Genetics of the Neuromuscular Junction: Mechanisms and Disease Models

神经肌肉接头的遗传学：机制和疾病模型

• 批准号: 9765407
• 负责人: Robert W Burgess
• 金额: $ 55.29万
• 依托单位: JACKSON LABORATORY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-05 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9765407
• 关键词: 4-thiouracil; Adopted; Affect; Alleles; Amino Acid Substitution; Amino Acids; Amino Acyl-tRNA Synthetases; Axon; Binding; Biochemical; Biology; Cells; Charcot-Marie-Tooth Disease; Charge; Clinical; Data; Defect; Development; Disease; Disease model; Dominant-Negative Mutation; Drosophila genus; Effectiveness; Embryo; Epitopes; Extracellular Domain; Facial motor nucleus; Functional disorder; GARS gene; Genes; Genetic; Genetic Transcription; Genetic study; Germany; Glycine; Glycine-Specific tRNA; Goals; Hereditary Disease; Housekeeping Gene; Imagery; Impairment; In Vitro; KDR gene; Knock-out; Knockout Mice; Lead; Mediating; Medical Genetics; Messenger RNA; Motor; Motor Neurons; Mus; Mutant Strains Mice; Mutation; Nerve Degeneration; Neuromuscular Junction; Neurons; Neuropathy; Pathogenicity; Peripheral; Peripheral Nervous System Diseases; Phenotype; Proteins; Public Health; RNA; Ribosomes; Role; Signal Transduction; Specificity; Testing; Therapeutic; Thiouracil; Transfer RNA; Transfer RNA Aminoacylation; Transgenic Organisms; Translations; Vascular Endothelial Growth Factors; Viral; Work; Yang; axonal degeneration; cell type; chemotherapy induced neuropathy; diabetic; effective therapy; gain of function; improved; in vivo; loss of function; mouse model; mutant; novel; novel therapeutic intervention; overexpression; postnatal; receptor; ribosome profiling; therapy development

ABSTRACT The goal of this project is to determine how dominant mutations in glycyl tRNA synthetase (GARS) cause peripheral axon degeneration in Charcot-Marie-Tooth disease (CMT) type 2D. Six tRNA synthetase genes are associated with CMT, but these are ubiquitous “housekeeping genes” that charge amino acids onto their cognate tRNAs, and why they cause such a specific disease is unclear. A loss of tRNA charging activity would not explain the disease specificity and is not completely consistent with clinical genetics. Our work in mouse models of CMT2D strongly suggests that a toxic gain-of-function causes neuropathy. Mice heterozygous for amino acid substitutions in Gars (C201R and P278KY) develop peripheral neuropathy, whereas mice heterozygous for a null allele of Gars are unaffected, arguing against a haploinsufficiency. Also, 10-20 fold overexpression of transgenic wild-type GARS does not suppress the neuropathy, arguing against a loss of function. Through collaborative work, we now have two intriguing gain-of-function mechanisms. First, mutant forms of GARS, but not wild type, bind Neuropilin1 (NRP1) in vitro and antagonize its activity as a VEGF receptor in vivo. The motor nucleus of the facial nerve does not migrate caudally in GarsP278KY/+ embryos, resembling Nrp1 or Vegf knockouts. Nrp1 and GarsP278KY also interact genetically, and viral delivery of Vegf partially rescues the GarsP278KY/+ phenotype. However, questions remain, and mutant GARS may have additional pathogenic activities. Deletion of Nrp1 at P10 does not cause neuropathy, suggesting the mutant GARS/NRP1 interaction must occur earlier, and how much of the phenotype this mechanism explains and the cell autonomy of these interactions are unclear. To investigate these issues, in Aim 1A, we will look for developmental defects in Gars mutant mice that are consistent with NRP1 antagonism, and attempt to delete Nrp1 earlier to assess if this is sufficient to cause neuropathy. In Aim 1B, we will examine the effectiveness, timing, and cell autonomy of NRP1 decoy receptors in mitigating the activity of mutant GARS. Our second novel mechanism comes from work in Drosophila, where transgenic expression of disease-associated GARS alleles causes axon degeneration without altering tRNA charging, also suggesting a gain-of-function. However, global translation in affected neurons was reduced, and this was shown to be sufficient for axon degeneration. We will use an in vivo analysis of transcription and translation with cell-type-specific approaches including 4-thiouracil tagging of RNA, HA-epitope tagging of ribosomes, and noncanonical amino acid tagging of newly synthesized proteins to analyze RNA and proteins in the cell bodies and axons of peripheral neurons of control and mutant mice. We will determine if translation is suppressed in our Gars mutant mice, identify the specific RNA and proteins affected, determine whether this results from changes in RNA or translation, and whether the changes are specific to cell bodies or axons. This analysis is relevant to both gain- and loss-of- function mechanisms, and will improve our understanding of CMT2D and suggest new therapeutic strategies.

