Effect Of Dehydroevodiamine On Alzheimer-like Tau Hyperphosphorylation And Its Mechanism

Alzheimer's disease is a common and progressive neurodegenerative disease in the eldly population. Current medications that have passed approval for treatment of AD seem to be able to produce modest symptomatic improvement in some of mild to moderate cases. None of the medications, however, appears to be able to cure AD or stop the disease progression. Because the etiology of AD is still unclear, the search for efficient drugs to prevent or treat AD is facing opportunity and will have nice prospect.Pathologically, AD is characterized by the formation of numerous intracellular neurofibrillary tangles (NFTs) and extracellular senile plaques (SPs) in the brain, which are respectively consisted of hyperphosphorylated tau andβ-amyloid (Aβ) peptide. Hyperphosphorylated tau is believed to be an early pathological event of AD and only the amount of NFTs is closely correlated with the clinical dementia degree of AD patients. Thus target the underlying pathogenic mechanisms of tau hyperphosphorylation in AD might be a potential therapeutic strategy for prevention or treatment of the diseae.Dehydroevodiamine (DHED) is a constituent of alkaloids isolated from the unripe fruit of a Chinese herb, namely Evodia rutaecarpa Bentham. In our present study, we have investigated the pharmacological effect of DHED on WT/GFX induced AD-like pathological models and its involved mechanism of the effection.The rats were injected through vena caudalis 6.25 mg/kg/day or 12.5 mg/kg/day of DHED or NS for 7 days. At the same time, the rats received water maze training. At the day 8, wortmannin (WT, a specific PI3K inhibitor) and GF-109203X (GFX, a specific PKC inhibitor) (100μM of each, total volume of 10μl) were co-injected into the left ventricle of the rats and the spatial memory retention was tested at 24 h after the brain injection. The samples were prepared for western blotting and immunocytochemistry research after the spatial memory retention test. The results are shown as follows.1. Effects of DHED on WT/GFX-induced spatial memory retention deficits of rats.It was shown that the latency increased significantly after the injection of WT/GFX, and pre-injection of DHED at both dosages significantly shortened the WT/GFX-induced increase of the latency. Instead of taking a tortuous swimming path to find the hidden platform as seen in WT/GFX-injected group, the pre-injection of DHED at both dosages significantly improved the searching strategy of the rats. These results suggest that pre-administration of DHED can effectively prevent the rats from WT/GFX-induced spatial memory deficits.2. Effects of DHED on WT/GFX-induced tau hyperphosphorylation in rat hippocampus.The immunoreactivity of PHF-1 and PS396 was enhanced and the immunoreactivity of tau-1 was decreased in WT/GFX-injected rats, suggesting that WT/GFX induces tau hyperphosphorylation at Ser396/404 (PHF-1 and PS396) and Ser199/202 (tau-1 reacts with non-phosphorylated tau). Pre-injection of DHED at 12.5 mg/Kg/day attenuated the WT/GFX-induced tau hyperphosphorylation at PHF-1, PS396 and tau-1 epitopes. When lower concentration of DHED (6.25 mg/Kg/day) was used, the attenuation of tau hyperphosphorylation was only seen at PS396 site but not at PHF-1 and tau-1 epitopes. We also tested the level of total tau using antibody R134d, but no significant difference was observed.The effect of DHED on WT/GFX-induced tau hyperphosphorylation was also studied by immunocytochemistry. In WT/GFX-injected rats, the immunoreaction of PHF-1 mainly in the mossy fibers of CA3 region of the hippocampus was enhanced, while the immunoreactivity of tau-1 in the mossy fibers of CA3 region was decreased, suggesting that WT/GFX induces tau hyperphosphorylation. Pre-injection of DHED significantly arrested the WT/GFX-induced tau hyperphosphorylation at PHF-1 and tau-1 epitopes.3. Effects of DHED on WT/GFX-induced overactivation of GSK-3βin rat hippocampus.we measured the total level and the activity-dependent Ser9-phosphorylated level of GSK-3βin the hippocampal extracts by western blotting. No obvious change was seen in total level of GSK-3βin WT/GFX-injected or DHED-preinjected rats. However, the level of Ser9-phosphorylated GSK-3β(p-S9-GSK-3β, representing the inactivated form of the kinase) was significantly decreased in WT/GFX-injected control group, and preinjection of DHED increased the level of the Ser9-phosphorylated GSK-3β. suggesting that the DHED could prevent GSK-3βfrom WT/GFX-induced overactivation.To further study the effect of DHED on tau phosphorylation and GSK-3βactivation in cell line, we pretreated the neuro2A (N2a) cells with different dose of DHED for 24h, and then treated 1μM each of WT and GFX for 1h. The main results are as followings:1. Effect of DHED on tau hyperphosphorylation induced by WT/GFX in N2a cells.Western blotting demonstrated that the immunoreactivity of tau Ser396/404 and Ser199/202 was significantly attenuated by 5 and 10μM DHED (p<0.01). No significant difference was observed between the two doses of DHED. The application of DHED alone did not change of tau phosphorylation level.2. Effect of DHED on overactivation of GSK-3βin N2a cells. We measured the total level and the activity-dependentSer9-phosphorylated level of GSK-3βin N2a cells by western blotting. No obvious change was seen in total GSK-3βwith or without DHED. However, the level of Ser9-phosphorylated GSK-3β(p-S9-GSK-3β) was significantly decreased to 41% after 1μM WT/GFX treatment 1h(p<0.01), and pretreatment of DHED 5μM and 10μM for 24h increased the level of the p-S9-GSK-3βto 81% and 87% respectively (p<0.01). Suggesting that the DHED could prevent GSK-3βfrom WT/GFX-induced overactivation.By immunofluorescence staining, a significantly decreased immunoreaction of p-S9-GSK-3βwith an enhanced staining of the phosphorylated tau at PHF-1 was seen after WT/GFX treatment, and pretreatment of the cells with 10μM DHED for 24h reversed the changes.We transiently transfected wtGSK-3βin N2a cells and treated the cells with DHED for 24h. The overexpression of the exogenous GSK-3βwas suppressed by DHED. Tau hyperphosphorylation following with overexpression of wtGSK-3βalso attenuated by add with DHED. These data confirmed that DHED down-regulating the activity of GSK-3β.3. DHED increases the phosphorylation level of Akt in N2a cells.The most important kinase responsible for Ser9-phosphorylation of GSK-Sβis Akt. It is to believe that Akt requires phosphorylation at Ser473 and Thr308 to be activated. We tested the activity-dependent Ser473- and Thr308-phosphorylated level of Akt in the N2a cells. No obvious change was seen in total level of Akt with or without DHED. However, the level of Ser473 and Thr308-phosphorylated Akt was significantly decreased after 1μM WT/GFX treatment 1h, and pretreatment of DHED for 24h increased the level of the Ser473 and Thr308-phosphorylated Akt.In summary, we have demonstrated in the present study that WT/GFX induced tau hyperphosphorylation through GSK-3βactivation in N2a cell line and rats. DHED attenuates tau hyperphosphorylation via GSK-3βinhibition. The effect of DHED on GSK-3βmay be involves Akt activation.