Understanding the Role of the Integrated Stress Response in tRNA Synthetase-associated Charcot-Marie-Tooth Disease

了解综合应激反应在 tRNA 合成酶相关夏科-马里-图思病中的作用

• 批准号: 10740335
• 负责人: Timothy Hines
• 金额: $ 12.22万
• 依托单位: JACKSON LABORATORY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10740335
• 关键词: Affect; Alleles; Amino Acids; Amino Acyl-tRNA Synthetases; Autopsy; Axon; Binding; Biological Models; Career Transition Award; Cell Fraction; Cell Line; Cells; Charcot-Marie-Tooth Disease; Charge; Clustered Regularly Interspaced Short Palindromic Repeats; Data; Defect; Demyelinations; Disease; Distal; Drug usage; EIF-2alpha; Engineering; Ensure; Enzymes; Event; FGF21 gene; Family; Functional disorder; GDF15 gene; Gene Expression; Gene Family; Genes; Genetic Engineering; Goals; Hereditary Disease; Histology; Human; Human Engineering; Immunofluorescence Immunologic; In Vitro; Inherited; Knock-out; Ligase; Link; Mammalian Genetics; Measures; Mediating; Mentors; Methods; Modeling; Morphologic artifacts; Motor; Motor Neurons; Mus; Mutation; Nerve Degeneration; Neuromuscular Diseases; Neuromuscular Junction; Neuronal Dysfunction; Neuropathy; Paper; Pathogenesis; Pathogenicity; Pathology; Pathway interactions; Patients; Peptide Initiation Factors; Peripheral Nerves; Peripheral Nervous System Diseases; Persons; Phase; Phenotype; Phosphorylation; Phosphotransferases; Physiology; Postdoctoral Fellow; Production; Protein Biosynthesis; Proteins; Proteomics; Publishing; Quantitative Reverse Transcriptase PCR; RNA Interference; Rare Diseases; Reagent; Resource Development; Review Literature; Ribosomes; Role; Sampling; Science; Series; Serum; Skeletal Muscle; Source; Spinal Cord; System; Testing; The Jackson Laboratory; Therapeutic; Therapeutic Intervention; Tissue Sample; Tooth Diseases; Training; Transfer RNA; Transgenic Mice; Translations; Tyrosine-tRNA Ligase; Up-Regulation; Variant; Western Blotting; Work; YARS gene; afferent nerve; biological adaptation to stress; career; career development; causal variant; data integration; differential expression; directed differentiation; disease phenotype; disease-causing mutation; dominant genetic mutation; experimental study; fibroblast growth factor 21; gene replacement; human disease; human stem cells; in vivo; induced pluripotent stem cell; mouse model; mutant; neuromuscular; neuronal cell body; novel therapeutics; overexpression; prevent; protein expression; response; sensor; stem cell model; targeted treatment; therapeutic evaluation; therapeutic target; tool; transcription factor; transcriptome sequencing; transcriptomics

