Multi-omics integration identifies VIM and LGALS3 as apoptosis-associated genes in astrocytes after traumatic brain injury.

Abstract

Apoptosis contributes substantially to neurological deficits after traumatic brain injury (TBI), yet the molecular drivers and cellular regulation of astrocyte-associated apoptosis remain poorly defined. We integrated bulk RNA-seq, single-cell RNA-seq, and spatial transcriptomics to identify apoptosis-related hub genes in TBI. Differentially expressed genes were intersected with apoptosis-related genes and ranked by protein-protein interaction network topology. Astrocyte heterogeneity was examined through sub-clustering, pseudotime, regulon, and in silico transcription-factor perturbation analyses. Findings were validated in vivo using a Feeney weight-drop model and in vitro using an astrocyte scratch model, with Western blot, immunofluorescence co-staining, siRNA knockdown, wound-healing, CCK-8, and flow-cytometric apoptosis assays. VIM and LGALS3 were identified as the apoptosis-related hub genes and showed reproducible injury-associated upregulation across multiple independent datasets. Single-cell analysis showed their expression in a reactive astrocyte subpopulation, with levels rising along the homeostatic-to-reactive trajectory. CEBPB, FOSL1/FOSL2, and MAFF showed higher regulon activity in TBI-reactive astrocytes, and there in silico knockout shifted reactive astrocytes toward the homeostatic state. Spatially, both transcripts accumulated in the lesion core and peri-lesional zones, coinciding with elevated apoptotic activity. Western blot confirmed their upregulation in vivo and in vitro, and immunofluorescence co-staining of injured cortical tissue showed their expression in GFAP-positive astrocytes; silencing either genes reduced astrocyte migration, and flow cytometry together with an increased cleaved-caspase-3/caspase-3 ratio indicated enhanced apoptotic signaling. In astrocytes after TBI, VIM and LGALS3 modulate migration and apoptotic signaling, representing candidate molecular markers and potential therapeutic targets for secondary brain injury that warrant further validation.