CNTF And Nrf2 Are Co-ordinately Involved In Regulating Self-renewal And Differentiation Of Neural Stem Cell During Embryonic Neural Development

Aims/hypothesisThere is high risk of fetal neurodevelopmental defects in pregestational diabetes mellitus(PGDM).However,the effective mechanism of hyperglycemia-induced neurodevelopmental negative effects,including neural stem cell self-renewal and differentiation,still remains obscure.Neuropoietic cytokines have been shown to play a vital part during nervous system development and in the coordination of neurogenesis,gliogenesis.Nuclear factor-E2-related factor-2(Nrf2)dysfunction might be related to a reduction of self-protective response in brain malformation induced by hyperglycemia.We therefore evaluated the role of Nrf2 and neuropoietic cytokines in neurodevelopmental defects and determined the mechanisms involved.MethodsNeural stem cell self-renewal and differentiation were assessed in two mouse models: control,PGDM,and five cell models: 5.5 mmol/L d-glucose,5.5 mmol/L d-glucose + CNTF(ciliary neurotrophic factor),25 mmol/L d-glucose,25 mmol/L d-glucose + CNTF,25 mmol/L d-glucose + CNTF + ML385(Nrf2 inhibitor).Reactive oxygen species(ROS)production,the expression of Nrf2 and neuropoietic cytokines were evaluated by immunofluorescent staining,quantitative PCR and western blot.ResultsThe expression of neuronal markers(NF-M,Tuj1)was decreased and the expression of glial cell markers(Olig2,GFAP)was enhanced in E12.5,E15.5 and E18.5 PGDM mouse neural tubes compared with control.Neural stem cell self-renewal in PGDM E15.5 and E18.5 neural tube was suppressed,which was also validated by primary culture of neurosphere assay and the hyperglycemia-induced reduction of neurosphere frequency-of-occurrence was partially rescued in presence of CNTF.Neurosphere assay data indicated that hyperglycemia increased ROS production and Nrf2 expression in neural stem cells,while the expression of Nrf2,HO1,SOD2,NQO1 and p62 was upregulated in PGDM E18.5 neural tubes.In cell culture assay,hyperglycemia inhibited NF-M expression and promoted Olig2 & GFAP expression in differentiated cells,resulting in an imbalance between neurons and gliocytes;on the contrary,CNTF addition upregulated NF-M expression,and suppressed Olig2 & GFAP expression in differentiated cells;blocking Nrf2 transcription activity with ML385 could intensify the imbalance in presence of hyperglycemia and CNTF,and this aggravating effect was not because of interfering with CNTF expression,although STAT3 signaling pathway was also observed to change spontaneously when Nrf2 inhibitor was applied.Conclusions/interpretationTaken together,our data reveal that PGDM dramatically impairs the developmental switch of neural stem cells from neurogenesis to gliogenesis,principally under the cooperative mediation of neuropoietic cytokine CNTF and Nrf2 antioxidative signaling.