Abstract 1354 The role of ATF4 in tRNA synthetase-associated forms of Charcot-Marie-Tooth disease

Charcot-Marie-Tooth disease (CMT) is a rare, genetically and phenotypically heterogeneous neuromuscular disorder with causative mutations found in over 100 different genes. The tRNA synthetases (aaRSes) are the largest gene family associated with CMT. These enzymes charge tRNAs with their cognate amino acid so the tRNA can participate in protein synthesis. Data from multiple mouse and cell-based models of aaRS-associated CMT show that the mutant aaRS sequesters tRNAs, causing ribosome stalling, and activation of the integrated stress response (ISR) in motor and sensory neurons through the activating kinase, GCN2. There are two major cellular consequences of ISR activation – shutdown of cap-dependent translation through eIF2α phosphorylation and selective translation of activating transcription factor 4 (ATF4), which induces the expression of stress response genes. The objective of this study is to determine how each of these events contribute to the CMT phenotype. To do this, I am generating transgenic mice with motor neuron (MN)-specific Atf4 overexpression (OE) and knockout (KO), and the latter are being crossed to Gars/CMT2D mice. Matings were set up so that all genetic controls are littermates of experimental animals and all mice in a litter were phenotyped at 9 or 26 weeks of age with the experimenter blind to genotype (mice were generally not genotyped until after tissue harvest). Each mouse is considered an experimental unit, no mice were excluded from the study, and reported sample sizes are not final, as experiments are ongoing. All animal procedures were reviewed and approved by the Animal Care and Use Committee at The Jackson Laboratory (protocol number AUS 01026, approved 7 November 2022). Transgenic, MN-specific ATF4-OE in WT Gars mice causes a neuropathy phenotype, with reduced nerve conduction velocity (NCV), body weight, and femoral motor nerve diameter. Conversely, MN-specific Atf4-KO ameliorates the NCV deficits, increases nerve diameter, and improves transmission at the neuromuscular junction, which is dysfunctional in Gars/CMT2D mice. Neuromuscular performance, nerve histology, changes in gene expression, and levels of translation are being assessed in these mice. These results suggest that ATF4 expression in motor neurons is pathogenic and could be a therapeutic target in CMT2D. This work was funded by Uplifting Athletes' Young Investigator Draft, the Charcot-Marie Tooth Association and NINDS 1K99NS130151-01A1 (TJH); NINDS R37 NS054154-14S1 Diversity Supplement (RWB/TJH); and NINDS R37 NS054154-14, R24 NS098523, and R01 NS113583 (RWB). NINDS R25 NS117356-3 Diversifying CNS program at University of Minnesota also provided support to TJH.