Mutual Action Of Proteasome And Tau Phosphorylation And The Underlying Mechanism

One of the major neuropathological features of Alzheimer disease (AD) is the formation of large amounts of neurofibrillary tangles (NFTs), which are composed of hyperphosphorylated microtubule-associated protein tau. The accumulation of abnormal hyperphosphorylated tau in AD brain is a main cause for the damaged neuronal functions, but the mechanism remains unclear. In vitro studies have shown that in the paired helical filaments (PHF) formed by hyperphosphorylated tau extracted from AD brains, tau is not only phosphorylated but also ubiquitinated, which is an important signal of protein degradation by ubiquitin-proteasome system. Further studies have discovered that the proteasome activity in the brain of AD patients is decreased and PHF can directly inhibit the proteasome activity in vitro. However, the relationship between proteasome and tau phosphorylation as well as the role of their mutual action in the development of AD pathology remains unclear. In the present study, we have investigated the effect of proteasome inhibition on tau phosphorylation and vise veso as well as the involved mechanism. The results are shown as follows.1. Proteasome inhibition increases total level of tau in HEK293 stably expressing human tau441 After treatment of the cells with 2.5 μM, 5 uM and 10 μM lactacystin for 24 h,we observed that the activity of proteasome decreased in a dose-dependent manner. Different dose of lactacystin decreased proteasome activity to 53%, 28%, 11% of the control, respectively. At the same time, we measured the total tau level by using Tau-5 a monoclonal antibody to total tau and R134d another polyclonal antibody to total tau. It was shown that the immunoreactivity of Tau-5 increased in accordance with a decreased proteasome activity induced by different dose of lactacystin (2.5μM,5μM,10μM). To exclude the possible effect of protein synthesis on the increased tau level, we treated the cells simultaneously with cycloheximide (50 μg/ml) to inhibit protein translation. In this condition, we still observed the same result.2. Proteasome inhibition increases phosphorylated tau in HEK293/441 cellsThe level of the phosphorylated tau was measured by using a panel of phosphorylation-dependent antibodies after treatment of the cells with 2.5 μM, 5 μM and 10 μM lactacystin for 24 h . It was shown that the immunoreactivity of tau at PHF-1 epitope increased to 1.5-, 1.7-, 1.9-folds, at Ser-262 site increased to 2.0-, 2.6-, 3.1-folds, at Thr-231 site increased to 1.6-, 1.9-, 2.0-folds, and at tau-1 epitope increased to 1.1-, 1.1-, 1.4-folds of the control levels, respectively. After normalized to total tau (tau-5), the relative level of the phosphorylated tau at PHF-1, Ser-262 and Thr-231 sites was still higher than that of the controls, but the relative level of tau at tau-1 sites was decreased .These results suggest that proteasome degrades both phosphorylated epitopes (PHF-1, PT231 and PS262) and nonphosphorylated epitope (Tau-1) of tau, These results suggest that proteasome degrades both phosphorylated epitopes (PHF-1, PT231 and PS262) and nonphosphorylated epitope (Tau-1) of tau, but it is more efficient to the phosphorylated tau, because the accumulation of the phosphorylated tau was more obvious when the proteasome was inhibited.3. The involvement of GSK-3 in the lactacystin-induced accumulation of the phosphorylated tau.As the accumulated tau is hyperphosphorylated at some of the GSK-3 favorite sites during proteasome inhibition, we studied the involvement of GSK-3. We observed that the activity of GSK-3 was increased significantly with a concomitant decrease in serine-9-phosphorylated GSK-3 (inactive form). The total level of GSK-3β is also decreased. To further explore the role of GSK-3 in the phosphorylation and accumulation of tau during proteasome inhibition, we used LiCl to inhibit GSK-3 in the study. It was shown that the lactacystin-induced suppression of serine-9-phosphorylated GSK-3 was not only restored but also significantly higher than normal control after LiCl treatment. At the same time, the accumulation of the hyperphosphorylated tau at PHF-1 and Thr-231 epitopes induced by lactacystin was also partially restored by LiCl. These results suggest that activation of GSK-3 is involved in the lactacystin-induced accumulation of the hyperphosphorylated tau in the cells. 