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

Abstract

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.

A critical role of TRPM2 channel in Aβ42‐induced microglial activation and generation of tumor necrosis factor‐α

“…In addition, Ab 42 -induced microglial activation (Figure 2d,e) and generation of nuclear PAR (Figure 5e,f) were strongly inhibited by PJ34, consistent with the reported role of PARP-1 in Ab-induced microglial activation (Kauppinen et al, 2011). Activation of these molecular mechanisms has been recently shown to be vital in TRPM2 channel activation induced by prolonged exposure to Zn 21 (Mortadza et al, 2017). As introduced above, it has been welldocumented that TNF-a is potent in inducing neurotoxicity and that Ab-induced generation of TNF-a from microglial cells significantly contributes to neuroinflammation in AD (Alam et al, 2016;Block et al, 2007;Doll et al, 2015;Heppner et al, 2015;Krabbe et al, 2017;Liu & Hong, 2003;Montgomery et al, 2011;Montgomery et al, 2013;Wyss-Coray & Rogers, 2012).…”

“…Cells were loaded with 5 mg/ml Fluo4/AM (Life Technologies) at room temperature in standard bath solution (SBS in mM: 134 NaCl, 5 KCl, 0.6 MgCl 2 , 1.5 CaCl 2 , 8 glucose and 10 HEPES, pH 7.4) as described in our recent study (Mortadza et al, 2017). Cells were loaded with 5 mg/ml Fluo4/AM (Life Technologies) at room temperature in standard bath solution (SBS in mM: 134 NaCl, 5 KCl, 0.6 MgCl 2 , 1.5 CaCl 2 , 8 glucose and 10 HEPES, pH 7.4) as described in our recent study (Mortadza et al, 2017).…”

“…Ab 42 -induced TNF-a release from WT microglial cells was strongly suppressed by treatment with 2-APB, PJ34 or DPQ, another PARP inhibitor (Figure 3d). It has been recently shown that ROS-induced activation of the TRPM2 channel can cause microglial cell death via necrosis (Mortadza et al, 2017). However, PI staining showed that exposure to Ab 42 up to 300 nM for 72 hr, the longest treatment used for TNF-a release, induced no significant necrotic cell death (Supporting Information, Figure S2), largely ruling out the possibility that Ab 42 -induced TNF-a release results from loss of membrane integrity accompanied with necrotic cell death.…”

“…(c) Summary of TNF-a release determined by ELISA from microglial cells without (CTL) or with exposure for 72 hr to 100 nM Ab 42 alone or treatment with PF or U0126 at indicated concentrations, 30 min prior to and during exposure to 100 nM Ab 42 , from three independent cell preparations . In addition, protein kinase C (PKC) and NOX have been reported for gangliosideinduced microglial activation (Min et al, 2004) and microglial cell death after prolonged exposure to Zn 21 (Mortadza et al, 2017). ###p < .005 compared with cells exposed to Ab 42 alone [Color figure can be viewed at wileyonlinelibrary.com] 3.3 | PKC/NOX-mediated generation of ROS is critical in Ab 42 -induced TRPM2 channel activation, microglial activation and generation of TNF-a NADPH oxidases (NOX) in microglial cells have a significant role in generation of ROS in AD and other neurodegenerative conditions (Block et al, 2007;Cheret et al, 2008;Harrigan, Abdullaev, Jourd'heuil, & Mongin, 2008).…”

“…The expression of TRPM2 channel in microglial cells is also welldocumented (Haraguchi et al, 2012;Jeong, Kim, Lee, Jung, & Oh, 2017;Miyake et al, 2014;Mortadza, Sim, Stacey, & Jiang, 2017). Poly (ADPR) polymerase-1 (PARP-1) has a crucial role in ROS-induced generation of ADPR and TRPM2 channel activation (Jiang et al, 2010).…”