细胞器膜蛋白ATP9A基因突变导致人遗传性智力障碍的作用机制研究

Organelle membrane proteins play important roles in the structure maintenance, membrane formation, substance transport and signal transduction of organelle, and dysfunction of the organelle membrane protein causes a variety of diseases. The phospholipid flippase ATP9A is a membrane protein involved in substance transport between organelles such as plasma membrane, Golgi apparatus, endosomes. However, its function in human diseases remains largely unknown. In the present study, we have found a pair of siblings with intellectual disability but their parents are normal in the clinical studies. Whole exon capture sequencing of the family showed that this kind of intellectual developmental disorder was caused by mutations of ATP9A gene at two sites, but the function and pathogenesis of this gene in the nervous system has not been reported. In this study, CRISPR/ CAS9 technique was used to achieve mouse models with gene knock-out and mutation knock-in of ATP9A. We try to reproduce the phenotype of the pathogenic gene in human disease in mouse model, and the effects of gene deletion and mutation on the behavior and brain function of mice will be also intensively analyzed. The subcellular localization, interaction proteins and signaling pathway of ATP9A will be further explored by immunoelectron microscopy, histology and omics study. Our project aims to revealing the pathogenesis of organelle membrane protein ATP9A in neurological diseases by investigating the potential function and molecular mechanism of ATP9A.

细胞器膜蛋白在细胞器结构维持、膜形成、物质转运和信号转导中发挥重要作用，若功能异常将导致疾病发生。磷脂转位酶ATP9A是一种参与质膜、高尔基体、内涵体等细胞器之间物质转运的膜蛋白，但其功能和在人类疾病中作用不明。申请人及合作者在临床研究中发现了一对患有智力发育障碍但父母均表现正常的兄妹，对其家系4人的基因进行全外显子捕获测序发现该疾病是由ATP9A单基因双位点突变引起，而该基因在神经系统中的功能和致病机制还未见报道。本项目将利用CRISPR/CAS9技术在小鼠模型上进行ATP9A基因的敲除和突变敲入工作，在小鼠模型模拟该致病基因在人类疾病上的表型，深入分析该基因缺失或突变对小鼠行为和大脑功能的影响，通过免疫电镜和组学等体外实验手段明确ATP9A在正常和病理状态的亚细胞定位，挖掘互作蛋白及信号通路，研究其潜在功能和作用机制，为揭示细胞器膜蛋白ATP9A在神经系统疾病中的致病机理提供有益线索。

遗传性智力障碍是一种伴有遗传因素的神经发育障碍，在儿童神经系统罕见病的表型中较为常见。我们在临床研究中发现一个常染色体隐性遗传性智力障碍家系携带ATP9A（NM_006045）基因的两种无义突变c.658C>T/c.433C>T（p.Arg220*/p.Arg145*），而子代中ATP9A的复合杂合突变导致其蛋白水平明显低于正常人，并伴有注意缺陷和多动障碍等神经疾病表型。而该蛋白的临床意义和在神经系统中的功能目前尚未被报道。本项目成功构建了能模拟人类神经系统疾病表型的ATP9A基因敲除和突变小鼠模型，结果显示ATP9A缺失的小鼠也出现明显学习记忆受损、过度运动和焦躁，并导致大脑皮质神经元突起受损和细胞活力降低，因此我们确定了ATP9A的无义突变是引起智力障碍的新致病因素。进一步研究表明，ATP9A是一个主要定位在晚期和循环内吞体的膜蛋白，而内吞体功能异常可导致多种神经系统疾病。因此，我们认为ATP9A的功能可能与内吞体介导的囊泡转运密切相关。内吞实验表明ATP9A缺失或突变干扰了转铁蛋白进入早期内吞体和从早期内吞体向循环内吞体的转运的运输过程，并导致循环内吞体分布改变，而对内吞降解途径无影响。由此我们得出ATP9A缺失或突变阻碍了神经细胞内吞循环途径的物质运输，内吞物质在胞内滞留引发神经毒性，继而引起神经元形态损伤和细胞死亡，这可能是 ATP9A突变在神经系统疾病中的致病机制。该研究为揭示ATP9A突变在神经系统疾病中的发病机理和潜在的治疗靶点提供了理论基础，并为临床罕见疾病的基因筛查提供了科学依据。

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