Early developmental mechanisms of Rett Syndrome

Rett综合征（RTT）是一种严重的神经发育障碍，主要影响女孩。在其经典形式中，RTT 主要由编码甲基CpG结合蛋白2（MECP 2）的基因突变引起。MeCP 2是 一种多功能基因表达调节因子，通过多种机制调节转录， 如DNA结合，与转录因子复合物的相互作用，染色质结构的调节和调控 在不同的发育阶段，这种机制是多效性的。MeCP 2是 被认为主要是通过后期发展到成年，但最近的临床研究RTT 儿童指出了这种疾病的早期症状。MeCP 2的早期发育机制很差， 明白我们之前使用RTT患者的iPSC显示MeCP 2的减少导致过表达 miRNA-199和miRNA-214的表达，神经祖细胞的增加，神经发生和神经元的减少， 皮质类器官中的迁移。我们现在建议详细分析移民赤字，并研究 赤字背后的机制。本研究的目的是开发一种新型的活细胞成像平台 融合3D干细胞技术、微流体和多光子显微镜，并将其联合收割机与最先进的 最先进的分子方法，包括质谱蛋白质组学和单细胞RNA测序， 与MECP 2中的RTT引起突变相关的神经元迁移缺陷的机制。目标1： 建议开发无标记的三次谐波产生三光子显微镜，并用它来表征 与同基因对照相比，RTT类器官中的神经元迁移缺陷。我们还将开发一个 这是一个基于微流体的实时成像平台，可以稳定地对类器官进行成像，并对神经元进行数天的跟踪。 在目标2中，我们将研究MECP 2突变对下游分子通路的影响。 在神经元分化和迁移中的作用我们将研究AKT异常过度表达的机制， RTT类器官和神经祖细胞，并使用蛋白质组学和磷酸蛋白质组学筛选来定义新的 RTT中神经元迁移的蛋白质和途径失调。我们将利用 单个细胞来揭示RTT和对照类器官之间的细胞类型、群体和转录组差异。 在目标3中，我们将使用目标1的技术和目标2的结果来研究牵连信号的作用。 神经元迁移的途径。我们将询问AKT和下游信号分子的功能， 和新蛋白质，包括从我们的研究中发现的细胞粘附和细胞骨架组织的调节剂， 屏幕，被预测为涉及迁移。我们将在小鼠体内验证特定途径的能力， 和粘着斑蛋白来挽救RTT神经元迁移缺陷。我们共同期待这些成果 将促进我们对RTT早期皮质发育缺陷机制的理解， 提出了针对这些阶段的潜在新疗法。

Rett syndrome (RTT) is a severe neurodevelopmental disorder primarily affecting girls. In its classical form, RTT is predominantly caused by mutations in the gene encoding methyl-CpG binding protein 2 (MECP2). MeCP2 is a multifunctional regulator of gene expression which regulates transcription through diverse mechanisms such as DNA-binding, interaction with transcription factor complexes, modulation of chromatin structure and regulation of miRNAs – mechanisms that are engaged pleiotropically through different developmental stages. MeCP2 was considered to act predominantly through late development into adulthood, but recent clinical studies of RTT children point to very early signs of the disorder. The early developmental mechanisms of MeCP2 are poorly understood. We previously used RTT patient iPSCs to show that reduction of MeCP2 leads to overexpression of miRNA-199 and miRNA-214, an increase in neural progenitors, and reduction in neurogenesis and neuronal migration in cortical organoids. We now propose to analyze the migration deficits in detail, and examine the mechanisms underlying the deficits. The objective of this proposal is to develop a novel live-cell imaging platform merging 3D stem cell technologies, microfluidics and multiphoton microscopy, and combine it with state-of-the- art molecular approaches, including mass spectrometry proteomics and single cell RNA sequencing, to examine mechanisms of neuronal migration deficits associated with RTT-causing mutations in MECP2. In Aim 1, we propose to develop label-free third-harmonic generation three-photon microscopy and use it to characterize neuronal migration deficits in RTT organoids compared to isogenic controls. We will additionally develop a microfluidics-based live imaging platform where organoids can be stably imaged and neurons tracked for days. In Aim 2, we will examine the consequence of MECP2 mutations on downstream molecular pathways involved in neuronal differentiation and migration. We will examine mechanisms of anomalous overexpression of AKT in RTT organoids and neural progenitors, and use a proteomic and phospho-proteomic screen to define new proteins and pathways of neuronal migration dysregulated in RTT. We will exploit the transcriptomic profile of single cells to reveal cell types, populations and transcriptomic differences between RTT and control organoids. In Aim 3, we will use the technologies of Aim 1, and results of Aim 2, to examine the role of implicated signaling pathways in neuronal migration. We will interrogate the function of AKT and downstream signaling molecules, and that of new proteins, including modulators of cell adhesion and cytoskeleton organization identified from our screens, that are predicted as involved in migration. We will validate in vivo in mice the ability of specific pathways and focal adhesion proteins to rescue RTT neuronal migration deficits. Together, we expect that these results will advance our understanding of mechanisms involved in deficits of early cortical development in RTT, and suggest potential novel therapeutics targeting these stages.