Profiling of cytosolic and mitochondrial H₂O₂ production using the H₂O_2-sensitive protein HyPer in LPS-induced microglia cells

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 H₂O_2-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.