DNA甲基化调控α-突触核蛋白的体内研究及其对帕金森病发病的影响

α-synuclein, encoded by the SNCA gene, is the core pathogenic protein leading to Parkinson’s disease (PD). Previous human results have suggested that the expression of SNCA is regulated by CpG island methylation in its intron 1, and the methylation level is decreased in brain tissues of PD patients. We further found that SNCA methylation was regulated by miR-17 mediated DNMT1. However, it is still in the blank whether the abnormal SNCA methylation plays a key role in vivo regulating α-synuclein expression and inducing PD pathogenesis. Unlike human, the SNCA gene in model organisms such as mouse does not contain the human-like CpG island, which limits the in vivo investigation of the above questions and related interventions. Therefore, on the basis of previous work, the current project aims to use CRISPR-Cas9 techniques to knock in multiple different CpG islands into murine SNCA intron 1 in murine cells, studying their regulating effects, and then to construct and identify the CpG island knockin mice which by methylation can regulate SNCA expression, thereafter to study the impact of SNCA methylation on PD pathogenesis, and last to study the miR-17 mediated in vivo regulation pathway as well as the impact of its interference on PD progression. Our findings will have great implications in understanding PD epigenetic mechanisms, and the constructed knockin mice can apply to related methylation and intervention studies.

α-突触核蛋白是帕金森病（PD）的核心致病蛋白，由SNCA基因编码表达。人源细胞研究显示，SNCA表达受其1号内含子CpG岛甲基化调控，且甲基化率在PD患者脑组织中降低。课题组进一步发现，细胞中SNCA甲基化受miR-17介导的DNMT1调控。但是在体内，SNCA甲基化降低是否对其表达和PD的发病起关键作用尚处空白。与人类不同，小鼠等模式动物的SNCA基因不含有类似CpG岛，限制了上述问题的体内探索和干预研究。因此，本项目拟在前期工作基础上，应用CRISPR-Cas9技术，先在鼠源细胞对其SNCA 1号内含子做多种CpG岛敲入及其调控作用研究，进而构建和鉴定可甲基化调控SNCA表达的CpG岛敲入小鼠，研究SNCA甲基化对PD发病的影响，阐明miR-17介导的调控途径及其干预对PD发病的作用。研究成果对认识PD的表观遗传调控具有重要的理论意义，所构建小鼠可应用于相关甲基化机制和干预研究。

帕金森病（Parkinson’s disease, PD）是一种常见的呈进行性发展的神经退行性疾病，其主要病理特征为黑质致密部多巴胺能神经元的选择性丢失和胞体内路易氏小体的形成（核心成分是α-synuclein）。PD的发病机制目前尚未完全清楚，普遍的观点认为多种环境和遗传因素共同参与PD的发病过程。表观遗传是介导环境因素和遗传因素发挥共同作用的中间环节，其中DNA甲基化被认为在调控PD相关基因的表达上发挥了重要的作用。α-synuclein是PD的核心致病蛋白，由SNCA基因编码表达。人源细胞水平相关研究表明SNCA基因的表达可受位于其1号内含子CpG岛的甲基化调控。尸检结果表明该CpG岛的甲基化水平在PD患者的脑组织中是降低的。对上述调控关系需进一步在动物体内进行实验研究。而小鼠等常用模式动物Snca 1号内含子并不存在与人类SNCA基因相似的甲基化岛, 限制了上述问题的体内探索和干预研究。本项目在小鼠神经母细胞瘤细胞系（N2a）中应用CRIPSR/Cas9系统构建了两种不同CpG岛敲入Snca 1号内含子模型，发现人源CpG岛敲入对Snca表达具有调控作用。基于细胞中的结果成功构建人源CpG岛敲入小鼠模型，初步的研究表明该CpG岛具有与人类体内相似的作用特征。该小鼠模型的成功构建对体内研究Snca的甲基化调控作用具有重要的意义。