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.

项目摘要/摘要 糖基TRNA合成酶（GARS）中的显性突变如何引起charcot-marie-tooth疾病2D （CMT2D）周围神经病仍然不清楚并且有争议。研究的技术挑战 体内哺乳动物外周轴突已导致缺乏疾病机制。长期目标 该项目是要了解无用表达的GARS中的突变如何导致特定和 周围轴突的进行性变性。直接目标是使用体内，细胞类型和 隔室特异性方法是检验以下假设，即GARS中的突变导致两种情况下的翻译受损 CMT2D的鼠标模型。证据指出突变gars的功能障碍是 神经病，但尚未排除主导的负面机制。在果蝇中，过表达 外周神经元中的人类突变体GARS导致神经变性并减少翻译而不会改变 氨基酰化。因为至少有四个其他tRNA合成酶与charcot-marie-tooth有关 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 实验将进一步阐明功能获得与主要负面问题：1）我们将测试 假设突变体使用两个体内细胞类型特异性的运动神经元中的翻译 技术；非典型氨基酸标签（NCAT）将提供新的位置，身份和数量 翻译的蛋白质和核糖体标记将分类核糖体相关的RNA。运动神经元细胞体是 从脊髓和轴突从坐骨神经中聚集，提供细胞室特异性。 尽管成人局部翻译，但尚未建立哺乳动物轴突，但正常需要 受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。野生型坐骨神经控制将用于建立轴突翻译。 2）我们将检验以下假设：翻译障碍与转录变化无关。 Thiouracil（4-Tu） - 标记，第三个体内，细胞类型特异性技术，将用于分类新转录 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 那个突变的GARS轴突尝试再生，但由于翻译的损害而失败。 ncat，核糖体 - ， 将使用野生型再生运动神经元和数据进行4-TU标记 GARS突变样品。提出的实验将测试假设的疾病机制并发现 新的运动神经元生物学。他们的完成将导致翻译的全面概况和 CMT2D，野生型和再生野生型运动神经元细胞体和轴突的转录。识别 外围轴突中的局部翻译将代表与当前轴突稳态模型的背离， 将轴突揭示为具有独特的翻译需求及其满足它们的方式的单元格。 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。