Drp1转录后修饰减轻线粒体损伤后的坏死性凋亡介导大鼠脑缺血耐受的分子机制

As a new form of necrotic cell death, necroptosis was demonstrated to be an important process that contributes to neuronal death and functional impairment after cerebral ischemic injury. How to regulate necroptosis and protect neuron from cerebral ischemia is regarded as an urgent and critical research topic.. Mitochondria are the critical organelles involved in regulating generation of reactive oxygen species (ROS), which are important in executing necroptotic cell death. Recently, it has been reported that mitochondria morphology is highly dynamic and that mitochondrial morphology affects the survival and death of cells after cerebral ischemia. Mitochondria continuously change their morphology by fusion and fission, and this process is mainly executed by dynamin super-family proteins, such as dynamin-related protein 1 (Drp1). In preliminary studies we demonstrated that ischemia led to numerous damaged mitochondria with vague cristae and dense membranous material in transient global cerebral ischemia (tGCI) rats, whereas the mitochondria with clear and complete double membrane and crista structures was observed in hypoxic preconditioning (HPC) animals. Additionally, we found that this ischemic challenge up-regulated the receptor-interacting protein kinase 3 (RIP3) protein levels in the CA1 regions of the hippocampus. However, the administration of necrostatin-1 (Nec-1), a necroptosis inhibitor, dramatically decreased neuronal damage induced by tGCI. Moreover, no difference at the protein level of Drp1 was observed between the tGCI and HPC groups. These results suggest that HPC induces neuroprotection against tGCI in rats via decreasing mitochondrial damage and inhibiting necroptosis of neuron in the hippocampal CA1 subregion. Whether post-transcriptional regulation of Drp1 plays a key role in the mitochondria morphology and which molecular mechanism is involved remain unknown. In this project, we intend to demonstrate that hypoxic preconditioning decreases the RIP1-RIP3 complex by inhibiting the activation of CaMKII, in turn blocks MLKL/PGAM5S pathway, prevents Drp1 dephosphorylation, and reduces mitochondrial degradation and ROS production, and finally reduces necroptosis in the hippocampal CA1 subregion after tGCI. . More insights will be obtained on the neuroprotective molecular mechanisms of post-transcriptional modification of Drp1 induced by HPC. Moreover, in a context of cerebral ischemia a complete understanding of the mechanisms of regulated necroptosis might provide new targets for the therapy of ischemic stroke.

坏死性凋亡是近年发现的脑缺血后神经元死亡的一种方式。如何减轻脑缺血后的坏死性凋亡，从而保护神经元，是当今脑缺血研究中的热点与挑战。线粒体是坏死性凋亡的最重要执行者，动力蛋白相关蛋白1（Drp1）是调节线粒体形态与功能的关键蛋白。我们的预实验发现低氧预处理通过减轻大鼠短暂全脑缺血后海马CA1区神经元的线粒体损伤，从而减少坏死性凋亡，而上述的神经保护作用不是通过降低缺血后的Drp1表达来实现，故推测可能与影响Drp1的转录后修饰有关。本研究拟应用多种分子生物学技术，依次研究低氧预处理通过抑制CaMKII，减少坏死小体的产生，进而抑制MLKL/PGAM5S通路，阻止Drp1去磷酸化，减少线粒体降解和ROS的产生，从而减轻大鼠脑缺血后CA1区神经元的坏死性凋亡。本研究将揭示Drp1转录后修饰减轻线粒体损伤后的坏死性凋亡，并介导脑缺血耐受的分子机制，为研发针对脑缺血具有特异性的治疗药物奠定理论基础。

坏死性凋亡是近年发现的脑缺血后神经元死亡的一种方式。如何减轻脑缺血后的坏死性凋亡，从而保护神经元，是当今脑缺血研究中的热点与挑战。本项目在成年大鼠低氧预处理短暂全脑缺血模型的基础上，主要采用免疫组化、免疫印迹、免疫共沉淀等方法，明确大鼠短暂全脑缺血（tGCI）后海马CA1区神经元是否发生坏死性凋亡，并探讨低氧预处理减轻大鼠tGCI后CA1区神经元坏死性凋亡的分子机制。我们的结果发现，（1）大鼠tGCI后再灌注早期海马CA1区RIP3的表达增加，RIP1-RIP3的相互作用增强；在缺血前给予低氧预处理或者通过侧脑室注射Nec-1，可使大鼠tGCI后CA1区RIP3的表达减少，RIP1-RIP3的相互作用减弱；（2）低氧预处理减少tGCI后CA1区caspase-8的表达和caspase-8活性，在缺血再灌注前24 h经侧脑室注射zVAD，可使大鼠tGCI后CA1区caspase-8的表达降低、caspase-8活性减弱、存活的神经元增加以及变性细胞减少。（3）低氧预处理使大鼠tGCI后CA1区CaMKIIɑ 降低，RIP1与CaMKIIɑ的相互作用减弱，抑制CaMKIIɑ活性可使大鼠tGCI后CA1区RIP1-RIP3的相互作用减弱，从而减轻神经元损伤。（4）低氧后处理抑制大鼠tGCI后CA1区MLKL的磷酸化，抑制Drp1的去磷酸化，进而减少Drp1向线粒体转位。在缺血再灌注前24 h经侧脑室注射Mdivi-1或者Nec-1，可抑制大鼠tGCI后CA1区Drp1的去磷酸化，进而减少Drp1向线粒体转位，从而减轻神经元死亡。本项目证实低氧预处理通过抑制CaMKIIα，减少坏死小体的产生，进而抑制MLKL的磷酸化，抑制Drp1的去磷酸化，从而减轻成年大鼠短暂全脑缺血后海马CA1区神经元的坏死性凋亡而发挥神经保护作用。本项目为研发针对脑缺血的具有特异性的治疗药物，减轻脑缺血再灌注损伤，改善缺血性卒中患者的存活及长期预后提供更充分的科学依据。