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 H2 O2 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. H2 O2 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. H2 O2 -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 H2 O2 -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.