Golden-talen技术构建斑马鱼稳定敲除品系研究Trx2功能缺陷在帕金森病诱发中的作用

Parkinson's disease (PD) belongs to the chronic neurodegenerative diseases, which has a high incidence in old people. To date, it has been demonstrated that the mitochondria dysfunction and the accumulated ROS account for the pathogenesis of PD. Trx2 is one member of Trxs family, which locates in mitochondria and is responsible for the ROS scavenging. In our preliminary study, we found that Trx2 had a high expression in the brain of zebrafish. By injection with morpholino (MO), we further found that the Trx2 morphants showed a phenotype same as the Pink1 morphants reported before. It has been reported that Pink1 mutations are responsible for recessive inherited PD. Meanwhile，Trx2 knockdown in zebrafish led to reduced ΔΨm and ROS accumulation. In this proposal, we will generate the Trx2 homozygous and heterozygous null zebrafish lines by the golden-talen technology. Utilizing the knock-out zebrafish strains, we will determine the role of Trx2 dysfunction in the pathogenesis of PD. We also assess how the oxidative stress and drugs play a role in the induction of PD and the aging factor will also be explored .The second aim in our study is to explore the relevance between Trx2 and Pink1 in the pathogenesis of PD. Because of the reported data of their function and our preliminary study, we infer that Trx2 may be phosphorylated by Pink1. The results from this proposal will provide reliable and innovated theoretical basis for PD therapeutic treatment.

帕金森病（Parkinson's disease，PD）属于神经退行性疾病，在中老年人中发病率较高。目前研究认为，帕金森病的发生与线粒体功能失调及ROS积累相关。Trx2是定位于线粒体中负责ROS清除的硫氧还蛋白。在前期工作中，我们发现Trx2在模式生物斑马鱼的脑部有非常高的表达。随后，利用Morpholino技术在斑马鱼中进行Trx2敲低实验，我们发现出现PD相关表型，该表型类似于遗传性PD致病基因Pink1在斑马鱼中敲低的表型。因此在本课题中，我们将利用golden talen技术建立稳定的Trx2敲除品系，分析PD相关表型，分析氧化压力，药物和年龄因素对该品系PD诱发的影响。其次，根据我们的工作基础和已报道的二者功能分析，我们认为Pink1可能对Trx2进行磷酸化修饰。我们将探讨Pink1与Trx2在帕金森病诱发中的关系。该课题的研究将为预防和治疗帕金森病提供新的思路和方向。

随着我国社会老龄化趋势越发明显，帕金森等神经退行性疾病问题日趋严重。研究表明，线粒体功能异常导致的ROS积累是帕金森疾病发生的重要原因。线粒体特异性的硫氧还蛋白Trx2在清除ROS，抵抗线粒体介导的凋亡等方面发挥重要作用，但其在帕金森疾病发生中的功能未知。前期实验中我们通过Morpholino技术在斑马鱼中进行Trx2敲低实验，发现斑马鱼出现帕金森疾病类似表型，但Morpholino技术的时效性等特点限制对其功能进行深入研究。该项目中，我们分别利用golden-talen和CRISPR-Cas9技术尝试构建Trx2的敲除斑马鱼模型，但均出现胚胎致死的现象。为克服这一问题，我们正构建组织特异性Trx2敲除的斑马鱼模型及Trx2活性位点C89/C92双突变的斑马鱼模型。同时，利用SY5Y细胞，我们发现①Trx2在SH-SY5Y细胞中抵抗TBH诱导的凋亡；②SH-SY5Y细胞中Trx2参与调控TBH诱导的内质网压力激活；③SH-SY5Y细胞中Trx2参与调控TBH诱导的自噬流过程中；④SH-SY5Y细胞中Trx2抵抗CCCP诱导的线粒体自噬过程；⑤小分子伴侣alpha B-crystallin上调Trx2的表达，二者可能参与线粒体凋亡信号通路中。总之，利用斑马鱼和SH-SY5Y细胞模型，我们研究了Trx2在过氧化刺激对凋亡、内质网压力、线粒体自噬及溶酶体活性等信号通路中的调控作用，为理解帕金森疾病的发生原因提供新的思路。