Metformin Activates AMPK to Restrain Mitochondrial ROS-Driven Necroptosis in Cadmium Neurotoxicity

Cadmium (Cd) exposure is an emerging risk factor for neurodegeneration, yet the contribution of necroptosis to Cd-induced neuronal loss and its tractability for pharmacologic intervention remain unclear. Here, we identify an AMP-activated protein kinase (AMPK)-mitochondrial reactive oxygen species (mtROS)-RIPK1/RIPK3-MLKL axis as a central driver of Cd neurotoxicity and a critical target of metformin. In a chronic Cd-exposed mouse model and in primary hippocampal neurons, Cd provoked prominent neuronal injury characterized by the activation of necroptotic signaling (elevated p-RIPK1, p-RIPK3, and p-MLKL), excessive mtROS production, and mitochondrial injury. Oral metformin preserved hippocampal neuronal integrity and suppressed necroptotic markers in vivo, while in cultured neurons and SH-SY5Y cells, it reduced mtROS, maintained mitochondrial morphology and membrane potential, and prevented necrosome assembly. Pharmacologic inhibition of RIPK1 (necrostatin-1) or RIPK3 (GSK-872), as well as CRISPR/Cas9-mediated deletion of RIPK1, RIPK3, or MLKL, attenuated Cd-induced necrosis and phenocopied or enhanced the anti-necroptotic effects of metformin. Mechanistically, metformin restored Cd-suppressed AMPK activity, whereas blockade of AMPK with compound C or expression of a dominant-negative AMPKα1 mutant abolished metformin-mediated suppression of mtROS, RIPK1/RIPK3-MLKL signaling, and necroptosis. Scavenging mtROS with Mito-TEMPO or MitoQ reduced necroptosis and synergized with metformin to disrupt RIPK3-MLKL complex formation. Collectively, these findings demonstrate that Cd triggers neuronal necroptosis via mtROS-driven activation of RIPK1/RIPK3-MLKL, and that metformin confers neuroprotection by activating AMPK to restore mitochondrial homeostasis and restrain necroptotic signaling. This work positions the AMPK-mtROS-necroptosis axis as a therapeutically actionable pathway in heavy-metal neurotoxicity and supports repurposing metformin for necroptosis-associated neurodegenerative conditions.