Institute of Parasitology and Biomedicine "López-Neyra", Consejo Superior de Investigaciones Científicas (CSIC), Avda del Conocimiento s/n, 18016, Granada, Spain.
Department of Experimental Tumorbiology, Westfälische Wilhelms University Münster, Münster, Germany.
Mol Neurodegener. 2018 Jan 23;13(1):3. doi: 10.1186/s13024-018-0235-y.
Mutations in LRRK2 are a common genetic cause of Parkinson's disease (PD). LRRK2 interacts with and phosphorylates a subset of Rab proteins including Rab8a, a protein which has been implicated in various centrosome-related events. However, the cellular consequences of such phosphorylation remain elusive.
Human neuroblastoma SH-SY5Y cells stably expressing wildtype or pathogenic LRRK2 were used to test for polarity defects in the context of centrosomal positioning. Centrosomal cohesion deficits were analyzed from transiently transfected HEK293T cells, as well as from two distinct peripheral cell types derived from LRRK2-PD patients. Kinase assays, coimmunoprecipitation and GTP binding/retention assays were used to address Rab8a phosphorylation by LRRK2 and its effects in vitro. Transient transfections and siRNA experiments were performed to probe for the implication of Rab8a and its phosphorylated form in the centrosomal deficits caused by pathogenic LRRK2.
Here, we show that pathogenic LRRK2 causes deficits in centrosomal positioning with effects on neurite outgrowth, cell polarization and directed migration. Pathogenic LRRK2 also causes deficits in centrosome cohesion which can be detected in peripheral cells derived from LRRK2-PD patients as compared to healthy controls, and which are reversed upon LRRK2 kinase inhibition. The centrosomal cohesion and polarity deficits can be mimicked when co-expressing wildtype LRRK2 with wildtype but not phospho-deficient Rab8a. The centrosomal defects induced by pathogenic LRRK2 are associated with a kinase activity-dependent increase in the centrosomal localization of phosphorylated Rab8a, and are prominently reduced upon RNAi of Rab8a.
Our findings reveal a new function of LRRK2 mediated by Rab8a phosphorylation and related to various centrosomal defects.
LRRK2 突变是帕金森病（PD）的常见遗传原因。LRRK2 与包括 Rab8a 在内的一组 Rab 蛋白相互作用并使其磷酸化，Rab8a 蛋白与各种中心体相关事件有关。然而，这种磷酸化的细胞后果仍不清楚。
使用稳定表达野生型或致病性 LRRK2 的人神经母细胞瘤 SH-SY5Y 细胞来测试中心体定位时的极性缺陷。瞬时转染 HEK293T 细胞以及源自 LRRK2-PD 患者的两种不同的周围细胞类型分析中心体粘连缺陷。激酶测定、共免疫沉淀和 GTP 结合/保留测定用于研究 LRRK2 对 Rab8a 的磷酸化及其在体外的作用。瞬时转染和 siRNA 实验用于研究 Rab8a 及其磷酸化形式在致病性 LRRK2 引起的中心体缺陷中的作用。
在这里，我们表明致病性 LRRK2 导致中心体定位缺陷，从而影响轴突生长、细胞极化和定向迁移。致病性 LRRK2 还导致中心体粘连缺陷，与健康对照相比，源自 LRRK2-PD 患者的周围细胞中可以检测到这种缺陷，并且在 LRRK2 激酶抑制后可以逆转。当与野生型 Rab8a 而不是磷酸化缺陷型 Rab8a 共表达时，可以模拟野生型 LRRK2 引起的中心体粘连和极性缺陷。致病性 LRRK2 诱导的中心体缺陷与激酶活性依赖性的磷酸化 Rab8a 在中心体定位的增加有关，并且在 Rab8a 的 RNAi 后明显减少。
我们的发现揭示了 LRRK2 通过 Rab8a 磷酸化介导的新功能，与各种中心体缺陷有关。
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