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Updated: Feb 28, 2026
Author Spotlight: Advancing Mitochondrial Research - mtHyper7 Biosensor for Subcellular Analysis
Published on: June 2, 2023
Profiling of cytosolic and mitochondrial H2O2 production using the H2O2-sensitive protein HyPer in LPS-induced
Junghyung Park1, Seunghoon Lee2, Hyun-Shik Lee3
1School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu, Republic of Korea; College of Natural Sciences, Kyungpook National University, Daegu, Republic of Korea; National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Chungcheongbuk-do, Republic of Korea.
Abstract:
Dysregulation of the production of pro-inflammatory mediators in microglia exacerbates the pathologic process of neurodegenerative disease. ROS actively affect microglia activation by regulating transcription factors that control the expression of pro-inflammatory genes. However, accurate information regarding the function of ROS in different subcellular organelles has not yet been established. Here, we analyzed the pattern of cytosolic and mitochondrial H2O2 formation in LPS-activated BV-2 microglia using the H2O2-sensitive protein HyPer targeted to specific subcellular compartments. Our results show that from an early time, cytosolic H2O2 started increasing constantly, whereas mitochondrial H2O2 rapidly increased later. In addition, we found that MAPK affected cytosolic H2O2, but not mitochondrial H2O2. Consequently, our study provides the basic information about subcellular H2O2 generation in activated microglia, and a useful tool for investigating molecular targets that can modulate neuroinflammatory responses.
Insights
Reactive oxygen species (ROS) impact microglia activation in neurodegenerative diseases. This study reveals distinct cytosolic and mitochondrial hydrogen peroxide (H2O2) patterns in activated microglia, offering insights into neuroinflammation.
Area of Science:
- Neuroimmunology
- Cellular Biology
- Biochemistry
Background:
- Microglia play a crucial role in neuroinflammation, and their activation is exacerbated by pro-inflammatory mediators.
- Reactive oxygen species (ROS), including hydrogen peroxide (H2O2), are implicated in microglia activation and the pathogenesis of neurodegenerative diseases.
- The precise role of ROS in different subcellular compartments of microglia remains incompletely understood.
Purpose of the Study:
- To investigate the spatiotemporal dynamics of hydrogen peroxide (H2O2) formation in cytosolic and mitochondrial compartments of activated microglia.
- To elucidate the involvement of mitogen-activated protein kinases (MAPK) in regulating subcellular H2O2 production in microglia.
Main Methods:
- Utilized the genetically encoded H2O2-sensitive fluorescent probe HyPer, targeted to specific subcellular compartments (cytosol and mitochondria).
- Analyzed H2O2 formation patterns in lipopolysaccharide (LPS)-activated BV-2 microglia cell line.
- Investigated the effect of MAPK signaling on cytosolic and mitochondrial H2O2 levels.
Main Results:
- Cytosolic H2O2 levels showed a continuous increase from an early time point following LPS activation.
- Mitochondrial H2O2 levels exhibited a rapid increase at a later stage compared to cytosolic levels.
- MAPK signaling pathways were found to influence cytosolic H2O2 production but not mitochondrial H2O2 generation.
Conclusions:
- This study provides fundamental insights into the distinct subcellular generation patterns of H2O2 in activated microglia.
- The findings highlight the differential regulation of H2O2 production in the cytosol versus mitochondria.
- The developed tool and findings can facilitate the investigation of molecular targets for modulating microglial neuroinflammatory responses.
