Related Experiment Video
Updated: Jul 22, 2025
Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures
Published on: July 25, 2022
NMDA receptor inhibitor MK801 alleviated pro-inflammatory polarization of BV-2 microglia cells
Chih-Cheng Wu1, Chung-Yuh Tzeng2, Cheng-Yi Chang3
1Department of Anesthesiology, Taichung Veterans General Hospital, Taichung City, 407, Taiwan; Department of Financial Engineering, Providence University, Taichung City, 433, Taiwan; Department of Data Science and Big Data Analytics, Providence University, Taichung City, 433, Taiwan.
Abstract:
Microglia have both protective and pathogenic properties, while polarization plays a decisive role in their functional diversity. Apart from being an energetic organelle, mitochondria possess biological capabilities of signaling and immunity involving mitochondrial dynamics. The N-methyl-D-aspartate (NMDA)-type glutamate receptor displays excitatory neurotransmission, excitatory neurotoxicity and pro-inflammatory properties in a membrane location- and cell context-dependent manner. In this study, we have provided experimental evidence showing that by acting on mitochondrial dynamics, NMDA receptors displayed pro-inflammatory properties, while its non-competitive inhibitor MK801 exhibited anti-inflammatory potential in Lipopolysaccharide (LPS)-challenged BV-2 microglia cells. LPS stimulation increased the protein phosphorylation of cells regarding their NMDA receptor component subunits and Calcium/Calmodulin-dependent Protein Kinase II (CaMKII), along with mobilizing intracellular calcium. Additionally, parallel changes occurred in the activation of Transforming Growth Factor-β (TGF-β)-Activated Kinase 1 (TAK1), NF-κB p65 and NF-κB DNA binding activity, acquisition of pro-inflammatory M1 polarization and expression of pro-inflammatory cytokines. LPS-treated cells further displayed signs of mitochondrial dysfunction with higher expressions of the active form of Dynamin-Related Protein 1 (Drp1), NADPH Oxidase-2 (NOX2) expression and the generation of DCFDA-/MitoSOX-sensitive Reactive Oxygen Species (ROS). NMDA receptor blockade by MK801, along with CaMKII inhibitor KN93, Drp1 inhibitor Mdivi-1 and antioxidant apocynin alleviated LPS-induced pro-inflammatory changes. Other than the reported CaMKII/TAK1/NF-κB axis, our in vitro study revealed the CaMKII/Drp1/ROS/NF-κB axis being an alternative cascade for shaping pro-inflammatory phenotypes of microglia upon LPS stimulation, and MK801 having the potential for inhibiting microglia activation and any associated inflammatory damages.
Insights
N-methyl-D-aspartate (NMDA) receptors promote inflammation in microglia by affecting mitochondrial dynamics. MK801, an NMDA receptor inhibitor, shows anti-inflammatory potential by blocking this pathway, offering therapeutic possibilities.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia, the immune cells of the central nervous system, exhibit diverse functions influenced by polarization.
- Mitochondria are crucial for cellular energy, signaling, and immunity, with their dynamics impacting cellular functions.
- N-methyl-D-aspartate (NMDA)-type glutamate receptors are implicated in neurotransmission, neurotoxicity, and inflammation.
Purpose of the Study:
- To investigate the role of NMDA receptors and mitochondrial dynamics in lipopolysaccharide (LPS)-induced microglial activation.
- To explore the anti-inflammatory potential of NMDA receptor inhibition in microglia.
Main Methods:
- Utilized BV-2 microglia cells stimulated with LPS.
- Examined protein phosphorylation, intracellular calcium mobilization, and inflammatory markers.
- Assessed mitochondrial dysfunction indicators, including Dynamin-Related Protein 1 (Drp1) activation and reactive oxygen species (ROS) generation.
- Investigated the effects of NMDA receptor blockade (MK801) and inhibitors of CaMKII, Drp1, and ROS.
Main Results:
- LPS stimulation activated NMDA receptors, leading to increased CaMKII phosphorylation, calcium influx, and subsequent activation of the TAK1/NF-κB pathway.
- LPS induced M1 pro-inflammatory polarization and cytokine expression, accompanied by mitochondrial dysfunction (increased Drp1, NOX2, ROS).
- MK801, KN93, Mdivi-1, and apocynin attenuated LPS-induced pro-inflammatory responses and mitochondrial dysfunction.
Conclusions:
- NMDA receptor activation, through mitochondrial dynamics, drives pro-inflammatory microglial responses via a CaMKII/Drp1/ROS/NF-κB axis.
- MK801 demonstrates potential in inhibiting microglial activation and mitigating inflammatory damage, suggesting a therapeutic target.
