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 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.

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 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.

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. Video abstract.

• Authors: Jae Ryong Lim, Hyun Jik Lee, Young Hyun Jung, Jun Sung Kim, Chang Woo Chae, Seo Yihl Kim, Ho Jae Han
• Journal: Cell communication and signaling : CCS (Cell Commun Signal) Vol. 18 Issue 1 Pg. 123 (08 12 2020) ISSN: 1478-811X [Electronic] England
• PMID: 32787872 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Cyclic AMP Response Element-Binding Protein
• Inflammasomes
• NLR Family, Pyrin Domain-Containing 3 Protein
• Reactive Oxygen Species
• Receptors, N-Methyl-D-Aspartate
• Ethanol
• Ubiquitin-Protein Ligases
• parkin protein
• Cyclic AMP-Dependent Protein Kinases
• Calcium-Calmodulin-Dependent Protein Kinase Type 2
• Mitogen-Activated Protein Kinase 8
• Caspase 1
• DNM1L protein, human
• Dynamins
• Calcium

Topics

• Apoptosis (drug effects)
• Calcium (metabolism)
• Calcium-Calmodulin-Dependent Protein Kinase Type 2 (metabolism)
• Caspase 1 (metabolism)
• Cell Line, Tumor
• Cyclic AMP Response Element-Binding Protein (metabolism)
• Cyclic AMP-Dependent Protein Kinases (metabolism)
• Dynamins (metabolism)
• Enzyme Activation (drug effects)
• Ethanol (pharmacology)
• Humans
• Inflammasomes (metabolism)
• Intracellular Space (metabolism)
• Mitochondria (drug effects, metabolism)
• Mitochondrial Dynamics (drug effects)
• Mitogen-Activated Protein Kinase 8 (metabolism)
• Mitophagy (drug effects)
• Models, Biological
• NLR Family, Pyrin Domain-Containing 3 Protein (metabolism)
• Neurons (drug effects, metabolism)
• Reactive Oxygen Species (metabolism)
• Receptors, N-Methyl-D-Aspartate (metabolism)
• Signal Transduction (drug effects)
• Ubiquitin-Protein Ligases (metabolism)