Aquaporin1 Silencing Activates The Wnt Signaling Pathway To Improve Cognitive Function In Mice With Alzheimer’s Disease

Background/Aim:Alzheimer’s disease（AD）is the most common form of dementia affecting a growing population of elderly individuals so that it lays a heavy burden on society and family.AD is a neurodegenerative disease,characterized by loss of neurons accompanied by astrocytosis in the cerebral cortex,AD which is associated with cognitive dysfunction and dementia,and irreversibly damages the brain normal ability to gain memory and make decisions.Promoting neurogenesis is a potential strategy for improving cognition function related to AD.Changing protein homeostasis is a prominent feature of AD,showing abnormalities in protein aggregation.Thus,the investigation to find a possible molecular target of protein might help us find a novel potential treatment for AD.Aquaporins（AQPs）are a family of small,integral transmembrane proteins forming water channels,existing in a variety of subcellular membranes of prokaryotic as well as eukaryotic cells.AQP1,the first discovered water channel protein,was found by Benga group in 1985.AQP1 has been demonstrated to be expressed in epithelial cells of the choroid plexus participate in the formation of cerebrospinal fluid and brain water homeostasis.AQPs are regarded as potential treatment of neurological conditions.More importantly,the astrocytic water channel proteins AQP1 is recognized to be altered in brains,affected by several neurodegenerative disorders.Moreover,it has been documented that AQP1 play a crucial role in pathogenesis of various diseases including AD and Parkinson’s Disease.Thus,attempts were carefully made in the current study to investigate the potential association among AQP1 and AD.It is widely known that the formation ofβ-amyloid（Aβ）is an initiating factor leading to AD,Aβable to induce Wnt signaling pathway has been proven.Thus,we speculated that the Wnt signaling pathway might play a crucial role in AD.Besides,increasing evidence supports a key role for Wnt signaling a possible therapeutic target in the treatment of AD.Therefore,we inferred that AQP1 could affect cognitive function in AD via Wnt signaling pathway.Our findings uncovered a paramount role of AQP1 by activation of the Wnt signaling pathway in the pathogenesis of AD,in a bid to offer a novel target for disease treatment.Methods:1.Microarray-based gene expression analysisAD-related gene expression profiles（GSE28146 and GSE18309）along with the expression matrix and annotated documents were retrieved from the Gene Expression Omnibus（GEO）database.Affy package was used for background correction,normalization and preprocessing on expression matrix in R language.Differentially expressed genes（DEGs）were analyzed and selected using limma package.DEGs were identified and recorded.Jvenn was used to compare DEGs between GSE28146 and GSE18309.Disease Gene Search Engine with evidence Sentences（DiGSeE）was applied to search AD-related genes.The protein-protein interaction network（PPI）was constructed after analyzing the interaction between AD-related gene and DEGs in String.The network was visualized through Cytoscape 3.6.0 software.2.Establishment of transgenic mouse model of ADA total of 80 healthy adult male NIH mice（aged 9 weeks,weighted 18-22 g）were provided by Guangdong Experimental Animal Center（Guangzhou,China）,70 of which were used to establish AD mouse models.Mice were injected with Aβ_1-42 solution（2μL,2g/L）in the bilateral hippocampal CA1 injection point.Another 10 mice were treated with an equal volume of normal saline as control.Mice were raised in laminar flow cabinets under specific pathogen free（SPF）conditions.The experiments involving animals were performed in compliance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.3.Construction of lentiviral vectors and animal groupingLentiviral vectors of pSIH1-H1-copGFP and pLV-EGFP-N were purchased from Shanghai GenePharma Co,Ltd.Lentiviral shRNA targeting AQP1（sh-AQP1）and lentiviral AQP1 overexpression vector（oe-AQP1）were constructed.High-Titer lentiviral particles packaged in vitro were injected into the hippocampus.Mice were transfected with oe-AQP1,oe-NC,sh-AQP1 and sh-NC respectively.4.Step-through testThis experiment was a mice behavioral experiment,which was carried