The Mechanism Of OTUD3 Regulating The Proliferation And Differentiation Of Neural Stem Cells Through PTEN

Neural stem cells(NSCs)are self-renewing and multipotent cells in the embryonic brain during development and in the subventricular zone(SVZ)and subgranular zone(SGZ)of adulthood,and they have the potential to differentiate into neurons,astrocytes and oligodendrocytes.Therefore,regulating the proliferation and directed differentiation of NSCs could promote neural cell renewal,repair damaged neural function,and provide new ideas for treating neurodegenerative diseases.NSCs are regulated by various factors during brain development,among which the ubiquitin-proteasome system(UPS),as one of the mechanisms of protein post-translational modification plays an important role in the regulation of NSCs proliferation and differentiation.UPS is a dynamic and reversible process,whose reverse regulation is controlled by deubiquitinases(DUBs),a variety of DUBs are involved in regulating the proliferation and differentiation of NSCs.OTU domain-containing protein 3(OTUD3)belongs to the OTU subfamily of DUBs,which is located in human chromosome 1p36.13,so far,there have been few studies on the function of OTUD3.Since its structure was analyzed in 2013,the substrates found include phosphatase and tension homologue deleted from chromosome 10(PTEN),glucose-regulated protein 78-kDa(GRP78),iron regulatory protein 2(IRP2),a-actinin-4(ACTN4)and p53,animal experiments confirmed that OTUD3 may specifically promote the development of lung cancer and inhibit the development of breast cancer.Our previous research found that OTUD3 can affect the survival and function of dopaminergic neurons in substantia nigra by regulating IRP2 ubiquitination,and reported its role in nervous system for the first time.However,the effect of OTUD3 on nerve development and its regulation mechanism have not been clarified.PTEN is a tumor suppressor gene located on human chromosome 10q23.3,which consists of 9 exons and encodes a protein with 403 amino acids.Studies have shown that PTEN participates in neuronal development,axonogenesis and synaptic plasticity,and plays an important role in nerve development.In adult neurogenic region,conditional deletion of PTEN gene can promote the proliferation and differentiation of NSCs in SGZ.Whether OTUD3 can regulate the biological behavior of NSCs by regulating PTEN and downstream signals remains to be further studied.The clarification of this question is of great significance for understanding the regulatory function of OTUD3 in brain development and nerve regeneration disorders.To investigate the effect of OTUD3 on the biological behaviors of NSCs proliferation and differentiation,in the present study,we used primary cell culture method to harvest embryonic day 14 wild type(WT)and OTUD3 knockout(OTUD3-/-)mice cortical NSCs,divided into WT-NSCs and OTUD3-/--NSCs groups.Cell counting,neurosphere diameter measurement,immunofluorescence staining,flow cytometry and western blotting were used to investigate the effects of OTUD3 knockout on the cell viability,proliferation,stem cell maintenance,early apoptosis and differentiation of NSCs in vitro.Furthermore,lentivirus infection technique was used to overexpress PTEN in WT-NSCs and OTUD3-/--NSCs,respectively,to observe the role of PTEN in the regulation of biological behavior of NSCs by OTUD3,and to explore the mechanism.The results were as follows:1.The diameter of neurosphere formed in the OTUD3-/--NSCs group increased by 42.70%and 62.65%respectively on the 3rd and 4th day of culture,compared with the WT-NSCs group at the same time(P<0.01).The cell viability of the OTUD3-/--NSCs group was increased by 24.55%,16.72%and 34.60%,on the 3rd,4th and 5th day of culture in vitro in comparsion with the WT-NSCs group,respectively(P<0.05,P<0.01,P<0.001).On the third day of culture in vitro,the proportion of Ki67-positive cells in the OTUD3-/--NSCs group increased by 15.84%in comparsion with the WT-NSCs group(P<0.05).2.After 7 days of induced differentiation,the number of OTUD3-/--NSCs Nestin-positive cells were increased by 89.03%in comparsion with WT-NSCs(P<0.001).3.After 7 days of induced differentiation,the number of OTUD3-/--NSCs differentiated into β-Tubulin Ⅲ-positive neurons was increased by 74.45%in comparion with WT-NSCs(P<0.001).The expression of β-tubulin Ⅲ and MAP2 protein in OTUD3-/--NSCs was up-regulated,which was increased by 59.14%and 58.17%in comparsion with WT-NSCs(P<0.05,P<0.01).4.After 7 days of induced differentiation,the numbers of GFAP-positive astrocytes and GFAP protein expression in WT-NSCs and OTUD3-/--NSCs showed no statistically significant difference(P>0.05).5.After 7 days of induced differentiation,the expression of Oligo proteins in OTUD3-/--NSCs was increased by 36.50%in comparion with WT-NSCs(P<0.05).6.The early apoptosis rate of OTUD3-/--NSCs was decreased by 40.01%in comparsion with WT-NSCs(P<0.05).7.The expression of PTEN protein in the cerebral cortex of mice with OTUD3-/embryo 14 days was down-regulated,which was decreased by 20.06%in comparion with WT group(P<0.05).8.PTEN was overexpressed in OTUD3-/--NSCs,NSCs showed a trend of growing along the wall,the diameter of neurosphere was decreased by 19.70%and the length of neurites was increased by 147.80%in comparsion with the control group(P<0.001).9.Compared with the WT-NSCs group,the expression of Cyclin D1 protein was upregulated in the OTUD3-/--NSCs group(P<0.01).After overexpression of PTEN,the protein expression of Cyclin D1 in OTUD3-/--NSCs group cells showed a significant downregulation,which was decreased by 59.08%in comparion with the control group(P<0.001);The PI3K pathway protein expression was upregulated,which was increased by 28.51%in comparion with the control group(P<0.05).The above results indicated that primary NSCs cultured in vitro have good proliferation and differentiation ability.Knockout OTUD3 promoted the proliferation of NSCs and inhibited early apoptosis.Knockout OTUD3 promoted the differentiation of NSCs into neurons and oligodendrocytes,but there was no significant effect on astrocytes differentiation in vitro.PTEN inhibits the proliferation of NSCs through cell cycle.Knockout OTUD3 upregulates the expression of the pathway signal protein PI3K of PTEN.This study indicated that knockout OTUD3 could affect the proliferation and differentiation of NSCs,and its regulatory mechanism may be related to PTEN.This study reveals the biological function of OTUD3 in neural development and provides new ideas for NSCs transplantation therapy for neurodegenerative diseases.