Project 1

项目1

基本信息

批准号：
10443847
负责人：
GARY J BASSELL
金额：
$ 53.7万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-25 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10443847
关键词：
3-Dimensional Address Adult Animal Model Automobile Driving Behavior Brain Cell Proliferation Cells Clinical Trials Collaborations Coupled Data Defect Development Disease Down-Regulation Embryo FMR1 Failure Fragile X Syndrome Genetic Translation Hippocampus (Brain)Human Impairment Joints Lentivirus Mediating Messenger RNA Modeling Molecular Molecular Target Morphology Mus Neuronal Differentiation Neurons Nuclear Pore Complex Organoids Outcome Pathway interactions Patients Phenotype Physiological Protein Biosynthesis Proteins RNA Regulation Reporting Retinoic Acid-Binding Proteins Role Signal Transduction Synapses Synaptic plasticity System Testing Therapeutic Therapeutic Intervention Tissues Translations Work base cell type comparative crosslinking and immunoprecipitation sequencing drug efficacy effective therapy experimental study fetal induced pluripotent stem cell insight migration monolayer mouse model neural model neurodevelopment neurogenesis neuron development neuronal excitability neurophysiology neurotransmission novel preclinical study primary endpoint relating to nervous system ribosome profiling single-cell RNA sequencing small molecule synergism three-dimensional modeling tool development transcriptome sequencing translatome

项目摘要

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.

项目概要先前对脆性 X 综合征 (FXS) 动物模型的研究为了解正常情况提供了宝贵的见解。脆性 X 智力低下蛋白 (FMRP) 的分子、细胞和生理功能；然而，一个有效的治疗仍然难以捉摸。尽管这些失败可能归因于多种因素，但现在显然，FXS 相关表型、药物疗效和救援策略势在必行在 FXS 动物模型中进行表征和/或在人类 FXS 患者中表征新表型衍生的、与疾病相关的细胞类型。一个关键的限制是缺乏可用的人类 FXS 患者衍生的神经网络模型研究 FMRP 介导的蛋白质合成和信号传导调节的作用。我们最近有开发了多个人类 iPSC 衍生的 2D 神经和 3D 皮质类器官模型来研究 FMRP 介导的大脑发育过程中蛋白质合成和信号传导的调节。的目标项目 1 将使用这些 FXS 患者 iPSC 衍生的 2D 单层以及 3D 皮质和海马类器官来解决三个具体目标所描述的问题。目标 1 是表征蛋白质合成特定细胞类型中的失调和相关的分子、细胞和神经生理表型人类 FXS iPSC 神经模型中的神经发育。我们的初步数据表明 FXS 患者细胞增加了蛋白质合成率，增加了增殖并改变了迁移，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会对神经元的发育和功能产生影响。目标 2 是确定 FMRP 目标并转化为在多个人类 FXS iPSC 神经模型中，大脑发育过程中 mRNA 失调。使用 CLIP-seq 我们已经在人类皮质类器官和小鼠胚胎皮质中以相似的速度鉴定了 FMRP 目标 mRNA。发展阶段。我们的比较分析揭示了三组 FMRP mRNA 靶标：人类仅、仅鼠标和共享。我们最近还使用核糖体分析来识别翻译成年小鼠大脑整个皮质中失调的 mRNA，其中一些是 FMRP 靶标。因此，核糖体分析将用于使用同基因组来表征 FXS 患者和对照的翻译组来自多个患者以及同基因 3D 皮质类器官的 i3Neurons 和 i3Neurons。用于比较在 FXS 模型之间，我们还将对 FXS 小鼠胚胎皮层进行核糖体分析。在目标 3 中，我们将制定有针对性的策略来拯救人类 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操纵失调的 FMRP 靶标的表达来拯救 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。