帕金森病致病基因LRRK2启动子关键顺式元件的确定及功能研究

Strong evidence has proved that a defect in the LRRK2 gene is a common, known genetic cause of Parkinson disease (PD). Mutations in LRRK2 are thus far the most prevalent genetic cause associated with autosomal dominant and idiopathic PD. Although the mechanism of how PD-associated LRRK2 mutations cause disease is yet unknown, It is thought that increased protein kinase activity and GTP bingding of LRRK2, and the changed function serve as a scaffolding protein contribute to the development of PD. several studies indicated that overexpress wildtype LRRK2 was found to be toxic in cultured cells and transgenic fly model. Becides, LRRK2 was found to be associated with abnormal protein deposition in the brains of patients with lowy body disease including PD. To overexpress wildtype LRRK2, G2019S mutant or kinase domain alone accelerate the formation of aggregates caused by α-synuclein A53T mutant. On the other hand, To knockout of LRRK2 and LRRK2 transgene animals results in the axonal growth damage, dysfunction of mitochondria and endocytosis, dysfunction of vesicles transportation and autophage. These results suggest that precise regulation of LRRK2 expression and function is necessary for maintaining homeostasis of the organism, and the disruption of the regulation mechanism may cause disease. Therefore, to elucidate the mechanism of LRRK2 transcriptional regulation is helpful to understand PD aetiology. In this study,we plan to isolate and characterize promoter, analyze cis-acting elements and their effects on promoter. We hope that results will elucidate the mechanism of activity regulation of LRRK2.

LRRK2基因是帕金森病（Parkinson Disease，PD）的重要致病基因，其导致PD发生的机制仍不清楚，目前认为LRRK2可能通过其激酶、GTP酶活性的改变，及支架蛋白功能的改变参与PD的发病。已有的研究发现过表达野生型LRRK2可引起细胞凋亡；敲除LRRK2基因或LRRK2转基因动物可导致其神经元轴突生长障碍、线粒体功能障碍、影响神经元胞吞作用、囊泡运输及自噬。说明LRRK2的表达水平决定神经元的细胞生物学功能。而转录调控机制决定LRRK2的表达水平，调控机制的异常可能导致LRRK2的表达和功能异常，最终导致疾病的发生。因此，有必要对LRRK2基因进行转录调控研究，并有望在转录中有目的地对该蛋白进行操控，人为干预其所涉及的多重功能。本课题组前期已经对LRRK2基因启动子的位置、活性和特征进行了分析，在本研究中，将进一步鉴定顺式元件并对其功能进行深入研究。

LRRK2是常染色体显性遗传性帕金森病（PD，Parkinson Disease）最常见的致病基因，该基因也与克罗恩病和麻风病的易感性相关。目前，我们对LRRK2基因突变导致PD发病机制了解甚少，此外对于LRRK2正常的生理功能以及其转录调控机制仍不清楚。在研究中我们分离了人类LRRK2基因核心启动子区，并鉴定转录因子Egr-1能够对LRRK2基因进行转录调节，LRRK2基因上游9个核苷酸GCGTGGGCG为Egr-1结合位点。进一步在EB病毒永生化的B淋巴细胞中干扰内源性Egr-1的表达后LRRK2的表达也降低，12-肉豆蔻佛波醇13乙酸酯（PMA）在细胞中能够快速诱导Egr-1的表达，进而诱导LRRK2基因表达上调。上述结果说明在B淋巴细胞中转录因子Egr-1对LRRK2基因具转录调节作用。.我们进一步研究了Egr-1和其靶基因LRRK2在B淋巴细胞中的功能，发现在EB病毒永生化的B淋巴细胞中LRRK2参与调节PMA诱导的IL-6的生产，表明在活化的B淋巴细胞中LRRK2参与调节炎症因子的生成。此外，干扰Egr-1或LRRK2的表达后细胞免疫球蛋白的分泌受到抑制，但不影响其生成，表明Egr-1/LRRK2通路对组成型免疫球蛋白的分泌有着重要调节作用，并且该调节功能与LRRK2的磷酸化活性相关。免疫荧光结果显示内源性LRRK2与晚期内吞体/溶酶体的标记物RAB7有较好的共定位，意味着LRRK2可能参与B淋巴细胞中的内吞/溶酶体途径。我们的研究表明LRRK2能够通过调节B细胞功能进而参与免疫反应，为进一步了解免疫系统中LRRK2的病理生理作用提供了新的研究方向。对来源于广西地区108例散发PD患者和197例健康人外周血gDNA对LRRK2基因ATG上游900bp区域进行突变筛查研究，共检出4个SNP位点、1个只仅出现在正常人对照中的变异和2个仅出现在PD患者中的变异，但所有变异基因型和等位基因频率在患者和正常人之间的并未见差别。