Abstract
Microglia, brain resident macrophages, are activated in brain injuries and several neurodegenerative diseases. However, microglial activators that are produced in the brain are not yet defined. In this study, we showed that gangliosides, sialic acid-containing glycosphingolipids, could be a microglial activator. Gangliosides induced production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) and expression of cyclooxygenase-2 (COX-2). The effect of gangliosides on NO release increased dose-dependently in the range of 10-100 microgram/ml; however, the effect decreased at concentrations higher than 200 microgram/ml. Specific types of gangliosides showed differential effects on microglial activation. Similar to gangliosides, GT1b induced production of NO and TNF-alpha and expression of COX-2. However, GM1 and GD1a induced expression of COX-2 but had little effect on NO and TNF-alpha release. The effect of gangliosides and GT1b on NO release was reduced in the presence of neuraminidase, which removes sialic acid residues from gangliosides and GT1b. Gangliosides activated extracellular signal-regulated kinase significantly but activated c-jun N-terminal kinase/stress-activated protein kinase and p38 relatively weakly. The inhibition of extracellular signal-regulated kinase by PD98059 reduced NO release from both gangliosides- and GT1b-treated microglia whereas inhibition of p38 by SB203580 increased it rather slightly. Gangliosides activated NF-kappaB, and N-acetyl cystein, an inhibitor of NF-kappaB, reduced NO release. These results suggest that gangliosides could be a microglial activator that functions via activation of mitogen-activated protein kinase and NF-kappaB.
Highlights
Microglia known as major inflammatory cells of the brain are activated in brain injuries and diseases such as trauma, ischemia, Alzheimer’s disease, and multiple sclerosis [1,2,3]
Gangliosides Induced nitric oxide (NO) Release—The effect of gangliosides on NO release was dose-dependent up to 100 g/ml: 0.8 Ϯ 2.2, 13.4 Ϯ 3.4, 27.0 Ϯ 2.1, and 29.4 Ϯ 1.0 M NO were released from cells (5 ϫ 104) treated with 1, 10, 50, and 100 g/ml gangliosides, respectively, whereas 2.3 Ϯ 0.8 M was released from untreated cells (Fig. 1A)
The decrease of NO release was not because of toxicity of gangliosides on these cells, which was confirmed by morphology of cells under a light microscope. iNOS expression was detected within 12 h after gangliosides treatment and was maintained until 48 h (Fig. 1A, inset)
Summary
From the ‡Department of Pharmacology and the ¶Department of Physiology, Ajou University School of Medicine, Suwon, 442–749, Korea. The inhibition of extracellular signal-regulated kinase by PD98059 reduced NO release from both gangliosides- and GT1b-treated microglia whereas inhibition of p38 by SB203580 increased it rather slightly. Activated microglia induced the expression of the genes encoding iNOS, TNF-␣, and COX-2, which are responsible for the production of inflammatory mediators, nitric oxide (NO), TNF-␣, and prostaglandins, respectively (4 – 6). GM1, GD1a, GD1b, GT1b, and GQ1b are major types of gangliosides found in the brain [12, 13] They are different in the number and sites of sialic acid residues attaching to the carbohydrates. We investigated whether gangliosides could activate microglia and whether gangliosides induced microglial activation via activation of MAPKs and NF-B as did LPS and A [20, 34]
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