Timed mRNA translation events in neocortical development and neurodevelopmental disorders

 描述(由申请者提供)：在大脑的新皮质内，起源于我们进化过程中最高级的复杂认知功能。支撑这些复杂回路的神经干细胞和有丝分裂后神经元的不同亚群是由它们的分子定义的 基因表达模式，其中功能基因的表达最终通过核糖体水平上的信使核糖核酸翻译来合成蛋白质。因此，mRNA翻译必须在发育过程中受到严格调控，以使特定的mRNAs产生分子定义的新皮质神经元亚群。自闭症谱系障碍是一种临床异质性疾病，具有复杂的认知功能。在过去的十年中，基因组和转录组分析表明，聚集在皮质蛋白质合成上的基因和途径是ASD病因学的靶点，并得到了以翻译异常为中心的多种综合征ASD亚型的加强，如脆性X综合征(FXS)。虽然ASD涉及核心翻译成分的调节，但特定mRNAs的异常翻译如何导致新皮质功能障碍仍未得到解答。此外，尚不清楚核糖体成分是否在新皮质发育过程中是动态的，也不知道这是否与新皮质神经元如何从特定mRNA转录本的翻译中进行分子定义有关。我们的初步数据表明，核心翻译机制的组件以及与之相关的胎儿新皮质中的mRNAs确实是动态的，特别是在神经发生中期-ASD基因组荟萃分析中高度涉及的敏感期。我们的数据显示，这种中间-神经性转变的显著特征是真核细胞延伸因子2(EEF2)的磷酸化水平显著增加，推测是通过其激酶eEF2K来调节延伸活性。有趣的是，在多项研究中，eEF2K作为一个可变的基因组位点与自闭症相关，一种跨越该基因座的独特的微缺失综合征的特征是严重的认知缺陷。EEF2K还与FMRP合作，调控FXS中特定mRNAs的翻译。我们发现在周期神经干细胞和跨越新皮质生成的分化神经元中都发生了动态的eEF2磷酸化，eEF2K的缺失导致了新皮质神经元的异常分化。我们推测，eEF2K在胎儿新皮质发育和功能障碍中调节mRNA翻译、神经干细胞周期和分化。这将通过首先分析我们在整个发育过程中在eEF2K、KO和WT新皮质中进行的初步研究确定的活跃翻译核糖体(多聚体)的动态候选蛋白质组分和mRNA载量来进行验证。其次，将在eEF2K KO与WT新皮质的发育中分析神经干细胞的周期和分化，以扩大我们对eEF2K耗竭的异常新皮质回路的初步发现。该项目旨在将我们对ASD的理解从以前的基因组和转录水平提高到ASD蛋白质组，利用先进的体内技术来识别特定的翻译治疗靶点。

 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.