Understanding the mechanisms of translational repression in motor axons of Charcot–Marie–Tooth disease model

腓骨肌萎缩症（Charcot-Marie-Tooth， CMT）是一种由控制运动和感觉的外周运动和感觉轴突进行性变性引起的不治之症。它会导致肌肉变弱、麻木和行走困难。CMT 2D型是由编码甘氨酸- trna合成酶（GARS）基因的毒性功能获得性突变引起的，GARS是一种将甘氨酸与其同源tRNAGly连接在一起的酶。直到最近，这种疾病的分子机制还不清楚。我们的最新工作（Mendonsa et al. 2021）使用异源测试系统显示突变体GARS对tRNAGly具有异常高的亲和力，这耗尽了可用于翻译的glyyl -tRNAGly库，并导致核糖体在甘氨酸密码子处暂停。虽然我们的工作为CMT的机制提供了重要的见解，但仍不清楚为什么无处不在的GARS突变主要影响患者的运动和感觉轴突。这种知识差距主要是由于缺乏敏感的方法和获得纯运动神经元群体和分离亚细胞神经元室的技术挑战。我们之前的工作已经建立了一种有效的方法来分离亚细胞神经元室-细胞体和轴突-用于组学分析（空间组学；Zappulo等人，2017；Ciolli等人，2019）。这包括高分辨率核糖体分析，这是一种解剖CMT主要翻译缺陷所需的技术（Mendonsa et al. 2021）。此外，我们还开发了一种强大的方案，从hipsc中产生纯运动神经元群体（> 90%效率）。与动物模型相比，hipsc衍生的运动神经元的本质优势在于其同质性、可扩展性和人类遗传背景。这使我的实验室处于一个独特的位置来剖析运动轴突对CMT的高易感性背后的机制。在目前的建议中，我们旨在确定限制运动轴突的翻译相关因素，并剖析其在CMT发病机制中的作用。为此，我们将(1)通过CRISPR-Cas9引入CMT引起的突变，生成hipsc衍生的CMT运动神经元模型。接下来，我们将(2)分离轴突和细胞体上的CMT运动神经元，然后对分离的亚细胞区室进行RNA-seq、质谱分析和核糖体分析。这些分析将识别翻译成分，这些成分根据我们之前的研究（如tRNAGly， GARS等）参与核糖体暂停，并且仅限于CMT运动轴突。最后，我们将(3)结合空间组学和功能分析，利用恢复和耗竭实验来剖析已确定的限制因素在轴突病理中的作用。了解CMT的轴突特异性机制将提示最有效的治疗方法。

Charcot-Marie-Tooth (CMT) is an incurable disease caused by progressive degeneration of peripheral motor and sensory axons, which control movement and sensation. It leads to weaker muscles, numbness and difficulty walking. CMT type 2D is caused by toxic gain-of-function mutations in the gene encoding glycyl-tRNA synthetase (GARS), an enzyme that ligates glycine to its cognate tRNAGly. The molecular mechanisms of this disease have been unclear until recently. Our latest work (Mendonsa et al. 2021) used a heterologous test system to show that mutant GARS acquires an abnormally high affinity to tRNAGly, which depletes the pool of glycyl-tRNAGly available for translation and causes ribosomes to pause at glycine codons.While our work has provided essential insights into the mechanism of CMT, it remains unclear why mutations in ubiquitously expressed GARS primarily affect motor and sensory axons in patients. This gap of knowledge is mostly due to a lack of sensitive approaches and the technical challenges in obtaining a pure population of motor neurons and isolating subcellular neuronal compartments. Our prior work has established an efficient method for separation of subcellular neuronal compartments – cell bodies and axons – for omics analyses (spatial omics; Zappulo et al. 2017; Ciolli et al. 2019). This includes high-resolution ribosome profiling, a technique required to dissect the primary translational defect in CMT (Mendonsa et al. 2021). Moreover, we have also developed a robust protocol to generate a pure motor neuron population (> 90% efficiency) from hiPSCs. Essential advantages of hiPSC-derived motor neurons over animal models are their homogeneity, scalability and human genetic background. This puts my lab in a unique position to dissect the mechanism behind higher susceptibility of motor axons to CMT.In the current proposal, we aim to identify translation-related factors which are limiting in motor axons and dissect their role in CMT pathogenesis. For that, we will (1) generate hiPSC-derived motor neuron model of CMT, by introducing CMT-causing mutations with CRISPR–Cas9. Next, we will (2) separate CMT motor neurons on axons and cell bodies, followed by RNA-seq, mass spectrometry and ribosome profiling of isolated subcellular compartments. These analyses will identify translation components, which are involved in ribosome pausing according to our prior studies (e.g. tRNAGly, GARS etc), and are limiting in CMT motor axons. Finally, we will (3) dissect the roles of identified limiting factors in axonal pathology using rescue and depletion experiments in combination with spatial omics and functional assays. Understanding the mechanisms of axonal specificity of CMT would suggest the most efficient approaches for treatment.