G(D3)/ cholesterol mixtures, and have determined the permeability of water across a membrane of G(D3)/cholesterol/phospholipid uni-lamellar vesicle. We have found that monovalent Na(+) and K(+) cations affect the permeability of water differently. We have found also that the permeability of water is greatly enhanced by the K(+) ion, suggesting the function of the G(D3)/cholesterol rich clusters on neuronal excitations by K(+). The complimentary use of small-angle X-ray and neutron scattering techniques is useful to determine the permeability of water across a vesicle membrane.

Chloroquine-induced endocytic pathway abnormalities: Cellular model of GM1 ganglioside-induced A beta fibrillogenesis in Alzheimer's disease
Kohei Yuyama, Naoki Yamamoto, Katsuhiko Yanagisawa
FEBS LETTERS, 580, 30, 6972, 6976, ELSEVIER SCIENCE BV, 2006年12月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, Endocytic pathway abnormalities were previously observed in brains affected with Alzheimer's disease (AD). To clarify the pathological relevance of these abnormalities to assembly of amyloid beta-protein (A beta), we treated PC12 cells with chloroquine, which potently perturbs membrane trafficking from endosomes to lysosomes. Chloroquine treatment induced accumulation of GM1 ganglioside (GM1) in Rab5-positive enlarged early endosomes and on the cell surface. Notably, an increase in GM1 level on the cell surface was sufficient to induce A beta assembly. Our results suggest that endocytic pathway abnormalities in AD brain induce GM1 accumulation on the cell surface, leading to amyloid fibril formation in brain. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Structure of raft-model membrane by using the inverse contrast variation neutron scattering method
Mitsuhiro Hirai, Harutaka Hirai, Masaharu Koizumi, Kohji Kasahara, Kohei Yuyama, Naoko Suzuki
PHYSICA B-CONDENSED MATTER, 385-86, 868, 870, ELSEVIER SCIENCE BV, 2006年11月, ［査読有り］
英語, 研究論文（学術雑誌）, By means of the inverse contrast variation method in small-angle neutron scattering, we have studied the structure of a small unilamellar vesicle (SUV) composed of ganglioside, cholesterol and dipalmitoyl-phosphocholine. The SUV treated has a similar lipid composition as in a plasma membrane with microdomains, so-called rafts. The present results indicate an asymmetric distribution of lipid components within the bilayer of the vesicle, that is, a predominant distribution of ganglioside and cholesterol at the outer leaflet of the vesicle bilayer. The deviation from the linearity in a pseudo-Stuhrmannplot strongly suggests the presence of a large heterogeneity of lipid composition in a bilayer, namely a clustering of ganglioside and cholesterol molecules. This deviation is enhanced by temperature elevation, meaning that ganglioside-cholesterol clusters become larger with holding liquid-ordered (L-o) phase. (c) 2006 Elsevier B.V. All rights reserved.

