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

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

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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.

项目摘要脆弱X综合征（FXS）动物模型的先前工作为正常提供了宝贵的见解脆弱X智力低下蛋白（FMRP）的分子，细胞和生理功能；但是，一个有效的治疗仍然难以捉摸。尽管这些失败可能归因于几个因素，但现在是显然，必须与FXS相关的表型，药物的功效和救援策略在FX的动物模型中的特征和/或人类FXS患者的特征是/或新表型衍生的，与疾病相关的细胞类型。关键的限制是缺乏可用的人FXS患者衍生的神经研究FMRP介导的蛋白质合成和信号传导调节的作用的模型。我们最近有开发了多个人IPSC衍生的2D神经和3D皮质器官模型，以研究 FMRP介导的蛋白质合成和信号传导调节。目标的目标项目1将使用这些FXS患者IPSC衍生的2D单层以及3D皮层和海马符合三个特定目的所描述的问题的器官。目标1是表征蛋白质合成的在特定细胞类型中人FXS IPSC神经模型中的神经发育。我们的初步数据表明FXS患者细胞蛋白质合成率增加，增殖增加和迁移改变，导致延迟细胞命运和神经元分化的获取。这些早期的神经发育缺陷预计对神经元的发育和功能产生影响。 AIM 2是识别FMRP目标和翻译多种人FXS IPSC神经模型中大脑发育过程中的mRNA失调。使用夹式我们已经确定了类似人类皮质器官和小鼠胚胎皮质中的FMRP靶标mRNA 发展阶段。我们的比较分析揭示了三组FMRP mRNA靶标，人类只有鼠标和共享的鼠标。我们最近还使用了核糖体分析来识别翻译成年小鼠脑的整体皮质中的mRNA失调，其中一些是FMRP靶标。因此，核糖体分析将用于表征FXS患者的翻译和对照组的转化来自多个患者的i3neurons和i3neuron，以及来自等源性3D皮质器官。进行比较在FXS模型之间，我们还将进行FXS小鼠胚胎皮质的核糖体分析。在AIM 3中，我们将在人FXS IPSC神经模型中设计有针对性的策略来挽救细胞和突触表型。我们将使用基于慢病毒的方法来挽救FXS-的方法来操纵失调的FMRP靶标表达相关的细胞和突触表型。该项目的实验结果，再加上与其他项目的协同作用将发现与FXS相关表型的新型机制和关键驱动因素在皮质开发中，使用我们新生成的人类IPSC衍生的2D和3D神经模型。