LRRK2 enhances oxidative stress-induced neurotoxicity via its kinase activity
LRRK2 enhances oxidative stress-induced neurotoxicity via its kinase activity
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DOI：
10.1016/j.yexcr.2009.09.014
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发表时间：
2010-02-15
影响因子：
3.7
通讯作者：
Seol, Wongi
中科院分区：
文献类型：
--
作者：
Heo, Hye Young;Park, Ji-Min;Seol, Wongi
LRRK 2是一种常染色体显性基因，其突变会导致家族性帕金森病（PD）。LRRK 2蛋白含有功能性激酶和GT3结构域。由LRRK 2突变引起的PD表型与特发性PD的表型相似，表明LRRK 2是PD发病机制的重要参与者。在LRRK 2的PD特异性突变中，G2019 S是最常见的突变。已知其过表达与野生型（WT）LRRK 2相比增加激酶活性和神经毒性。在这里，使用一个简单的比色细胞活力测定，我们分析了LRRK 2的多巴胺能SN 4741细胞与过氧化氢处理后的神经毒性。当WT、G2019 S或空载体在SN 4741细胞中表达时，细胞死亡以G2019 S> WT >载体的顺序适度且显著地增加。当这些转染细胞用过氧化氢处理以模拟氧化应激时，细胞神经毒性以相同的顺序增强（即C2019 S> WT >载体）。此外，将SN 4741细胞与来自表达G2019 S并经受过氧化氢处理的细胞的条件培养基一起孵育，显示出比来自用载体或WT转染的细胞的条件培养基多10-15%的细胞死亡，表明表达G2019 S的细胞分泌影响邻近细胞活力的因子。激酶结构域被映射为负责氧化应激诱导的神经毒性。此外，WT和C2019 S LRRK 2的过表达导致细胞内活性氧（ROS）以G2019 S> WT的顺序微弱但显著地增加，如在存在和不存在H2 O2处理的情况下通过DCFH-DA测定所测量的。此外，在表达G2019 S的细胞中，抗氧化剂蛋白DJ-I或ERK抑制剂处理的共表达将存活率恢复到与在H2 O2处理下用对照载体转染的细胞相似的水平。综上所述，我们的数据表明，LRRK 2激酶结构域增加ROS的产生，并在H2 O2处理下引起增强的神经毒性，这至少部分地被DJ-I或ERK抑制剂所挽救。(C)2009 Elsevier Inc. All rights reserved.
LRRK2 is art autosomal dominant gene whose mutations cause familial Parkinson's disease (PD). The LRRK2 protein contains a functional kinase and a GTPase domain. PD phenotypes caused by LRRK2 Mutations are similar to those of idiopathic PD, implying that LRRK2 is an important participant in PD pathogenesis. Of LRRK2's PD-specific mutations, the G2019S is the most frequently observed one. Its over-expression is known to increase kinase activity and neurotoxicity compared to wild type (WT) LRRK2. Here, using a simple colorimetric cell viability assay, we analyzed LRRK2's neurotoxicity in dopaminergic SN4741 cells following treatment with hydrogen peroxide. When WT, G2019S, or empty vector was expressed in SN4741 cells, cell death was modestly and significantly increased in the order of G2019S > WT > vector. When these transfected cells were treated with hydrogen peroxide to mimic oxidative stress, cellular neurotoxicity was enhanced in the same order (i.e. C2019S > WT > vector). Moreover, incubation of SN4741 cells with conditioned medium from cells expressing G2019S and Subjected to hydrogen peroxide treatment exhibited 10-15% more cell death than conditioned medium from cells transfected with vector or WT, suggesting that G2019S-expressing cells secrete a factor(s) affecting viability of neighboring cells. The kinase domain was mapped to be responsible for oxidative stress-induced neurotoxicity. In addition, over-expression of WT and C2019S LRRK2 lead to a weak, but significant, increase in intracellular reactive oxygen species (ROS) in the order of G2019S > WT as measured by DCFH-DA assay in both the presence and absence of H2O2 treatment. Furthermore, in G2019S-expressing cells, co-expression of the anti-oxidant Protein DJ-I OF ERK inhibitor treatment restored survival rate to a level similar to that of cells transfected with control vector under H2O2 treatment. Taken together, our data suggest that the LRRK2 kinase domain increases the generation of ROS and causes enhanced neurotoxicity under H2O2 treatment, which call be at least partially rescued by DJ-I or the ERK inhibitor. (C) 2009 Elsevier Inc. All rights reserved.