Interrogating Parkinson's disease LRRK2 kinase pathway activity by assessing Rab10 phosphorylation in human neutrophils.
Interrogating Parkinson's disease LRRK2 kinase pathway activity by assessing Rab10 phosphorylation in human neutrophils.
复制标题
DOI：
10.1042/bcj20170803
复制
发表时间：
2018-01-02
期刊：
影响因子：
--
通讯作者：
Sammler EM
中科院分区：
文献类型：
--
作者：
Fan Y;Howden AJM;Sarhan AR;Lis P;Ito G;Martinez TN;Brockmann K;Gasser T;Alessi DR;Sammler EM
有令人信服的证据表明富亮氨酸重复激酶2 （LRRK2）的作用，特别是其激酶功能在帕金森病中的作用。口服生物可利用、脑渗透和有效的LRRK2激酶抑制剂正处于临床开发的后期阶段。在这里，我们描述了一种简单而强大的方法，通过测量LRRK2介导的Rab10在人外周血中性粒细胞中的磷酸化，来量化LRRK2激酶途径的活性。我们使用选择性MJFF-pRab10单克隆抗体识别被LRRK2磷酸化的Rab10 Thr73磷酸化表位。通过研究少数G2019S LRRK2相关和散发性帕金森病患者以及健康对照，我们强调了在临床环境中使用我们的检测方法的可行性和实用性。我们认为外周血中性粒细胞是LRRK2研究的宝贵资源，应考虑将其纳入帕金森病生物库收集，因为它们丰富、同质且表达相对较高水平的LRRK2和Rab10。相比之下，广泛使用的外周血单核细胞是异质的，只有少数细胞（单核细胞和污染中性粒细胞）表达LRRK2。虽然我们的LRRK2激酶途径分析可以帮助基于LRRK2激酶活性的患者分层，但我们设想它可能在未来LRRK2抑制剂试验的药效学和靶标参与研究中找到更大的用途。
There is compelling evidence for the role of the leucine-rich repeat kinase 2 (LRRK2) and in particular its kinase function in Parkinson's disease. Orally bioavailable, brain penetrant and potent LRRK2 kinase inhibitors are in the later stages of clinical development. Here, we describe a facile and robust assay to quantify LRRK2 kinase pathway activity by measuring LRRK2-mediated phosphorylation of Rab10 in human peripheral blood neutrophils. We use the selective MJFF-pRab10 monoclonal antibody recognising the Rab10 Thr73 phospho-epitope that is phosphorylated by LRRK2. We highlight the feasibility and practicability of using our assay in the clinical setting by studying a few patients with G2019S LRRK2 associated and sporadic Parkinson's as well as healthy controls. We suggest that peripheral blood neutrophils are a valuable resource for LRRK2 research and should be considered for inclusion in Parkinson's bio-repository collections as they are abundant, homogenous and express relatively high levels of LRRK2 as well as Rab10. In contrast, the widely used peripheral blood mononuclear cells are heterogeneous and only a minority of cells (monocytes and contaminating neutrophils) express LRRK2. While our LRRK2 kinase pathway assay could assist in patient stratification based on LRRK2 kinase activity, we envision that it may find greater utility in pharmacodynamic and target engagement studies in future LRRK2 inhibitor trials.