Abstract
BackgroundLymphotoxin (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.MethodsThe 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.ResultsIn 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.ConclusionThis 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.
Highlights
Lymphotoxin (LT) is a lymphokine mainly expressed in lymphocytes
Lymphotoxin α1β2 (LTα1β2) activates classical and nonclassical nuclear factor κB (NFκB) signaling pathways in neural stem/progenitor cells In our previous study, we found that LTα1β2 stimulates activation of NFκB-luciferase reporter in mouse embryonic/neonatal subventricular zone (SVZ) Neural stem cells (NSC)/Neural progenitor cells (NPC) and enteric neuronal cell line [19], indicating the immunological impact on the neurogenesis in the developmental period [59]
During our investigation on NFκB signaling in initiating NSC differentiation, we surprisingly found that LTα1β2 induces potent activation of NFκB signaling in embryonic/neonatal NSCs, with similar potency to Tumor necrosis factor-α (TNFα) [19]
Summary
Lymphotoxin (LT) is a lymphokine mainly expressed in lymphocytes. LTα binds one or two membraneassociated LTβ to form LTα2β1 or LTα1β2 heterotrimers. The predominant LTα1β2 binds to LTβ receptor (LTβR) primarily expressed in epithelial and stromal cells. We investigated the role of LTβR-mediated NFκB signaling in regulating neural lineage differentiation. Neurogenesis involves the proliferation, migration, and lineage differentiation of neural stem/progenitor cells (NSCs/NPCs) during development and adult life. NSCs are characterized by their self-renewal and multipotent differentiation into various neural cell lineages, which depend upon the orchestral interplay between the intrinsic cellular properties and extrinsic environmental factors including immunity-related cytokines and chemokines [11, 12]. Little is known about the role of NFκB signaling in regulating neural differentiation of NSCs. Activation of NFκB by Toll-like receptor 2 (TLR2) induces neuronal differentiation whereas TLR4 activation inhibits neuronal differentiation of NSCs [17]. Our previous study showed that selective inhibition of the classical NFκB signaling retains the tripotent ability of differentiation and promotes self-renewal capability of NSCs, suggesting a role of NFκB signaling in mediating the differentiation of NSCs into NPCs at the very early stage [19]
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