Differential Roles of Notch Receptors in Regulating Activation of Intact Adult Mouse Spinal Cord-derived NSCs

Neural stem cells (NSCs) play a crucial role in neural regeneration following spinal cord injury (SCI) owing to their self-proliferative and multidirectional differentiation capabilities. This study examined the biological properties of adult spinal cord-derived NSCs (sp-NSCs) and the role of Notch receptors in regulating their activation. NSCs were isolated from the spinal cords of 8-week-old C57/BL6 mice, and their biological properties, including the gene expression profile of Notch receptors, were subsequently analyzed using single-cell RNA sequencing (scRNA-Seq) and bioinformatics. The NSCs were subsequently infected with lentiviral vectors encoding Notch1 shRNA, Notch2 shRNA, and a combination of Notch1 and Notch2 shRNA sequences, and evaluated using Sphere assays and EdU staining to determine their activation effects. The expression levels of downstream genes, NICD and Rbpj, in the Notch signaling pathway, as well as the related target genes, Hes1 and Hey1, were subsequently quantified using Western blot analysis. Intact adult mouse sp-NSCs predominantly existed in a quiescent state, with their population increasing significantly with age. Notch receptors served as critical regulators of adult sp-NSC activation. Notably, Notch1 expression was significantly elevated compared to Notch2 and Notch3, demonstrating its predominant role in sustaining NSC activation. In contrast, Notch2 and Notch3 were primarily responsible for maintaining NSCs in a quiescent state. Overall, both Notch1 and Notch2 signals are involved in different regulatory roles that facilitate the activation and fate determination of NSCs via NICD-Rbpj.