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Published on: August 15, 2012
Production and release of neuroprotective tumor necrosis factor by P2X7 receptor-activated microglia
Tomohisa Suzuki1, Izumi Hide, Katsutoshi Ido
1Department of Pharmacology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan.
Abstract:
After a brain insult, ATP is released from injured cells and activates microglia. The microglia that are activated in this way then release a range of bioactive substances, one of which is tumor necrosis factor (TNF). The release of TNF appears to be dependent on the P2X7 receptor. The inhibitors 1,4-diamino-2,3-dicyano-1,4-bis[2-amino-phenylthio]butadiene (U0126), anthra[1,9-cd]pyrazol-6(2H)-one (SP600125), and 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)IH-imidazole (SB203580), which target MEK (mitogen-activated protein kinase kinase), JNK (c-Jun N-terminal kinase), and p38, respectively, all potently suppress the production of TNF in ATP-stimulated microglia, whereas the production of TNF mRNA is strongly inhibited by U0126 and SP600125. SB203580 did not affect the increased levels of TNF mRNA but did prevent TNF mRNA from accumulating in the cytoplasm. The ATP-provoked activation of JNK and p38 [but not extracellular signal-regulated kinase (ERK)] could be inhibited by brilliant blue G, a P2X7 receptor blocker, and by genistein and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, which are general and src-family-specific tyrosine kinase inhibitors, respectively. Most important, we found that treatment of the microglia in neuron-microglia cocultures with the P2X7 agonist 2'-3'-O-(benzoyl-benzoyl) ATP led to significant reductions in glutamate-induced neuronal cell death, and that either TNF-alpha converting enzyme inhibitor or anti-TNF readily suppressed the protective effect implied by this result. Together, these findings indicate that both ERK and JNK are involved in the regulation of TNF mRNA expression, that p38 is involved in the nucleocytoplasmic transport of TNF mRNA, and that a PTK (protein tyrosine kinase), possibly a member of the src family, acts downstream of the P2X7 receptor to activate JNK and p38. Finally, our data suggest that P2X7 receptor-activated microglia protect neurons against glutamate toxicity primarily because they are able to release TNF.
Insights
Activated microglia release tumor necrosis factor (TNF) after brain injury, a process dependent on the P2X7 receptor. Inhibiting specific kinases (MEK, JNK, p38) suppressed TNF production and protected neurons from glutamate toxicity.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Brain injury triggers ATP release, activating microglia.
- Activated microglia release tumor necrosis factor (TNF), crucial in neuroinflammation.
- P2X7 receptor signaling is implicated in microglial activation and TNF release.
Purpose of the Study:
- To elucidate the signaling pathways regulating TNF production by activated microglia.
- To investigate the role of P2X7 receptor in microglial activation and neuroprotection.
- To determine the mechanisms by which microglia protect neurons from glutamate toxicity.
Main Methods:
- Utilized specific kinase inhibitors (U0126, SP600125, SB203580) targeting MEK, JNK, and p38.
- Assessed TNF and TNF mRNA levels in ATP-stimulated microglia.
- Employed P2X7 receptor blockers and tyrosine kinase inhibitors.
- Investigated neuronal protection in neuron-microglia cocultures using P2X7 agonists and TNF inhibitors.
Main Results:
- MEK, JNK, and p38 inhibitors suppressed TNF production; MEK and JNK inhibitors reduced TNF mRNA levels, while p38 inhibition affected TNF mRNA cytoplasmic accumulation.
- P2X7 receptor activation led to JNK and p38 activation, modulated by tyrosine kinases.
- P2X7 receptor activation in microglia significantly reduced glutamate-induced neuronal death.
- Inhibition of TNF or its converting enzyme abolished the neuroprotective effect.
Conclusions:
- ERK and JNK regulate TNF mRNA expression, while p38 controls TNF mRNA transport.
- A protein tyrosine kinase, potentially src-family, acts downstream of P2X7 to activate JNK and p38.
- P2X7 receptor-activated microglia confer neuroprotection against glutamate toxicity via TNF release.
