The Effects And Mechanisms Of PIP5KIγ On Li₂CO₃-induced NTDs Murine Model

Background:Neural tube defects(NTDs)are the most serious congenital malformation of central nervous system in fetus and newborn.It can lead to death or varying degrees of disability,placing a heavy burden on families and society.Lithium carbonate,as an effective mood stabilizer,is the first-line treatments for mania and bipolar disorder and so on.Even though many studies have shown that lithium has a profound effect on early embryonic development,taking lithium carbonate remains an indispensable treatment for patients with bipolar disorder during pregnancy.A series of studies have focused on the teratogenicity of lithium ions in the development of human and mammalian embryos,including affecting the development of the embryonic nervous system,the most serious of which is NTDs.It is reported that Li2CO3 specifically inhibits inositol monophosphatase(IMPase)and glycogen synthase kinase 3β(GSK-3β),which is related to inositol metabolism,and nutrient inositol deficiency is closely related to the occurrence of NTDs.Our previous study had preliminarily established a NTDs murine model induced by Li2CO3 and found that the low inositol level in NTDs pregnant mice.The nutrient inositol and its metabolic substances were closely related to the occurrence of NTDs.Type ⅠPhosphatidylinositol 4-phosphate 5-kinase γ gene(PIP5KIγ)encodes the key enzyme for in the synthesis of phosphatidylinositol 4,5-bisphosphate(PIP2)in the inositol pathway,which is most abundant in brain tissue.PIP5KIy was located in the primary cilia and regulated the formation of cilia.It has been reported that primary cilia were closely related to NTDs.In our previous study,we found that there were differences in the expression of cilia-related genes between the NTDs and the control group,as well as the occurrence of abnormal primary cilia formation.Previous studies have shown that the PIP5KIγ-/-mice and the abnormalities of primary cilia can result in NTDs.However,the effects and mechanisms of PIP5KIy on Li2CO3-induced NTDs remain unclear.Objective:This study was aim to systematically investigate the effects and mechanisms of PIP5KIγ on Li2CO3-induced NTDs murine model and to explore the molecular mechanism of maternal lithium exposure on the development of offspring,which would provide experimental basis for the molecular mechanism of lithium-induced NTDs and further clarify the pathogenesis of NTDs and provide control new ideas.Method:1.C57BL/6 mice were injected with 350mg/kg Li2CO3 intraperitoneally on embryonic day 7.5(E7.5),and embryos collected during E8.5-E13.5 to establish the Li2CO3-induced NTDs dynamic development murine model.The quantitative real-time PCR(qRT-CR),western blotting(WB),immunohistochemistry(IHC)and EdU labeling were used to detect the proliferation and apoptosis-related proteins,and to explore the effect of Li2CO3 on embryonic neural development in E13.5 embryos;The enzymatic method,enzyme-linked immunosorbent assay(ELISA),and gas chromatography-mass spectrometry(GC-MS)were used to identify the action target of Li2CO3 during the embryonic neural development in pregnant mice and embryos.The pregnant mice with 400mg/kg inositol supplementation after Li2CO3 intervention were used to establish the inositol-replenished Li2CO3 murine model and verified the effectiveness of inositol supplementation.2.The expression level of PIP5KIγ and its product PIP2 in the Li2CO3-induced NTDs dynamic development murine model and inositol-replenished Li2CO3 murine model were explored by qRT-PCR,WB and IHC.3.The mouse embryonic stem cell(mESCs)models of Li2CO3 and different inositol levels were established by using Ui2CO3 and different cell media with different inositol contents.The optimal concentration was screened by thiazolyl tetrazolium(MTT)method,and the cell inositol content treated with Li2CO3 was detected by GC-MS.MTT,EdU labeling and flow cytometry were used to detect the effect of Li2CO3 and different inositol levels on mESCs.WB,qRT-PCR,immunofluorescence(IF)and confocal microscopy techniques were used to explore the effect of Li2CO3 and inositol on the expression levels of PIP5KIγ and PIP2 in mESCs.4.PIP5KIγ knockout and overexpression mESCs models were established by using CRISPR-Cas9 and plasmid transfection technology.PIP5KIγ specific inhibitor UNC3230 was used to interfere with mESCs and the optimal intervention concentration was screened by MTT.Then PIP5KIy functional inhibition mESCs model was established.The overexpression,knockout and functional inhibition were detected by Sanger sequencing,qRT-PCR,IF,confocal microscopy and WB.The cell growth of each group was observed under a light microscope.5.The effects of the expression level of PIP5KIγ on the proliferation in mESCs were observed on the established mESCs model of knockout,overexpression and functional inhibition of PIP5KIγ by light microscopy,MTT and flow cytometry.The PIP5KIγ specific interaction protein was screened in normal and no-inositol treated mESCs using IP/MS technology.The screened interaction proteins were analyzed by using GO,KEGG and STRING databases,and the PIP5KIγ-specific primary binding protein was screened out.Finally,based on the abnormal primary cilia formation discovered by our previous research cilia chip screening,all the above genes were verified at the mRNA level on animal and cell models to obtain the possible molecular mechanism of PIP5KIγ in the process of Li2CO3-induced inositol deficiency producing NTDs.6.The statistical difference between the two groups was analyzed by independent sample t test,and the analysis of variance was used to test the statistical difference between the two groups,with P<0.05 as the statistical difference.Results:1.The effects of Li2CO3 on mouse embryonic development were dynamically observed.by dissecting the E8.5-E13.5 embryos.The closure of the neural tube of E8.5E9.5 was found delayed and the failure of