Sphk1和TRAF2相互结合介导缺血神经元necroptosis的分子机制

Necrosis is traditionally recognized as a process of cell death that is not regulated and programmed. In contrast to the conventional view, necroptosis is a newly identified type of programmed necrosis regulated by specific signaling cascades. Necroptosis essentially contributes to neuronal cell death following cerebral ischemia, although the mechanisms by which necroptosis is elicited in post-ischemic neurons remain unclear. We found that sphingosine kinase 1 (SphK1), a kinase converting sphingosine to sphingosine-1-phosphate, was significantly induced in post-ischemic neurons. SphK1 is well acknowledged as a kinase that inhibits apoptosis and promotes cell survival under various pathophysiological conditions. However, to our surprise, we observed that the specific SphK1 inhibitor and siRNA reduced infarction in mice following experimental stroke and protected primary neurons from oxygen-glucose deprivation. Furthermore, we found that ischemia-induced Sphk1 directly interacted with TRAF2 (Tumor-necrosis factor receptor-associated factor 2) and thus enhanced the stability of TRAF2 following cerebral ischemia. TRAF2 is reported to promote caspase-8 degradation by acting as a ubiquitin ligase and degradation of caspase-8 is a key step in intiating necroptosis. Thus, we propose in this project to test the following hypotheses: 1) post-ischemic enhancement of TRAF2 stability by Sphk1 via its direct interaction with TRAF2 is a molecular switch that initiates post-ischemic neuronal death; 2)ischemia-induced SphK1 elicits neuronal necroptosis by acting through the cascade SphK1-TRAF2-Caspase-8. This proposed study will shed some light on the molecular mechanisms by which SphK1 acts through TRAF2 to promote necroptosis and will identify novel targets for stroke therapies based on the necroptosis mechanisms.

程序化坏死(Necroptosis)是脑缺血后神经元死亡的重要机制，但相关分子信号通路尚不清楚。我们发现鞘氨醇激酶1(SphK1)在缺血神经元中诱导表达。但与SphK1公认的抗凋亡功能相矛盾的是：我们发现SphK1抑制剂和siRNA在动物脑缺血和原代神经元缺氧缺糖模型中具有保护作用。我们的前期研究还表明脑缺血后SphK1与TRAF2直接结合而提高TRAF2的稳定性。TRAF2可通过泛素化修饰导致Caspase8的降解，而Caspase8的降解是启动程序化坏死的关键步骤。本课题拟在细胞和动物模型上探索：1）SphK1与TRAF2相互结合提高TRAF2稳定性是缺血神经元死亡的关键调控步骤；2)SphK1-TRAF2-caspase 8信号通路导致缺血神经元necroptosis的分子机制。本课题将从程序化坏死的角度揭示SphK1通过TRAF2调控缺血神经元死亡的机制，将为脑缺血治疗发现新靶点。

细胞程序性坏死（necroptosis）是介导缺血性脑损伤的重要病理分子机制。但至今对脑缺血后necroptosis的调控机制尚不清楚。肿瘤坏死因子受体相关蛋白2（TRAF2）抑制necroptosis，但目前尚不清楚TRAF2是否介导脑缺血后的病理机制并调控脑缺血导致的necroptosis。本课题在细胞及小鼠大脑中动脉堵塞模型（MCAO）探讨了TRAF2在缺血性脑损伤病理机制及脑缺血导致的necroptosis中的作用。Western blot、RT-PCR及免疫组化结果表明MCAO后TRAF2在神经元和小胶质细胞中表达水平显著上升。但是与野生型小鼠相比，MCAO后鞘氨醇激酶1（Sphk1）基因敲除小鼠脑内TRAF2蛋白的诱导表达水平并未明显下降。这表明缺血后脑内TRAF2蛋白诱导表达并非由Sphk1介导。MCAO后，以慢病毒shRNA敲减内源性TRAF2在小鼠纹状体的诱导表达导致脑梗死体积增加、细胞死亡增加及脑内炎症应答上调。在原代小胶质细胞以神经元缺氧缺糖条件上清和zVAD共处理导致的necroptosis模型中，以及海马HT22神经元以TNF-α和zVAD共处理导致的necroptosis模型中，TRAF2也被诱导表达。敲减内源性TRAF2的诱导表达也在这两种细胞模型中加剧necroptosis。另外，necroptosis特异性抑制剂Nec-1可在小鼠MCAO模型显著抑制TRAF2敲减加剧的细胞死亡。这进一步表明TRAF2可抑制脑缺血导致的necroptosis。在分子机制方面，脑缺血后TRAF2通过增强与MLKL（mixed lineage kinase domain like protein）的结合从而抑制necroptosis。总之，我们的研究揭示了脑缺血后调控necroptosis的内源性机制：即TRAF2诱导表达是抑制脑缺血后神经元和小胶质细胞necroptosis的内源性保护机制，从而降低缺血性脑损伤。