Thursday, April 18, 2024
4/18/2024
PROJECT SUMMARY/ABSTRACT This project examines dominant mutations in tRNA synthetase genes that cause inherited peripheral neuropathy. We seek to understand the biochemical and cellular basis for these diseases, to test mechanisms that could explain their specificity for motor and sensory neurons, and to test possible pharmacological and gene therapy-based treatments. Charcot-Marie-Tooth disease (CMT) is a collection of inherited diseases of the peripheral nervous system, and close to 100 genes are associated with CMT. The largest gene family associated with CMT is the tRNA synthetases. These enzymes charge amino acids onto their cognate tRNAs, and mutations in at least six cause peripheral neuropathy. Our preliminary data show that mutations in glycyl- and tyrosyl-tRNA synthetase (Gars and Yars, respectively) in mouse models lead to the activation of the integrated stress response (ISR) through the kinase GCN2. Genetic deletion of Gcn2 alleviates the phenotype of Gars/CMT2D mouse models. GCN2 is activated by uncharged tRNAs and also stalled ribosomes, and genetic interaction studies are consistent with ribosome stalling in vivo. Furthermore, uncharged tRNAs should be rescued by overexpression of wild-type synthetase, and we have shown this does not happen in Gars mouse models. Instead we favor a mechanism in which the tRNA substrate becomes limiting. We propose the following model: The mutant synthetases have aberrantly increased affinity for their cognate tRNAs, effectively sequestering them. This leads to ribosome stalling at the relevant codons during translation, which activates GCN2 and the ISR. The chronic activation of the ISR contributes to disease. We propose three aims to test this model. In Aim 1, we will directly measure the affinities of wild-type and mutant synthetases for their cognate tRNAs, anticipating that mutant synthetases will have higher affinities and slower off rates. We will also develop cell-based models using pluripotent human cell lines engineered to carry neuropathy-associated tRNA synthetase alleles. Differentiating these cells to motor neurons will validate our model in human cells and enable studies ribosome stalling and other relevant cell biology. We can also combine these systems predictively to create pathogenic or protective mutations, correlating tRNA affinity, ribosome stalling, induction of the ISR, and severity of neuropathy. In Aim 2, we will test possible mechanisms underlying the specificity of the disease. The tRNA synthetase genes are ubiquitously expressed, and the change in affinity should not be cell-type specific. Therefore, motor and sensory neurons may be particularly susceptible to ribosome stalling and the induction of the ISR, or may be particularly sensitive to tRNA sequestration due to high expression of the synthetase genes or poor expression of tRNAs. In Aim 3, we will test whether pharmacological inhibition of the ISR is beneficial, as suggested by our genetic studies with Gcn2 knockout mice. We will also test if the phenotype is rescued (which we anticipate) or exacerbated using AAV9 vectors to increase tRNA expression. These studies are potentially translational, and also directly test our proposed tRNA sequestration mechanism.
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