TRPM2 ion channel trafficking and its role in mitochondrial fragmentation and cell death

Mitochondria play a central role in oxidative stress-induced cell death. By increasing the production of reactive oxygen species, such as H2O2, oxidative stress causes mitochondrial fragmentation and apoptosis. It was hypothesised that Transient Receptor Potential Melastatin 2 (TRPM2) channels play a role in mitochondrial fragmentation and cell death. The rationale behind this hypothesis was the published evidence that oxidative stress stimulates TRPM2 channels, resulting in an increase in the cytosolic levels of Ca2+ and Zn2+, and that both these ions are detrimental to mitochondrial health and cell survival. To test the hypothesis, human umbilical vein endothelial cells (HUVECs) and endothelial cells isolated from wild-type and TRPM2 knock-out mice were used. TRPM2 actions were suppressed using pharmacological agents and small interfering RNA (siRNA). Fluorescent reporters were used to examine changes in intracellular ion distribution and organelle morphology. Molecular biology, biochemical and imaging techniques were used to examine the dynamics of ions and organelles. Exposure of HUVECs to H2O2 or high glucose stress led to TRPM2 activation, resulting in extracellular Ca2+ entry, lysosomal membrane permeability (LMP) and the release of lysosomal free Zn2+. Unexpectedly, this was accompanied by the accumulation of Zn2+ in the mitochondria. The rise in mitochondrial Zn2+ led to extensive mitochondrial fragmentation, mitochondrial outer membrane permeabilisation (MMP) and cell death. Silencing of TRPM2 channels with siRNA prevented intracellular Zn2+ redistribution, mitochondrial fragmentation and cell death. Endothelial cells derived from TRPM2 knock-out mice were resistant to oxidative stress-induced mitochondrial fragmentation. Biochemical and immunostaining experiments revealed an unexpected presence of TRPM2 channels in mitochondria, where they mediated mitochondrial Zn2+ uptake. Accumulation of Zn2+ in the mitochondria led to mitochondrial fragmentation by promoting the recruitment of cytoplasmic Drp1, an enzyme responsible for mitochondrial fission. Taken together, the results of this thesis revealed a novel mechanism for how oxidative stress can cause excessive mitochondrial fragmentation and cell death: the mechanism involves activation of TRPM2 channels leading to increased Ca2+ entry, LMP and release of lysosomal Zn2+; Zn2+ thus released is taken up by the mitochondria, leading to Drp1 recruitment, mitochondrial fragmentation and finally cell death. Since mitochondrial fragmentation is associated with several age-related chronic illnesses, including neuronal (Alzheimer’s, Parkinson’s), cardiovascular (atherosclerosis, myocardial infarction) and metabolic/inflammatory (diabetes) disorders, these results suggest that the TRPM2 channel is a novel target that could be explored for therapeutic intervention of age-related illnesses.