PKD致病基因PRRT2通过RIM-BP1调控突触囊泡释放的分子机制研究

Paroxysmal kinesigenic dyskinesia (PKD) is a paroxysmal movement disorder with complex clinical heterogeneity. To date, there are only a few reports about the pathogenesis of PKD. In our previous study, we identified PRRT2 as the causative gene of PKD. We have generated PRRT2 knock-out mice by CRISPR/Cas9 system and found that SNARE complexes in PRRT2 KO mice were increased. In addition, the mRNA and protein level of RIM-BP1, a component of active zones complexes that regulates neurotransmitter releasing, were also increased significantly in the PRRT2 KO mice. Based on our previous studies, this project will investigate the specific molecular mechanism how PRRT2 regulates the SNARE complexes assembly, vesicle docking, Ca2+ channels localization and transients and neurotransmitter releasing through the transcription of RIM-BP1. The proposed study aims to provide new information in the pathogenesis of PKD.

发作性运动诱发性运动障碍（paroxysmal kinesigenic dyskinesia，PKD）是一种具有高度临床异质性的运动障碍性疾病。目前国际上有关PKD的发病机制报道较少。本项目前期研究证明了PRRT2基因是PKD的致病基因，利用CRISPR/Cas9技术我们构建了PRRT2基因敲除小鼠，发现PRRT2功能缺失导致SNARE复合物表达显著增加。有意思的是，参与调节神经递质释放的RIM-BP1基因的mRNA和蛋白水平在PRRT2基因敲除小鼠中也显著升高。本项目将在上述研究的基础上，进一步研究PRRT2是否通过调节RIM-BP1的转录来调控囊泡锚靠、钙离子通道定位与开放以及递质释放，以期阐明PRRT2基因的具体分子调控机制，对揭示PKD的致病机理具有重要的意义。

发作性运动诱发性运动障碍（paroxysmal kinesigenic dyskinesia, PKD）是一种具有高度临床异质性的运动障碍性疾病。我们在前期研究中证明了PRRT2基因是PKD的致病基因，本项目在此基础上，进一步构建了PRRT2敲除和PRRT2 C.649dupC敲入两种PKD疾病小鼠模型。行为学分析发现，四周龄的PRRT2敲除和敲入小鼠均表现出一定的运动缺陷，且药物诱发的癫痫易感性增加。病理学分析发现，PRRT2敲除和敲入小鼠中突触前膜囊泡总数和锚定在活性区的囊泡数目都显著高于对照组；SNARE复合物的表达显著高于野生型小鼠，而组成复合物的单体SNAP25的表达显著低于野生型小鼠。同时，本项目研究发现PRRT2 C.649dupC突变导致其mRNA稳定性降低从而引起PRRT2蛋白失去功能，PRRT2的功能缺失引起RIM-BP1转录和翻译的上调，导致钙离子通道开放异常引起胞内钙离子释放过度，本项目为阐明PKD发生的病理机制提供了较强的理论依据。