The Study Of Memory Related Gene Kibra Inhibits Amyloid ?-induced Neuronal Apoptosis

Background:Alzheimer's disease(AD)is a neurodegenerative disease that hazard to human health,characterized by progressive cognitive deficits,with the decline and loss of daily living capacity.The WHO and Alzheimer's Disease International shows that about 47 million people worldwide suffer from dementia,which is expected to 1 billion by 2050.AD has been one of the worldwide health issue threatening human health.Recently,a series of attempts at therapeutically targeting AD have not been successful.The reason may be that the underlying mechanisms of AD are still unknown.Thus,it is significance for the treatment of AD to look for effective targets that can delay the development of AD.The gene KIBRA was first described in 2003 and the name was given for its predominant mRNA expression in kidney and brain.The KIBRA gene,a member of the WWC family of proteins that encodes postsynaptic skeletal protein,is highly expressed in the kidney(glomerular podocytes,tubules and the collecting ducts)and memory-related brain regions(such as the hippocampus and cortex).KIBRA has recently been first shown to be significantly associated with human memory performance through genome-wide single nucleotide polymorphism screening.Thus,KIBRA is also known as memory-related gene.Memory impairment is one of the most important clinical features of AD,KIBRA becomes another important candidate gene for AD.And KIBRA,a postsynaptic scaffold protein that connecting cytoskeletal and signaling molecules,is involved in a variety of cellular function,such as cell polarity and migration,vesicle transport,transcriptional regulation,especially playing an important role in synaptic plasticity and memory formation.KIBRA knockdown accelerates the rate of AMPAR recycling following Nmethyl-D-aspartate receptor induced internalization.Genetic deletion of KIBRA in mice impairs both long-term depression and long-term potentiation at hippocampal Schaffer collateral-CA1 synapses.Moreover,KIBRA knockout mice have severe deficits in learning and memory.It is well known that the accumulation of amyloid-?(A?)plaques and the formation of neurofibrillary tangles(NFTs)play a core role in the pathogenesis of AD.The abnormal deposition of A? in the brain could induce synaptic degeneration,oxidative stress,inflammation,neuronal degeneration and apoptosis,and ultimately lead to the occurrence and development of AD.In Drosophila,KIBRA,a new autophagy regulator;,is required for autophagy to function properly.The absence of KIBRA caused defectsin the formation of autophagic vesicles and autophagic degradation.Once the autophagic vesicles produce or release obstacles,leading to a large numberof false folding protein abnormal accumulation and cells apoptosis.Therefore,the lack of KIBRA and cell apoptosis are closely related.Current study has confirmed that KIBRA is related to AD.A?-induced neuronal apoptosis plays an important role in the pathogenesis of AD,suggesting that KIBRA may be involved in A?-induced neuronal apoptosis,so our study mainly focuses on the molecular mechanism of KIBRA in AD.Aims:1.To study the specific expression pattern of KIBRA in APP/PS1 mouse.2.To investigate the crucial role of KIBRA on neuronal apoptosis.3.To study the mechanism of KIBRA on A?-induced neuronal apoptosis.Methods1.AnimalsWe used APPswe/PSEN1dD9(APP/PS1)transgenic mouse and KIBRA-knockout(KIBRA-KO)mouse.APP/PS1 transgenic mouse is the most widely used animal model in the study of AD which was purchased from the Jackson Laboratory.KIBRA-knockout mouse is ideal animal model to study the biological functions of KIBRA,which was gift from Dr.jixin Dong.And we employed the littermate with wild type as the control.Mice were housed with a 12/12 hr light/dark cycle and with ad libitum access to food and water.2.Behavioral testDifferent age of APP/PS1 mice were treated with Morris water maze to test cognitive function.Briefly,mice were trained to find a submerged escape platform in an open swimming arena.Each trial lasted 60 seconds with an additional 30 seconds learning time where mice were allowed to remain on the platform.After 5 days learning,mice were subjected to a probe test in which the platform was removed.Mice were analyzed for escape time(latency),exploration distance,target quadrant exploration time and distance and the number of times they passed.Behavior data was analyzed using Smart 3.0 software.3.Cell culture and transfectionMouse hippocampal neuronal cell line(HT22)was obtained from University of California,Los Angeles.Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10%fetal bovine serum,100 U/ml penicillin and 100 ?g/ml streptomycin at 37 ? in humidified atmosphere containing 5%CO2.Cells were passaged,cryopreserved or treated according to the growth of cells.Cells transfection:The lentiviral vector were transfected to HT22 cells in good conditions.Cells were cultured for 12-24 h and then replaced with complete medium.After 72 h of infection,the expression of GFP was observed under fluorescence microscope.4.A?1-42 oligomers preparationA?-42 oligomers were prepared as described by Professor William J.Bowers in Rochester medicine center.In brief,human synthetic A?1-42 was suspended in hexafluoroisopropanol to reach 1 mM and stored as a dried film at-20? after evaporation of solvent using a Speed-Vac.To form A?1-42 oligomers,peptide films were re-suspended to 5 mM in dimethyl sulfoxide(DMSO).Samples generated above were allowed to proceed for 24 h at 4? and then further diluted with cold PBS to 11.1 nM before incubation for 2 weeks at 40C.And prior to use,the oligomer solution was centrifuged at 4?,13,000 rpm for 10 min to remove foreign substance.5.ImmunohistochemistryTissues were fixed and embedded in paraffin blocks.The following primary antibodies:rabbit anti-?-Amyloid,1:400;mouse anti-Tau,1:800;rabbit anti-P-Tau Thr181,1:400;rabbit anti-KIBRA,1:100;application of DAB system.Antibodies used in the experiments:mouse anti-beta ??Tubulin,1:1000;rabbit anti-KIBRA,1:100;Alexa Fluor 488 conjugated goat anti-rabbit IgG and Alexa Fluor 594 conjugated goat anti-mouse IgG.TUNEL assay for detection and quantification of apoptosis(programmed cell death)at single cell level,based on labeling of DNA strand breaks(TUNEL technology).All images were analyzed by Image J software.6.Western blotProteins in the mouse tissues homogenate were extracted with RIPA buffer.Equal amounts of protein lysed in RIPA were separated on 8%,10%or 12%SDS-PAGE gels.The membranes were blocked with 5%milk and then incubated with primary antibodies.Western blotting was employed to determine the protein levels of KIBRA,Tau,P-Tau Thrl81,Tuj1,PARP,caspase 3,Akt,P-Akt Ser473,P-ERK1/2 Thr202/Tyr204,ERK1/2,P-PKC?/?Thr410/403,PKC?,?-actin.Image J software was used to analysis gray scale of western blot bands.7.Statistical analysisStatistical analysis was performed by GraphPad Prism 5.Data were expressed as means± SEM.Two-way ANOVA was used to compare differences between multiple groups.And t-test was used to compare differences between two groups.Differences were considered significant when p<0.05.Results:1.Cognitive function,A? deposition and tau protein level in different ages of APP/PSl mice.Morris water maze showed that the spatial learning and memory ability in 9-month-old and 12-month-old of APP/PS1 mice were significantly decreased than those of the control mice(p<0.05,p<0.01).Compared with the control groups,more A? deposition was observed in the brain of 5-month-old,9-month-old and 12-month-old APP/PS1 mice by immunohistochemical staining.The expression of p-Tau Thrl81 and total Tau in the brain of 12-month-old of APP/PS1 mice were significantly higher than those in the control group(p<0.01,p<0.05).2.The region-specific expression of KIBRA in APP/PS1 mice brain.Compared with the control groups,the expression of KIBRA in the hippocampus,both in the CA1 and CA3,was significantly decreased in 9-month-oldand 12-month-old APP/PS1 mice,especially at 12-month of age(p<0.01,p<0.001).Western blot confirmed that the expression of KIBRA in hippocampus was significantly reduced(p<0.001),while no difference of KIBRA expression in cortex(p=0.0937).Moreover,KIBRA mainly localized in neurons and the proportion of KIBRA+/total neurons(Tuj1+)was significantly reduced in the hippocampus of APP/PS1 mice by immunofluorescence staining(p<0.05).While more KIBRA positive staining was observed in the cortex of APP/PS1 mice(p<0.01).KIBRA expressed the region-specific and age-dependent decreased in the hippocampus of APP/PS1 mice,suggesting that KIBRA expression was closely related to learning and memory impairment of AD.3.Unique