STUB1 stabilizes sirtuin 6 via K63-linked ubiquitination to promote NRF2 deacetylation for enhanced ferroptosis resistance after spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) is a severe traumatic condition with limited treatments. Ferroptosis, an iron-dependent type of controlled cell death, participates in SCI pathology. Nonetheless, the molecular mechanisms governing ferroptosis after SCI remain incompletely understood. Therefore, this study aims to elucidate the regulatory role of SIRT6 in neuronal ferroptosis post-SCI, identify its upstream modulator and downstream target, and evaluate the therapeutic potential of targeting this pathway for functional recovery. METHODS: Ferroptosis markers were assessed in SCI mouse models and primary neurons. Sirtuin 6 (SIRT6) expression was regulated via lentiviral vectors and adeno-associated viral vectors. Protein interactions were identified by immunoprecipitation coupled with mass spectrometry and co-immunoprecipitation. Ubiquitination assays were conducted using ubiquitin mutants. Nuclear transcription factor erythroid 2-related factor 2 knockout (NRF2-KO) mice were used for in vivo validation. Functional recovery was evaluated by Basso Mouse Scale score, rotarod test, footprint analysis, and electromyography. RESULTS: SIRT6 expression was downregulated following SCI and during erastin-induced ferroptosis. SIRT6 overexpression inhibited ferroptosis and triggered functional recovery, while SIRT6 suppression exacerbated these effects. Mechanistically, SIRT6 interacted with and deacetylated NRF2, promoting its nuclear translocation and antioxidant gene activation. STIP1 homology and U-box containing protein 1 (STUB1) was identified as an upstream regulator that physically interacted with SIRT6 via its U-box domain. STUB1 stabilized SIRT6 by promoting K63-linked ubiquitination, protecting it from proteasomal degradation. STUB1 overexpression inhibited ferroptosis and improved functional recovery, effects that were abolished by SIRT6 knockdown or NRF2-KO. CONCLUSIONS: The STUB1/SIRT6/NRF2 axis represents a novel regulatory pathway that suppresses neuronal ferroptosis and promotes functional recovery post-SCI. Targeting this axis may offer a prospective therapeutic strategy for SCI.