Department of Neurosurgery, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010, China.
Center for Metabolic Disease Research, Department of Pathology and Laboratory Medicine, Temple University Lewis Katz School of Medicine, 3500 N Broad Street, Philadelphia, PA, 19140, USA.
J Neuroinflammation. 2018 Feb 20;15(1):49. doi: 10.1186/s12974-018-1074-z.
Lymphotoxin (LT) is a lymphokine mainly expressed in lymphocytes. LTα binds one or two membrane-associated LTβ to form LTαβ or LTαβ heterotrimers. The predominant LTαβ 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.
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.
In cultured NSCs/NPCs, LTαβ 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. LTαβ 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.
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.
淋巴毒素（LT）是一种主要在淋巴细胞中表达的淋巴因子。LTα 结合一个或两个膜相关的 LTβ 形成 LTαβ 或 LTαβ 三聚体。主要的 LTαβ 结合主要在上皮细胞和基质细胞中表达的 LTβ 受体（LTβR）。大多数关于 LTβR 信号的研究都集中在淋巴组织的组织、发育和维持上。然而，LTβR 信号在神经系统中的作用，特别是在神经发生中的作用尚不清楚。在这里，我们研究了 LTβR 介导的 NFκB 信号在调节神经谱系分化中的作用。
使用 C57BL/6J 野生型和 GFAP-dnIκBα 转基因小鼠。从小鼠胚胎干细胞中培养无血清类胚体，并进一步诱导为神经干细胞/祖细胞（NSCs/NPCs）。从小鼠胚胎和成年大脑中培养原代神经球，然后进行单层培养扩增/传代。通过腺病毒介导的 NFκB-萤火虫荧光素酶报告基因测定和 p65/RelB/p52 核易位测定来确定 NFκB 的激活。通过定量 RT-PCR 评估 LTβR mRNA 的表达，通过免疫组织化学和 Western blot 分析来确定 LTβR 蛋白的表达。进行多标记免疫细胞化学或免疫组织化学，然后通过荧光共聚焦显微镜和神经谱系分化的定量分析。使用 GraphPad Prism 软件进行绘图和统计分析。
在培养的 NSCs/NPCs 中，LTαβ 刺激诱导经典和非经典 NFκB 信号的激活。在成年小鼠大脑中已建立的神经发生区鉴定出 GFAP/Sox2 NSCs 中 LTβR 样免疫反应性的表达。定量 RT-PCR 和 Western blot 分析验证了培养的 NSCs/NPCs 和脑神经发生区中 LTβR 的表达。LTβR 的表达在神经诱导过程中显著增加。LTαβ 刺激培养的 NSCs/NPCs 促进星形胶质细胞和少突胶质细胞谱系分化，但抑制神经元谱系分化。GFAP-dnIκBα 转基因小鼠中的星形胶质细胞 NFκB 失活挽救了 LTβR 介导的培养 NSCs/NPCs 的异常表型。
本研究首次提供了 LTβR 信号在 NSCs/NPCs 中的表达和功能的证据。LTβR 信号的激活促进了神经胶质谱系的分化。我们的结果表明，神经发生受适应性免疫和炎症反应的调节。
