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

基本信息

批准号：
10313796
负责人：
Debra Silver
金额：
$ 20.13万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
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/10313796
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 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

项目摘要

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（一种 X 连锁 RNA 解旋酶）的从头突变导致女性智力障碍 (ID) 的 1-3% 并与广泛的表型相关，包括发育迟缓、癫痫、自闭症和大脑畸形。大约一半的突变是无义的，而另外 50% 是错义的突变。初步报告表明 DDX3X 突变主要导致功能丧失表型。然而，我们的合作者在 DDX3X 个体中发现了反复出现的错义突变，这些突变始终与与其他 DDX3X 中未观察到的更严重的临床结果和特定脑畸形相关个人。这些数据强烈表明一些 DDX3X 错义突变发挥显性失活表型。我们进一步发现 DDX3X 是大脑正常发育所必需的；具体来说，Ddx3x 的损失小鼠胚胎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-蛋白质颗粒和异常的 DDX3X 蛋白质相互作用可能会导致疾病的严重程度。然而，这些机制尚未经过审查。在本提案中，我们试图阐明分子发病机制 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 错义突变扰乱了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，我们将表征神经祖细胞和神经元表达中的 DDX3X 颗粒轻度和重度 DDX3X 错义突变。我们还将确定颗粒是否与受损有关神经发生。其次，我们将在神经祖细胞和神经元中使用无偏蛋白质组筛选来确定不同的 DDX3X 错义突变如何损害 WT 蛋白相互作用组。这些研究对于了解与 DDX3X 相关的显性失活表型的分子基础综合症。此外，我们的研究将广泛建立一个理解其他神经发育的范例 RNA 调节在其中发挥核心作用的疾病。