The Expression And Significance Of Protein 4.1 In The App Transgenic Mouse Brain Tissue

PURPOSEAlzheimer's disease(AD) is a progressivly degenerative disease of the neural system,seriously affact- ting the quality of life of older people and even their lives,with the aging of the world's population,the incidence of the process was rising.The main pathological features of AD is a large number of amyloid senile plaques(SPs) deposite between the neurons and neural fibrillary tangles(NFTs) in neurons of the brain.NFTs are insoluble compounds of the abnormal accumulation of neurons,mainly formainted by the hyperphosphorylation of protein Tau,which is a neuron cell skeleton protein.Studies showed that one major reason of AD was the abnormal metabolites of the protein APP,Which is namedβ-amyloid peptide(Aβ) depositting in the brain and neuron cells.Because Aβcunld produce toxic which effects on neurons and affact theirs degenerat-ion,or cause them to wither and death,especially the cortical neurons and neuron synapses of cholinergic receptor loss,reduction in cerebral cortical neurons,and caused cortical arterioles of the vascular amyloidosis.In recent years,the pathogenesis of the toxic mechanism of Aβ-related research on AD has been a hot spot and made great achievements,but has not yet been clarified,the clinical treatment is not ideal.Protein 4.1 is an essential element of membrane skeleton proteins,its family including protein 4.1R(red cell expression),4.1N(neuron-specific expression),4.1G (wide expression)and 4.1B(wide expression,the expression of brain-based),they have some of the same domain:SABD domain,FERM domain or the membrane binding domain and the CTD domain.4.1N was found expressed widely in the central nervous system and peripheral neurons,and were found in the mitosition of early embryonic neurons.Family protein 4.1 members not only have the functions of maintainning the cell membrane structure,regulating avariety of transmembrane protein function and activity of membrane-bound enzymes,but also play a key role in the procession of protein sorting,cell mitosion regulation and cell signal transduction.It was proved the neurotoxicity of Aβplays a critical role in the process of the occurrence and development of AD lesions.The over-expression of APP can produce excessive aggregation of neurotoxic and the APP gene mutations results in the changing in restriction sites,leading to the overexpression of Aβ.Research shows that brain protein 4.1 with neurofibrillary tangles have a total of positioning in AD patients,the former have a combination sequence(Y687 KFFEQMQN) with the protein APP in carboxy-terminal,which is close to the bit(Met671-Asp672) ofβ-secretase hydrolysis and the necessary anchoring fragment(Tyr653-664) of protein APP,andβ-secretase is considered to be the rate limiting enzyme of Aβproduce. Therefore,the 4.1 protein may affect the metabolism of APP and the generation of Aβ, There may be mutation or abnormal protein expression of 4.1 family gene existence in AD patients brain.In this study,we explore the relationship between protein APP and 4.1 protein family,also the relationship between the metabolites Aβof APP and 4.1 protein family by study the cell position,the level of protein and gene expression characteristics of 4.1 family members in APP transgenic mouse brain tissue,in order to explore the pathogenesis better prevention and treatment of of AD and other Aβ-related disease.METHODSWe extracted DNA of new born APP transgenic(human APP695/770) mice tail,using the PCR technology to detect the APP gene,according to whether APP transgenic mice carrying APP gene,the APP transgenic mice are divided into two groups:APP(+) group and APP(-) group.From 12-month-old APP transgenic mouse brain tissue to detect the expression of APP and Aβby immunohistochemical staining and Congored staining.We study in order to detecte the expression in protein and mRNA level of 4.1 protein family members in the hippocampus,cortex,and cytoplasm neural cells of APP(+) group and APP(-) mice by immunohistochemistry,Western-blot and RT-PCR technology.Statistic analysis:all the experimental results were analyzed by spss 10.0 software with independent samples t statistical methods test,the differences between the two groups in every brain regions to P＜0.05 indicated statistical significance.RESULTS1.The identification results of DNA by PCR technology showed that the high expression of APP DNA in APP transgenic mice known as APP positive mice group the,but no APP DNA expression in APP transgenic mice known as the APP negative mice.2.Immunohistochemistry results showed that there are high expression of protein APP in cytoplasmic of noural cell,and Aβhigh expressed in cytoplasmic and stromal of hippocampus and cortex cells of 12-month-old APP transgenic positive mice,but no expression in APP transgenic negative mice;the results of Congo red staining showed that pink colored homogeneous mass of amorphous material can be seen in the positive APP transgenic mice cortex and hippocampus interstitial,but no expression in negative APP transgenic mice.3.The expression of the family proteins 4.1 by DAB immunohistochemical staining of APP(+) group and APP(-) group mice brain tissue showed that the two groups both have the expression of protein 4.1 family members.APP(+) group:the protein 4.1N express in the cerebral cortex and hippocampus neurol cell membrane, connecting parts of axons and cell distribution of interstitial plaque can also be positive staining in the two brain regions.Protein 4.1B was filamentous distribution mainly in cerebral cortex and hippocampus neuron cells membrane,and strong expression in dentate gyru;Protein 4.1G mainly express in the cerebral cortex and hippocampal neural cells membrane,and express strongly in hippocampal dentate gyrus.Protein 4.1R mainly express in the cortex and hippocampal neurons memb- rane,strong expression in hippocampus.APP(-) group:every protein 4.1 family member shows a weak positive staining(P＜0.05 n=6).4.The results of Western-blot detection showed that 4.1 protein family member 4.1N,4.1G,4.1B and 4.1R express in the cerebellum,hippocampus and cortex tissues in mice brain tissue of the APP(+) and APP(-) groups,the former express higher than the latter,the difference had statistically significant(P＜0.05,n=6).5.The results of RT-PCR detection showed that mRNA of 4.1 protein family member 4.1N,4.1G,4.1B and 4.1R express in the cerebellum,hippocampus and cortex tissue in mice brain tissue of the APP(+) and APP(-) groups,but the expression of the two groups has no difference(P＞0.05,n=6).CONCLUSIONThere are no significant differences in the level of gene expression of protein 4.1 family members between the negative and positive APP transgenic mice brain tissue.But in the level of protein expression,the 4.1 family members express significantly higher in positive APP transgenic mice than in the negative APP transgenic mice,these suggesting that there may be abnormalities in 4.1 protein translation or metabolic process in APP transgenic mice brain,such abnormalities may be caused by protein APP high expression,while 4.1 protein abnormalities metabolic may also be involved in abnormal metabolism of protein APP and Aβgeneration.