| BackgroundAlzheimer’s disease(AD)is characterized by neurodegeneration of the cerebral cortex,hippocampus and other subcortical structures,and massive cell death in the frontal and temporal cortices,ultimately leading to memory and cognitive dysfunction.Therefore,an in-depth investigation of the molecular mechanisms that delay neuronal death or increase neurogenesis will provide new strategies for the prevention and treatment of AD.Previous studies by our group found that the deacetylase SIRT3 induces neural stem cells to differentiate into neurons in an inflammatory environment caused by Aβ activation of microglia,and that glycogen synthase kinase 3β(GSK3β)protein expression is downregulated and p-GSK3βexpression is increased during this process,suggesting that GSK3 may be involved in GSK3 is a serine/threonine kinase involved in a variety of cellular processes and has two isoforms,GSK3α and GSK3β,both of which play different but overlapping functions in many biological processes.Most current studies emphasize the role of GSK3β in cellular functions and diseases,while underestimating the function of GSK3α.ObjectivesTo investigate the possible involvement of GSK3 isoenzymes in mediating AD neurogenesis and regulating the biological functions of neural stem cells,with the aim of elucidating the different roles of GSK3 isoenzymes in regulating AD neurogenesis,and also to provide an experimental basis for the function of GSK3α in neurogenesis.MethodsA Transwell system was constructed to co-culture neural stem cells C17.2 and microglia BV.2.C17.2 cells in the co-culture model were subjected to GSK3α/βoverexpression and its gene silencing to up-or down-regulate GSK3α/β expression,and CCK8,EdU,Western blot and immunofluorescence techniques were used to explore the effects of GSK3α/β on NSC proliferation,differentiation and apoptosis.Results1.GSK3α and GSK3β inhibit the proliferation and apoptosis of NSC in co-culture modelCCK8,EdU and flow cytometry results showed that compared with the blank control,the microenvironment of microglia activated by Aβ led to decreased NSC proliferation and promoted apoptosis;compared with the co-culture model group,overexpression of GSK3α/β further inhibited NSC proliferation and promoted NSC apoptosis in the co-culture model;while inhibition of GSK3α/β promoted NSC proliferation and inhibited its apoptosis.Among them,the effect of GSK3α on NSC proliferation and apoptosis in the co-culture model was more significant compared with GSK3β.2.GSK3α and GSK3β inhibited NSC differentiation in the co-culture modelThe results of immunofluorescence staining and Western blot showed that the microenvironment of microglia activated by Aβ inhibited the differentiation of NSC to neurons and promoted their differentiation to glial cells compared with the blank control;compared with the co-culture model group,overexpression of GSK3α/βinhibited the differentiation of NSC to neurons in the co-culture model,while glial cell differentiation was not affected.Silencing GSK3α/β promoted NSC differentiation to neurons and inhibited their differentiation to glial cells in the co-culture model.Among them,the role of GSK3β in regulating NSC differentiation was more significant compared with that of GSK3α.ConclusionIn summary,overexpression of GSK3 isoenzymes inhibits NSC proliferation and promotes NSC apoptosis in an inflammatory environment caused by microglia activation by Aβ.Among them,GSK3α had a more significant regulatory role in NSC proliferation and apoptosis compared with GSK3β;in terms of NSC differentiation,silencing GSK3α/β promoted NSC differentiation to neurons in a co-culture model while inhibiting their differentiation to glial cells.Among them,GSK3β plays a dominant role in regulating NSC differentiation.In this study,we preliminarily investigated that GSK3α and GSK3β differentially regulate the developmental process of NSC in AD,and provided experimental and theoretical support for the future development of more specific drugs selectively targeting GSK3 isoenzymes. |
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