鞘氨醇激酶2在脑预适应激活自噬信号通路中的作用及机制

Our previous results showed that sphingosine kinase 2(SPK2) and autophagy both are involved in cerebral preconditioning, but whether preconditioning- induced SPK2 upregulation contributes to autophagy activation remains to be elucidated. Activation and upregulation of SPK2 may release S1P to induce autophagy via acting on S1P receptors. But, SPK2 upregulation might also mediate autophagy activation via its BH3 domain in a S1P-independent manner. This proposal will test the novel hypothesis that preconditioning stimuli up-regulates SPK2 via HIF1α, and that the BH3-domains of SPK2 then displace Beclin1 from the Bcl-2/Beclin 1 or Bcl-XL/Beclin 1 complexes, resulting in release of Beclin1 and activation of mitophagy or reticulophagy. In this project, we will establish isoflurane, hypoxic and ischemic preconditioning models in mice, primary murine cortical neurons and HT22 hipocampal neuronal cells to explore the role of sphingosine kinase 2 in preconditioning- induced autophagy activation. First, we intend to demonstrate whether Parkin related- mitophagy or GRP78 dependent reticulophagy participates in the neuroprotection of cerebral preconditioning. This study will not only provide experimental evidence for the treatment with isoflurane and other anesthetics on cerebral ischemia, but also extend our knowledge of the regulation, mechanisms and bioactivity of mitophagy and reticulophagy. Second, we will investigate the relationship between SPK2 and autophagy activation during preconditioning to elucidate the novel signal pathway by which preconditioning induces autophagy. Finally, we want to characterize the molecular mechanisms by which SPK2 contributes to autophagy activation. Furthermore,we will use drugs acting SPK2 or autophagy to mimic preconditioning-induced neuroprotection, thus proposing a therapeutic strategy for ischemic cerebrovascular diseases.

我们的前期研究发现鞘氨醇激酶2(SPK2)和自噬都参与脑预适应过程，但预适应上调SPK2是否与自噬相关尚不可知。我们推测预适应经HIF1α上调SPK2，一方面，SPK2催化S1P生成，可能作用于S1P受体引起自噬；另外，SPK2结构中BH3 domain亦可能从Bcl-2(Bcl-XL)/Beclin1复合物中取代Beclin1，诱发Parkin相关线粒体自噬或GRP78相关内质网自噬。本课题建立小鼠和神经细胞异氟醚、缺氧和缺血预适应模型，应用分子生物学方法研究脑预适应过程中SPK2和自噬激活的关系，旨在：①研究线粒体自噬或内质网自噬是否参与脑预适应，揭示预适应神经保护的机制。②深入研究SPK2是否参与脑预适应激活自噬，阐明预适应激活自噬的新信号通路。③探索神经细胞内SPK2激活自噬的分子机制，探讨SPK2蛋白与酶活性无关的新功能，为治疗脑缺血提供靶向SPK2或自噬的候选保护药物。

鞘氨醇激酶2(SPK2)和自噬都参与了预适应过程，但预适应引起SPK2上调是否与自噬相关尚不可知。一方面，SPK2上调和激活可能使S1P生成释放增加，通过作用于S1P受体引起自噬。另一方面，SPK2激活自噬的作用亦可能与S1P无关，由其结构中的BH3 domain介导，SPK2的BH3 domain从Bcl-2/Beclin1或Bcl-XL/Beclin1的复合物中取代Beclin1，诱发自噬。本课题通过建立小鼠和神经细胞异氟醚和缺氧预适应模型，在整体和细胞水平应用多种分子生物学方法研究预适应过程中SPK2和自噬激活的关系，研究发现：①.证实Beclin1依赖的自噬途径参与异氟醚和缺氧预适应的神经保护作用，为开发异氟醚等麻醉剂防治缺血性脑中风提供实践经验，并扩展自噬生物活性的研究领域；②. 证明SPK2参与了脑预适应激活自噬，阐明预适应激活自噬的新颖信号通路； ③. 探索神经细胞内SPK2激活自噬的分子机制，证实SPK2诱导自噬的作用与其催化活性无关，而是通过其BH3 domain从Bcl-2/Beclin1的复合物中取代Beclin1，释放出游离的Beclin1，诱发自噬。根据SPK2的BH3 domain设计了Tat-SPK2肽，可能为防治缺血性脑中风提供靶向自噬的候选保护药物。