The 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: 10303668
• 负责人: Robert W Burgess
• 金额: $ 27.78万
• 依托单位: JACKSON LABORATORY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10303668
• 关键词: Affect; Afferent Neurons; Affinity; Alleles; Amino Acids; Amino Acyl-tRNA Synthetases; Biochemical; Biological; Cell Differentiation process; Cells; Cellular biology; Charcot-Marie-Tooth Disease; Charge; Chronic; Codon Nucleotides; Collection; Data; Disease; Disease model; Engineering; Enzymes; Gene Family; Genes; Genetic; Genetic study; Health; Hereditary Disease; Human; Human Cell Line; Inherited; Knockout Mice; Lead; Ligase; Measures; Modeling; Motor Neurons; Mus; Mutation; Nerve; Neuromuscular Junction; Neuropathy; Pathogenicity; Patients; Peripheral Nervous System Diseases; Pharmacology; Phenotype; Phosphotransferases; Ribosomes; Severities; Specificity; System; Testing; Transfer RNA; Translations; Tyrosine-tRNA Ligase; Work; base; biological adaptation to stress; cell type; gene therapy; in vivo; mouse model; mutant; overexpression; parent grant; treatment strategy; vector; Affect; Afferent Neurons; Affinity; Alleles; Amino Acids; Amino Acyl-tRNA Synthetases; Biochemical; Biological; Cell Differentiation process; Cells; Cellular biology; Charcot-Marie-Tooth Disease; Charge; Chronic; Codon Nucleotides; Collection; Data; Disease; Disease model; Engineering; Enzymes; Gene Family; Genes; Genetic; Genetic study; Health; Hereditary Disease; Human; Human Cell Line; Inherited; Knockout Mice; Lead; Ligase; Measures; Modeling; Motor Neurons; Mus; Mutation; Nerve; Neuromuscular Junction; Neuropathy; Pathogenicity; Patients; Peripheral Nervous System Diseases; Pharmacology; Phenotype; Phosphotransferases; Ribosomes; Severities; Specificity; System; Testing; Transfer RNA; Translations; Tyrosine-tRNA Ligase; Work; base; biological adaptation to stress; cell type; gene therapy; in vivo; mouse model; mutant; overexpression; parent grant; treatment strategy; vector

PROJECT SUMMARY/ABSTRACT – from parent grant R37NS054154 This project examines dominant mutations in tRNA synthetase genes that cause inherited peripheral neuropathy. We seek to understand the biochemical and cellular basis for these diseases, to test mechanisms that could explain their specificity for motor and sensory neurons, and to test possible pharmacological and gene therapy-based treatments. Charcot-Marie-Tooth disease (CMT) is a collection of inherited diseases of the peripheral nervous system, and close to 100 genes are associated with CMT. The largest gene family associated with CMT is the tRNA synthetases. These enzymes charge amino acids onto their cognate tRNAs, and mutations in at least six cause peripheral neuropathy. Our preliminary data show that mutations in glycyl- and tyrosyl-tRNA synthetase (Gars and Yars, respectively) in mouse models lead to the activation of the integrated stress response (ISR) through the kinase GCN2. Genetic deletion of Gcn2 alleviates the phenotype of Gars/CMT2D mouse models. GCN2 is activated by uncharged tRNAs and also stalled ribosomes, and genetic interaction studies are consistent with ribosome