Low concentrations of nitric oxide (NO) induced cell death in PC12 cells through activation of p38 mitogen-activated protein kinase (p38 MAPK) but not via extracellular signal-regulated kinases (ERK1/2) or c-Jun N-terminal protein kinase (JNK)
T Yamamoto, K Yuyama, H Yamamoto
NEUROSCIENCE LETTERS, 392, 3, 170, 173, ELSEVIER IRELAND LTD, 2006年01月, ［査読有り］
英語, 研究論文（学術雑誌）, Nitric oxide (NO), a highly reactive gaseous molecule, has been previously reported to induce apoptosis-like cell death even at a low concentration in PC12 cells. In this study, we examined NO-induced activation of members of the mitogen-activated protein kinase (MAPK) family, i.e., p38 MAPK, extracellular signal-regulated kinases (ERK1/2), and c-Jun N-terminal protein kinase (INK). Following the exposure of PC12 cells to an NO donor, (+)-(E)-4-ethyl-2-[hydroxyimino]-5-nitro-3-hexenaniide (NOR3; 100 mu M), the phosphorylation level of p38 MAPK increased time dependently from 2 to 6 h, but that of both ERK1/2 and INK did not. Treatment with a p38 MAPK inhibitor SB203580 partially blocked the NOR3-induced cell death. Neither PD98059, U0126 (inhibitors of ERK1/2) nor SP600125 (a specific inhibitor of JNK) treatments had any significant effect on the NOR3-induced cell death. These findings suggest that the activation of a p38 MAPK pathway, but not that of ERK1/2 or INK, plays an essential role in the apoptosis-like cell death induced by low concentrations of NO. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Structures and dynamics of glycosphingolipid-containing lipid mixtures as raft models of plasma membrane
M Hirai, M Koizumi, H Hirai, T Hayakawa, K Yuyama, N Suzuki, K Kasahara
JOURNAL OF PHYSICS-CONDENSED MATTER, 17, 31, S2965, S2977, IOP PUBLISHING LTD, 2005年08月, ［査読有り］
英語, 研究論文（学術雑誌）, The structure and function of mammalian plasma membrane microdomains, so-called rafts, are among the hot topics in cell biology, since it is suggested that these domains are involved in important membrane-associated events, especially ones such as signal transduction, which were frequently seen in physiological and immunological studies. In spite of the accumulation of large amounts of evidence, results on physical properties of the structure and dynamics of membranes such as those in intact cells are less abundant. In this report we treat the structure and dynamics of glycosphingolipid (ganglioside)-cholesterol and glycosphingolipid (ganglioside)-cholesterol-phospholipid mixtures used as models of rafts and plasma membranes. The present results clearly show that the incorporation of cholesterol with ganglioside aggregates is limited to a maximum miscibility of the molar ratio between the ganglioside and cholesterol ranging from similar to 1/1 to 1/3 and that small vesicles with diameters of about 250-300 angstrom form. These molar ratios and sizes agree well with the reported constituent ratio and minimum size for the rafts. In the vesicle systems containing ganglioside, cholesterol, and phospholipid (PC, DSPC, DOPC, POPC), the bending modulus tends to take the smallest value at the molar ratio of [gang] / [chol] / [phospholipid] = 0.1/0.1/1. The present results would strongly support a functional physical property of the raft model: sphingolipids and cholesterol clustering to form rafts that move within the fluid lipid bilayer.

[Lipid microdomains in nervous system].
Sekino-Suzuki N, Yuyama K, Sanai Y, Kasahara K
Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 49, 2397, 2403, 15 Suppl, 2004年11月, ［査読有り］

Caspase-independent cell death by low concentrations of nitric oxide in PC12 cells: Involvement of cytochrome c oxidase inhibition and the production of reactive oxygen species in mitochondria
K Yuyama, H Yamamoto, Nishizaki, I, T Kato, Sora, I, T Yamamoto
JOURNAL OF NEUROSCIENCE RESEARCH, 73, 3, 351, 363, WILEY-BLACKWELL, 2003年08月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, We reported previously that low levels of nitric oxide (NO) induced cell death with properties of apoptosis, including chromatin fragmentation and condensation in undifferentiated PC12 pheochromocytoma cells. The present study demonstrates that cytotoxicity of low concentrations of NO is mediated by inhibition of mitochondrial cytochrome c oxidase and generation of reactive oxygen species (ROS). An NO donor, (+/-)-(E)-4-ethyl-2-[ (E)-hydroxyimino]-5-nitro-3-hexenamide (NOR3) induced cell death even at low concentrations (10-100 muM), whereas peroxynitrite and a peroxynitrite generator, 3-(4-morpholinyl)-sydnonimine (SIN-1), did not have a significant effect on cell viability up to a concentration of 0.5 mM. The NOR3-induced cell death was unaffected by pretreatment with superoxide dismutase (SOD) or its mimetic peroxynitrite scavenger, manganese(III) tetrakis-(benzoic acid)porphyrin chloride (Mn-TBAP), or with uric acid. These findings indicate that peroxynitrite does not contribute to this cell death. Furthermore, neither the release of cytochrome c from mitochondrial membranes, the cleavage of poly-ADP ribose polymerase (PARP), nor the activation of caspase-3-like activities was observed. Inhibitors of PARP, benzamide, and aminobenzamide had no effect on the NOR3-induced cell death. In addition: pretreatment with general or selective caspase inhibitors, benzyloxy-carbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-fmk), N-acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), and benzyloxycarbonyl-Asp-2,6-dichloro-benzoyloxymethylketone (Z-Asp-Ch(2)-DCB) did not prevent NOR3-induced cell death. Taken together, these findings suggest that cell death induced by NOR3 occurs by a caspase-independent mechanism. In contrast, we found an early increase in mitochondrial H(2)O(2) production during NOR3 exposure using the fluorescent dye 2',7'-dichlorofluorescin-diacetate (DCFH-DA) and dihydrorohdamine123 (DHR123), and these events were accompanied by strong inhibition of cytochrome c oxidase activity in the cells. Furthermore, we observed that several antioxidants, such as ascorbate, glutathione (GSH), cysteine, tetrahydrobiopterin, and dithiothreitol (DTT), all effectively prevented the NOR3-induced cell death. NOR3 treatment decreased the level of total intracellular GSH, but did not affect the activities of antioxidant enzymes SOD, GSH-peroxidase (GPX), and catalase. These results suggest that cell death induced at physiologically low concentrations of NO is mediated by ROS production in mitochondria, most likely resulting from the inhibition of cytochrome c oxidase, with ROS acting as an initiator of caspase-independent cell death. (C) 2003 Wiley-Liss, Inc.

