Function And Mechanism Of Tmem72 And Sdpr Regulating Proliferation And Differentiation Of Neural Stem Cells

Objective To explore the function and mechanism of Tmem72 regulating proliferation and differentiation of mouse neural stem cells.Methods(1)The expression level of Tmem72 protein in different ages and brain regions of wild mice were determined by Western blot.(2)The Tmem72 knockdown plasmid was transiently transfected in N2a cells,and the ability of proliferation in Tmem72 deficient N2a cells was determined by CCK8,RTqPCR and Western blot.The proliferation and apoptosis changes were determined by flow cytometry.(3)Mouse neural stem cells were infected with packaged lentivirus.The knockdown efficiency and the expression changes of proliferation and differentiation related markers were determined by RT-qPCR and Western blot.(4)Differentially expressed genes and gene-enriched signaling pathways between control and Tmem72 knockdown NSCs were analyzed by RNA-seq,and the signal pathways were preliminarily verified by Western blot.Results(1)Tmem72 was expressed in different age groups in mouse brain and different regions of the whole brain.(2)Compared with the control group,the expression of Tmem72 in N2a cells and neural stem cells in the Tmem72 knockdown group was significantly down-regulated(P<0.05).(3)After Tmem72 knockdown,the proliferation ability and activity of N2a cells were significantly up-regulated(P<0.05),while the apoptosis ability was significantly decreased(P<0.01).(4)After Tmem72 knockdown,The differentiation ability of neural stem cells was promoted towards astrocytes and neurons(P<0.05).(5)Transcriptome sequencing analysis revealed differential gene enrichment in transsynaptic signaling conditions,regulation of glial cell differentiation,PI3K-Akt,MAPK signaling pathways,and ECM-receptor interactions.(6)Tmem72 may regulate the differentiation of NSCs by regulating p21,Akt,and Erk signaling pathways.Conclusions Tmem72 knockdown promoted proliferation and inhibited apoptosis of N2a cells;Tmem72 knockdown promoted the differentiation of neural stem cells into astrocytes and neurons;Tmem72 may regulate the differentiation of NSCs by regulating p21,Akt,and Erk signaling pathways.Objective To explore the function of Sdpr regulating proliferation and differentiation of mouse neural stem cells.Methods(1)The expression levels of Sdpr in different age groups and brain regions of wild mice were determined by RT-qPCR and Western blot.(2)The expression levels of Sdpr in Sdpr knockout and transgenic mice were determined by RT-qPCR and Western blot.(3)NSCs of wild,Sdpr knockout and transgenic mice at 8 weeks were isolated,and the neurospheres-forming ability of NSCs,including the size and number of spheres were determined.(4)The expression of markers on proliferation and differentiation of NSCs were determined by RT-qPCR,western blot and immunofluorescence.(5)The behavioral changes of wild,Sdpr knockout and transgenic mice were determined by behavioral techniques.Results(1)Sdpr was expressed in different age groups,with the highest expression at 4 months old and decreased at 6 months old.Sdpr was expressed in all brain regions of mice,and was highly expressed in SVZ,the main ecological niche of NSCs.(2)The expression levels of Sdpr in SVZ and hippocampus of gene knockout mice were significantly lower than those of wild mice;Sdpr expression levels in SVZ and hippocampus of transgenic mice were significantly higher than those of wild mice.Sdpr knockout and transgenic mice were successfully constructed.(3)The number of secondary neurospheres of NSCs increased slightly and the size decreased significantly in Sdpr knockout mice;the proliferation ability of NSCs in Sdpr knockout mice was decreased;and differentiation towards astrocytes and immature neurons was reduced.(4)The number of primary and secondary spheres of NSCs in Sdpr transgenic mice increased significantly,and the size of secondary spheres decreased;The proliferation ability of NSCs in Sdpr transgenic mice was enhanced,but differentiation towards astrocytes and neurons was reduced.(5)The total distance and velocity of Sdpr transgenic mice increased significantly in open field test;The duration of "depression immobility" increased significantly in the tail suspension test;The time of immobilization of conditioned response to scene and sound fear was significantly reduced;Sdpr transgenic mice spent more time in the nontarget quadrant during the exploration plateau of Morris water maze.Conclusions(1)Sdpr knockout inhibited the self-renewal potential ability and proliferation ability of NSCs,and inhibited the differentiation into neurons and astrocytes.(2)Overexpression of Sdpr promoted the self-renewal potential ability and proliferation ability of NSCs and inhibited the differentiation into neurons and astrocytes.(3)After Sdpr overexpression,the anxiety level and depression-related behaviors of mice were significantly increased,and the memory ability was impaired.