Functional Characterization of ASD-Associated EEF1A2 Mutations in Human Neurons

人类神经元中 ASD 相关 EEF1A2 突变的功能表征

基本信息

批准号：
10311035
负责人：
Muhaned Mohamed
金额：
$ 4.42万
依托单位：
NEW YORK UNIVERSITY SCHOOL OF MEDICINE
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：
2020-09-04 至 2025-09-03
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10311035
关键词：
Actins Affect Alleles Amino Acyl Transfer RNA Astrocytes Behavior Biological Assay Brain CRISPR/Cas technology Cell Line Cells Child Complex Defect Development Diagnosis Disease Dominant-Negative Mutation EEF1A1 gene EEF1A2 gene Electrophysiology (science)Elongation Factor Epilepsy Exhibits Fragile X Syndrome Freezing Frequencies Functional disorder Gait abnormality Genes Genetic Guanosine Triphosphate Heart Human Impairment Induced pluripotent stem cell derived neurons Intellectual functioning disability Knock-out Lead Learning Location Measures Memory Messenger RNA Microtubules Missense Mutation Modeling Morphology Mus Muscle Muscle Cells Muscular Atrophy Mutate Mutation Nerve Degeneration Neurites Neurodevelopmental Disorder Neurons Pathologic Pathway interactions Patients Peptide Elongation Factor 1 Phenotype Play Prevalence Process Protein Biosynthesis Protein Isoforms Proteins Regulation Research Ribosomes Role Shapes Signal Pathway Site Social Behavior Stains Synapses Synapsins Syndrome System Tissues Translating Translations Tremor Tuberous sclerosis protein complex United States Variant Vertebral column Weaning autism spectrum disorder density excitatory neuron experimental study gain of function genetic variant induced pluripotent stem cell inhibitor/antagonist insight interest live cell imaging migration motor neuron degeneration mouse model muscle form mutant neurite growth neurodevelopment neuron development neuronal cell body neuronal survival patch clamp progenitor repetitive behavior ribosome profiling social communication synaptic function synaptogenesis transcription factor translatome

项目摘要

Project Summary: Protein synthesis is a fundamental process in all living cells and is highly regulated to accommodate the specific needs of each cell. Dysregulated protein synthesis has been demonstrated to underlie many of syndromic forms of autism such as Fragile X syndrome (FXS) and Tuberous Sclerosis Complex (TSC), both of which result from defects in genes that regulate protein synthesis. Moreover, mouse models of FXS and TSC exhibit defective synaptic function, and ASD-like behaviors. Recent studies have shown that Eukaryotic Elongation Factor 1A2 (EEF1A2), a protein responsible for GTP-dependent transport of aminoacyl-tRNAs to the elongating ribosome, is mutated in patients with autism, intellectual disability and epilepsy. Elongation Factor 1A has two isoforms, one that is ubiquitously expressed, EEF1A1, and another, EEF1A2 that is expressed only in neurons and myocytes. It is unclear why another isoform is needed in these specific cells; however, it has been found that EEF1A2 is critical for neuronal survival. The wasted mouse, a mouse model with a homozygous deletion of mouse Eef1a2, has been found to exhibit neuron degeneration, tremors, loss of muscle bulk and gait abnormalities after weaning. EEF1A2 has been also shown to bundle actin and microtubules independently of translation, a process known to be critical for neuronal development and migration. This evidence suggests a critical role played by EEF1A2 in neuronal development and function. This proposal aims to uncover how ASD-associated mutations in EEF1A2 results in deficits in neuronal development and autism pathophysiology. Using human iPSC (induced pluripotent stems cells) derived neurons as a model, the CRISPR-Cas9 system will be used to recapitulate patient mutations. These iPSCs will then be differentiated into neurons using neurogenin-2, a master transcription factor capable of inducing differentiation into excitatory neurons in under 2 weeks. Using this platform, the effect of ASD-associated mutations on neuronal function will be studied. The first aim examines the effect of ASD-associated EEF1A2 mutations on protein synthesis in neurons, given the central role that EEF1A2 plays in protein synthesis. Furthermore, the changes to the translatome profile, elongation rate and translational efficiency in these cells will be identified. The second aim will explore changes to neuronal morphology. function and development. After differentiation, induced neurons with ASD-associated EEF1A2 mutations will be examined for altered morphology using immunocytochemical analysis. During differentiation, live cell imaging will be used to track neurite growth and the aberrant signaling pathways involved in actin dynamics and cytoskeletal regulation will be studied. Finally, electrophysiology will used to assess synapse function and strength by measuring excitatory post synaptic currents. The proposed research will advance our understanding of the role translation control plays in neuronal development, and how its dysregulation leads to ASD pathophysiology.

项目概要：蛋白质合成是所有活细胞的基本过程，并且受到高度调节以适应每个细胞的特定需求。蛋白质合成失调已被证明是许多疾病的根源自闭症的综合征形式，例如脆性 X 综合征 (FXS) 和结节性硬化症 (TSC)，两者均这是由调节蛋白质合成的基因缺陷引起的。此外，FXS和TSC的小鼠模型表现出有缺陷的突触功能和类似 ASD 的行为。最近的研究表明，真核生物延伸因子 1A2 (EEF1A2)，一种负责 GTP 依赖性氨酰 tRNA 转运至延长核糖体在自闭症、智力障碍和癫痫患者中发生突变。伸长率1A 有两种异构体，一种广泛表达，EEF1A1，另一种，EEF1A2，仅在神经元和肌细胞。目前还不清楚为什么这些特定细胞需要另一种同种型。然而，它已经发现 EEF1A2 对于神经元的存活至关重要。废弃小鼠，纯合子小鼠模型小鼠 Eef1a2 缺失，被发现会表现出神经元变性、震颤、肌肉体积和步态丧失断奶后出现异常。 EEF1A2 也被证明可以独立地捆绑肌动蛋白和微管翻译，已知对神经元发育和迁移至关重要的过程。这一证据表明 EEF1A2 在神经元发育和功能中发挥关键作用。该提案旨在揭示 EEF1A2 中与 ASD 相关的突变如何导致神经元缺陷发育和自闭症病理生理学。使用人类 iPSC（诱导多能干细胞）衍生的神经元作为模型，CRISPR-Cas9 系统将用于重现患者突变。这些 iPSC 将被使用neurogenin-2分化为神经元，neurogenin-2是一种能够诱导分化的主转录因子在不到两周的时间内进入兴奋性神经元。使用这个平台，ASD 相关突变对神经元的影响函数将被研究。第一个目标是检查 ASD 相关 EEF1A2 突变对蛋白质的影响鉴于 EEF1A2 在蛋白质合成中发挥的核心作用，神经元中的合成。此外，这些变化根据翻译组图谱，将确定这些细胞的延伸率和翻译效率。第二个目的将探索神经元形态的变化。功能和发展。分化后诱导将使用以下方法检查具有 ASD 相关 EEF1A2 突变的神经元的形态改变免疫细胞化学分析。在分化过程中，活细胞成像将用于跟踪神经突生长和将研究参与肌动蛋白动力学和细胞骨架调节的异常信号通路。最后，电生理学将通过测量兴奋性突触后来评估突触功能和强度电流。拟议的研究将增进我们对翻译控制在神经元中的作用的理解发育，以及其失调如何导致 ASD 病理生理学。