Overexpression of V-1 prevents nitric oxide-induced cell death: Involvement of enhanced tetrahydrobiopterin biosynthesis
K Yuyama, H Yamamoto, K Nakamura, Nishizaki, I, T Yamakuni, SY Song, Sora, I, T Nagatsu, T Yamamoto
JOURNAL OF NEUROSCIENCE RESEARCH, 72, 6, 716, 725, WILEY-LISS, 2003年06月, ［査読有り］
英語, 研究論文（学術雑誌）, Previously we reported that the synthesis of catecholamines, dopamine, and noradrenaline was enhanced by overexpression of V-1 protein, a neuronal protein active in the initial stage of development of the rat cerebellum, in the neuronal cell line PC12D, a model of dopamine cells (Yamakuni et al. [1998] J. Biol. Chem. 273:27051-27054). To investigate the physiological role of this protein, we examined the effect of V-1 overexpression on cell toxicity induced by nitric oxide (NO) used at low concentrations. Two clones of PC12D cells overexpressing V-1, transfectants termed V1-46 and V1-69, were significantly more resistant to NOR3 (an NO donor) but not to etoposide (an inhibitor of topoisomerase II)-induced apoptotic cell death than the control cells (termed C-7 and C-9) that had been transfected with the vector alone. The addition of L-DOPA, dopamine, or noradrenaline to the medium did not abolish NOR3-induced cell death in PC12D cells. Moreover, pretreatment of V1-46 and V1-69 cells with L-alpha-methyl-p-tyrosine (alpha-MPT), an inhibitor of tyrosine hydroxylase, to inhibit catecholamine biosynthesis did not affect the resistance to NO toxicity. These results indicate that the catecholamine levels increased by V-1 overexpression did not produce the protection against NOR3-induced toxicity. We further showed that overexpression of V-1 enhanced the synthesis of (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4). In addition, pretreatment with BH4 or with sepiapterin, which is converted to BH4 intracellularly, significantly protected PC12D cells in a dose-dependent manner. The increased BH4 synthesis by V-1 overexpression was dose dependently inhibited by pretreatment with diaminohydroxypyrimidine (DAHP), an inhibitor of GTP-cyclohydrolase 1, which is the rate-limiting enzyme for the biosynthesis of BH4, concomitantly with the loss of protective effect afforded by V-1 overexpression. Furthermore, the addition of BH4 or sepiapterin to DAHP-pretreated V146 and V1-69 cells restored cell vi-ability. Taken together, these results indicate that V1 protein plays an important role in protection against cell death induced by NO at low levels by promoting the synthesis of BH4. Moreover, these findings suggest the up-regulation of V1 expression as a possible therapeutic target for protection against the insult of NO-induced oxidative stress. (C) 2003 Wiley-Liss, Inc.

