The Protective Effects Of The Exogenous Brain-derived And Neurotrophic Factor On Alzheimer’s Disease

Objective: Alzheimer’s disease(AD)is a common neurodegenertive disease in old people,and is the most common reason for dementia.The pathogenesy of AD is not very clear.The classic pathologic characteritics of AD are senile plagues(SP),neurofibrillarytangle(NTF)and the selective neuron lossing.In addition,NTF is closely rellated to the degree of dementia.NTFs mainly composed of double helix fibrillary,which consists of abnormally hyperphosphorylated tau protein.Tau protein is a kind of phosphatide protein which normally presents in the axon and cytoplasm,blonging to the microtuble-associated protein family.It mainly promotes the microtuble protein assembling into microtuble,and enhance its stability,maintain the growth and development of cell.The activity of tau is modified by phosphorylation and dephosphorylation.Protein phosphatase 2A(PP2A)is the most importment phosphoesterase in our brain that can make tau dephosphorylate.It is reported that the activity and expression of PP2 A is down regulated in the AD’s brain,and inhibition of PP2 A in many modles can induce the abnormally hyperphosphorylation and aggregation of tau like AD.The reducing of methylation at L309 or phosphorylation at Y307 of catalytic subunit of PP2 A both can induce the hyperphosphorylation of tau.Many studis shows that the abnormally hyperphosphorylation of tau is the early stage of AD,so inhibition of tau hyperphosphorylation is the key to prevent and heal AD.Brain-derived neurotrophic factor(BDNF)is a kind of alkalinous protein which was derived from pig brain by Barde ect.al in 1982.The positive immunity cell of BDNF are widely distributed in the brain,such as cerebral cortex,hippocampus,basal forebrain,corpus striatum,septal area,hypothalamus and cerebellum,but the most concentrated area is the pyramidal cell layer of hippocampus CA2 and CA3 driction and the granular cell layer of dentate gyrus.BDNF can improve many kinds of neurons to surivive and develop;enhance the activity of antioxidase to against the free radical;prevent the cell appoptosis by impressing the cytotoxicity of excitatory amino acids;and it is very importment for cognition,learning,and memory formation.While,the expression of BDNF and it’s receptor are significantly decreased in the related area of AD’s brain.At present,studyshows that neurodegentive dementia,like AD,is closely related to the lack of axonal transport of neurontrophins,and is related to the imblance and defunction of neurontrophins.Here we make exogenous BDNF deal with the SH-SY5 Y cell which was treated with Okadaic acid(OA),a kind of inhibitor of PP2 A,and make brain injection at the same time,to study the effects of BDNF on tau hyperphosphorylation,cell appoptosis and action of rat’s which were induced by OA,and research the possible mechanism.Methods:Animal experiment: 60 healthy adult male Wistar rats were derived into 6 groups at random:(1)Control group(n=10);(2)Sham group(n=10);(3)OA group(n=10);(4)BDNF group(n=10);(5)PI3K inhibitor group(n=10);and(6)TrkB inhibitor group(n=10).Establish the AD animal model by hippocampus stereotaxis injection methods : 2μl Sodium Chloride was injected into the double hippocampus of sham operation group;2 ul OA(0.2 μmol/L)was injected into the OA group;2 μl OA(0.2 μmol/L)+2 μl BDNF(50 ng/ml)for BDNF group;(0.2 μmol/L)+2 μl BDNF(50 ng/ml)+2μl LY294002 for PI3 K inhibitor group;and 2μl OA(0.2 μmol/L)+ 2μl BDNF(50 ng/ml)+2 μl K252 a for trkB inhibitor group.Examine the successful model through Morris water maze 24 h after the injection.Choose 3 rats each group for TUNEL stain to examine the cell appoptosis,and for immunohistochemistry study to examine the expression of caspase-3,bcl-2,bax;the other 3 rats were used for Western blot to examine the expression of tau-1、tau-5、PHF-6、PP2A、PP2Ac-Yp307、GSK3β、PI-3K、AchE and for RT-PCR to examine the expression of BDNF mRNA、trkB mRNA、SYN mRNA.Cell culture:SH-SY5 Y cell was treated with OA(40 nmol/L)for 24 h,then BDNF,LY294002 and K252 a was added into it for 15 min,and then was collected for immunofluorescence and MTT and flow cytometer.Results:1.Morris Water Maze experimentDelitescence of OA and trkB