Project 1

项目摘要 以前在脆性X综合征（FXS）动物模型中的工作为正常的 脆性X智力低下蛋白（FMRP）的分子，细胞和生理功能;然而， 有效的治疗仍然是难以捉摸的。虽然这些失败可以归因于几个因素，但现在 很明显，FXS-associated表型、药物疗效和救援策略是必要的， 在FXS动物模型中表征的新表型和/或在人FXS患者中表征的新表型- 衍生的疾病相关细胞类型。一个关键的限制是缺乏可用的人FXS患者来源的神经细胞。 模型研究FMRP介导的蛋白质合成和信号传导调节的作用。我们最近 开发了多种人类iPSC衍生的2D神经和3D皮质类器官模型，以研究 脑发育过程中FMRP介导的蛋白质合成和信号传导调节。的目标 项目1是使用这些FXS患者iPSC衍生的2D单层以及3D皮质和海马 类器官解决三个具体目标中描述的问题。目的1是表征蛋白质合成 调节异常和相关的分子，细胞和神经生理表型在特定的细胞类型， 人FXS iPSC神经模型中的神经发育。我们的初步数据表明FXS患者细胞 增加蛋白质合成率，增加增殖和改变迁移，导致延迟 获得细胞命运和神经元分化。这些早期神经发育缺陷预计将 对神经元的发育和功能有影响。目标2是确定FMRP目标， 在多种人FXS iPSC神经模型中，在脑发育期间mRNA失调。使用CLIP-seq，我们 在人类皮质类器官和小鼠胚胎皮质中， 发育阶段我们的比较分析揭示了三组FMRP mRNA靶点，人类， 只有鼠标和共享的。我们最近还使用核糖体分析来识别 在成年小鼠大脑的整个皮质中，FMRP靶基因表达失调。因此，核糖体 将使用同源基因和同源基因分析来表征FXS患者和对照的翻译组。 i3神经元和来自多个患者的i3神经元，以及来自同基因3D皮质类器官。为了比较 在FXS模型之间，我们还将进行FXS小鼠胚胎皮层的核糖体分析。在目标3中，我们 设计有针对性的策略，以拯救人类FXS iPSC神经模型中的细胞和突触表型。我们 将使用基于慢病毒的方法来操纵失调的FMRP靶点的表达，以拯救FXS- 相关的细胞和突触表型。该项目的实验结果，加上 与其他项目的协同作用，将揭示FXS相关表型的新机制和关键驱动因素 使用我们新生成的人类iPSC衍生的2D和3D神经模型进行皮质发育。

Project Summary Previous work in animal models of fragile X syndrome (FXS) has provided invaluable insight into the normal molecular, cellular, and physiological functions of fragile X mental retardation protein (FMRP); however, an effective treatment remains elusive. Although these failures could be attributed to several factors, it is now apparent that it is imperative that FXS-associated phenotypes, the efficacy of drugs, and rescue strategies characterized in animal models of FXS be validated and/or new phenotypes characterized in human FXS patient- derived, disease-relevant cell types. A critical limitation is lack of an available human FXS patient-derived neural model to investigate the role of FMRP-mediated regulation of protein synthesis and signaling. We have recently developed multiple human iPSC-derived 2D neural and 3D cortical organoid models to investigate the role of FMRP-mediated regulation of protein synthesis and signaling during brain development. The objectives of Project 1 are to use these FXS patient iPSC-derived 2D monolayers as well as 3D cortical and hippocampal organoids to address questions delineated in three specific aims. Aim 1 is to characterize protein synthesis dysregulation and associated molecular, cellular and neurophysiological phenotypes in specific cell types across neural development in human FXS iPSC neural models. Our preliminary data indicate that FXS patient cells have increased protein synthesis rates, increased proliferation and altered migration, resulting in delayed acquisition of cell fate and neuronal differentiation. These early neurodevelopmental defects are anticipated to have consequences on neuronal development and function. Aim 2 is to identify FMRP targets and translationally dysregulated mRNAs during brain development in multiple human FXS iPSC neural models. Using CLIP-seq we have identified FMRP target mRNAs in both human cortical organoids and mouse embryonic cortex at similar developmental stages. Our comparative analyses have revealed three groups of FMRP mRNA targets, human only, mouse only and shared ones. We have also recently used ribosome profiling to identify translationally dysregulated mRNAs, some of which are FMRP targets, in whole cortex in the adult mouse brain. Thus, ribosome profiling will be applied to characterize the translatomes of FXS patients and controls using both isogenic i3Neurons and i3Neurons from multiple patients, as well as from isogenic 3D cortical organoids. For comparison between FXS models, we also will conduct ribosome profiling of FXS mouse embryonic cortex. In Aim 3, we will devise targeted strategies to rescue cellular and synaptic phenotypes in human FXS iPSC neural models. We will manipulate expression of dysregulated FMRP targets using lentivirus-based approaches to rescue FXS- associated cellular and synaptic phenotypes. The outcome of the experiments in this Project, coupled with synergy with the other projects, will uncover novel mechanisms and key drivers of FXS-associated phenotypes in cortical development using our newly generated human iPSC-derived 2D and 3D neural models.