The Effects And Mechanisms Of TOX3 And TIGAR On The Proliferation And Differentiation Of Neural Stem Cells

Neural stem cells(NSCs)have the potential to self-renew and differentiate into neurons,astrocytes and oligodendrocytes.NSCs can generate different types of nerve cells in the course of normal physiological activity in both developing and adult mammalian brains to guarantee the integrity and precision of neural connection and cerebral function.The abnormal development of NSCs in embryonic stage will lead to many birth defects of newborns,including neural tube malformation,microcephaly,etc.,which will bring heavy burden to family and society.In addition,NSCs can differentiate into different types of neurons under specific conditions and factors,which provides a new treatment for neurodegenerative diseases(such as Parkinson's disease,Alzheimer's disease)and nerve injury(such as brain inj ury,cerebral ischemic disease).Therefore,the study of the regulation mechanism of proliferation and differentiation of NSCs is the key to reveal the pathogenesis of common diseases of central nervous system development defects,and also provides new ideas for the treatment of some neurological disorders.Our laboratory has been devoted to the establishment of embryonic development defect disease model and the study of pathogenesis.Previous work screened the protein TOX3 containing the HMG-box domain,which belongs to the TOX(thymocyte selection-associated HMG-box)family.TOX3 is highly expressed in the brain,but there are few studies on the development of nervous system.In addition,the p53-induced glycolysis inhibitor TIGAR(TP53-induced glycolysis and apoptosis regulator)has also aroused our great interest.TIGAR is widely distributed in neurons and can rescue neuron injury induced by ischemia/reperfusion in ischemic stroke model.However,the role of TIGAR in NSCs during embryonic development remains unclear.This study focused the above two key scientific issues.In the first part of this paper,we investigated the roles and mechanisms of TOX3 in the proliferation and differentiation of NSCs during the development of cerebral cortex by using gene knockout mice.In the second part,the role and mechanism of glycolysis inhibitor TIGAR in regulating the differentiation of NSCs were discussed based on the relationship between energy metabolism and the development of NSCs.Part ? The role and mechanism of TOX3 in the regulation of neural stem cell proliferation and differentiation BackgroundThe formation of cerebral cortex is a highly ordered and strictly regulated development process,involving the production of various nerve cells and the formation of nerve networks.The proliferation,differentiation,migration and stratification of NSCs are the cytological basis of cerebral cortex development.Therefore,it is important to understand the potential molecular mechanism of proliferation and differentiation of NSCs.However,the underlying molecular mechanisms regulating neurogenesis in the cerebral cortex are not yet fully understood.The transcription factor TOX3 is a nuclear protein containing nuclear localization signal(NLS)and HMG-box domain.It belongs to the TOX family and has four members:TOX1,TOX2,TOX3 and TOX4.The members of the family are highly conservative.TOX3 contains specific CAG repeats,and CAG repeats are associated with some neurological diseases such as schizophrenia,autism,fragile X syndrome,suggesting that TOX3 may play an important role in the nervous system.As a calcium-dependent transcription factor,TOX3 is reported to be highly expressed in the brain and participate in many biological processes,including neuron survival,tumorigenesis and transcriptional regulation.However,the detailed role of TOX3 in the development of cerebral cortex in vivo remains unclear.In this study,we explored the exact role and epigenetic mechanism of transcription factor TOX3 in the development of cerebral cortex from the perspective of proliferation and differentiation of NSCs by using TOX3 knockout mouse.The regulatory network of transcription factors and epigenetic modification in the development of cerebral cortex was established to provide theoretical guidance for the pathogenesis of common developmental defects of central nervous system in clinic.Objective1.Detect