Microglial NCAM1 attenuates ischemic brain injury by inhibiting NF-κB-driven neuroinflammation through IκBα stabilization

Ischemic stroke is a devastating condition with limited treatment options, where neuroinflammation plays a pivotal role in secondary brain injury. Neural cell adhesion molecule 1 (NCAM1) is implicated in neural development and plasticity, but its specific role in microglia during ischemic stroke remains unclear. Here, we demonstrate that microglial NCAM1 expression is significantly downregulated in the acute phase of ischemic stroke in both human patients and a mouse transient middle cerebral artery occlusion (tMCAO) model. Using microglia/macrophage-specific NCAM1-overexpressing CX3CR1Cre/ERT2 mice, we show that NCAM1 overexpression reduces infarct volume, improves neurological deficits, and enhances long-term functional recovery. Mechanistically, NCAM1 directly interacts with the E3 ubiquitin ligase TRIM67 via its cytoplasmic domain. This NCAM1-TRIM67 complex enhances K63-linked ubiquitination while suppressing K48-linked ubiquitination of IκBα, thereby stabilizing IκBα protein, preventing NF-κB p65 nuclear translocation, and ultimately inhibiting NF-κB-driven neuroinflammation and apoptosis. Furthermore, through molecular docking and high-throughput screening, we identified DB07993 as a potent NCAM1 agonist. DB07993 treatment mimicked the neuroprotective effects of NCAM1 overexpression in vitro and in vivo, primarily through activation of the NCAM1-TRIM67-IκBα axis. Our study unveils a novel regulatory mechanism where microglial NCAM1 serves as a critical brake on post-stroke neuroinflammation and identifies DB07993 as a promising lead compound for developing NCAM1-targeted stroke therapies. Under ischemic conditions, activated microglia promote NF-κB nuclear translocation and pro-inflammatory gene expression, leading to neuronal death. NCAM1 overexpression or DB07993 treatment initiates a protective mechanism through direct interaction with TRIM67. This NCAM1-TRIM67 complex enhances K63-linked and suppresses K48-linked ubiquitination of IκBα, resulting in IκBα stabilization and subsequent inhibition of NF-κB signaling. This molecular cascade ultimately attenuates neuroinflammation and creates a favorable environment for neuronal repair.