基于iPS技术的FOXP1综合征细胞模型的建立及其影响神经干细胞功能的机制研究

Characterized by mental retardation with language impairment and with or without autistic features, FOXP1 syndrome is a kind of global developmental delay-related disorder. There are some differences in interpreting the function of Foxp1 between mice model and human model. And the molecular mechanism of Foxp1 on neurodevelopment remains unknown. Foxp1 expresses in the striatum. It has been reported that it would lead to Ncor2 and Snw1 up-regulation, while Ncor2- and Snw1-related Notch signal pathway plays an essential role in the development of neural stem cells (NSCs) and maintains its normal function. Therefore, we suspect that transcription factor Foxp1 would regulate the function of NSCs through the Notch signal pathway. We identified a novel de novo variant in the FOXP1 gene (c.1652+5 G>A) in an adult patient with FOXP1 syndrome. RNA analysis revealed that this variant would impact splicing of the FOXP1 and result in a truncated protein. In this project, we aim to generate iPSCs from patient’s cells, investigate the impacts of the mutated Foxp1 on iPSC induced NSCs, study the effect of Foxp1 on the Notch signal pathway, identify the target gene and reveal the molecular mechanism of transcriptional regulation by some cytological and molecular biological experiments. With the implementation of this project, the molecular mechanism of FOXP1 syndrome resulting in mental retardation could be demonstrated and new thoughts for clinical intervention and therapy of FOXP1 syndrome could be provided.

FOXP1综合征是一种以精神发育迟缓，语言障碍及自闭症为特征的疾病。现有小鼠模型在揭示Foxp1功能时与人源模型还存在一定差异，且Foxp1作用于神经发育的分子机制尚不明确。Foxp1表达于纹状体且会上调Notch信号通路中的Ncor2和Snw1，而该通路参与和调控神经干细胞（NSCs）的发育与功能。因此我们推测转录因子Foxp1通过Notch信号通路调控NSCs的发育及功能。前期研究中我们在患者中发现一影响剪接的新突变（FOXP1:c.1652+5G>A）。因此，本项目拟以该患者淋巴细胞构建iPS细胞模型并诱导为NSCs，通过一系列细胞学及分子生物学技术研究Foxp1对NSCs功能及Notch通路信号的影响，明确Foxp1调控的目标基因并揭示转录调控机制。本项目的实施对阐明Foxp1在FOXP1综合征中导致智力障碍相关表型的分子机制有重要的意义，将为临床干预及诊治提供新思路。

FOXP1综合征（MIM #613670）是一种以轻到中度智力障碍，语言障碍及自闭症谱系为特征的疾病，呈常染色体显性遗传。研究表明该蛋白在大脑特定区域中影响神经元发育，进而影响智力语言等。目前相关研究主要集中于鼠源细胞，对于人源特别是特定疾病来源的细胞研究较少。因鼠源与人源存在的差异，聚焦于人源细胞的功能研究能更准确全面地揭示Foxp1对神经系统功能的影响。本项目收集FOXP1综合征患者外周血单个核细胞（PBMCs），RT-PCR结果提示突变会造成编码氨基酸的移码，但移码的mRNA含量非常低，疑似被降解；进一步行TA克隆证实该突变会影响剪接，进而引起移码形成终止密码子。为研究该突变对神经系统的影响，我们将患者PBMCs诱导为多能干细胞（iPS），免疫染色法、体内畸胎瘤实验和体外三胚层分化等实验都表明该细胞系具有全能性。至此，我们利用iPS 技术成功构建FOXP1 综合征细胞模型，该模型可用于相关致病机制研究及潜在的药物干预研究。随后我们利用IDT Alt-R® CRISPR/Cas9系统将iPSCs中的突变位点进行修复，成功构建野生型对照株。采用拟胚体方案将iPSCs诱导分化为神经前体细胞，NPC特异性抗原免疫荧光染色表明新建立的细胞系具有NPC的特性，为研究Foxp1对神经系统的影响提供基础。在细胞层面对NSCs进行相关功能检测及差异分析，结果提示突变会降低细胞增殖速度和迁移能力，mRNA被降解无法形成相应的蛋白，进一步揭示该突变会导致Foxp1单倍剂量不足。对已有的ChIP-Seq和RNA-Seq数据进行分析，在分子水平揭示Foxp1对Notch信号通路的调控不显著，但Foxp1通过PI3K-AKT通路影响NPCs的生长、增殖、迁移等细胞功能，其目标基因为PIK3CA基因，揭示了FOXP1综合征可能的分子致病机制。

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