Abstract Sat404: TRIM3-Mediated Ubiquitination of GPX4 Exacerbates Neuronal Ferroptosis After Cardiac Arrest

Background: Ferroptosis, an iron-dependent form of regulated cell death characterized by lipid peroxidation, plays a key role in neuronal injury after cardiac arrest (CA) and cardiopulmonary resuscitation (CPR). Glutathione peroxidase 4 (GPX4) is a critical inhibitor of ferroptosis, but mechanisms driving its degradation in post-CA brain injury are unclear. TRIM3, an E3 ubiquitin ligase implicated in neurodegeneration, may regulate GPX4 turnover. Objective: We investigated whether TRIM3 promotes ferroptosis via ubiquitination-dependent degradation of GPX4, contributing to hippocampal damage after CA-CPR, and whether TRIM3 suppression offers neuroprotection. Methods: A rat model of CA-CPR was used to assess hippocampal injury. Serum LDH and NSE levels quantified acute neuronal damage. Time-specific inhibition of necrosis, apoptosis, and ferroptosis helped define cell death dynamics. Immunoblotting, qPCR, immunofluorescence, and MDA assays evaluated ferroptotic markers. TRIM3–GPX4 interaction was confirmed via co-immunoprecipitation and mass spectrometry. AAV9-mediated TRIM3 knockdown assessed therapeutic potential. Neurological function was tested with NDS, Y-maze, Morris water maze, and open-field assays. PBMCs from rats and human CA-CPR patients were analyzed for TRIM3 expression. Results: Necrosis and apoptosis contributed to early injury (4 h post-CPR), while ferroptosis dominated at 24 h. GPX4 levels declined, with increased MDA in hippocampal CA1. TRIM3 expression rose in neurons and directly bound GPX4, promoting its K48-linked ubiquitination and degradation. TRIM3 knockdown preserved GPX4, suppressed ferroptosis, and improved cognitive outcomes. TRIM3 was also elevated in PBMCs from rats and patients post-CA. Conclusion: TRIM3 drives ferroptosis by mediating GPX4 degradation, worsening neuronal injury and cognitive decline after CA. Targeting TRIM3 may offer a novel therapeutic approach for post-resuscitation brain injury.