Integrative analysis and experimental validation identify the role of CD44 and Nucleolin in regulating gliogenesis following spinal cord injury

Spinal cord injury (SCI) triggers a complex cascade of cellular and molecular responses, yet the complex cellular communication remains incompletely understood. This study explored how intercellular communication contributes to the activation of microglia and astrocytes after SCI. Here, we integrated four datasets using single-cell RNA sequencing (scRNA-seq) or single-nucleus RNA sequencing (snRNA-seq) and constructed a comprehensive cellular atlas of the injured spinal cord. Transcriptomic changes in microglia and astrocytes were analyzed. We identified CD44 as a key receptor in SPP1-mediated microglial activation, which represented a subpopulation involved in inflammatory response in microglia. We defined a gliogenesis subpopulation of astrocytes that emerged at 3 dpi, which became the predominant cell type in the injured spinal cord. These astrocytes highly expressed the Nucleolin (Ncl) gene and interacted via the Pleiotrophin (Ptn) signaling pathway, which is associated with astrocyte proliferation. To validate these findings, we utilized a crush injury model. Flow cytometry of isolated microglia and astrocytes confirmed the upregulation of CD44 in microglia and NCL in astrocytes in response to SCI. In vivo results confirmed that the CD44 positive microglia accumulated and PLA results further confirmed the combination of SPP1 with CD44. In parallel, the upregulated expression of NCL in astrocytes facilitated their proliferation, underscoring the role of the NCL receptor in gliogenesis after SCI. In vitro validation demonstrated that exogenous SPP1 upregulates CD44 expression by promoting the phosphorylation of p65 and activating the NF-κB pathways in BV2 microglia, and that high expression of IL-6 indicates the activation of inflammation. PTN may enhance NCL expression and thus facilitates astrocyte proliferation. Collectively, our study identified key receptors that regulated inflammation responses and gliogenesis. Targeting the CD44 and NCL receptors may provide promising therapeutic strategies to modulate inflammation and promote tissue repair after SCI.