NEDD8 promotes the ferritinophagy and ferroptosis of neurons in ischemic stroke via mediating neddylation of NRF2.

PURPOSE: Ischemic stroke (IS) causes severe brain tissue damage characterized by focal neuronal injury. Emerging evidence implicates ferritinophagy-mediated ferroptosis as a critical contributor to ischemic neuronal damage. This study aimed to investigate the role of NEDD8 in regulating ferritinophagy and ferroptosis in neurons under IS, with particular emphasis on elucidating the NEDD8/NRF2/FTH1 signaling axis in IS. METHODS: Murine hippocampal neuronal cells (HT22) were subjected to OGD to simulate ischemic injury in vitro. NEDD8 expression was suppressed using siRNA-mediated knockdown. Cell viability, oxidative stress parameters (ROS, MDA, SOD, GSH, GSSG, GSH/GSSG ratio), and intracellular Fe²⁺ levels were measured. The expression of ferroptosis- and ferritinophagy-related proteins (NCOA4, ACSL4, and GPX4) was evaluated by Western blotting. The interaction between NEDD8 and NRF2 was examined by Co-IP, and NRF2 neddylation was assessed by immunoprecipitation. NRF2 protein stability was assessed using CHX chase assays. NRF2 binding to the FTH1 promoter was predicted using the JASPAR database and experimentally validated by dual luciferase reporter gene assay and ChIP. Functional interactions within the signaling axis were further verified using combination experiments. RESULTS: OGD treatment significantly upregulated NEDD8 expression in HT22 cells and promoted ferritinophagy and ferroptosis. NEDD8 knockdown alleviated OGD-induced neuronal injury by reducing oxidative stress, iron accumulation, and ferroptosis-associated protein alterations. Mechanistically, NEDD8 facilitated NRF2 degradation by enhancing its neddylation, thereby decreasing NRF2 protein stability. Silencing NRF2 abolished the inhibitory effects of NEDD8 knockdown on ferritinophagy and ferroptosis. Furthermore, NRF2 directly bound to the FTH1 promoter and positively regulated FTH1 transcription. Knockdown of FTH1 reversed the protective effects of NEDD8 silencing, restoring ferritinophagy activation and ferroptotic cell death. CONCLUSION: NEDD8 promotes neuronal ferritinophagy and ferroptosis in ischemic conditions by accelerating NRF2 degradation via neddylation, thereby suppressing NRF2-mediated FTH1 transcription. Targeting the NEDD8/NRF2/FTH1 axis may represent a novel therapeutic strategy for mitigating ferroptosis-associated neuronal injury in ischemic stroke.