富含亮氨酸的重复激酶2(LRRK2)与多巴胺能神经元对氧化应激(OS)的脆弱性有关,线粒体损伤,特发性和家族性帕金森病(PD)的细胞死亡增加。然而,这种激酶究竟是如何参与OS-线粒体-凋亡连接的,目前尚不清楚.我们在人胚肾细胞系293(HEK-293)中使用成簇的规则间隔短回文重复序列(CRISPR)/Cas9LRRK2敲除(KO)来评估对线粒体抑制剂复合物I鱼藤酮(ROT)的细胞反应,众所周知的OS和细胞死亡诱导剂。我们报告了使用CRISPR编辑在HEK-293细胞中成功敲除LRRK2基因(ICE,约60%)和流式细胞术(81%)分析。我们发现HEK-293LRRK2WT细胞暴露于鱼藤酮(ROT,50μM)导致细胞内活性氧(ROS,+7400%);氧化的DJ-1-Cys106-SO3(+52%);LRRK2(+70%)和c-JUN(+171%)的磷酸化;肿瘤蛋白表达增强(TP53,+2000%),p53上调细胞凋亡调节剂(PUMA,+1950%),和帕金(PRKN,+22%);caspase3的激活(CASP3,+8000%),DNA片段化(+35%)和线粒体膜电位降低(ΔkW,-58%)和PTEN诱导的推定激酶1(PINK1,-49%),与未处理的细胞相比。通过与外膜20(TOM20)的转位酶共定位,还观察到了细胞质裂变蛋白动力蛋白相关蛋白1(DRP1)向线粒体的易位。出色的,用ROT处理的HEK-293LRRK2KO细胞显示未改变的OS和凋亡标志物。我们得出结论,LRRK2的丢失导致HEK-293对ROT诱导的OS具有抗性,线粒体损伤,和体外凋亡。我们的数据支持以下假设:LRRK2通过调节线粒体蛋白充当促凋亡激酶(例如,PRKN,PINK1、DRP1和PUMA),转录因子(例如,c-JUN和TP53),和CASP3在应激条件下的细胞。一起来看,这些观察结果表明,LRRK2是PD发病机制中的重要激酶。
Leucine-rich repeat kinase 2 (LRRK2) has been linked to dopaminergic neuronal vulnerability to oxidative stress (OS), mitochondrial impairment, and increased cell death in idiopathic and familial Parkinson\'s disease (PD). However, how exactly this kinase participates in the OS-mitochondria-apoptosis connection is still unknown. We used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 LRRK2 knockout (KO) in the human embryonic kidney cell line 293 (HEK-293) to evaluate the cellular response to the mitochondrial inhibitor complex I rotenone (ROT), a well-known OS and cell death inducer. We report successful knockout of the LRRK2 gene in HEK-293 cells using CRISPR editing (ICE, approximately 60%) and flow cytometry (81%) analyses. We found that HEK-293 LRRK2 WT cells exposed to rotenone (ROT, 50 μM) resulted in a significant increase in intracellular reactive oxygen species (ROS, +7400%); oxidized DJ-1-Cys106-SO3 (+52%); phosphorylation of LRRK2 (+70%) and c-JUN (+171%); enhanced expression of tumor protein (TP53, +2000%), p53 upregulated modulator of apoptosis (PUMA, +1950%), and Parkin (PRKN, +22%); activation of caspase 3 (CASP3, +8000%), DNA fragmentation (+35%) and decreased mitochondrial membrane potential (ΔΨm, -58%) and PTEN induced putative kinase 1 (PINK1, -49%) when compared to untreated cells. The translocation of the cytoplasmic fission protein dynamin-related Protein 1 (DRP1) to mitochondria was also observed by colocalization with translocase of the outer membrane 20 (TOM20). Outstandingly, HEK-293 LRRK2 KO cells treated with ROT showed unaltered OS and apoptosis markers. We conclude that loss of LRRK2 causes HEK-293 to be resistant to ROT-induced OS, mitochondrial damage, and apoptosis in vitro. Our data support the hypothesis that LRRK2 acts as a proapoptotic kinase by regulating mitochondrial proteins (e.g., PRKN, PINK1, DRP1, and PUMA), transcription factors (e.g., c-JUN and TP53), and CASP3 in cells under stress conditions. Taken together, these observations suggest that LRRK2 is an important kinase in the pathogenesis of PD.