Cdk5磷酸化Sirt2介导NLRP3炎症小体激活在帕金森氏病神经元焦亡中的机制研究

The substantial loss of dopaminergic neurons in substantia nigra is the most important pathological basis of Parkinson's disease (PD), a variety of environmental toxins and genetic factors can lead to specific neuronal cell death in the substantia nigra. Recent studies have found that, in addition to the classic form of cell death, necrosis and apoptosis, a new type of programmed cell death, caspase-1-dependent pyroptosis were proved. Pyroptosis are confirmed to participate in the progression of infectious diseases and atherosclerosis, but whether pyroptosis is involved in neuronal death in PD, and its mechanism has yet to be confirmed. The current project follows the concept of translational medicine. Firstly, we evaluate the key role of caspase-1-dependent NLRP3 inflammasome activation and neuronal pyroptosis in the cellular models, animal models and clinical specimens of Parkinson's disease. Thus, we deeply explore the molecular mechanisms of the neuronal inflammasome activation, and we focus on the phosphorylation of deacetylase Sirtuin-2 by Cdk5, one of the critical kinase in nervous system, which lower the deacetylase activity of Sirtuin-2, resulting in excessive acetylation of α-tubulin, and consequent NLRP3 inflammasome activation and neuronal pyroptosis. Finally, we verify whether the intervention of Cdk5 phosphorylation Sirt2 could have protective effects of neuronal cell death in PD models. This project focus on the provision of new research ideas and therapeutic targets for dopaminergic neuronal death in PD.

中脑黑质多巴胺能神经元大量丢失是帕金森氏病（PD）最重要的病理基础，多种环境毒素和遗传因素可引发黑质神经元特异性死亡；近年研究发现，坏死和凋亡之外的新型死亡途径——细胞焦亡（Pyroptosis）参与多种疾病发生发展，但其在PD中的作用机理尚待证实。本项目遵循转化医学理念，首先在细胞—组织—动物—临床PD样品中验证NLRP3炎症小体激活介导的神经元焦亡在PD发病中的关键作用，检测脑脊液IL-1β、IL-18可否作为PD诊断指标；进而深入研究PD神经元炎症小体激活分子机制，探讨神经系统关键激酶Cdk5是否可以磷酸化Sirt2，下调其去乙酰化酶活性导致微管蛋白α-tubulin过度乙酰化，通过促进微管转运，引发NLRP3炎症小体组装和激活，诱发IL-1β、IL18释放和神经元焦亡；最后验证干预Cdk5磷酸化Sirt2可否保护PD神经元细胞焦亡，为PD神经元死亡的机制研究提供新思路和治疗靶点。

SIRT2是NAD+ 依赖的去乙酰化酶Sirtuin 家族的主要成员，在神经系统特异性的高表达。SIRT2可以通过去乙酰化多种底物发挥调控细胞周期、抗氧化、抑制肿瘤细胞生长等作用。众多国内外学者和我们实验室的研究发现敲除或者抑制SIRT2，可以减轻帕金森病（Parkinson's disease，PD）发病时的多巴胺能神经元的丢失和运动功能障碍，对PD具有保护作用，但是其具体机制尚不明确。本研究探讨SIRT2参与PD发病的分子机制。研究发现：（1）在PD的动物和细胞模型中，SIRT2 mRNA和总蛋白表达没有改变，但亚细胞定位改变，发生核转位；（2）SIRT2蛋白的核转位引起神经元的死亡；（3）免疫共沉淀和GST-pulldown结果显示，CDK5和SIRT2存在相互作用，CDK5抑制剂Roscovitine可阻止MPP+处理原代神经元中SIRT2蛋白的核转位；（4）应用多种生物信息学软件联合预测结果提示SIRT2蛋白的S331位点和S335位点有可能是CDK5的磷酸化位点，体外激酶反应及磷酸化质谱等方法在体内体外验证CDK5可在体外及细胞中直接磷酸化SIRT2蛋白的S331位点和S335位点，磷酸化的SIRT2发生核转位；（5）在PD细胞模型和动物模型中通过添加MYR-SIRT2 328-339肽（MYR-TSASPKKSPPPA），干扰CDK5磷酸化SIRT2蛋白的Ser331位点和Ser335位点可减轻PD时神经元的死亡和运动功能障碍。本研究发现，在PD中SIRT2 mRNA和总蛋白表达没有改变，但发生SIRT2蛋白核转位，其核转位导致神经元的死亡；在PD中，神经系统关键激酶CDK5可直接磷酸化SIRT2蛋白的S331位点和S335位点，介导SIRT2发生核转位；干扰CDK5磷酸化SIRT2，可抑制SIRT2核转位，并减轻PD发病时的神经元死亡和运动功能障碍。本研究为揭示PD的发病机理和临床治疗提供了新的靶点和思路。