Lymphotoxin β receptor-mediated NFκB signaling promotes glial lineage differentiation and inhibits neuronal lineage differentiation in mouse brain neural stem/progenitor cells

摘要

Abstract Background Lymphotoxin (LT) is a lymphokine mainly expressed in lymphocytes. LTα binds one or two membrane-associated LTβ to form LTα2β1 or LTα1β2 heterotrimers. The predominant LTα1β2 binds to LTβ receptor (LTβR) primarily expressed in epithelial and stromal cells. Most studies on LTβR signaling have focused on the organization, development, and maintenance of lymphoid tissues. However, the roles of LTβR signaling in the nervous system, particularly in neurogenesis, remain unknown. Here, we investigated the role of LTβR-mediated NFκB signaling in regulating neural lineage differentiation. Methods The C57BL/6J wild-type and GFAP-dnIκBα transgenic mice were used. Serum-free embryoid bodies were cultured from mouse embryonic stem cells and further induced into neural stem/progenitor cells (NSCs/NPCs). Primary neurospheres were cultured from embryonic and adult mouse brains followed by monolayer culture for amplification/passage. NFκB activation was determined by adenovirus-mediated NFκB-firefly-luciferase reporter assay and p65/RelB/p52 nuclear translocation assay. LTβR mRNA expression was evaluated by quantitative RT-PCR and LTβR protein expression was determined by immunohistochemistry and Western blot analysis. Multilabeled immunocytochemistry or immunohistochemistry followed by fluorescent confocal microscopy and quantitative analysis of neural lineage differentiation were performed. Graphing and statistical analysis were performed with GraphPad Prism software. Results In cultured NSCs/NPCs, LTα1β2 stimulation induced an activation of classical and non-classical NFκB signaling. The expression of LTβR-like immunoreactivity in GFAP+/Sox2+ NSCs was identified in well-established neurogenic zones of adult mouse brain. Quantitative RT-PCR and Western blot analysis validated the expression of LTβR in cultured NSCs/NPCs and brain neurogenic regions. LTβR expression was significantly increased during neural induction. LTα1β2 stimulation in cultured NSCs/NPCs promoted astroglial and oligodendrocytic lineage differentiation, but inhibited neuronal lineage differentiation. Astroglial NFκB inactivation in GFAP-dnIκBα transgenic mice rescued LTβR-mediated abnormal phenotypes of cultured NSCs/NPCs. Conclusion This study provides the first evidence for the expression and function of LTβR signaling in NSCs/NPCs. Activation of LTβR signaling promotes glial lineage differentiation. Our results suggest that neurogenesis is regulated by the adaptive immunity and inflammatory responses.