the expression patterns of TOX3 in the development of embryonic cerebral cortex,and clarify the role of TOX3 in the development of cerebral cortex.2.Explore the role of TOX3 in the proliferation and differentiation of neural stem cells.3.Clarify the mechanism of TOX3 in regulating the development of cerebral cortex at the level of neural stem cells.Results?.The expression patternof TOX3 in embryonic brainThe temporal expression patterns of TOX3 mRNA/protein were detected by qPCR and Western blot in cerebral cortex at different stages(E10.5 to El 8.5).The results showed that TOX3 was highly expressed in the cortex,and the expression of TOX3 increased significantly during the peak period of proliferation and differentiation of NSCs(E12.5 and E14.5).Different tissues of mice on P1 were taken and the expression profiles of TOX3 mRNA/protein were detected by qPCR and Western blot.The results showed that TOX3 was highly expressed in the cortex.?.The role of TOX3 in the development of cerebral cortex1.The construction and phenotypic observation of TOX3 knockout miceIn order to explore the roles of TOX3 in embryonic brain development,TOX3 knockout mice were constructed.qPCR and Western blot showed that the knockout efficiency of TOX3 knockout mice was 80%.TOX3 heterozygote mice could survive and have normal body shape and fertility,while the homozygote mice had smaller body size and died within one day to one week after birth.On the first day after birth,body weight and brain weight was reduced in knockout mice.2.TOX3 knockout leads to thinning of cerebral cortexHistological staining and statistical analysis showed that the thickness of cortex was thinner in TOX3 knockout mice than the WT mice.Immunofluorescence staining was performed with different specific markers of cortical neurons,including Satb2(layer II-IV),Ctip2(layer V)and Tbrl(layer VI).The results showed that the number of TBR1+,Ctip2+ and Satb2+ neurons in TOX3 KO mice decreased significantly compared with WT.These results suggest that TOX3 is essential for brain development.?.The effect of TOX3 deletion on the proliferation and differentiation of neural stem cells1.Loss of TOX3 results in a decrease in the number of cortical neural stem cellsTo test whether the decreased number of cortical neurons could be caused by reduced numbers of NPCs in the TOX3 KO brains,we used Pax6 and Tbr2 to examine NSCs and INPs,respectively,in the VZ/SVZ of the developing cerebral cortex.The results showed that the number of Pax6+ NSCs and Tbr2+ INP were significantly decreased in the cortical sections of KO mice than WT mice at both E17.5 and P1.2.Loss of TOX3 does not impair cell survivalTUNEL and active caspase-3 staining were used to detect late apoptosis and early apoptosis respectively.The results showed that there was no significant difference in the number of TUNEL positive cells and active caspase-3 positive cells in the cerebral cortex of WT and TOX3 KO mice at E15.5,E17.5 and P1.3.Loss of TOX3 results in reduced proliferation of cortical neural stem cellsThe proliferation marker Ki67 was used for fluorescence staining,which could label cells in all cell cycles except Go.Compared with WT,the number of Ki67+cells decreased significantly in TOX3 KO mice at E15.5.EdU labeling assay was used to detect the number of S phase cells.After 2 hours of EdU labeling,the number of cells labeled with EdU was significantly reduced in TOX3 KO mice compared with WT.Phosphorylated histone 3(PHH3)labeled mitotic proliferating cells.PHH3 labeled cells in the VZ/SVZ of TOX3 KO mice decreased significantly at E15.5.4.Loss of TOX3 results in enhanced cell cycle exit of cortical neural stem cells24-h EdU labeling and Ki67 co-staining were used to detect the cell cycle exit of E15.5 and E16.5.Immunofluorescence results showed that TOX3 knockout resulted in a significant increase in the number of EdU+/Ki67-cells exiting cell cycle and a decrease in the number of EdU+/Ki67+ cells in the cell cycle.The expression of cyclin-dependent kinase inhibitors(CKIs)such as p16,p21,p27 and p57 was detected by qPCR.The expression of P57 in the cortex of TOX3 KO mice increased significantly compared with WT.The protein expression levels of Tuj 1 and Pax6 in the cerebral cortex of TOX3 KO mice were also decreased.At the same time,qPCR