Ethanol-activated CaMKII signaling induces neuronal apoptosis through Drp1-mediated excessive mitochondrial fission and JNK1-dependent NLRP3 inflammasome activation.

Abstract

Background: Neurodegeneration is a representative phenotype of patients with chronic alcoholism. Ethanol-induced calcium overload causes 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. Methods: 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. Results: Ethanol increased cleaved caspase-3 levels and mitochondrial reactive oxygen species (ROS) generation associated with neuronal apoptosis. In addition, ethanol increased PKA activation and CREB phosphorylation, which increased NMDA receptor (NMDAR) expression. Ethanol-increased NMDAR induced intracellular calcium overload and CaMKII activation leading to phosphorylation of dynamin-related protein 1 (Drp1) and 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. Conclusions: 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.

RCAN1 deficiency aggravates sepsis-induced cardiac remodeling and dysfunction through accelerating mitochondrial pathological fission

“…In this study, we found that RCAN1 de ciency resulted in increased mitochondrial ROS production and a rapid loss of mitochondrial membrane potential. Inhibition of excessive ssion by blocking the interaction between Drp1 and mitochondrial adaptor ssion 1 (Fis1) rescued mitochondrial function, promoted survival of cardiomyocytes and reduced morbidity and mortality of septic cardiomyopathy [13,21]. By Western blotting, we found that the phosphorylation level of Drp1, the major pro-ssion marker located at the outer mitochondrial membrane, was signi cantly increased in LPS-induced RCAN1 −/− mice.…”

“…Previous studies showed that CaMKII determines mitochondrial stress responses in heart [14]. Mitochondrial translocation of CaMKII leads to increased ROS production, enhanced mitochondrial fragmentation and mitochondrial ΔΨm deterioration, thereby promoting cell remodeling and death in septic heart [21,[28][29][30]. Unlike the effect of Drp1 dephosphorylation at Ser637 triggering its mitochondrial translocation, CaMKII induced Drp1 phosphorylation at Ser616 further activated Drp1dependent mitochondrial ssion, fragmentation and mPTP opening [16,31].…”

“…Under high resolution of confocal microscope, we observed that RCAN1 de ciency also enhanced the interaction between CaMKII and Drp1 in LPS-induced cardiomyocytes and increased hypertrophic response and cell death. Excessive ethanol uptake promoted CaMKII-dependent Drp1 activation and Drp1-related mitochondrial fragmentation and dysfunction in neuronal cells [21]. Therefore, RCAN1 de ciency-mediated calcineurin/CaMKII pathway and impaired Ca 2+ handling might be the cause of cardiac remodeling at least partly due to CaMKII-dependent Drp1 activation and pathological mitochondrial ssion [16,31].…”

Loss of hepatic DRP1 exacerbates alcoholic hepatitis by inducing megamitochondria and mitochondrial maladaptation

“…Our results thus support a novel concept that chronic accumulation of megamitochondria induced by alcohol consumption may lead to mitochondrial maladaptation resulting in liver injury. Alcohol has been shown to affect DRP1‐mediated mitochondria dynamics in cardiomyocytes, neuroblastoma cells, and engineered hepatoma cells, although the exact mechanisms remain elusive 23,36,37. TFEB is a master regulator of lysosomal biogenesis, which is impaired by alcohol consumption in hepatocytes 20.…”

Polydatin attenuates chronic alcohol consumption-induced cardiomyopathy through a SIRT6-dependent mechanism

“…3,37 Therefore, we further evaluated mitochondrial dynamics, mitophagy and mitochondrial functional changes in alcohol-treated myocardium. Previously, over-activated mitochondrial fission and its detrimental effects have been reported to be a key response in various organs or cell lines exposed to alcohol such as liver tissue, 57,58 brain tissue, 59 neural cells, 60 retinal pigment epithelial cells 61 and alveolar macrophages 62 exposed to ethanol challenge. In the cardiovascular system, less evidence has been reported concerning the modulatory effect of alcohol on mitochondrial dynamics although mitochondrial fragmentation has been observed in alcohol-treated myocardium.…”