Abstract
BackgroundNeurodegeneration is a representative phenotype of patients with chronic alcoholism. Ethanol-induced calcium overload causes NOD-like receptor protein 3 (NLRP3) inflammasome formation and an imbalance in mitochondrial dynamics, closely associated with the pathogenesis of neurodegeneration. However, how calcium regulates this process in neuronal cells is poorly understood. Therefore, the present study investigated the detailed mechanism of calcium-regulated mitochondrial dynamics and NLRP3 inflammasome formation in neuronal cells by ethanol.MethodsIn this study, we used the SK-N-MC human neuroblastoma cell line. To confirm the expression level of the mRNA and protein, real time quantitative PCR and western blot were performed. Co-immunoprecipitation and Immunofluorescence staining were conducted to confirm the complex formation or interaction of the proteins. Flow cytometry was used to analyze intracellular calcium, mitochondrial dysfunction and neuronal apoptosis.ResultsEthanol increased cleaved caspase-3 levels and mitochondrial reactive oxygen species (ROS) generation associated with neuronal apoptosis. In addition, ethanol increased protein kinase A (PKA) activation and cAMP-response-element-binding protein (CREB) phosphorylation, which increased N-methyl-D-aspartate receptor (NMDAR) expression. Ethanol-increased NMDAR induced intracellular calcium overload and calmodulin-dependent protein kinase II (CaMKII) activation leading to phosphorylation of dynamin-related protein 1 (Drp1) and c-Jun N-terminal protein kinase 1 (JNK1). Drp1 phosphorylation promoted Drp1 translocation to the mitochondria, resulting in excessive mitochondrial fission, mitochondrial ROS accumulation, and loss of mitochondrial membrane potential, which was recovered by Drp1 inhibitor pretreatment. Ethanol-induced JNK1 phosphorylation activated the NLRP3 inflammasome that induced caspase-1 dependent mitophagy inhibition, thereby exacerbating ROS accumulation and causing cell death. Suppressing caspase-1 induced mitophagy and reversed the ethanol-induced apoptosis in neuronal cells.ConclusionsOur results demonstrated that ethanol upregulated NMDAR-dependent CaMKII phosphorylation which is essential for Drp1-mediated excessive mitochondrial fission and the JNK1-induced NLRP3 inflammasome activation resulting in neuronal apoptosis.EBGddboEPhESjQm19dbAFrVideo abstractGraphical abstract
Highlights
Neurodegeneration is a representative phenotype of patients with chronic alcoholism
Ethanol-increased N-methyl-D-aspartate receptor (NMDAR) induced intracellular calcium overload and calmodulin-dependent protein kinase II (CaMKII) activation leading to phosphorylation of dynamin-related protein 1 (Drp1) and c-Jun N-terminal protein kinase 1 (JNK1)
Our results demonstrated that ethanol upregulated NMDAR-dependent CaMKII phosphorylation which is essential for Drp1-mediated excessive mitochondrial fission and the JNK1-induced NOD-like receptor protein 3 (NLRP3) inflammasome activation resulting in neuronal apoptosis
Summary
Neurodegeneration is a representative phenotype of patients with chronic alcoholism. Ethanolinduced calcium overload causes NOD-like receptor protein 3 (NLRP3) inflammasome formation and an imbalance in mitochondrial dynamics, closely associated with the pathogenesis of neurodegeneration. The present study investigated the detailed mechanism of calcium-regulated mitochondrial dynamics and NLRP3 inflammasome formation in neuronal cells by ethanol. An increase in excitatory amino acids, such as glutamate, which induces calcium-dependent excitotoxicity through N-methyl-D-aspartate receptor (NMDAR) overstimulation promoting neuronal cell death, was observed in patient with chronic alcoholism [6,7,8]. A recent study suggested that chronic ethanol treatment induced calcium overload by increasing NR1 expression and neuronal apoptosis [9]. Calcium overload via NMDAR was found to play an important role in ethanol-induced neuronal apoptosis, the detailed mechanisms need to be investigated further. A previous report indicated that an imbalance of mitochondrial dynamics, such as excessive mitochondrial fission, induced mitochondria damage leading to neuronal apoptosis [13]. The study on the mechanism of mitochondrial fission via calcium signaling will be help to understand ethanol-induced neuronal apoptosis
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