results showed that TOX3 KO resulted in the decrease of SOX2,MAP2 and neuronal differentiation-related transcription factors Ngnl and NeuroDl.?.Loss of TOX3 impaired NSCs proliferation and differentiation in vitro.NSCs were isolated from the forebrain of E12.5 TOX3 KO and WT fetal mice.Comparing WT NSCs,the size of neurospheres were smaller in TOX3 KO NSCs.MTT assay showed that TOX3 KO reduced NSCs cell viability.The expression of SOX2,Nestin and Ki67 in TOX3 KO NSCs decreased significantly.EdU incorporation assay showed that the proliferation of TOX3 KO NSCs was significantly reduced,and TUNEL staining showed no significant difference in apoptosis.Western blot and immunofluorescence showed that Tujl,a neuronal differentiation marker,was significantly decreased in the TOX3 KO group.qPCR results showed that the expression levels of MAP2,NeuroD1 and Ngn1 decreased significantly.?.TOX3 regulates gene transcription by the acetylation of Histone H3.1.Loss of TOX3 leads to increased heterochromatinChromatin structure is closely related to transcriptional regulation.Transmission electron microscopy(TEM)was used to detect the effect TOX3 on the nuclear chromatin structure in the E14.5 cerebral cortex and NSCs cultured in vitro.TEM showed an increase in heterochromatin in TOX3 KO mice or NSCs.2.Loss of TOX3 leads to decreased H3K9ac levels.Histone acetylation modification is one of the important regulatory mechanisms of chromatin structure.Western blot analysis showed that there was no significant difference in the expression of H3K27ac,H3K14ac,H3K56ac and H3K18ac in TOX3 KO mice,but the expression of H3K9ac decreased significantly.Chromatin immunoprecipitation(ChIP)combined with qPCR analysis showed a decrease of H3K9ac at the Ngn1,NeuroD1 and Tuj1 promoters after TOX3 knockout.These data suggest that TOX3 knockout may inhibit the expression of genes related to neuronal differentiation by H3K9ac.?.TOX3 directly binds to the promoter of GCN5 and regulates its transcription.1.Loss of TOX3 leads to decreased GCN5 levels.Histone acetylation is related to histone acetyltransferase(HATs).The changes of HATs related to H3K9ac were analyzed by qPCR.The results showed that GCN5 mRNA level decreased significantly,while HATs such as CBP,P300,ACTR and SRC-1 did not change significantly.Western blot results showed that the protein levels of GCN5 also decreased.2.TOX3 directly binds to the promoter of GCN5 and regulates its transcription.To determine whether the expression of GCN5 is directly regulated by TOX3,ChIP-qPCR and luciferase reporter assays were performed.ChIP-qPCR results showed that TOX3 binds to the promoter of GCN5 and Nestin is a positive control.The promoter region of GCN5 was linked to the plasmid of luciferase reporter gene.TOX3 was found to activate the transcription of GCN5 gene by luciferase reporter assay.Conclusion and innovation1.The present study confirms the role of TOX3 in the development of embryonic cerebral cortex.TOX3 knockout mice exhibited the phenotype of microcephaly,such as reduced brain volume,thinned cerebral cortex and disordered structure.It provides a new model for the study of pathogenesis of some common developmental defects of the central nervous system.2.TOX3 knockout reduced the proliferation and differentiation of NSCs.The present study further clarified that the normal process of proliferation and differentiation of NSCs is the basis of cerebral cortex development.Therefore,the study of the regulation mechanism of NSCs development is the key to reveal the pathogenesis of congenital malformations caused by some developmental defects of the central nervous system.3.In the mechanism study,we found that TOX3 knockout can inhibit the transcription of NSCs differentiation-related transcription factors NeuroDl and Ngnl by reducing the acetylation level at H3K9,and TOX3 can regulate the expression of GCN5.4.It is the first time to find that TOX3 can participate in the development of cerebral cortex and the proliferation and differentiation of NSCs through epigenetic regulation mechanism,which provides a new idea for the study of the role and mechanism of TOX3 in the nervous system,and provides a new theoretical basis and research basis for the treatment of neurological developmental deficits.Part ? TIGAR promotes