High mobility group box-1 (HMGB1), a nonhistone chromatin DNA-binding protein, is released from neurons into the extracellular space under ischemic, hemorrhagic, and traumatic insults. However, the details of the time-dependent translocation of HMGB1 and the subcellular localization of HMGB1 through the release process in neurons remain unclear. In the present study, we examined the subcellular localization of HMGB1 during translocation of HMGB1 in the cytosolic compartment using a middle cerebral artery occlusion and reperfusion model in rats. Double immunofluorescence microscopy revealed that HMGB1 immunoreactivities were colocalized with MTCO1(mitochondrially encoded cytochrome c oxidase I), a marker of mitochondria, and catalase, a marker of peroxisomes, but not with Rab5/Rab7 (RAS-related GTP-binding protein), LC3A/B (microtubule-associated protein 1 light chain 3), KDEL (KDEL amino acid sequence), and LAMP1 (Lysosomal Associated Membrane Protein 1), which are endosome, phagosome, endoplasmic reticulum, and lysosome markers, respectively. Immunoelectron microscopy confirmed that immune-gold particles for HMGB1 were present inside the mitochondria and peroxisomes. Moreover, HMGB1 was found to be colocalized with Drp1 (Dynamin-related protein 1), which is involved in mitochondrial fission. These results revealed the specific subcellular localization of HMGB1 during its release process under ischemic conditions.
Keywords: high-mobility group box 1; middle cerebral artery occlusion; subcellular localization and subcellular organelle.