Fan Ying, Tonelli Francesca, Padmanabhan Shalini, Baptista Marco A S, Riley Lindsey, Smith Danielle, Marras Connie, Howden Andrew, Alessi Dario R, Sammler Esther
MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee.
The Michael J. Fox Foundation for Parkinson's Research.
J Vis Exp. 2020 Mar 21(157). doi: 10.3791/58956.
The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson' disease (PD) and all pathogenic LRRK2 mutations result in hyperactivation of its kinase function. Here, we describe an easy and robust assay to quantify LRRK2 kinase pathway activity in human peripheral blood neutrophils by measuring LRRK2-controlled phosphorylation of one of its physiological substrates, Rab10 at threonine 73. The immunoblotting analysis described requires a fully selective and phosphospecific antibody that recognizes the Rab10 Thr73 epitope phosphorylated by LRRK2, such as the MJFF-pRab10 rabbit monoclonal antibody. It uses human peripheral blood neutrophils, because peripheral blood is easily accessible and neutrophils are an abundant and homogenous constituent. Importantly, neutrophils express relatively high levels of both LRRK2 and Rab10. A potential drawback of neutrophils is their high intrinsic serine protease activity, which necessitates the use of very potent protease inhibitors such as the organophosphorus neurotoxin diisopropylfluorophosphate (DIFP) as part of the lysis buffer. Nevertheless, neutrophils are a valuable resource for research into LRRK2 kinase pathway activity in vivo and should be considered for inclusion into PD biorepository collections.
富含亮氨酸重复激酶2（LRRK2）是遗传性帕金森病（PD）中最常发生突变的基因，所有致病性LRRK2突变都会导致其激酶功能的过度激活。在此，我们描述了一种简便且可靠的检测方法，通过测量LRRK2控制的其生理底物之一Rab10在苏氨酸73处的磷酸化，来定量人外周血中性粒细胞中的LRRK2激酶途径活性。所描述的免疫印迹分析需要一种完全选择性的磷酸特异性抗体，该抗体能够识别由LRRK2磷酸化的Rab10 Thr73表位，例如MJFF-pRab10兔单克隆抗体。该方法使用人外周血中性粒细胞，因为外周血易于获取，且中性粒细胞是一种丰富且同质的成分。重要的是，中性粒细胞表达相对高水平的LRRK2和Rab10。中性粒细胞的一个潜在缺点是其较高的内在丝氨酸蛋白酶活性，这就需要使用非常有效的蛋白酶抑制剂，如有机磷神经毒素二异丙基氟磷酸酯（DIFP）作为裂解缓冲液的一部分。尽管如此，中性粒细胞仍是研究体内LRRK2激酶途径活性的宝贵资源，应考虑将其纳入PD生物样本库。
