NF-κB and TNF Affect the Astrocytic Differentiation from Neural Stem Cells

Abstract

The NF-κB signaling pathway is crucial during development and inflammatory processes. We have previously shown that NF-κB activation induces dedifferentiation of astrocytes into neural progenitor cells (NPCs). Here, we provide evidence  that the NF-κB pathway plays also a fundamental role during the differentiation of NPCs into astrocytes. First, we show that the NF-κB pathway is essential to initiate astrocytic differentiation as its early inhibition induces NPC apoptosis and impedes their differentiation. Second, we demonstrate that persistent NF-κB activation affects NPC-derived astrocyte differentiation. Tumor necrosis factor (TNF)-treated NPCs show NF-κB activation, maintain their multipotential and proliferation properties, display persistent expression of immature markers and inhibit astrocyte markers. Third, we analyze the effect of  NF-κB activation on the main known astrocytic differentiation pathways, such as NOTCH and JAK-STAT. Our findings suggest that the NF-κB pathway plays a dual fundamental role during NPC differentiation into astrocytes: it promotes astrocyte specification, but its persistent activation impedes their differentiation.

Keywords: NF-κB; apoptosis; astrocyte; differentiation; neural progenitor cell; tumor necrosis factor.

Figure 1

Inhibition of the NF-kB pathway by JSH-23 obstructs the astrocytic differentiation via Caspase 3 cleavage. (A) JSH-23 (1, 5, 10, 20 mM) induces the loss of cell viability by MTT assay after a 48 h treatment (n = 3, mean ± SEM). (B) JSH-23 decreases the cell viability during the first step of astrocyte differentiation. JSH-23 (10 mM) was added at different time points during astrocytic differentiation (0, 24, 48, 72 h) and the MTT assay was performed after 96 h (n = 3, mean ± SEM). * p < 0.05 (C) Analysis of the expression levels of Caspase3 after treatment with JSH-23 at different concentrations by RT-PCR (6 h) (C, n = 5, mean ± SEM) and by Western blot (3 and 6 h) (D, n = 3). α-tubulin was used as a loading control. * p < 0.05 and ** p < 0.01 (D) TUNEL assay was performed to explore cell death induced by JSH-23 (10, 20 mM) at 3 and 6 h. Nuclei were stained with DAPI (blue) and TUNEL positive cells display green nuclei. Scale bar: 50 µm.

Figure 2

Snake plot. Top 25 and bottom 25 genes were extracted according to the log₂ fold change value and plotted on the y-axis. Each gene dot is filled with its q-value and using the viridis color gradient (viridis R package, GarnierSversion0.4.0. https://CRAN.Rproject.org/package=viridis, accessed on 5 April 2021). Gene symbols are reported next to each dot.

Figure 3

TNF tends to increase immaturity markers. Kinetic of Cd133 (A) and Cd44 (C) gene expression obtained by RT-PCR. Each time point is normalized to its 0 h of differentiation. (A) Effect of TNF treatment on Cd133 (A) and Cd44 (C) gene expression obtained by RT-PCR. Each time point is normalized to its FBS control (Ctrl = 1) * p < 0.05 and ** p < 0.01. Results are given as mean ± SEM (n = 4). (B) Immunocytochemistry showing CD133 (B) and CD44 (D) protein expression (green) in TNF treated cells at 6, 24, and 72 h. Nuclei were counterstained with DAPI (blue). Scale bar: 50 µm.

Figure 4

TNF modulates astrocytic differentiation through the classical Gfap marker. (A) Kinetic of Gfap mRNA gene expression obtained by RT-PCR. Each time point is normalized to its 6 h of differentiation. Results are given as mean ± SEM (n = 3) * p < 0.05 and ** p < 0.01. (B) Effect of TNF treatment on Gfap gene expression obtained by RT-PCR. Each time point is normalized to its FBS control (Ctrl = 1) ** p < 0.01. Results are given as mean ± SEM (n = 4). (C) Immunocytochemistry showing GFAP protein expression (red) in TNF treated cells at 6, 24, and 72 h. Nuclei were counterstained by DAPI (blue). Scale bar: 50 µm. (D) Immunoblots from TNF treated and untreated at 0, 24, and 72 h of differentiation. GFAP protein expression is affected by TNF, ** p < 0.01.

Figure 5

TNF increases neural progenitor cell proliferation. (A) Kinetic of mKi67 mRNA gene expression obtained by RT-PCR. Each time point is normalized to its 6 h of differentiation (Ctrl = 1). Results are given as mean ± SEM (n = 3) * p < 0.05, ** p < 0.01 and *** p < 0.001. (B) Effect of TNF treatment on mKi67 gene expression obtained by RT-PCR. Each time point is normalized to its FBS control (Ctrl = 1) * p < 0.05. Results are given as mean ± SEM (n = 4). (C) Immunocytochemistry showing KI67 protein expression (red) in TNF treated cells at 6, 24, and 72 h. Nuclei were counterstained by DAPI (blue). Scale bar: 50 µm. (D) TNFα induces the NSP reformation. After 6, 24, and 48 h of NSP-derived astrocyte differentiation treated with TNFα (50 ng/mL), an NSP reformation assay was performed in presence of EGF and FGF (20 ng/mL both, 10,000 cells/well). Ten days after, the number of reformed NSP at 6, 24, and 48 h was counted (n = 3, mean ± SEM).

Figure 6

Effect of NF-kB activation on the main astrocytic signaling pathways during NSPs differentiation into astrocytes. Effect of TNF treatment on Hes1 (A), Mash1 (B), and NeuroD1 (C) genes expression obtained by RT-PCR. Each time point is normalized to its FBS control (Ctrl = 1). Results are given as mean ± SEM (n = 4). Kinetic of Socs3 mRNA gene expression obtained by RT-PCR. Each time point is normalized to 6 h of differentiation. Results are given as mean ± SEM (n = 3) (D). Effect of TNF treatment on Socs3 gene expression obtained by RT-PCR. Results are given as mean ± SEM (n = 6) (E). * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 7

Proposed model for the role of NF-kB in the generation of astrocyte during neural stem cell differentiation.