Abstract
Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease characterized by progressive degeneration of motor neurons in the central nervous system. Prostaglandin E2 (PGE2) plays a pivotal role in the degeneration of motor neurons in human and transgenic models of ALS. We have shown previously that PGE2 directly induces neuronal death through activation of the E-prostanoid (EP) 2 receptor in differentiated NSC-34 cells, a motor neuron-like cell line. In the present study, to clarify the mechanisms underlying PGE2-induced neurotoxicity, we focused on generation of intracellular reactive oxygen species (ROS) and examined the effects of N-acetylcysteine (NAC), a cell-permeable antioxidant, on PGE2-induced cell death in differentiated NSC-34 cells. Dichlorofluorescein (DCF) fluorescence analysis of PGE2-treated cells showed that intracellular ROS levels increased markedly with time, and that this effect was antagonized by a selective EP2 antagonist (PF-04418948) but not a selective EP3 antagonist (L-798,106). Although an EP2-selective agonist, butaprost, mimicked the effect of PGE2, an EP1/EP3 agonist, sulprostone, transiently but significantly decreased the level of intracellular ROS in these cells. MTT reduction assay and lactate dehydrogenase release assay revealed that PGE2- and butaprost-induced cell death were each suppressed by pretreatment with NAC in a concentration-dependent manner. Western blot analysis revealed that the active form of caspase-3 was markedly increased in the PGE2- and butaprost-treated cells. These increases in caspase-3 protein expression were suppressed by pretreatment with NAC. Moreover, dibutyryl-cAMP treatment of differentiated NSC-34 cells caused intracellular ROS generation and cell death. Our data reveal the existence of a PGE2-EP2 signaling-dependent intracellular ROS generation pathway, with subsequent activation of the caspase-3 cascade, in differentiated NSC-34 cells, suggesting that PGE2 is likely a key molecule linking inflammation to oxidative stress in motor neuron-like NSC-34 cells.
Highlights
Amyotrophic lateral sclerosis (ALS) is a type of motor neuron disease characterized by progressive atrophy of skeletal muscle resulting from selective degeneration of motor neurons
To identify endogenous inducers of intracellular oxidative stress and clarify the molecular mechanism underlying the interaction of oxidative stress with the inflammatory response in ALS neurodegeneration, we examined the ability of prostaglandin E2 (PGE2) to induce intracellular reactive oxygen species (ROS) production in the mouse motor neuron-like cell line NSC-34, and found that EP2 receptor-dependent ROS production contributes to PGE2-induced cytotoxicity
We first examined the effect of PGE2 on DCF fluorescence intensity as a marker of intracellular ROS production in differentiated NSC-34 cells preloaded with DCFH-DA
Summary
Amyotrophic lateral sclerosis (ALS) is a type of motor neuron disease characterized by progressive atrophy of skeletal muscle resulting from selective degeneration of motor neurons. Inhibition of mPGES-1 by AAD-2004, a dual-function drug derived from aspirin and sulfasalazine, has been reported to exhibit significant neuroprotective effects and to prolong survival in ALS model mice [9]. We reported that positivefeedback regulation of EP2 in spinal motor neurons may exacerbate PGE2-induced damage to neurons during the progression of ALS in the murine model [10]. These results suggest that PGE2-EP2 signaling is a critical mediator of motor neuron death in the pathogenesis of ALS
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