No Dopamine Cell Loss or Changes in Cytoskeleton Function in Transgenic Mice Expressing Physiological Levels of Wild Type or G2019S Mutant LRRK2 and in Human Fibroblasts

Mutations within the LRRK2 gene have been identified in Parkinson’s disease (PD) patients and have been implicated in the dysfunction of several cellular pathways. Here, we explore how pathogenic mutations and the inhibition of LRRK2 kinase activity affect cytoskeleton dynamics in mouse and human cell systems. We generated and characterized a novel transgenic mouse model expressing physiological levels of human wild type and G2019S-mutant LRRK2. No neuronal loss or neurodegeneration was detected in midbrain dopamine neurons at the age of 12 months. Postnatal hippocampal neurons derived from transgenic mice showed no alterations in the seven parameters examined concerning neurite outgrowth sampled automatically on several hundred neurons using high content imaging. Treatment with the kinase inhibitor LRRK2-IN-1 resulted in no significant changes in the neurite outgrowth. In human fibroblasts we analyzed whether pathogenic LRRK2 mutations change cytoskeleton functions such as cell adhesion. To this end we compared the adhesion characteristics of human skin fibroblasts derived from six PD patients carrying one of three different pathogenic LRRK2 mutations and from four age-matched control individuals. The mutant LRRK2 variants as well as the inhibition of LRRK2 kinase activity did not reveal any significant cell adhesion differences in cultured fibroblasts. In summary, our results in both human and mouse cell systems suggest that neither the expression of wild type or mutant LRRK2, nor the inhibition of LRRK2 kinase activity affect neurite complexity and cellular adhesion.LRRK2基因的突变已在帕金森病（Parkinson’s disease，PD）患者中被检出，并被证实与多条细胞通路的功能异常相关。本研究旨在探究致病性LRRK2突变与LRRK2激酶活性抑制，对小鼠及人类细胞系统中细胞骨架动态的影响。我们构建并表征了一种新型转基因小鼠模型，该模型可表达生理水平的人类野生型与G2019S突变型LRRK2。在该模型12月龄时，未在中脑多巴胺能神经元中检测到神经元丢失或神经退行性病变。从该转基因小鼠中分离的产后海马神经元，经高内涵成像（high content imaging）自动采集数百个神经元的神经突生长相关参数，共检测7项指标，未发现任何显著变化。使用激酶抑制剂LRRK2-IN-1处理后，神经突生长亦未出现显著改变。在人类成纤维细胞实验中，我们分析了致病性LRRK2突变是否会改变细胞黏附等细胞骨架相关功能。为此，我们比较了6名携带3种不同致病性LRRK2突变的PD患者，以及4名年龄匹配的健康对照个体的皮肤成纤维细胞的黏附特性。突变型LRRK2变体以及LRRK2激酶活性抑制，均未在培养的成纤维细胞中引发显著的细胞黏附差异。综上，我们在人类与小鼠细胞系统中的研究结果表明，无论是表达野生型或突变型LRRK2，还是抑制LRRK2激酶活性，均不会影响神经突复杂性与细胞黏附功能。