The Effect Of AMDA10 Deletion On Processing Of APP In Adult Murine Brain

Aims:ADAM10 is a member of A Disintegrin And Metalloproteinase family.Up to date,many studys have confirmed that ADAM10 is the genuine constitutive ?-secretase and participates in APP processing in neurons.There are two APP processing pathways.One is called “amyloidogenic pathway”,in which the amyloid precursor protein(APP)is sequentially cleaved by ?-secretase/BACE1 and ?-secretase to produce A? peptides,including the most abundant isoforms A?1-40 and A?1-42.The amyloid-?(A?)peptides represent the core components of senile plaques(SP).The over-production and accumulation of A? peptides is thought to be the main cause of Alzheimer's disease(AD).The other processing way is called “none-amyloidogenic pathway”,which is the main way of APP processing.In this way,APP is cleaved by ?-secretase and ?-secretase to produce sAPP?,P3 peptides and intracellullar domain AICD.Importantly,the site of APP ?-secretase cleavage occurs in the center of the A? peptides sequence between Lys-16 and Leu-17.Thus,?-secretase can preclude A? generation.ADAM10 is a candidate of ?-secretase and is also a key enzyme in the metabolism of APP.However,the effect of ADAM10 gene deletion in neurons of adult mice is not completely clear.Therefore,we established an conditional ADAM10 gene knockout(ADAM10 cKO)mice,which can survive to adulthood,to study the effect of ADAM10 gene deletion on APP metabolism.Methods:Genotypes of transgenic mice were determined by PCR.Homozygous ADAM10 cKO mice were used as experimental groups in the following experiments.ADAM10Fl/Fl mice were used as controls.Analysis of APP processing was performed by western bloting using cortex and hippocanpus lysis of 12 month old ADAM10 cKO mice(n=4)and control mice(n=4).To make the effect of ADAM10 gene deletion on APP metaboliasm clear,the amount of FL-APPtotal,mAPP,imAPP,sAPPtotal,sAPP?,s APP?,CTF?,CTF?,A?total,A?37,A?38,A?39,A?40,and A?42/43 were tested in this study.Levels of A?40 and A?42,in cortex and hippocampus of 12 moth old ADAM10 cKO mice and controls,were detected by using ELISA kits,respectively.The grey value of Western bloting exposure was analysed by Image J software.The significance was calculated by Student's t test using SPSS software.Results:Western blotting shows that Compared with the control mice,the amount of sAPP total and FL-APP,including mAPP and imAPP,have no obvious changes in cortex and hippocampus of ADAM10 cKO mice(n=4,P>0.5).However,the amount of A?total in ADAM10 cKO mice has increased by 30.98%(n=4,P<0.01)and 58.97%(n=4,P<0.001)in cortex and hippocampus compared with controls,respectively.Levels of A?37,38 and 39 in the brain of ADAM10 cKO mice have no obvious changes compared with controls(n=4,P>0.05).Levels of A?40 in cortex and hippocampus of ADAM10 cKO mice have increased by 9.60%(n=4,P<0.05)and 17.91%(n=4,P<0.01)compared witn controls,respectively.Levels of A?42/43 in cortex and hippocampus of ADAM10 cKO mice have increased by 31.70%(n=4,P<0.01)and 76.93%(n=4,P<0.001)compared witn controls,respectively.At the same time,in cortex and hippocampus of ADAM10 cKO mice,the amount of sAPP? has decreased by 8.38%(n=4,P<0.001)and 10.15%(n=4,P<0.01),while the amount of sAPP? has increased by10.31%(n=4,P<0.05)and 12.54%(n=4,P<0.001)compared with controls,respectively.The amount of CTF?(C99)in cKO cortex and hippocampus has increased by 16.64%(n=4,P<0.01)and 21.55%(n=4,P<0.001),and the amount of CTF?(C83)has decreased by 13.98%(n=4,P<0.01)and 18.93%(n=4,P<0.01)compared with the controls,respectively.ELISA shows that levels of A?40 in the cortex and hippocampus of ADAM10 cKO mice have increased by 17.91%(n=4,P<0.05)and 39.53%(n=4,P<0.01),and levels of A?42 in the cortex and hippocampus of ADAM10 cKO mice have increased by 55.49%(n=4,P<0.01)and 70.96%(n=4,P<0.001),which are in accordance with the results of Western blotting.Conclusion:The results show that the deletion of ADAM10 gene has caused a decrease of theamount of sAPP? and CTF?,while the amout of sAPP? and CTF?,the products of?-secretase,have increased.At the same time,the amount of A?total has also increased,for example,A?40 and A?42 have increased at different extent.But the deletion of ADAM10 gene has no significant effect on FL-APP and sAPPtotal productions.The conditional knockout of ADAM10 gene in neurons decreased ?-secretase products and increased ?-secretase products directly,but APP and sAPPtotal had no obvious changes.That is to say,ADAM10,as an ?-secretase,has a great effect on APP processing,and to a certain extent,there is a competitive relationship between ?-and?-secretase.