Preconditioning Stimuli Induce Autophagy via Sphingosine Kinase 2 in Mouse Cortical Neurons*
Preconditioning Stimuli Induce Autophagy via Sphingosine Kinase 2 in Mouse Cortical Neurons*
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DOI：
10.1074/jbc.m114.578120
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发表时间：
2014-06
期刊：
影响因子：
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通讯作者：
Rui Sheng;Tong-tong Zhang;Valeria D. Felice;T. Qin;Zheng-Hong Qin;Charles D Smith;E. Sapp;M. Difiglia;C. Waeber
中科院分区：
文献类型：
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作者：
Rui Sheng;Tong-tong Zhang;Valeria D. Felice;T. Qin;Zheng-Hong Qin;Charles D Smith;E. Sapp;M. Difiglia;C. Waeber
背景：预处理提供了对内源性机制的深入了解，可用于保护脑免受损伤。结果：预处理刺激上调鞘氨醇激酶2，导致自噬。结论：鞘氨醇激酶2可能通过阻断Beclin 1/Bcl-2相互作用介导自噬和预适应。重要性：新的独立于SPK 2催化活性的信号传导的发现为药物化学家提供了对神经保护重要的新的“可药用”靶标。鞘氨醇激酶2（SPK 2）和自噬都参与脑预适应，但是否预适应诱导的SPK 2上调和自噬激活的机制仍有待阐明。在这项研究中，我们使用体外和体内模型来探讨SPK 2介导的自噬在异氟醚和低氧预适应中的作用。在原代小鼠皮层神经元中，异氟醚和低氧预处理均诱导自噬。异氟烷和低氧预处理保护随后的氧葡萄糖剥夺或谷氨酸损伤，而预处理与自噬抑制剂（3-甲基腺嘌呤或KU 55933）取消预处理诱导的耐受。用SPK 2抑制剂（ABC 294640和SKI-II）或SPK 2敲低预处理防止预处理诱导的自噬。异氟烷还诱导小鼠体内自噬，如LC 3和p62的蛋白质印迹、LC 3免疫染色和电子显微镜所示。异氟烷诱导缺乏SPK 1亚型（SPK 1 −/−）的小鼠自噬，但在SPK 2 −/−小鼠中没有。鞘氨醇1-磷酸和鞘氨醇1-磷酸受体激动剂FTY 720没有保护对氧葡萄糖剥夺培养的神经元，并没有改变LC 3和p62的表达，这表明SPK 2介导的自噬和保护不是S1 P依赖的。Beclin 1敲除消除了预处理诱导的自噬，SPK 2抑制剂消除了异氟烷诱导的Beclin 1/Bcl-2结合的破坏。这些结果强烈表明，自噬参与异氟烷预处理在体内和体外，SPK 2有助于预处理诱导的自噬，可能是通过破坏Beclin 1/Bcl-2的相互作用。
Background: Preconditioning provides insights into endogenous mechanisms that could be used to protect brain from injury. Results: Preconditioning stimuli up-regulate sphingosine kinase 2, leading to autophagy. Conclusion: Sphingosine kinase 2 mediates autophagy and preconditioning, possibly by disrupting Beclin 1/Bcl-2 interaction. Significance: The discovery of new signaling independent of SPK2 catalytic activity provides medicinal chemists with novel “druggable” targets important for neuroprotection. Sphingosine kinase 2 (SPK2) and autophagy are both involved in brain preconditioning, but whether preconditioning-induced SPK2 up-regulation and autophagy activation are linked mechanistically remains to be elucidated. In this study, we used in vitro and in vivo models to explore the role of SPK2-mediated autophagy in isoflurane and hypoxic preconditioning. In primary mouse cortical neurons, both isoflurane and hypoxic preconditioning induced autophagy. Isoflurane and hypoxic preconditioning protected against subsequent oxygen glucose deprivation or glutamate injury, whereas pretreatment with autophagy inhibitors (3-methyladenine or KU55933) abolished preconditioning-induced tolerance. Pretreatment with SPK2 inhibitors (ABC294640 and SKI-II) or SPK2 knockdown prevented preconditioning-induced autophagy. Isoflurane also induced autophagy in mouse in vivo as shown by Western blots for LC3 and p62, LC3 immunostaining, and electron microscopy. Isoflurane-induced autophagy in mice lacking the SPK1 isoform (SPK1−/−), but not in SPK2−/− mice. Sphingosine 1-phosphate and the sphingosine 1-phosphate receptor agonist FTY720 did not protect against oxygen glucose deprivation in cultured neurons and did not alter the expression of LC3 and p62, suggesting that SPK2-mediated autophagy and protections are not S1P-dependent. Beclin 1 knockdown abolished preconditioning-induced autophagy, and SPK2 inhibitors abolished isoflurane-induced disruption of the Beclin 1/Bcl-2 association. These results strongly indicate that autophagy is involved in isoflurane preconditioning both in vivo and in vitro and that SPK2 contributes to preconditioning-induced autophagy, possibly by disrupting the Beclin 1/Bcl-2 interaction.