out 11 to 12days after surgery.The mice were placed in the test chamber for 3 min,and then placed in the illuminated room.Those mice who subjected to electric shock when they entered the dark room were extracted and the test was repeated after 24 h.The latency（the first shocked time within 300 s）and numbers of errors（electric shocks）were recorded.5.Water mazeThe learning trials were conducted over 5 days,with 4 trials per day.After mice were sequentially released from 4 designated points,the time taken to reach the platform（latency）was recorded.On the 6th day,the platform was withdrawn.Times that the mice entered the original platform quadrant within 1 min was recorded.6.Reverse transcription quantitative polymerase chain reaction（RT-qPCR）Total RNA from hippocampus tissues and cells was extracted using Trizol.The concentration and purity of RNA were measured by Nanodrop 2000 spectrophotometer.Reverse transcription was performed to generate cDNA according to the instructions of TaqMan MicroRNA Assays Reverse Transcription primer.The synthesis of primer of AQP1 was conducted by TaKaRa.Real-Time Quantitative fluorogenic PCR assay was developed using ABI7500 PCR instrument.Glyceraldehyde-3-phosphate dehydrogenase（GAPDH）was used as an internal reference.The relative expression of target genes was measured by previously established 2^-ΔΔCt method.7.Western blot analysisTotal proteins from tissue and cells were extracted by whole cell lysis buffer and quantified using BCA method.50μg of protein from each sample was electrophoresed by 12%SDS PAGE and transferred to PVDF membranes.The PVDF membranes were incubated overnight at 4℃ with the primary antibody.Then the PVDF membranes were washed three times with TBST,and incubated with the secondary antibody at 37℃ for 1h.Protein expression was visualized with a chemiluminescence ECL kit.GAPDH served as a loading control,and band intensity was quantified using Imagine J software.The image was photographed with Bio-Rad image analysis system（Bio-Rad,Hercules,CA,USA）and subjected to gray scale analysis using Quantity One v4.6.2 4.0（Media Cybernetics,USA）.The relative ratio was calculated with GAPDH as the internal reference.8.Hematoxylin-eosin staining（HE staining）After mice were anesthetized with 3%sodium pentobarbital,the hippocampus tissues were taken for gradient ethanol dehydration,xylene transparent and paraffin embedding.Slices was dewaxed,dehydrated,stained with hematoxylin and eosin respectively followed by the dehydration,transparentization and mounting.9.ImmunohistochemistryThe slices were incubated with methanol H₂O₂,washed with distilled water and PBS,followed by the addition of normal goat serum blocking buffer at room temperature.Then,the slices were incubated with antibody to caspase-3 overnight and supplemented with goat anti-rabbit IgG followed by an incubation with horseradish peroxidase-labeled streptavidin.Diaminobenzidine（DAB）was used to develop the color.Reaction time was controlled under the microscope.The slices were counterstained with hematoxylin for 20s.The mounted sections were observed under the microscope.10.Isolation and culture of hippocampal neuronsHippocampus of 1-day-old mice were isolated,cut into pieces with 0.4 mm diameter and treated with trypsin and DNase.After cells were dispersed,the cell suspension was cultured with minimal essential medium（MEM）supplemented.Filter liquor was transferred onto a culture dish of 12 mm diameter with poly-L-lysine-coated coverslip and cultured in an incubator at 37℃with 5%CO₂ and 95%O₂ with 0.6×10⁹/L cell density.On the 3th day,5μmol/L cytarabine was added to the culture dish.And after 24h,it was changed to Dulbecco’s modified Eagle medium（DMEM）culture solution containing 10%FBS and penicillin/streptomycin.On the seventh day,immunocytochemical staining for neuron-specific enolase（NSE）of hippocampal neural cells was performed using immunocytochemical staining for substance P（SP）.11.Hippocampal neural cells grouping and treatmentAfter 7 days of incubation,a set of hippocampal neural cells were cultured with a in serum-free medium supplemented with 5μmol/L Aβ_1-42 Oligomers for 24 h.With pretreatment with 0.5 nmol/L Wnt3a protein for 2 h.a set of hippocampal neural cells then were cultured with a in serum-free medium supplemented with 5μmol/L