Magic™ Anti-CaMKII polyclonal antibody

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.

The physiological and motivational effects of heroin and other abused drugs become associated with environmental (contextual) stimuli during repeated drug use. As a result, these contextual stimuli gain the ability to elicit drug-like conditioned effects. For example, after context-heroin pairings, exposure to the heroin-paired context alone produces similar effects on peripheral immune function as heroin itself. Conditioned immune effects can significantly exacerbate the adverse health consequences of heroin use. Our laboratory has shown that exposure to a heroin-paired context suppresses lipopolysaccharide (LPS)-induced splenic nitric oxide (NO) production in male rats, and this effect is mediated in part by the dorsal hippocampus (dHpc). However, specific dHpc output regions, whose efferents might mediate conditioned immune effects, have not been identified, nor has the contribution of ventral hippocampus (vHpc) been investigated. Here, we evaluated the role of CaMKII alpha-expressing neurons in the dHpc and vHpc main output regions by expressing Gi-coupled designer receptors exclusively activated by designer drugs (DREADDs) under a CaMKII alpha promoter in the dorsal subiculum and CA1 (dSub, dCA1) or ventral subiculum and CA1 (vSub, vCA1). After context-heroin conditioning, clozapine-N-oxide (CNO, DREADD agonist) or vehicle was administered systemically prior to heroin-paired context (or home-cage control) exposure and LPS immune challenge. Chemogenetic inhibition of CaMKII alpha-expressing neurons in dHpc, but not vHpc, output regions attenuated the expression of conditioned splenic NO suppression. These results establish that the main dHpc output regions, the dSub and dCA1, are critical for this context-heroin conditioned immune effect.