The Role And Mechanism Of Topoisomerase?a In The Adult Neurogenesis In Mouse SVZ

Background: Adult neural stem cells(NSCs)were a flock of immature cells aggregating in special regions of central nervous system(CNS),and they have unique biological characteristics such as continuous proliferation and multi-potential differentiation.These adult NSCs played important roles in maintaining tissue function and integrity of an organism.In the brain of adult mouse,NSCs and its offspring were mainly located in subventricular zone(SVZ),and these cells can proliferate continuously and migrate to the Olfactory bulb(OB),turned into the interneurons for complements.Lots of the extracellular and intracellular signals that control adult NSCs activity have been identified.For example,the BMP and Notch signaling pathways can maintain adult NSCs in a quiescent state,and the Wnt pathway can promote adult NSCs divisions and activate neurogenesis.However,how the key genes or transcription factors regulate the activity of adult neurogenesis remain poorly defined.Topoisomerase IIa(Top2a)is a type of enzyme widely present in the cell nucleus of mammalian.Its catalysis can induce the cleavage and reconnection of DNA double strands,thereby altering the topology of DNA strands.This function plays an important role in DNA replication,RNA transcription and DNA damage repair.Topoisomerase IIa is extremely important for cell division and proliferation processes.During embryonic development,knockout of topoisomerase IIa can lead to the arrest of embryonic development in a 4-cells or 8-cells state.Until now,little is known,however,of the function of topoisomerase IIa during neurodevelopment,not to mention in adult neurogenesis.Methods: First,we used the methods of bioinformatics to screen out multiple candidate genes that may play a role in adult neurogenesis.And then we verified the expression of these candidate genes by RT-PCR and Western Blotting using cultured NSCs.Based on the results,Top2a was selected as the main goal molecular for our research.Next,we examined the expression pattern of Top2a in the adult mouse CNS by immunofluorescence staining.Then,we used Top2a shRNA,specific inhibitors,and conditional knockout mice to investigate the function of Top2a in NSCs.Finally,CHIP-seq and RNA-seq were used to explore the mechanism of Top2a,and seek for the directly regulated genes of the enzyme.Results: According to microarray and sequencing data in public database,we screened out several candidate genes specific for NSCs: Aurka,Aurkb,Birc5,Bub1 b,Ccna2,Cdc20,Cdc25c,Cdc45,Cdc6,Cdk1,Cdt1,Cenpa,Cenpf,Dbf4,Dlgap5,Kif23,Kif2 c,Mcm3,Mcm5,Mcm6,Mcm7,Plk1,Top2a,Ube2c.Among these genes,we verified the expression of Kif2 c and Top2a at the protein level.In a result,we finally selected Top2a as the target molecule for our study in adult neurogenesis.By investigating the expression pattern of Top2a,we found that it was specifically expressed in SVZ and RMS of adult mice,and in other brain regions the expression of Top2a can be hardly found,which had been determined early in neural development.In terms of the cellular localization at SVZ,we found Top2a was mainly expressed in NPCs and activated NSCs.In vitro,we inhibited the function of Top2a and found both the growth and the proliferative ability of NSCs was significantly restrained.At the same time,we also constructed conditional knockout mice for Top2a.Results showed that knockout of Top2a in vivo can significantly restrict the neurogenic process in SVZ and reduce the number of proliferating cells in situ,leading to a significant reduction for the amount of neonatal interneurons in the olfactory bulb.At last,from the CHIP-seq and RNA-seq data,the regulation effect of Top2a on the gene transcription was announced,and meanwhile 310 target genes which can be considered as the directly regulated genes of Top2a were also identified.This indicated the intrinsic mechanism of Top2a in adult neurogenesis.Conclusion: Based on the current results,we can get the following conclusions:1.Top2a is a characteristic molecule of NSCs.2.Top2a possesses a timely and spatially specific expression pattern,compared to Top2b(isomer of Top2a).For example,Top2a mainly expressed in adult SVZ and RMS.3.After the inhibition of Top2a in vitro,the normal growth of NSCs was impeded,indicating that Top2a is necessary for normal proliferation of NSCs.4.After conditional knockout of Top2a in transgene mice,adult neurogenesis activity in SVZ and the production of neonatal interneurons in the olfactory bulb were both obstructed.5.Based on the binding sites of Top2a on the genome and the RNA-seq data,we inferred that nucleoporin Nu153 and the transcription factor Sox2 were downstream target genes that mediate Top2a function.