Microglial TRPM2 Channel Activation and Its Relationship to Neurodegenerative Diseases

Microglial cell plays a key role in neuroinflammation induced by diverse danger associated molecular patterns (DAMP) molecules, such as Zn2+, Aβ42 and TNF-α, and strongly implicated in neurodegenerative diseases. The molecular mechanisms for neuroinflammation are however not fully defined. Reactive oxygen species (ROS) production is critical in DAMP-induced microglial cell activation and cytokine production. Studies presented in this thesis aimed to investigate, using immunocytochemistry, single cell imaging, cell death and ELISA assays in combination with genetic and pharmacological interventions, the role of ROS-sensitive TRPM2 channel in cell death, cell activation and production of TNF-α in primary microglial cells in response to Zn2+, Aβ42 and TNF-α as well as H2O2. H2O2 (10-300 mM) and Zn2+ (10-300 mM) induced concentration-dependent increases in the intracellular Ca2+ concentration ([Ca2+]i) via Ca2+ influx, which were prevented by TRPM2 knockout (TRPM2-KO) or treatment with TRPM2 inhibitor 2-APB or PARP inhibitor PJ34. Pathological concentrations of H2O2 (100-300 mM) and Zn2+ (100-300 mM) induced substantial cell death that was ablated by TRPM2-KO and treatment with 2-APB or PJ34. Zn2+ also induced ROS production and PARP-1 activation. All these Zn2+-induced effects were suppressed by treatment with PKC inhibitor chelerythrine, NOX inhibitors DPI, GKT137831 or Phox-I2. Zn2+-induced PARP-1 stimulation, increase in the [Ca2+]i and cell death were also inhibited by PYK2 inhibitor PF431396 or MEK/ERK inhibitor U0126. Exposure to Aβ42 (30-300 nM) and TNF-α (10-100 ng/ml) resulted in concentration-dependent TRPM2-mediated Ca2+ influx and increases in the [Ca2+]i, microglial cell activation and TNF-α production. Aβ42 and TNF-α stimulated ROS production and PARP-1 activation. These effects induced by Aβ42 or TNF-α were suppressed by inhibiting PKC and NOX. Moreover, Aβ42/TNF-α induced PARP-1 activation, increase in the [Ca2+]i, microglial cell activation and TNF-α production were attenuated by inhibiting PYK2 and MEK/ERK. In summary, studies provide strong evidence to reveal a critical role for the TRPM2 channel in Ca2+ signalling in microglial cells induced by Zn2+, Aβ42 and TNF-α. TRPM2 channel activation by Zn2+, Aβ42 and TNF-α depends on PKC/NOX-mediated ROS production and PARP-1 activation and is additionally enhanced by the PYK2-MEK-ERK signalling pathway. Such mechanisms are critically involved in cell death in response to Zn2+, or microglial cell activation and TNF-α production in response to Aβ42 and TNF-α. These findings provide novel insights into the role of microglial cells in neuroinflammation and in the pathogenesis of neurodegenerative diseases.