Melatonin Directly Binds And Inhibits Death-associated Protein Kinase 1 Function In Alzheimer’s Disease

Objective:According to reports,melatonin can decrease tau hyperphosphorylation,β-amyloid(Aβ)deposition and other lesions of Alzheimer’s disease(AD),but the molecular mechanism is unclear.In addition,many research teams and previous studies of our research group have proved that death-associated protein kinase 1(DAPK1)plays an important role in the occurrence and development of sporadic AD,and that DAPK1 inhibition reduces tau phosphorylation,Aβ secretion,neuronal death,and improve the cognitive dysfunction of AD mice.However,it is unknown whether and how DAPK1 expression or activity is regulated in AD.Therefore,this study will explore the molecular mechanism of melatonin in alleviating AD by regulating DAPK1,as well as the role of melatonin and DAPK1 inhibitor in the occurrence and development of AD.This project would provide a novel therapeutic option for AD treatments.Methods:1.I treated indole hormones such as melatonin to neuroblastoma cells and primary neurons and detected protein and m RNA levels of DAPK1 by Western blot and q RT-PCR to explore whether and how melatonin regulate the expression of DAPK1.2.I treated cycloheximide,protein synthesis inhibitors,and proteasome inhibitor MG-132 to primary neurons and detected the expression level of DAPK1 by Western blot,to explore whether and how melatonin degrades DAPK1.Flag-DAPK1 and His-ubiquitin plasmids were co-transfected into He La cells,and melatonin and MG-132 were used for intervention.The protein level of DAPK1 was detected by Western blot to explore the effect of melatonin on the ubiquitination process of DAPK1.3.Biotin-melatonin competition experiment,pulldown and in vitro kinase assay were performed to explore the interaction between melatonin and DAPK1,and its effect on DAPK1 kinase activity.Primary neurons were treated with specific inhibitors of melatonin receptors to explore whether the interaction depends on the melatonin receptors.4.Brain slices from human tau-overexpression mice and SK-N-BE(2)which were transfected with GFP-Tau were treated with melatonin and/or DAPK1 inhibitor.The cell lysates were subjected to Western blot and immunofluorescence analyses to explore the effects of melatonin and DAPK1 inhibitor on the phosphorylation level and biological functions of tau.5.The expression levels of melatonin and DAPK1 in brain tissues of normal people and AD patients were checked by immunohistochemistry to explore the correlation between DAPK1 and melatonin expression levels in AD patients.Results:1.Melatonin can significantly reduce the protein level of endogenous DAPK1,and is dose-and time-dependent,while melatonin has no significant effect on the m RNA level of DAPK1.Moreover,other indole hormones(tryptophan and serotonin)have no significant effect on the protein and m RNA levels of DAPK1.2.Melatonin reduces the stability of DAPK1 after treatment with CHX.MG-132 can reverse the down-regulation of DAPK1 protein stability by melatonin.Melatonin can promote ubiquitination of DAPK1 through ubiquitination proteasome pathway.3.Melatonin can directly binds to the ankyrin repeat domain of DAPK1 independent of melatonin receptors.Melatonin does not inhibit DAPK1 activity in vitro kinase assay system.4.Melatonin and DAPK1 inhibitor reduce the phosphorylation level of tau and Pin1-Ser71,increase neurite outgrowth and microtubule assembly.Moreover,the effect is more significant for combined treatment of melatonin and DAPK1 inhibitor.5.The results showed that,compared with the sample of normal people,the expression level of DAPK1 in the brain tissue of AD patients was significantly increased,while the expression level of melatonin was significantly reduced.Conclusion:Melatonin directly binds to DAPK1 and reduces its stability by promoting the ubiquitin-mediated proteasome degradation pathway,thereby reducing the level of Pin1-Ser71 phosphorylation mediated by DAPK1.In addition,melatonin and DAPK1 inhibitor synergistically reduce tau hyperphosphorylation and increase neurite outgrowth and microtubule assembly.Therefore,the combined intervention of melatonin and DAPK1 inhibitor is expected to become a new method of AD treatment.