Functions Of AF1q In Neurodevelopment And The Regulation Mechanism Of AF1q Gene Transcription

Background:Alzheimer’s disease (AD), is the most common reason of dementia, accounting for about two-thirds in the patients suffering from dementia. The morbidity of ADin China is increasingbecause of the issue of population aging. AD has become a serious danger to public health. AD patients will finally lose theliving ability. AD is a chronic and progressiveneurodegenerative disease characterized by cognitive and memorial dysfunctions, with the typical neuropathological characteristics of senile plaques, neurofibrillary tangles and neuronal loss.Although spending lots of effort, little progress is made to reveal the mechanism of AD.Research foradult neural regeneration is on theway recently. Now, it’s generally agreedthat adult brain stem cells have the abilities of proliferation, migration and differentiation, and can finally be integrated into the neural network of adult brain. So it is believed that dult brain stem cellsplay an important role in nervous degenerative diseases, such as AD. A large number of studies have shown that in the process of AD, the nerve regenerationof hippocampusis changed. Nerve regeneration can compensate the injured neurons function and provide a new choice for the treatment of AD. The treatments of AD by neural stem cellsinclude transplantation of xenogenousstem cell and the activation of autologous neural stem cells. Exogenous neural stem cell transplantation has made great progress in the treatment of AD, but it is limited due to torture of ethics, stem cells deficiency and immune rejection after transplantation. Autologous neural stem cells have become the focus in the treatment of AD because of its special advantages, such as stable source, immunogenicity-free, tumorigenicity-free, harmless to the stable internal environment and easy to operate. The activation of autologous neural stem cells for reparing nervous system is a very promising for treatment for AD. This research will provide a novel perspective for the mechanism exploration and treatment of neurodegenerative diseases, such as AD.Adult hippocampal neurogenesis is regulated by a variety of endogenous and exogenous factors, and some signal molecules can also regulate hippocampal neural regeneration, such as the Wnt signaling pathway. Wnt signaling pathwayplays an important role in the development of nervous system, proliferation and differentiation of the hippocampus regeneration. Wnt signaling can enhance proliferation of neural stem cells from the brain, while suppressing the Wnt signaling pathway would inhibitthe proliferation of hippocampus neural stem cells. Otherwise, Wnt signaling pathway is reported to participates in the pathology of AD. To sum up, Wnt-related proteins or compoundswill be of great potential for the treatment of AD bystimulatingactivation of autologous neural stem cell.Section 1The regulatory mechanisms of AF1q gene transcriptionObjectiveThe aim of this study was to identify weather the expression of AF1q is specific in Alzheimer’s disease; Furthermore, we will elucidate the underlyingmolecularregulatory mechanisms of AF1q gene transcription by REST. Our original researchwill provide new relationship between AF1q and REST.Materials and methods1. Real time RT-PCR:The mice brain tissues from B6C3-Tg(APPswe, PSEN1dE9)miceand normal control were obtained from Nanjing University Laboratory Animal Center. Total RNA was extracted from samplesusing TRIzol kit. Reverse transcription was performed with Takara PrimeScriptTM.The expression of Af1q mRNA was quantified by TOYOBO SYBR Green gene Expression Analysis kit.2. Detect the regulation mechanism of REST to AF1q gene transcription.2.1 Transfection:HEK293 cell lines were transfected with pREST or psiREST to elevated or decreased the expression of REST using Lipofectamine 2000 reagent according to themanufacturer’s instructions. RT-PCR was applied to detect theexpression ofAF1q mRNA.2.2 Construction of pAF1qpromoter plasmid:The promoter region of the AF1q gene obtained by PCR was cloned into the promoter-less plasmid pGL-3Basic. The promoter construct was transfected into HEK293 cells using Lipofectamine 2000 reagent according to themanufacturer’s instructionsand