Effect Of OTUD3 On Proliferation And Differentiation Of Neural Stem Cells

Neural stem cells（NSCs）are self-renewing and multipotent cells capable of differentiating into neurons,astrocytes and oligodendrocytes,mainly found in embryonic brain and adult mammalian neurogenic areas.In the case of disease or injury,NSCs repair the injury by proliferating and differentiating into new nerve cells,providing a new therapeutic strategy for neurodegenerative diseases and nerve injuries.Biobehaviors of NSCs are controlled and regulated by various factors.Therefore,understanding the mechanisms regulating the proliferation and differentiation of NSCs is of great importance for the clinical application of NSCs therapy.As one of the mechanisms of post-translational modification of proteins,the ubiquitin-proteasome system plays a key role in regulating the self-renewal,proliferation and differentiation of NSCs.Ubiquitin ligase E3 and deubiquitinase are key enzymes involved in the reversible processes of ubiquitination and deubiquitination.Various deubiquitinases have been found to regulate self-renewal,proliferation,and differentiation of NSCs.For example,deubiquitinase USP7 can stabilize the transcription factor c-Myc and maintain the undifferentiated state of NSCs;deubiquitinase OTUD7B knockdown induces neuronal differentiation.OTU domain protein 3（OTUD3）is a member of the deubiquitinases OTU subfamily.So far,four substrates,phosphatase and tension homologue deleted from chromosome ten（PTEN）,glucose-regulated protein 78（GRP78）and p53,have been found to have important regulatory effects on neural development.Among them,PTEN,as the target protein of OTUD3,plays an important role in the regulation of neural development.The loss of PTEN promoted the proliferation of NSCs,inhibited apoptosis,increased brain volume and abnormal tissue structure.Studies have found that Toxoplasma gondii OTUD3A（Tg OTUD3）,which has homologous structure with human OTUD3,was expressed at a low level in the cytoplasm of G1 phase and increased with the progress of cell cycle,indicating that OTUD3 played an important role in the regulation of cell cycle and proliferation.However,the effect of OTUD3 on the development and neurogenesis of NSCs and its regulatory mechanism are still largely unknown.In order to further study the effect of OTUD3 on the proliferation and differentiation of NSCs,this study aims to investigate the effects of OTUD3 on the proliferation and differentiation of NSCs.Firstly,the proliferation and differentiation of NSCs in vivo were observed in OTUD3 transgenic overexpression mice(OTUD3^TG)and OTUD3 knockout mice(OTUD3^-/-)by immunofluorescence staining and western blots,and the effects of OTUD3 overexpression and knockout on the development of NSCs were analyzed.Furthermore,cerebral cortex OTUD3^TG-NSCs were cultured in vitro,and the effects of OTUD3 on the proliferation ability of NSCs were studied by measuring the diameter of neurospheres,detecting cell viability with CCK-8 kit,and counting of Ki67-positive cells.The expression ofβ-tubulin III and GFAP were detected by immunofluorescence staining and western blots,and the effect of OTUD3 on differentiation of NSCs was studied.The experimental results were as follows:1.Compared with WT mice,brain length,width and volume of OTUD3^TGmice were decreased,and cerebral cortex thickness of OTUD3^TGmice was significantly lower than that of WT mice.The brain length,width and volume of OTUD3^-/-mice were increased,and the cerebral cortex thickness of OTUD3^-/-mice was significantly greater than that of WT mice.2.The expression of Nestin and the number of Ki67-positive cells in the neurogenic region of OTUD3^TGnewborn mice were significantly decreased,which was statistically significant compared with that of WT mice in the same litter（P＜0.05）.3.On the 3rd,4th and 5th day of NSCs proliferation cultured in vitro,the diameter of OTUD3^TGneurospheres was decreased by 7.586%,5.862%and 17.199%,respectively,which was highly statistically significant compared with the control group（P＜0.01,P＜0.01,P＜0.0001）.On the 3rd,4th and 5th day of proliferation,cell viability of OTUD3^TG-NSCs was decreased by 28.89%,45.92%and 68.86%, respectively,which was highly statistically significant compared with WT-NSCs （P＜0.01,P＜0.0001,P＜0.0001）.Ki67-positive cells in OTUD3^TG-NSCs were decreased by 12.35%,which was statistically significant compared with WT-NSCs （P＜0.05）.4.There was no significant difference in the differentiation ofβ-tubulin III-positive neurons between the OTUD3^TGnewborn mice and WT mice（P＞0.05）.5.The percentage of differentiation intoβ-tubulin III-positive neurons of WT-NSCs and OTUD3^TG-NSCs was 11.06±0.405%and 11.29±5.706%,respectively,which had no statistical significance（P＞0.05）.There was no significant difference inβ-tubulin III-protein expression between WT-NSCs and OTUD3^TG-NSCs after induced differentiation for 7 days（P＞0.05）.6.There was no significant difference in the differentiation of GFAP-positive astrocytes between the OTUD3^-/-newborn mice and WT mice（P＞0.05）.7.The differentiation percentage of WT-NSCs and OTUD3^TG-NSCs into GFAP-positive astrocytes was 46.71±0.033%and 46.00±16.866%,respectively,which has no statistical significance（P＞0.05）.There was no significant difference in GFAP protein expression between WT-NSCs and OTUD3^TG-NSCs after induced differentiation for7 days（P＞0.05）.8.Compared with WT-NSCs,the proportion of NSCs in G1 phase of OTUD3^-/--NSCs decreased,the proportion of OTUD3^-/--NSCs in S and G2 phases increased significantly.The difference was statistically significant（P＜0.05,P＜0.01,P＜0.05）.In conclusion,the abnormal expression of OTUD3 affected the development of cerebral cortex in mice.The overexpression of OTUD3 inhibited the development of cerebral cortex,while the knockout of OTUD3 promoted the development of cerebral cortex.In vitro experiments showed that the overexpression of OTUD3 inhibited the proliferation of NSCs,but did not affect their differentiation.OTUD3 inhibited the proliferation of NSCs by regulating the cell cycle.This study reveals the biological function of OTUD3,from a new aspect,by studying the regulatory effect and mechanism of OTUD3 in neural development,and also provides new ideas for the treatment of some neurological disorders.