EPAC2Downregulation Induce Tau Hyperphosphorylation By Activate Cyclin Dependent Kinase5(cdk5)

[Background]Alzheimer’s Disease (AD) is one of the most common progressive neurodegenerative disease, the clinical symptoms of which are intracellular neurofibrillary tangles (NFTs) and extracellular senile plaques(SPs) in neurons in the brain. NFTs are composed of abnormal hyperphosphorylated microtubule-associated protein Tau. Hyperphosphorylated Tau loss the ability of promoting tublins assembly and stability, disaggregation of microtubules and forms paired helical filament(PHF), forming NFTs, inducing synapses degeneration and neuron loss, playing an important role in the process of AD.As the direct activation of cAMP downstream protein, EPAC(guanine nucleotide exchange protein) plays an important role in regulation of biological processes. The EPAC1/2mice exhibit learning and memory impairment and social interaction disorder. The expression of the EPAC2mRNA level was decreased in the brain of AD patients, suggesting that EPAC2downregulation participate in AD pathogenesis. However, the EPAC2signaling pathway in vivo which affects learning and memory is still unclear.[Objective]To explore the role of EPAC2pathway act in Tau hyperphosphorylation. To explore which molecular in the signaling pathway can the reverse of the learning and memory impairment induced by EPAC2downregulation.[Methods]To explore the role of EPAC2pathway in Tau hyperphosphorylation, we applied the EPAC2-/-mice and si-EPAC2plasmids. With the western bolt, we detected the tau phosphorylation related antibodies on both the animal and the cell levels. To explore whether the EPAC2downregulation have an influence on the solubility of Tau, we isolated the soluble section and insoluble section. To confirm whether EPAC2downregulation formed the NFTs, Bielschowsky stain was used.To explore the mechanism of the above results, we screened a serious of Tau phosphorylation related kinase and esterase with western blot, and discovered the activation of the key kinase CDK5.According to the documents published, we discovered that Calpain can activate CDK5, then we tested the activity of Calpain in EPAC2-/-mice. To explain the connection between EPAC2downregulation and Calpain activation, we screened a serious of inflammatory factor and finally find TGF β1, which is the intermediate of EPAC2and Calpain.To reverse the Tau lesion induced by EPAC2downregulation, we applied the Calpain inhibitor(CI-Ⅳ) and the CDK5inhibitor Roscovitine, stereotaxic injected into paracele of EPAC2-/-mice. With the western blot detecting whether the inhibitors can renovate the EPAC2downregulation induced Tau phosphorylation.[Results]1.EPAC2downregulation induce Tau lesion like Tau hyperphosphorylation, the increase of insoluble Tau and NFTs formation.To explore the influence of EPAC2downregulation on Tau, we build the EPAC2downregulation environment with the EPAC2-/-mice and si-EPAC2plasmids, then take samples to analyses the phosphorylation level of Tau. The results indicated that since the6m, EPAC2downregulation induces the phosphorylation level of Tau, along with the increase of phosphorylation at the sites of pT205、pT231、pS396、pS404.We then separated the brain tissues of EPAC2+/+and EPAC2-/-mice into soluble section and insoluble section, discovered that the Tau significantly increased in insoluble section of EPAC2-/-mice. With the Bielschowsky silver stain, we found the NFTs in the EPAC2-/-mice since9m.2.EPAC2downregulation induce Tau hyperphosphorylation through the activation of CDK5.The phosphorylation of Tau is result of the imbalance of related kinase and esterase. To find out the key impact factor which can promoting Tau phosphorylation after EPAC2downregulation, we screened related kinase and esterase, finally found that CDK5was activated after the downregulation of EPAC2.Downregulation of CDK5can reverse the Tau hyper phosphorylation induced by EPAC2downregulated.3. EPAC2downregulation activate CDK5through Calpain.Calpain is the key molecular which can activate CDK5.We detected a higher activity in EPAC2-/-mice compared with the EPAC2+/+mice. Downregulated Calpain in EPAC2-/-mice decreased the activity of CDK5and reversed the Tau lesion.4. EPAC2downregulation though TGF-β1/Calpain/CDK5pathway induce Tau hyperphosphorylation.Experiments certificated that the EPAC2downregulation activated Calpain and CDK5wasn’t through the traditional Ca2+activity. To identify the way EPAC2act on CDK5, we screened a serious of inflammatory factors and finally found TGF-β1.We then focus on the TGF-β1/Calpain/CDK5to conduct the future research.[Conclusion]1.EPAC2downregulation induce Tau lesion like Tau hyperphosphorylation, the increase of insoluble Tau and NFTs formation.2.EPAC2downregulation induce Tau hyperphosphorylation through the activation of CDK5.3. EPAC2downregulation activate CDK5through Calpain.4. EPAC2downregulation though TGF-β1/Calpain/CDK5pathway induce Tau hyperphosphorylation.