neural stem cell differentiation through acetyl-CoA-mediated histone acetylationBackgroundTIGAR is generally regarded as an antiapoptotic gene expressed in response to p53-induced cell death.As a bisphosphatase,TIGAR reduces intracellular fructose-2,6-bisphosphate levels,resulting in an inhibition of glycolysis.TIGAR is highly expressed in human breast cancer cells.Recent evidence showed that overexpression of TIGAR in carcinoma cells altered metabolic compartmentalization to a mitochondrial metabolic phenotype and ultimately increased tumor growth.More importantly,TIGAR is widely distributed in neurons and plays a vital role in the central nervous system.For example,in ischemic stroke model,TIGAR protects against ischemic/reperfusion-induced injury via glucose 6-phosphate dehydrogenase-enhanced pentose phosphate pathway(PPP)flux.However,in the developing brain,the effect of TIGAR in NSCs is largely unknown.In this study,we mainly explored the role of TIGAR in the proliferation and differentiation of NSCs and its metabolic regulation mechanism in inducing differentiation of NSCs.Exploring the role and mechanism of TIGAR in the differentiation of NSCs will help to provide new strategies for the treatment of neurodegenerative diseases.Objective1.Clarify the roles of TIGAR in the NSCs development of embryonic cerebral cortex.2.Explore the mechanism of TIGAR in regulating the differentiation of NSCs.Results?.The expression pattern of TIGAR in embryonic neocortex and neural stem cells1.TIGAR is highly expressed in the embryonic ventricular/subventricular and neocortexFetal brains at different developmental stages(E10.5 to E18.5)were harvested and the expression patterns of TIGAR mRNA/protein were detected by qPCR and Western blot.The results showed that the expression of TIGAR increased significantly during cortical development,especially at the peak stage of NSCs differentiation(E14.5 and E16.5).At the same time,immunofluorescence staining also showed that TIGAR was widely distributed in the cerebral cortex at E16.5,especially in the ventricular zone(VZ),subventricular zone(SVZ)and cortical plate(CP).In order to confirm the expression of TIGAR in NSCs,nestin and sox2,two markers of NSCs,were used for immunofluorescence staining with TIGAR.The results showed that TIGAR was co-localized with nestin and sox2,and TIGAR was localized in the cytoplasm of NSCs.2.The expression of TIGAR was increased during the differentiation process of NSCsNSCs were isolated and cultured in proliferation and differentiation medium respectively.The expression of TIGAR in the proliferation and differentiation of NSCs was analyzed by qPCR.The results showed that the expression of TIGAR increased during the differentiation of NSCs,which was consistent with the expression of MAP2(a neuronal marker)and GFAP(an astrocyte marker),while the expression of TIGAR decreased gradually during proliferation.TIGAR may be an important regulator of NSCs differentiation during embryonic development.?.TIGAR regulates the differentiation of NSCs1.TIGAR is necessary and sufficient for the differentiation of NSCsTo investigate the role of TIGAR in the differentiation of NSCs,we used lentiviruses to knock down or overexpress TIGAR.qPCR results showed that knockdown of TIGAR significantly reduced the mRNA levels of MAP2,GFAP and transcription factors Ngn1 and NeuroD1,and increased the level of Rest in the differentiation process.Western blot also showed that the expression levels of Tujl and GFAP were decreased.In immunofluorescence staining,the number of Tuj1 and GFAP positive cells also decreased significantly.These results suggest that TIGAR is necessary for differentiation of NSCs.In addition,overexpression of TIGAR increased the expression of MAP2,GFAP,Tuj1 NeuroD1 and Ngn1,suggesting that TIGAR is sufficient of NSCs differentiation.2.TIGAR has no effect on the cell survival or proliferation of NSCs in differentiated stateUnder proliferation conditions,overexpression of TIGAR decreased the expression of Nestin and increased the expression of GFAP,but the expression of SOX2 and Ki67 was unchanged,suggesting that the role of TIGAR in proliferation is slight.The effects of TIGAR on cell survival and proliferation at differentiation stage were further examined by TUNEL staining and EdU