Signalling mechanisms mediating Zn2+-induced TRPM2 channel activation and cell death in microglial cells

Excessive Zn2+ causes brain damage via promoting ROS generation. Here we investigated the role of ROS-sensitive TRPM2 channel in H2O2/Zn2+- induced Ca2+ signalling and cell death in microglial cells. H2O2/Zn2+ induced concentration- dependent increases in cytosolic Ca2+ concentration ([ Ca2+] (c)), which was inhibited by PJ34, a PARP inhibitor, and abolished by TRPM2 knockout ( TRPM2- KO). Pathological concentrations of H2O2/Zn2+ induced substantial cell death that was inhibited by PJ34 and DPQ, PARP inhibitors, 2- APB, a TRPM2 channel inhibitor, and prevented by TRPM2- KO. Further analysis indicate that Zn2+ induced ROS production, PARP- 1 stimulation, increase in the [ Ca2+] c and cell death, all of which were suppressed by chelerythrine, a protein kinase C inhibitor, DPI, a NADPH- dependent oxidase ( NOX) inhibitor, GKT137831, a NOX1/ 4 inhibitor, and Phox-I2, a NOX2 inhibitor. Furthermore, Zn2+- induced PARP- 1 stimulation, increase in the [ Ca2+] c and cell death were inhibited by PF431396, a Ca2+- sensitive PYK2 inhibitor, and U0126, a MEK/ERK inhibitor. Taken together, our study shows PKC/NOX- mediated ROS generation and PARP- 1 activation as an important mechanism in Zn2+- induced TRPM2 channel activation and, TRPM2- mediated increase in the [ Ca2+] c to trigger the PYK2/ MEK/ ERK signalling pathway as a positive feedback mechanism that amplifies the TRPM2 channel activation. Activation of these TRPM2- depenent signalling mechanisms ultimately drives Zn2+- induced Ca2+ overloading and cell death.