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Project 1

项目1

基本信息

批准号：
10271307
负责人：
GARY J BASSELL
金额：
$ 53.67万
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/10271307
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。