Pyroptosis is a caspase-1-dependent inflammatory form of cell death. The adapter protein ASC binds directly to caspase-1 and is critical for caspase-1 activation in response to a broad range of stimuli. To elucidate the mechanism of activation of caspase-1 by ASC and its exact role in macrophage pyroptosis, we performed time-lapse confocal bioimaging analysis on human THP-1 macrophages stably expressing an ASC-GFP fusion protein. We show that stimulation of these cells with several proinflammatory stimuli trigger the formation of a large supramolecular assembly of ASC, termed here pyroptosome. Only one distinct pyroptosome in each stimulated cell is formed, which rapidly recruits and activates caspase-1 resulting in pyroptosis and the release of the intracellular proinflammatory cytokines. The pyroptosome is largely composed of oligomerized ASC dimers. Dimerization of ASC is driven by subphysiological concentrations of potassium as in vitro incubation of purified recombinant ASC in the presence of subphysiological concentrations of potassium induces the assembly of a functional pyroptosome. Furthermore, stimulation of potassium efflux in THP-1 cells with potassium-depleting agents induces formation of the pyroptosome, while increasing potassium concentrations in the culture medium or pharmacological inhibition of this efflux inhibits its assembly. Our results establish that macrophage pyroptosis is mediated by a unique pyroptosome, distinct from the inflammasome.

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TRPM2 is a Ca 2؉ -permeable channel that is activated by oxidative stress and confers susceptibility to cell death. Here, an isoform of TRPM2 was identified in normal human bone marrow that consists of the TRPM2 N terminus and the first two predicted transmembrane domains. Because of alternative splicing, a stop codon (TAG) is located at the splice junction between exons 16 and 17, resulting in deletion of the four C-terminal transmembrane domains, the putative calcium-permeable pore region, and the entire C terminus. This splice variant was found in other hematopoietic cells including human burst forming unit-erythroid-derived erythroblasts and TF-1 erythroleukemia cells. Endogenous expression of both the short form of TRPM2 (TRPM2-S) and the full length (TRPM2-L) was determined by reverse transcriptase-PCR, and localization of endogenous TRPM2 to the plasma membrane was demonstrated by confocal microscopy.

Arachidonic acid is released by phospholipase A2 when activation of N-methyl-D-aspartate (NMDA) receptors by neurotransmitter glutamate raises the calcium concentration in neurons, for example during the initiation of long-term potentiation and during brain anoxia. Here we investigate the effect of arachidonic acid on glutamate-gated ion channels by whole-cell clamping isolated cerebellar granule cells. Arachidonic acid potentiates, and makes more transient, the current through NMDA receptor channels, and slightly reduces the current through non-NMDA receptor channels. Potentiation of the NMDA receptor current results from an increase in channel open probability, with no change in open channel current. We observe potentiation even with saturating levels of agonist at the glutamate- and glycine-binding sites on these channels; it does not result from conversion of arachidonic acid to lipoxygenase or cyclooxygenase derivatives, or from activation of protein kinase C. Arachidonic acid may act by binding to a site on the NMDA receptor, or by modifying the receptor's lipid environment. Our results suggest that arachidonic acid released by activation of NMDA (or other) receptors will potentiate NMDA receptor currents, and thus amplify increases in intracellular calcium concentration caused by glutamate. This may explain why inhibition of phospholipase A2 blocks the induction of long-term potentiation.

Background: TRPM2 channels play an essential role in cell death following oxidative stress. Results: Dominant negative TRPM2-S decreases growth of neuroblastoma xenografts and increases doxorubicin sensitivity through modulation of HIF-1/2␣ expression, mitophagy, and mitochondrial function. Conclusion: TRPM2 is important for neuroblastoma growth and viability through modulation of HIF-1/2␣. Significance: Modulation of TRPM2 may be a novel approach in cancer therapeutics.

Amyloid b-peptide (Ab) is the main component of senile plaques which characterize Alzheimer's disease and may induce neuronal death through mechanisms which include oxidative stress. To date, the signalling pathways linking oxidant stress, a component of several neurodegenerative diseases, to cell death in the CNS are poorly understood. Melastatin-like transient receptor potential 2 (TRPM2) is a Ca 2+ -permeant non-selective cation channel, which responds to increases in oxidative stress levels in the cell and is activated by oxidants such as hydrogen peroxide. We demonstrate here that Ab and hydrogen peroxide both induce death in cultured rat striatal cells which express TRPM2 endogenously. Transfection with a splice variant that acts as a dominant negative blocker of TRPM2 function (TRPM2-S) inhibited both hydrogen perox-

Dentate status is an important risk factor for clinically significant weight loss among community-dwelling older adults.

Background: TRPM2 channels are present in the heart, but their function is unknown. Results: Genetic ablation of TRPM2 results in cardiac mitochondrial dysfunction, enhanced ROS production, and exacerbated cardiac ischemic injury. Conclusion: TRPM2 channels preserve cardiac mitochondrial bioenergetics and protect cardiac myocytes from ischemic injury. Significance: TRPM2 is a rational target for treatment of ischemic heart disease.