A critical role of the transient receptor potential melastatin 2 channel in a positive feedback mechanism for reactive oxygen species‐induced delayed cell death

Transient receptor potential melastatin 2 (TRPM2) channel activation by reactive oxygen species (ROS) plays a critical role in delayed neuronal cell death, responsible for postischemia brain damage via altering intracellular Zn2+ homeostasis, but a mechanistic understanding is still lacking. Here, we showed that H2O2 induced neuroblastoma SH‐SY5Y cell death with a significant delay, dependently of the TRPM2 channel and increased [Zn2+]i, and therefore used this cell model to investigate the mechanisms underlying ROS‐induced TRPM2‐mediated delayed cell death. H2O2 increased concentration‐dependently the [Zn2+]i and caused lysosomal dysfunction and Zn2+ loss and, furthermore, mitochondrial Zn2+ accumulation, fragmentation, and ROS generation. Such effects were suppressed by preventing poly(adenosine diphosphate ribose, ADPR) polymerase‐1‐dependent TRPM2 channel activation with PJ34 and 3,3′,5,5′‐tetra‐tert‐butyldiphenoquinone, inhibiting the TRPM2 channel with 2‐aminoethoxydiphenyl borate (2‐APB) and N‐(p‐amylcinnamoyl)anthranilic acid, or chelating Zn2+ with N,N,N,N‐tetrakis(2‐pyridylmethyl)‐ethylenediamine (TPEN). Bafilomycin‐induced lysosomal dysfunction also resulted in mitochondrial Zn2+ accumulation, fragmentation, and ROS generation that were inhibited by PJ34 or 2‐APB, suggesting that these mitochondrial events are TRPM2 dependent and sequela of lysosomal dysfunction. Mitochondrial TRPM2 expression was detected and exposure to ADPR‐induced Zn2+ uptake in isolated mitochondria, which was prevented by TPEN. H2O2‐induced delayed cell death was inhibited by apocynin and diphenyleneiodonium, nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase (NOX) inhibitors, GKT137831, an NOX1/4‐specific inhibitor, or Gö6983, a protein kinase C (PKC) inhibitor. Moreover, inhibition of PKC/NOX prevented H2O2‐induced ROS generation, lysosomal dysfunction and Zn2+ release, and mitochondrial Zn2+ accumulation, fragmentation and ROS generation. Collectively, these results support a critical role for the TRPM2 channel in coupling PKC/NOX‐mediated ROS generation, lysosomal Zn2+ release, and mitochondrial Zn2+ accumulation, and ROS generation to form a vicious positive feedback signaling mechanism for ROS‐induced delayed cell death.