Towards a Better Understanding of Fragile X Syndrome Using Human Brain Organoids

项目摘要 脆性X综合征(FXS)是最常见的遗传性智力残疾形式，也是一种主要的遗传性疾病 自闭症谱系障碍(ASD)的病因。功能性脆性X智力低下引起的FXS 蛋白质(FMRP)。FMRP由FMR1基因编码，是一种选择性的RNA结合蛋白，与 翻译多聚核糖体。以前的工作主要集中在FMRP作为翻译调节因子和 FMRP的许多mRNA靶点已被证明是与ASD相关的基因。表观遗传调控起着关键作用 在神经发育和神经精神障碍中的作用，包括自闭症。除了DNA和组蛋白 修饰，150多个转录后修饰的核糖核苷已经在不同的 核糖核酸的类型。在不同的RNA修饰中，N6-甲基腺苷(M6A)是迄今为止最知名的 对信使核糖核酸和核糖核糖核酸的修饰。M6A通过专用写入器、橡皮擦和读取器进行动态调节 蛋白质。M6A显著影响RNA的剪接、输出、定位、翻译效率和稳定性。我们 发现神经发育过程中m6A的时空分布是高度动态的，并且 M6A标记的转录本在与ASD相关的基因以及脆性X的mRNA靶标中都是丰富的 智力低下蛋白(FMRP)。我们的生化分析以及其他人的工作都发现 FMRP可与其mRNA靶分子(m6A阅读器)的m6A位点结合。此外，我们还发现FMRP可以 维持其mRNA靶标的稳定性，提示FMRP在基因调控中具有新的生物学作用。 人类诱导的多能干细胞(IPSCs)是多能的，能够产生许多不同的细胞 类型。IPSCs的三维(3D)聚集培养是从类胚体培养演变而来的， 忠实地跟踪人类器官发生，为研究人脑提供了一个新的平台 在培养皿中培养，否则无法进行实验。我们的初步数据显示， FMRP可改变人前脑器官的发育。此外，最近公布的数据 提示在人类神经发育过程中，m6A基因的修饰比小鼠更为普遍。在这 在拟议的研究中，我们将开发和表征FXS的人前脑有机体，并确定 人脑发育过程中潜在的人类特异性FMRP的mRNA靶点。我们建议的工程将会 导致了FXS的人类前脑器官模型的发展，并有可能识别人类- 具体的FMRP目标。

Project Summary Fragile X syndrome (FXS) is the most common inherited form of intellectual disability and a leading genetic cause of autism spectrum disorders (ASD). FXS is caused by the loss of functional fragile X mental retardation protein (FMRP). FMRP, encoded by the FMR1 gene, is a selective RNA-binding protein associated with translating polyribosomes. Previous works have focused on the role of FMRP as translational regulator and many mRNA targets of FMRP have been shown to be ASD-linked genes. Epigenetic regulation plays a pivotal role in neurodevelopment and neuropsychiatric disorders, including ASD. In addition to DNA and histone modifications, more than 150 post-transcriptionally modified ribonucleosides have been identified in various types of RNA. Among different RNA modifications, N6-methyladenosine (m6A) is by far the best-known modification on mRNA and lncRNA. m6A is dynamically regulated with dedicated writer, eraser, and reader proteins. m6A significantly affects RNA splicing, export, localization, translation efficiency and stability. We have found that the temporal and spatial distribution of m6A during neurodevelopment is highly dynamic, and the m6A-marked transcripts are enriched among ASD-linked genes as well as the mRNA targets of fragile X mental retardation protein (FMRP). Our biochemical analyses as well as the work of others have found that FMRP could bind to the m6A sites of its mRNA targets (m6A reader). Furthermore, we found that FMRP could maintain the stability of its mRNA targets, suggesting a new biological role of FMRP in gene regulation. Human-induced pluripotent stem cells (iPSCs) are pluripotent and are able to generate many different cell types. Three-dimensional (3D) aggregate culture of iPSCs has evolved from embryoid body culture, quite faithfully following human organogenesis, and provides a new platform to investigate human brain development in a dish, otherwise inaccessible to experimentation. Our preliminary data suggest that the loss of FMRP could alter the development of human forebrain organoids. Furthermore, recently published data suggest that m6A modification is more pervasive during neurodevelopment in human than mouse. In this proposed study, we will develop and characterize the human forebrain organoids of FXS and identify the potential human-specific mRNA targets of FMRP during human brain development. Our proposed works will lead to the development of the human forebrain organoid model for FXS and potentially identify the human- specific FMRP targets.