Neutrophil extracellular traps aggravate neuronal apoptosis and neuroinflammation via neddylation after traumatic brain injury

Rationale: Neddylation, akin to ubiquitination, regulates various cellular processes by attaching neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8) to target proteins. Its specific role in traumatic brain injury (TBI) remains poorly defined. Neutrophil extracellular traps (NETs), which accumulate at injury sites in TBI models, are linked to poor outcomes, yet the connection between NETs and neddylation remains unclear. Methods: We analyzed contused brain tissues from TBI patients and mice subjected to controlled cortical impact (CCI) using Western blotting, immunofluorescence, and immunohistochemistry. Neddylation was inhibited using MLN4924, a small-molecule NEDD8-activating enzyme (NAE) inhibitor. We conducted short-term neurobehavioral tests, Fluoro-Jade C staining, TUNEL assay, and Evans blue extravasation. Additionally, co-immunoprecipitation (Co-IP) and mass spectrometry were employed to explore the mechanisms of neddylation post-TBI. Results: Neddylation increased in neurons during the acute phase of TBI. Inhibition of neddylation with MLN4924 reduced neuronal death, mitigated proinflammatory polarization of microglia, and maintained the integrity of the blood-brain barrier (BBB). MLN4924 treatment also decreased cerebral lesion volume and improved short-term neurological outcomes. NETs induced neuronal neddylation and apoptosis, while MLN4924 rescued neurons from NET-induced apoptosis. Mechanistically, NETs promoted NEDD8 binding to the ubiquitin ligase TRIM56, facilitating STING K63-linked ubiquitination and activating the NF-kB pathway, leading to neuroinflammation and neuronal death. Conclusions: Our study revealed that NETs trigger neuronal death and neuroinflammation via neddylation. We propose that inhibiting neddylation could offer therapeutic benefits in TBI.