The Screening And Function Study On ADAM10 MicroRNA Targets In A YXQN Drug Attenuating The Cognitive Impairment Model

Alzheimer’s disease(AD),with amyloid-β(Aβ)deposition in the brain as a crucial pathologicalhallmark,is a serious disease in aging society.Under the pathology conditions,pathological cleavage of amyloid precursor protein(APP)produces pathogenic Aβ.Unfortunately,all the medicines designed to prevent thepathological cleavage of APP and production andaggregation of Aβhave invariably failed.Therefore,a traditional Chinese medicine YXQN augmenting CBF has been studied and discovered that significantly improved AD pathology and the cognitive function of AD mice(APPswe/PS1 d E9 mice).YXQN consists of 11 Chinese herbs,many of which have effects of blood circulation and neuroprotection.We found that YXQN drugs can improve the cognitive impairment and reduced Aβ pathology of AD micemodel,in which ADAM10 shows the most significant enhancement induced by YXQN.The therapeutic effect of YXQN on AD is achieved by improving the physiological pathway of APPwith manifestation of5-fold enhancement expression of ADAM10(α-secretase)in YXQN drug treated AD mice.The physiological cleavage of APP,mediated by ADAM10(α-secretase)and PS1(γ-secretase),has an antagonism against itspathological cleavage producing Aβ.The promotion of physiological cleavage of APP by enhancing the expression of physiological cleavage enzyme(ADAM10)will be a potential AD therapy.MicroRNAs(miRNAs),endogenous non-coding RNAs of about 22 nucleotides in length,inhibit m RNA expression at transcriptional and post-transcriptional levels to participate in vital life regulation.MiRNAs targeting ADAM10 werescreened and functional studied to identify novel miRNA targets in the course of AD based on the effects of YXQN on ADpathology and cognitive function in APP/PS1 transgenic mice.The miRNA microarray was used to identify the miRNA associated with AD by comparing the hippocampus tissues of normal mice,AD mice(APPswe/PS1 d E9mice)and YXQN drug treated AD mice,and 32 miRNAs were obtained.Using ADAM10 detection in Aβ-generating cell model and bioinformatics prediction methods,we discovered two novel miRNAs targeting ADAM10.At the level of cytology and molecular biology,it was demonstrated that these miRNAs inhibit the transcription and protein expression of ADAM10.Further,with the construction of miRNA targeting ADAM10 virus and intracerebroventricular injection in mice,we found that these miRNAs significantly reduced the expression of ADAM10 in the brain,inhibited the physiological cleavage of APP in the brain,especially the hippocampus,while the pathological cleavage of APP was promoted leading to the generation of much more Aβ,whichseverely accelerated cognitive impairment in AD mouse.By these means,the discovery of new AD disease miRNA targets were achieved.To identify the key miRNAs associated with AD,using miRNA microarray,we compared the hippocampus tissues of normal mice,AD mice(APPswe/PS1 d E9 mice),and YXQN drug treated AD mice(by using miRNA microarray)and obtained 10 miRNAs with down-regulated expression in the AD group and up-regulated expression in the drug treatment group.The accuracy of the microarray results was verified by real-time PCR.ADAM10 is a key secretory enzyme in the physiological cleavage pathway of Aβ.To obtain miRNAs that has an inhibitory effect on ADAM10 expression,we transfected miRNA mimics in SH-SY5 Y cells.Western blot and real-time PCR showed thatmiR-31-5p and miR-212-5p reduced ADAM10 protein levels and m RNA levelssignificantly,which were increased after transfection of miR-31-5p and miR-212-5p inhibitors.This part of the results demonstrated that miR-31-5p and miR-212-5p can inhibit the expression of ADAM10 at the post-transcriptional level.The physiological cleavage of APP has an antagonism against its pathological cleavage.The s APPαcleavaged by ADAM10 has a neuroprotective effect to antagonizing Aβ formation.Therefore,we detected the production of carboxy-terminal cleaved product s APPα and that of pathological Aβ.The miRNA mimics were transfected into SH-SY5 Y cells and western blot experiments were done,which showed miR-31-5p