Multi-omics integration reveals TIM-4 as a master regulatory hub of neuroinflammation and neuronal cell death

Traumatic brain injury (TBI) is a leading cause of severe disability, frequently resulting in persistent cognitive dysfunction. Microglial M1/M2 polarization is critically involved in TBI pathogenesis, yet its molecular regulatory mechanisms remain poorly understood. TIM-4, a TIM family member implicated in cerebral ischemia-reperfusion injury, has an unknown function in TBI-particularly regarding its regulation of neuronal death.

We employed a comprehensive multi-omics strategy integrating bulk RNA-seq, publicly available single-cell RNA sequencing (scRNA-seq; GEO: GSE101901), weighted gene co-expression network analysis (WGCNA), quantitative proteomics, phosphoproteomics, and epigenomic profiling (ATAC-seq and H3K27ac ChIP-seq) to systematically investigate TIM-4 in TBI. Functional validation included TIM-4 knockdown experiments, TUNEL apoptosis detection, Golgi staining for dendritic spine analysis, and behavioral assessments.

TIM-4 was the most significantly upregulated gene and protein across all omics layers, with expression positively correlated with pro-inflammatory factors and negatively correlated with anti-inflammatory markers. ScRNA-seq revealed TIM-4 upregulation was restricted to activated M1-like microglia, and pseudotime trajectory analysis demonstrated TIM-4-driven M1 polarization. WGCNA identified a TIM-4-associated co-expression module strongly correlated with TBI severity and behavioral outcomes (r = 0.92, p < 0.001). Phosphoproteomics identified TIM-4 Y⁴⁶ hyper-phosphorylation as a key post-translational regulatory event, and ATAC-seq/ChIP-seq revealed NF-κB-driven chromatin remodeling at the TIM-4 locus. Multi-omics integration ranked TIM-4 as the master regulatory hub (composite evidence score = 0.89). Functionally, TIM-4 knockdown promoted microglial M2 polarization, reduced neuronal apoptosis and dendritic spine loss, and significantly improved spatial memory deficits and motor coordination following TBI.

TIM-4 is established as a critical driver of neuroinflammation-associated neuronal death in TBI, representing a promising therapeutic target for TBI-related cognitive dysfunction.