Aβ_1-42Oligomers for 24 h.With pretreatment with 1.0μg·m L-1 dickkopf-1（Dkk1）protein for2 h,another set of hippocampal neural cells were cultured in a serum-free medium supplemented with 5μmol/L Aβ_1-42 Oligomers for 24 h.To brief,neurons were treated with Aβ_1-42（the Aβgroup）,Wnt pathway inhibitors（the Dkk1+Aβgroup）and Wnt pathway stimulator（the Wnt3a+Aβgroup）respectively.12.Terminal deoxynucleotidyl transferase（TDT）-mediated dUTP-biotin nick end-labeling（TUNEL）Slices were stained according to the instructions of the apoptosis kit.Sections were observed and photographed under the microscope.Twenty high-power fields randomly selected from each slice with 400 cells per field were chosen to count the apoptosis index（AI）.13.Statistical analysisThe data were processed using SPSS 19.0 statistical software（IBM SPSS Statistics,Chicago,IL,USA）.Measurement data were expressed as mean±standard deviation.Unpaired data in compliance with normal distribution and homogeneity between two groups were compared using unpaired t-test.Comparisons among multiple groups were conducted by one-way（ANOVA）with Tukey’s post hoc test.A value of p<0.05indicated significant difference.Results:1.AQP1 was highly expressed in AD.In order to find the DEG in AD,microarray-based analysis was conducted,showing that 757 and 770 DEGs were screened from GSE28146 and GSE18309 by using R language,and the top 400 genes of the two profiles were selected for comparison.Venn diagram was drawn,revealing 12 intersected genes.The top 20 AD-related genes were retrieved from DiGSeE.The PPI network of AD genes and DEGs were constructed.The results showed that AQP1 was the gene with highest relevance.The heatmap illustrating the expression of the top 100 DEGs in the GSE28146 was plotted and shown.It revealed a high expression of AQP1 in AD.Besides,the expression of AQP1 was displayed in GSE18309 suggested that the expression of AQP1 in patients with AD was significantly higher than that in healthy people.Thus,it was proved that the expression of AQP1increased in AD.Detection of AQP1 levels in hippocampal neurons in AD mice by RT-qPCR and western blot,we found that AQP1 expression was higher in AD mice than in the control group.2.AQP1 silencing improved cognitive function in AD mice.After 12 days of modeling,the AD group had shorter electric shock latency,increased number of errors,longer escape latency,and fewer times of crossings across the platform,compared with the sham group.RT-qPCR and Western blot analysis were applied to show the expression of AQP1 in AD mice,which showed that the expression of AQP1 was significantly higher in the AD mice than the mice in the sham group.Thus,the aforementioned results indicated that AQP1 was highly expressed in AD mice.3.Silencing AQP1 had protective effect on hippocampal neurons in AD mice.The results showed AQP1 silencing improves cognitive function in AD mice by Step-through test and Water maze.The results of RT-qPCR and Western blot analysis showed silencing AQP1 could improve cognitive function in AD mice.4.It is suggested that AQP1 inhibited the Wnt signaling pathway.The results of HE staining analysis immunohistochemistry assay results indicated that silencing AQP1 could inhibit neuronal apoptosis in AD mice,maintain normal morphology of hippocampal neurons and protect hippocampal neurons.5.AQP1 promoted neuronal apoptosis by inhibiting the Wnt signaling pathway,thereby damaging the cognitive function of AD mice.AD Western blot analysis assay results indicated that the Wnt signaling pathway was inhibited in AD,and silencing AQP1 was able to activate the Wnt signaling pathway.Apoptosis of neurons was detected by TUNEL assay,which suggested that activating Wnt signaling pathway activity was beneficial to alleviate Aβ_1-42 induced neuronal apoptosis.Conclusion:1.AQP1 was highly expressed in AD.2.AQP1 silencing improved cognitive function in AD mice.3.Silencing AQP1 had protective effect on hippocampal neurons in AD mice.4.It is suggested that AQP1 inhibited the Wnt signaling pathway.5.AQP1 promoted neuronal apoptosis by inhibiting the Wnt signaling pathway,thereby damaging the cognitive function of AD mice.6.Silencing AQP1 enhanced cognitive function of AD by activating the Wnt signaling pathway,suggesting a potential therapeutic strategy for new targets of AD.