神経保護
Neuroprotection
O3-9-1-1
NecdinによるPGC-1αの安定化を介したニューロン内ミトコンドリア生合成の促進
Necdin promotes mitochondrial biogenesis via PGC-1α stabilization in neurons

○長谷川孝一1, 白石千夏1, 藤原一志郎1, 吉川和明1
○Koichi Hasegawa1, Chinatsu Shiraishi1, Kazushiro Fujiwara1, Kazuaki Yoshikawa1
阪大・蛋白研・神経発生制御1
Lab. of Regulation of Neuronal Development, Inst. for Protein Res., Osaka Univ.1

Postmitotic neurons need to survive against various intrinsic and extrinsic stresses throughout the lifetime of animals. Neurons require a large amount of ATP produced in the mitochondria to maintain their function and survival. Necdin, a mammalian-specific protein, is predominantly expressed in postmitotic neurons and regulates neuronal differentiation and survival. Our previous study has shown that necdin forms a ternary complex with the tumor suppressor protein p53 and the NAD-dependent protein deacetylase Sirt1, and that necdin represses p53-induced apoptosis by enhancing Sirt1-dependent deacetylation of p53 in cortical neurons. In the present study, we examined whether necdin affects mitochondrial biogenesis and function in neurons prepared from the necdin gene-knockout mice. Quantitative real time-PCR analysis demonstrated that mitochondrial biogenesis-related transcription factors, such as Nrf2 and Tfam, and mitochondria-specific DNA levels were decreased in necdin-deficient cortical neurons. FACS analysis using MitoTracker revealed that the number of mitochondria was also decreased in necdin-deficient cortical neurons. Expression of the Nrf2 and Tfam genes is upregulated by the transcriptional coactivator PGC-1α, which plays a pivotal role in energy homeostasis and mitochondrial activity. Thus, we examined the physical and functional interactions between necdin and PGC-1α. Coimmunoprecipitation assay demonstrated that necdin interacted with PGC-1α and markedly increased the PGC-1α protein level. Because PGC-1α is degraded by the ubiquitin-proteasome pathway, we examined whether necdin affects the ubiquitination of PGC-1α. We found that necdin inhibited PGC-1α ubiquitination by interacting with E3-ligase Rnf34. These results suggest that necdin enhances mitochondrial biogenesis in neurons by stabilizing PGC-1α to promote their activity and survival.
O3-9-1-2
水溶性αリポ酸DM-His.Znは,フリーラジカルを除去し組織の過酸化を抑制することにより,虚血-再灌流負荷に対する脳保護作用を示す
Water-soluble α-lipoic acid derivative, DM-His.Zn, scavenges free radicals, inhibits peroxidation, and protects brain tissue from ischemia-reperfusion injury

○徳丸治1, 水谷有輝1,2, 島田真樹1,2, 尾方和枝1, 北野敬明3, 横井功1
○Osamu Tokumaru1, Yuki Mizutani1,2, Masaki Shimada1,2, Kazue Ogata1, Takaaki Kitano3, Isao Yokoi1
大分大・医・神経生理1, 大分大・医・医学科学生2, 大分大・医・医学教育センター3
Dept Neurophysiol, Oita Univ Facult Med, Yufu, Oita1, Med School, Oita Univ Facult Med, Yufu, Oita2, Med Edu Ctr, Oita Univ Facult Med, Yufu, Oita3

Sodium zinc histidine dithiooctanamide (DM-His.Zn) is a newly developed water-soluble α-lipoic acid derivative. Neuprotective effects of DM-His.Zn against ischemia-reperfusion injury were investigated in terms of energy metabolism of rat brain by phosphorous nuclear magnetic resonance spectroscopy (31P-NMR). Brain slices (400 μm-thick) were incubated in a NMR sample tube superfused with standard artificial cerebrospinal fluid (ACSF) with DM-His.Zn (0, 0.01, 0.1, 1 mM) at 27.5°C and inserted into the superconducting magnet of the NMR spectrometer. Brain slices were exposed to ischemia-reperfusion injury (IRI) by halting the perfusion for 1 hr, followed by the reperfusion for 3 hrs. High-energy phosphates in brain slices, phosphocreatine (PCr) and γ-ATP, were serially measured by 31P-NMR. Radical scavenging activity of DM-His.Zn was evaluated by electron spin resonance spectrometry (ESR) by spin trapping method with DMPO or g-CYPMPO. Antioxidant activity of DM-His.Zn on peroxidation in brain homogenate was also evaluated by thiobarbituric acid reactive substances (TBARS) assay. At concentrations of 0.1 and 0.01 mM, but not of 1 mM, DM-His.Zn demonstrated neuroprotective effect against IRI in terms of energy metabolism; PCr levels had significantly better recovery than control (p < 0.05). In ESR study, DM-His.Zn scavenged multiple radicals examined (superoxide anions, ascorbate free radicals, DPPH radicals and methyl radicals) but hydroxyl radicals with EC50's on the order of mM. DM-His.Zn of 1 mM rather increased the production of hydroxyl radicals. Meanwhile, DM-His.Zn dose-dependently inhibited peroxidation of brain homogenate by hydroxyl radicals (EC50 0.7 mM) as well as by carbon-centered radicals (EC50 >1mM). It is speculated that DM-His.Zn might be neuroprotective against IRI in a limited range of concentration through its antioxidant activity attributable to its free radical scavenging activity against multiple free radicals.
O3-9-1-3
血小板由来増殖因子β受容体は虚血脳における脳血液関門の機能に重要である
PDGFR-β is involved in BBB function after cerebral ischemia

