Abstract
Rare inherited diseases caused by mutations in the copper transporters SLC31A1 (CTR1) or ATP7A induce copper deficiency in the brain and throughout the body, causing seizures and neurodegeneration in infancy. The mechanistic underpinnings of such neuropathology remains unclear. Here, we characterized the molecular mechanisms by which neuronal cells respond to copper depletion in multiple genetic model systems. Targeted deletion of CTR1 in neuroblastoma clonal cell lines produced copper deficiency that was associated with compromised copper-dependent Golgi and mitochondrial enzymes and a metabolic shift favoring glycolysis over oxidative phosphorylation. Proteomic and transcriptomic analysis revealed simultaneous upregulation of mTORC1 and S6K signaling, along with reduced PERK signaling in CTR1 KO cells. Patterns of gene and protein expression and pharmacogenomics show increased activation of the mTORC1-S6K pathway as a pro-survival mechanism, ultimately resulting in increased protein synthesis as measured by puromycin labeling. These effects of copper depletion were corroborated by spatial transcriptomic profiling of the cerebellum of Atp7a flx/Y :: Vil1 Cre/+ mice, in which copper-deficient Purkinje cells exhibited upregulated protein synthesis machinery and expression of mTORC1-S6K pathway genes. We tested whether increased activity of mTOR in copper-deficient neurons was adaptive or deleterious by genetic epistasis experiments in Drosophila. Copper deficiency dendritic phenotypes in class IV neurons are partially rescued by increased S6k expression or 4E-BP1 (Thor) RNAi, while epidermis phenotypes are exacerbated by Akt, S6k, or raptor RNAi. Overall, we demonstrate that increased mTORC1-S6K pathway activation and protein synthesis is an adaptive mechanism by which neuronal cells respond to copper depletion.