NOTCH1-STAT3 signaling axis regulates astrocytic differentiation of hippocampal neural stem/progenitor cells

Abstract

Hippocampal neural stem/progenitor cells (HNPCs) in the hippocampus can differentiate into neurons and astrocytes, and are regulated by complex signaling pathways, such as the NOTCH1–signal transducer and activator of transcription 3 (STAT3) axis, which are crucial for cell fate determination. However, the exact molecular mechanism underlying HNPC differentiation remains unclear. This study investigated the role of NOTCH1–STAT3 pathway in HNPC differentiation into neuronal and astrocytic lineages during embryonic development. Mouse HNPCs were cultured with basic fibroblast and epidermal growth factors to promote proliferation. Differentiation was assessed using western blotting, immunofluorescence, RNA sequencing (RNA-seq), and reverse transcription-quantitative PCR to analyze gene expression. The roles of NOTCH1 and STAT3 in cell fate were assessed using their respective inhibitors, DAPT and Stattic, respectively. Immunoprecipitation was performed to investigate the interactions between NOTCH1 and STAT3. Proliferative conditions induced a shift from neurogenesis to astrocytic differentiation in HNPCs, as demonstrated by the increased GFAP and decreased TUJ1 levels. RNA-seq and gene ontology analyses revealed the upregulation of astrocyte-related genes and suppression of neurogenesis. NOTCH1 signaling promoted astrocytic differentiation through elevated DLL1 expression. Additionally, the inhibition of STAT3 or NOTCH1 reduced GFAP expression, whereas NOTCH1 knockdown reduced STAT3 activation, suggesting that NOTCH1 regulates astrocytic fate via STAT3 in proliferating HNPCs. These findings reveal the regulatory mechanisms of neural differentiation, emphasizing the critical role of the NOTCH1–STAT3 signaling axis in astrocytic differentiation of HNPCs, thereby enhancing our understanding of the molecular basis of neural cell fate decisions during brain development.