NEP1-40-overexpressing Neural Stem Cells Enhance Axon Regeneration by Inhibiting Nogo-A/NgR1 Signaling Pathway

Nogo-66 antagonistic peptide (NEP1-40) offers the potential to improve spinal cord injury (SCI).To explore the effect of NEP1-40 overexpression on neural stem cells (NSCs) regulating the axon regeneration of injured neurons.We isolated NSCs from brain tissues of pregnant rat fetuses and used Nestin immunofluorescence to identify them. The NEP1-40 overexpressing NSCs were constructed by transfection with the NEP1-40-overexpressing vector. The expression of NSCs differentiation associated markers, including Tuj-1, GFAP, Oligo2, and MBP, were detected by RT-PCR, western blotting, and immunofluorescence. NeuN immunofluorescence staining was used to measure the number of neurons. And western blotting was used to detect the phosphorylation levels of LIMK1/2, cofilin, and MLC-2 and the protein levels of GAP-43, MAP-2, and APP.The NEP1-40 overexpression promoted the expression level of Tuj-1, Oligo2, and MBP, and increased the number of Tuj-1, Oligo2, and MBP positive cells. NEP1-40-overexpressing NSCs (NEP-NSCs) improved NeuN positive cells of co-culture with injured neurons. And NEP-NSCs also increased the protein levels of axon regeneration indicators (GAP-43, MAP-2) and decreased APP protein level. In addition, the phosphorylation level of LIMK1/2, cofilin, and MLC-2 were markedly decreased in NEP-NSCs.NEP1-40 overexpression enhanced the ability of NSCs differentiation into neurons and promoted axon regeneration by inhibiting the Nogo-A/NgR1 signaling pathway. This study provides an alternative gene modified transplantation NSCs for the SCI treatment.