Resistance of PC12 cells against nitric oxide (NO)-induced toxicity in long-term culture: implication of neuronal NO synthase expression
K Yuyama, H Yamamoto, K Nakamura, T Kato, Sora, I, T Yamamoto
NEUROSCIENCE LETTERS, 309, 3, 169, 172, ELSEVIER SCI IRELAND LTD, 2001年08月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, We have previously reported that a nitric oxide (NO)-donor, (+/-)-(E)-4-ethyl-2-[ (E)-hydroxyimino]-5-nitro-3-hexenamide (NOR3) induced cell death even at a low concentration in undifferentiated PC12 cells. In the present study, we found that PC12 cells which were cultured long-term for over 80 passages acquired resistance to the NOR3-induced cell toxicity. After 24 h exposure to 10-100 muM NOR3, a concentration-dependent cell death was observed in short-term cultured PC12 cells (8-30 passages), but not in long-term cultured cells (over 80 passages). In the cells cultured short-term, the cell death was accompanied by nuclear condensation and fragmentation. We further examined the alterations in total glutathione (GSH) levels, and activities of antioxidant enzymes, superoxide dismutase (SOD) and catalase in the short- and long-term cultured PC12 cells. SOD activity decreased in the long-term cultured cells, while catalase activity did not change. The GSH content significantly increased in the cells cultured long-term. Furthermore, the long-term but not the short-term cultured cells, expressed neuronal NO synthase (nNOS), but neither endothelial nor inducible NOS. These findings suggest that the PC12 cells acquire resistance to the NO-induced toxicity, accompanied by an increase in the GSH level and the expression of nNOS after long-term culture. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

Kinetic characterization of the nitric oxide toxicity for PC12 cells: effect of half-life time of NO release
T Yamamoto, K Yuyama, K Nakamura, T Kato, H Yamamoto
EUROPEAN JOURNAL OF PHARMACOLOGY, 397, 1, 25, 33, ELSEVIER SCIENCE BV, 2000年05月, ［査読有り］
英語, 研究論文（学術雑誌）, We investigated the effects of low concentrations of nitric oxide (NO) on cell viability using NO donors, (+/-)-(E)-4-methyl-2-[ (E)-hydroxyimino]-5-nitro-6-methoxy-3-hexenamide (NOR1), (+/-)-(E)-4-methyl-2-[ (E)- hydroxyimino]-5-nitro-3-hexenamide (NOR2), (+/-)-(E)-4-ethyl-2-[ (E)-hydroxyimino]-5-nitro-3-hexamide (NOR3) and (+/-)-N-[ (E)-4-ethyl-2-[ (Z)-hydroxyiminol-5-nitro-3-hexenamide (NOR4) and (+/-)-N-[ (E)-4-ethyl-2- [ (Z)-hydroxyimino]-5-nitro-3-hexane-1-yl]-3-pyridine (NOR4). The half-life times of the NO release from these four NOR analogs, NOR1, NOR2, NOR3 and NOR4, were determined (6.5, 84, 105 and 340 min, respectively) by using 4,5-diaminofluorescein (DAF-2), a newly developed indicator of NO. Exposure of undifferentiated PC12 cells to low concentrations of NO donors, NOR2 or NOR3 (1-100 mu M), but not NORI nor NOR4, resulted in cell death in a dose- and time-dependent manner, as determined from cell viability assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. After 24 h exposure to 50 mu M NOR2 or NOR3, more than 90% of PC12 cells had died. Furthermore, while the toxic effect of NOR3 was attenuated by replacing the medium at 20 min, 1 or 2 h after drug addition, it was continued by replacing the medium at 3 h or later after drug addition. The cell death was characterized by DNA degradation, nuclear condensation and fragmentation, suggesting apoptosis-like cell death. Pretreatment with an antioxidant ascorbic acid (0.1-0.5 mM) completely prevented the cell death caused by NOR3, while glutathione (0.1-0.2 mM) and cysteine (0.2-0.4 mM) provided partial protection. These findings suggest that the cell toxicity induced by NO at low concentrations strongly depends upon the duration of expose to NO from NO donors, and these toxic effects are effectively prevented by the antioxidant, ascorbic acid. (C) 2000 Elsevier Science B.V. All rights reserved.

