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.

Neuroinflammation is a fundamental feature in the pathogenesis of amyotrophic lateral sclerosis (ALS) and arises from the activation of astrocytes and microglial cells. Previously, we reported that Miyako Bidens pilosa extract (MBP) inhibited microglial activation and prolonged the life span in a human ALS-linked mutant superoxide dismutase-1 (SOD1^G93A) transgenic mouse model of ALS (G93A mice). Herein, we evaluated the effect of MBP on microglial activation in the spinal cord of G93A mice and lipopolysaccharide-stimulated BV-2 microglial cells. The administration of MBP inhibited the upregulation of the M1-microglia/macrophage marker (interferon-γ receptor (IFN-γR)) and pro-inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6) in G93A mice. However, MBP did not affect the increase in the M2-microglia/macrophage marker (IL-13R) and anti-inflammatory cytokines (transforming growth factor (TGF)-β and IL-10) in G93A mice. BV-2 cell exposure to MBP resulted in a decrease in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) reduction activity and bromodeoxyuridine incorporation, without an increase in the number of ethidium homodimer-1-stained dead cells. Moreover, MBP suppressed the production of lipopolysaccharide-induced pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in BV-2 cells. These results suggest that the selective suppression of M1-related pro-inflammatory cytokines is involved in the therapeutic potential of MBP in ALS model mice.

Chronic stress is widely recognized as a risk factor for the development of major depression and anxiety disorders. Recently, we reported that yokukansan (YKS), a traditional Japanese herbal medicine, alleviated emotional abnormality in stress-maladaptive mice. The aim of the present study was to examine the effect of YKS on the expression of excitatory amino acid transporter (EAAT) 1-4 in the prefrontal cortex and hippocampus in stress-maladaptive mice. Mice were chronically exposed to inadaptable stress, i.e. repeated restraint stress for 240 min/day for 14 days. After the final exposure to stress, brains of mice were rapidly removed and the hippocampus and prefrontal cortex were dissected. Expressions of EAAT1-4 and glial fibrillary acidic protein (GFAP), a marker of astrocytes, in the brain tissues were analyzed by western blotting. Western blot analysis revealed that the expression level of EAAT2 was specifically decreased in the hippocampus of stress-maladaptive mice while there were no changes in the level of GFAP, and this change was inhibited by chronic treatment with YKS. In contrast, no changes were observed in the levels of EAAT1, EAAT3 or EAAT4 in stress-maladaptive mice. These results suggest that YKS may protect against the decrease in hippocampal EAAT2 expression induced by stress maladaptation, and this may contribute, at least in part, to the improvement of emotional abnormality.

Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disorder characterized by progressive paralysis resulting from the death of upper and lower motor neurons. There is currently no effective pharmacological treatment for ALS, and the two approved drugs riluzole and edaravone have limited effects on the symptoms and only slightly prolong the life of patients. Therefore, the development of effective therapeutic strategies is of paramount importance. In this study, we investigated whether Miyako Island Bidens pilosa (MBP) can alleviate the neurological deterioration observed in a superoxide dismutase-1 G93A mutant transgenic mouse (G93A mouse) model of ALS. We orally administered 2 g/kg/day of MBP to G93A mice at the onset of symptoms of neurodegeneration (15 weeks old) until death. Treatment with MBP markedly prolonged the life of ALS model mice by approximately 20 days compared to that of vehicle-treated ALS model mice and significantly improved motor performance. MBP treatment prevented the reduction in SMI32 expression, a neuronal marker protein, and attenuated astrocyte (detected by GFAP) and microglia (detected by Iba-1) activation in the spinal cord of G93A mice at the end stage of the disease (18 weeks old). Our results indicate that MBP administered after the onset of ALS symptoms suppressed the inflammatory activation of microglia and astrocytes in the spinal cord of the G93A ALS model mice, thus improving their quality of life. MBP may be a potential therapeutic agent for ALS.

Prostaglandins are a group of physiologically active lipid compounds derived from arachidonic acid. Our previous study has found that prostaglandin E₂ promotes neurite outgrowth in NSC-34 cells, which are a model for motor neuron development. However, the effects of other prostaglandins on neuronal differentiation are poorly understood. The present study investigated the effect of prostaglandin D₂ (PGD₂) on neuritogenesis in NSC-34 cells. Exposure to PGD₂ resulted in increased percentages of neurite-bearing cells and neurite length. Although D-prostanoid receptor (DP) 1 and DP2 were dominantly expressed in the cells, BW245C (a DP1 agonist) and 15(R)-15-methyl PGD₂ (a DP2 agonist) had no effect on neurite outgrowth. Enzyme-linked immunosorbent assay demonstrated that PGD₂ was converted to 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) under cell-free conditions. Exogenously applied 15d-PGJ₂ mimicked the effect of PGD₂ on neurite outgrowth. GW9662, a peroxisome proliferator-activated receptor–gamma (PPARγ) antagonist, suppressed PGD₂-induced neurite outgrowth. Moreover, PGD₂ and 15d-PGJ₂ increased the protein expression of Islet-1 (the earliest marker of developing motor neurons), and these increases were suppressed by co-treatment with GW9662. These results suggest that PGD₂ induces neuritogenesis in NSC-34 cells and that PGD₂-induced neurite outgrowth was mediated by the activation of PPARγ through the metabolite 15d-PGJ₂.