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.

项目总结/摘要-来自母基金R37 NS 054154 该项目研究了导致遗传性外周血淋巴细胞癌的tRNA合成酶基因的显性突变， 神经病变我们试图了解这些疾病的生化和细胞基础， 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 基于基因疗法的治疗。腓骨肌萎缩症（CMT）是一种遗传性疾病， 周围神经系统，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。这些酶将氨基酸装载到它们的同源tRNA上， 并且至少六个基因的突变导致周围神经病变。我们的初步数据显示甘氨酰- 和酪氨酰-tRNA合成酶（分别为加尔斯和亚尔斯）的激活， 通过激酶GCN 2整合应激反应（ISR）。Gcn 2基因缺失导致表型异常 加尔斯/CMT 2D小鼠模型。GCN 2被不带电荷的tRNA和停滞的核糖体激活， 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 可以通过野生型合成酶的过度表达来拯救，我们已经证明这在加尔斯中不会发生 小鼠模型。相反，我们倾向于tRNA底物变得有限的机制。我们提出 以下模型：突变合成酶对其同源tRNA具有异常增加的亲和力， 有效地隔离他们。这导致核糖体在翻译过程中在相关密码子处停滞， 启动GCN 2和ISR ISR的慢性激活有助于疾病。我们提出三个目标 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的亲和力。 同源tRNA，预期突变的合成酶将具有更高的亲和力和更慢的解离速率。我们将 还开发了基于细胞的模型，使用多能人类细胞系， 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 并使核糖体停滞和其他相关细胞生物学的研究成为可能。我们也可以将这些系统联合收割机 预测性地产生致病性或保护性突变，使tRNA亲和力、核糖体停滞、诱导 ISR和神经病变的严重程度。在目标2中，我们将测试以下特异性的可能机制： 这种疾病tRNA合成酶基因普遍表达，亲和力的变化不应被忽视。 细胞类型特异性。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，或者由于以下的高表达而对tRNA螯合特别敏感： 合成酶基因或低表达的tRNA。在目标3中，我们将测试是否药理学抑制 ISR是有益的，正如我们对Gcn 2敲除小鼠的遗传研究所表明的。我们还将测试 使用AAV 9载体来增加tRNA表达可以挽救（我们预期）或加剧表型。 这些研究是潜在的翻译，也直接测试我们提出的tRNA螯合机制。

项目成果

The integrated stress response contributes to tRNA synthetase-associated peripheral neuropathy.

• DOI: 10.1126/science.abb3414
• 发表时间: 2021-09-03
• 期刊: Science (New York, N.Y.)
• 作者: Spaulding EL;Hines TJ;Bais P;Tadenev ALD;Schneider R;Jewett D;Pattavina B;Pratt SL;Morelli KH;Stum MG;Hill DP;Gobet C;Pipis M;Reilly MM;Jennings MJ;Horvath R;Bai Y;Shy ME;Alvarez-Castelao B;Schuman EM;Bogdanik LP;Storkebaum E;Burgess RW
• 通讯作者: Burgess RW

Genetic analysis of Pycr1 and Pycr2 in mice.

小鼠 Pycr1 和 Pycr2 的遗传分析。

• DOI: 10.1093/genetics/iyab048
• 发表时间: 2021
• 期刊: Genetics
• 影响因子: 3.3
• 作者: Stum,MorganeG;Tadenev,AbigailLD;Seburn,KevinL;Miers,KathyE;Poon,PakP;McMaster,ChristopherR;Robinson,Carolyn;Kane,Coleen;Silva,KathleenA;Cliften,PaulF;Sundberg,JohnP;Reinholdt,LauraG;John,SimonWM;Burgess,RobertW
• 通讯作者: Burgess,RobertW

tRNA overexpression rescues peripheral neuropathy caused by mutations in tRNA synthetase.

• DOI: 10.1126/science.abb3356
• 发表时间: 2021-09-03
• 期刊: Science (New York, N.Y.)
• 作者: Zuko A;Mallik M;Thompson R;Spaulding EL;Wienand AR;Been M;Tadenev ALD;van Bakel N;Sijlmans C;Santos LA;Bussmann J;Catinozzi M;Das S;Kulshrestha D;Burgess RW;Ignatova Z;Storkebaum E
• 通讯作者: Storkebaum E

T Cells from NOD-PerIg Mice Target Both Pancreatic and Neuronal Tissue.

• DOI: 10.4049/jimmunol.2000114
• 发表时间: 2020-10-15
• 期刊: Journal of immunology (Baltimore, Md. : 1950)
• 作者: Racine JJ;Chapman HD;Doty R;Cairns BM;Hines TJ;Tadenev ALD;Anderson LC;Green T;Dyer ME;Wotton JM;Bichler Z;White JK;Ettinger R;Burgess RW;Serreze DV
• 通讯作者: Serreze DV

Two new mouse models of Gjb1-associated Charcot-Marie-Tooth disease type 1X.

Gjb1 相关 1X 型腓骨肌萎缩症的两种新小鼠模型。

• DOI: 10.1111/jns.12588
• 发表时间: 2023
• 期刊: Journal of the peripheral nervous system : JPNS
• 作者: Tadenev,ALD;Hatton,CL;Pattavina,B;Mullins,T;Schneider,R;Bogdanik,LP;Burgess,RobertW
• 通讯作者: Burgess,RobertW