Gangliosides Trigger Inflammatory Responses via TLR4 in Brain Glia

Abstract

Gangliosides participate in various cellular events of the central nervous system and have been closely implicated in many neuronal diseases. However, the precise molecular mechanisms underlying the pathological activity of gangliosides are poorly understood. Here we report that toll-like receptor 4 (TLR4) may mediate the ganglioside-triggered inflammation in glia, brain resident immune cells. Gangliosides rapidly altered the cell surface expression of TLR4 in microglia and astrocytes within 3 hours. Using TLR4-specific siRNA and a dominant-negative TLR4 gene, we clearly demonstrate the functional importance of TLR4 in ganglioside-triggered activation of glia. Inhibition of TLR4 expression by TLR4-siRNA suppressed nuclear factor (NF)-B-binding activity, NF-B-dependent luciferase activity, and transcription of inflammatory cytokines after exposure to gangliosides. Transient transfection of dominant-negative TLR4 also attenuated NF-B-binding activity and interleukin-6 promoter activity. In contrast, these activities were slightly elevated in cells with wild-type TLR4. In addition, CD14 was required for ganglioside-triggered activation of glia, and lipid raft formation may be associated with ganglioside-stimulated signal propagation. Taken together, these results suggest that TLR4 may provide an explanation for the pathological ability of gangliosides to cause inflammatory conditions in the brain.

GM1 induces p38 and microtubule dependent ramification of rat primary microglia in vitro

“…We previously showed that gangliosides are microglial activators and these effects are manifested via TLR4 (Jou et al, 2006;Pyo et al, 1999). Considering the previous notion that ramified microglia are associated with a resting phenotype, whereas ameboid microglia are associated with an activated phenotype, our present observation appears to be somewhat inconsistent with our previous observations.…”

“…In the present study, we observed that Gmix induced ramified morphological changes in cultured rat primary microglia, which were different from those induced by other agents, such as LPS or IFN-γ, and that the effect of Gmix on the ramified morphological changes of microglia was due to GM1, one of the gangliosides in Gmix. Approximate content of each ganglioside in Gmix used in the current study was as follows: 18% GM1, 55% GD1a, 15% GD1b, 10% GT1b, and 2% others (Jou et al, 2006). Therefore, it is of interest to note that GM1 which comprises a small fraction of Gmix could induce typical ramified morphological changes by overpowering the effect of other gangliosides.…”

“…In our previous studies with cultured rat primary microglia (Jou et al, 2006;Kim et al, 2002;Pyo et al, 1999), we observed that brain gangliosides mixture (Gmix) induced morphological changes in cultured rat primary microglia which were different from those induced by other agents such as LPS or IFN-γ. Therefore, to further pursue this observation, we plated cultured rat primary microglia onto 35 mm dishes, incubated them with either Gmix (50 μg/ml), LPS (100 ng/ml), or IFN-γ (10 U/ml) for 3 days, and observed morphological changes using phasecontrast microscopy.…”

“…Hakomori, 2000). Furthermore, gangliosides have previously been shown to induce the phagocytosis of microglia (Bocchini et al, 1988) and activate microglia through various signaling pathways (Jou et al, 2006;Kim et al, 2002;Min et al, 2004;Pyo et al, 1999). In the present study, therefore, we investigated whether exogenously added brain gangliosides mixture (Gmix) enhanced microglial ramification and underlying mechanism of how Gmix enhanced microglial ramification.…”

Dual Functionality of Myeloperoxidase in Rotenone-Exposed Brain-Resident Immune Cells

“…After removal of the microglia, primary astrocytes were prepared using trypsinization. 25 Murine BV2 microglial cells were maintained in DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 g/mL streptomycin at 37°C in a humidified incubator under 5% CO 2 .…”