PROJECT SUMMARY This proposal will address the mechanisms underlying neuromuscular degeneration in Charcot-Marie-Tooth disease (CMT). CMT is a genetically and phenotypically heterogeneous neuromuscular disorder with causative mutations found in over 100 genes. While considered a rare disease, CMT is the most common inherited disorder of the peripheral nervous system, affecting ~1 in 3,500 people worldwide. Dominant mutations in 6 different tRNA synthetases (aaRSs) cause forms of CMT (aaRS-CMT), making them the largest family of CMT-associated genes. Each of these genes is involved in protein synthesis suggesting a common mechanism that leads to defects in protein production and ultimately CMT pathologies. Our recently published work uncovered a potential mechanism underlying aaRS-CMT. We found that mutant aaRSs inappropriately sequester tRNAs from the ribosome, which stalls ribosome function and activates an integrated stress response (ISR) via a sensor protein, GCN2. ISR activation causes two major cellular events: 1) shutdown of a major form of protein synthesis and, 2) upregulation of the transcription factor, ATF4, and its target genes. The relative contributions of each of these events is currently unknown. One goal of this project is to determine the role of ATF4 and target genes in the pathophysiology observed in aaRS-CMT. Preliminary results show that ATF4 overexpression is toxic to motor neurons and produces a CMT- like phenotype in mice, evidence that ATF4 could be a viable therapeutic target for aaRS-CMT. In Aim 1 we will manipulate ATF4 expression levels in validated mouse models of aaRS-CMT to determine whether the disease pathology is driven by decreased protein translation or by increased expression of the ATF4 gene. To advance toward therapeutic applications we need to establish that human motor neurons also activate the ISR in response to aaRS-CMT mutations. Therefore, in Aim 2 we will establish and validate human induced pluripotent stem cell (hiPSC)-derived motor neuron cultures which have been genetically engineered to model aaRS-CMT. We will also test therapeutic strategies in these human cell-based models. Interestingly, ATF4 expression is common in many different types of neurodegeneration. Therefore, in Aim 3, we will integrate data from ATF4 mice in Aim 1, and hiPSC-derived motor neurons in Aim 2 to identify common genes and cellular pathways involved in ATF4-mediated neurodegeneration. These hiPSC-based models will be a powerful tool to help identify and develop new targets or pathways for potential therapeutic interventions. This MOSAIC (Maximizing Opportunities for Scientific and Academic Independent Careers) Postdoctoral Career Transition Award to Promote Diversity will be supported by excellent career development resources and a mentoring team of globally recognized experts in CMT (R.W. Burgess) and human stem cells (M.F. Pera) at The Jackson Laboratory for Mammalian Genetics.

项目摘要 这项提案将解决的机制，神经肌肉变性在腓骨肌萎缩症 疾病（CMT）。CMT是一种遗传和表型异质性神经肌肉疾病， 在100多个基因中发现了突变。虽然被认为是一种罕见的疾病，但CMT是最常见的遗传性疾病 周围神经系统，影响到全球约3，500人中的1人。6种不同tRNA的显性突变 合成酶（aaRSs）导致CMT形式（aaRS-CMT），使其成为CMT相关的最大家族。 基因.这些基因中的每一个都参与蛋白质合成，这表明了一种共同的机制， 蛋白质生产缺陷和最终的CMT病理学。 我们最近发表的工作揭示了aaRS-CMT的潜在机制。我们发现那个变种人 aaRSs不适当地从核糖体中隔离tRNA，这会阻止核糖体功能并激活一个 整合应激反应（ISR）通过传感器蛋白，GCN 2。ISR激活导致两个主要的细胞事件： 1)关闭主要形式的蛋白质合成，2）上调转录因子，ATF 4， 靶基因这些事件中每一个的相对贡献目前尚不清楚。 该项目的一个目标是确定ATF 4和靶基因在观察到的病理生理学中的作用。 aaRS-CMT。初步结果表明，ATF 4过表达对运动神经元有毒性，并产生CMT-1。 类似于小鼠中的表型，证明ATF 4可以是aaRS-CMT的可行治疗靶标。在目标1中， 在经验证的aaRS-CMT小鼠模型中操纵ATF 4表达水平，以确定疾病是否 病理学是由蛋白质翻译减少或由ATF 4基因表达增加驱动的。 为了推进治疗应用，我们需要确定人类运动神经元也激活了 ISR对aaRS-CMT突变的应答。因此，在目标2中，我们将建立并验证人类诱导的 多能干细胞（hiPSC）衍生的运动神经元培养物，其已被遗传工程化以模拟 aaRS-CMT。我们还将在这些基于人类细胞的模型中测试治疗策略。有趣的是，ATF 4 表达在许多不同类型的神经变性中是常见的。因此，在目标3中，我们将整合数据 Aim 1中的ATF 4小鼠和Aim 2中的hiPSC衍生的运动神经元，以鉴定共同的基因和细胞 参与ATF 4介导的神经变性的途径。这些基于hiPSC的模型将是一个强大的工具， 有助于识别和开发潜在治疗干预的新靶点或途径。 MOSAIC（最大化科学和学术独立职业的机会） 促进多元化的过渡奖将得到优秀的职业发展资源和 由全球公认的CMT专家组成的指导团队（R.W. Burgess）和人干细胞（M.F. Pera）在The 杰克逊哺乳动物遗传学实验室。