ROCK1激活Drp1诱导的神经细胞线粒体异常分裂在帕金森病发病中的作用机制研究

Dopamine deficiency caused by midbrain nigra striatum dopaminergic nerve cell apoptosis is the main pathological basis of Parkinson's disease (PD). The Current study has shown that dynamin-related protein 1 (Drp1)-induced mitochondrial aberrant fission plays an important role in dopaminergic neural cell apoptosis. However, the upstream regulatory mechanism of its action remains unclear. Previously we have successfully established cell and animal models of PD and confirmed that the mitochondrial aberrant fission regulated by Drp1 was involved in the process of PD, in which ROCK1 was activated and the specific ROCK1 inhibitor Y-27632 could inhibit mitochondrial aberrant fission through suppressing CaN-induced Drp1 dephosphorylation/activation, decrease dopaminergic nerve cell apoptosis and significantly improve symptoms of PD in mice. Our preliminary experiments revealed that ROCK1 could dephosphorylate/activate Drp1 through CaN, resulting in mitochondria aberrant fission, and eventually leading to PD. In this proposal, we aim to elucidate the molecular mechanism of ROCK1 via activation of Drp1-mediated neural cell mitochondrial aberrant fission in PD in vitro and in vivo, and confirm the therapeutic effect of ROCK1 inhibitor on PD. Our study will strengthen the understanding of the pathogenesis of PD and provide experimental evidence for the application of ROCK1 inhibitor in treating PD.

中脑黑质纹状体中多巴胺能神经细胞凋亡导致多巴胺分泌不足是帕金森病（PD）的主要病理基础。目前研究表明，动力相关蛋白1（Drp1）诱导的线粒体异常分裂在多巴胺能神经细胞凋亡中发挥了重要作用，但其上游调控机制尚不清楚。在我们前期构建的PD细胞和动物模型中，证实了Drp1调控的线粒体异常分裂参与了PD发生，并发现PD中ROCK1被激活，而ROCK1活化抑制剂（Y-27632）可通过抑制CaN介导的Drp1去磷酸化激活抑制线粒体异常分裂和神经细胞凋亡，改善模型小鼠的PD症状。我们前期实验结果提示：ROCK1可能是通过CaN去磷酸化激活Drp1，从而参与PD的发生。本项目拟通过体内外研究，阐明ROCK1激活Drp1诱导的神经细胞线粒体异常分裂在PD发病中的作用机制，同时明确ROCK1抑制剂对PD的治疗作用。本项目将加深对PD发病机制的理论认识，为推动ROCK1抑制剂成为PD治疗药物提供实验依据。

中脑黑质纹状体中多巴胺能神经细胞凋亡导致多巴胺分泌不足是帕金森病（PD）的主要病理基础。最近的研究表明动力相关蛋白1 (Drp1)诱导的线粒体异常分裂在多巴胺能神经细胞凋亡中发挥了重要作用，但其上游调控机制尚不清楚。我们分别采用MPP+和MPTP构建了PD的细胞和动物模型，研究结果表明，基因敲降Drp1显著抑制MPP+诱导的线粒体异常分裂和细胞凋亡。 重要的是，我们发现在PD细胞模型中ROCK1被激活，ROCK1敲降和ROCK1活化抑制剂Y-27632均可通过抑制Drp1去磷酸化/活化介导的多巴胺能神经细胞线粒体分裂和细胞凋亡。在PD 模型小鼠上，Y-27632也可显著逆转PD小鼠步态蹒跚、动作迟缓震颤、对外界反应低下等反应，以及明显增加在转棒上的停留时间，其运动协调性明显改善。同时，酪氨酸羟化酶（体内合成多巴胺的限速酶） 阳性表达的细胞数显著增加，表明 Y-27632可显著逆转模型小鼠的PD症状。同时， Y-27632可阻断PD小鼠中ROCK1的激活和显著抑制PD小鼠中脑黑质致密部和纹状体中细胞凋亡以及Drp1线粒体转位和Drp1去磷酸化，表明Y-27632是通过抑制Drp1介导的线粒体异常分裂和细胞凋亡显著改善模型小鼠的PD症状。综上,我们的研究结果表明，ROCK1通过激活Drp1诱导的线粒体异常分裂调控的多巴胺能神经细胞凋亡是PD发病的重要分子机制之一， 同时明确了ROCK1抑制剂对PD的治疗作用，为开发ROCK1为PD治疗靶点提供依据。本项目将加深对PD发病机制的理论认识，为推动ROCK1抑制剂成为PD治疗药物提供实验依据。