Dan-Dan Ruan ¹ Li-Sheng Liao ¹ Jing Zou ¹ Ming-Dong Ji ¹ Ruo-Li Wang ¹ Jian-Hui Zhang ¹ Li Zhang ¹ Mei-Zhu Gao ¹ Qian Chen ¹ Hong-Ping Yu ¹ Wen Wei ² Yun-Fei Li ¹ Hong Li ¹ Fan Lin ¹ Jie-Wei Luo ^1* Xin-Fu Lin ¹

• ¹ Fujian provincial hospital, Fuzhou, China
• ² Ganzhou Municipal Hospital, Ganzhou, Jiangxi Province, China

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. In this study, ATP1A3 wild-type and mutant HeLa cell lines were constructed. 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. Additionally, we also constructed zebrafish models with ATP1A3 wild-type (WT) overexpression and p.Ala275Pro mutant (MUT) overexpression. 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. 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.