Neddylation-dependent CUL3-KLHL12 E3 ligase drives microglial oxidative stress and neuroinflammation in traumatic brain injury by targeting GCLM for degradation

BACKGROUND: Microglia-mediated neuroinflammation and oxidative stress are pivotal drivers of secondary injury following traumatic brain injury (TBI). While neddylation governs essential cellular functions, its specific contribution to microglial activation and TBI pathology remains poorly understood. METHODS: We integrated bulk microglial RNA sequencing profiles with single-cell RNA sequencing (scRNA-seq) datasets from TBI mouse brains. To assess therapeutic potential, we employed a controlled cortical impact mouse model and treated animals with the neddylation inhibitor MLN4924. The role of microglia was validated using microglia-depleted mice. Mechanistically, a combinatorial approach utilizing AlphaFold 3 molecular docking predictions, quantitative proteomics, and immunoprecipitation-mass spectrometry was performed to identify molecular targets. RESULTS: We revealed a specific and robust up-regulation of neddylation exclusively within microglial clusters. Pharmacological inhibition of neddylation using MLN4924 significantly ameliorated neurological deficits, attenuated brain edema, and preserved blood-brain barrier integrity. Crucially, these neuroprotective benefits were abrogated in microglia-depleted mice, pinpointing microglia as the primary cellular target. We identified the glutamate-cysteine ligase modifier subunit (GCLM) as a novel substrate of the CUL3-KLHL12 E3 ligase complex. MLN4924 inhibits CUL3 neddylation, thereby impeding the CUL3-KLHL12-mediated ubiquitination and degradation of GCLM. Consequently, GCLM stabilization restores intracellular glutathione synthesis, effectively scavenging reactive oxygen species and mitigating neuroinflammation. CONCLUSIONS: Our findings characterize the Neddylation-CUL3-KLHL12-GCLM axis as a critical regulator of microglial redox homeostasis and highlight this pathway as a promising therapeutic target for TBI intervention.