Cell biological and proteomic investigation of pathogenic DDX3X missense mutations during neurogenesis

神经发生过程中致病性 DDX3X 错义突变的细胞生物学和蛋白质组学研究

• 批准号: 10474429
• 负责人: Debra Silver
• 金额: $ 24.15万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10474429
• 关键词: Address; Biochemical; Biological; Biological Assay; Brain; Cells; Clinical; Co-Immunoprecipitations; Collaborations; Cytoplasmic Granules; Data; Development; Developmental Delay Disorders; Diagnostic; Disease; Dominant-Negative Mutation; Embryo; Epilepsy; Etiology; Female; Fluorescence Recovery After Photobleaching; Generations; Genetic; Image; Impairment; Individual; Intellectual functioning disability; Investigation; Label; Lead; Lentivirus; Link; Mass Spectrum Analysis; Measures; Mediating; Microcephaly; Microscopy; Missense Mutation; Molecular; Monitor; Mus; Mutation; Nature; Neurodegenerative Disorders; Neurodevelopmental Disorder; Neurologic Deficit; Neurons; Nonsense Mutation; Outcome; Pathogenesis; Pathogenicity; Pathway interactions; Phenotype; Plant Roots; Play; Proteins; Proteomics; RNA; RNA Helicase; RNA-Binding Proteins; Recurrence; Regulation; Reporting; Role; Severities; Severity of illness; Syndrome; Testing; Therapeutic; Therapeutic Intervention; Time; Toxic effect; Translations; autism spectrum disorder; brain dysfunction; brain malformation; cohort; de novo mutation; falls; helicase; insight; knock-down; loss of function; loss of function mutation; mouse model; mutant; nerve stem cell; nervous system disorder; neurodevelopment; neurogenesis; progenitor; protein aggregation; tool; virtual

Abstract De novo mutations in DDX3X, an X-linked RNA helicase, account for 1-3% of intellectual disability (ID) in females and are associated with a broad range of phenotypes, including developmental delay, epilepsy, autism and brain malformations. Approximately half of the mutations are nonsense, whereas the other 50% are missense mutations. Initial reports suggested that DDX3X mutations primarily cause loss-of-function phenotypes. However, our collaborators identified recurrent missense mutations in DDX3X individuals that were consistently associated with more severe clinical outcomes and specific brain malformations not observed in other DDX3X individuals. These data strongly suggest some DDX3X missense mutations exert dominant negative phenotypes. We have further discovered that DDX3X is required for proper brain development; specifically, loss of Ddx3x in the embryonic mouse brain impairs the ability of neural progenitors to make neurons. We also demonstrated that DDX3X missense mutations associated with severe impairment have nearly complete loss of helicase activity, impaired translation of key targets, and form ectopic RNA-protein granules. These findings indicate that ectopic RNA-protein granules and aberrant DDX3X protein interactions may contribute to disease severity. However, these mechanisms have not been examined. In this proposal, we seek to elucidate the molecular pathogenesis of DDX3X missense mutations linked to severe clinical deficits in DDX3X syndrome. We will test the hypothesis that clinically severe DDX3X missense mutations perturb its cellular dynamics and protein interactome during neurogenesis. First, we will characterize DDX3X granules in neural progenitors and neurons expressing mild and severe DDX3X missense mutations. We will also determine whether granules are linked to impaired neurogenesis. Second, we will use an unbiased proteomic screen in neural progenitors and neurons to determine how different DDX3X missense mutations impair the WT protein interactome. These studies are critical for understanding the molecular underpinnings of dominant negative phenotypes associated with DDX3X syndrome. Further, our studies will broadly establish a paradigm for understanding other neurodevelopmental disorders in which RNA regulation plays a central role.

抽象的 DDX3X中的从头突变（一种X连锁RNA解旋酶）占女性智障（ID）的1-3％ 并与广泛的表型有关，包括发育延迟，癫痫，自闭症和大脑 畸形。大约一半的突变是胡说八道，而其他50％是错过的 突变。最初的报告表明，DDX3X突变主要导致功能障碍表型。 但是，我们的合作者确定了DDX3X个体中的经常性错义突变，这些突变始终是 与其他DDX3X中未观察到的更严重的临床结果和特定的脑畸形相关 个人。这些数据强烈表明某些DDX3X错义突变具有主导的负面表型。 我们进一步发现，适当的大脑发育需要DDX3X。具体而言，DDX3X在 胚胎小鼠大脑会损害神经祖细胞制造神经元的能力。我们还证明了 与严重损伤相关的DDX3X错义突变几乎完全丧失了解旋酶活性， 关键靶标的翻译受损并形成异位RNA-蛋白颗粒。这些发现表明异位 RNA蛋白颗粒和异常DDX3X蛋白相互作用可能导致疾病严重程度。然而， 这些机制尚未进行检查。在此提案中，我们试图阐明分子发病机理 DDX3X错义突变与DDX3X综合征的严重临床缺陷有关。我们将检验假设 临床上严重的DDX3X错义突变扰动其细胞动力学和蛋白质相互作用组 在神经发生过程中。首先，我们将表征在表达神经祖细胞和神经元中的DDX3X颗粒 轻度和严重的DDX3X错义突变。我们还将确定颗粒是否与受损相关 神经发生。其次，我们将在神经祖细胞和神经元中使用公正的蛋白质组学筛选来确定 不同的DDX3X错义突变如何损害WT蛋白相互作用。这些研究对于 了解与DDX3X相关的主要负表型的分子基础 综合征。此外，我们的研究将广泛建立一个范式来理解其他神经发育 RNA调节起着核心作用的疾病。