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Updated: Nov 6, 2025
Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures
Published on: July 25, 2022
Methylmercury induces neuronal cell death by inducing TNF-α expression through the ASK1/p38 signaling pathway in
Takashi Toyama1,2, Takayuki Hoshi1,2,3, Takuya Noguchi4
1Laboratory of Molecular and Biochemical Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aoba-ku, Aramaki, Sendai, Miyagi, 980-8578, Japan.
Abstract:
We recently found that tumor necrosis factor-α (TNF-α) may be involved in neuronal cell death induced by methylmercury in the mouse brain. Here, we examined the cells involved in the induction of TNF-α expression by methylmercury in the mouse brain by in situ hybridization. TNF-α-expressing cells were found throughout the brain and were identified as microglia by immunostaining for ionized calcium binding adaptor molecule 1 (Iba1). Methylmercury induced TNF-α expression in mouse primary microglia and mouse microglial cell line BV2. Knockdown of apoptosis signal-regulating kinase 1 (ASK1), an inflammatory cytokine up-regulator that is responsible for reactive oxygen species (ROS), decreased methylmercury-induced TNF-α expression through decreased phosphorylation of p38 MAP kinase in BV2 cells. Suppression of methylmercury-induced reactive oxygen species (ROS) by antioxidant treatment largely abolished the induction of TNF-α expression and phosphorylation of p38 by methylmercury in BV2 cells. Finally, in mouse brain slices, the TNF-α antagonist (WP9QY) inhibited neuronal cell death induced by methylmercury, as did the p38 inhibitor SB203580 and liposomal clodronate (a microglia-depleting agent). These results indicate that methylmercury induces mitochondrial ROS that are involved in activation of the ASK1/p38 pathway in microglia and that this is associated with induction of TNF-α expression and neuronal cell death.
Insights
Methylmercury triggers microglial cells to produce tumor necrosis factor-α (TNF-α), leading to neuronal cell death. Inhibiting this pathway protects against methylmercury-induced brain damage.
Area of Science:
- Neuroscience
- Toxicology
- Immunology
Background:
- Methylmercury exposure is a significant neurotoxicant.
- Tumor necrosis factor-alpha (TNF-α) is implicated in methylmercury-induced neuronal cell death.
- The specific cellular mechanisms and pathways involved remain to be fully elucidated.
Purpose of the Study:
- To identify the specific brain cells responsible for TNF-α induction by methylmercury.
- To investigate the signaling pathways, including reactive oxygen species (ROS) and p38 MAP kinase, involved in methylmercury-induced TNF-α expression.
- To evaluate the therapeutic potential of targeting these pathways against methylmercury neurotoxicity.
Main Methods:
- In situ hybridization to detect TNF-α expression in mouse brain.
- Immunostaining using ionized calcium binding adaptor molecule 1 (Iba1) to identify microglia.
- Primary microglia and BV2 cell cultures treated with methylmercury.
- Knockdown of apoptosis signal-regulating kinase 1 (ASK1) and assessment of p38 MAP kinase phosphorylation.
- Antioxidant treatment to suppress ROS.
- Administration of TNF-α antagonist, p38 inhibitor, and microglia-depleting agent in mouse brain slices.
Main Results:
- Methylmercury induced TNF-α expression predominantly in microglia throughout the mouse brain.
- Methylmercury increased TNF-α expression and p38 MAP kinase phosphorylation in microglia, mediated by ROS and ASK1.
- Inhibition of TNF-α, p38, or microglia significantly reduced methylmercury-induced neuronal cell death.
Conclusions:
- Microglia are key mediators of methylmercury neurotoxicity through TNF-α production.
- The ASK1/p38 pathway, activated by mitochondrial ROS, is crucial for methylmercury-induced TNF-α expression in microglia.
- Targeting microglial activation and the TNF-α/p38 pathway offers a potential therapeutic strategy against methylmercury poisoning.
