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

• 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
• 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

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.

项目摘要： 蛋白质合成是所有活细胞中的基本过程，并且被高度调节以适应细胞的生长。 每个细胞的具体需求。蛋白质合成失调已被证明是许多疾病的基础。 自闭症的综合征形式，如脆性X综合征（FXS）和多发性硬化症（TSC），两者都是 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ASD样行为。最近的研究表明， 延伸因子1A 2（EEF 1A 2），一种负责氨酰-tRNA依赖GTP转运至 在自闭症、智力障碍和癫痫患者中，延伸系数1A 有两种亚型，一种是普遍表达的，EEF 1A 1，另一种是仅表达于 神经元和肌细胞。目前还不清楚为什么在这些特定的细胞中需要另一种同种型;然而，它已经被证明是一种特异性的蛋白质。 EEF 1A 2对神经元的存活至关重要。浪费的老鼠，一个纯合子的老鼠模型 已经发现小鼠Eef 1a 2的缺失表现出神经元变性、震颤、肌肉体积和步态的损失 断奶后出现异常。EEF 1A 2也被证明可以独立地捆绑肌动蛋白和微管， 翻译，这是一个已知对神经元发育和迁移至关重要的过程。这些证据表明， EEF 1A 2在神经元发育和功能中发挥关键作用。 该提案旨在揭示ASD相关的EEF 1A 2突变如何导致神经元损伤。 发展和自闭症病理生理学。使用人iPSC（诱导多能干细胞）衍生的神经元 作为一个模型，CRISPR-Cas9系统将被用来概括患者的突变。这些iPSC将被 使用能够诱导分化的主转录因子neurogenin-2分化成神经元 转化为兴奋性神经元使用这个平台，研究ASD相关突变对神经元的影响。 功能将被研究。第一个目的是检查ASD相关EEF 1A 2突变对蛋白表达的影响。 考虑到EEF 1A 2在蛋白质合成中发挥的核心作用，EEF 1A 2在神经元中的蛋白质合成中发挥重要作用。此外，变化 将鉴定这些细胞中的延伸率和翻译效率。第二 目的是探索神经元形态的变化。功能与发展。分化后，诱导 将使用以下方法检查具有ASD相关EEF 1A 2突变的神经元的形态学改变： 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， 将研究涉及肌动蛋白动力学和细胞骨架调节的异常信号通路。最后， 电生理学将用于通过测量兴奋性突触后神经元的兴奋性来评估突触功能和强度。 水流这项研究将促进我们对翻译控制在神经元细胞中所起作用的理解。 发展，以及其失调如何导致ASD病理生理学。