The Role Of NF-κB Pathway In Autophagy Activation And Differentiation Of Neural Stem Cells

Aim: A number of experimental methods have been developed to promote the differentiation of neural stem cells (NSCs) toward specific lineages. Neuronal differentiation is often associated with cell cycle arrest, altered rate of macromolecule synthesis and degradation as well as remodeling of cytoskeletal structures. NF-κB plays important roles in proliferation of lymphocytes, tumor cells and NSCs, however, its possible role in NSCs differentiation has nerve been explored. In this study, we studied if inhibiting NF-κB signaling pathway is involved in NSCs differentiation in vitro.Method: After treatment with SN50, the growth inhibition of NSCs was assessed by MTT colorimetric assay and the cytotoxicity of SN50 was measured by lactose dehydrogenase (LDH) leakage. Immunohistochemistry and Western blot analysis were used to study the mechanisms involved in NSCs differentiation in vitro by inhibiting NF-κB signaling pathway. MDC staining was used to examine autophagy induced by SN50 in NSCs. To examine the involvement of autophagy in SN50-induced differentiation of NSCs, the autophagy specific inhibitor 3-methyadenine (3-MA) was added with SN50. Immunohistochemistry and Western blot analysis were used to study the autophagic mechanisms involved in differentiation of NSCs.Results: NSCs cultured from mouse embryonic brains exposed to SN50, a recombinant cell-permeable peptide blocking nuclear translocation of NF-κB, resulted in the decrease in proliferation of NSCs. Meanwhile, SN50 promoted migration of NSCs away from neurospheres and growth of neuritis in a dose- and time-dependent fashion. Biochemical analysis revealed increases in the expression of protein levels of MAP-2, NeuN and GAP-43, indicating enhanced differentiation of NSCs towards neuronal lineage. Activation of autophagy during SN50-induced differentiation of NSCs was evidenced by presence of increased number of acidic vacuoles in the cytoplasm and increased expression of membrane form of microtubule associate protein light chain 3 (LC3-II) and becllin 1. Inhibition of autophagy by 3-methyladenine (3-MA) abrogated SN50-induced NSCs differentiation.Conclusion: These results suggest that blocking NF-κB transcriptional activation activity arrests cell cycle and promotes stem cell differentiation. Autophagy activation is associated with NF-κB inhibition-evoked stem cell differentiation.