Previous studies have shown that alcohol abuse enhances neuroinflammation in vivo [15, 28, 29], with specific impairment of immune responses in an animal model of Human Immunodeficiency Virus-1 (HIV1) Encephalitis [28] and of neurological recovery after traumatic brain injury [29], thus suggesting that ethanol mediated-toxicity can exacerbate neuronal injury. Since damaging reactive oxygen species are generated during ethanol metabolism [30], and since the inflammasome pathway has recently been identified as player in a signaling response to a double challenge [24], we hypothesized that the ethanol-mediated activation of the inflammasome in iPS cells and NPCs would make them more vulnerable to a second toxic insult. To test this hypothesis in our system, we challenged both iPS cells and NPCs with peroxide (5 and 10 μM for iPS cells, and 100 and 500 μM for NPCs; concentrations were determined by the lethality of the exposure) for 14hr on day 7 after ethanol pre-exposure. The morphology of the cells that were challenged by peroxide was remarkably altered, becoming round and shrunken. This was accompanied by lysosomal and mitochondrial distributions that appeared clustered and inhomogeneous (Fig. 6b). Remarkably, this effect was enhanced in cells that had undergone both ethanol and peroxide treatments.