and miR-212-5p reduced the expression of s APPα.Furthermore,we overexpressed APPswe,BACE1,and PS1 plasmids in the SH-SY5 Y cell line to construct AD cell models with a large number of Aβexpression.To detect the expression of pathological Aβ40 and Aβ42,we used ELISA assay and found that miR-31-5p and miR-212-5p inhibited the production of APP biocleavage product s APPα and promoted the production of APP pathological cleavage product Aβ.On this basis,we further examined the binding sites of miR-31-5p and miR-212-5p with ADAM10 3’-UTR.First,preliminary predictions of binding sites using bioinformatics analysis software revealed that miR-31-5p can bind to 7 bp from3143 bp to3149 bp in ADAM10 3’-UTR,and miR-31-5p can bind to 6 bp from 3281 bp to 3286 bp.Next,the binding sites were detected by constructing the ADAM10 3’-UTR luciferase reporter(wild-type)and the corresponding site-mutant plasmid.As a result,the expression of miR-31-5p and miR-212-5p was significantly reduced after transfection with the wild-type plasmid,which was reduced by 30% and74%respectively,compared with the control,but not in the mutant group.It was demonstrated that the negative regulatory targets of miR-31-5p and miR-212-5p on the ADAM10 geneare located in the 3’-UTR region of the m RNA.On one hand,through cell experiments,we found that miR-31-5p and miR-212-5p can inhibit the expression of ADAM10 at the post-transcriptional level,thereby inhibiting the production of s APPα,a physiological cleavage product of APP,which generate more Aβ.On the other hand,we further studied the role of miR-31-5p and miR-212-5p in the physiological cleavage pathway of APP in vivo,with the construction of the two miRNAs virus and intracerebroventricular injection in mice.Firstly,in order to examine the effect of miRNAs on learning and memory and behavioral cognitive function in AD model mice,the positioning navigation experiment and space exploration experiment of the Morris water maze showed mice in the lentivirus injection group searched for a much longer path of the underwater platform,and the search time of the correct platform quadrant was extended up to 48%compared with the control group.The Y-maze test also found that the accuracy of mice in the lentiviral injection group was reduced by about 33%.The results suggest that miR-31-5p and miR-212-5p significantly aggravate behavioral cognitive impairment in AD mice.To examine the mechanism by which miR-31-5p and miR-212-5p aggravate the congnitive impairment of AD mice,we examined the expression of ADAM10 in the cortex and hippocampus of the mouse at m RNA and protein levels.Real-time PCR and immunohistochemical staining showed that theexpression of ADAM10 at m RNA and protein levels in the virus injection group was significantly reduced by approximately 60%.These results suggest that miR-31-5p and miR-212-5p degrade ADAM10 m RNA at the post-transcriptional level,thereby reducing the expression of ADAM10 protein.Next,to confirm the effect of the two miRNAs on pathological lesions of AD mice,we used thioflavine-S staining,Congo red staining,and Aβ-specific antibody immunohistochemical staining to detect the effect of miRNA on AD brain pathology.The results showed a 4.7-fold increase of amyloid deposition in the hippocampus and cortex in the miR-31-5p virus-injected group,and a 4.2-fold increase in the miR-212-5p virus-injected group,compared with the control group.These results suggest that miR-31-5p and miR-212-5p significantly promote the generation of Aβ in the brain of AD mice,leading to the aggravation AD symptoms.In summary,we confirmed the negative regulation of miR-31-5p and miR-212-5p on ADAM10 3’-UTR at the celluar and animal model levels.The negative regulation inhibits the physiological cleavage of APP and promotes the pathological cleavage of APP,which promoted the generation of Aβ leading to the deterioration of AD.Overall,it is clarified that the behavioral cognitive effect of YXQN drugs on APPswe/PS1 d E9 double transgenic mice is achieved by reducing expression levels of miR-31-5p and miR-212-5p.The discovery of the new miRNA target for AD contributes to a new mechanism for drug therapy of Alzheimer’s disease.