NADH‐Reductive Stress Induced by Dihydrolipoamide Dehydrogenase Activation Contributes to Cuproptosis

Abstract Copper (Cu) is an essential trace element for cellular metabolism, while excessive Cu accumulation leads to neurotoxicity. Current therapeutic strategies for Cu overload remain inadequate in mitigating neurological symptoms. The recently discovered Cu‐dependent mitochondrial cell death pathway, cuproptosis, offers novel insights into Cu‐mediated neurotoxicity. In this study, the mechanistic link between mitochondrial respiration and cuproptosis is elucidated. The current study demonstrates that activated dihydrolipoamide dehydrogenase (DLD), induced by excess Cu under alkaline mitochondrial pH conditions, drives nicotinamide adenine dinucleotide (NADH) accumulation. Cu mediated mitochondrial permeability transition pore (mPTP) opening that facilitates NADH translocation to the cytosol, triggering NADH‐reductive stress. This promotes aberrant purine biosynthesis, leading to severe adenosine triphosphate depletion and energy stress. Pharmacological interventions targeting DLD activity, cytosolic NADH, mPTP opening, purine biosynthesis, or energy stress effectively rescued Cu‐induced cell death in SH‐SY5Y neuroblastoma cells. Collectively, these findings reveal characteristics of NADH‐reductive stress under excessive Cu exposure, establishing cuproptosis as a novel NADH‐reductive stress‐dependent cell death pathway. This mechanistic insight provides new therapeutic avenues for Cu‐associated neurological pathologies and new aspects to explore Cu cellular physiology.