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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

基本信息

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
项目类别：
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

项目摘要

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