PLA2G6基因突变所致的炎症反应在帕金森病发病机制中的作用

PLA2G6 was reported to be the causative gene for a form of autosomal recessive early-onset parkinsonism and sporadic early-onset parkinsonism in our previous studies, but the exact mechanism of PLA2G6 mutation is still unknown. It was reported that the inflammatory response exist in the brain of patients with PLA2G6 mutation and PLA2G6 knockout mouse, which suggested that inflammation played an important role in the pathogenesis of PLA2G6 mutation, but so far the exact mechanism of inflammation and the downstream signaling pathways remain unclear. In this study, we will verify the inflammatory response by detecting the PLA2G6 knockout mouse (PLA2G6-/-) which we already purchased last year. And then we will verify whether TLRs are involved in the astrocytes and microglia which were isolated from PLA2G6-/- mice and looking for the downstream signaling pathways. We will further explore whether Nurr1-CoREST, DRD2-CRYAB and NLRs participate the inflammatory response and whether these anti-inflammatory medium antagonize the pro-inflammatory factors which are lethal to dopamine neurons. The main objective of this study is to further study the inflammation in the pathogenesis of PD caused by PLA2G6 dysfunction.

前期本研究小组发现PLA2G6基因突变可导致常染色体隐性遗传早发性帕金森综合征及散发早发性帕金森综合征。研究发现PLA2G6基因突变患者脑组织及敲除小鼠模型脑组织中均存在炎症反应，提示炎症在PLA2G6基因突变的致病机制中存在作用，但其具体致病机制仍不明确。本研究拟在前期研究基础上，利用已获得的PLA2G6基因敲除小鼠模型进一步验证小鼠脑内炎症反应，明确PLA2G6-/-小鼠原代培养的星形胶质细胞和小胶质细胞炎症反应是否存在TLRs的参与，以及其下游激活蛋白，在此基础上筛选出TLRs相关的信号转导通路，进一步探讨Nurr 1-CoREST、DRD2-CRYAB、NLRs是否参与PLA2G6所致的炎症反应及其对多巴胺能神经元的损伤作用，深入探讨PLA2G6所致炎症反应在帕金森病发病机制中的作用。

前期本研究小组发现PLA2G6基因突变可导致常染色体隐性遗传性早发性帕金森综合征及散发性早发性帕金森综合征。研究发现PLA2G6基因突变患者脑组织及敲除小鼠模型脑组织中均存在炎症反应，提示炎症在PLA2G6基因突变的致病机制中存在作用，但至今其具体致病机制仍不明确。本项目利用CRISPR/Cas9打靶技术成功构建了PLA2G6基因羧基端KO小鼠模型，并在DNA、RNA和蛋白水平上获得证实，行为学上发现13月龄小鼠开始出现类帕金森病样表现，并逐渐加重。应用qRT-PCR对6月龄PLA2G6基因KO小鼠大脑皮层、海马、中脑、小脑组织进行促炎因子和抗炎因子的检测，发现促炎因子GFAP、iba1、IL-1β、IL-6等表达上调，而抗炎因子IL-14、TGF-β等存在表达下调，提示PLA2G6基因KO小鼠中存在炎症反应；应用qRT-PCR、Western Blot等定量检测方法发现TLRs家族中TLR7和TLR9表达异常；TLRs下游蛋白筛选中发现MyD88、MAL和TRAM表达不存在差异，而TRAM2表达存在异常；TLRs相关信号转导通路筛选中发现NF-kB、ERK1/2、JNK2、AKT1/3、JAK1和STAT1/5B表达存在差异；Nurr 1、CRYAB、NLRX1表达不存在差异，NLRC5存在下调，以上结果提示PLA2G6基因KO小鼠中iPLA2β的缺失可改变TLR7和TLR9的表达，从而诱导TRAM2，激活NF-κB信号转导通路、MAP激酶通路、PI3K-Akt信号转导通路和JAK/STAT信号转导通路，共同参与神经炎症的发生，而Nurr 1-CoREST、DRD2-CRYAB、NLRs等抗炎蛋白未被激活。此外，应用芯片技术对PLA2G6基因KO小鼠模型黑质基因表达谱进行研究，发现存在钙离子信号通路、PI3K-Akt信号通路、神经营养因子信号通路等通路的异常，进一步支持炎症信号通路参与了PLA2G6基因突变的致病过程，为深入探讨PLA2G6基因突变的致病机制提供了线索。