The Role Of GSK3 In The Development Of Neural Stem Cells

GSK3 is a serine protein kinase that plays a central role in a variety of intracellular signaling pathways,including activation by Wnt/β-catenin,sonic hedgehog,Notch,growth factor/receptor tyrosine kinase and G protein-coupled receptors.In mammals,GSK3 is encoded by two homologous genes,GSK3αand GSK3β,whose amino acid sequences within the kinase domain are about 90%identical.Previous studies have shown that GSK3αand GSK3βhave overlapping functions in regulating Wnt/β-catenin signaling pathway.On the other hand,knockout of GSK3αand GSK3βproduced different phenotypes in mice.Although GSK3βknockdown results in embryonic lethality,GSK3αknockout mice are alive and have only minor defects in hepatic glycogen metabolism.These results indicate that the two GSK3 isozymes have a non-redundant role in the development process,but the basis is not clear.GSK3 is an attractive drug target because the dysregulation of GSK3 activity is related to the pathogenesis of various diseases.The importance of GSK-3 for various aspects of neurodevelopment,including neural progenitor self-renewal,neurogenesis,neuronal migration,and neural differentiation.However,the role of individual GSK3isoenzymes in the pathogenesis of these diseases remains unclear.Previous studies in our laboratory have shown that CHIR as a non-selective inhibitor can promote long-term self-renewal of embryonic-derived neural stem cells in vitro.By using Sox1 as a marker for naive mouse NSCs,a combination of growth factors and different small molecules was screened and found that NSCs can stably self-renew by activating the bFGF signaling pathway and binding to the GSK-3 inhibitor CHIR99021.These NSCs can be amplified from individual cell clones.More importantly,compared to previous neural stem cell culture protocols,this laboratory culture protocol retains high-efficiency neuronal differentiation and exhibits specificity for the forebrain and midbrain regions.Although the embryonic-derived neural stem cells cultured by this protocol have strong cell proliferation ability,some neural stem cells may still differentiate,and the expression is negative in Sox1.This study envisages a protocol that can maintain Sox1-positive neural stem cells more stably and efficiently.Our laboratory previously applied the Chemical-Genetic method and found that in mouse embryonic stem cells,selective inhibition of GSK3βis sufficient to maintain mouse ESC self-renewal,while GSK3αinhibition promotes mouse ESC differentiation into the neural lineage.It is suggested that genome-wide transcriptional analysis shows that GSK3αand GSK3βhave different downstream target groups.Therefore,this experiment aims to investigate the role of inhibition of two isoenzymes in the self-renew and differentiation of neural stem cells and to find more effective neural stem cell differentiation and culture protocols.Since there is currently no small molecule inhibitor that can well distinguish between two isozymes.Understanding the mechanisms by which GSK3αand GSK3βdifferentially regulate cellular processes can establish more efficient in vitro culture of neural stem cells and promote the development of more specific drugs to target a single GSK3 isoenzyme for the treatment of GSK3 activity disorders.a disease.Studies of GSK3 isoenzymes by gene knockout methods can lead to very different results.Because gene knockouts can alter protein structure,leading to bias in research results.Our previous study found that in mouse embryonic stem cells,GSK3αor GSK3βwas knocked out by gene knockout,and the other could replace each other’s functions.If GSK3αor GSK3βalone is inhibited by a novel chemical-genetic method by gene knockout,since the other party does not lose its functional structure,the other part cannot replace its function,thereby realizing different mechanisms of action of two isozymes of GSK3.Here,we show that GSK3αand GSK3βcan be selectively inhibited using the chemical-genetic method.Part Ⅰ Inhibits the role of Gsk3αand GSK3βin the development of neuralstem cellsObjective:To study the role of non-selective inhibition and selective inhibition in the development of neural stem cellsMethods:ES cells were diferentiate into Sox1^GFP-positive neural stem cells using a single-layer differentiation protocol,and neural stem cells were cultured and identified in vitro for a long time.Non-selective inhibition and selective inhibition of CHIR in neural stem cells were used to investigate the role of GSK3 in neural stem cell development,and to compare the different mechanisms of non-selective inhibition and selective inhibition.Results:1.Mouse ES cells were seeded in gelatin-coated 6-well plates in mESC medium.The LIF was removed the next day and the LIF-free medium was replaced every 2 days.After 4 days,Sox1^GFP-positive neural stem cells were observed under a fluorescence microscope.It reached its peak after 6 days.At this stage,neural stem cells can be cultured in vitro with ABC（cocktail）in N2B27 medium and kept self-renewing.During the screening with puromycin for 1 week,more than 99%of Sox1^GFP-positive neural stem cells can be obtained.2.The expression level of NSC-specific molecular