inhitibor group is prolonged compared with normal control and sham operation group,and diminish in BDNF and PI3 K inhibitor group compared with OA and trkB inhibitor group,but it is still longer than normal control and sham operation group(p﹤0.05).2.TUNEL stain of hippocampusThe positive cell is rearly seen in the CA3 region of hippocampus of normal control,but increased obviously in OA,PI3 K and trkB inhitibor group;the possitive staining was diminished in BDNF group but still more than the normal obviously(p﹤0.05).3.Caspase-3 immunohistochemistry of hippocampus.There area a few caspase-3 positive cells in hippocampus CA3 region of normal control group,but there are significant increasing in the OA,trkB inhitibor and PI3 K inhibitor group.BDNF can make it decrease,but still more than the normal(p﹤0.05).4.The expression of synaptophysin in hippocampusA great deal of synaptophysin is expressed in CA3 region of normal control gruop,but obviously less in the OA,PI3 K inhibitor and trkB inhibitor group(p﹤0.05);while BDNF can promote it’s experssion nearly normal.5.The expression of BDNF mRNA,trkB mRNA and SYN mRNA in hippocampus.The expression of BDNF mRNA,trkB mRNA and and SYN mRNA is obviously decreased in OA and trkB inhibitor group compared with nornal control(p﹤0.05),but in BDNF group,they nearly reach the level of normal control.6.The phosphorylation detection of tau protein in hippocampusThere is only microamount hyperphosphorylated tau protein was detected AT8(p-Ser199/202),PHF-1(p-Ser396/404),PHF-6(p-Thr231)in normal control,but it is increased obviously in OA,PI3 K inhitibor and trkB inhibitor group,exogenous BDNF can decrease the phosphorylation of above-mentioned situs of tau protein;the expression of tau-1(Ser199/202)in OA group is less than BDNF group and normal control(p﹤0.05),there is no difference between BDNF group and normal control(p﹥0.05);and there is no difference of expression among all the groups in tau-5(total tau)(p﹥0.05).7.Tau phosphorylation and it’s enzyme in rat’s brain.The expression of PP2A、p-AKT is diminished obviously in OA,PI3 K inhibitor and trkB inhibitor group compared with normal control,but PP2Ac-Yp307 、 GSK-3βincreased obviously;the expression of PP2Ac-Yp307 and GSK-3β is diminished while PP2 A and p-AKT is increased in BDNF group(p﹤0.05).8.Tau protein phosphorylation and correlated enzyme in SH-SY5 Y cell.The detected phosphorylated situs of cells of each group is the same as the rats measurement situs,the results of tau protein phosphorylation expression is the same as results of rat’s hippocampus,but the contribution of BDNF is cancelled when GSK-3βinhibitor was used.9.The immunofluorescence detection of AT8 and PP2Ac-Yp307 in SH-SY5 Y cellsThere is rarely positive expression of AT8 and PP2Ac-Yp307 in normal control cells,but there’s a large amount of positive expression of AT8(cytoplasm green fluorescence)and PP2Ac-Yp307(cytoplasm red fluorescence)in OA group,and the both expression is highly accompanied.The fluorescence of BDNF group is attenuated compared with OA group.10.MTT experiment detect cell survival rate of SH-SY5 Y cells.The survival rate of OA treated cells is lower than normal control,while BDNF group is higher.11.Flow cytometry detect apoptosis after PI simple stain.There is a little apoptosis in normal control,apoptosis rate raise obviously in OA group,and BDNF can decrease the apoptosis rate.Conclusion:1.Injecting OA can induce hyperphosphorylation of sensitive situs AT8、PHF-1、PHF-6 of tau protein,exogenous BDNF can make it decrease.2.The increase of phosphorylation of catalytic subunit of PP2 A reduce it’s activity,and increased of GSK-3β after injecting OA;BDNF inhibits the activity of GSK-3βthrough activiting PI3K/AKT pathway,at the same time it reduce the phosphorylation of catalytic subunit and increase the activity of PP2 A,that decrease the phosphorylation of tau protein.3.The ability of stereo-spatialization of rats was injured obviously after stereotaxis injection of OA.Exogenous BDNF can improve its ability through dehyphosphorylateing tau,inhibiting the activity of GSK-3β to reduce the expression of caspase-3,which can reduce the cell apoptosis,increasing the expression of BDNF mRNA,trkB mRNA,SYN mRNA,and other many ways.