4. The involvement of PP-2A in lactacystin-induced accumulation of thephosphorylated tauPP-2A is the key phosphatase in tau dephosphorylation, therefore, we measured the activity of PP-2A by γ- ³²P-labeling assay and found that it was decreased when proteasome activity was inhibited by lactacystin (2.5 μM, 5 μM and 10 μM).To explore the possible mechanism leading to the inhibition of PP-2A, we measured the level of I1-PP-2A. And we found coincidently that the level of I1-PP-2A was increased upon lactacystin (2.5 μM and 10 μM) treatment (Fig.4C). These results suggest that proteasome may inhibit PP-2A through up-regulating I1-PP-2A.The above data have demonstrated that inhibition of proteasome can cause tau accumulation and GSK-3 and PP-2A are involved. To further study the effect of tau phosphorylation on the proteasome activity, we stably transfected the longest human isoform of tau (tau441) into human embryonic kidney cell(HEK293), which has no endogenous tau expression, and we used Foskorlin (foskorlin, an activitor of PKA) to induce tau hyperphosphorylation and measured the activity of proteasome. The main results are as followings:1. Induction of tau phosphorylation by forskolinForskolin treatment at different concentrations (0.5, 1, 2 and 4 μM) for 2 h The changes in tau phosphorylation were examined by Western blotting analysis using a panel of site-specific tau antibodies (PS214, PS262, PHF-1 and Tau-1). Quantitative results demonstrated that the levels of phosphorylated tau at various sites detected by these antibodies were significantly increased after forskolin incubation. Western blotting results demonstrated that the level of total tau was not altered significantly 2 h after forskolin (0.5, 1, 2 and 4 μM) treatment.2. Induction of tau aggregation by forskolinWe then questioned whether the increased phosphorylation of tau might aggregate in HEK293/tau441 by staining the cells with PS214 antibody and thioflavin-S simultaneously. In control culture, the intensity of both PS214 and thioflavin-S staining were dim and thioflavin-S-positive deposits were rare(<5% of the cells). After 2 μM forskolin treatment, the intensity of PS214 staining increased evidently and the phosphorylated tau is distributed in the cell soma and the fibers. Thioflavin-S positive deposit was also increased(>20% of the cells), however, the extend of this increase was not as evident as that of phosphorylated tau. The merged micrograph showed that the area of thioflavin-S positive deposit was much smaller than that of phosporylated tau, suggesting that tau aggregation was confined in some area of phosphorylated tau. Treating HEK293/tau441 with 4 μM of forskolin slightly enhanced the intensity of PS214 staining while the thioflavin-S positive aggregates became obvious(>60% of the cells) (Fig. 3g). Thioflavin-S positive aggregates overlapped well with phosphorylated tau in both cell soma and fibers, suggesting a widespread tauaggregation upon its phosphorylation.3. Dual effects of tau phosphorylation on proteasome activityThen, we studied the effects of tau phosphorylation on the trypsin-like proteasome activity by using Boc-Leu-Ser-Thr-Arg-AMC as substrate. The result showed that low degree of tau phosphorylation induced by foskorlin could activate the proteasome, however, when the phosphorylation of tau was increased to certain level, the proteasome activity was inhibited obviously. It suggests that the proteasome inhibition might be a later event in the pathogenesis of AD, which is possibly followed by tau hyperphosphorylation.In summary, we have demonstrated in the present study that lactacystin inhibits proteasome activity and increases the levels of tau with a dose-dependent manner in HEK293/tau441 cell line. The activation of GSK-3 and inhibition of PP-2A also contributes to the lactacystin-induced accumulation of the hyperphosphorylated tau. The inhibition of PP-2A by lactacystin involves up-regulation of I₁-PP-2A. Tau hyperphosphorylation also regulates the activity of proteasome.