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LIM kinase-2 induces programmed necrotic neuronal death via dysfunction of DRP1-mediated mitochondrial fission.

ABSTRACT: Although the aberrant activation of cell cycle proteins has a critical role in neuronal death, effectors or mediators of cyclin D1/cyclin-dependent kinase 4 (CDK4)-mediated death signal are still unknown. Here, we describe a previously unsuspected role of LIM kinase 2 (LIMK2) in programmed necrotic neuronal death. Downregulation of p27(Kip1) expression by Rho kinase (ROCK) activation induced cyclin D1/CDK4 expression levels in neurons vulnerable to status epilepticus (SE). Cyclin D1/CDK4 complex subsequently increased LIMK2 expression independent of caspase-3 and receptor interacting protein kinase 1 activity. In turn, upregulated LIMK2 impaired dynamic-related protein-1 (DRP1)-mediated mitochondrial fission without alterations in cofilin phosphorylation/expression and finally resulted in necrotic neuronal death. Inhibition of LIMK2 expression and rescue of DRP1 function attenuated this programmed necrotic neuronal death induced by SE. Therefore, we suggest that the ROCK-p27(Kip1)-cyclin D1/CDK4-LIMK2-DRP1-mediated programmed necrosis may be new therapeutic targets for neuronal death.

SUBMITTER: Kim JE

PROVIDER: S-EPMC4207472 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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LIM kinase-2 induces programmed necrotic neuronal death via dysfunction of DRP1-mediated mitochondrial fission.

Kim J-E JE Ryu H J HJ Kim M J MJ Kang T-C TC

Cell death and differentiation 20140221 7

Although the aberrant activation of cell cycle proteins has a critical role in neuronal death, effectors or mediators of cyclin D1/cyclin-dependent kinase 4 (CDK4)-mediated death signal are still unknown. Here, we describe a previously unsuspected role of LIM kinase 2 (LIMK2) in programmed necrotic neuronal death. Downregulation of p27(Kip1) expression by Rho kinase (ROCK) activation induced cyclin D1/CDK4 expression levels in neurons vulnerable to status epilepticus (SE). Cyclin D1/CDK4 complex ...[more]

PMID: 24561342

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Project description:Background & aimsIn cirrhosis, astrocytic swelling is believed to be the principal mechanism of ammonia neurotoxicity leading to hepatic encephalopathy (HE). The role of neuronal dysfunction in HE is not clear. We aimed to explore the impact of hyperammonaemia on mitochondrial function in primary co-cultures of neurons and astrocytes and in acute brain slices of cirrhotic rats using live cell imaging.MethodsTo primary cocultures of astrocytes and neurons, low concentrations (1 and 5 μM) of NH4Cl were applied. In rats with bile duct ligation (BDL)-induced cirrhosis, a model known to induce hyperammonaemia and minimal HE, acute brain slices were studied. One group of BDL rats was treated twice daily with the ammonia scavenger ornithine phenylacetate (OP; 0.3 g/kg). Fluorescence measurements of changes in mitochondrial membrane potential (Δψm), cytosolic and mitochondrial reactive oxygen species (ROS) production, lipid peroxidation (LP) rates, and cell viability were performed using confocal microscopy.ResultsNeuronal cultures treated with NH4Cl exhibited mitochondrial dysfunction, ROS overproduction, and reduced cell viability (27.8 ± 2.3% and 41.5 ± 3.7%, respectively) compared with untreated cultures (15.7 ± 1.0%, both p <0.0001). BDL led to increased cerebral LP (p = 0.0003) and cytosolic ROS generation (p <0.0001), which was restored by OP (both p <0.0001). Mitochondrial function was severely compromised in BDL, resulting in hyperpolarisation of Δψm with consequent overconsumption of adenosine triphosphate and augmentation of mitochondrial ROS production. Administration of OP restored Δψm. In BDL animals, neuronal loss was observed in hippocampal areas, which was partially prevented by OP.ConclusionsOur results elucidate that low-grade hyperammonaemia in cirrhosis can severely impact on brain mitochondrial function. Profound neuronal injury was observed in hyperammonaemic conditions, which was partially reversible by OP. This points towards a novel mechanism of HE development.Lay summaryThe impact of hyperammonaemia, a common finding in patients with liver cirrhosis, on brain mitochondrial function was investigated in this study. The results show that ammonia in concentrations commonly seen in patients induces severe mitochondrial dysfunction, overproduction of damaging oxygen molecules, and profound injury and death of neurons in rat brain cells. These findings point towards a novel mechanism of ammonia-induced brain injury in liver failure and potential novel therapeutic targets.

Project description: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.

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LIM kinase-2 induces programmed necrotic neuronal death via dysfunction of DRP1-mediated mitochondrial fission.

Publications

LIM kinase-2 induces programmed necrotic neuronal death via dysfunction of DRP1-mediated mitochondrial fission.

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