Melatonin-mediated MiR-504-3p Upregulation Inhibits Tau Hyperphosphorylation In Alzheimer’s Disease

Objective:It has been reported that phosphorylation of tau protein is regulated by many protein kinases.Moreover,dysregulation of phosphorylated kinases and phosphatases of tau protein leads to Alzheimer’s disease(AD).micro RNA(miRNA)expression is dysregulated in brain tissue of AD patients.miRNAs affect the phosphorylation of tau by regulating the protein level or activity of protein kinases.Melatonin can regulate a variety of miRNAs and participate in biological processes such as neural differentiation.we have found that melatonin can reduce tau hyperphosphorylation in our latest study.Therefore,we would like to investigate whether melatonin regulates tau protein phosphorylation kinase by targeting miRNAs,which inhibits the occurrence and development of AD.This project is expected to provide new targets for the treatment of AD.Methods:1.C57BL/6 wild-type mice and h Tau transgenic mice were selected as animal models.h Tau transgenic mice were intervened with melatonin(treatment group)and saline(control group),respectively,and C57BL/6 wild-type mice were untreated(healthy group).The phosphorylation levels of tau in mouse brain tissue were detected by immunohistochemical staining.Moreover,the soluble and insoluble tau proteins were separated by 1%(w/v)N-lauryl sarcosine sodium salt,and the phosphorylation levels of tau were detected by Western blot to explore the effect of melatonin on tau hyperphosphorylation in AD.2.I also explored the effect of melatonin on tau-related neuropathology in AD by detecting neurolfibrillary tangles(NFTs)in mouse brain tissues through thioredoxin-S staining.3.The sequence of miRNAs was performed in the treatment and control groups by high-throughput sequencing technologyn Candidate miRNAs and their candidate target genes were identified by literature reports and database analysis.4.P39 3’UTR-WT and 3’UTR-Mut were ligated to the dual luciferase reporter gene vector,and these two plasmids were cotransfected with miR-504-3p mimics into 293 T cells to detect the relative luciferase activity and to explore the the interaction of miR-504-3p with p39.5.Mi R-504-3p mimics were transfected into N2 a cells,and the protein level of p39 was examined by Western blot to detect the phosphorylation levels of tau protein by Cdk5.The m RNA level of p39 was examined by q RT-PCR to explore whether miR-504-3p affects the expression level of p39 and kinase activity of Cdk5.6.The miR-504-3p knockdown cells were treated with melatonin.And then the protein level of p39 and the phosphorylation levels of tau protein by Cdk5 were detected by Western blot.The m RNA level of p39 was detected by q RT-PCR to explore whether melatonin attenuates the pathology of AD by increasing the expression level of miR-504-3p,and whether inhibition of miR-504-3p reverses the effect of melatonin on tauopathies in AD.Results:1.The results of both immunohistochemical staining and Western blot showed that the phosphorylation levels of tau were significantly lower in the brain tissue of h Tau mice in the melatonin-treated group compared with the control group.The levels of insoluble total tau were reduced whereas there was no change for the levels of soluble total tau.2.The number of NFTs significantly reduced in the brain tissue of h Tau mice in the melatonin-treated group compared to the control group.3.Sequencing results revealed that the expression of 12 miRNAs was significantly upregulated and 5 were significantly downregulated in the brain tissue of h Tau mice in the melatonin-treated group compared to the control group.Moreover,the candidate miRNA miR-504-3p and its candidate target gene p39 were identified.4 The results of dual luciferase reporter assay showed that the relative luciferase activity was significantly reduced in the p39 3’UTR-WT group.While the relative luciferase activity of the p39 3’UTR-Mut group was not changed significantly.5.The results showed that the m RNA,protein levels of p39 and the phosphorylation levels of tau protein were significantly reduced in miR-504-3p overexpressed N2 a cells.6.The results showed that the m RNA,protein levels of p39 and the phosphorylation levels of tau protein were significantly increased in miR-504-3p knockdown N2 a cells with melatonin intervention.Conclusion:Melatonin significantly upregulated the expression level of miR-504-3p.The miR-504-3p reduced the expression level of p39 by targeting to the p39 3’UTR,which decreased Cdk5 activity.Thus,the phosphorylation levels of Cdk5 kinase-associated tau were reduced,thereby the formation of.NFTs was decreased.