抽象的 该项目的目标是确定甘氨酰 tRNA 合成酶 (GARS) 的显性突变如何导致 2D 型腓骨肌萎缩症 (CMT) 中的外周轴突变性有 6 个 tRNA 合成酶基因。 与 CMT 相关，但这些是无处不在的“管家基因”，它们将氨基酸装载到它们的基因上 同源 tRNA，以及它们为何会导致这种特定疾病尚不清楚。 不能解释疾病的特异性，并且与我们在小鼠中的临床遗传学工作不完全一致。 CMT2D 模型强烈表明，毒性功能获得会导致杂合子小鼠的神经病变。 Gars 中的氨基酸替代（C201R 和 P278KY）会导致周围神经病变，而小鼠 Gars 无效等位基因的杂合子不受影响，也反对单倍体不足。 转基因野生型 GARS 的过度表达并不能抑制神经病变，反对丧失 通过协作工作，我们现在有了两种有趣的功能获得机制：首先，突变。 GARS 形式（而非野生型）在体外结合 Neuropilin1 (NRP1) 并拮抗其作为 VEGF 的活性 GarsP278KY/+ 胚胎中面神经的运动核不会向尾部迁移， 重组 Nrp1 或 Vegf 敲除 Nrp1 和 GarsP278KY 也会在基因上相互作用，以及 Vegf 的病毒传递。 部分拯救了 GarsP278KY/+ 表型然而，问题仍然存在，突变体 GARS 可能具有。 P10 处 Nrp1 的缺失不会引起神经病变，表明该突变体。 GARS/NRP1 相互作用必须更早发生，该机制解释了多少表型以及 这些相互作用的细胞自主性尚不清楚。为了研究这些问题，在目标 1A 中，我们将寻找 Gars突变小鼠的发育缺陷与NRP1拮抗作用一致，并尝试删除 尽早评估 Nrp1 是否足以引起神经病变，在目标 1B 中，我们将检查其有效性， NRP1 诱饵受体的时间和细胞自主性在减轻突变体 GARS 的活性方面是我们的第二个。 新机制来自果蝇研究，其中与疾病相关的 GARS 进行转基因表达 等位基因导致轴突变性而不改变 tRNA 充电，这也表明功能获得。 然而，受影响神经元的全局翻译减少，这对于轴突来说已经足够了 我们将通过细胞类型特异性方法对转录和翻译进行体内分析。 包括 RNA 的 4-硫尿嘧啶标签、核糖体的 HA-表位标签和非规范氨基酸标签 新合成的蛋白质用于分析周围神经元细胞体和轴突中的 RNA 和蛋白质 我们将确定 Gars 突变小鼠的翻译是否受到抑制，并确定 受影响的特定 RNA 和蛋白质，确定这是否是 RNA 或翻译变化的结果，以及 这些变化是否特定于细胞体或轴突该分析与增益和损失相关。 功能机制，并将提高我们对 CMT2D 的理解并提出新的治疗策略。