PDF(Pubmed)
Abstract：
Parkinson\'s disease (PD) is a progressive late-onset neurodegenerative disease leading to physical and cognitive decline. Mutations of leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of PD. LRRK2 is a complex scaffolding protein with known regulatory roles in multiple molecular pathways. Two prominent examples of LRRK2-modulated pathways are Wingless/Int (Wnt) and nuclear factor of activated T-cells (NFAT) signaling. Both are well described key regulators of immune and nervous system development as well as maturation. The aim of this study was to establish the physiological and pathogenic role of LRRK2 in Wnt and NFAT signaling in the brain, as well as the potential contribution of the non-canonical Wnt/Calcium pathway. In vivo cerebral Wnt and NFATc1 signaling activity was quantified in LRRK2 G2019S mutant knock-in (KI) and LRRK2 knockout (KO) male and female mice with repeated measures over 28 weeks, employing lentiviral luciferase biosensors, and analyzed using a mixed-effect model. To establish spatial resolution, we investigated tissues, and primary neuronal cell cultures from different brain regions combining luciferase signaling activity, immunohistochemistry, qPCR and western blot assays. Results were analyzed by unpaired t-test with Welch\'s correction or 2-way ANOVA with post hoc corrections. In vivo Wnt signaling activity in LRRK2 KO and LRRK2 G2019S KI mice was increased significantly ~ threefold, with a more pronounced effect in males (~ fourfold) than females (~ twofold). NFATc1 signaling was reduced ~ 0.5-fold in LRRK2 G2019S KI mice. Brain tissue analysis showed region-specific expression changes in Wnt and NFAT signaling components. These effects were predominantly observed at the protein level in the striatum and cerebral cortex of LRRK2 KI mice. Primary neuronal cell culture analysis showed significant genotype-dependent alterations in Wnt and NFATc1 signaling under basal and stimulated conditions. Wnt and NFATc1 signaling was primarily dysregulated in cortical and hippocampal neurons respectively. Our study further built on knowledge of LRRK2 as a Wnt and NFAT signaling protein. We identified complex changes in neuronal models of LRRK2 PD, suggesting a role for mutant LRRK2 in the dysregulation of NFAT, and canonical and non-canonical Wnt signaling.
摘要：
帕金森病(Parkinson’sdisease,PD)是一种进行性迟发性神经退行性疾病,可导致身体和认知功能下降。富含亮氨酸的重复激酶2(LRRK2)的突变是PD的最常见的遗传原因。LRRK2是一种复杂的支架蛋白，在多种分子途径中具有已知的调节作用。LRRK2调节途径的两个突出实例是无翼/Int(Wnt)和活化T细胞的核因子(NFAT)信号传导。两者都被描述为免疫和神经系统发育以及成熟的关键调节因子。本研究的目的是建立LRRK2在脑内Wnt和NFAT信号传导中的生理和致病作用,以及非经典Wnt/钙途径的潜在贡献。在LRRK2G2019S突变体敲入（KI）和LRRK2敲除（KO）雄性和雌性小鼠中定量体内脑Wnt和NFATc1信号传导活性，并在28周内重复测量，采用慢病毒荧光素酶生物传感器，并使用混合效应模型进行分析。要建立空间分辨率，我们调查了组织，和来自不同大脑区域的原代神经元细胞培养物，结合了荧光素酶信号传导活性，免疫组织化学，qPCR和蛋白质印迹测定。结果通过非配对t检验与Welch's校正或双向方差分析与事后校正。LRRK2KO和LRRK2G2019SKI小鼠体内Wnt信号活性显著增加~3倍,对男性的影响（〜四倍）比女性（〜两倍）更明显。在LRRK2G2019SKI小鼠中,NFATc1信号传导降低约0.5倍。脑组织分析显示Wnt和NFAT信号传导成分的区域特异性表达变化。这些作用主要在LRRK2KI小鼠的纹状体和大脑皮层的蛋白质水平上观察到。原代神经元细胞培养分析显示在基础和刺激条件下Wnt和NFATc1信号传导的显著基因型依赖性改变。Wnt和NFATc1信号分别主要在皮质和海马神经元中失调。我们的研究进一步基于LRRK2作为Wnt和NFAT信号蛋白的知识。我们在LRRK2PD的神经元模型中发现了复杂的变化，表明突变体LRRK2在NFAT失调中的作用，以及规范和非规范Wnt信号。