○笹原正清1, , 石井陽子1, 山本誠士1, 濱島丈1松島貴子1, 倉茂洋一1
○Masakiyo Sasahara1, Jie Shen1, Yoko Ishii1, Seiji Yamamoto1, Takeru Hamashima1, Chung T Dang1, Takako Matsushima1, Yoichi Kurashige1
富山大学大学院 医学薬学研究部 病態病理学1
Dept Pathology, Univ of Toyama, Toyama, Japan1

Although platelet-derived growth factors (PDGFs) and receptors (PDGFRs) are abundantly expressed in central nervous system, their functions largely remain elusive. We investigated the role of PDGFR-β in tissue responses and functional recovery after photothrombolic middle cerebral artery occlusion (MCAO). In normal adult mouse brain, PDGFR-β was mainly localized in neuron and pericyte/vascular smooth muscle cell (PC/vSMC). Apparent abnormalities were not detected in the mutant mouse in which systemic PDGFR-β gene knockout was postnatally induced (Esr-KO). From 3 to 28 days after MCAO, Esr-KO exhibited the delayed recovery of body weight and behavior, and larger infarction volume than PDGFR-β preserving control mouse (Floxed). In the ischemic lesion, angiogenesis was comparable, but the recruitment of PC/vSMC to the blood vessels was severely decreased in the Esr-KO than in Floxed throughout experiment. Severe brain edema and decrease of tight junction proteins including claudin 5 and occludin were detected in Esr-KO after MCAO. Furthermore, blood vessels in the ischemic lesion of Esr-KO showed increased leakage of infused fluorescent-labeled albumin in laser confocal microscopy, increased endothelial transcytosis of infused horse-radish peroxidase in electron microscopy (EM), and altered tight junction structures of endothelial cells in EM. In another PDGFR-β conditional knockout mouse (Nestin-KO), PDGFR-β was deleted in neuron and glial cell from embryonic day 10.5, but was preserved in PC/vSMC. After MCAO, vascular leakage and infarction volume in Nestin-KO were worse than controls, but partly improved compared with Esr-KO. In summary, it was suggested that the PDGFR-β- dependent PC/vSMC recruitment to newly formed blood vessel was important for the restoration of blood brain barrier function in the ischemic lesion, and for the tissue remodeling after stroke.
O3-9-1-4
kATPチャネルの開放は脳特異的なミトコンドリア蛋白を誘導する
kATP-channel opening up-regulates brain specific mitochondrial protein

○山本清二1, , 小泉慎一郎1, 井原勇人1
○Seiji Yamamoto1, Min Thura1, Shinichiro Koizumi1, Hayato Ihara1
浜松医科大学 メディカルフォトニクス研究センター1, 和歌山県立医科大学・RI実験施設2
Med Photonics Res Ctr, Hamamatsu Univ Sch Med1, RI Center, Wakayama Med Univ, Wakayama, Japan2

Objective: We have previously observed, in rats, that the electrical stimulation of cerebellar fastigial nucleus (FN) induces ischemia tolerance and up-regulates brain specific mitochondrial protein, uncoupling protein 4 (UCP4). Since the kATP-channel opening blocker inhibits the effects induced by FN stimulation, kATP-channel opening plays a key role. In the present study, we tested the hypothesis that kATP-channel opening up-regulates UCP4.
Method:
Neuro2A (N2A) cells, neuroblastoma cell line, were used. UCP4 mRNA in N2A cells was examined by RT-PCR 24 h and 48 h after the administration of 500 μM diazoxide, a kATP-channel opener. N2A cells were transfected by combination of UCP4 promoter and TOMATO (red fluorescence protein) plasmid and purified by puromycin to get N2A-UCP4-TOMATO cell line. Using this cell line, we monitored the increase in red fluorescence intensity, that is, UCP4 up-regulation, by Biostation IM (Nikon) following the administration of 500 μM diazoxide. In SHR rats (12 w), UCP4 promoter and TOMATO (red fluorescence protein) was transfected by intraventricular injection of plasmid. 72 h after the intraperitoneal (ip) injection of diazoxide (10 mg/kg), brain slices were sectioned, and observed under the fluorescence microscope.
Results:
UCP4 mRNA level increased 3.8 times 48 h after (n=3) the administration of 500 μM diazoxide, while it did not change 24 h later (n=3). N2A-UCP4-TOMATO cells showed the significant (p<0.05, n=3) increase in fluorescence intensity from 6 to 48 h after 500 μM diazoxide application. In SHR, diazoxide (10 mg/kg ip, n=3) increased fluorescence intensity of UCP4 promoter and TOMATO as compared with that in the control brain (n=3).
Conclusion:
kATP-channel opening up-regulates UCP4 in the brain, which can be a component of neuroprotective mechanism.Supported by the grant from JSPS (KAKEN #23390345)
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