Role Of PTEN In The Development Of Cellular Models Of Alzheimer's Disease

Alzheimer's disease(AD), is a kind of primary neurodegenerative diseases which is highly correlated with the age , the main feature of this disease are the formation of neurofibrillary tangle(NFT) and senile plaque (SP) . At present we are not clear about its etiology and pathogenesis and we still lack effective treatment for this disease. Therefore, it is the recent hot point in the AD research regarding the exploration of AD pathogenesis, the targets of new drug and the search of effective intervention areas.PTEN (Phosphatase and tensin homologue deleted from chromosome 10) is a protein tyrosine phosphatase with dual specificity lipid and protein phosphatase activity. It is an important regulating molecular in signaling pathways, and also it connects with the growth, differentiation and apoptosis regulation of cell closely. In recent years, with the increasing understanding of PTEN, more and more attention has been paid to the function of PTEN in central nervous system, especially in the diseases of central nervous system injury. However, more research needs to be done in regard to the significance and association between PETN and the development of AD meaning.Our previous research worked on the changes of PTEN in the development of the cellular models of Alzheimer's disease. It was first time that we demonstrated PTEN altering in the cellular models of Alzheimer's disease induced by Okadaic acid(OA). With the severity of cell injury and changes in tau hyperphosphorylation levels, levels of PTEN protein show the phenomenon of increasing first and then decreasing. Therefore, this study first established a variety of different cellular models of Alzheimer's disease that induced by four different inducing factors. We confirmed the changes of PTEN in the development of AD by the variations of PTEN protein and PTEN mRNA. Then we analyzed the effects and role of PTEN downregulation in the cellular injuries and hyperphosphorylation of tau protein with RNA interference (RNAi) in the cellular models. On this basis, we further study the possible molecular mechanisms, aiming to further understand the pathogenesis of AD. It would provide new direction of treatment to AD and new therapeutic drug targets in exploring new drug for AD. The research included two parts:1 The changes of PTEN in the development of cellular models of Alzheimer's diseaseSH-SY5Y cells or primary cultured cortical neurons of SD rats were induced by OA and Aβ_25-35 respectively. Cell morphology and p-tau (Ser³⁹⁶) protein level were used to determine the establishment and identification of cellular models of Alzheimer's disease. In the mean time, we observed the changes of PTEN protein and expression of PTEN mRNA and discussed the relevance with AD. The studies show that cell shrinkage, cell neurite disruption and hyperphosphorylation of tau protein were found in the all four experimental model, indicating the success of model building. 1) In the cellular model induced by 40 nmol/L OA, the level of PTEN protein increased after 3 hours and reached the peak after 6 hours. Then it declined and lower than the level of control after 48 hours (P>0.05). The expression of PTEN mRNA increased after 3 hours and reached the peak after 12 hours, and then it declined but higher than the expression of control after 48 hours (P<0.01). 2) In the cellular model induced by 40μmol/L Aβ_25-35, the level of PTEN protein increased after 3 hours and reached the peak after 12 hours. Then it declined and was about the level of control after 48 hours. The expression of PTEN mRNA declined after 6 hours and reached the peak after 12 hours, Then it declined but higher than the expression after of control 48 hours(P<0.01). 3) In the cellular model of primary cultured cortical neurons induced by 20 nmol/L OA, the level of PTEN protein increased after 3 hours and reached the peak after 12 hours. Then it declined and lower than the level of control after 48 hours. 4) In the cellular model of primary cultured cortical neurons induced by 20μmol/L Aβ_25-35, the level of PTEN protein increased and reached the peak after 6 hours. Then it declined and was about the level of control after 24 hours. Furthermore, the levels of PTEN protein increased precede the level of phosphorylation tau protein. It indicates that PTEN may have certain relevance with AD; the expression of PTEN may increase in the early development of AD.2 Effect of PTEN downregulation on tau hyperphosphorylation in SH-SY5Y cell which induced by OA and its possible mechanismsThe RNA interference lentiviral particles for human PTEN gene (PTEN-RNAi-LV) and the negative lentiviral particles with GFP (NC-GFP-LV) transfected into SH-SY5Y cells. The lentiviral effectively silenced the PTEN expression and the inhibitory efficiency of RNAi is 86.7% determined by western-blot and real-time quantitative PCR. Cells were treated with 40 nmol / L OA to make cellular model of AD. Compared with control group(CON) and negative control group (NC) group, vitality of knockdown (KD) group was improved and phosphorylated tau protein were significantly reduced when cells were incubated with OA for 24h. Then we link the relation of PTEN and tau phosphorylation to the PI3K/Akt signaling pathway. We found that PTEN downregulation activated PI3K/Akt signaling pathway by increasing p-Akt(Ser473) and inhibited GSK-3βactivity, then reduced tau hyperphosphorylation. The experimental results show that PTEN downregulation can antagonized the hyperphosphorylation of tau protein induced by OA and its mechanism may be related with the activation of PI3K/Akt signaling pathway.According to the results, we have these conclusions:1 Level of PTEN protein and expression of PTEN mRNA were dynamic changed in the development of cellular models of Alzheimer's disease. And the changes of PTEN protein happened ahead of that of tau hyperphosphorylation. It indicates that PTEN may have certain relevance with AD; the expression of PTEN may increase in the early development of AD. 2 PTEN downregulation can antagonized the hyperphosphorylation of tau protein induced by OA, it indicates that expression of PTEN increasing may contribute to the development of AD.3 The effects of PTEN on tau hyperphosphorylation may be related with the regulation of PI3K/Akt signaling pathway and its downstream GSK-3β.