Early Growth Response-1 Regulates Acetylcholinesterase And The Sex Differences In Alzheimer’s Disease Pathology

Part Ⅰ:Early Growth Response-1 Regulates Acetylcholinesterase in the Course of Alzheimer’s DiseaseOur previous studies showed that the transcription factor early growth response-1(EGR1)may play a role in keeping the brain cholinergic function intact in the preclinical stages of Alzheimer’s disease(AD).In order to elucidate the mechanisms involved,we first performed data mining on the microarray data of our previous postmortem human prefrontal cortex(PFC)study,to reveal the changes in the expression of EGR1 and acetylcholinesterase(AChE)and their relationship during the course of AD.The study contained 49 patients,ranging from non-demented controls(Braak stage 0)to late AD patients(Braak stage VI).We found that EGR1-mRNA was high in early AD and decreased in late AD stages,while AChE-mRNA was stable in preclinical AD and slightly decreased in late AD stages.A significant positive correlation was found between the mRNA levels of these two molecules,especially in Braak VI.In addition,we studied the relationship between EGR1-and AChE-mRNA levels in the frontal cortex of 3-12-months old triple-transgenic AD(3xTg-AD)mice.EGR1-and AChE-mRNA were lower in 3xTg-AD mice compared with wild type(WT)mice.A significant positive correlation between these two molecules was present in the entire group and in each age group of either WT or 3xTg-AD mice.Therefore,the 3xTg-AD mouse is not a proper model for studying early brain activation and its related gene expression in preclinical AD.Subsequently,AChE expression was determined following up-or down-regulating EGR1 by plasmid transfection in SY5Y cell lines and the EGR1 levels were found to regulate AChE at both the mRNA and protein level.Dual-luciferase assay,mutation analysis of the potential binding sites and electrophoretic mobility shift assay in the EGR1-overexpressing cells were performed to determine the functionally effective binding sites of the EGR1 on the AChE gene promoter.We observed 2 sites that are close to the transcription starting point are functionally effective,which may contribute significantly to the changes in cholinergic function in the course of ADPart Ⅱ:Sex Differences in Alzheimer’s Disease Pathological Hallmarks Accumulation in the Entorhinal Cortex of Preclinical StageClear sex differences are present in the pathogenesis of Alzheimer’s disease(AD).Women are more vulnerable than men for AD,but the mechanism involved is not known.One important reason of this gap in information is the absence of molecular pathological observations on sex differences in AD brains.We aimed to observe whether there are sex differences in AD-related proteins,i.e.β-amyloid(Aβ)deposition and hyperphosphoiylated Tau(p-Tau)accumulation,in postmortem human brains,which may lay a basis for further exploration of the mechanism of sex differences in AD.We first investigated the neuropathological scores,i.e.Braak stages and amyloid scores,in 648 individuals from the Netherlands Brain Bank,ranging from non-demented controls(Braak stage 0)to late AD patients(Braak stage VI).We found that female AD patients showed higher neuropathological scorings than male AD patients did.In addition,we did in the entorhinal cortex of non-demented(prechnical AD)individuals,an immunohistochemical staining for Aβ and p-Tau,using antibodies 4G8 and AT8,respectively and quantified the amounts by image analysis.We observed that females showed significantly higher p-Tau accumulation than male subjects.Subsequently,we constructed a p-Tau overexpressing environment by infecting TauV337M/R406W harbored lentivirus in a SY5Y cell line(Taumut-SY5Y),which resulted in increased EGR1 and AChE expression levels.These results indicates that more p-Tau accumulates in the entorhinal cortex of female than male individuals in early AD,which may contribute to upregulated neuronal activity including the increased expression of EGR1 and AChE,as we also observed in the early AD stages.These data deserve further studies on the involvement of EGR1 in the dynamic changes of cholinergic system activity(including acetylcholine production and breakdown)in the sexual dimorphic mechanism of the AD process,especially in the early stage of AD.