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

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 – 来自家长拨款 R37NS054154 该项目检查了 tRNA 合成酶基因的显性突变，这些突变导致遗传性外周血 我们寻求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，并测试可能的药理学和 基于基因治疗的治疗方法。腓骨肌萎缩症 (CMT) 是一系列遗传性疾病。 周围神经系统，近 100 个基因与 CMT 相关，是最大的基因家族。 与 CMT 相关的是 tRNA 合成酶，这些酶将氨基酸装载到其同源 tRNA 上。 我们的初步数据表明，至少有六种突变会导致周围神经病变。 小鼠模型中的酪氨酰-tRNA 合成酶（分别为 Gars 和 Yars）导致激活 通过激酶 GCN2 进行整合应激反应 (ISR) Gcn2 的基因删除可缓解表型。 Gars/CMT2D 小鼠模型的 GCN2 被不带电的 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，不带电荷的 tRNA 应该是一致的。 可以通过野生型合成酶的过度表达来拯救，并且我们已经证明这不会发生在 Gars 中 相反，我们建议采用一种 tRNA 底物受到限制的机制。 以下模型：突变合成酶对其同源 tRNA 的亲和力异常增加， 有效地隔离它们，这导致核糖体在翻译过程中停滞在相关密码子处。 激活 GCN2 和 ISR ISR 的长期激活会导致疾病。 为了测试这个模型，我们将直接测量野生型和突变型合成酶的亲和力。 同源 tRNA，预计突变合成酶将具有更高的亲和力和更慢的解离速率。 还开发了基于细胞的模型，使用经过改造的多能人类细胞系来携带神经病相关的 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。 并能够研究核糖体停滞和其他相关的细胞生物学。我们还可以将这些系统结合起来。 预测产生致病性或保护性突变，关联 tRNA 亲和力、核糖体停滞、诱导 在目标 2 中，我们将测试 ISR 特异性的可能机制。 tRNA合成酶基因普遍表达，亲和力的变化不应该发生。 因此，运动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 tRNA 的高表达而对 tRNA 隔离特别敏感 在目标 3 中，我们将测试是否有药物抑制作用。 正如我们对 Gcn2 敲除小鼠的基因研究所表明的，ISR 是有益的，我们还将测试 ISR 是否有益。 使用 AAV9 载体增加 tRNA 表达可以挽救（我们预计）或加剧表型。 这些研究具有潜在的转化性，也直接测试了我们提出的 tRNA 隔离机制。

项目成果

Dominant, toxic gain-of-function mutations in gars lead to non-cell autonomous neuropathology.

gars 中显性的有毒功能获得突变导致非细胞自主神经病理学。

• 发表时间: 2015-08-01
• 影响因子: 3.5
• 作者: Grice, Stuart J;Sleigh, James N;Motley, William W;Liu, Ji;Burgess, Robert W;Talbot, Kevin;Cader, M Zameel
• 通讯作者: Cader, M Zameel

Charcot-Marie-Tooth-linked mutant GARS is toxic to peripheral neurons independent of wild-type GARS levels.

Charcot-Marie-Tooth 连锁突变体 GARS 对周围神经元具有毒性，与野生型 GARS 水平无关。

• 发表时间: 2011-12
• 影响因子: 4.5
• 作者: Motley, William W;Seburn, Kevin L;Nawaz, Mir Hussain;Miers, Kathy E;Cheng, Jun;Antonellis, Anthony;Green, Eric D;Talbot, Kevin;Yang, Xiang;Fischbeck, Kenneth H;Burgess, Robert W
• 通讯作者: Burgess, Robert W

Trk receptor signaling and sensory neuron fate are perturbed in human neuropathy caused by Gars mutations.

Gars 突变引起的人类神经病中，Trk 受体信号传导和感觉神经元命运受到干扰。

• 发表时间: 2017-04-18
• 影响因子: 11.1
• 作者: Sleigh, James N;Dawes, John M;West, Steven J;Wei, Na;Spaulding, Emily L;Gómez;Zhang, Qian;Burgess, Robert W;Cader, M Zameel;Talbot, Kevin;Yang, Xiang;Bennett, David L;Schiavo, Giampietro
• 通讯作者: Schiavo, Giampietro

An active dominant mutation of glycyl-tRNA synthetase causes neuropathy in a Charcot-Marie-Tooth 2D mouse model.

甘氨酰-tRNA 合成酶的活性显性突变会导致 Charcot-Marie-Tooth 2D 小鼠模型出现神经病变。

• 发表时间: 2006-09-21
• 影响因子: 16.2
• 作者: Seburn, Kevin L;Nangle, Leslie A;Cox, Gregory A;Schimmel, Paul;Burgess, Robert W
• 通讯作者: Burgess, Robert W

Gene therapies for axonal neuropathies: Available strategies, successes to date, and what to target next.

轴突神经病的基因疗法：可用策略、迄今为止的成功以及下一步的目标。

• 发表时间: 2020
• 影响因子: 2.9
• 作者: Morelli, Kathryn H;Hatton, Courtney L;Harper, Scott Q;Burgess, Robert W
• 通讯作者: Burgess, Robert W