Kinetic analysis of nitric oxide (NO) release from several NO-donors using fluorescence dye, diaminofluorescein-2 (DAF-2).
C Tamura, N Tanaka, K Yuyama, T Kato, H Yamamoto, T Yamamoto
FRONTIERS OF THE MECHANISMS OF MEMORY AND DEMENTIA, 1200, 91, 92, ELSEVIER SCIENCE BV, 2000年, ［査読有り］
英語, 研究論文（国際会議プロシーディングス）, We measured the half-life time of the nitric oxide (NO) release from several NO donors, NOR-, NOC- and NONOate-analogs by using 4,5-diaminofluorescein (DAF-2), a newly developed indicator of NO. Furthermore, we determined the toxic effect of NO donors on PC12 cells. Both NOR2 and NOR3 (1 similar to 100 mu M), but not the other NO donors, resulted in cell death in a dose- and time-dependent manner. These results indicate that the cell toxicity induced by NO donors strongly depend upon its half-life time of the NO release.

脳アミロイド蓄積検出のための細胞外小胞バイオマーカー研究　　　　　　　　　　　　　　　
湯山耕平, 孫慧, 門出健次, 平瀬匠, 牧野洋一, Precision Medicine, 7, 8, 663, 667, 2024年07月
日本語, 速報，短報，研究ノート等（学術雑誌）

細胞外小胞のデジタル検出法開発―アルツハイマー病早期検出への応用―　　　　　　　　　　　　　　　
湯山 耕平, 孫 慧, 門出 健次, 平瀬 匠, 牧野 洋一, Medical Science Digest, 49, 432, 435, 2023年08月
記事・総説・解説・論説等（学術雑誌）

Linking glycosphingolipids to Alzheimer's amyloid-ß: extracellular vesicles and functional plant materials.
Kohei Yuyama, Yasuyuki Igarashi, Glycoconjugate journal, 39, 5, 613, 618, 2022年10月, ［国際誌］
Glycosphingolipids (GSLs) are a specialized class of membrane lipids composed of a ceramide and a carbohydrate head group. GSLs are localized in cell membranes and were recently found to be enriched in the membrane of neuron-derived exosomes, which are a type of extracellular vesicle. Our studies demonstrated that exosomal GSLs may be associated with the amyloid-ß (Aß) peptide, a principal agent of Alzheimer's disease (AD), and act to clear Aß by transporting Aß into brain phagocytic microglia. In this review, we summarize and discuss the function of exosomal GSLs in Aß homeostasis in AD pathology. Improvement in Aß clearance is a potent strategy for AD prevention and therapy. Dietary glucosylceramides (GlcCer) isolated from plants are absorbed into the body as various metabolites, including ceramides. Our recent work demonstrated that dietary GlcCer accelerates neuronal exosome production, which facilitates Aß clearance in mice. Furthermore, studies of AD model mice and human clinical trials have found that oral administration of plant-type GlcCer attenuates the Aß burden in the brain. We also introduce the development of plant-type GlcCer as functional food materials to prevent AD., 英語

神経変性疾患におけるスフィンゴ脂質の役割　　　　　　　　　　　　　　　
湯山耕平, 五十嵐靖之, 生化学, 92, 5, 640, 648, 2020年10月, ［査読有り］, ［招待有り］, ［筆頭著者, 責任著者］
日本語, 記事・総説・解説・論説等（学術雑誌）

広がりを見せる食餌性植物セラミドの生体調節機能　　　　　　　　　　　　　　　
湯山耕平, 江口晃一, 五十嵐靖之, バイオサイエンスとインダストリー, 78, 4, 302, 307, 2020年07月, ［招待有り］, ［筆頭著者］
日本語, 記事・総説・解説・論説等（学術雑誌）

こんにゃくセラミドによるエクソソーム産生誘導と認知症予防効果　　　　　　　　　　　　　　　
江口晃一, 向井克之, 湯山耕平, 五十嵐靖之, FOODSTYLE21, 38, 41, 2020年02月, ［招待有り］
日本語, 記事・総説・解説・論説等（学術雑誌）

