Glutamine synthetase deficiency triggers mitochondrial oxidative stress and a ribotoxic stress response during acute hepatic metabolic crisis.

Background

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Aims

Chlorfenapyr (CHL) is associated with high acute lethality in humans, with a case fatality rate exceeding 80%. However, the mechanisms underlying its hepatotoxicity remain poorly understood.

Methods

Transcriptomic, proteomic, and metabolomic analyses were combined with assessments of mitochondrial morphology and function in cultured hepatocytes. Rescue experiments were performed using MitoTEMPO, glutamine, and GSH. Glutamine synthetase (GLUL) function was investigated through overexpression of wild-type and catalytically inactive mutants, and CRISPR knockout. ZAKα siRNA and c-Jun N-terminal kinase (JNK) inhibitors were used to examine downstream signaling mechanisms. In vivo, the effects of MitoTEMPO treatment and hepatocyte-specific GLUL reconstitution were evaluated in mice.

Results

Multi-omics profiling identified mitochondria and ribosomes as the primary organelles affected by CHL. CHL induced mitochondrial damage and oxidative stress; MitoTEMPO restored viability, confirming mtROS causality. GLUL emerged as a key downregulated target. CHL destabilized GLUL, thereby disrupting the glutamine-glutamate axis, and supplementation with glutamine or GSH restored cell viability and redox homeostasis. Wild-type GLUL overexpression restored viability and suppressed mtROS, whereas the inactive mutant or knockout did not. Mechanistically, mtROS activated the ribotoxic stress response (RSR) through the ZAKα-JNK pathway. Critically, ZAKα depletion abolished CHL-induced JNK phosphorylation and apoptosis, an effect mirrored by JNK inhibition. In vivo, both MitoTEMPO treatment and GLUL reconstitution alleviated hepatic injury and attenuated RSR signaling.

Conclusion

These findings delineate a GLUL-mtROS-RSR signaling axis driving CHL hepatotoxicity and positioning GLUL stabilization as a potential therapeutic strategy for this highly lethal poisoning.