neural tube closure could be seen from E10.5,leading to various NTDs phenotypes.The most common phenotype was encephalocele.Li2CO3 affected the growth and development of the embryo.EdU results showed that the cells in the E13.5 NTDs group exhibited abnormal proliferation as compared with that in the control group(P<0.01).The results of qRT-PCR,IHC and WB showed that the expression level of PH3 protein was significantly increased in the E13.5 NTDs group(P<0.01),while the expressions of P53 and Caspase3 proteins were significantly decreased(P<0.01),indicating that cell proliferation was promoted and apoptosis was inhibited in embryonic neural tissues.After intervention with Li2CO3,the activity levels of IMPase and GSK-3β in the pregnant mice brain of E7.5-E13.5 were detected.The activity level of IMPase in the pregnant mice brain was the lowest at 8h after intervention(P<0.01),and then it began to rise gradually at 8h-16h,and maintained at a low level at E8.5-E10.5(P<0.01),then it gradually rose,and it was close to the same level as that in the normal pregnant mice brain after E12.5,with significant changes in IMPase activity(P<0.01).The activity of GSK-3β decreased slightly 4h-8h after Li2CO3 intervention,and returned to the normal level after 16h,but it decreased slightly after E12.5(P<0.01).The detection of IMPase and GSK-3β in the embryonic neural tissue of E9.5-E13.5 showed that IMPase in the embryonic neural tissue of NTDs group was continuously lower than that of the control group after Li2CO3 intervention(P<0.01).The enzyme activity of GSK-3β had no significant difference in E9.5-E10.5,and was higher than that of NTDs group after E11.5(P<0.01).The change trend of plasma inositol in E7.5-E13.5 pregnant mice was consistent with that of IMPase.The PIP2 content in the neural tissues of NTDs embryos in E13.5 was significantly lower than that in the control group(P<0.01),indicating the inhibition of IMPase by Li2CO3 played a dominant role in embryonic neural development,which caused abnormal inositol metabolism.The NTDs rate was reduced(14.9%)and the offspring type was alleviated in the 400mg/kg inositol replenishing lithium carbonate mice model.2.The expression of PIP5KIy on E10.5-E13.5 was continuously increased compared with that of the control group in the Li2CO3-induced NTDs model(P<0.01),while the expression of PIP2 was increased in E10.5-E11.5(P<0.01),while it was decreased significantly on E12.5-E13.5(P<0.01).The metabolic pattern was different from that of the control group,which confirmed that Li2CO3 induced phosphatidylinositol metabolic disorder in vivo.Inositol supplementation saved the expression levels of PIP5KIγ and PIP2 in embryonic neural tissue on E13.5.3.The effect of Li2CO3 on mESCs was dose-dependent,and it promoted the mESCs proliferation at low dose level(<5mM,P<0.01),while it inhibited the proliferation at high dose level(≥5mM,P<0.01).1.75mM Li2CO3 was the optimal intervention dose.The decreased inositol level promoted the proliferation of mESCs,and the abnormal cell proliferation was most significant in the non-inositol group(P<0.01).The proliferation of mESCs was significantly inhibited under high inositol(P<0.01).The inositol supplementation after intervention with Li2CO3 could alleviate the excessive proliferation of mESCs.With the decrease of inositol level,the expression of PIP5KIγ was increased(P<0.01).The expression of PIP5KIγ was inhibited under high inositol(P<0.01)and inositol supplementation saved its expression.The changes of PIP2 and PIP5KIγexpression levels were basically consistent.4.Sequencing of the knockout group revealed a 95bp deletion mutation and a 110bp deletion mutation at the two sgRNA cleavage sites of PIPSKIγ,respectively,followed by homozygous frameshift knockout mutations.Validation at mRNA and protein levels showed that there was little expression of PIP5KIγ in the knockout group compared with the control group(P<0.01).The cells were regular in morphology and stable in growth state.The model of PIP5KIγ-knockout mESCs model was successfully established.It was found that the expression levels of PIP5KIγ in the overexpression group were significantly higher than those in the no-load and control groups at the mRNA and protein levels(P<0.01).Moreover,it had regular morphology and stable growth state.The mESCs model for PIP5KIy overexpression was successfully established.After intervention with mESCs at the optimal concentration of the screened inhibitor UNC3230,the expression of PIP5KIy was not affected compared with the control group at the protein level,but the mRNA level was slightly decreased and the PIP2 expression was significantly decreased(P<0.01),proving that the inhibitor UNC3230 inhibited the catalytic activity of PIP5KIγ to block the production of PIP2,but it did not affect the expression of PIP5KIγ at protein level.The PIP5KIγ function inhibited mESCs model was successfully established.5.The overexpression of PIP5KIγ caused abnormal proliferation of mESCs(P<0.01),while the knockout and functional inhibition groups of PIP5KIγ inhibited the proliferation of mESCs(P<0.01).Four interacting proteins with PIP5KIy were screened by IP/MS(Tuba1c,Actg1,and Actn4),which were obtained with analyzed by GO,KEGG,and STRING databases,all of which were related to actin.Based on the previous screening results of cilia,which were verified in animal and cell models,the results showed that the expression of PIP5KIγ was increased under inositol deficiency,with the expressions of actin gene Actn4,cilia genes IFT80 and Smo were decreased after intervention with Li2CO3(P<0.01).Conclusion:Li2CO3 mainly inhibited the inositol deficiency by IMPase with the high expression of PIP5KIγ and PIP2 metabolism imbalance,which down-regulated actin gene Actn4,cilia gene IFT80 and Smo.Thereby inhibiting the formation of primary cilia caused abnormal cell proliferation,which made embryonic neurodevelopmental disorders,leading to the occurrence of NTDs.