Institute for Brain Science and Technology, Busan, South Korea.
Exp Cell Res. 2010 Feb 15;316(4):649-56. doi: 10.1016/j.yexcr.2009.09.014. Epub 2009 Sep 19.
LRRK2 is an 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. G2019S>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 G2019S 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 H(2)O(2) treatment. Furthermore, in G2019S-expressing cells, co-expression of the anti-oxidant protein DJ-1 or ERK inhibitor treatment restored survival rate to a level similar to that of cells transfected with control vector under H(2)O(2) treatment. Taken together, our data suggest that the LRRK2 kinase domain increases the generation of ROS and causes enhanced neurotoxicity under H(2)O(2) treatment, which can be at least partially rescued by DJ-1 or the ERK inhibitor.
LRRK2 是一种常染色体显性基因，其突变导致家族性帕金森病（PD）。LRRK2 蛋白包含一个功能性激酶和 GTP 酶结构域。由 LRRK2 突变引起的 PD 表型与特发性 PD 相似，这表明 LRRK2 是 PD 发病机制的重要参与者。在 LRRK2 的 PD 特异性突变中，G2019S 是最常见的一种。与野生型（WT）LRRK2 相比，其过表达已知会增加激酶活性和神经毒性。在这里，我们使用简单的比色细胞活力测定法，分析了过氧化氢处理后多巴胺能 SN4741 细胞中 LRRK2 的神经毒性。当 WT、G2019S 或空载体在 SN4741 细胞中表达时，细胞死亡按 G2019S>WT>载体的顺序适度且显著增加。当这些转染细胞用过氧化氢处理以模拟氧化应激时，细胞神经毒性也按相同顺序增强（即 G2019S>WT>载体）。此外，用来自表达 G2019S 并接受过氧化氢处理的细胞的条件培养基孵育 SN4741 细胞比用载体或 WT 转染的细胞的条件培养基表现出 10-15%更多的细胞死亡，表明表达 G2019S 的细胞分泌一种影响邻近细胞活力的因子（s）。激酶结构域被定位为负责氧化应激诱导的神经毒性。此外，用 DCFH-DA 测定法在存在和不存在 H（2）O（2）处理的情况下，WT 和 G2019S LRRK2 的过表达均导致细胞内活性氧（ROS）的弱但显著增加，其顺序为 G2019S>WT。此外，在 G2019S 表达细胞中，抗氧化蛋白 DJ-1 的共表达或 ERK 抑制剂处理可将存活率恢复至与 H（2）O（2）处理下转染对照载体的细胞相似的水平。总之，我们的数据表明，LRRK2 激酶结构域增加了 ROS 的产生，并导致 H（2）O（2）处理下神经毒性增强，这至少可以部分通过 DJ-1 或 ERK 抑制剂得到挽救。
