Transcriptional and Translational Profiling of Motor Neurons in Two Mouse Models of Charcot-Marie-Tooth Disease Type 2D

两种 2D 型腓骨肌萎缩症小鼠模型运动神经元的转录和翻译分析

• 批准号: 9256641
• 负责人: Emily Louisa Spaulding
• 金额: $ 2.85万
• 依托单位: JACKSON LABORATORY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-01 至 2020-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9256641
• 关键词: 4-thiouracil; Adult; Affect; Afferent Neurons; Alpha Cell; Amino Acids; Amino Acyl-tRNA Synthetases; Aminoacylation; Axon; Binding; Biology; Catalogs; Cell Compartmentation; Cells; Charcot-Marie-Tooth Disease; Data; Defect; Dependence; Disease; Dominant-Negative Mutation; Drosophila genus; GARS gene; Genes; Genetic; Genetic Transcription; Genotype; Goals; Homeostasis; Human; Impairment; Inherited; Injury; KDR gene; Lead; Link; Location; Messenger RNA; Modeling; Motor; Motor Neurons; Mus; Mutant Strains Mice; Mutation; Natural regeneration; Nature; Nerve Crush; Nerve Degeneration; Neurons; Neuropathy; Neuropilin-1; Pathologic; Patients; Peripheral; Peripheral Nervous System Diseases; Phenotype; Process; Proteins; Public Health; RNA; RNA Transport; Ribosomes; Role; Sampling; Specificity; Spinal Cord; Techniques; Testing; Thiouracil; Translating; Translations; Work; cell type; disease heterogeneity; experimental study; gain of function; in vivo; meetings; mouse model; mutant; novel therapeutics; overexpression; regenerative; sciatic nerve

PROJECT SUMMARY/ABSTRACT How dominant mutations in glycyl tRNA synthetase (GARS) cause Charcot-Marie-Tooth disease Type 2D (CMT2D) peripheral neuropathy is still unclear and controversial. The technical challenge of studying the mammalian peripheral axon in vivo has contributed to the lack of a disease mechanism. The long-term goal of this project is to understand how mutations in ubiquitously expressed GARS lead to the specific and progressive degeneration of peripheral axons. The immediate objective is to use an in vivo, cell type- and compartment-specific approach to test the hypothesis that mutations in Gars cause impaired translation in two mouse models of CMT2D. Evidence points to a toxic gain-of-function of mutant GARS as the cause of neuropathy, but a dominant negative mechanism has not been ruled out. In Drosophila, overexpression of human mutant GARS in peripheral neurons causes neurodegeneration and reduced translation without altering aminoacylation. Because at least four other tRNA synthetases are linked to Charcot-Marie-Tooth, impaired translation is an attractive potential disease mechanism, and its detailed testing through the following experiments will further elucidate the gain-of function vs. dominant negative question: 1) We will test the hypothesis that mutant GARS impairs translation in motor neurons using two in vivo, cell type-specific techniques; non-canonical amino acid-tagging (NCAT) will provide the location, identity, and quantity of newly translated proteins, and ribosome-tagging will catalog ribosome-associated RNA. Motor neuron cell bodies are gathered from the spinal cord and axons from the sciatic nerve, providing cell compartment-specificity. Although local translation in adult, mammalian axons has not been established, it is required for normal regeneration after injury and preliminary data show that ribosomes are present and associated with mRNA in motor axons of the sciatic nerve. Wild-type sciatic nerve controls will be used to establish axonal translation. 2) We will test the hypothesis that translational impairments are independent of transcriptional changes. Thiouracil (4-TU)-tagging, a third in vivo, cell type-specific technique, will be used to catalog newly transcribed RNA in motor neuron cell bodies and newly transported RNA in axons. 3) Finally, we will test the hypothesis that mutant Gars axons attempt regeneration, but fail because of impairments in translation. NCAT, ribosome-, and 4-TU-tagging will be performed using wild-type regenerating motor neurons and the data compared to Gars mutant samples. The proposed experiments will test a hypothesized disease mechanism and uncover new motor neuron biology. Their completion will result in a comprehensive profile of translation and transcription in CMT2D, wild-type, and regenerating wild-type motor neuron cell bodies and axons. Identifying local translation in peripheral axons will represent a departure from the current model of axonal homeostasis, revealing the axon as a cell compartment with unique translational needs and its own ways of meeting them. Dependence of axons on local translation could explain their sensitivity to mutations in tRNA synthetases.

项目概要/摘要 甘氨酰 tRNA 合成酶 (GARS) 的显性突变如何导致 2D 型腓骨肌萎缩症 (CMT2D) 周围神经病变仍不清楚且有争议。研究的技术挑战 哺乳动物体内外周轴突的缺乏导致了一种疾病机制。长期目标是 该项目旨在了解普遍表达的 GARS 中的突变如何导致特定的和 周围轴突进行性退化。直接目标是使用体内、细胞类型和 隔室特异性方法来检验 Gars 突变导致两种细胞翻译受损的假设 CMT2D 小鼠模型。有证据表明突变体 GARS 的毒性功能获得是导致 神经病变，但尚未排除显性负性机制。在果蝇中，过度表达 外周神经元中的人类突变体 GARS 会导致神经变性并减少翻译，但不会改变 氨酰化。因为至少有四种其他 tRNA 合成酶与 Charcot-Marie-Tooth 相关，受损 翻译是一种有吸引力的潜在疾病机制，其详细测试通过以下 实验将进一步阐明增益函数与显性否定问题的关系：1）我们将测试 假设突变体 GARS 使用两种体内细胞类型特异性损害运动神经元的翻译 技术；非规范氨基酸标签 (NCAT) 将提供新氨基酸的位置、身份和数量 翻译的蛋白质，核糖体标签将对核糖体相关的 RNA 进行分类。运动神经元细胞体是 从脊髓收集，从坐骨神经轴突收集，提供细胞区室特异性。 虽然成人、哺乳动物轴突的局部翻译尚未建立，但它是正常的所必需的。 损伤后的再生和初步数据表明核糖体存在并与 mRNA 相关 坐骨神经的运动轴突。野生型坐骨神经对照将用于建立轴突翻译。 2）我们将检验翻译损伤与转录变化无关的假设。 硫尿嘧啶 (4-TU) 标签是第三种体内细胞类型特异性技术，将用于对新转录的基因进行编目 运动神经元细胞体中的 RNA 和轴突中新运输的 RNA。 3）最后，我们将检验假设 突变的 Gars 轴突尝试再生，但由于翻译缺陷而失败。 NCAT，核糖体-， 和 4-TU 标记将使用野生型再生运动神经元进行，并将数据与 Gars突变体样本。拟议的实验将测试假设的疾病机制并揭示 新运动神经元生物学。他们的完成将产生一个全面的翻译概况和 CMT2D、野生型和再生野生型运动神经元细胞体和轴突中的转录。识别 外周轴突的局部翻译将代表与当前轴突稳态模型的背离， 揭示了轴突作为一个具有独特翻译需求的细胞区室以及它自己的满足这些需求的方式。 轴突对局部翻译的依赖性可以解释它们对 tRNA 合成酶突变的敏感性。