Integrative single-cell and bulk transcriptomic analyses identify a microglia-associated NAGLU signature linked to disulfidptosis-associated transcriptional patterns after spinal cord injury

Abstract

Background Spinal cord injury (SCI) induces profound neuroinflammatory responses and extensive cellular remodeling within the injured spinal cord. Disulfidptosis, a recently identified form of regulated cell death associated with disulfide stress, has been implicated in cellular stress responses and tissue injury. However, the transcriptional characteristics of disulfidptosis-related genes (DRGs) and their associations with the cellular microenvironment of SCI remain poorly understood. Methods Single-cell RNA sequencing data from GSE162610 were analyzed to characterize cellular heterogeneity in DRG-associated transcriptional signatures. AUCell was used to calculate DRG signature scores at the single-cell level, and differentially expressed genes associated with DRG signatures were identified by comparing cells with high and low DRG signature scores. Bulk transcriptomic data from GSE47681 were analyzed using differential expression analysis and weighted gene co-expression network analysis (WGCNA) to identify SCI-associated differentially expressed genes and gene modules correlated with DRG signature scores. Candidate genes were identified by integrating single-cell marker genes, bulk differentially expressed genes, and genes from DRG signature-associated WGCNA modules. A random forest algorithm was applied to prioritize key candidate genes, followed by validation in the independent dataset GSE45006. The expression pattern of the selected candidate gene was further examined at single-cell resolution and validated at the protein level in a rat SCI model 3 days after injury. Results DRG signature scores exhibited marked heterogeneity across spinal cord cell types. Integrative analyses identified 24 candidate genes associated with DRG-related transcriptional alterations. Functional enrichment analyses highlighted lysosome-related pathways, efferocytosis, macrophage activation, and glycosaminoglycan degradation. Random forest analysis prioritized three genes for external validation, among which Naglu showed consistent differential expression in the independent dataset. Single-cell analysis indicated that Naglu was predominantly expressed in microglia. Protein-level validation further demonstrated increased NAGLU expression in spinal cord tissues from SCI rats at 3 days post-injury compared with Sham controls. Conclusions This study characterizes DRG-associated transcriptional heterogeneity in SCI and identifies Naglu as a microglia-associated lysosomal gene upregulated after spinal cord injury. These findings suggest that DRG-associated transcriptional states are linked to microglial lysosomal responses after SCI and provide a basis for future mechanistic studies investigating neuroinflammation and tissue repair.