CDR1as与Cyrano在帕金森病模型中调控α-突触核蛋白积聚的作用及机制研究

In Parkinson's disease (PD), aggregated alpha-synuclein (α-syn) causes dopaminergic neuronal loss in the substantia nigra. It is reported that microRNA-7(miR-7) plays an important role in inhibiting the abnormal accumulation of α-syn and dopaminergic neuronal loss in PD. Both circular RNA CDR1as and long non-coding RNA Cyrano are identified as the two most important regulatory factors of miR-7 by CLIP-Seq technology. Circular RNA CDR1as, which functions as miR-7 sponge, is found to storage miR-7, while long non-coding RNA Cyrano is found to degrade miR-7 via base pairing. In our previous studies, significantly decreased miR-7 was observed in the substantia nigra of PD mice with α-syn overexpression. However, the roles of CDR1as and Cyrano in PD are still unclear. In this project, we will construct the PD cell and mice models with α-syn overexpression and further investigate that CDR1as was degraded by the upstream molecule microRNA-671 and release miR-7, which inhibited dopaminergic neuronal loss in substantia nigra and improved animal behavioral disorders. Also, we will explore whether decreased Cyrano promote the expression of miR-7, then produce the same effects as above. All these experiments will elucidate the upstream molecular mechanism that contributes to the accumulation of α-syn in dopaminergic neuronal loss, which will provide new thoughts and new targets to develop drugs for PD treatment.

帕金森病（PD）中α-突触核蛋白（α-syn）积聚导致黑质多巴胺能神经元（DNs）变性丢失。miR-7是抑制α-syn积聚与DNs变性丢失的关键分子；CDR1as作为miR-7海绵贮存miR-7，而Cyrano靶向降解miR-7，且二者被CLIP-Seq技术证实为miR-7最重要的两个调控因素。前期研究表明α-syn过表达的PD小鼠中脑黑质区miR-7表达显著降低，但CDR1as与Cyrano是否通过miR-7调控α-syn的积聚并参与PD发病，目前尚不清楚。本研究在α-syn过表达的PD细胞模型和小鼠模型中，探讨CDR1as的上游分子miR-671降解CDR1as，释放miR-7抑制α-syn积聚，从而改善DNs变性丢失和动物行为学障碍；并探讨降低Cyrano的表达也可上调miR-7发挥上述调节作用；阐明miR-7调控α-syn积聚的上游分子机制，为寻找防治PD新靶点提供实验依据。

帕金森病（PD）中α-突触核蛋白（α-syn）积聚导致黑质多巴胺（DA）能神经元变性丢失。miR-7是抑制α-syn聚集与DA能神经元变性丢失的关键分子；CDR1as作为miR-7海绵贮存miR-7，而 Cyrano靶向降解miR-7，且二者被CLIP-Seq技术证实为miR-7最重要的两个调控因素，而ZSWIM8蛋白也是调控miR-7表达的重要因子。因此阐明miR-7调控α-syn聚集的上游分子机制有利于寻找防治PD的新靶点。在MPTP模型小鼠黑质与MPP+处理的小鼠神经母细胞瘤N2a细胞中检测到miR-7显著下降，在MPP+处理的N2a细胞中检测到CDR1as随着MPP+处理时间的延长先降低后升高，MPTP模型小鼠黑质中ZSWIM8表达明显升高，提示PD发病过程中miR-7的表达降低可能与CDR1as基因和ZSWIM8蛋白表达变化有关，进而参与PD发病。抑制线粒体电压依赖性阴离子通道1VDAC1的表达可阻断MPP+诱导的线粒体损伤，在MPP+处理的N2a细胞中高表达miR-7后观察到线粒体VDAC1和Bax蛋白水平下降，SOD表达增加，ROS释放减少，提示miR-7的神经保护作用机制可能与减少线粒体氧化应激有关。IL-1β、TNFα等促炎因子可加剧PD发病进程，在LPS处理的小鼠小胶质细胞系BV2细胞中，高表达miR-7后观察到Nod样受体蛋白3(NLRP3)、TNF-α和IL-1β蛋白水平显著下降，提示miR-7可能通过靶向抑制NF-kappaB(P65)/NLRP3的表达间接下调IL-1β、TNFα等促炎因子的表达，推测miR-7的神经保护作用机制可能与抑制神经炎症有关。除此以外，本项目还探究了降低α-syn聚集的制剂通过诱导自噬来保护DA能神经元，细胞外α-syn通过内质网应激调节DA能神经元的铁代谢相关蛋白。本项目利用RNA测序技术与生物信息学相结合的方法，在SNCA基因高表达的PD大鼠黑质中筛选得到了新的非编码长链RNA与环状RNA，以期为PD的防治提供靶点。