after transfection transfection with the plasmid for 24h, the promoter activity was determined by Luciferase.2.3 Transfection:HEK293 cell lines were co-transfected with pREST or psiREST and pAFlqpromoter using Lipofectamine 2000 reagent according to themanufacturer’s instructions. Luciferase was applied to detect thepromoter activity.3. The determination of the functional NRSE site in AF1q promoter region.3.1 In Jaspar analysis of AF1q promoter region revealed 2 putative NRSE sites.3.2 Construction of AF1q promoter deletion plasmid:Various deletion plasmids of AF1q promoter region were obtained and measured the promoter activity by Luciferase. Then HEK293 cell lines were co-transfected with pRESTand various deletion plasmids of AF1q promoter region using Lipofectamine 2000 reagent according to themanufacturer’s instructions. Luciferase was applied to detect thepromoter activity to indicate the possible position of NRSE site.3.3 Electrophoretic mobility shift assay (EMS A) and Chromatin Immunoprecipitation (ChIP) were used to confirm in vitro and in vivo the functional NRSE site found in the above experiments.4. Detect the expression pattern of AFlq and REST in normal adult C57BL/6 mice brains during neurodevelopment. Total RNA was extracted from samplesusing TRIzol kit. Reverse transcription was performed with Takara PrimeScriptTM.The mRNA levels of Af1q and Rest from normal mice brain aging at embryonic day 13.5 (E13.5),18 (E18), postnatal dayl (P1),7 (P7),14 (P14) and adult were quantified by TOYOBO R SYBR Green gene Expression Analysis kit.5.4. Statistical analysis:Statistical analysis was carried out using SPSS software(version 17.0). Data are expressed as means ± standard errors of at least 3independent experiments. Student’s t tests and Spearman.correlation analysiswereused to determine significance between groups. Differences with P-values of lessthan 0.05 were considered statistically significant.Results1.Real-time PCR results:The mRNA levels ofAF1q in APPswe/PSEN1dE914 mice were significant downregulation compared with normal controls.2. REST can inhibit AF1q gene transcription.2.1 REST overexpression decreased AF1q mRNA, while REST knockdown significantly increased AF1q mRNA as confirmed by RT-PCR analysis.2.2 The activity of the promoter was significantly higher than pGL-3Basic.2.3 The activity of the promoter was remarkably reduced by REST.3. The functional NRSE site was confirmed.3.1 In Jaspar analysis of AF1q promoter region revealed 2 putative NRSE sites:-383～-363bp and +422～+442;3.2 All the deletion plasmids of AF1q promoter region had the promoter activity. REST expression can decrease the promoter activity of AF1q deletion constructs containing the NRSE site from-383 to-363bp.3.3 EMSA and ChIP also revealed that -383 to -363bp is the functional NRSE site.4. The real-time RT-PCR results showed that mRNA levels of Aflq and Rest were coordinately expressed, with significant negative correlation during neurodevelopment (by Spearman Correlation), indicating that Af1q gene expression was negatively regulated by Rest in neurodevelopment.Conclusion:1. The mRNA levels ofAF1q ware significant downregulation in APPswe/PSEN1dE914 mice compared with normal controls.2. REST inhibited human AF1q gene transcription.3. The functional NRSE site in AF1q gene promoter was from-383 to -363bp.4. Af1q expression was negatively correlated with Rest in mice neurodevelopment.Section 2AFlq play a role in neurodevelopment throughactivating Wnt/β-catenin pathway viabinding of T-cell factor-7ObjectiveThe aim of this study was to identify the function of AF1q which involved in the neurodevelopment. Furthermore, we revealed the mechanism of AF1q in the regulation of Wnt signaling pathway. Additionally, we intended to investigate the biologicalinteraction between AF1q and TCF7 to improve Wnt signaling pathway.Materials and methods1. The biological effects of AFlq in SH-SY5Y cell lines.1.1 Construction of plasmids and Transfection:The pcDNA3.1-AF1q-6myc plasmid and pEGFP-AF1q-C3were transfected into SH-SY5Y cells using Lipofectamine 2000 reagent according to themanufacturer’s instructions.The expression of AF1q was detected by RT-PCR and Western blot after transfection with the plasmid for48h.1.2 AF1q lentivirus production and