The Role Of MOF In The Proliferation And Differentiation Of Neural Stem Cells

Neural stem cells(NSC)have the ability to self-renew and can differentiate into neurons,astrocytes and oligodendrocytes.During the development of the embryonic nervous system,neural stem cells first undergo symmetrical division to expand their population,then undergo neurogenesis to produce neurons,and secondly undergo glialogenesis to produce astrocytes and oligodendrocytes.Whether it is premature neurogenesis or the sequence of neurogenesis and glialogenesis is broken,it will cause the nervous system to develop poorly and then produce neurological diseases,and severely lead to embryonic death.Premature neurogenesis in the embryonic stage leads to hypoplasia of the brain,and premature glial formation can impair neurogenesis and is accompanied by autism symptoms.There are neural stem cells in the subgranular area of the hippocampal dentate gyrus and the subependymal area around the lateral ventricle of adult mammals.The stemness of neural stem cells in these areas is weakened.And neurogenesis is reduced,nerve damage repair function is weakened.The risk of degenerative diseases is increased.Alzheimer's disease,Huntington's disease,Parkinson's disease and other neurodegenerative diseases have not yet developed effective therapeutic drugs.Therefore,hope that neural stem cell transplantation therapy will help.This requires a deeper understanding of the regulation of neural stem cell stems and the mechanism of maintenance and differentiation.Although neural stem cell transplantation has made some progress in animal experiments,it can significantly improve the behavior and cognitive ability of animals,there is still a risk of tumor formation.It is necessary to further study the regulation mechanism of neural stem cell differentiation in vivo and the efficiency of neural circuit integration.MOF is also called MYST1 or KAT8.MOF was originally identified as a component of the X chromosome compensation complex in male fruit flies.The specific acetylation of H4K16 doubled the expression of X-linked genes in male fruit flies.MOF is involved in a variety of cell biology processes,including gene transcription,DNA damagerepair,embryonic stem cell development,cancer,etc.MOF also plays an important role in the development of the nervous system.The absence of MOF and H4K16ac in Purkinje cells can cause the neurological abnormal phenotype of ataxia telangiectasia in mice,and EMX1-Cre MOFfl/fl knockout mice showed defects in brain development and premature neurodevelopment.Sequencing showed that there were point mutations in the MOF in 9 patients with mental retardation,and these point mutations all affected the acetylation function of MOF.We are committed to studying the mechanism of MOF in the proliferation and differentiation of neural stem cells,deepening our understanding of brain tissue development,and providing theoretical support for neural stem cell therapy.Therefore,we combined in vitro and in vivo experiments to show that MOF inhibits the differentiation of neural stem cells.In mice that knocked out MOF under nestin-Cre conditions,the expression of TUBB3 increased,indicating that neural stem cells began to differentiate prematurely.Moreover,the stemness of neural stem cells of knocked out MOF is weakened.Based on all the experimental results,we found that MOF plays an important role in maintaining the stemness of neural stem cells.Method:1.Neural stem cells from the cerebral cortex of fetal rats at E13.5 days were extracted and isolated.Their spheroidizing ability was observe in vitro,and were performed immunofluorescence staining on Nestin,Ki67,TUBB3,and GFAP to identify their neural stem cell characteristics,proliferation characteristics and differentiation ability.2.The expression of MOF in Nestin,TUBB3,and GFAP positive cells was identified by immunofluorescence;MOF,TUBB3,GFAP,OLIG2,H4K16ac protein was detected during brain tissue development at E10.5,E13.5,E15.5,E17.5,and P1 by WB;MOF TUBB3 mRNA level was detected by qPCR.3.In vitro differentiation of neural stem cells 0,4,6 days,MOF,TUBB3,GFAP,SOX2,H4K16ac protein was detected by WB;MOF,TUBB3,SOX2,GFAP mRNA levels was detected by qPCR.4.Overexpression of MOF in neural stem cells in vitro,MOF,TUBB3,GFAP,H4K16ac protein was detected by WB;overexpression of MOF at the same time differentiation and culture for 4 days,TUBB3,GFAP protein was detected by WB.The mRNA level of TUBB3,GFAP was detected by qPCR.5.MOFfl/fl,Nestin-Cre E13.5 days of brain tissue protein and RNA was extracted,MOF,TUBB3,GFAP,H4K16ac protein was detected by WB.MOF,TUBB3 mRNA level was detected by qPCR.And in vitro spheroidization experiment was performed by MOFfl/fl,Nestin-Cre E13.5 days of neural stem cells.6.Neural stem cells from MOFfl/fl E13.5 day fetal mice was isolated,and neural stem cells were infected with Ad-Cre to mediate MOF knockout,proliferation was detected by EdU proliferation test.Overexpression of MOF in neural stem cells,proliferation was detected by EdU.Results:1.The isolated neural stem cells formed neurospheres,which were Nestin and Ki67 positive,and differentiated into TUBB3 and GFAP positive cells in the differentiation medium.2.Immunofluorescence showed that MOF was expressed in Nestin,TUBB3,and GFAP positive cells.Expression of MOF in brain tissues at the E10.5,E13.5,E15.5,E17.5,and P1 stages hardly changed.The qPCR results showed that the level of MOF mRNA was the highest on day E10.5.In addition,the changes of TUBB3,GFAP and other proteins were consistent with the differentiation process of neural stem cells.3.WB results showed that the protein level of MOF gradually decreased during 0,4,and 6 days of differentiation in vitro,the protein level of TUBB3 was the highest on day 4,the protein of GFAP was higher on day 4 and day 6,and SOX2 protein only expressed in undifferentiated neural stem cells.While the mRNA of MOF hardly changed during the differentiation process,the mRNAs of TUBB3 and GFAP were significantly increased compared to day 0.4.Overexpression of MOF in neural stem cells reduced TUBB3 protein.After 4 days of differentiation,both TUBB3 and GFAP proteins decreased,but the mRNA changes of TUBB3 and GFAP were inconsistent with protein changes.5.MOFfl/fl,Nestin-Cre E13.5 days of brain tissue of MOF,H4K16ac protein significantly decreased,TUBB3 protein increased,qPCR results showed that the mRNA level of MOF also significantly decreased,but TUBB3 mRNA almost did not change,GFAP protein is almost undetectable;neural stem cells of knocked out MOF had almost no spheroidizing ability.6.Ad-Cre mediated MOF knockout significantly reduced the proliferation ability of neural stem cells,while overexpression of MOF enhanced the proliferation ability of neural stem cells.Conclusions:1.Successfully isolated neural stem cells.2.MOF expressed in Nestin,TUBB3,GFAP positive cells,and highly expressed during brain tissue development.3.The protein level of MOF gradually decreased during the differentiation of neural stem cells in vitro.4.Overexpression of MOF inhibited the differentiation of neural stem cells.5.Knock out MOF promoted the differentiation of neural stem cells.MOF maintained neural stem cells stemness.6.MOF promoted cell proliferation of neural stem cells.