Transcriptional Regulation Of VGF By Psychiatric Candidate Gene NPAS3 And Their Molecular Mechanism In The Process Of Adult Neurogenesis

Objective: To explore the molecular mechanism of the psychiatric candidate gene NPAS3 in the regulation of VGF,and their role in the process of neuronal cell proliferation and the potential molecular mechanism of this process.Method: Transfect the cells with eukaryotic expression vector,short hairpin RNA or small interfering RNA by using lipofectamine 3000,to overexpress or knock down the expression of NPAS3 or VGF.Immunofluorescence technique was used to detect the location of NPAS3 and VGF in hippocampus of rats.Using rat neural stem cells as an in vitro model,q PCR and Western blot were used to detect the regulation of VGF by NPAS3 at the m RNA level and protein level,respectively.Construct a VGF promoter(990 bp)without the ?B binding site,to identify the activity of full-length(1237bp)and truncated(990bp)VGF promoters induced by NPAS3 by using luciferase reporter assay.Use NF-?B inhibitor Caffeic Acid Phenethyl Ester(CAPE)to stimulate cells and / or change the expression level of NPAS3,then check the expression of VGF,NF-?B p65 and NF-?B p52 by Western blot.Treat the cells with different methods and the cell proliferation was measured by CCK8.Overexpress NPAS3 and / or use metabotropic glutamate receptor 5(m Glu R5)inhibitor 2-methyl-6-(phenylethynyl)-pyridine(MPEP)to stimuate cells,knock down VGF and / or overexpress NPAS3 followed by western blot to detect the phosphorylation level of protein kinase D(PKD).Check the activation of calcium / calmodulin-dependent protein kinase II(Ca MKII)using western blot after overexpressing NPAS3 and / or using the N-methyl-D-aspartate receptor(NMDA receptor)(one of the ionic glutamate receptors)inhibitor Dizocilpine(MK-801),knocking down VGF and / Or overexpression NPAS3.Results: After transfecting plasmid pc DNA3.1-NPAS3 for 48 hours,the content of NPAS3 protein in 293 T cells was significantly increased;The results of western blot showed that the effect of NPAS3-sh-892 was the best one to knock down the expression of NPAS3 after transfecting the four small interfering RNA plasmids,and this plasmid will be used in the following experiments.Immunofluorescence results showed that NPAS3 co-located with VGF,mainly distributed in the subgranular zone of the dentate gyrus with processes radiating into the granule cell proper.In rat neural stem cells,overexpress NPAS3 can upregulate the m RNA level of VGF(P <0.01)by q PCR assay;In contrast,the transcription level of VGF was significantly decreased(P <0.01)when knockdown of NPAS3 protein,these results were consistent with the early results of gene chip.At the protein level,the expression of VGF protein was significantly increased after overexpression of NPAS3 protein(P <0.001),and decreased after knock down NPAS3(P <0.01).Luciferase reporter assay check the VGF promoter activity at different levels of NPAS3,and the results showed that regardless of whether the VGF promoter contains the ?B binding site,high level of NPAS3 can activate the VGF promoter.The activity of the VGF promoter(1237bp)containing the ?B binding site was significantly higher than the VGF promoter without ?B binding site(990bp)(P <0.01);In contrast,after knocking down NPAS3,although the activity of the VGF promoter was attenuated compared to the control group,the VGF promoter with the ?B binding site(1237bp)was still stronger than that without the ?B binding site(990bp)(P <0.05).these results demonstrate that NPAS3 can enhance the activity of VGF promoter through the ?B site.The expression of p65 and p52 protein involved in NF-?B family was further to detect by using CAPE.Western blot showed that VGF expression was decreased(P <0.001)when CAPE was used;Compared with DMSO group,the promotion of VGF induced by NPAS3 could be antagonized by CAPE(P <0.01);For NF-?B p65 protein,up regulate NPAS3 can promote its expression,and after the use of CAPE,the expression of p65 is same with the change of VGF;besides p52 protein content have no change with a variety of treatment.These fundings indicate that NPAS3 can indirectly affect the expression of VGF through p65.CCK8 results showed that NPAS3 could promote cell proliferation in neural cell lines(P <0.05);Compared with the control group when knockdown VGF,the cell proliferation showed a decreased trend;the cell proliferation of overexpression of NPAS3 plus down regulation of VGF group was significantly higher than the only down regulation of VGF group(P <0.05). futher to analyze the molecular mechanism by which NPAS3 contribute to cell proliferation,we chose two pathway to explore.In the m Glu R5 pathway,the content of PKD can be regulated by NPAS3(P <0.001);Compared with DMSO group,the levels of phosphorylated PKD were decreased(P <0.05)after using MPEP.However,overexpressing of NPAS3 at the same time giving MPEP stimulation,the phosphorylation level of PKD was no longer elevated,indicate that NPAS3 can regulate PKD through m Glu R5.After knocking down VGF,the content of phosphorylated PKD was decreased(P <0.001),forthermore,compared with the control group,the phosphorylation of PKD was significantly decreased after overexpression of NPAS3(P <0.001),indicating that NPAS3 regulates downstream PKD by activating m Glu R5 though VGF.At the same time,we detect the NMDA receptor pathway,consistent with the results of m Glu R5 pathway,overexpression of NPAS3 could promote Ca MKII phosphorylation(P <0.001),and this effect can be blocked by NMDA receptor inhibitor Dizocilpine MK-801.Similarly,knockdown of VGF reduced the phosphorylated Ca MKII,whereas overexpression of NPAS3 could not reverse this phenomenon(P <0.001).Conclusion: 1.The psychiatric candidate gene NPAS3 and neurotrophic factor VGF were co-located in the hippocampal dentate gyrus subgranular zone and granule cell proper.2.In the rat neural stem cells,VGF could be regulated by NPAS3 at the transcription and expression level.3.NPAS3 could regulate VGF through the NF-?B(p65)pathway.4.NPAS3 promotes neuronal proliferation through VGF.5.NPAS3 and VGF are involved in cell proliferation by activating downstream molecular protein kinase D(PKD)and calcium / calmodulin-dependent protein kinase II(Ca MKII)through the metabotropic glutamate receptor 5 and N-methyl-D-aspartate receptor pathway.