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Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation
Published on: February 16, 2018
High mobility group box 1, a novel serotonin receptor-7 negative modulator, contributes to M2 microglial ferroptosis
Ou Du1, Chao Wu2, Yu-Xin Yang3
1Department of Pharmacology, Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan, China.
Abstract:
Emerging evidence points to a pivotal role of microglial polarization in the neuroinflammatory processes linked to post-stroke depression (PSD), with both high mobility group box 1 (HMGB1) and serotonin receptor-7 (5-HT7R) being implicated in the microglial inflammatory response. However, the underlying mechanisms remain unclear. This study investigated the potential roles of HMGB1 and 5-HT7R in PSD. We first examined the interaction between HMGB1 and 5-HT7R in HEK293 cells using bioluminescence resonance energy transfer (BRET) technology. The expression of 5-HT7R was most significantly increased in microglia in vitro upon HMGB1 stimulation and in a transient middle cerebral artery occlusion (tMCAO) model, although it is also enhanced in astrocytes and neurons to some extent. Subsequently, the interaction between HMGB1 and 5-HT7R in primary microglia was examined using co-immunoprecipitation (Co-IP) and pull-down assays. Meanwhile, we found that HMGB1 upregulated the expression of 5-HT7R and downregulated the accumulation of second messenger cyclic adenosine monophosphate (cAMP) in HEK293 cells and primary microglia. Additionally, employing a 4-week tMCAO mice model, we found that 5-HT7R deletion or HMGB1 inhibition alleviated ischemic brain injury, depressive-like behaviors, M2 microglial ferroptosis, and neuroinflammation, meanwhile upregulating the cAMP/PKA and Nrf2/xCT/GPX4 pathways. Moreover, in vitro, recombinant HMGB1 (rHMGB1) effectively induced M2 microglial ferroptosis and neuroinflammation and suppressed the cAMP/PKA and Nrf2/xCT/GPX4 pathways in primary microglia, which were markedly attenuated by 5-HT7R deletion. Finally, cAMP analog dibutyryl cAMP (db-cAMP) or Nrf2 activator sulforaphane (SFN) attenuates ferroptosis in M2-polarized BV2 microglial cells and neuroinflammation induced by rHMGB1. Altogether, these results suggest that HMGB1 functions as a novel negative regulator of 5-HT7R, contributing to ischemic brain injury and PSD by promoting M2 microglial ferroptosis and neuroinflammation.
Insights
High mobility group box 1 (HMGB1) negatively regulates serotonin receptor-7 (5-HT7R), exacerbating post-stroke depression by promoting microglial ferroptosis and neuroinflammation. Inhibiting this interaction may offer therapeutic benefits for stroke recovery.
Area of Science:
- Neuroscience
- Immunology
- Molecular Biology
Background:
- Microglial polarization is crucial in neuroinflammation following stroke, with High Mobility Group Box 1 (HMGB1) and Serotonin Receptor-7 (5-HT7R) implicated.
- The precise mechanisms linking HMGB1 and 5-HT7R in the context of post-stroke depression (PSD) remain incompletely understood.
Purpose of the Study:
- To investigate the interaction between HMGB1 and 5-HT7R and their roles in post-stroke depression (PSD).
- To elucidate the molecular pathways involved in HMGB1-induced microglial activation and ferroptosis.
Main Methods:
- Bioluminescence Resonance Energy Transfer (BRET) and co-immunoprecipitation (Co-IP) assays to examine HMGB1-5-HT7R interaction.
- Transient middle cerebral artery occlusion (tMCAO) mouse model to study in vivo effects.
- Analysis of microglial ferroptosis, neuroinflammation markers, and cAMP/PKA and Nrf2/xCT/GPX4 pathways.
Main Results:
- HMGB1 stimulation upregulated 5-HT7R expression and downregulated cyclic adenosine monophosphate (cAMP) accumulation.
- 5-HT7R deletion or HMGB1 inhibition in a tMCAO model alleviated brain injury, depressive behaviors, M2 microglial ferroptosis, and neuroinflammation.
- HMGB1 induced M2 microglial ferroptosis and neuroinflammation, suppressed cAMP/PKA and Nrf2/xCT/GPX4 pathways, effects attenuated by 5-HT7R deletion.
Conclusions:
- HMGB1 acts as a novel negative regulator of 5-HT7R, contributing to ischemic brain injury and PSD.
- The HMGB1-5-HT7R axis promotes M2 microglial ferroptosis and neuroinflammation, offering potential therapeutic targets for PSD.
