Induction of autophagy contributes to the neuroprotection of nicotinamide phosphoribosyltransferase in cerebral ischemic stroke
Induction of autophagy contributes to the neuroprotection of nicotinamide phosphoribosyltransferase in cerebral ischemic stroke
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DOI：
10.4161/auto.8.1.18274
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发表时间：
2012-01-01
期刊：
影响因子：
13.3
通讯作者：
Miao, Chao-Yu
中科院分区：
文献类型：
--
作者：
Wang, Pei;Guan, Yun-Feng;Miao, Chao-Yu
最近的研究表明，自噬作为一种应激反应，可能参与脑缺血的病理生理过程。烟酰胺磷酸核糖转移酶（Nampt，也称为visfatin）是哺乳动物NAD+生物合成的限速酶，通过抑制神经元凋亡和坏死来预防缺血性卒中。本研究旨在确定自噬参与Nampt在脑缺血中的神经保护作用。采用大鼠大脑中动脉闭塞（MCAO）和培养的皮层神经元氧糖剥夺（OGD）模型。在体内和体外使用慢病毒介导的基因转移过表达或敲低Nampt。进行免疫化学（LC 3-II）、电子显微镜和免疫印迹测定（LC 3-II、Beclin 1、雷帕霉素的哺乳动物靶标[mTOR]、S6 K1和结节性硬化症复合物-2 [TSC 2]）以评估自噬。我们发现Nampt的过表达在MCAO后2 h增加了体内和体外的自噬（LC 3斑点免疫化学染色，LC 3-II/Beclin 1表达和自噬体数量）。在OGD早期，自噬诱导剂雷帕霉素可保护Nampt基因敲低引起的神经元损伤，而自噬抑制剂3-甲基腺嘌呤可部分消除Nampt的神经保护作用。过表达或敲低Nampt可通过增强TSC 2 Ser 1387位点的磷酸化而非Thr 1462位点的磷酸化来调节OGD应激时mTOR和S6 K1信号通路的磷酸化。此外，在培养的SIRT 1敲除神经元中，Nampt对自噬蛋白LC 3-II和Beclin 1的调节被取消。我们的研究结果表明，Nampt通过诱导自噬促进神经元的存活，在脑缺血过程中，通过调节TSC 2-mTOR-S6 K1信号通路以SIRT 1依赖的方式。
Recent reports indicate that autophagy serves as a stress response and may participate in the pathophysiology of cerebral ischemia. Nicotinamide phosphoribosyltransferase (Nampt, also known as visfatin), the rate-limiting enzyme in mammalian NAD+ biosynthesis, protects against ischemic stroke through inhibiting neuronal apoptosis and necrosis. This study sought to determine the involvement of autophagy in neuroprotection of Nampt in cerebral ischemia. Middle cerebral artery occlusion (MCAO) in rats and oxygen-glucose deprivation (OGD) in cultured cortical neurons were performed. Nampt was overexpressed or knocked down using lentivirus-mediated gene transfer in vivo and in vitro. Immunochemistry (LC3-II), electron microscope and immunoblotting assays (LC3-II, Beclin 1, mammalian target of rapamycin [mTOR], S6K1 and tuberous sclerosis complex-2 [TSC2]) were performed to assess autophagy. We found that overexpression of Nampt increased autophagy (LC3 puncta immunochemistry staining, LC3-II/Beclin 1 expression and autophagosomes number) both in vivo and in vitro at 2 h after MCAO. At the early stage of OGD, autophagy-inducer rapamycin protected against neuronal injury induced by Nampt knockdown, whereas autophagy-inhibitor 3-methyladenine partly abolished the neuroprotective effect of Nampt. Overexpression or knockdown of Nampt regulated the phosphorylation of mTOR and S6K1 signaling pathway upon OGD stress through enhancing phosphorylation of TSC2 at Ser1387 but not Thr1462 site. Furthermore, in cultured SIRT1-knockout neurons, the regulation of Nampt on autophagic proteins LC3-II and Beclin 1 was abolished. Our results demonstrate that Nampt promotes neuronal survival through inducing autophagy via regulating the TSC2-mTOR-S6K1 signaling pathway in a SIRT1-dependent manner during cerebral ischemia.