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.

项目概要 腓骨肌萎缩症 (CMT) 是一系列遗传性周围神经病，具有累积性 发病率~1：2500人。目前还没有针对 CMT 100 种遗传亚型中任何一种的批准治疗方法， 提出了巨大的未满足的临床需求。至少五种形式的 CMT 是由 tRNA 显性突变引起的 合成酶基因，将氨基酸装载到其 tRNA 上进行翻译的管家酶。我们 最近提出了一种治疗这种疾病的机制，其中突变酶结合其 tRNA，但不释放 它与核糖体结合，有效地隔离底物。这导致核糖体停滞并激活 通过传感器激酶 GCN2 进行综合应激反应 (ISR)。 ISR 的激活有助于 疾病严重程度，以及当 ISR 激活被基因删除或药理学抑制所阻断时 GCN2，2D 型 CMT 小鼠模型中的神经病变要轻得多，由甘氨酰显性突变引起 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 (eIF2α) 被磷酸化，抑制帽依赖性翻译，并且 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。该项目的目标是 确定 ISR 激活的这两个结果中哪一个加剧了 Gars1/CMT2D 的神经病变 鼠标模型。为此，我们提出两个目标。在目标 1 中，我们将检查以下内容的翻译水平： 具有和不具有 Gcn2 缺失的 Gars1/CMT2D 小鼠。我们之前已经证明运动神经元 Gars1 突变小鼠的翻译水平降低，但是当 ISR 未激活时，翻译水平是否仍然较低， 或者它是否会随着神经病变表型的改善而恢复，尚不清楚。我们将使用 荧光非规范氨基酸标记用于分析运动神经元和其他脊髓细胞的翻译 这些小鼠中的类型。在目标 2 中，我们将讨论 ATF4 的可能作用。在一项实验中，我们将过度表达 在野生型背景下运动神经元中的条件 ATF4 转基因。我们的初步数据 表明这概括了 Gars1 突变小鼠中观察到的一些表型，暗示了 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 条件性敲除 Atf4，从 Gars1/CMT2D 遗传背景的运动神经元中删除该基因 看看消除 ATF4 靶基因表达是否可以减轻神经病变表型，正如我们的 ATF4 所预测的那样 过度表达初步数据。在目标 2 中，将使用行为学、神经生理学、 我们已经建立了与临床相关且至关重要的组织病理学和基因表达测定法 Gars1 小鼠的疾病过程。完成后，这些实验将表明它是否减少 ATF4 靶基因（或组合）的翻译或表达，有助于 ISR 介导的 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点 在这种疾病中。