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

项目摘要/摘要 甘氨酰tRNA合成酶(GARS)显性突变如何导致2D型Charcot-Marie-Tooth病 (CMT2D)周围神经病仍不清楚，也存在争议。研究这一问题的技术挑战 哺乳动物体内的外周轴突缺乏致病机制。的长期目标是 这个项目是为了了解普遍表达的GAR的突变是如何导致特定的和 外周轴突进行性变性。目前的目标是使用体内的细胞类型--以及 用特定隔室的方法检验GAR突变导致两个基因的翻译受损的假说 CMT2D小鼠模型。有证据表明，突变的GARS的毒性功能增强是导致 神经病，但并不排除一种显性的负面机制。在果蝇中，过度表达 外周神经元中人突变型GARS导致神经变性和翻译减少而不改变 氨酰化。因为至少有四个其他tRNA合成酶与Charcot-Marie-Tooth相连，受损 翻译是一种有吸引力的潜在疾病机制，它通过以下几个方面进行了详细的测试 实验将进一步阐明增益函数与显性负问题：1)我们将测试 突变型GARS在体内使用两种细胞类型特异性损伤运动神经元翻译的假说 技术；非规范氨基酸标记(NCAT)将提供新的 翻译的蛋白质和核糖体标记将对核糖体相关的RNA进行分类。运动神经元胞体 从脊髓和来自坐骨神经的轴突收集，提供细胞隔室特异性。 虽然成年哺乳动物轴突的局部平移尚未建立，但它是正常所必需的。 损伤后再生和初步数据显示核糖体存在并与mRNA相关 坐骨神经的运动神经轴突。野生型坐骨神经控制将用于建立轴突平移。 2)我们将检验翻译障碍与转录变化无关的假设。 硫尿嘧啶(4-TU)标记是体内第三种特定细胞类型的技术，将用于对新转录的基因进行分类 运动神经元胞体的RNA和轴突中新运输的RNA。3)最后，我们将检验假设 突变的Gars轴突试图再生，但由于翻译障碍而失败。NCAT，核糖体， 和4-TU标记将使用野生型再生运动神经元和数据比较 GARS突变样本。拟议中的实验将测试一种假设的疾病机制，并揭示 新的运动神经元生物学。它们的完成将导致全面的翻译和 在CMT2D、野生型和再生野生型运动神经元胞体和轴突中转录。识别 外周轴突的局部平移将代表与当前轴突内稳态模型的背离， 揭示了轴突作为一个细胞隔间具有独特的翻译需求及其满足这些需求的方式。 轴突对局部翻译的依赖可以解释它们对tRNA合成酶突变的敏感性。

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.