Mutations in glycyl-tRNA synthetase activate the integrated stress response in motor neurons through GCN2 kinase and ribosome stalling


What’s New in ALS and CMT - molecular pathways, mutations, phenotype-genotype correlations

Poster Number: 214


Timothy Hines, PhD, Emily Spaulding, Abigail Tadenev, Courtney Hatton, Robert Burgess, PhD


1. The Jackson Laboratory, 2. The Jackson Laboratory, 3. The Jackson Laboratory, 4. The Jackson Laboratory, 5. The Jackson Laboratory

Charcot-Marie-Tooth type 2D (CMT2D) is a dominant axonal neuropathy caused by mutations in glycyl tRNA-synthetase (GARS). The GARS enzyme charges glycine onto its cognate tRNAs for translation, but like many neurodegenerative diseases, this “housekeeping gene” causes a very specific disease phenotype when mutated. To better understand the disease mechanism underlying mutations in GARS and CMT2D, we performed gene expression profiling, using ribosome tagging to specifically examine the motor neuron “translatome,” and also using whole spinal cord RNAseq. Both approaches revealed signatures consistent with activation of the integrated stress response (ISR). In situ hybridization for upregulated ATF4 targets showed the ISR was selectively activated in alpha-, but not gamma-motor neurons, and in medium and large sensory neurons that constitute a mix of mechanoreceptors and proprioceptors. We postulated that the ISR was activated through GCN2, one of four kinases that serve as sensors of various cell stresses. To test this, we combined the Gars/CMT2D mouse model with a knockout of Gcn2. Double mutant mice no longer had gene expression changes indicating activation of the ISR. In addition, the phenotype of the double mutant mice was much milder than Gars mutations alone, suggesting that chronic activation of the ISR contributes to the severity of the neuropathy. Activation of the ISR via GCN2 can be caused by translation defects that lead to ribosome stalling. To test if stalled ribosomes are contributing to the disease phenotype, we crossed the Gars/CMT2D mice to mice with homozygous loss-of-function mutations in the ribosome rescue factor, Gtpbp2. Double mutant mice displayed an exacerbated neuropathy phenotype. This genetic interaction suggests ribosomes do indeed stall in the Gars mutant mice. To confirm these results, we are currently investigating if a loss-of-function mutation in another ribosome quality control protein, NEMF, has a similar effect.