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: 9083289
• 负责人: Robert W Burgess
• 金额: $ 61.25万
• 依托单位: JACKSON LABORATORY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-12-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9083289
• 关键词: 4-thiouracil; Address; Adopted; Affect; Alleles; Amino Acid Substitution; Amino Acids; Amino Acyl-tRNA Synthetases; Axon; Binding; Biochemical; Biology; Cell Nucleus; Charcot-Marie-Tooth Disease; Charge; Defect; Development; Disease; Disease model; Dominant-Negative Mutation; Drosophila genus; Embryo; Epitopes; Essential Genes; Facial motor nucleus; Functional disorder; Genes; Genetic; Genetic Transcription; Genetic study; Germany; Glycine; Glycine-Specific tRNA; Glycine-tRNA Ligase; Goals; Health; Heterozygote; Housekeeping Gene; Imagery; In Vitro; Inborn Genetic Diseases; Inherited; Knock-out; Knockout Mice; Lead; Medical Genetics; Messenger RNA; Motor; Motor Neurons; Mus; Mutant Strains Mice; Mutation; Nerve; Nerve Degeneration; Neuromuscular Junction; Neurons; Neuropathy; Peripheral; Peripheral Nervous System; Peripheral Nervous System Diseases; Phenotype; Proteins; RNA; Ribosomes; Role; Signal Transduction; Specificity; Testing; Therapeutic; Transfer RNA; Transfer RNA Aminoacylation; Transgenic Organisms; Translations; Uracil; VEGFA gene; Vascular Endothelial Growth Factor Receptor; 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; neuronal cell body; novel therapeutics; overexpression; postnatal; therapy development

 DESCRIPTION (provided by applicant): 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 candidate 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. Also, this mechanism has only been tested in vivo in GarsP278KY/+, and it is unclear if it generalizes. 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 interactions of Nrp1 and GarsC201R/+ in vivo and in vitro to determine if this is a general mechanism. The other candidate 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. These studies will improve our understanding of CMT2D and suggest new therapeutic strategies.

 描述（由申请人提供）：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) 的外周轴突变性 6 个 tRNA 合成酶基因与 CMT 相关。 ，但这些是无处不在的“看家基因”，它们将氨基酸装载到其同源 tRNA 上，并且它们导致这种特定疾病的原因尚不清楚。 tRNA 充电活性不能解释疾病特异性，并且与 CMT2D 小鼠模型的临床遗传学不完全一致，强烈表明毒性功能获得导致 Gars 中氨基酸取代杂合的小鼠。 ) 会出现周围神经病变，而 Gars 无效等位基因杂合的小鼠则不受影响，这表明单倍体不足，也有 10-20 倍的影响。转基因野生型 GARS 的过度表达不会抑制神经病变，通过合作研究，我们现在有了两种候选的功能获得机制：首先，突变形式的 GARS 结合 Neuropilin1，但野生型不结合。 (NRP1) 在体外并拮抗其作为 VEGF 受体的活性 在 GarsP278KY/+ 胚胎中，面神经的运动核不会向尾部迁移，类似于 Nrp1 或Nrp1 和 GarsP278KY 也在基因上相互作用，Vegf 的病毒传递部分挽救了 GarsP278KY/+ 表型。然而，问题仍然存在，突变体 GARS 在 P10 处的 Nrp1 缺失可能不会引起神经病变。 GARS/NRP1 相互作用必须更早发生，而且这种机制仅在 GarsP278KY/+ 中进行了体内测试，尚不清楚。为了研究这些问题，在目标 1A 中，我们将寻找与 NRP1 拮抗作用一致的 Gars 突变小鼠的发育缺陷，并尝试尽早删除 Nrp1 以评估这是否足以导致神经病变。我们将在体内和体外检查 Nrp1 和 GarsC201R/+ 的相互作用，以确定这是否是一种通用机制。另一种候选机制来自果蝇的研究，其中 Nrp1 的转基因表达。与疾病相关的 GARS 等位基因在不改变 tRNA 充电的情况下导致轴突变性，这也表明受影响神经元的整体翻译减少，这被证明足以导致轴突变性。使用细胞类型特异性方法进行转录和翻译，包括 RNA 的 4-硫尿嘧啶标记、核糖体的 HA 表位标记以及新合成蛋白质的非规范氨基酸标记，以分析 RNA 和蛋白质我们将确定 Gars 突变小鼠的外周神经元的细胞体和轴突中的翻译是否受到抑制，识别受影响的特定 RNA 和蛋白质，确定这是否是 RNA 或翻译变化的结果，以及是否是由 RNA 或翻译变化引起的。这些变化与细胞体或轴突相关，这些研究将提高我们对 CMT2D 的理解并提出新的治疗策略。