Activating Transcription Factor 6 Reduces Toxic A?1-42 And Restores Memory In Alzheimer's Disease Model Mice

BackgroundAlzheimer's disease(AD)is a typical neurodegenerative diseasec[1].Clinically,it is characterized by memory impairment,aphasia,indiscriminateness,loss of recognition,visual spatial impairment,executive dysfunction,and changes in personality and behavior.But the etiology of AD has not been known so far.Alzheimer's disease is the most common form of dementia,and its main pathological features include extracellular amyloid deposition and neurofibrillary tangles formed by intracellular abnormally phosphorylated Tau proteins.There were Familial Alzheimer's disease and sporadic Alzheimer's disease.Familial Alzheimer's disease is mainly caused by genetic factors,while sporadic Alzheimer's disease is mainly caused by genetic and environmental factors.World Alzheimer's Disease Report shows that the number of patients with Alzheimer's disease will reach 130 million by 2050.Therefore,it is extremely urgent for Alzheimer's research and searching the treatment.The study of the molecular mechanism of AD was very important for AD treatment.The endoplasmic reticulum is an important organelle that regulates protein folding and modification.When misfolded and unfolded protein aggregates in the endoplasmic reticulum or disordered calcium ion balance,endoplasmic reticulum stress(ER stress)is triggered,which activates unfolded protein response(UPR).As we all known,the transcriptional activator 6(ATF6)is one of the major signal transduction molecules in the unfolded protein response.Studies have shown that the deposition of Ap in Alzheimer's disease may lead to endoplasmic reticulum stress in neuron,further leading to UPR.And there is a phenomenon of elevated Ca2+concentration in the cells of AD patients,probably caused by A?.There are three main signaling pathways for endoplasmic reticulum stress activation,including the PERK pathway,the IRE1 pathway,and the ATF6 pathway.The first two pathway studies are more extensive,while the research on ATF6 pathway and Alzheimer's disease is relatively rare.Therefore,our study is based on the relationship between Alzheimer's disease and endoplasmic reticulum stress,focusing on the mechanism of ATF6 signaling pathway in Alzheimer's disease,and is devoted to exploring the role of ATF6 in the pathogenesis of AD.Hope to find new potential targets for the treatment of AD.MethodsIn this study we explored the expression of ATF6 between wild type and APP/PS1 model mice firstly.And then the effect of ATF6 on A?1-42 was detected by ELISA in LN229 cell line.We detect the expression of AD-related candidate genes using qPCR and Western blot.Secondly,the gene promoter plasmid was constructed,and the effect of ATF6 on the promoter of the gene was determined by co-transfection of the ATF6 plasmid with the promoter plasmid.Finally,the effects of ATF6 were verified at the animal level in Alzheimer mice model.We used stereotaxic injection to deliver a eukaryotic expression plasmid encoding human activating ATF6 into the lateral ventricle in APP/PS1 mice aged 12 months.And some behavioral experiments like Y maze and Morris Water Maze test indicated that ATF6 could protect retention of spatial memory in APP/PS1 mice.An ELISA and thioflavin-S staining were performed,the results suggested that injected ATF6 could reduce the A?1-42 and alleviate the A? plaque load in APP/PS1 mice cortex.Results1.The expression of ATF6 in brain tissue of AD model mice and significantly decreased than wild type mice(P<0.05).2.Overexpression of ATF6 in LN229 cell,the level of A?1-42 in the supernatant was significantly decreased([<0.01),indicating that AIF6 inhibited amyloid formation pathway.3.The overexpression of ATF6 reduced the APP protein levels in protein level and increased the AD AM17 both in protein and RNA levels(P<0.05),and BACE1 was significantly decreased in both protein and RNA levels(P<0.001).Knokdown the ATF6,we got the same results.4.The effect of ATF6 on the promoter activities of AD AM17 and BACE1 was detected by Luciferase assay.It was found that ATF6 had no significant effect on the promoter region of AD AM17,but significantly inhibited the BACE1 promoter activity(P<0.001).Though promoter prediction software and literature review,we found that the transcription factors affecting the activity of BACE1 promoter are mainly SP1,P53,PPAR?.But it is proved by experiments that ATF6 acts on the promoter region of BACE1 does not through these transcription factors.5.AD model mice were injected ATF6 into the lateral ventricle,then the Y maze and Morris water maze test indicated that ATF6 could protect retention of spatial memory in APP/PS1 mice(P<0.01).Western blotting analysis showed that the level of BACE1 was reduced after ATF6 plasmid injection.An ELISA and thioflavin-S staining were performed,the results suggested that injected ATF6 could reduce the A?1-42 and alleviate the A? plaque load in APP/PS1 mice brain.6.ATF6 could promote the production of H2S(P<0.001)and promote autophagy.The H2S in the central nervous system is mainly produced by CBS.It was found that CBS can promote autophagy by promoting ATG5 sulfhydration,thereby alleviating AD.But the experiment proves that ATF6 has no effect on CBS.7.The Cre/loxP recombinase system was used to construct the cerebral cortex and hippocampus specific ATF6 knockout AD model mice.Firstly,the cerebral cortex and hippocampus specific ATF6 knockout mice were hybridized with APP/PS1 mice to obtain the APP/PS1 model of cerebral cortex and hippocampal specific ATF6 knockout.At present,the mice model has been successfully constructed,laying the foundation for subsequent experiments.ConclusionThe expression of ATF6 in the brain of AD model mice was significantly decreased.ATF6 affected the production of amyloid precursor protein and affected the enzymes in the process of amyloid formation,mainly though affecting the expression of BACE1 and inhibiting the promoter activity of BACE1.Finally,it affects the production of A?.Injection of ATF6 into the lateral ventricle of AD mouse model can reduce the deposition of plaques in the brain of mice,and has a positive effect on the learning and memory ability of model mice,and also alleviates the cognitive impairment of model mice.It provide the basis that ATF6 may be a potential molecular target for the treatment of AD.