The Genetics of the Neuromuscular Junction: Mechanisms and Disease Models

项目摘要/摘要-来自母公司赠款R37NS054154 这个项目检查了导致遗传性外周血细胞的tRNA合成酶基因的显性突变。 神经病。我们试图了解这些疾病的生化和细胞基础，测试机制。 这可以解释它们对运动神经元和感觉神经元的特异性，并测试可能的药理学和 以基因治疗为基础的治疗。Charcot-Marie-Tooth病(CMT)是一组遗传性疾病 外周神经系统和近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。这些酶将氨基酸充电到它们的同源tRNA上， 至少有6个基因的突变会导致周围神经病变。我们的初步数据显示，甘氨酰- 和酪氨酰-tRNA合成酶(分别为Gars和Yars)在小鼠模型中导致激活 整合应激反应(ISR)通过激酶GCN2。GCN2基因缺失可缓解表型 GARS/CMT2D小鼠模型。GCN2被不带电荷的tRNA和停滞的核糖体激活，并且 遗传相互作用研究与体内核糖体停滞一致。此外，未充电的tRNA应该 通过野生型合成酶的过度表达而获救，我们已经证明这在GARS中不会发生 老鼠模型。相反，我们倾向于一种tRNA底物变得有限的机制。我们建议 以下模型：突变的合成酶异常地增加了对其同源tRNA的亲和力， 有效地隔离了它们。这导致核糖体在翻译过程中在相关密码子处停滞，这 激活GCN2和ISR。ISR的慢性激活导致了疾病。我们提出了三个目标 来测试这个模型。在目标1中，我们将直接测量野生型和突变型合成酶对其 同源tRNA，预期突变的合成酶将具有更高的亲和力和更慢的关闭速度。我们会 还利用设计成携带神经病变相关基因的多潜能人类细胞系开发基于细胞的模型 TRNA合成酶等位基因。将这些细胞分化为运动神经元将在人类细胞中验证我们的模型 并使研究核糖体停滞和其他相关细胞生物学成为可能。我们也可以将这些系统结合起来 预测性地产生致病或保护性突变，与tRNA亲和力、核糖体停滞、诱导 以及神经病变的严重程度。在目标2中，我们将测试潜在的可能的机制 这种疾病。TRNA合成酶基因普遍表达，亲和力的变化不应 特定于单元格类型。因此，运动神经元和感觉神经元可能特别容易受到核糖体停滞的影响。 和ISR的诱导，或者可能由于高表达的tRNA而对tRNA隔离特别敏感 合成酶基因或tRNAs的低表达。在目标3中，我们将测试药物抑制是否 ISR是有益的，正如我们对GCN2基因敲除小鼠的遗传学研究所表明的那样。我们还将测试是否 使用AAV9载体来增加tRNA的表达可以挽救(我们预期的)或加剧表型。 这些研究可能是翻译的，也直接测试了我们提出的tRNA隔离机制。

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.