Dissecting the Integrated Stress Response in tRNA Synthetase-Associated Neuropathies

剖析 tRNA 合成酶相关神经病的综合应激反应

• 批准号: 10647281
• 负责人: Robert W Burgess
• 金额: $ 21.64万
• 依托单位: JACKSON LABORATORY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10647281
• 关键词: Affect; Amino Acids; Amino Acyl-tRNA Synthetases; Axon; Behavioral; Binding; Body Weight; Breeding; Cells; Cellular Stress; Charcot-Marie-Tooth Disease; Charge; Clinical; Codon Nucleotides; Collection; Data; Defect; Disease; Dominant Genes; Double-Stranded RNA; Enzymes; Eukaryotic Initiation Factor-2; Eukaryotic Initiation Factors; Frequencies; GARS gene; Gene Deletion; Gene Expression; Gene Expression Profiling; Gene Family; Genes; Genetic; Glycine; Glycine-Specific tRNA; Goals; Housekeeping; Housekeeping Gene; Human; Incidence; Inherited; Mediating; Motor; Motor Neurons; Mus; Mutant Strains Mice; Mutation; Neural Conduction; Neurons; Neuropathy; Outcome; Paper; Peripheral; Peripheral Nervous System; Peripheral Nervous System Diseases; Persons; Phenotype; Phosphorylation; Phosphotransferases; Process; Proteins; Reproducibility; Research; Ribosomes; Role; Severities; Severity of illness; Spinal; Spinal Cord; Supportive care; Therapeutic Intervention; Traction; Transfer RNA; Transfer RNA Aminoacylation; Transgenes; Transgenic Organisms; Translating; Translations; Vertebral column; Virus Diseases; axonal degeneration; biological adaptation to stress; cell type; clinically relevant; conditional knockout; dominant genetic mutation; experimental study; improved; in vivo; misfolded protein; mouse model; mutant; neurophysiology; neuroprotection; novel; overexpression; pharmacologic; primary outcome; reduced muscle mass; sensor; sensory neuropathy; stressor; therapeutic target; transcription factor; translation assay

PROJECT SUMMARY Charcot-Marie-Tooth disease (CMT) is a collection of inherited peripheral neuropathies with a cumulative incidence of ~1:2500 people. There is no approved treatment for any of the 100 genetic subtypes of CMT, presenting a large unmet clinical need. At least five forms of CMT are caused by dominant mutations in tRNA synthetase genes, the housekeeping enzymes that charge amino acids onto their tRNAs for translation. We recently proposed a mechanism for this disease wherein the mutant enzyme binds its tRNA, but does not release it to the ribosome, effectively sequestering the substrate. This leads to ribosome stalling and activation of the integrated stress response (ISR) through the sensor kinase GCN2. The activation of the ISR contributes to the disease severity, and when ISR activation is blocked by genetically deleting or pharmacologically inhibiting GCN2, the neuropathy is much milder in mouse models of CMT type 2D, caused by dominant mutations in Glycyl tRNA synthetase (Gars1). The activation of the ISR has two primary effects: 1) eukaryotic initiation factor 2- alpha (eIF2) is phosphorylated, suppressing cap-dependent translation, and 2) the transcription factor ATF4 is selectively translated, promoting expression of cellular stress response genes. The goal of this project is to determine which of these two outcomes of ISR activation are exacerbating the neuropathy in Gars1/CMT2D mouse models. Towards this, we propose two aims. In Aim 1, we will examine the levels of translation in Gars1/CMT2D mice with and without Gcn2 deletion. We have previously shown that motor neurons have reduced translation in the Gars1 mutant mice, but whether translation remains low when the ISR is not activated, or whether it recovers, paralleling the improvement in the neuropathy phenotype, is unknown. We will use fluorescent non-canonical amino acid tagging to assay translation in motor neurons and other spinal cord cell types in these mice. In Aim 2, we will address the possible role of ATF4. In one experiment, we will overexpress a conditional ATF4 transgene in motor neurons in an otherwise wild-type background. Our preliminary data suggest this recapitulates some phenotypes seen in Gars1 mutant mice, implicating ATF4 target gene expression as a way in which ISR worsens the Gars1 phenotype. In the second experiment, we will use a conditional knockout of Atf4 to delete the gene from motor neurons in a Gars1/CMT2D genetic background to see if eliminating ATF4 target gene expression alleviates the neuropathy phenotype, as predicted by our ATF4 overexpression preliminary data. In Aim 2, the mice will be evaluated using behavioral, neurophysiological, histopathological, and gene expression assays that we have established as clinically relevant and central to the disease process in the Gars1 mice. Upon completion, these experiments will indicate whether it is decreased translation or expression of ATF4 target genes (or a combination) that is contributing to the ISR-mediated neuropathy in the CMT2D mice. These results may reveal novel, more focused points of therapeutic intervention in this disease.

项目总结 Charcot-Marie-Tooth病(CMT)是一组遗传性周围神经病变，具有累积性 发病率约为1：2500人。目前还没有针对CMT的100种遗传亚型中的任何一种获得批准的治疗方法， 呈现出大量未得到满足的临床需求。至少有五种类型的CMT是由tRNA的显性突变引起的 合成酶基因是一种内务酶，负责将氨基酸充电到tRNA上进行翻译。我们 最近提出了一种治疗这种疾病的机制，即突变的酶与其tRNA结合，但不释放 它与核糖体结合，有效隔离底物。这会导致核糖体停滞和激活 整合的应激反应(ISR)通过传感器激酶GCN2。ISR的激活有助于 疾病严重性，以及ISR激活通过基因删除或药物抑制而被阻止时 GCN2，在CMT 2D型小鼠模型中，GCN2的神经病要轻得多，这是由甘氨酸显性突变引起的 TRNA合成酶(Gars1)。ISR的激活有两个主要作用：1)真核起始因子2- α(eIF2)被磷酸化，抑制帽依赖的翻译，以及2)转录因子ATF4是 选择性翻译，促进细胞应激反应基因的表达。这个项目的目标是 确定ISR激活的这两种结果中的哪一种加剧了Gars1/CMT2D的神经病变 老鼠模型。为此，我们提出了两个目标。在目标1中，我们将检查中的翻译水平 GCN2缺失和不缺失的Gars1/CMT2D小鼠。我们之前已经证明运动神经元 Gars1突变小鼠的翻译减少，但当ISR没有激活时，翻译是否保持低水平， 或者，它是否会恢复，与神经病变表型的改善同步，还是个未知数。我们将使用 荧光非标准氨基酸标记检测运动神经元和其他脊髓细胞的翻译 这些老鼠的类型。在目标2中，我们将讨论ATF4可能的作用。在一个实验中，我们将过度表达 在野生型背景下运动神经元中有条件的ATF4转基因。我们的初步数据 提示这概括了在Gars1突变小鼠中看到的一些表型，涉及ATF4靶基因 表达是ISR恶化Gars1表型的一种方式。在第二个实验中，我们将使用 有条件地敲除ATF4以删除Gars1/CMT2D遗传背景中运动神经元的基因 看看消除ATF4靶基因表达是否像我们的ATF4预测的那样减轻神经病变的表型 过度表达初步数据。在目标2中，将使用行为、神经生理学、 组织病理学和基因表达分析，我们已经建立了作为临床相关和中心的 Gars1小鼠的疾病过程。完成后，这些实验将显示是否减少了 参与ISR介导的ATF4靶基因(或其组合)的翻译或表达 CMT2D小鼠的神经病变。这些结果可能揭示新的、更有针对性的治疗干预要点。 在这种疾病中。