Abstract
BackgroundNeuroinflammation and microglial activation have been implicated in both alcohol use disorders (AUD) and fetal alcohol spectrum disorders (FASD). Chemokine monocyte chemoattractant protein 1 (MCP-1) and its receptor C-C chemokine receptor type 2 (CCR2) are critical mediators of neuroinflammation and microglial activation. FASD is the leading cause of mental retardation, and one of the most devastating outcomes of FASD is the loss of neurons in the central nervous system (CNS). The underlying molecular mechanisms, however, remain unclear. We hypothesize that MCP-1/CCR2 signaling mediates ethanol-induced neuroinflammation and microglial activation, which exacerbates neurodegeneration in the developing brain.MethodsC57BL/6 mice and mice deficient of MCP-1 (MCP-1−/−) and CCR2 (CCR2−/−) were exposed to ethanol on postnatal day 4 (PD4). Neuroinflammation, and microglial activation, and neurodegeneration in the brain were evaluated by immunohistochemistry and immunoblotting. A neuronal and microglial co-culture system was used to evaluate the role of microglia and MCP-1/CCR2 signaling in ethanol-induced neurodegeneration. Specific inhibitors were employed to delineate the involved signaling pathways.ResultsEthanol-induced microglial activation, neuroinflammation, and a drastic increase in the mRNA and protein levels of MCP-1. Treatment of Bindarit (MCP-1 synthesis inhibitor) and RS504393 (CCR2 antagonist) significantly reduced ethanol-induced microglia activation/neuroinflammation, and neuroapoptosis in the developing brain. MCP-1−/− and CCR2−/− mice were more resistant to ethanol-induced neuroapoptosis. Moreover, ethanol plus MCP-1 caused more neuronal death in a neuron/microglia co-culture system than neuronal culture alone, and Bindarit and RS504393 attenuated ethanol-induced neuronal death in the co-culture system. Ethanol activated TLR4 and GSK3β, two key mediators of microglial activation in the brain and cultured microglial cells (SIM-A9). Blocking MCP-1/CCR2 signaling attenuated ethanol-induced activation of TLR4 and GSK3β.ConclusionMCP-1/CCR2 signaling played an important role in ethanol-induced microglial activation/neuroinflammation and neurodegeneration in the developing brain. The effects may be mediated by the interaction among MCP-1/CCR2 signaling, TLR4, and GSK3β.
Highlights
Neuroinflammation and microglial activation have been implicated in both alcohol use disorders (AUD) and fetal alcohol spectrum disorders (FASD)
Since microglia are the major source of monocyte chemoattractant protein 1 (MCP-1), we examined the effect of ethanol on a cultured microglial cell line (SIM-A9)
We have recently shown that ethanol stimulated MCP-1/CCR-2 signaling in the spinal cord of early postnatal mice, and knocking out MCP-1 or chemokine receptor type 2 (CCR2) made mice resistant to ethanol-induced apoptosis of spinal cord neurons [70], indicating the involvement of MCP-1/CCR2 signaling in ethanol-induced neurodegeneration in the developing spinal cord
Summary
Neuroinflammation and microglial activation have been implicated in both alcohol use disorders (AUD) and fetal alcohol spectrum disorders (FASD). We hypothesize that MCP-1/CCR2 signaling mediates ethanol-induced neuroinflammation and microglial activation, which exacerbates neurodegeneration in the developing brain. A neuronal and microglial co-culture system was used to evaluate the role of microglia and MCP-1/CCR2 signaling in ethanol-induced neurodegeneration. Increased MCP-1 expression and microglial activation have been observed in the brain of human alcoholics [11]. It has been hypothesized that ethanol exposure may sensitize the immune system to subsequent insults and lead to excess neuroinflammation in alcohol use disorders (AUD) and FASD [12, 13]. We sought to determine whether MCP-1/CCR2 signaling is involved in ethanol-induced microglial activation and neuroinflammation in the context of neurodegeneration in the developing brain. We showed that inhibiting MCP-1/CCR2 signaling significantly reduced ethanol-induced microglial activation/neuroinflammation and ameliorated neurodegeneration in the developing brain. It appeared that ethanol-induced microglial activation/neuroinflammation was mediated by the interaction among MCP-1/CCR2 signaling, TLR4, and GSK3β
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