The Influence Of DISCI Gene Function Interference In Mouse Embryonic Brain On Adult Behavior, Brain Metabolism And Interneuron Development

Mental illnesses like schizophrenia(SCZ)and major depression disorder(MDD)are devastating brain disorders.The SCZ risk gene,disrupted in schizophrenia 1(DISC1),has been associated with neuropsychiatric conditions.However,little is known regarding the long-lasting impacts on brain metabolism and behavioral outcomes from genetic insults on fetal NPCs during early life.We have established a new mouse model that specifically interrupts DISC1 functions in NPCs in vivo by a dominant-negative DISC1(DN-DISC1)with a precise temporal and spatial regulation.Interestingly,prenatal interruption of mouse Disc1 function in NPCs leads to abnormal depression-like deficit in adult mice.Here we took a novel unbiased metabonomics approach to identify brain-specific metabolites that are significantly changed in DN-DISC1 mice.Surprisingly,the inhibitory neurotransmitter,GABA,is augmented.Consistently,parvalbumin(PV)interneurons are increased in the cingulate cortex,retrosplenial granular cortex,and motor cortex.Interestingly,somatostatin(SST)positive and neuropeptide Y(NPY)interneurons are decreased in some brain regions,suggesting that DN-DISC1 expression affects the localization of interneuron subtypes.To further explore the cellular mechanisms that cause this change,DN-DISC1 suppresses proliferation and promotes the cell cycle exit of progenitors in the medial ganglionic eminence(MGE),whereas it stimulates ectopic proliferation of neighboring cells through cell non-autonomous effect.Mechanistically,it modulates GSK3? activity and interrupts Dlx2 activity in the Wnt activation.In sum,our results provide evidence that specific genetic insults on NSCs at a short period of time could lead to prolonged changes of brain metabolism and development,eventually behavioral defects.Our study is included three chapters:PART1: ESTABLISHMENT OF NEW NES-DN-DISC1 TRANSGENIC MICE MODEL AND BEHAVIOR TESTObjective To establish a new Nes-DN-DISC1 transgenic mouse model;to test the Western blot of Nes-DN-DISC1 mice;and to test the behavior of the Nes-DN-DISC1 transgenic mouse model.Methods 1.Nes-rt TA-GFP mice were mated with teto-DN-DISC1 mice to create the Nes-DN-DISC1 double transgenic lines with Nestin-rt TA-GFP and tetoDN-DISC1.For Nes-rt TA-GFP mice,genotyping was performed using rt TA primers(5'-GGA CAA GAG CAA AGT CAT AAA CGG-3' and 5'-TTC GTA CTG TTT CTC TGT TGG GC-3'),and for Teto-DN-DISC1 mice,genotyping was performed using TRE-DISC1 primers(TRE-CM V-F4: 5'-gacctccatagaagacaccgggac-3' and TRE-h DISC1-R2: 5'-tgagctgaat cccaaagtgcgccg-3').2.The brain sections of E17 embryos were immunohistochemically stained;Brain cell lysates from E17-induced or non-induced brain were lysed and blotted with mouse anti-myc(DSHB)and mouse anti-actin(Gen Script)antibodies.All images were acquired using a Zeiss LSM Pascal confocal microscope and analyzed using Zeiss LSM image browsing analysis software and image analysis software.Data Analysis was performed with T-test and all histograms were shown as mean ± standard deviation.In all analyses,p<0.05 indicates a statistically significant difference.3.Behavioral tests: open field test,forced swim test,high plus maze,novelty inhibition feeding,fear condition test,grooming test.We recorded each experiment with the Etho Vision XT video tracking system and software(Noldus).Data was analyzed using Excel and was expressed as mean ± standard error of the mean(SEM).The statistical significance between the experimental and control groups was analyzed by Student's t-test and ANOVA.Results 1.We established a novel Nes-DN-DISC1 transgenic mouse model by crossing Nes-rt TA transgenic mice.GFP and rt TA are driven by the nestin promoter in this mouse model and DN-DISC1 in the teto-DN-DISC1 mouse strain is controlled by the doxycycline(Dox)inducible promoter(teto).The mouse strain provides a spatial control because the transgene is only turned on in the NPC controlled by the nestin promoter.Dox also provides time control of DN-DISC1 expression.2.The Nes-rt TA mouse strain provides GFP reporter to specifically label NPCs in embryonic brains.Neocortical NPCs in the ventricular zones(VZs)and subventricular zones(SVZs)produce cortical projection neurons.Cortical interneurons of the interneuron progenitor cells(IPC)in the ganglionic eminence(GE)migrate tangentially to the boundaries of the developing cortex where they mature