E3 Ubiquitin Ligase NEDD4-Mediated CTSB Ubiquitination Prevents Microglial Ferroptosis After Subarachnoid Hemorrhage

To investigate the role of cathepsin B (CTSB) in microglial ferroptosis after subarachnoid hemorrhage (SAH) and its upstream regulatory mechanisms. A SAH mouse model was established, and BV2 microglial cell line was utilized. Gene expression was detected by qRT-PCR and western blot. Co-immunoprecipitation was performed to verify the interaction between NEDD4 and CTSB. CCK-8 and PI staining were used to evaluate cell viability and death. Immunofluorescence was employed to observe protein localization and oxidative stress marker levels. TUNEL staining was conducted to detect neuronal apoptosis. CTSB was specifically upregulated in microglia after SAH. CTSB knockdown significantly enhanced cell viability, reduced cell death, and reversed the abnormal changes in ferroptosis-related proteins and oxidative stress markers induced by hemin/RSL3. Mechanistically, NEDD4 was identified as an E3 ubiquitin ligase for CTSB, capable of directly binding and ubiquitinating CTSB protein for degradation. NEDD4 overexpression effectively reduced CTSB protein levels in SAH mouse brains while simultaneously alleviating brain edema, neuronal apoptosis, and neurological deficits, significantly inhibiting ferroptosis after SAH. NEDD4 attenuates secondary brain injury after SAH by ubiquitinating and degrading CTSB, thereby inhibiting microglial ferroptosis.