■我们以前报道过ATP1A3c.823G>C(p。Ala275Pro)突变体会导致同一家庭中儿童期替代偏瘫和快速发作性肌张力障碍-帕金森病的表型不同。本研究旨在研究ATP1A3c.823G>C的功能(p。Ala275Pro)在细胞和斑马鱼模型中的突变体。
■构建了ATP1A3野生型和突变型Hela细胞系,和ATP1A3mRNA表达,ATP1A3蛋白表达与定位,检测各组细胞的Na+-K+-ATP酶活性。此外,我们还构建了ATP1A3野生型过表达(WT)和p.Ala275Pro突变体过表达(MUT)的斑马鱼模型。随后,我们检测了多巴胺信号通路相关基因的mRNA表达,帕金森病相关基因,和每组斑马鱼的凋亡相关基因,观察生长,发展,和斑马鱼的运动行为。
■携带p.Ala275Pro突变的细胞显示较低水平的ATP1A3mRNA,ATP1A3蛋白表达降低,与野生型细胞相比,Na-K-ATPase活性降低。免疫荧光分析显示ATP1A3主要位于细胞质中,突变前后ATP1A3蛋白定位差异无统计学意义。在斑马鱼模型中,WT和MUT组均显示较低的大脑和身体长度,多巴胺神经元荧光强度,逃生能力,游泳距离,和平均游泳速度与对照组相比。此外,野生型和突变型ATP1A3的过表达导致斑马鱼多巴胺信号通路和帕金森病相关基因的mRNA表达异常,并显著上调凋亡信号通路中bad和caspase-3的转录水平,同时降低bcl-2的转录水平和bcl-2/bax比值。
■本研究揭示p.Ala275Pro突变体降低ATP1A3蛋白表达和Na+/K+-ATP酶活性。野生型或突变型ATP1A3基因的异常表达会损害生长,发展,和斑马鱼的运动行为。
UNASSIGNED: We previously reported that
ATP1A3 c.823G>C (p.Ala275Pro) mutant causes varying phenotypes of alternative hemiplegia of childhood and rapid-onset dystonia-parkinsonism in the same family. This study aims to investigate the function of
ATP1A3 c.823G>C (p.Ala275Pro) mutant at the cellular and zebrafish models.
UNASSIGNED: ATP1A3 wild-type and mutant Hela cell lines were constructed, and
ATP1A3 mRNA expression, ATP1A3 protein expression and localization, and Na+-K+-ATPase activity in each group of cells were detected. Additionally, we also constructed zebrafish models with ATP1A3 wild-type overexpression (WT) and p.Ala275Pro mutant overexpression (MUT). Subsequently, we detected the mRNA expression of dopamine signaling pathway-associated genes, Parkinson\'s disease-associated genes, and apoptosisassociated genes in each group of zebrafish, and observed the growth, development, and movement behavior of zebrafish.
UNASSIGNED: Cells carrying the p.Ala275Pro mutation exhibited lower levels of
ATP1A3 mRNA, reduced ATP1A3 protein expression, and decreased Na+-K+-ATPase activity compared to wild-type cells. Immunofluorescence analysis revealed that ATP1A3 was primarily localized in the cytoplasm, but there was no significant difference in
ATP1A3 protein localization before and after the mutation. In the zebrafish model, both WT and MUT groups showed lower brain and body length, dopamine neuron fluorescence intensity, escape ability, swimming distance, and average swimming speed compared to the control group. Moreover, overexpression of both wild-type and mutant ATP1A3 led to abnormal mRNA expression of genes associated with the dopamine signaling pathway and Parkinson\'s disease in zebrafish, and significantly upregulated transcription levels of bad and caspase-3 in the apoptosis signaling pathway, while reducing the transcriptional level of bcl-2 and the bcl-2/bax ratio.
UNASSIGNED: This study reveals that the p.Ala275Pro mutant decreases ATP1A3 protein expression and Na+/K+-ATPase activity. Abnormal expression of either wild-type or mutant ATP1A3 genes impairs growth, development, and movement behavior in zebrafish.