to form functional networks with excitatory neurons.It was confirmed that GFP and Nestin showed overlapping expression.All GFP-positive cells in VZs of neocortex and GE were confirmed to be Sox2 + NPC.DN-DISC1 expression on NPC was confirmed.And Dox can passed the placenta and successfully induced DN-DISC1 expression in the Nes-DN-DISC1 mouse embryonic brain.In contrast,mice that received Dox-free conventional foods did not express detectable DN-DISC1.3.The test results showed that the mice showed normal motor function,and there was no significant difference in total travel time in OFT.However,the Nes-DN-DISC1 group spent far less time in the center than the control group,indicating the presence of anxiety-like behavior.Nevertheless,we did not detect significant differences between the two groups in the EPM test(P=0.97).Since the DISC1 variant is associated with MDD,we examined whether NES-DN-DISC1 mice showed any depressive behavior using the FST test and the NSF test.Interestingly,although Nes-DN-DISC1 mice did not show a depressive phenotype under FST acute stress,chronic stress-induced depression was predisposed in NSF test(P<0.001).Nes-DN-DISC1 mice did not show fear memory defects at 8 weeks of age,with no regular grooming behavior.Conclusions 1.A new Nes-DN-DISC1 transgenic mouse model was established.2.DN-DISC1 in Nes-DN-DISC1 mice is induced at E17.3.Early genetic damage in NPC may present long-term behavioral abnormalities in adulthood,even if the risks are eliminated.PART2: EFFECTS OF DISC1 PRENATAL FUNCTIONAL INTERFERENCE ON METABONOMICS AND INTERNEURONS IN NPCObjective To examine the effect of DISC1 prenatal functional interference in NPC on the metabonomics;to detect the changes of interneurons in Nes-DN-DISC1 mice;and to examine the effect of DN-DISC1 expression on the development of neural networks in embryonic NPC.Methods The brain and liver are harvested immediately after CO2 asphyxiation on day 1 or day 30.Samples were prepared for 1H NMR spectroscopy,spectral data processing and multivariate data analysis.We used a no-deviation metabolomics method(NMR)to determine detailed metabolite changes.Orthogonal projection to latent structures with discriminant analysis(OPLS-DA)was performed using liver and brain tissue extracts from control mice and Nes-DN-DISC1 mice of different ages.Data was collected from 8 mice per genotype.We performed DN-DISC1 induction from E0 to P0 and then examined the total brain structure of two-month-old transgenic mice.The brain of 16-day-old embryonic mice was collected,then slice fixation and freezing,immunohistochemical staining of brain slices were performed to detect the expression of interneurons in cerebral cortex of embryonic mice.Results 1.Compared with the control mice,the levels of fumaric acid,choline and glucose were lower in the liver of Nes-DN-DISC1 on the first day after birth(P1).However,on P30,no significant differences in metabolites were observed between control mice livers and Nes-DN-DISC1 mice livers.2.Interestingly,Nes-DN-DISC1 mice showed significant increases in lipid,3-HB,creatine,unsaturated fatty acids and some amino acid levels,including glutamate,glycine,tyrosine,histidine and phenylalanine,whereas hypoxanthine level and alanine,glutathione,choline,glucose and AMP levels in the liver decreases.3.In the brain,Nes-DN-DISC1 mice had higher levels of GABA,citric acid and inosine on P30 than control littermates,but cholesterol and ADP/AMP levels were lower.Compared with the control mice of P1,the Nes-DN-DISC1 mice of P1 had lower choline level and higher branch chain amino acid branch amino acids(BCAA)level.On P1,GABA,NAA(N-acetylaspartate),glutamate,aspartate,creatine,fumarate and inosine levels were higher than that of P30 in Nes-DN-DISC1 mice,while on P1,choline,taurine,AMP/UMP levels are lower.4.In contrast,SST interneurons were significantly reduced in hippocampal nerve fibers and dentate gyrus(DG)of the hippocampus,while the number of interneurons in the SST did not change in other brain regions.The NPY interneurons were reduced in DG but not in the thalamus and somatosensory cortex.The number of PV interneurons increased in some brain regions,while the number of PV interneurons did not change in other brain regions.Conclusions It is confirmed that NMR can detect the age-related changes of mouse liver metabolites.DN-DISC1 expression in embryonic NPC alters metabolites in the brain and liver and has a long-term effect on adult behavior.DN-DISC1 expression does not