TRPM2 Channel-Mediated Signalling Mechanisms for Neuronal Cell Death

Transient receptor potential melastatin-related 2 (TRPM2) channel is gated by ADP-ribose (ADPR) and potently activated by reactive oxygen species (ROS) through stimulating ADPR-generating mechanisms. Recent studies provide evidence to show a crucial role for TRPM2 in neuronal death and cognitive impairment associated with ischemic stroke and Alzheimer’s disease. However, the underlying mechanisms are poorly understood. Studies described in this thesis adopted genetic and pharmacological interventions, in conjunction with immunofluorescent and live cell imaging, to investigate TRPM2-dependent cell death induced by H2O2 and the 42-residue of amyloid β (Aβ42) in cultured hippocampal neurons. H2O2 and Aβ42 induced significant neuronal death, which was reduced or prevented by TRPM2 knock-out (TRPM2-KO), TRPM2 channel inhibitors, or Zn2+ chelator TPEN. H2O2 and Aβ42 induced intracellular Zn2+ increase, lysosomal dysfunction and Zn2+ release, mitochondrial Zn2+ accumulation, dysfunction and ROS generation. Bafilomycin A1-induced lysosomal dysfunction also resulted in mitochondrial Zn2+ accumulation and ROS generation. These events were abolished by TRPM2-KO or suppressed by inhibiting poly(ADP-ribose) polymerase-1 (PARP-1) or TRPM2 channel. Immunofluorescent imaging suggests mitochondrial localization of TRPM2. ADPR enhanced Zn2+ accumulation in isolated mitochondria from wild-type (WT) but not TRPM2-KO neurons. Finally, the inhibition of protein kinase C (PKC) and NADPH oxidases (NOX), particularly NOX1/4, suppressed H2O2/Aβ42-induced neuronal death and Aβ42-induced intracellular Zn2+ increase, lysosomal and mitochondrial dysfunction, and mitochondrial ROS generation. The inhibition of the proline-rich tyrosine kinase 2 (Pyk2) and the downstream MEK/ERK kinases protected against Aβ42-induced neuronal death. Taken together, these results provide evidence to support a vicious positive feedback signalling loop that drives hippocampal neuronal death in response to ROS and Aβ42, in which the TRPM2 channel in mitochondria integrates multiple mechanisms comprising PKC/NOX-mediated ROS generation, lysosomal dysfunction and Zn2+ release, mitochondrial Zn2+ accumulation, mitochondrial dysfunction and ROS generation. In addition, the Pyk2-MEK-ERK signalling pathway is critically involved in Aβ42-induced TRPM2-dependent neuronal death. These findings provide novel insights into the mechanisms underlying neuronal death and cognitive impairment related to ischemic stroke and AD.