TNFα-induced Up-regulation of Ascl2 Affects the Differentiation and Proliferation of Neural Stem Cells - PubMed (original) (raw)
TNFα-induced Up-regulation of Ascl2 Affects the Differentiation and Proliferation of Neural Stem Cells
Zhongfeng Liu et al. Aging Dis. 2019.
Abstract
The molecular mediators underlying the effects of inflammation on neural stem cells (NSCs) are not fully characterized. In this study, we identified Ascl2 as a downstream basic helix-loop-helix (bHLH) transcription factor in NSCs following exposure to TNFα. Under normal conditions, Ascl2 expression is inhibited at post-transcriptional levels by miR-26a, which targets the 3' untranslated region (UTR) of Ascl2. Upon exposure to TNFα, miR-26a expression is reduced, which leads to up-regulation of Ascl2. Overexpression of Ascl2 promotes neuronal differentiation, reduces proliferation, and increases the level of cleaved CASPASE 3 in NSCs, as observed in the in vitro and in ovo experiments. Ascl2 may serve in NSCs as a standby factor that readily responds to TNFα, which is often induced in inflammatory situations. In a chronic inflammatory condition with consistent up-regulation of TNFα, overexpression of Ascl2 may inhibit neurogenesis as a net result.
Keywords: Ascl2; TNFα; in ovo; miR-26a; neural stem cells.
Copyright: © 2019 Liu et al.
Conflict of interest statement
Conflict of interest The authors declare no competing financial interests.
Figures
Figure 1.
Exposure to TNFα up-regulates Ascl2 expression in neural stem cells (NSCs). (A-B) Mouse NSCs were treated with different concentrations of TNFα in proliferation (A) and differentiation (B) medium for 48 h, and Ascl2 mRNA expression levels were examined. The results were normalized to NSCs without TNFα. See also Figure S2. Pro: proliferation, Dif: differentiation. (n=3). (C-D) NSCs were treated with 20 ng/ml TNFα for various lengths of time in proliferation (C) and differentiation (D) medium, and Ascl2 mRNA expression levels were examined. The results were normalized to NSCs in differentiation medium without TNFα for 5 h. (n=3). (E-F) NSCs were treated with TNFα of different doses for 48 h in proliferation (E) and differentiation (F) medium, and the protein levels of ASCL2 were examined. Data in (A), (B), (C) and (D) are represented as the means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 by one-way analysis of variance.
Figure 2.
MiR-26a regulates Ascl2 expression. (A) The alignments of 3’UTR of Ascl2 and miR-26a/b as predicted by TargetScan analysis. The sequences show a high level of complementarity (indicated by vertical bars) and sequence conservation between mice and rats. (B) Expression levels of different miR-26 family members following TNFα treatment for 5 h in NSCs. The results were normalized to NC. (n=3). (C) NIH3T3 cells were transfected with empty control vectors, Ascl2 3’UTR luciferase construct or Ascl2 3’UTR mutation luciferase construct together with miR-26a-5p mimics. Forty-eight hours following transfection, cells were collected and lysed. Firefly luciferase activities were examined and normalized to Renilla luciferase activities. (n=6). Similar results were obtained in 293T cell line as shown in Figure S4C. The results were normalized to vector + NC. (D) NSCs were transfected with miR-26a-5p mimics or miR-26a-5p inhibitor with or without TNFα for 48 h, and ASCL2 protein levels were examined. Control means NSCs culture in proliferation medium without TNFα or MicroRNA. (n=3). Data in (A), (C) and (D) are represented as the means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 by Student’s t test for comparison (B) and one-way analysis of variance (C) and (D).
Figure 3.
Ascl2 affects the differentiation of NSCs in vitro. (A) Monolayer murine NSCs were infected with retrovirus encoding pBMN-_Ascl2_-GFP or pBMN-GFP, which were then subjected to a differentiation or proliferation condition for 1, 2 and 4 days. Early neuronal markers TUJ-1 and DCX, and mature neuron marker NEUN were stained. Pro, proliferation; Dif, differentiation. Scale bars, 50 μm. (B-D) The proportions of TUJ-1-, DCX- and NEUN-positive cells among GFP+ cells were scored. (n=10-18). Data in (B-D) are represented as the means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 by Student’s t test for comparison.
Figure 4.
The impact of Ascl2 overexpression on the proliferation of NSCs. (A) Schematic representation of the experimental procedures. (B) The flow cytometer-sorted single cells were cultured in a proliferation medium to test the ability to form neurospheres. Representative pictures 5 days after flow cytometer sorting were shown. (C) The sphere sizes were measured from day 2 through day 7 after flow cytometer sorting. (n=17-47). (D) Cell cycle progression in proliferation condition was analyzed by PI staining and flow cytometry. (n=3). (E-F) The proliferative capacity was tested by BrdU pulsing for 1 h and immunostaining 14 h later. The proportions of BrdU+ and DCX+ cells among GFP+ cells were scored respectively. (n=3). (G) Co-labeling of BrdU with DCX. Scale bars, 100 μm. Data in (C), (D), (E) and (F) are represented as the means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 by Student’s t test for comparison.
Figure 5.
Overexpression of Ascl2 induces expression of cleaved CASPASE 3 in neurospheres. (A) NSCs infected with retrovirus encoding Ascl2 and GFP or GFP only were sorted by using flow cytometry, 24 h later, the spheres were immunostained for cleaved CASPASE 3 and DCX. Scale bars, 50 μm. (B-C) The cells positive for cleaved CASPASE 3 and DCX were quantified respectively. (n=9-11). (D) The proportion of cleaved CASPASE 3-positive cells co-expressing DCX was scored. (n=9-11). Data in (B), (C) and (D) are represented as the means ± SEM. **p < 0.01; ***p < 0.001 by Student’s t test for comparison.
Figure 6.
Overexpression of Ascl2 induces expression of cleaved CASPASE 3 in ovo. (A) PCIG-_Ascl2_-GFP vectors and the control vectors pCIG-GFP were electroporated into chicken embryo neural tubes, 24 h and 48 h later, the chicken embryos were fixed and sliced for staining of cleaved CASPASE 3 and DCX. Scale bars, 200 μm. (B) The proportions of GFP+ cells co-expressing DCX were scored 24 h and 48 h after electroporation. (n=6). (C) The proportions of cleaved CASPASE3-positive cells co-expressing DCX were scored 24 h and 48 h after electroporation. (n=6). (D) The proportions of cleaved CASPASE3-positive cells that were GFP-positive were scored 24 h and 48 h after electroporation. (n=6). (E) The proportions of DCX-positive cells that co-expressed cleaved CASPASE3 were scored 24 h and 48 h after electroporation. (n=6). (F) Distribution of GFP+ cells in the neural tubes. (n=6). Data in (B), (C), (D) and (E) are represented as the means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 7.
The impact of Ascl2 overexpression on differentiation _in ov_o. Related to Figure S6. (A) Expression of neural stem cell marker SOX2 and mature neuron marker NEUN at 48 h post-electroporation. (B) Rate of NEUN-positive cells of genetically modified (GM) side over non-genetically modified (non-GM) side 48 h post-electroporation. (n=6). (C) The proportion of NEUN- and GFP-double positive cells in GFP+ cells 48 h post-electroporation. (n=6). (D) The proportion of SOX2- and GFP-double positive cells in GFP+ cells 48 h post-electroporation. (n=6). Data in (B), (C) and (D) are represented as the means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 by Student’s t test for comparison.
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