Expression Of LC-3B And Activation Of Caspase-3 In Alzhemer's Disease Models

Background:Alzheimer's disease(AD)is a degenerative disease of the central nervous system characterized by progressive cognitive dysfunction and behavioral damage,and is the principal cause of dementia.Senile plaques(SP)formation,neurofibrillary tangles(NFT),neuronal loss and glial cell hyperplasia are pathologic features of AD..The main component of SP is amyloid-beta(A?),which is cleaved by ?-amyloid precursor protein(APP).A? can cause chronic inflammation of CNS and neuronal dysfunction.Autophagy is the main pathway for A? degradation and is essential for maintaining environmental homeostasis in the CNS.Microtuble-associated protein 1 light chain 3B(LC3B),a specific marker for autophagosomes,is involved in the formation of autophagosomes,and its expression increases significantly when autophagy occurs.Whether the autophagy function in AD is enhanced or decreased is still inconclusive.Autophagic failure can cause deposition of some proteins abnormally in cells and promote apoptosis.Tau is included in the family of microtubule-associated proteins(MAPs),maintaining the cytoskeleton.Hyperphosphorylated tau lose its biological activity and accumulates abnormally in the cell.The aggregation of hyperphosphorylated tau has been described to lead to neuronal cell apoptosis and promote neurodegenerative diseases AT-8 is commonly used as an experimental indicator to reflect the hyperphosphorylation of Tau protein(identify the phosphorylation site of S202/T205),and its expression level can reflect the level of hyperphosphorylated tau.Caspase-3 is known to be the terminal splicing enzyme of apoptosis,which exists in cells in the form of non-active proenzyme(pro-caspase-3).It is cleaved into active caspase-3(MW of 19 kDa and 17kDa),under the activation of apoptosis signal,and then cleaved various cell substrates to perform apoptosis.Active caspase-3 is irreplaceable in cell apoptosis and can be used as a marker to evaluate the apoptosis.Autophagy might be defective in AD,leading to A? deposition,abnormal aggregation of intracellular hyperphosphorylated tau,and apoptosis.It is known that APP and PS1(presenilin 1)double transgenic mice are common animal models for studying AD.Therefore,this study intends to use APP/PS1 double transgenic mice and BV2 microglia as the research object to verify the changes of autophagy and apoptosis in AD,and the possible relationship between them and hyperphosphorylated tau.Objective:This study aimed to investigate the expression of LC3 B and the activation of caspase-3 in both animal and cell models of AD,as well as their possible relationships with AT-8 in AD,in order to provide a new theoretical basis and action target for the clinical treatment of AD.Methods:1.Animal experimentsThe DNA of mice bred from APP/PS1 double transgenic mice and wild-type mouse were extracted at 4-5 weeks postpartum with genomic DNA extraction kit.the target fragment was amplified by PCR and mice genotypes were identified by agarose gel electrophoresis..Thirteen mice of each age groug from 3 months old(3M),9 months old(9M)and 17 months old(17M)APP/PS1 double transgenic mice and wild type mice of the same age were selected randomly(all experimental mice were male),of which 7 were used to prepare frozen sections and 6 were used to extract brain tissue proteins.The deposition of senile plaques and the number and morphological changes of microglia were detected by immunohistochemistry.The expression levels of LC3 B,active caspase-3 and AT-8 were detected by immunofluorescence and Western-blot method.2.Cell experiments5?M,10?M and 20?M A?1-42 were used to treat BV2 microglia,accompanied with a blank control.BV2 microglia were cultured under the condition of 37 °C,5% CO2 for 6h with different concentration of A?1-42.Then cell were harvested and the cell proteins were extracted for Western-blot to examine the expression of LC3 B,caspase-3 and AT-8 proteins.3.Statistical analysisThe analyses were performed using SPSS 19.0 software.A independent-samples t-test was used when two values were compared.Parametric data were compared using one-way analysis of variance(ANOVA)to compare several independent groups.There was statistically significant heterogeneity if P? 0.05.Results:1.Animal experimental results1.1 The number of SP in APP/PS1 double transgenic mice was significantly higher(3M: P ?0.001,9M: P?0.001,17M: P?0.001),and the area became significantly larger(3M: P ? 0.001,9M: P ? 0.001,17M: P ? 0.001)than in the wild-type mice of the same age group,and with aging,the number and the area of SP in the APP/PS1 double transgenic mice increased.1.2 There was no significant difference of the number and the soma area of microglia cells between APP/PS1 double transgenic mice and wild-type mice at 3 months old(quantity: P =0.403,soma area: P =0.258).The number of microglia cells in APP/PS1 double transgenic mice at 9 months old and 17 months old was more(9M: P =0.03,17M: P =0.003),and the soma area became significantly larger(9M: P =0.022,17M: P =0.022)than in the wild-type mice of the same age group,and with aging,the number of microglia in APP/PS1 double transgenic mice increased,and the soma area became larger.1.3 The expression of LC3 B in APP/PS1 double transgenic mice at 3 months old and 9 months old was significantly higher(3M: P ? 0.001,9M: P ? 0.001)than in the wild-type mice of the same age group,The expression level of LC3 B in APP/PS1 double transgenic mice at 17 months old was significantly lower(P ? 0.01)than in the wild-type mice of the same age group,and the results were statistically significant.1.4 There was no significant difference of the expression of active caspase-3 between APP/PS1 double transgenic mice and wild-type mice at 3 months old(P >0.05),and the expression of active caspase-3 in APP/PS1 double transgenic mice at 9 months old and 17 months old was significantly higher than in the wild-type mice of the same age group(9M: P?0.01,17M: P?0.01),and the expression of active caspase-3 in APP/PS1 double transgenic mice increased with aging.1.5 There was no significant difference of AT-8 expression between APP/PS1 double transgenic mice and wild-type mice at 3 months old(P >0.05),and the expression level of AT-8 in APP/PS1 double transgenic mice at 9 months old and 17 months old was significantly higher than in the wild-type mice of the same age group(9M: P?0.001,17M: P?0.001),and the expression level of AT-8 in APP/PS1 double transgenic mice increased with aging.2.Cell experiment resultsCompared with the blank control group,The LC3B-II expression level of BV2 cells dealt with 5?M A?1-42 increased significantly(P ? 0.001),and the expression levels of active caspase-3 and AT-8 were no significant change(P >0.05).Compared with the 5 ?M experimental group,the LC3B-II expression level decreased significantly in the 10 ?M and 20 ?M experimental groups(P ? 0.001),and the expression levels of active caspase-3 and AT-8 increased significantly(active caspase-3: P ? 0.01,AT-8: P ? 0.001),and the difference was statistically significant.the LC3B-II expression level was negatively correlated with A?1-42 concentration,and the expression levels of active caspase-3 and AT-8 were positively correlated with A?1-42 concentration.Conclusion:In AD,Amyloid-? deposits with aggregation of hyperphosphorylated tau and apoptosis enhancement.,which is potentially supported by autophagic failure of microglial cells.