incorporation assay.TUNEL staining showed that knockdown of TIGAR had little effect on cell survival.EdU incorporation assay showed that the percentage of EdU positive cells was similar between the Lenti-siTigar and Lenti-siSCR treated groups.?.TIGAR promotes the transition of glucose metabolism to mitochondrial metabolism during NSC differentiation1.Knockdown of TIGAR promotes glycolysis and inhibits mitochondrial oxidative phosphorylationTo investigate the effect of TIGAR on glycolysis in NSCs,we analyzed the expression levels of transporters and metabolic enzymes after knockdown of TIGAR.The results showed that the mRNA and protein expression levels of GLUTs,MCTs,PFKFB3 and LDHA were not changed after TIGAR knockdown.However,knockdown of TIGAR decreased the expression of LDHB.2-NBDG was used to the measure glucose uptake of NSCs.knockdown of TIGAR had no significant effect on glucose uptake.The lactate production,as a measure of glycolysis,was increased.In addition,we examined the expression of the mitochondrial biogenesis markers PGC-l? and NRF1,and the OXPHOS markers MitoNEET and Tomm20 in differentiated NSCs.The results showed that TIGAR knockdown decreased the expression of PGC-1?a,NRF1 and MitoNEET,while overexpression of TIGAR increased the expression of these genes.Overexpression of TIGAR increased the oxygen consumption rate,and knockdown of TIGAR could significantly reduce ATP level.These results indicate that TIGAR can increase the conversion of lactic acid to pyruvate and promote oxidative phosphorylation during the differentiation of NSCs.2.Knockdown of TIGAR decreases the levels of acetyl-CoAMetabolic reprogramming can induce alteration of cellular acetyl-CoA levels.The results showed that the levels of acetyl-CoA in total cell lysate and isolated mitochondria were decreased significantly after TIGAR knockdown.ACSS2 and ACLY are two important enzymes involved in the production of acetyl-CoA.qPCR showed that the knockdown of TIGAR significantly reduced the level of ACLY mRNA.Overexpression of TIGAR-TM mutant lost its effect on glycolysis and oxidative phosphorylation.More importantly,blocking the activity of TIGAR metabolic enzymes could inhibit its effect on ACLY expression.?.TIGAR promotes NSC differentiation by upregulating the level of the acetylation of H3K9.1.Knockdown of TIGAR decreases the levels of H3K9acAcetyl-CoA,a central metabolic intermediate,has been shown to be an important second messenger that regulates histone acetylation.Western blot showed that TIGAR knockdown did not change the levels of H3K18ac,H3K14ac or H3K27ac,but the level of H3K9ac decreased significantly.Overexpression of TIGAR increased the level of H3K9ac.ChIP-qPCR showed that the location of H3K9ac in the promoters of Ngn1,NeuroDl and GFAP genes decreased significantly after knockdown of TIGAR.2.Acetate reverses the phenotype induced by TIGAR knockdownAcetyl-CoA can be generated from acetate by ACSS2,which is independent of citrate.Supplementation with acetate reversed Lenti-siTigar-induced decrease of acetyl-CoA level.Importantly,acetate significantly increased the level of H3K9 acetylation and rescued the effect of Lenti-siTigar on the decrease in H3K9 acetylation in NSCs.qPCR and Western blot showed that acetate reversed the decrease in MAP2,Tuj1 and GFAP expression caused by knockdown of TIGAR.Conclusion and innovation1.The present study found that TIGAR was highly expressed during cortical development,especially at the peak stage of NSCs differentiation(E14.5 to E16.5).2.TIGAR could inhibit or promote the differentiation of primary NSCs by knocking down or overexpressing TIGAR by virus transfection.TIGAR is necessary and sufficient for NSCs differentiation.3.TIGAR can promote the transition from glucose metabolism to mitochondrial metabolism and increase the level of acetyl-CoA during the differentiation of NSCs.4.TIGAR regulates the level of H3K9ac through acetyl-CoA,and promotes the differentiation of NSCs by regulating the expression of differentiation-related genes.5.It is the first time to find that the role of TIGAR in the proliferation and differentiation of NSCs and its metabolic regulation mechanism in inducing differentiation of NSCs,which will help to provide a new strategy for the treatment of neurodegenerative diseases.