transduction:Packaging of the AF1q constructs inpseudoviral particles according to the manufacturer’s instructions. SH-SY5Y cells were stably transducted with lenti-AF1q,and maintained under blasticidin selection. Empty lentivirus was used as a controlfor the experiments. The expression of AF1q was detected by RT-PCR and Western blot after transduction.1.3 Plasmid pEGFP-AF1q-C3 and its empty control were transfected into SH-SY5Y cells using Lipofectamine 2000 reagent according to themanufacturer’sinstructions. Observe the location of AF1q by confocal laser scanning microscope.1.4 MTT assay:SH-SY5Y cells were stably transducted with lenti-AF1q or the empty lentivirus. The proliferation of cellgrowth was analyzed by MTT assay for 5days.1.5 EdU assay:SH-SY5Y cells were stably transducted with lenti-AF1q or the empty lentivirus. The proliferation of cellwas analyzed by EdU assay.1.6 Cell cycle:SH-SY5Y cells were stably transducted with lenti-AF1q or the empty lentivirus. The cells were stained with PI. Then cells wereimmediately fixed by 70%ethanol under-20℃ for 1h then measured by FACS Calibur.2. The functions of AF1q in proliferation and differentiation of neural stem cells.2.1 Neural stem cells:Primary NSCs were isolated from 13-day-old embryos of rat.Rat forebrains were minced with scalpels and digested in stempro accutase. Primary neurospheres were formed after 5-7 days of culture. Identify these were neural stem cells by Nestin immunofluorescence staining.2.2 Transduction:Neural stem cells were stably transducted with lenti-AF1q. Empty lentivirus was used as a controlfor the experiments.The expression of AF1q was detected by RT-PCR after transduction.2.3 Measurement of neural stem cells size and numbers:the neural stem cells size and numbers were observed under microscope after transducted by lenti-AF1q or its control for 3days.2.4 Neural stem cells differentiation:neural stem cells were stably transfected with lenti-AF1q or its control. The expression of ngn1 was detected by RT-PCR. Then cells were exposed to DMEM containing 2% fetal bovine serum (FBS) for 1 week and analyzed MAP2 and GFAP by immunofluorescence.3. The mechanism of AF1q activating Wnt signaling pathway.3.1 TOP-Flash&FOP-Flash assay:The pcDNA3.1-AF1q-6myc plasmid and control were transfected into HEK293 cells using Lipofectamine 2000 reagent according to themanufacturer’s instructions.The activation of Wnt signaling pathway was detected by TOP-Flash&FOP-Flash assay after transfection with the plasmid for 24h.3.2Co-IP assay was applied to detect the combination between AF1q and TCF7: The pcDNA3.1-AF1q-6myc plasmid and TCF7 or LEFlor β-catenin plasmid were co-transfected into HEK293 cells using Lipofectamine 2000 reagent according to themanufacturer’s instructions.The combination betweenthem was detected by Co-IP assay.3.3 Western blot:The pcDNA3.1-AF1q-6myc plasmid and TCF7 plasmid were co-transfected into HEK293 cells using Lipofectamine 2000 reagent according to themanufacturer’s instructions. After transfected 48h, cells were treated with CHX (100ug/ml) for 0h、3h、6h、9h、12h and 24h.The expression of TCF7 protein was detected by Western blot.3.4 Nuclear transcription:The pcDNA3.1-AF1q-6myc plasmid and TCF7 plasmid were co-transfected into HEK293 cells using Lipofectamine 2000 reagent according to themanufacturer’s instructions. Nuclear protein wasextractedafter transfected 48h.The expression of TCF7 protein was detected by Western blot.3.5 EdU assay:The TCF7-shRNA plasmid wastransfected into HEK293 cells using Lipofectamine 2000 reagent according to themanufacturer’s instructions. The expression of TCF7 was detected by Western blot after transfection with the TCF7 plasmid for48h.Then the pcDNA3.1-AFlq-6myc plasmid andTCF7-shRNA plasmid were co-transfected into SH-SY5Y cells.The proliferation of cellwas analyzed by EdU assay.3.6 Co-IP assay was applied to detect the combination site between AF1q and TCF7:The deletion plasmid including 1-30aa、30-90aa、 1-60aa、60-90aa、 1-20aa、 20-80aa、 1-10aa,10-90aa、20-30aa and 1120aa were transfected into HEK293 cells using Lipofectamine 2000 reagent according to themanufacturer’s instructions. The expression of these deletion plasmids were detected by Western blot after transfection for48h. Then these deletion plasmid and