stalling in vivo. Furthermore, uncharged tRNAs should be rescued by overexpression of wild-type synthetase, and we have shown this does not happen in Gars mouse models. Instead we favor a mechanism in which the tRNA substrate becomes limiting. We propose the following model: The mutant synthetases have aberrantly increased affinity for their cognate tRNAs, effectively sequestering them. This leads to ribosome stalling at the relevant codons during translation, which activates GCN2 and the ISR. The chronic activation of the ISR contributes to disease. We propose three aims to test this model. In Aim 1, we will directly measure the affinities of wild-type and mutant synthetases for their cognate tRNAs, anticipating that mutant synthetases will have higher affinities and slower off rates. We will also develop cell-based models using pluripotent human cell lines engineered to carry neuropathy-associated tRNA synthetase alleles. Differentiating these cells to motor neurons will validate our model in human cells and enable studies ribosome stalling and other relevant cell biology. We can also combine these systems predictively to create pathogenic or protective mutations, correlating tRNA affinity, ribosome stalling, induction of the ISR, and severity of neuropathy. In Aim 2, we will test possible mechanisms underlying the specificity of the disease. The tRNA synthetase genes are ubiquitously expressed, and the change in affinity should not be cell-type specific. Therefore, motor and sensory neurons may be particularly susceptible to ribosome stalling and the induction of the ISR, or may be particularly sensitive to tRNA sequestration due to high expression of the synthetase genes or poor expression of tRNAs. In Aim 3, we will test whether pharmacological inhibition of the ISR is beneficial, as suggested by our genetic studies with Gcn2 knockout mice. We will also test if the phenotype is rescued (which we anticipate) or exacerbated using AAV9 vectors to increase tRNA expression. These studies are potentially translational, and also directly test our proposed tRNA sequestration mechanism.

项目摘要/摘要 - 来自父母赠款R37NS054154 该项目考试在引起遗传外周的tRNA合成酶基因中的显性突变 神经病。我们试图了解这些疾病的生化和细胞基础，以测试机制 这可以解释它们对运动和感觉神经元的特异性，并测试可能的药物和 基于基因治疗的治疗。 Charcot-Marie-Tooth病（CMT）是一系列遗传性疾病 周围神经系统和接近100个基因与CMT相关。最大的基因家族 与CMT相关的是TRNA合成酶。这些酶将氨基酸加到其同源trnas上， 至少六个中的突变引起周围神经病。我们的初步数据表明，糖基的突变 小鼠模型中分别 通过激酶GCN2进行综合应力反应（ISR）。 GCN2的遗传缺失减轻了表型 GARS/CMT2D鼠标型号。 GCN2被无负荷的TRNA和停滞的核糖体激活，以及 遗传相互作用研究与体内的核糖体失速一致。此外，无需充电的trnas应该 通过野生型合成酶的过表达来挽救，我们表明这在GARS中不会发生 鼠标模型。取而代之的是，我们偏爱一种机制，其中tRNA底物变得有限。我们建议 以下模型：突变合成酶异常增加了对其同源trNA的亲和力， 有效地隔离它们。这会导致翻译过程中相关密码子的核糖体停滞 激活GCN2和ISR。 ISR的慢性激活有助于疾病。我们提出三个目标 测试此模型。在AIM 1中，我们将直接测量野生型和突变合成酶的亲和力 Cognate trnas，预计突变型合酶的亲和力将较高，并且降低了速率。我们将 还使用多元能力的人类细胞系开发基于细胞的模型 tRNA合成酶等位基因。将这些细胞区分为运动神经元将验证我们在人类细胞中的模型 并启用研究核糖体失速和其他相关细胞生物学。我们还可以结合这些系统 预测创建致病性或保护突变，将tRNA亲和力相关，核糖体停滞，诱导 ISR和神经病的严重程度。在AIM 2中，我们将测试特异性基础的可能机制 疾病。 tRNA合成酶基因普遍表达，亲和力的变化不应是 细胞类型的特异性。因此，运动和感觉神经元可能特别容易受到核糖体的损失 以及ISR的诱导，或者由于高表达的高表达，可能对tRNA隔离特别敏感 合成酶基因或TRNA表达不佳。在AIM 3中，我们将测试药物抑制是否 正如我们对GCN2基因敲除小鼠的遗传研究所暗示的那样，ISR是有益的。我们还将测试是否 使用AAV9向量响应表型（我们预计）或加剧了表型，以增加tRNA表达。 这些研究具有潜在的转化，也直接测试了我们提出的tRNA隔离机制。