Exosomes as Carriers of Alzheimer's Amyloid-beta
Kohei Yuyama, Yasuyuki Igarashi, FRONTIERS IN NEUROSCIENCE, 11, 2017年04月
The intracerebral level of the aggregation-prone peptide, amyloid-beta (A beta), is constantly maintained by multiple clearance mechanisms, including several degradation enzymes, and brain efflux. Disruption of the clearance machinery and the resultant A beta accumulation gives rise to neurotoxic assemblies, leading to the pathogenesis of Alzheimer's disease (AD). In addition to the classic mechanisms of A beta clearance, the protein may be processed by secreted vesicles, although this possibility has not been extensively investigated. We showed that neuronal exosomes, a subtype of extracellular nanovesicles, enwrap, or trap A beta and transport it into microglia for degradation. Here, we review A beta sequestration and elimination by exosomes, and discuss how this clearance machinery might contribute to AD pathogenesis and how it might be exploited for effective AD therapy., FRONTIERS MEDIA SA, 英語, 書評論文,書評,文献紹介等

Physiological and pathological roles of exosomes in the nervous system
Kohei Yuyama, Yasuyuki Igarashi, Biomolecular Concepts, 7, 1, 53, 68, 2016年02月01日
Exosomes represent a subtype of extracellular nanovesicles that are generated from the luminal budding of limiting endosomal membranes and subsequent exocytosis. They encapsulate or associate with obsolete molecules to eliminate or to transfer their cargos in intercellular communication. The exosomes are also released and transported between neurons and glia in the nervous system, having a broad impact on nerve development, activation and regeneration. Accumulating evidence suggests that the exosomes are attributed to the pathogenesis of several neurodegenerative diseases such as prion disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, as well as aging, in which the exosomes lack the capacity for cellular self-repair and spread their enclosed pathological agents among neurons. In this article, we review the current proposed functions of exosomes in physiological and pathological processes in the nervous system., Walter de Gruyter GmbH, 英語, 書評論文,書評,文献紹介等

エクソソームによるアミロイドβ蛋白質除去作用
湯山耕平, 五十嵐靖之, 細胞, 48, 1, 44, 47, 2016年01月, ［招待有り］, ［筆頭著者, 責任著者］
ニューサイエンス社, 日本語, 記事・総説・解説・論説等（学術雑誌）

神経細胞由来エクソソームのアミロイドβクリアランス促進機能
湯山耕平, 五十嵐靖之, Dementia Japan, 30, 3, 358, 366, 2016年, ［招待有り］, ［筆頭著者, 責任著者］
日本認知症学会, 日本語, 記事・総説・解説・論説等（学術雑誌）

Pathological roles of ceramide and its metabolites in metabolic syndrome and Alzheimer's disease.
Yuyama K, Mitsutake S, Igarashi Y, Biochimica et biophysica acta, 1841, 5, 793, 798, 2014年05月, ［筆頭著者］
The public health burden of metabolic syndrome (MetS), a multiplex risk factor that arises from insulin resistance accompanying abnormal adipose conditions, and Alzheimer's disease (AD), the most common form of dementia, continues to expand. Current available therapies for these disorders are of limited effectiveness. Recent findings have indicated that alternations in sphingolipid metabolism contribute to the development of these pathologies. Sphingolipids are major constituents of the plasma membrane, where they are known to form several types of microdomains, and are potent regulators for a variety of physiological processes. Many groups, including ours, have demonstrated that membrane sphingolipids, especially ceramide and its metabolites such as ceramide 1-phosphate, have roles in arteriosclerosis, obesity, diabetes, and inflammation associated with MetS. Aberrant sphingolipid profiles have been observed in human AD brains, and accumulated evidence has demonstrated that changes in membrane properties induced by defective sphingolipid metabolism impair generation and degradation of amyloid-beta peptide (A beta), a pathogenic agent of AD. In this review, we summarize current knowledge and pathophysiological implications of the roles of SLs in MetS and AD, to provide insight into the SL metabolic pathways as potential targets for therapy of these diseases. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology. (C) 2013 Elsevier B.V. All rights reserved., ELSEVIER SCIENCE BV, 英語

三量体G蛋白質と脂質ラフト
湯山耕平, 鈴木直子, 笠原浩二, 生体の科学, 60, 3, 181, 186, 2009年, ［招待有り］, ［筆頭著者］
金原一郎記念医学医療振興財団, 日本語, 記事・総説・解説・論説等（学術雑誌）