TCF7plasmid were co-transfected into 293 cells.The combination betweenthem was detected by Co-IP assay.3.7 The relationship between phosphorylation-AF1q and TCF7.3.7.1 Construction of pAF1qS11F plasmid:The pAF1qS11F plasmid wastransfected into HEK293 cells. The expression of AF1q was detected by Western blot after transfection with the pAF1qS11F plasmid for48h.3.7.2 Western blot:The pAFlq plasmid or pAF1qS11Fplasmid and TCF7 plasmid were co-transfected into HEK293 cells using Lipofectamine 2000 reagent according to themanufacturer’s instructions. After transfected 48h, cells were treated with CHX (100ug/ml) for 0h、3h、6h 、9h、12h and 24h.The expression of AF1q protein was detected by Western blot.3.7.3 TOP-Flash&FOP-Flash assay:The pAF1q plasmid or pAF1qS11Fplasmid and TCF7 plasmid were co-transfected into HEK293 cells using Lipofectamine 2000 reagent according to themanufacturer’s instructions. After transfected 48h, the activation of Wnt signaling pathway was detected by TOP-Flash&FOP-Flash assay.3.7.4 EdU assay:The pAFlq plasmid or pAF1qS11Fplasmid and TCF7 plasmid were co-transfected into SH-SY5Y cells using Lipofectamine 2000 reagent according to themanufacturer’s instructions.The proliferation of cellwas analyzed by EdU assay.4. Statistical analysis:Statistical analysis was carried out using SPSS software(version 17.0). Data are expressed as means ± standard errors of at least 3independent experiments. Student’s t test wasused to determine significance between groups. Differences with P-values of lessthan 0.05 were considered statistically significant.Results:1. AF1q promoted the proliferation of SH-SY5Y cells.1.1 RT-PCR confirmed that pcDNA3.1-AF1q-6myc and pEGFP-AF1q-C3 effectively enhanced theexpression of AF1q.1.2 RT-PCR confirmed that lenti-AFlq effectively enhanced theexpression of AF1q.1.3 Observed by confocal laser scanning microscope:AF1q mainly located in cytoplasm and less in nucleus, and AFlq packed with granular around the nuclear membrane in the state of the cell division.1.4 MTT assay showed that elevated AFlq can promote the proliferation of SH-SY5Y cells.1.5 EdU assay showed that elevated AFlq can promote the proliferation of SH-SY5Y cells.1.6 FACS Calibur showed elevated AF1q resulted in an increase of S phase cells, G2 phase cells, and a decrease of Gl phase cellsin SH-SY5Y cells.2 AF1q promoted the proliferation of neural stem cells and inhibited the differentiation of neural stem cells.2.1Nestin immunofluorescence staining identified neural stem cells.2.2 RT-PCR confirmed that lenti-AF1q effectively enhanced theexpression of AF1q in neural stem cells.2.3 Elevated AF1q can promote cells size and numbers of the neural stem cells.2.4 RT-PCR confirmed that elevated AF1q effectively decreased theexpression of ngnl in neural stem cells. And elevated AF1q effectively decreased MAP-2 postive cells.3. The mechanism of AF1q activating Wnt signaling pathway.3.1 TOP-Flash & FOP-Flash assay showed elevated AF1q can activate Wnt signaling pathway.3.2 Co-IP assay showed that AF1q can combination with TCF73.3 Western blot showed that elevated AF1q can promote the expression of TCF7 protein and maintain stability of TCF7 protein.3.4 Elevated AF1q can promote the expression of TCF7 protein in nucleus.3.5 EdU assay showed that TCF7-shRNA plasmid can dismiss theproliferation of cell by AF1q.3.6 The combination site between AFlq and TCF7 was located in 11-20aa detected by Co-IP assay.3.7 The relationship between phosphorylation-AF1q and TCF7.3.7.1 Western blot detected the expression of AF1qand pAF1qS11F.3.7.2 Western blot showed that pAF1q protein had stronger stability than pAF1qS11F protein.3.7.3 TOP-Flash&FOP-Flash assay showed that pAF1qS11F had weakenesseffect to activate the Wnt signaling pathway compared with pAF1q.3.7.4 EdU assay showed that pAF1qS11F had weakenesseffect to promote cell proliferation compared with pAF1q.Conclusion:1. AF1q can promote the proliferation of SH-SY5Y cells.2. AF1q stimulates the proliferation of neural stem cells and inhibited the differentiation of neural stem cells.3. AF1q activated Wnt signaling pathway through interaction with TCF7.4. The binding site between AF1q and TCF7 was located in 11-20 amino acids.5. The phosphorylation of AF1q11 site was the key functional site.