cause dramatic changes in brain volume,cortical lamination and total cell density in the cortex and hippocampus.Depression in Nes-DN-DISC1 mice is not caused by abnormalities in adult neurogenesis.Expression of DN-DISC1 in Embryonic NPC Alters GABA in the adult brain and inhibits neuronal distribution.PART3: MOLECULAR MECHANISM OF DN-DISC1 REGULATING THE DEVELOPMENT OF INTERMEDIATE NEURONSObjective To investigate how DN-DISC1 expression affects the development of intermediate neural progenitor cells;in order to investigate the mechanism of DN-DISC1 regulating the proliferation of MGE progenitor cells,we examined the cell cycle exit index;to detect the effect of DN-DISC1 expression on Wnt signal.Methods 1.DNA Construct Full-length DN-human DISC1 was amplified by PCR and subcloned into the 3×FLAG expression vector.Super 8×TOP FLASH,which contains 8 TCF/LEF binding sites,and a Renilla-Luc TK reporter(p RL-TK,Promega)were used to test TCF transcriptional activity.2.Luciferase Assay N2 a cells were seeded into 24-well plates and the cells were transfected with polyethylenimine,0.2?g of 8×TOPFLASH reporter,0.05?g of p RL-TK,0.4 ?g of WT-DISC1 or DN-DISC1 and 0.4?g of mouse DLX2.Twenty-four hours after transfection,TCF reporter activity was measured using a dual luciferase assay system(Promega).3.Immunohistochemistry Dye-treated brain sections were mounted on glass slides and photographed with a Zeiss Pascal confocal microscope(Carl Zeiss,USA).Zeiss LSM image viewer software(Carl Zeiss,USA)and Image J are used for image analysis.The number of p H3-positive cells was counted from the sections,expressed as a percentage of GFP-labeled cells.The number of PV-positive cells was counted in the section,and the density was expressed as the number of cells divided by the area of the region.Stereological analysis was used to examine cell distribution.Basically,brain slices are separated from every 6 slices in the entire brain and stained with different antibodies.More than 200 positive cells were calculated for each brain(n = 3-6 brains).4.Western blot detection N2 a cells were transfected with vector,flag-tagged WT-DSC1 and DN-DISC1 for 48 hours,and the protein concentration was measured using an assay kit(Bio-Rad Laboratories).Western blot was performed using rabbit anti-p Y216GSK3?,rabbit anti anti-whole GSK3?(cell signaling)and mouse anti-FLAG epitope(Sigma).Results 1.One of the consistent findings in the brain after SCZ death is that PV interneurons are reduced,which comes from the medial GE(MGE).Compared to control mice,the number of GFP-positive NPCs in MGE of Nes-DN-DSIC1 mice was significantly reduced.Disturbance of DISC1 function greatly reduced NPC proliferation(>60%)in the MGE region.We observed that GFP-negative(eg,nestin-negative)ectopic p H3-positive cells increased by more than 2-fold in MES of Nes-DN-DISC1 mice.2.GFP+/Brd U+/Ki67+cells are in S phase on E15 and remain circulating on E16.GFP+/Brd U+/Ki67-cells(arrows)of E15 are in S phase but exit the cell cycle when up to E16.The cell cycle exit index indicates the ratio of GFP+/ Brd U+/Ki67-cells to total GFP+/Brd U+ cells.We observed a 2-fold increase in the cell cycle exit index of DN-DISC1-expressing embryonic brains,suggesting that the reduction in proliferating progenitor cells in the Nes-DN-DISC1 brain may be due to an increased number of cell cycle exits.4.Our preliminary results show that DN-DISC1 adversely affects NPCs in GE.This effect is unexpected because DISC1 is highly expressed in VZ/SVZ and GE's VZ/SVZ in all three regions of the neocortex.This suggests that IPC in GE expresses some of the unique factors that interact with DN-DISC1 function and exert this beneficial effect on the development of interneurons.Our data show that WT-DISC1 inhibits GSK3? activity by decreasing p Y216 levels,whereas DN-DISC1 significantly increases the level of p Y216.5.DN-DISC1 changes in Wnt activity,and Dlx2 can increase 3 times the basic level of reporter gene activity and can play a synergistic effect with WT-DISC1 in Wnt activation.However,DN-DISC1 blocks this enhancement.Conclusions DN-DISC1 expression has an autonomic effect on cells that inhibit the proliferation of GFP+ NPC,but shows a non-autonomous effect on cells that promotes the proliferation of adjacent progenitor cells;DN-DISC1 inhibits Wnt activation through its dominant negative effect on endogenous DISC1;DN-DISC1 negatively regulates Dlx2-mediated Wnt activation,hindering the role of Dlx2 in the development of interneurons.