[Deubiquitinase USP30 alleviates brain injury after intracerebral hemorrhage by regulating MFN2 ubiquitination].

objectivesIntracerebral hemorrhage (ICH) is a type of stroke associated with high rates of disability and mortality. Mitochondrial dysfunction plays an important role in secondary brain injury following ICH, and preservation of mitochondrial function is considered a potential therapeutic strategy. Ubiquitin-specific protease 30 (USP30) is a deubiquitinase located on the outer mitochondrial membrane and plays an important role in regulating mitochondrial homeostasis. This study aims to investigate the role of USP30 after ICH and its potential molecular mechanisms.

methodsIn vitro and in vivo experiments were conducted using a hemin-induced SH-SY5Y cell model and a type IV collagenase-induced rat model of ICH. For the in vitro experiments, SH-SY5Y cells were divided into a control group, a 24-h hemin treatment group, and a 48-h hemin treatment group. Western blotting and co-immunoprecipitation (Co-IP) were performed to determine the expression and ubiquitination levels of mitofusin 2 (MFN2). To verify USP30 overexpression efficiency, SH-SY5Y cells not exposed to hemin were transfected with either an empty vector or a USP30-Flag plasmid for 48 h, designated as the control+vector and control+USP30 groups, respectively, and USP30 protein expression was examined by Western blotting. Subsequently, cells were divided into a control group, a hemin group, a hemin+vector group, and a hemin+USP30-overexpression group. In the latter 2 groups, cells were treated with hemin 48 hours after transfection with USP30-Flag plasmid or empty vector, respectively. Cell viability was assessed using the cell counting kit-8 (CCK-8) assay. Mitochondrial function was evaluated using adenosine triphosphate (ATP), reactive oxygen species (ROS), and MitoSOX assay kits. Mitochondrial morphology was examined using Mito-Tracker staining and transmission electron microscopy. Western blotting and Co-IP were used to detect the expression of related proteins and the ubiquitination level of MFN2. For the in vivo experiments, rats were divided into a sham-operated group and a 3-day ICH group. Western blotting and Co-IP were performed to determine MFN2 expression and ubiquitination in perihematomal tissues. Subsequently, the rats were divided into a sham-operated group, an ICH group, an ICH plus negative-control AAV (AAV-NC) group, and an ICH plus adeno-associated virus expressing USP30 (AAV-USP30) group. In the latter 2 groups, adeno-associated virus (AAV)-USP30 or AAV-NC was injected into the lateral ventricle 20 days before ICH induction. On day 3 after ICH, neurological function was assessed using the modified Garcia score. Histological injury and apoptosis were evaluated using hematoxylin and eosin (HE) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Mitochondrial function and ultrastructure were assessed by measuring ATP and ROS levels, ATP5H immunofluorescence, and transmission electron microscopy. Western blotting and Co-IP were used to determine the expression of related proteins and the ubiquitination level of mitochondrial MFN2.

resultsIn the in vitro experiments, compared with the control group, SH-SY5Y cells treated with hemin for 24 or 48 h exhibited significantly increased MFN2 ubiquitination and significantly decreased MFN2 protein expression (all

conclusionsAcute ICH is accompanied by increased MFN2 ubiquitination and decreased MFN2 protein expression, which may contribute to mitochondrial structural and functional impairment. USP30 preserves mitochondrial structural and functional integrity by suppressing MFN2 ubiquitination and degradation, thereby alleviating ICH-induced brain injury. USP30 may therefore represent a potential molecular target for the treatment of ICH.