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

点击翻译按钮获取中文摘要

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.