アルツハイマー病におけるエンドサイトーシス障害　　　　　　　　　　　　　　　
湯山耕平, 柳澤勝彦, Dementia Japan, 23, 1, 47, 54, 2009年, ［招待有り］, ［筆頭著者］
日本語, 記事・総説・解説・論説等（学術雑誌）

脂質ラフトと３量体G蛋白質
湯山耕平, 鈴木直子, 笠原浩二, 蛋白質 核酸 酵素, 53, 1558, 1563, 2008年, ［招待有り］, ［筆頭著者］
12 Suppl, 日本語

Signal transduction of heterotrimeric G proteins in lipid rafts
Kohei Yuyama, Naoko Sekino-Suzuki, Kohji Kasahara, TRENDS IN GLYCOSCIENCE AND GLYCOTECHNOLOGY, 19, 105, 19, 27, 2007年01月, ［招待有り］, ［筆頭著者］
Lipid rafts, glycosphingolipid and cholesterol-rich microdomains of plasma membrane, are implicated in signal transduction because a variety of signaling molecules, such as heterotrimeric G proteins and G protein-coupled receptors, are associated with them. Dynamic assemblies of lipid rafts with these molecules by extracellular events conduct signaling and sequentially regulate certain physical responses. This review focuses on the role of lipid rafts as a spatial compartment enriched in components of heterotrimeric G protein signaling., GAKUSHIN PUBL CO, 英語, 書評論文,書評,文献紹介等

神経細胞接着分子TAG-1の脂質ラフトを介するシグナル伝達　　　　　　　　　　　　　　　
湯山耕平, 鈴木直子, 佐内豊, 笠原浩二, MEMBRANE, 30, 91, 93, 2005年, ［筆頭著者］
日本語, 記事・総説・解説・論説等（学術雑誌）

神経系における脂質マイクロドメイン　　　　　　　　　　　　　　　
鈴木直子, 湯山耕平, 佐内豊, 笠原浩二, 蛋白質 核酸 酵素, 49, 2397, 2403, 2004年, ［招待有り］
日本語, 記事・総説・解説・論説等（学術雑誌）

Lipid rafts in cellular signaling and disease
K Yuyama, N Sekino-Suzuki, Y Sanai, K Kasahara, TRENDS IN GLYCOSCIENCE AND GLYCOTECHNOLOGY, 15, 83, 139, 151, 2003年05月, ［招待有り］, ［筆頭著者］
Lipid rafts are cholesterol and glycosphingolipid-rich subdomains of the plasma membrane. Various molecules including those involved in signal transduction and pathology localize into the microdomain. Dynamic assemblies of lipid rafts with these molecules by intracellular or extracellular events conduct signaling and sequentially regulate certain physical responses. Furthermore, several microorganisms have been found to handle lipid rafts for their infections. This review provides several examples of dynamic raft functions in signaling regulation and in the molecular mechanisms in diseases such as Alzheimer's disease and viral infections., GAKUSHIN PUBL CO, 英語, 書評論文,書評,文献紹介等

セラミド研究の新展開 : 基礎から応用へ
湯山耕平, 五十嵐靖之, 第20章 細胞外小胞の生産・機能に関わるセラミド関連脂質のはたらき
食品化学新聞社, 2019年06月01日, 9784916143358, 337p, 日本語, ［分担執筆］

認知症の早期診断技術と進行抑制/予防薬・機能性食品の開発
湯山耕平、五十嵐靖之, 6章5節 認知症病理形成にかかわるエクソソームの働きとその予防・治療標的可能性
技術情報協会, 2019年04月26日, 9784861047442, 486p, 日本語, ［分担執筆］

認知症の早期診断技術と進行抑制/予防薬・機能性食品の開発
高橋香織, 湯山耕平, 向井克之, 五十嵐靖之, 9章12節 セラミドを利用したアルツハイマー病予防法の研究開発
技術情報協会, 2019年04月, 9784861047442, 486p, 日本語, ［分担執筆］

医療を変えるエクソソーム : 生体機能から疾患メカニズム、臨床応用まで
湯山耕平, 五十嵐靖之, 第４章 神経変性疾患の病態発現にかかわるエクソソームの働き
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