Timed mRNA translation events in neocortical development and neurodevelopmental disorders

新皮质发育和神经发育障碍中的定时 mRNA 翻译事件

• 批准号: 8930467
• 负责人: Matthew Lee Kraushar
• 金额: $ 3.97万
• 依托单位: RBHS-ROBERT WOOD JOHNSON MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-28 至 2019-08-27
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8930467
• 关键词: Animal Model; Behavior; Brain; Cell Cycle; Cell Differentiation process; Cells; Cognitive deficits; Complex; Data; Density Gradient Centrifugation; Development; Disease; Elongation Factor; Embryo; Etiology; Event; FMRP; Fragile X Syndrome; Functional disorder; Gene Expression; Gene Expression Profile; Genes; Genetic Translation; Genomics; Health; Human; Immunohistochemistry; Lead; Measures; Messenger RNA; Meta-Analysis; Mitotic; Molecular; Molecular Target; Mus; Neocortex; Neurodevelopmental Disorder; Neuronal Differentiation; Neurons; Pathway interactions; Peptide Elongation Factor 2; Phosphorylation; Phosphotransferases; Polyribosomes; Predisposition; Protein Biosynthesis; Protein Dynamics; Proteins; Proteome; Regulation; Reverse Transcriptase Polymerase Chain Reaction; Ribosomal Proteins; Ribosomes; Role; Specificity; Staging; Stem cells; Sucrose; Susceptibility Gene; Syndrome; Techniques; Testing; Thymidine; Time; Transcript; Translating; Translations; Western Blotting; Work; autism spectrum disorder; base; cognitive function; disorder subtype; fetal; in vivo; microdeletion; neocortical; nerve stem cell; neurogenesis; neuronal circuitry; new therapeutic target; progenitor; small hairpin RNA; stem cell differentiation; stem cell fate; therapeutic target; transcriptomics; vector control

 DESCRIPTION (provided by applicant): Within the brain's neocortex originates our most evolutionarily advanced complex cognitive functions. The diverse subpopulations of neural stem cells and post-mitotic neurons underlying these intricate circuits are molecularly defined by their patterns of gene expression, where functional gene expression culminates in protein synthesis via mRNA translation at the level of the ribosome. Therefore, mRNA translation must be tightly regulated in development for specific mRNAs to generate molecularly defined subpopulations of neocortical neurons. Autism Spectrum Disorders (ASDs) are clinically heterogeneous disorders of complex cognitive functions. In the past decade, genomic and transcriptomic analyses have implicated genes and pathways that converge on protein synthesis in the cortex as a target in ASD etiology, reinforced by multiple syndromic ASD subtypes where translation abnormalities are central, such as Fragile-X Syndrome (FXS). While the regulation of core translation components has been implicated in ASDs, how abnormal translation of specific mRNAs leads to neocortical dysfunction remains unanswered. Furthermore, it is not known if ribosomal components are dynamic during neocortical development, nor whether this relates to how neocortical neurons are molecularly defined from the translation of specific mRNA transcripts. Our preliminary data suggest that components of the core translation machinery and the mRNAs that associate with them in the fetal neocortex are indeed dynamic, with a particular transition occurring at mid- neurogenesis - a susceptibility period highly implicated in ASD genomic meta-analyses. Our data show that this mid-neurogenic transition is marked by a dramatic increase in the phosphorylation of eukaryotic elongation factor 2 (eEF2), putatively modulating elongation activity via its kinase, eEF2k. Interestingly, eEF2k has been associated with ASDs as a variable genomic locus in multiple studies, and a unique microdeletion syndrome spanning this locus is characterized by severe cognitive deficits. eEF2k also partners with FMRP to regulate the translation of specific mRNAs in FXS. We found that dynamic eEF2 phosphorylation occurs in both cycling neural stem cells and differentiated neurons spanning neocorticogenesis, and loss of eEF2k results in abnormal neocortical neuron differentiation. We hypothesize that eEF2k regulates mRNA translation, neural stem cell cycling, and differentiation in fetal neocortical development and dysfunction. This will be tested by first analyzing the dynamic candidate protein components and mRNA cargo of actively translating ribosomes (polysomes) identified by our preliminary studies in eEF2k KO and WT neocortices throughout development. Second, neural stem cell cycle and differentiation will be analyzed in developing eEF2k KO vs. WT neocortices to extend our preliminary finding of abnormal neocortical circuits with eEF2k depletion. This project aims to advance our understanding of ASDs from previous studies of the genomic and transcriptomic levels towards an ASD proteome, employing advanced in vivo techniques to identify specific translational therapeutic targets.

 描述（由申请人提供）：在大脑的新皮层内，产生了我们进化上最先进的复杂认知功能。神经干细胞和有丝分裂后神经元的不同亚群构成了这些复杂回路的基础，其分子定义是由它们的 基因表达模式，其中功能性基因表达在蛋白质合成中通过核糖体水平的mRNA翻译达到高潮。因此，mRNA翻译在发育过程中必须受到严格调节，以使特定mRNA产生分子定义的新皮质神经元亚群。自闭症谱系障碍（ASD）是临床上复杂认知功能的异质性障碍。在过去的十年中，基因组学和转录组学分析表明，在ASD病因学中，聚集在皮质中的蛋白质合成的基因和途径，通过翻译异常为中心的多种综合征ASD亚型（如脆性X综合征（FXS））得到加强。虽然核心翻译组分的调节与ASD有关，但特定mRNA的异常翻译如何导致新皮质功能障碍仍然没有答案。此外，目前尚不清楚核糖体成分在新皮层发育过程中是否是动态的，也不知道这是否与新皮层神经元如何从特定mRNA转录物的翻译中分子定义有关。我们的初步数据表明，核心翻译机制的组成部分和与它们相关的mRNA在胎儿新皮层中确实是动态的，在神经发生中期发生了一个特定的过渡-ASD基因组荟萃分析中高度涉及的易感期。我们的数据表明，这种中期神经源性的转变是由真核细胞延伸因子2（eEF 2）的磷酸化的显着增加，pupillary通过其激酶，eEF 2k调节延伸活性。有趣的是，在多项研究中，eEF 2k作为可变基因组位点与ASD相关，并且跨越该位点的独特微缺失综合征的特征在于严重的认知缺陷。eEF 2k还与FMRP合作调节FXS中特定mRNA的翻译。我们发现，动态eEF 2磷酸化发生在循环神经干细胞和分化的神经元跨越新皮质，和eEF 2k的损失导致异常的新皮质神经元分化。我们假设eEF 2k调节mRNA翻译，神经干细胞周期，并在胎儿新皮质发育和功能障碍的分化。这将通过首先分析我们在整个发育过程中在eEF 2k KO和WT新皮质中的初步研究中鉴定的主动翻译核糖体（多核糖体）的动态候选蛋白组分和mRNA货物来进行测试。其次，将在开发eEF 2k KO与WT新皮层中分析神经干细胞周期和分化，以扩展我们对eEF 2k耗尽的异常新皮层回路的初步发现。该项目旨在推进我们对ASD的理解，从以前的基因组和转录组水平的ASD蛋白质组的研究，采用先进的体内技术，以确定特定的翻译治疗靶点。