Neurodegeneration 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. In 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. Ethanol 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. Our 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.

中文翻译：

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乙醇激活的 CaMKII 信号通过 Drp1 介导的过度线粒体裂变和 JNK1 依赖性 NLRP3 炎性体激活诱导神经元凋亡。


神经变性是慢性酒精中毒患者的代表性表型。乙醇诱导的钙超载会导致 NOD 样受体蛋白 3 (NLRP3) 炎性体形成和线粒体动力学失衡，与神经退行性疾病的发病机制密切相关。然而，人们对钙如何调节神经元细胞中的这一过程知之甚少。因此，本研究研究了乙醇在神经元细胞中钙调节线粒体动力学和NLRP3炎症小体形成的详细机制。在本研究中，我们使用 SK-N-MC 人神经母细胞瘤细胞系。为了确认mRNA和蛋白质的表达水平，进行了实时定量PCR和蛋白质印迹。进行免疫共沉淀和免疫荧光染色以确认蛋白质的复合物形成或相互作用。采用流式细胞术分析细胞内钙、线粒体功能障碍和神经元凋亡。乙醇增加了与神经元凋亡相关的 caspase-3 裂解水平和线粒体活性氧 (ROS) 的产生。此外，乙醇增加了蛋白激酶 A (PKA) 的激活和 cAMP 反应元件结合蛋白 (CREB) 的磷酸化，从而增加了 N-甲基-D-天冬氨酸受体 (NMDAR) 的表达。乙醇增加的 NMDAR 诱导细胞内钙超载和钙调蛋白依赖性蛋白激酶 II (CaMKII) 激活，导致动力相关蛋白 1 (Drp1) 和 c-Jun N 末端蛋白激酶 1 (JNK1) 磷酸化。 Drp1磷酸化促进Drp1易位至线粒体，导致线粒体过度裂变，线粒体ROS积累，线粒体膜电位丧失，通过Drp1抑制剂预处理可恢复。 乙醇诱导的 JNK1 磷酸化激活了 NLRP3 炎症小体，从而诱导 caspase-1 依赖性线粒体自噬抑制，从而加剧 ROS 积累并导致细胞死亡。抑制 caspase-1 诱导的线粒体自噬并逆转乙醇诱导的神经元细胞凋亡。我们的结果表明，乙醇上调 NMDAR 依赖性 CaMKII 磷酸化，这对于 Drp1 介导的过度线粒体裂变和 JNK1 诱导的 NLRP3 炎性体激活（导致神经元凋亡）至关重要。