SENP6依赖的Annexin-A1去SUMO化介导其核转位在脑缺血神经元凋亡中的作用及机制研究

Cerebral ischemia is one of the major diseases threatening the health of Chinese residents. A large number of studies have shown that apoptosis of neurons caused by nuclear translocation of Annexin-A1 (ANXA1) plays an important role in the development of cerebral ischemia. According to the previous studies, the SUMOylation level of ANXA1 decreased significantly after cerebral ischemia. On this basis, we further found that SENP6 mediated ANXA1 de-SUMOylation and participated in the process of neuronal injury after ischemia. But its molecular mechanism is still not clear. In this research, based on the de-SUMOylation of ANXA1 induced by cerebral ischemia, we will study the molecular mechanism of de-SUMOylation involved in ischemic injury of ANXA1 at the molecular-cellular-animal level. Firstly, the de-SUMOylation site of ANXA1 will be screened based on the confirmation of de-SUMOylation of ANXA1 by SENP6. Next, by studying the changes of phosphorylation level, we would find that de-SUMOylation of ANXA1 by SENP6 could increase the phosphorylation level of ANXA1, which leading to the accumulation of ANXA1 in the nucleus, activating the apoptosis pathway and ultimately, triggering neuronal apoptosis. Finally, we will verify whether the intervention of SENP6-mediated de-SUMOylation of ANXA1 could have protective effects against neuronal cell apoptosis in cerebral ischemia and reperfusion injury. In summary, this project would put forward a novel pathophysiological mechanism and therapeutic target under the neuronal apoptosis in cerebral ischemia.

缺血性脑卒中是威胁我国居民健康的重大疾病之一，Annexin-A1（ANXA1）核转位所引发的神经元凋亡在脑缺血发生发展过程中起重要作用。前期研究发现，脑缺血后ANXA1的SUMO化水平显著下降。在此基础上，我们进一步发现SENP6介导ANXA1去SUMO化并参与脑缺血神经损伤过程，但其分子机制尚不明确。本研究拟在分子—细胞—动物水平，以脑缺血诱导ANXA1的SUMO化水平下降为切入点，首先在确认SENP6对ANXA1去SUMO化的基础上，筛选出ANXA1去SUMO化修饰位点；进一步，通过研究ANXA1磷酸化水平变化，探明SENP6对ANXA1的去SUMO化能引起ANXA1磷酸化水平增加，进而导致ANXA1核转位增加，激活凋亡通路，引发神经元凋亡；最终验证干预SENP6去SUMO化ANXA1对脑缺血神经元凋亡的保护作用，为阐明脑缺血神经元凋亡的分子机制和干预治疗提供新的研究思路和药物靶点。

缺血性脑卒中是威胁人类健康的重大疾病之一，膜联蛋白-A1 (ANXA1)在脑缺血再灌注损伤后神经元凋亡中发挥关键作用。我们最近的研究表明，ANXA1蛋白能被SUMO化修饰，并且这种修饰在脑缺血再灌注后明显减弱，但其对神经元死亡的影响及其潜在的分子机制尚未完全阐明。在本项研究中，我们首先筛选发现sentrin/SUMO特异性蛋白酶6 (SENP6)为ANXA1蛋白SUMO化修饰的去苏木化酶。在此基础上，进一步研究发现SENP6介导ANXA1蛋白的去SUMO化修饰可诱导其核转位，并能够引发脑缺血再灌注后神经元凋亡。机制研究表明，SENP6介导ANXA1蛋白的去SUMO化能上调磷酸化激酶TRPM7和PKC依赖的ANXA1磷酸化修饰水平。此外，抑制SENP6介导的ANXA1蛋白去SUMO化修饰能抑制转录因子p53的转录活性，降低促凋亡基因Bid的表达，进而抑制caspase-3细胞凋亡通路的活化，并减少受氧-葡萄糖剥夺和再复氧引发的神经元凋亡。更为重要的是，通过在体在神经元中特异性过表达SENP6酶活性缺失型突变体来抑制内源性SENP6的活性，能明显阻断脑缺血再灌注损伤导致的海马和皮质区域神经元凋亡，减小脑缺血梗死灶体积，改善脑缺血再灌注损伤后小鼠的神经功能及空间学习记忆能力与运动功能。该研究为阐明SENP6介导的ANXA1蛋白去苏木化修饰在脑缺血再灌注损伤中的作用提供了新的理论基础和分子机制，进一步的研究可以为缺血性脑卒中的临床治疗提供新的研究策略和分子靶点。

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