Ethanol-induced ceramide production causes neuronal apoptosis by increasing MCL-1S-mediated ER-mitochondria contacts

Heavy alcohol consumption causes neuronal cell death and cognitive impairment. Neuronal cell death induced by ethanol may result from increased production of the sphingolipid metabolite ceramide. However, the molecular mechanisms of neuronal cell death caused by ethanol-induced ceramide production have not been elucidated. Therefore, we investigated the mechanism through which ethanol-induced ceramide production causes neuronal cell apoptosis using human induced-pluripotent stem cell-derived neurons and SH-SY5Y cells and identified the effects of ceramide on memory deficits in C57BL/6 mice. First, we found that ethanol-induced ceramide production was decreased by inhibition of the de novo synthesis pathway, mediated by serine palmitoyltransferase (SPT). The associated alterations of the molecules related to the ceramide pathway suggest that the elevated level of ceramide activated protein phosphatase 1 (PP1), which inhibited the nuclear translocation of serine/arginine-rich splicing factor 1 (SRSF1). This led to aberrant splicing of myeloid cell leukemia 1 (MCL-1) pre-mRNA, which upregulated MCL-1S expression. Our results demonstrated that the interaction of MCL-1S with the inositol 1, 4, 5-trisphosphate receptor (IP3R) increases calcium release from the endoplasmic reticulum (ER) and then activated ER-bound inverted formin 2 (INF2). In addition, we discovered that F-actin polymerization through INF2 activation promoted ER-mitochondria contacts, which induced mitochondrial calcium influx and mitochondrial reactive oxygen species (mtROS) production. Markedly, MCL-1S silencing decreased mitochondria-associated ER membrane (MAM) formation and prevented mitochondrial calcium influx and mtROS accumulation, by inhibiting INF2-dependent actin polymerization interacting with mitochondria. Furthermore, the inhibition of ceramide production in ethanol-fed mice reduced MCL-1S expression, neuronal cell death, and cognitive impairment. In conclusion, we suggest that ethanol-induced ceramide production may lead to mitochondrial calcium overload through MCL-1S-mediated INF2 activation-dependent MAM formation, which promotes neuronal apoptosis.