DAPK1磷酸化BRCC3参与帕金森病中NLRP3炎症小体激活的作用机制

Neuroinflammation associated with Parkinson's disease (PD) process. The activation of inflammasomes is crucial to the occurrence of inflammation. Recent studies have found NLRP3 can be regulated by BRCC3, which is one of the deubiquitinases, to be involved in the activation of inflammasome. And it is reported that the expression of death associated protein kinase 1 (DAPK1) increased in neurodegenerative diseases. But whether BRCC3 and DAPK1 are involved in the activation of inflammasome in PD, and its mechanism has not yet to be confirmed. We have proved in the prior period that the key molecules in the inflammasome pathway express in neurons and involve in the pathogenesis of PD, NLRP3 inflammasome in PD models are activated. And the preliminary experimental results suggest that BRCC3 can be phosphorylated by DAPK1, moreover, the suppression of DAPK1 inhibit the activation of inflammasome in PD. The current project, following the concept of translation medicine, is to explored BRCC3 phosphorylation by DAPK1 and the mechanism of which participates in the regulation of the activation of inflammasome in the cellular and animal models of Parkinson's disease. Firstly, we evaluate the expression of DAPK1 or BRCC3 in PD models and verify its role of neuronal inflammasome activation. Then we deeply explore whether BRCC3 be phosphorylated by DAPK1 and its regulating mechanism on the neuronal inflammasome activation in PD. We focus on the phosphorylation of BRCC3 by DAPK1 whether increased the deubiquitinase activity of BRCC3, and whether resulted in excessive deubiquitination of NLRP3, consequent NLRP3 inflammasome activation, and whether induced IL-1β and IL-18 release. Finally, we verify whether the intervention of BRCC3 phosphorylation by DAPK1 could have suppressed the activation of neuronal inflammasome in PD models. This project has provided new research ideas and potential therapeutic targets for inflammation occurrence in PD.

神经免疫炎症与帕金森病(PD)进程相关，炎症小体激活对炎症发生至关重要。研究发现，去泛素化酶BRCC3可调节NLRP3去泛素化而参与炎症小体活化，死亡相关蛋白激酶1(DAPK1) 在神经退行性病变中表达增多，但其在PD中的作用机理尚待证实。课题组前期证明炎症小体通路关键分子在神经元中表达，NLRP3炎症小体在PD模型中被激活，且预实验结果提示DAPK1可以磷酸化BRCC3，抑制DAPK1活性可抑制PD中炎症小体激活。本项目遵循转化医学理念，从分子-细胞-动物水平深入探讨DAPK1磷酸化BRCC3参与PD中NLRP3炎症小体激活的调控机制:首先在PD模型中验证DAPK1和BRCC3的表达及其在炎症小体激活中的作用；进而探讨DAPK1磷酸化BRCC3并参与PD中炎症小体激活的调节机制；最后验证干预DAPK1磷酸化BRCC3将抑制炎症小体活化。本课题为PD中炎症发生的研究提供新的思路和治疗靶点。

神经免疫炎症与帕金森病(PD)进程密切相关，炎症小体激活对炎症发生至关重要。已知去泛素化酶BRCC3和死亡相关蛋白激酶1(DAPK1) 在脑组织表达丰富，但迄今未见BRCC3和DAPK1在PD中的作用机制研究。课题组针对DAPK1和BRCC3这两个重要分子在PD炎症发生的作用中进行了深入研究。首先，我们利用PD细胞和动物模型证明了死亡相关蛋白激酶 DAPK1 在PD中高表达，并应用DAPK1抑制剂在PD细胞模型中抑制DAPK1的蛋白表达，发现其可降低NLRP3炎症小体复合物的表达，提示DAPK1在PD炎症发生中的正向调控作用；其次，我们证明了去泛素化酶 BRCC3在PD细胞和动物模型中表达升高，应用BRCC3抑制剂G5以及shBRCC3慢病毒在PD细胞模型中抑制BRCC3蛋白表达后，结果显示，NLRP3炎症小体表达下降，神经元分泌的炎性因子IL-1β减少，说明BRCC3参与PD中NLRP3炎症小体的激活，该研究成果已公开发表于SCI刊物上；再次，通过生物信息学软件以及体外激酶实验证明了 DAPK1 对BRCC3的磷酸化作用，磷酸化位点可能发生在BRCC3蛋白的Ser101位点；最后，进一步在体内外验证DAPK1对BRCC3的正向调节作用，可能是通过DAPK1蛋白的CaM结构域与BRCC3结合从而调节的活化，提示PD中的炎症可能是通过DAPK1-BRCC3通路调控NLRP3炎症小体激活而的。该研究成果可为PD中炎症发生的研究提供新的思路和治疗靶点。