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.

项目摘要 charcot-marie-tooth病（CMT）是累积的遗传周围神经病的集合 〜1：2500人的发病率。 CMT的100个遗传亚型中的任何一个均未得到批准的治疗 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中的显性突变引起的 合成酶基因，氨基酸在其TRNA上充电的管家酶进行翻译。我们 最近提出了这种疾病的机制，其中突变酶结合其tRNA，但不释放 它是核糖体，有效地隔离了底物。这导致核糖体的失速和激活 通过传感器激酶GCN2进行综合应力响应（ISR）。 ISR的激活有助于 疾病的严重程度，当ISR激活被遗传删除或药理抑制所阻断何时。 GCN2，神经病是CMT 2D型小鼠模型中的米勒，是由糖中显性突变引起的 tRNA合成酶（GARS1）。 ISR的激活具有两个主要影响：1）真核起始因子2- α（EIF2）是磷酸化的，抑制帽依赖性翻译，2）转录因子ATF4 IS 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激活的这两个结果中的哪个正在加剧GARS1/CMT2D中的神经病 鼠标模型。在此方面，我们提出了两个目标。在AIM 1中，我们将研究翻译的水平 带有和没有GCN2删除的GARS1/CMT2D小鼠。我们以前已经表明运动神经元有 减少了GARS1突变小鼠的翻译，但是当未激活ISR时，翻译是否保持较低， 或者是否恢复，与神经病变表型的改善并联是未知的。我们将使用 在运动神经元和其他脊髓细胞中的荧光非典型氨基酸标记以测定翻译 这些小鼠的类型。在AIM 2中，我们将解决ATF4的可能作用。在一个实验中，我们将过表达 在原本野生型背景下，有条件的ATF4在运动神经元中转化。我们的初步数据 建议这概括了在GARS1突变小鼠中看到的一些表型，隐含的ATF4靶基因 表达是ISR使GARS1表型恶化的一种方式。在第二个实验中，我们将使用 在GARS1/CMT2D遗传背景下从运动神经元中删除基因的条件敲除将基因删除 如我们的ATF4所预测的那样，查看消除ATF4靶基因表达是否减轻了神经病表型 过表达初步数据。在AIM 2中，将使用行为，神经生理学， 组织病理学和基因表达分析，我们已确定为临床相关且与 GARS1小鼠的疾病过程。完成后，这些实验将指示是否减少 有助于ISR介导的ATF4靶基因的翻译或表达 CMT2D小鼠中的神经病。这些结果可能揭示了新颖，更集中的热干预点 在这种疾病中。