The Study On Leptin Ameliorate Alzheimer’s Disease-like Tau Protein Hyperphosphorylation Via Cdk5Pathway

Alzheimer’s disease（AD), is a kind of neurodegenerative diseases which ishighly correlated with the age, the main feature of this disease are the formationof neurofibrillary tangle(NFT) and senile plaque(SP), thereinto the essence ofneurofibrillary tangle is hyperphosphorylated tau accumulation. At present we arenot clear about its etiology and pathogenesis and we still lack effective mdecinefor this disease. Therefore, it is the recent hot point in the AD research regardingthe exploration of AD pathogenesis, the targets of new drug and the search ofeffective intervention areas.Leptin acts on hypothalamic neuronal targets to regulate energy balance andneuroendocrine function. However, recent research has shown that leptin is alsoinvolved in neuroprotection. In order to understand the role and molecularmechanism of leptin in AD damage, this study aims to establish AD model inneuroblastoma cell line (SH-SY5Y), primary hippocampal neurons andorganotypic cerebral slice with okadaic acid (OA), which is used to explore theeffect of leptin on AD injury and its mechanism of regulation. And observe thepathological changes in the autopsy brain tissue of patients with AD.The4parts of this study includes: Part Ⅰ The protective role of leptinplayed in tau hyperphosphorylation of neuroblastoma cell line via inhibition ofcyclin-dependent kinase5(Cdk5); Part ⅡThe neuroprotective effect of leptinplayed in primary hippocampal neurons tau hyperphosphorylation and thepossible molecular mechanism; Part Ⅲ The neuroprotective effect of leptinplayed in AD model of organotypic brain slice; Part Ⅳ Pathological observationand detection on autopsical hippocampus tissue of AD patient. The results are asfollows:1Establish AD cell model in the level of SH-SY5Y cell line(1) OA, as inducer, can successfully establish tau protein hyperphosphorylation of AD cell model;400ng/mL leptin significantly increasedcell viability in6h; it suggested that leptin has a protective effect on SH-SY5Y.(2) Double immunofluorescence stain showed there were co-expression ofObR and Cdk5, ObR and p-tau. There may be interactions between ObR and thetwo.(3) Leptin reduced p-tau protein level in a dose dependent manner, the sameas Cdk5expression (resulting in tau hyperphosphorylation as a direct factor)400ng/mL leptin significantly reduced the expression of Cdk5, ObR specific sealantcan reverse the effects above; it suggested that leptin, binding to its receptor, canreduce the level of p-tau by inhibiting Cdk5expression;(4) Cdk5SiRNA treatment decreased p-tau, while its specific inhibitorRoscovitine could not;2Establish AD cell model in the level of primary hippocampal neurons(1) Main lesion areas of AD concentrated in the hippocampus, the useage ofprimary hippocampal neurons was aim to explore the protective effect of leptin oncell from lesions. ObRb mRNA expression was significantly increased after OAinduction, suggesting that leptin may exert its effect through its long formreceptor in AD model;(2) The same with cell line level,400ng/mL leptin significantly reduced theexpression of Cdk5and p-tau, ObR specific sealant can reverse the effects above;Cdk5SiRNA treatment decreased p-tau, while Roscovitine could not;(3) The excessive activation of ERK1/2(extracellular regulated proteinkinases1/2) was induced by OA, while leptin can down-regulate the activation;inhibition of ERK1/2by U0126can down-regulate Cdk5and p-tau; it suggestedthat ERK1/2play an important role in the protection of leptin on AD-like injury(4) ERK1/2can be activated by Roscovitine, peaked during3h-6h;6h to12h began to decrease; this may explain why it can not reduce the level of p-tau.3Establish AD tissue model in the level of organotypic brain slice(1) Organotypic slice belonged to organization level, it could better simulatethe influence between cells, and it was closer to the state in vivo. Real-time PCRshowed that ObRb mRNA expression was significantly increased after OAinduction;(2) The same with cell level,400ng/mL leptin significantly reduced the expression of Cdk5and p-tau, ObR specific sealant can reverse the effects above;Cdk5SiRNA treatment decreased p-tau, while Roscovitine could not;4Pathological Change in autopsical brain tissue of AD patient(1) Autopsical pathological sections can be very intuitive to observepathological changes of hippocampal tissue in patients with AD, the results weremost real. HE stain showed that cells arranged was in disorder, amount of cellsbecame into degeneration and necrosis, cytoplasm was loose, significant vacuolargranules was observed, cell volume was significantly reduced in vacuole;(2) Double immunofluorescence stain showed ObR and Cdk5, ObR andp-tau were colocalized and fluorescence intensity were all higher in lesion areas,while the normal regions were relatively low;(3) Immunohistochemistry stain showed that, ObR was widely distributed inthe area of lesion, while normal tissue was less. The positive cells of p-tau andCdk5were mainly located in vacuolar granules, less distribution in normal tissue.In summary, this study shows that:(1) Exogenous leptin exerts neuroprotective effects in AD-like tauhyperphosphorylation by binding to its long form receptor ObRb;(2) Leptin decreases Cdk5expression in the AD model induced by OA,which decreased tau phosphorylation level;(3) During AD occurrence, ERK1/2is excessively activated and exogenousleptin can down-regulate the activation;(4) Inhibition of ERK1/2contributes to down-regulation of Cdk5and p-taulevel;(5) SiRNA makes directly interference of the Cdk5gene, which plays a rolein the down-regulation of p-tau, and its chemical inhibitors can not achieve thiseffect due to the activation of ERK1/2.(6) Pathological slices of autopsical brain tissue shows that ObR, Cdk5andp-tau expression were increased in the lesion areas because the endogenous leptincontent is low, last two of which led to a significant pathological changes.