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INTRODUCTION

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 c.823G>C (p.Ala275Pro) mutant at the cellular and zebrafish models.

METHODS

ATP1A3 wild-type and mutant Hela cell lines were constructed, and ATP1A3 mRNA expression, protein expression and localization, and Na-K-ATPase activity in each group of cells were detected. Additionally, we also constructed zebrafish models with 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.

RESULTS

Cells carrying the p.Ala275Pro mutation exhibited lower levels of mRNA, reduced protein expression, and decreased Na-K-ATPase activity compared to wild-type cells. Immunofluorescence analysis revealed that was primarily localized in the cytoplasm, but there was no significant difference in 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 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 and in the apoptosis signaling pathway, while reducing the transcriptional level of and the ratio.

CONCLUSION

This study reveals that the p.Ala275Pro mutant decreases protein expression and Na/K-ATPase activity. Abnormal expression of either wild-type or mutant genes impairs growth, development, and movement behavior in zebrafish.

INTRODUCTION

METHODS

RESULTS

CONCLUSION

引言

我们之前报道过，ATP1A3基因c.823G>C（p.Ala275Pro）突变在同一家族中导致了儿童交替性偏瘫和快速起病的肌张力障碍-帕金森综合征的不同表型。本研究旨在探讨c.823G>C（p.Ala275Pro）突变在细胞和斑马鱼模型中的功能。

方法

构建了ATP1A3野生型和突变型Hela细胞系，检测每组细胞中ATP1A3 mRNA表达、蛋白质表达和定位以及钠钾ATP酶活性。此外，我们还构建了野生型过表达（WT）和p.Ala275Pro突变型过表达（MUT）的斑马鱼模型。随后，我们检测了每组斑马鱼中多巴胺信号通路相关基因、帕金森病相关基因和凋亡相关基因的mRNA表达，并观察了斑马鱼的生长、发育和运动行为。

结果

与野生型细胞相比，携带p.Ala275Pro突变的细胞表现出较低水平的mRNA、降低的蛋白质表达和钠钾ATP酶活性。免疫荧光分析显示，其主要定位于细胞质，但突变前后蛋白质定位无显著差异。在斑马鱼模型中，与对照组相比，WT和MUT组的脑和体长、多巴胺神经元荧光强度、逃避能力、游泳距离和平均游泳速度均较低。此外，野生型和突变型的过表达均导致斑马鱼中与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相关基因的转录水平，同时降低相关基因的转录水平和比例。