Institute of Parasitology and Biomedicine López-Neyra (IPBLN), Consejo Superior de Investigaciones Científicas (CSIC), Granada, Spain.
Department of Pathology, Stanford University, Stanford, CA, USA.
J Parkinsons Dis. 2022;12(5):1423-1447. doi: 10.3233/JPD-213128.
Coding variation in the Leucine rich repeat kinase 2 gene linked to Parkinson's disease (PD) promotes enhanced activity of the encoded LRRK2 kinase, particularly with respect to autophosphorylation at S1292 and/or phosphorylation of the heterologous substrate RAB10.
To determine the inter-laboratory reliability of measurements of cellular LRRK2 kinase activity in the context of wildtype or mutant LRRK2 expression using published protocols.
Benchmark western blot assessments of phospho-LRRK2 and phospho-RAB10 were performed in parallel with in situ immunological approaches in HEK293T, mouse embryonic fibroblasts, and lymphoblastoid cell lines. Rat brain tissue, with or without adenovirus-mediated LRRK2 expression, and human brain tissues from subjects with or without PD, were also evaluated for LRRK2 kinase activity markers.
Western blots were able to detect extracted LRRK2 activity in cells and tissue with pS1292-LRRK2 or pT73-RAB10 antibodies. However, while LRRK2 kinase signal could be detected at the cellular level with over-expressed mutant LRRK2 in cell lines, we were unable to demonstrate specific detection of endogenous cellular LRRK2 activity in cell culture models or tissues that we evaluated.
Further development of reliable methods that can be deployed in multiple laboratories to measure endogenous LRRK2 activities are likely required, especially at cellular resolution.
与帕金森病（PD）相关的富含亮氨酸重复激酶 2 基因（LRRK2）的编码变异可促进编码 LRRK2 激酶的活性增强，尤其是 S1292 自身磷酸化和/或异源底物 RAB10 的磷酸化。
使用已发表的方案，确定野生型或突变型 LRRK2 表达情况下细胞 LRRK2 激酶活性测量的实验室间可靠性。
使用已发表的方案，在 HEK293T、鼠胚胎成纤维细胞和淋巴母细胞系中进行磷酸化 LRRK2 和磷酸化 RAB10 的基准 Western blot 评估，并进行原位免疫方法学评估。还评估了携带或不携带腺病毒介导的 LRRK2 表达的大鼠脑组织以及患有或不患有 PD 的人类脑组织中的 LRRK2 激酶活性标志物。
Western blot 能够使用 pS1292-LRRK2 或 pT73-RAB10 抗体检测细胞和组织中的提取 LRRK2 活性。然而，虽然在用细胞系中转染的突变型 LRRK2 可以在细胞水平上检测到 LRRK2 激酶信号，但我们无法证明在我们评估的细胞培养模型或组织中能够特异性检测内源性细胞 LRRK2 活性。
可能需要进一步开发可在多个实验室中部署以测量内源性 LRRK2 活性的可靠方法，特别是在细胞分辨率水平。