markers was examined by immunostaining and qPCR.In addition to Sox1,other NSC-specific markers（including Sox2,Nestin,and Pax6）are expressed in long-term self-renewing NSCs,and these cells also show positive staining for some rosette-specific markers,including ZO1,PLZF.The results confirmed that under our culture conditions,neural stem cells are in the Rosettes phase and maintain stable self-renewal.3.Neural stem cells are seeded into the laminin-coated 6-well plate in N2B27medium,ABC is removed,half of the medium is replaced every 2 days,and the neural stem cells will differentiate into neuron cells with neurites within 7-10 days.There will be very few astrocytes.Under this culture condition,99%of neural stem cells differentiated into TUJ1-positive（immature neurons）and 1%differentiated into astrocytes.4.In the presence of bFGF,non-selective inhibition of GSK3 by CHIR can significantly increase cell proliferation of neural stem cells.At the same time,the positive proportion of Sox1 in neural stem cells in long-term culture is improved,and the neural stem cells are maintained at an early stage.5.In the presence of bFGF,global inhibition of Gsk3αand GSK3βsignificantly increased NSC proliferation compared with inhibition alone.Inhibition of GSK3βalone is more pronounced in terms of increasing cell proliferation than inhibition of Gsk3αalone.Inhibition of GSK3βalone can increase the positive proportion of Sox1 in neural stem cells compared with CHIR,and it can be stably maintained in long-term culture.However,inhibition of Gsk3αalone has no obvious effect.6.In the case of bFGF withdrawal,inhibition of GSK3βalone promoted neurite formation in NSC,and CHIR inhibition of GSK3 inhibited axonal formation.Inhibition of GSK3βalone promotes neural differentiation of NSCs and is more likely to differentiate into Neuron.Part Ⅱ Overexpression of GSK3αand GSK3βin the development of neuralstem cellsObjective:To study the role of overexpression of GSK3αand GSK3βin the development of neural stem cellsMethods:The neural stem cells transfected with the expression plasmid were cultured in N2B27 medium containing ABC cocktail using the neural stem cell induction and culture protocol mentioned in the first part.Inoculate neural stem cells into a laminin-coated6-well plate.Immunofluorescence was performed 2 days after the addition of DOX.Selective overexpression of GSK3αand GSK3β,respectively,to explore its role in neural stem cell development.Results:1.GSK3αand GSK3βwere added to DOX and differentiated in N2B27 for 3 days.The cells were then subjected to TUNEL assay.The number of TUNEL-positive cells was comparable between the control group and the GSK3αand GSK3βgroups,indicating that apoptosis was not significantly affected.FACS analysis examined the effect of GSK3isoforms on NSC proliferation.In the control group,the GSK3αand GSK3βgroups were added to DOX for 2 days,and the changes in the number of RFP+NSC were measured.In all three groups,the number of RFP+cells on the second day was doubled to a similar level,and there was no significant difference between the three groups.These data indicate that GSK3 does not affect the proliferative potential of NSC.Overexpression of GSK3αand GSK3β,respectively,had no effect on neural stem cell proliferation and apoptosis.2.Overexpression of GSK3βinhibits NSCs neural differentiation,while overexpression of GSK3αpromotes NSCs neural differentiation to some extent.Selective overexpression of GSK3βinhibits NSCs neural differentiation.Such as increasing Nestin expression,reducing the expression of TuJ1.In cells transduced with only control vectors expressing RFP,⁵0%of RFP positive cells were positive for TuJ1.Higher levels of TuJ1positive cells were observed in GSK3αtransduced cells at 61%,whereas in GSK3βtransduced cells the levels were reduced to 20%.Similar to the control,approximately 38%of GSK3αtransduced cells were positive for Nestin.However,overexpression of GSK3βincreased the percentage of Nestin positive cells to 75%..We found that overexpression of GSK3αin neural stem cells has a reverse trend relative to overexpression of GSK3β.Although this difference is not as obvious as overexpression of GSK3β,we still observed that overexpression of GSK3αincreased the expression of TuJ1（P<0.01）.3.Overexpression of GSK3βinhibits neural differentiation and promotes the differentiation of“rosette”cells into flat cells.Immunofluorescence staining showed its characteristic P75.We used differential digestion to separate the two forms of cells.qRT-PCR suggested that the squamous cells were neural crest cells.Overexpression of GSK3βpromotes the differentiation of NSCs into neural crest cells.Conclusion:Selective inhibition of GSK3βbut not GSK3αcan promote neural differentiation of neural stem cells.Overexpression of GSK3βcan inhibit the differentiation of neural stem cells and promote the differentiation of neural crest cells.Future development of small molecule drugs targeting GSK3βcan improve self-renewal of early neural stem cells in vitro.Our study opens up new avenues for defining GSK3 isoenzyme-specific functions in various cellular processes and demonstrates the different roles of GSK3 in neurodevelopmental processes.