Determining ribosome-associated mRNA in motor neurons after nerve crush [15NGS-001-rwb]. Determining ribosome-associated mRNA in motor neurons after nerve crush [15NGS-001-rwb]

Determining ribosome-associated mRNA in motor neurons of wild-type adult mice 4 days after unilateral sciatic nerve crush How mutations in broadly expressed housekeeping genes lead to neurodegeneration in specific cell types remains unclear. Mutations in ubiquitously expressed tRNA synthetase genes cause axonal peripheral neuropathy, accounting for at least six forms of Charcot-Marie-Tooth disease. Genetic evidence in mouse and Drosophila models suggests a neomorphic gain-of-function mechanism. Here, we use in vivo, cell-type-specific transcriptional and translational profiling of affected peripheral neurons to show that mutant tRNA synthetases impair translation and activate the integrated stress response (ISR) through the sensor kinase, GCN2. The chronic activation of the ISR contributes to the pathophysiology, and genetic deletion of Gcn2 alleviates the peripheral neuropathy. The activation of GCN2 by tRNA synthetase mutations indicates their neomorphic activity is still related to translation and suggests inhibiting GCN2 or the ISR as a therapeutic strategy. Overall design: RiboTagging was performed on spinal cords from HARPL22;ChAT-Cre;Gars+/+ crushed or uncrushed and corresponding negative controls (no Cre) and differential expression analyis was run.

单侧坐骨神经挤压损伤后4天的野生型成年小鼠运动神经元内核糖体结合信使核糖核酸（mRNA）的鉴定
广泛表达的管家基因发生突变为何会在特定细胞类型中引发神经退行性病变，目前仍不明确。普遍表达的氨酰-tRNA合成酶（tRNA synthetase）基因突变可引发轴索性周围神经病，目前该类突变已至少导致六种腓骨肌萎缩症（Charcot-Marie-Tooth disease）亚型。小鼠和果蝇（Drosophila）模型中的遗传学证据提示，此类突变存在新功能获得性致病机制。本研究通过对受累外周神经元开展活体内细胞类型特异性转录组与翻译组分析，证实突变型氨酰-tRNA合成酶会损伤翻译过程，并通过传感激酶GCN2激活整合应激反应（integrated stress response, ISR）。整合应激反应的慢性激活参与了疾病病理过程，而Gcn2基因的遗传敲除可缓解周围神经病症状。氨酰-tRNA合成酶突变可激活GCN2，这表明其新功能活性仍与翻译过程相关，同时提示靶向抑制GCN2或整合应激反应可作为潜在治疗策略。
实验整体设计：对HARPL22;ChAT-Cre;Gars+/+小鼠的脊髓组织开展核糖体标签（RiboTagging）实验，分组包括坐骨神经挤压损伤组、未损伤组以及对应的阴性对照（无Cre重组酶表达），并进行差异表达分析。