Pathogenic role of EP2 receptors up-regulation in motor neuron-like NSC-34 cells and lumbar motor neurons in ALS model mice

抄録

Amyotrophic lateral sclerosis (ALS) is a devastating progressive neurodegenerative disease characterized by selective loss of motor neurons in the cortex and spinal cord. Recently, inflammation in the spinal cord has been highlighted as an important pathogenic mechanism for ALS. Prostaglandin E2 (PGE2) and its corresponding four E-prostanoid receptors have shown to play roles in the pathophysiology of ALS both in humans and animal models. However, little information is available for the contributions of EP receptors in motor neurons in ALS. The present study was designed to investigate the levels of expression of EP receptors in the spinal motor neurons of ALS model mice and to examine whether PGE2 alters the expression of EP receptors in differentiated NSC-34 cells, a motor neuron-like cell line.

　Spinal cord tissue was obtained from symptomatic and end-stage stages of superoxide dismutase 1 (SOD1)-G93A mice. Protein expression levels of EP were assessed by immunohistochemistry and immunoblotting. To enhance cell differentiation, NSC-34 cells were cultured in DMEM/F12 supplemented with 0.5% fetal bovine serum. Cell viability was measured by the MTT reduction assay.

　Immunohistochemical analysis demonstrated that EP2 and EP3 immunoreactivity was localized in NeuN-positive large cells showing the typical morphology of motor neurons in mice. Semi-quantitative analysis showed that the intensity of EP2 immunoreactivity in motor neurons was significantly increased in the early symptomatic stage in ALS model mice. In contrast, the level of EP3 expression remained constant, irrespective of age. In differentiated NSC-34 cells, exposure to PGE2 resulted in a concentration-dependent decrease of MTT reduction. Although PGE2 had no effect on cell survival at concentrations of less than 10 µM, pretreatment with 10 µM PGE2 significantly up-regulated EP2 and concomitantly potentiated cell death induced by 30 µM PGE2. The upregulation of EP2 induced by PGE2 at 10 µM was significantly suppressed by PF-04418948, an EP2 selective antagonist, but not by L-798106, an EP3 selective antagonist.

　These results suggest that PGE2 is an important effector for induction of the EP2 subtype in differentiated NSC-34 cells, and that EP2 up-regulation in motor neurons plays a pivotal role in the vulnerability of motor neurons in ALS model mice.