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Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates
Published on: January 30, 2014
Microglial alpha7 nicotinic acetylcholine receptors drive a phospholipase C/IP3 pathway and modulate the cell
Tomohisa Suzuki1, Izumi Hide, Akiyo Matsubara
1Department of Pharmacology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan.
Abstract:
Microglia perform both neuroprotective and neurotoxic functions in the brain, with this depending on their state of activation and their release of mediators. Upon P2X(7) receptor stimulation, for example, microglia release small amounts of TNF, which protect neurons, whereas LPS causes massive TNF release leading to neuroinflammation. Here we report that, in rat primary cultured microglia, nicotine enhances P2X(7) receptor-mediated TNF release, whilst suppressing LPS-induced TNF release but without affecting TNF mRNA expression via activation of alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs). In microglia, nicotine elicited a transient increase in intracellular Ca(2+) levels, which was abolished by specific blockers of alpha7 nAChRs. However, this response was independent of extracellular Ca(2+) and blocked by U73122, an inhibitor of phospholipase C (PLC), and xestospongin C, a blocker of the IP(3) receptor. Repeated experiments showed that currents were not detected in nicotine-stimulated microglia. Moreover, nicotine modulation of LPS-induced TNF release was also blocked by xestospongin C. Upon LPS stimulation, inhibition of TNF release by nicotine was associated with the suppression of JNK and p38 MAP kinase activation, which regulate the post-transcriptional steps of TNF synthesis. In contrast, nicotine did not alter any MAP kinase activation, but enhanced Ca(2+) response in P2X(7) receptor-activated microglia. In conclusion, microglial alpha7 nAChRs might drive a signaling process involving the activation of PLC and Ca(2+) release from intracellular Ca(2+) stores, rather than function as conventional ion channels. This novel alpha7 nAChR signal may be involved in the nicotine modification of microglia activation towards a neuroprotective role by suppressing the inflammatory state and strengthening the protective function.
Insights
Nicotine modulates microglial responses via alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs), enhancing neuroprotective pathways while suppressing inflammatory ones. This suggests a potential role for nicotine in promoting brain health by altering microglial activation states.
Area of Science:
- Neuroscience
- Immunology
- Pharmacology
Background:
- Microglia exhibit dual neuroprotective and neurotoxic functions, influenced by activation state and mediator release.
- P2X7 receptor stimulation and lipopolysaccharide (LPS) trigger differential Tumor Necrosis Factor (TNF) release from microglia, impacting neuronal health and neuroinflammation.
- Alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs) are implicated in modulating microglial responses.
Purpose of the Study:
- To investigate the role of nicotine and alpha7 nAChRs in modulating microglial TNF release and activation pathways.
- To elucidate the signaling mechanisms downstream of alpha7 nAChR activation in microglia.
- To explore the potential of nicotine in shifting microglial function towards a neuroprotective phenotype.
Main Methods:
- Primary rat microglia cultures were used to study nicotine's effects on TNF release.
- Experiments involved stimulation with P2X7 receptor agonists and LPS, with measurements of TNF mRNA and protein expression.
- Nicotine's impact on intracellular calcium (Ca2+) levels, phospholipase C (PLC) activity, IP3 receptor signaling, and MAP kinase activation (JNK, p38) was assessed using specific inhibitors and blockers.
Main Results:
- Nicotine enhanced P2X7 receptor-mediated TNF release but suppressed LPS-induced TNF release, without altering TNF mRNA levels.
- Nicotine-induced Ca2+ transients in microglia were dependent on alpha7 nAChRs and involved PLC and IP3 receptor signaling, independent of extracellular Ca2+.
- Nicotine suppressed LPS-induced JNK and p38 MAP kinase activation, while enhancing Ca2+ responses in P2X7 receptor-activated microglia.
Conclusions:
- Microglial alpha7 nAChRs activate a signaling cascade involving PLC and intracellular Ca2+ release, functioning distinct from conventional ion channels.
- This novel alpha7 nAChR signaling pathway may underlie nicotine's ability to shift microglia towards a neuroprotective role.
- Nicotine's modulation of microglia may suppress inflammatory responses and bolster neuroprotective functions, offering therapeutic potential.