expression pattern of KIBRA in the enteric nervous system of APP/PS1 mice.A? plaques and NFTs were not only deposited in the brain of AD patients,but also deposited in the bowel of AD patients,which could cause mitochondrial dysfunction,oxidative stress,loss of neurons,and intestinal nerve dysfunction.The immunofluorescence staining of A? showed significantly higher amyloid plaque burden in APP/PS1 mice enteric canal(p<0.001).We examined the distribution of KIBRA in the enteric nervous system(ENS)with novel whole-mount staining method.Compared with the control group,KIBRA positive staining was significantly increased in the ENS of 12-month-old APP/PS1 mice(p<0.05).Western blot result showed the increased level of KIBRA in the intestinal tissue of APP/PS1 mice compared with the controls(p<0.05);however,there was no difference of Tuj1 expression levels between the two groups(p=0.9023).Different from the region-specific decreased of KIBRA in the hippocampus of APP/PS1 mice,higher expression of KIBRA was observed in the ENS of APP/PS1 mice.4.The indispensable role of KIBRA in the neuron apoptosis.We firstly performed the TUNEL assay on the brain samples.Compared with the wild type mice,a higher number of TUNEL positive neurons was observed in both hippocampus area and cortex area of APP/PS1 mice brain(p<0.01,p<0.01).Similarly,4-month-old KIBRA-KO mice brain showed significantly increased TUNEL positive neurons(p<0.05),suggesting that KIBRA plays an indispensable role in the neuron apoptosis.5.The protective function of KIBRA in cell proliferation and against A?-induced neuron apoptosis.The mice hippocampal neuronal cell line-HT22 cells were used to establish KIBRA knockdown and overexpression cell models,respectively.Interestingly,we found that KIBRA knockdown significantly suppressed proliferation at 96 h after seeding(p<0.001),whereas overexpressing of KIBRA stimulated cell proliferation(p<0.01).Taken together,these data revealed that KIBRA was a positive regulator in cell proliferation.We treated HT22 cell with A?1-42 oligomers to establish A? neurotoxic cell model.CCK8 and western blot result showed that 1 ?M AP1-42 oligomers induced significant reduction of cell viability(p<0.01),higher levels of apoptosis-related proteins(activated caspase 3 and cleaved PARP)in CRISPR KIBRA cells than that of the control(p<0.05,p<0.01),whereas there was an apparent reduction in apoptosis-associated signal molecules in KIBRA overexpression cells(p<0.05).These data clearly indicated that KIBRA significantly suppressed A?-induced neuron apoptosis,further promoted cell survival.6.The function of KIBRA on against A?-induced neuron apoptosis through targeting key downstream effectors of Akt signalingWe screened several survival-related signaling pathways.In the CRISPR control group,the phosphorylated Akt(p-Akt)Ser473 rapidly activated with peak time at 1 min with 1?M A?-42 oligomers treatment;however,the p-Akt Ser473 level delayed its peak time at 2 min in the CRISPR KIBRA cell(p<0.05).Both the KIBRA overexpression group and control group quickly reached their peak time of p-Akt Ser473 levels at 1 min,whereas the overexpression of KIBRA significantly increased the level of p-Akt Ser473 1 min after the treatment of 1 ?M A?-42 oligomers(p<0.01).The prior treatment of a specifically allosteric Akt inhibitor-MK2206 significantly inhibited p-Akt Ser473 signaling(p<0.05)after treatment with A?1-42 oligomers.Furthermore,MK2206 leaded to higher level of cleaved PARP compared with vehicle-treated control in KIBRA-overexpressed cells(p<0.05).The prior treatment of MK2206 significantly increased cell death(p<0.05),suggesting that KIBRA inhibited neuron apoptosis by targeting key downstream effectors of Akt signaling pathway.Conclusions:1.KIBRA expressed the region-specific and age-dependent decreased in the hippocampus of APP/PS1 mice,suggesting that KIBRA expression was closely related to learning and memory impairment of AD.Therefore,KIBRA,a memory related gene,plays an important role in the development and progression of AD.2.KIBRA also plays an indispensable role in the neuron apoptosis.3.Our data suggest that KIBRA functions as a neuroprotective gene in promoting neuron survival and inhibiting A?-induced apoptosis.Understanding the significance of KIBRA could contribute to the ongoing efforts to develop novel therapies in AD.