Effect And Mechanism Of PROG On Tau Hyperphosphorylation In AD Cellular Models And Animal Models

Objective: The intracellular neurofibrillary tangles(NFTs) consisting of abnormally hyperphosphorylated tau is one of the core mechanisms of Alzheimer’s disease(AD). The evidences have suggested that many factors could induce tau hyperphosphorylation. The most significant research is β-amyloid(Aβ). Studies have shown that PI3K/AKt-GSK3β signaling pathway is a major pathway regulating tau protein phosphorylation. Clinically, progesterone(PROG) is used to treat female reproductive system diseases, and it is an important endogenous neurosteroids that plays an important role in the brains. Studies have shown that progesterone play a neuroprotective effect by inhibiting neuronal excitotoxicity, improving AD animal models learning and memory function, inhibition of neuronal apoptosis and other mechanisms. In this study, we established AD cell model by Aβ25-35 inducing rat primary cultured cortical neurons in vitro and used APP/PS1 transgenic mice as AD animal model, which investigated the effect and mechanisms of progesterone on tau hyperphosphorylation at the cellular level and animal level.Methods: 1 Primary culture of rat cortical neurons Postnatal SD rats within 24 h were decapitated at sterile conditions. The cortex section was taken out, moved to 1.25 g/L trypsin, and digested. The cells were terminated digestion by complete medium containing 10% FBS, inoculated in cell culture plate, adjusted the cell density of 1.0×109/L. Cells were cultured at 5% CO2 and 37℃conditions. After 4 hours, the neurobasal complete culture medium(100 ml plus B27 2 m1) was completely exchanged. After 48 hours, Ara-C was added to the medium to inhibit the growth of glial cells at a final concentration of 5 μM, and the cells were cultured for 8~10 day.2 Preparation of Aβ25-35 The Aβ25-35 powder was dissolved in sterile distilled water to make the stock solution at a final concentration of 1 mmol/L, then mixed, packaged, placed at 37℃ and sterile environment to age for 3 days. The Aβ25-35 solution was frozen and set aside at-20℃. 3 grouping 3.1 Effect of Aβ25-35 on tau hyperphosphorylation in neurons The neurons were randomly divided into Control, Aβ25-35(20 μM, 40 μM), and co-cultured for 24 h. Western blot assay was used to detect tau phosphorylation sites at Ser404 and Thr231. 3.2 Effect of progesterone on tau protein hyperphosphorylation in neurons induced by Aβ25-35 The neurons were randomly divided into Control, Aβ25-35(20 μM), Aβ25-35+PROG(1 μM), and co-cultured for 24 h. Western blot assay and Immunofluorescence were used to detect tau phosphorylation sites at Ser404 and Thr231. 3.3 Effect and mechanisms of PROG on tau hyperphosphorylation in APP/PS1 transgenic mouse APP/PS1 transgenic mice were randomly divided into APP/PS1 model group and PROG treatment group with each group of six mice. PROG treatment groups were subcutaneously injected of progesterone one month. The process was that the mice were continuously injected for five days with an interval of two days, and continued injection. APP/PS1 model group were injected with the same dose of saline. Immunohistofluorescence was used to detect the effect of PROG on tau phosphorylation sites Ser404, Thr231 and the influence of PROG on PI3K/AKt-GSK-3β signal pathway in the hippocampus and cortex of APP/PS1 mouse. 3.4 Effect of progesterone on GSK-3β in neurons induced by Aβ25-35 The neurons were randomly divided into Control, Aβ25-35, Aβ25-35+PROG, Aβ25-35+Li Cl(5 m M, specific inhibitor of GSK-3β), and co-cultured for 24 h. Western blot assay was used to detect tau phosphorylation sites at Ser404,Thr231 and p-GSK-3β(Tyr216) protein expression levels.. 3.5 Effect of progesterone on PI3K/AKt in neurons induced by Aβ25-35 The neurons were randomly divided into Control, Aβ25-35, Aβ25-35+PROG, Aβ25-35+PROG+LY294002(10 μM, specific inhibitor of PI3K/AKt), and co-cultured for 24 h. Western blot assay was used to detect tau phosphorylation sites at Ser404, Thr231, p-GSK-3β(Tyr216) and p-AKt(Ser473) protein expression levels. 3.6 The role of PGRMC1 in progesterone inhibiting tau protein hyperphosphorylation The neurons were randomly divided into Control, Aβ25-35, Aβ25-35+PROG, Aβ25-35+PROG+AG205(5 μM, membrane receptor inhibitor of PROG), Aβ25-35+PROG+RU486(5 μM, nuclear receptor inhibitor of PROG), and cocultured for 24 h. Western blot assay was used to detect tau phosphorylation sites at Ser404 and Thr231. 3.7 Effect of PGRMC1 on PI3K/AKt signal pathway The neurons were randomly divided into Control, Aβ25-35, Aβ25-35+PROG, Aβ25-35+PROG+AG205, and co-cultured for 24 h. Western blot assay was used to detect p-GSK-3β(Tyr216) and p-AKt(Ser473) protein expression levels. 