The Neuroprotective Effects And The Mechanisms Of Melatonin Protects Primary Cortical Neurons Against A?25-35 Induced Neuronal Injury

Alzheimer's disease(AD)is a kind of neurodegenerative disorder associated with age,which characterized by progressive memory decline and cognitive deficits ranging from mild to severe dementia.Given the severity of the problem,elucidating the underlying mechanisms of AD and finding an effective therapeutic method is pressing.Investigations suggest that amyliod-P(A?)is implicated in the pathogenesis of AD,and accumulating evidences indicate that A? is a crucial factor in the pathogenesis of AD.Studies suggest oxidative stress,neuroinflammation and neuroimmune are involved in A?-induced neurotoxicity,and oxidative stress may play a critical role on AP-induced synaptic toxicity which leads to synaptic dysfunction and neuronal death.AP can cause neurons to produce excessive reactive oxygen species(ROS)and mitochondrial dysfunction which may contribute to the synaptic malfunction and neuron loss in AD.Therefore,antioxidant should be effective in treating AD and melatonin is one of them.Melatonin exerts multiple biological effects,including anti-apoptosis,anti-oxidation and protecting against mitochondrial dysfunction.Furthermore,it is suggested that the declination of melatonin is parallel with the neuropathological progression of AD.In vitro experiments,melatonin exerts neuroprotective effect in treating neurodegenerative disorders by against oxidative stress and mitochondrial dysfunction.However,the molecular mechanisms of A?-induced neurotoxicity and melatonin treatment remain elusive.miRNAs are critical for most biological processes as well as the pathogenesis of diseases.So,we supposed that miRNAs maybe involved in the molecular mechanisms of A?-induced neurotoxicity and melatonin treatment.miR-132,a brain-enriched miRNA,is a critical factor characterized of modulating neuronal plasticity and maintaining neuronal survival.Recent studies suggest that miR-132 expression is markedly down-regulated in human AD brain,including hippocampus and prefrontal cortex.Moreover,miR-132 participates in the regulation of the pathological processes in AD.In addition,PTEN and FOXO3a are two key targets of miR-132.PTEN can antagonize PI3K-AKT signaling cascade(a crucial pro-survival pathway).Whereas,F0X03a a pro-apoptotic factor triggers the cell-death genes cascade by nuclear translocation.PI3K/AKT and miR-132/PTEN/FOXO3a signals play an important role in AD,however,there are no reports about the relation between the neuroprotective of melatonin and miR-132 in AD.Based on the above investigations,we supposed that miR-132/PTEN/FOXO3a and PI3K/AKT pathway are involved in the effect of melatonin against A?-induced neurotoxicity.Here,we exposed the primary cultured cortical neurons with different concentrations of A?25-35,and detected the cell viability with MTT,determined the optimal density.Then,using different concentration of melatonin to protect the neuron,and identified the optimal concentration for protection.In our experiment,we designed three groups:control,A?25-35 induced damage,AP25-35 induced damage plus melatonin protection.Next,we detected the cell apoptosis and cell death via Hoechst/PI staining,the levels of intracellular ROS via ROS assay,and the mitochondrial membrance potential(A(?)m)via JC-l kit.The results indicated that A?25-35 exposure significantly increase the rate of cell apoptosis and cell death,the elevation of ROS,and depolarize A(?)m.Whereas melatonin could greatly counteract these neurotoxicity effects of AP25-35-induced.In order to demonstrate the molecular mechanisms of A?25-35 induced neurotoxicity and the neuroprotective effect of melatonin.Firstly,we detected the expression of miR-132 via qPCR.We found that A?25-35 exposure significantly decreased the expression of miR-132,whereas melatonin treatment could rescue the expression of miR-132.Next,we also detected the expression of downstream targets of miR-132,such as PTEN and FOXO3a,with western blot.The results indicated that A(325-35 exposure promoted both the expression of PTEN and FOXO3a,while melatonin treatment down-regulate the level of PTEN and F0X03a.Moreover,we detected the expression of p-AKT,p-FOXO3a and cleaved Caspase3 with western blot.We found that A?25-35 exposure significantly suppressed the expression of p-AKT and p-FOXO3a,while promoted the expression of cleaved Caspase3.Whereas,melatonin could increase the levels of p-AKT and p-FOXO3a,and decrease the levels of cleaved Caspase3.Moreover,we demonstrate that melatonin could block the A?-induced nuclear translocation of FOXO3a,and thereby suppressed its pro-apoptotic pathways.Furthermore,we investigated the relation of miR-132/PTEN/AKT/FOXO3a and the intervention of melatonin on A?325-35 induced neurotoxicity.Firstly,we increased the levels of miR-132 in primary neurons by transfecting neurons with lentivirus,and then the neurons were exposed with A?25-35.We identified that neurons over-expressing miR-132 were more resistant to A?-induced neurotoxicity.Then,we used the specific PTEN inhibitor VO-OHpic after A? exposure and PI3K-AKT signal pathway inhibitor LY294002 to treat neurons separately after melatonin treatment plus A? exposure.We found that LY294002 could inhibit the protective effects of melatonin on A?-induced neurotoxicity,whereas VO-OHpic exerted neural protective effects as melatonin.Together,our investigation based on the characters of neurotoxicity of A? and neuroprotective effect of melatonin.Here,in order to elucidate the molecular mechanisms of A?-induced toxicity and melatonin treatment,we exposed the primary cultured cortical neurons with A?25-35 and treated with melatonin.Importantly,we firstly demonstrated the role of miR-132 in A?-induced toxicity and melatonin treatment.Our investigations demonstrated that A?25-35 exposure significantly decreased the expression of miR-132 and elevated the expression of PTEN and FOXO3a.Whereas,melatonin treatment could rescue the expression of miR-132 and down-regulate the level of PTEN and FOXO3a.Moreover,melatonin blocked the nuclear translocation of FOX03a and thereby suppressed its pro-apoptotic pathways.In addition,our investigations suggested that the over-expression of miR-132 could block A?-induced neurotoxicity.We also found that VO-OHpic(PTEN inhibitor)could counteract A?-induced neuronal damage,and LY294002(AKT inhibitor)suppressed the protective effect of melatonin.Together,these results indicate that melatonin exerts its neuroprotective effect in A?-induced neurotoxicity via miR-132/PTEN/AKT/FOXO3a pathway.Together,our investigations provide new clues for the mechanisms of melatonin intervention on A?-induced neurotoxicity.