Janssen Pharmaceutical Companies of Johnson and Johnson, Department of CNS Research, Turnhoutseweg30, 2340 Beerse, Belgium.
J Pharm Biomed Anal. 2013 Mar 25;76:49-58. doi: 10.1016/j.jpba.2012.12.002. Epub 2012 Dec 16.
After the discovery of kinase activating mutations in leucine-rich repeat kinase 2 (LRRK2) as associated with autosomal dominant forms of Parkinson's disease, inhibition of the kinase is being extensively explored as a disease modifying strategy. As signaling properties and substrate(s) of LRRK2 are poorly documented, autophosphorylation has been an important readout for the enzyme's activity. Western blotting using anti-phospho-S910 or S935 LRRK2 antibodies showed effectiveness in demonstrating inhibitory effects of compounds. In this communication we describe two types of enzyme-linked immunosorbent assays (ELISA) to determine LRRK2 protein levels and kinase activity. Both assays take advantage of the sensitivity of the earlier described total and pS935 antibodies for detection (Nichols et al., Biochem. J. 2010) [10]. The first assay is based on anti-GFP-based capturing of overexpressed LRRK2 and is highly suitable to show cellular effects of kinase inhibitors in a 96-well format. In the other platform anti-LRRK2-based capturing allows detection of endogenously expressed LRRK2 in rat tissue with no significant signal in tissue from LRRK2 knockout rats. Furthermore, both assays showed a significant reduction in pS935 levels on cellular and transgenic R1441C/G LRRK2. With the anti-LRRK2 ELISA we were able to detect LRRK2 phosphorylation in human peripheral blood mononuclear cells (PBMC). To conclude, we report two sensitive assays to monitor LRRK2 expression and kinase activity in samples coming from cellular and in vivo experimental settings. Both can show their value in drug screening and biomarker development but will also be useful in the elucidation of LRRK2-mediated signaling pathways.
在发现富含亮氨酸重复激酶 2（LRRK2）中的激酶激活突变与常染色体显性形式的帕金森病相关后，抑制激酶作为一种疾病修饰策略正在被广泛探索。由于 LRRK2 的信号转导特性和底物（substrate）记录不佳，自身磷酸化一直是该酶活性的重要指标。使用抗磷酸化 S910 或 S935 LRRK2 抗体的 Western blot 显示了化合物的抑制效果。在本通讯中，我们描述了两种酶联免疫吸附测定（ELISA）来确定 LRRK2 蛋白水平和激酶活性。这两种测定都利用了之前描述的总抗体和 pS935 抗体的敏感性进行检测（Nichols 等人， Biochem. J. 2010）[10]。第一种测定基于 GFP 基的过表达 LRRK2 的捕获，非常适合在 96 孔格式中显示激酶抑制剂的细胞效应。在另一个平台上，基于抗 LRRK2 的捕获可以检测内源性表达的 LRRK2，而在 LRRK2 敲除大鼠的组织中没有明显的信号。此外，两种测定均显示在细胞和转基因 R1441C/G LRRK2 中 pS935 水平显著降低。使用抗 LRRK2 ELISA，我们能够检测到人外周血单核细胞（PBMC）中的 LRRK2 磷酸化。总之，我们报告了两种敏感的测定法，用于监测来自细胞和体内实验设置的样本中的 LRRK2 表达和激酶活性。这两种方法都可以在药物筛选和生物标志物开发中显示其价值，但对于阐明 LRRK2 介导的信号通路也将非常有用。
