Abstract
The hippocampus is an important region in the brain, responsible for learning, memory, and emotion. The hippocampus is composed of abundant neuronal cells, of which maturation is critical to physiological function and neural disease occurrence. Although the factors affecting neuronal maturation in the hippocampus has been widely studied, the specific mechanism involved in this process is still elusive to us. In the current study, Stat3 silencing and overexpression was achieved through lentivirus and adenovirus system. We found that hippocampal neuronal maturation was enhanced when Stat3 was downregulated. By contrast, formation of neurosphreres was observed in hippocampal cultures due to the overexpression of Stat3. In addition, these neuropheres had the capacity to differentiate into different cell subtypes, indicating the acquisition of multipotency when Stat3 was overexpressed in hippocampal cells. These processes were correlated with MAPK signaling, indicating the potential linkage among Stat3 expression, MAPK activation, and neuronal maturation. Above all, this study demonstrated the role of Stat3 in hippocampal neuronal maturation and differentiation. Also, the molecular mechanism was explored through the MAPK signal manipulation.
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Acknowledgments
This work was supported by the Heilongjiang province government postdoctoral scientific initial research funds(LBH-Q15091) and the foundation of the Fourth Affiliated Hospital of Harbin Medical University Outstanding Youth Project.
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Supplementary Figure 1
Expression change of Stat3 in the presence of Stat3-shRNA in hippocampal neurons. a Stat3 expression (red) was significantly decreased in Stat3-shRNA-infected neurons, while the DAPI (green) worked as an internal control. b Western blotting indicated the decrease in Stat3 and Tuj1 expression as well as increase in MAP2 expression. The statistical significance was determined by unpaired Student’s t-tests. **p < 0.01, ***p < 0.001. Scale bar was 10 μm. (DOCX 135 kb)
Supplementary Figure 2
Stat3 downregulation did not affect the phosphorylation of Stat3. a Western blotting showed that infection with lenti-Stat3-shRNA reduced the expression level of Stat3, but not the phospho-Stat3 at Ser727 and Tyr705. b Statistical analysis of the expression intensity of Stat3, p-Stat3 (Tyr705) and p-Stat3 (Ser727). Statistical significance was determined by unpaired Student’s t-tests. ‘ns’ represented not significant. (DOCX 56 kb)
Supplementary Figure 3
Stat3 downregulation did not alter the cell death and proliferation. a Neurons infected with lenti-Stat3-shRNA did not show a significant difference in ratio of TUNEL-positive cells compared to that of the neurons infected with control virus. b Ratio of BrdU-positive cells was not significantly changed after Stat3 silencing in hippocampal neurons. Statistical significance was determined by unpaired Student’s t-tests. ‘ns’ represented not significant. (DOCX 25 kb)
Supplementary Figure 4
MAPK inhibitor or activator altered the phosphorylation of Stat3 and downstream effectors. a MAPK inhibitor U0126 decreased the phosphorylation level of Stat3 at Ser727 in Stat3-downregulated neurons. b MAPK activator anisomycin increased the phosphorylation level of Stat3 at Ser727 and ERK1/2, but not p38 and JNK in Stat3-overexpressed neurons. Statistical significance was determined by unpaired Student’s t-tests. ‘ns’ represented not significant, *p < 0.05. (DOCX 50 kb)
Supplementary Figure 5
Effect of ERK1/2 activator Icaritin on the maturation of hippocampal neurons. a Real-time PCR analysis showed that lcaritin treatment increased the expression of MAP2, and decreased the expression of Tuj1 in Stat3-overexpressed neurons. Icaritin significantly increased the ratio of neurite bearing cells b and neurite length c in Stat3-overexpressed neurons. by unpaired Student’s t-tests. *p < 0.05, **p < 0.01. (DOCX 49 kb)
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Ma, X., Zhou, Y., Chai, Y. et al. Stat3 Controls Maturation and Terminal Differentiation in Mouse Hippocampal Neurons. J Mol Neurosci 61, 88–95 (2017). https://doi.org/10.1007/s12031-016-0820-x
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DOI: https://doi.org/10.1007/s12031-016-0820-x