4 Western blot analysis was used to detect protein levels of p-tau(Ser404), p-tau(Thr231), T-tau, p-GSK-3β(Tyr216), p-AKt(Ser473), T-GSK-3β and T-AKt. The treated cells were collected, and total protein was extracted. The supernatant is protein samples which were quantified by BCA. Equal amounts of protein were separated by SDS-PAGE and electrotransferred to a PVDF membrane. Membranes were blocked with skimmed milk powder, and incubated with the primary antibodies, 1:1000 rabbit anti-p-Tau(Ser404), p-Tau(Thr231),T-tau, p-GSK-3β(Tyr216), T-GSK-3β, p-AKt(Ser473), T-AKt overnight at 4℃. Membranes were blocked with secondary antibody, luminescenced and exposure. Results were scanned by gel imaging analysis system to measure each strip integral optical density, and represented by the ratio of protein and β-actin integrated optical density values.5 Immunofluorescence was used to detect protein levels of p-tau(Ser404) and p-tau(Thr231). The neurons on the slide were washed with PBS, fixed with 4% paraformaldehyde, punched with 0.05% Triton-X100, blocked with 3% BSA(diluted with PBS) for, and incubated with the primary antibodies, 1:200 rabbit anti-p-Tau(Ser404) and p-Tau(Thr231) overnight at 4℃. The next day, the cells were incubated with secondary antibody at 37 ℃cartridge for 2 h, and observed by fluorescence microscopy. 6 Immunohistofluorescence was used to detect protein levels of p-tau(Ser404), p-tau(Thr231), p-GSK-3β(Tyr216) and p-AKt(Ser473) in APP/PS1 transgenic mouse brain The 8 months mice in each group were anesthetized. Brains were rapidly removed, placed on ice boxes, fixed in 4% paraformaldehyde, and conventionally prepared frozen sections. The slices were blocked with goat serum for 30 min, and incubated with the primary antibodies, 1:200 rabbit anti-p-Tau(Ser404), p-Tau(Thr231), T-GSK-3β and p-AKt(Ser473) overnight at 4℃. The next day, the slices were incubated with secondary antibody at 37 ℃cartridge for 2 h, Hoechst 33258 stained for 5 min, washed with PBS 3 × 10 min times, and observed by fluorescence microscopy. 7 Statistical Analysis Experimental data were analyzed with SPSS 16.0 software, and given as the mean±SD( x ± s). Significance between the groups was analyzed by independent samples t test or ANOVA. P<0.05 was considered statistical significance.Results: 1 Effect of Aβ25-35 on tau hyperphosphorylation in neurons Western Blot showed that compared with the Control group, both 20 μM and 40 μM Aβ25-35 significantly increased phosphorylated tau at Thr231 and Ser404(P<0.05) in rat cortical neurons. However, Aβ25-35 had no effect on total tau level. Concerning the toxicity of Aβ25-35, we chose 20 μM as experimental concentration.2 Effect of PROG on tau hyperphosphorylation induced by Aβ25-35 in neurons Western Blot showed that compared with the Aβ25-35 group, tau protein phosphorylation levels in PROG group significantly decreased(P<0.05). Immunofluorescence showed that compared with the Control group, Aβ25-35 can significantly increase the expressions of tau protein phosphorylated sites at Thr231 and Ser404(P<0.05). PROG treatment significantly decreased tau phosphorylation levels compared with the Aβ25-35 group(P<0.05). 3 Effect of PROG on tau hyperphosphorylation in APP/PS1 transgenic mouse brain Immunofluorescence showed that tau protein phosphorylation sites at Thr231 and Ser404 in cerebral cortex and hippocampus of APP/PS1 transgenic mouse markedly colored. After treatment by progesterone, tau protein phosphorylation levels in APP/PS1 transgenic mouse significantly reduced(P<0.05). 4 Effect of PROG on GSK-3β induced by Aβ25-35 in neurons Western Blot showed that the expressions of tau phosphorylation in PROG group and Li Cl group significantly increased and p-GSK-3β level significantly decreased compared with the Aβ25-35 group(P<0.05). 5 Effect of PROG on PI3K/AKt induced by Aβ25-35 in neurons Western Blot showed that Aβ25-35 can significantly decrease the expressions of p-AKt compared with the Control group(P<0.05). However, PROG treatment significantly increased the expressions of p-AKt compared with the Aβ25-35 group(P<0.05). At the same time, compared with PROG group, tau protein phosphorylation levels at Thr231 and Ser404 significantly increased in LY294002 group(P<0.05). 6 The role of PGRMC1 in PROG attenuating tau hyperphosphorylation Western Blot showed that compared with PROG group, the expressions of tau protein phosphorylation at Thr231 and Ser404 in AG205 group significantly increased(P<0.05). However, RU486 group did not cause a significant change.7 Effect of PGRMC1 on PI3K/AKt-GSK-3β signal pathway Western Blot showed that compared with PROG group, the expressions of p-GSK-3β in AG205 group significantly increased(P<0.05), but the expressions of p-AKt significantly decreased(P<0.05). 8 Effect of PROG on GSK-3β in APP/PS1 transgenic mouse brain Immunofluorescence showed that p-GSK-3β in cerebral cortex and hippocampus of APP/PS1 transgenic mouse significantly colored. After treatment by progesterone, p-GSK-3β levels in APP/PS1 transgenic mouse significantly reduced(P<0.05). 9 Effect of PROG on PI3K/AKt in APP/PS1 transgenic mouse brain Immunofluorescence showed that p-AKt in cerebral cortex and hippocampus of APP/PS1 transgenic mouse expressed. However, after treatment by progesterone, p-AKt levels in APP/PS1 transgenic mouse significantly increased(P<0.05).Conclusions: 1 Progesterone could inhibit Aβ25-35-induced tau hyperphosphorylation in primary cultured rat cortical neurons and brains of APP/PS1 transgenic mouse 2 Progesterone could inhibit Aβ25-35-induced tau hyperphosphorylation in primary cultured rat cortical neurons and brains of APP/PS1 transgenic mouse by regulating the PI3K/AKt-GSK-3β signal pathway. 3 PGRMC1 may media progesterone inhibiting Aβ25-35-induced tau hyperphosphorylation and regulating the PI3K/AKt-GSK-3β signal pathway in primary cultured rat cortical neurons.