Nix介导的线粒体自噬在缺血性脑损伤中的作用及与Synphilin-1相关性研究

Mitophagy is activated along with cerebral ischemia. Our previous research demonstrated that mitophagy protected against focal brain ischemia/reperfusion (I/R) in mice. These results suggested that mitophagy could be a potential therapeutic target in ischemic brain injury. The mechanisms underlying mitophagy in this context are still unclear, our preliminary data found that Nix was recruited to mitochondira along with reperfusion. Knockdown of Nix significantly decreased the I/R-induced mitophagy, suggesting a key role of Nix in this process. Furthermore, we found a novel Nix-binding protein, Synphilin-1 by carring out tandem affinity purification assay. Our preliminary data interestingly find that knockdown of SYL-1 reduced Nix-induced mitophagy in Neuro2a cells. However, it is unclear whether SYL-1 is involved in the Nix-related mitophagy in ischemic brains. For this purpose, we aimed to fully identify the involvement and contribution of Nix-related mitophagy in the context of brain I/R. In addition, we intend to explore the underlying mechanisms by which SYL-1 regulates mitophagy in these systems. These investigations will provide significant experimental evidences in taking mitophagy as a novel therapeutic target in cerebral ischemia.

脑缺血过程伴随线粒体自噬。课题组前期研究表明脑缺血/复灌过程中线粒体自噬发挥了重要的神经保护作用，提示其是一种潜在的脑缺血治疗靶点。线粒体自噬的机制尚未阐明，预实验初步发现Bcl-2家族蛋白Nix在脑缺血/复灌过程中向线粒体聚集，小干扰Nix表达可显著降低缺血/复灌引起的线粒体自噬。提示Nix可能是脑缺血情况下介导线粒体自噬的关键蛋白。为阐明Nix的调控机制，申请人利用TAP结合蛋白质谱技术，发现并鉴定了一种新的Nix结合蛋白Synphilin-1(SYL-1)。小干扰SYL-1降低Nix诱发的线粒体自噬，但在脑缺血条件下SYL-1是否参与上述调控过程有待研究。因此，本研究拟采用体内、外脑缺血模型，利用Nix敲除小鼠，结合药理学、RNA干扰等手段，从多个层面明确Nix介导的线粒体自噬在脑缺血过程中的作用，并阐明其中SYL-1的调控机制，研究结果将为利用线粒体自噬作为脑缺血治疗靶点提供依据。

缺血性脑卒中是一种因循环暂停或急性局灶性脑血供障碍引起的脑损伤。自噬在脑缺血过程中发挥了关键的作用。申请人前期工发现脑缺血后神经元发生线粒体自噬，进而防止线粒体依赖的细胞凋亡，最终发挥了重要的神经保护作用。然而，脑缺血过程中线粒体自噬发生的机制及其作用尚未明确，是本领域研究的热点问题。Nix是一种位于线粒体外膜的自噬受体蛋白，但其是否在病理过程中发挥作用并不清楚。本研究利用Nix-/-小鼠进行MCAO手术，发现Nix敲除抑制缺血复灌引起的线粒体自噬，给予GFP-Nix腺相关病毒（GFP_Nix AAVs）可以逆转Nix敲除引起的线粒体自噬障碍，同时也逆转了Nix敲除引起的脑梗死增加。提示Nix参与了脑缺血复灌后的线粒体自噬并发挥保护作用。利用线粒体自噬相关蛋白Parkin基因敲除动物，有趣地发现Nix通过Parkin非依赖途径介导的脑缺血复灌过程中的线粒体自噬。我们首次发现并证明了Nix第81位丝氨酸的磷酸化修饰是Nix介导线粒体自噬的关键。上述结果阐明了脑缺血过程中Nix介导线粒体自噬的分子机制，并首次发现了Nix的一个新的磷酸化调节位点。这些研究将为进一步利用Nix，尤其是其磷酸化，调控脑缺血过程线粒体自噬进而干预缺血性脑损伤提供重要的实验依据。