Effect Of TOP2a On The Acquirement And Maintainance Of Stem Cell Properties Of Reactive Astrocytes After CNS Injury

The injury and repair of Central Nervous System（CNS）is still a clinical challenging problem in the field of neurology.Astrocytes are the most abundant and widely distributed glial cells which play multiple key roles in maintaining homeostasis of the CNS in physiological condition.In response to injury,astrocytes turn into an active form and proliferate.These reactive astrocytes constitute a major cellular component of scar formation which is critical for wound healing and tissue remodeling.Recent studies have shown that,unlike microglia and oligodendrocytes,some astrocytes acquire stem cell characteristics after CNS injury.However,the mechanism of astrocyte requiring stem cell characteristics is still unclear.By analyzing the gene expression of normal astrocytes and neural stem cells in GO、KEGG and GSEA,we screened out several genes which were specifically expressed in neural stem cells,and found that topoisomerase 2a was very important for maintaining the characteristics of neural stem cells.Topoisomerase 2a is one of the members of Topoisomerases family.It plays an important role in DNA replication,transcription,recombination and chromosome separation.Topoisomerase I（TOP1）and Topoisomerase II（TOP2）are two types of Topoisomerases in mammals,which respectively contribute to the production of single-stranded and double-stranded DNA gaps.TOP2 have two subtypes:TOP2a and TOP2b.TOP2b mainly exists in terminal differentiated cells while TOP2a is indispensable for the survival of mitotic cells.In addition,during the process of embryonic stem cell-neuron transformation,the expression of TOP2 changed from subtype A to subtype B.In addition to focusing on the role of TOP2a in physiological conditions,researchers mostly focus on the effect of TOP2a in the field of cancer.A large amount of studies have shown that TOP2a is closely related to the proliferation of cancer cells such as breast cancer,urothelial tumors,leiomyosarcoma and hepatocellular carcinoma.Drugs targeted on TOP2a have also shown effective.However,there are small number of studies regarding TOP2a in the nervous system.In order to explore the role of TOP2a in acquisition and maintenance of stem cell characteristics of reactive astrocytes,we firstly established a cortical injury model and detected the expression of stem cell markers of astrocytes.Then we cultured astrocyte around the injury area in vitro.Results have shown that the expression of stem cell markers of astrocytes were increased after injury,and the formation of neural stem cell spheres with self-renewal and differentiation characteristics could also be detected.Subsequently,we observed the expression of TOP2a in the whole brain of uninjury adult mice,and it was found that TOP2a was only expressed in the subependymal ventricular zone（SVZ）.In the injury model,we investigated the expression of TOP2a by immunofluorescence and surprisingly found that TOP2a began to re-express in the cortex and its expression changed dynamically with the time course after injury,that is,TOP2a could be detected on the 3rd day after injury,and the expression of TOP2a gradually decreased on the 5th and 7th day after injury,then were undetectable on the 14th day after injury.In this process,we detected the expression of TOP2a with reactive astrocytes labeled with stem cell markers,and found that some astrocytes with stem cell characteristics were also positive for TOP2a.In order to further explore the function of TOP2a on reactivated astrocytes,we constructed GFAP-CreER^T2;TOP2a ^f/f conditioned knockout mice.It was showed that the number of astrocytes around the injury was decreased and the multipotency of reactivated astrocytes was also impaired after knockout of TOP2a in reactivated astrocytes.Using TOP2a knockdown virus,we found that the number and the diameter of neural stem cell spheres decreased significantly.With the treatment of TOP2a specific inhibitors,the self-renew ability of neural stem cells was completely damaged.RNA-seq assay of neural stem cell spheres showed that the multipotency genes of astrocytes were decreased after treated with TOP2a specific inhibitors,and this down-regulation may be related to the decrease of transcription factor Pax6.In conclusion,our study suggested that TOP2a was involved in regulating the acquisition and maintenance of multipotency of reactive astrocytes,which might be mediated by transcription factor Pax6.These studies could help us understand the pathological process of central nervous system injury further and provide new target for the repair of central nervous system.