Study On The Expression Of Tau's Phosphorylation At Ser396 And Thr205 Sites Caused By Chronic Ethanol Exposure

Objective:With the rapid development of society,people's living standard is gradually improving.The consumption of alcoholic beverages is increasing.Extensive drinking seriously damages public health.A series of social behaviors after drinking also produce social public safety problems.Long term drinking can cause multi system and multi organ damage,especially for central nervous system damage,the damage shows neurodegenerative symptoms,such as calculation ability decline,memory decline,slow reaction,cognitive impairment,and even alcoholic dementia.Tau protein is a microtubule associated proteins(MAPs)widely existed in the nervous system.It mainly exists in cytoplasm and axons of neurons,and also in other neurocyte.It plays a role in promoting microtubule formation and stabilizing microtubule structure,thereby maintaining normal axonal transport of nutrients and metabolites.At the same time,Tau protein is a necessary microtubule associated protein in the growth and development of the nervous system and axonal signal transduction.It plays a role in promoting the growth and development of neurons,and participates in the formation of polar neurons and the process of axonal growth.But when Tau protein is induced to be phosphorylated,its ability to combine with microtubule decline causing decreasing of microtubule stability,and easy to accumulate into a double spiral filament abnormal structure,leading to the normal axonal transport barriers,thereby affecting the morphology of neurons,damaging neuronal function,causing neurodegeneration.The abnormal phosphorylation sites of Tau protein are more,and the level of phosphorylation of Ser396,Ser202 and Thr205 loci in neurodegenerative changes is higher.AT8 is a PHF-Tau specific monoclonal antibody,identifying abnormal phosphorylated Tau(Ser202/Thr205).It is a commonly used marker of neuropathology,and also one of the anti Tau antibodies widely used in the evaluation of the Tauopathy.Under normal circumstances,protein phosphokinase and PPs(phosphatases PPs)play a direct role in the phosphorylation process of Tau protein.They have opposite functions to ensure Tau protein normal level and play Tau protein normal biological functions.Glycogen synthase kinase-3(GSK-3)and cyclin dependent kinase 5(CDK5)are the most important enzymes that directly induce Tau protein phosphorylation.P35 is one of the specific activators of CDK5.The protein phosphatases that inhibit phosphorylation of Tau protein,that is,promote its dephosphorylation,mainly includes PP1,PP2 A,PP2B and PP5.In the process of regulating the dephosphorylation of Tau protein,the relative activity of PP2 A is the highest.Therefore,PP2 A is the main phosphatase.The results of the research group showed that chronic ethanol exposure can cause intracellular calcium influx and overload,and inositol-1,4,5-triphosphosate receptor(IP3R)and N-methyl-D-aspartic acid receptor(NMDAR)expression,induced apoptosis of mouse nerve cells,the impaired spatial memory ability mouse.However,whether chronic ethanol exposure causes Tau hyperphosphorylation and which phosphokinase and/or phosphatase play a major role in the process needs further study.In order to study the Tau phosphorylation during chronic ethanol exposure,we used a chronic ethanol exposure animal model to observe the effect of ethanol on total Tau,phosphorylated Tau,the proportion of phosphorylated Tau and total Tau,GSK-3? activity,CDK5 activity and PP2 A activity in mouse hippocampus in order to elucidate the changes of total Tau and phosphorylated Tau level during chronic ethanol exposure and provide experimental data for chronic ethanol induced neurotoxicity.Methods:This study used animal experiment: This study selected 120 male C57BL/6 mice,according to the concentration of ethanol drinking were randomly divided into 3 groups: control group(no ethanol),10% ethanol group and 20% ethanol group;in accordance with the ethanol drinking time were randomly divided into 4 groups: 30 d,60d,90 d,180d group.A total of 12 groups,each group of 10 mice.The expression of Tau5,AT8,p-Tau(Ser396),d-PP2 A,CDK5,GSK-3?,p-GSK-3?(Ser9),p-GSK-3?(Tyr216),P35 was detected by Western blot.The expressions of Tau5,p-Tau(Thr205),p-Tau(Ser396)were detected by immunohistochemistry.Results:The results of immunohistochemistry showed that the intensity of p-Tau(Thr205)and p-Tau(Ser396)staining in each ethanol-drinking group was higher than that of the control group,the expression of p-Tau(Thr205)and p-Tau(Ser396)was enhanced and brownish yellow;Tau5 staining compared with the control group the expression of signal strength is basically unchanged,no obvious change;The expression of p-GSK-3?(Ser9)was significantly lower than that of the control group,and the expression of p-GSK-3?(Ser9)was weak and light brown.The results of Western Blot showed that the expression of AT8 and p-Tau(Ser396)increased gradually and the expression of Tau5 did not change significantly in all alcohol-drinking groups with ethanol concentration increasing.With the increase of ethanol concentration,The expression level of p-GSK-3?(Ser9)decreased gradually,and there was no significant change in the expression of d-PP2 A,CDK5,GSK-3?,p-GSK-3?(Tyr216),P35.Conclusion:1.Chronic ethanol exposure causes Tau hyperphosphorylation at Ser396 and Thr205 sites in the hippocampus cells of mice.2.Chronic ethanol exposure can reduce the phosphorylation of GSK-3? at Ser9 in the hippocampus cells of mice.3.Tau hyperphosphorylation at Ser396 and Thr205 sites caused by chronic ethanol exposure may be related to the increased activity of GSK-3?.