Up-regulation of phosphorylated/activated p70 S6 kinase and its relationship to neurofibrillary pathology in Alzheimer's disease

Abstract

The ribosomal S6 protein kinase p70 S6 kinase is known for its role in modulating cell-cycle progression, cell size, and cell survival. In response to mitogen stimulation, p70 S6 kinase activation up-regulates ribosomal biosynthesis and enhances the translational capacity of the cell. In Alzheimer's disease (AD), there is a marked increase in total tau protein in the form of abnormally hyperphosphorylated tau (PHF-tau) in neurons with neurofibrillary tangles (NFTs). In the present study, we investigated whether p70 S6 kinase activation is associated with PHF-tau accumulation in AD. By immunohistochemistry, we found that the levels of phosphorylated p70 S6 kinase (at Thr389 or at Thr421/Ser424) were increased in accordance with the progressive sequence of neurofibrillary changes according to Braak's criteria. Confocal microscopy showed that in AD brain, phosphorylated p70 S6 kinase appeared especially in neurons that are known to later develop NFTs. This pattern of neurons showed dot-like structures of phosphorylated p70 S6 kinase and hyperphosphorylated tau, which partially correlated with rab5 (endosome marker), lamp-1 (lysosome marker), and ubiquitin (ubiquitin-proteasomal system marker). By indirect enzyme-linked immunosorbent assay, phosphorylated p70 S6 kinase (Thr389 or Thr421/Ser424), total tau, and PHF-tau were found to be significantly increased in AD brain as compared to control cases. The levels of total p70 S6 kinase and p70 S6 kinase phosphorylated at Thr421/Ser424 showed significant correlations with the levels of both total tau and PHF-tau. Regression analyses revealed a significant dependence of total tau or PHF-tau on p70 S6 kinase phosphorylated at Thr421/Ser424 rather than at Thr389. The levels of ribosomal protein S6 as well as the levels of markers for the proteolytic system were also significantly increased in AD as compared to control brain. Using a SH-SY5Y neuroblastoma cell model, we found that 100 micro mol/L zinc sulfate could induce p70 S6 kinase phosphorylation and activation, in particular at Thr421/Ser424. This up-regulation of the activated kinase resulted in an increased expression and phosphorylation of tau. Pretreatment of cells with rapamycin (an inhibitor of FRAP/mTOR which is the immediate upstream kinase of the p70 S6 kinase) attenuated the effects induced by zinc. In primary cultured neurons of rat cortical cortex, zinc sulfate treatment could repeat p70 S6 kinase phosphorylation and activation at Thr421/Ser424, followed by increased expression and phosphorylation of tau. Taken together, these data suggest that activated p70 S6 kinase could mediate an up-regulation of tau translation. The partial co-localization of phosphorylated p70 S6 kinase with rab5, lamp-1 and ubiquitin, or PHF-tau with ubiquitin suggests that the activated proteolytic system might not be sufficient to degrade the over-produced and over-phosphorylated tau protein. A p70 S6 kinase modulated up-regulation of tau translation might contribute to PHF-tau accumulation in neurons with neurofibrillary changes.

Figure 1.

Characterization of antibodies to p70 S6 kinase and detection of p70 S6 kinase immunoreactivity in human brain homogenates by Western blots. Blots of homogenates from AD and control brains (12.5 μg protein/lane), AD p-tau (2 μg protein/lane), PHF-tau (2 μg protein/lane), and extracts from non-treated and serum-treated NIH-3T3 cells (negative and positive controls, respectively) were developed with antibodies to total p70 S6 kinase, p70 S6 kinase phosphorylated at either T389 or T421/S424. The p70 S6 kinase antibodies consistently identified p70/p85 kd S6 kinase in homogenates and NIH-3T3 cell lysates. None of the p70 S6 kinase antibodies showed cross-reaction with either AD p-tau or PHF-tau (A and B). No difference between AD and control cases was observed with total p70 S6 kinase (p70 S6K) whereas antibody to the p70 S6 kinase T421/S424 showed increase in most AD cases (C).

Figure 2.

Immunohistochemical staining with antibody to activated p70 S6 kinase (T389) in brains with different degrees of neurofibrillary degeneration according to Braak’s staging criteria. The normal control that does not have any neurofibrillary involvement did not reveal any immunostaining (a). The early transentorhinal stage (I-II) that is characterized by mild neurofibrillary pathology in the transentorhinal region showed a few immunopositive neurons (b). The moderate limbic stage (III-IV) which is marked by a moderate involvement of neurofibrillary pathology in the entorhinal region, and the involvement of a few or many CA1 cells in the hippocampal region and temporal lobes showed immunostaining in several tangle-bearing neurons (c). The late isocortical stage (V-VI) which is characterized by the involvement of severe neurofibrillary pathology in the entorhinal cortex, the hippocampus, and isocortex showed the most robust immunostaining of neurons (d). Following the sequence of neurofibrillary degeneration from normal control to isocortical stage (V/VI), antibody to p70 S6 kinase phosphorylated at T389 showed progressively increased numbers of tangle-like inclusions and granular structures. Area 1 shows transentorhinal region in a and b or entorhinal region in c and d. Areas 2 and 3 show hippocampal CA1 and temporal isocortex, respectively in a, b, c and d. Bars in 3, 100 μm; bars in insets, 10 μm.

Figure 3.

Immunohistochemical staining with antibody to activated p70 S6 kinase (T421/S424) in brains with different degrees of neurofibrillary degeneration according to Braak’s staging criteria. Following the sequence of neurofibrillary degeneration (a to d, same as in Figure 2 ▶ ) from normal control to Stage V/VI, antibody to p70 S6 kinase (T421/S424) showed progressively increased numbers of tangle-like inclusions and granular structures. Bars in 3, 100 μm; bars in insets, 10 μm.

Figure 4.

Immunohistochemical co-localization of active p70 S6 kinase (T389) with PHF-tau (labeled by AT8). Active p70 S6 kinase (red) was partially or fully co-localized with AT8-labeled PHF-tau (green) in neuronal cytoplasm (A1, A2, and A3), and in a few dystrophic neurites (B1, B2, and B3). Bar, 20 μm.

Figure 5.

Immunoreactivity of active P70 S6 kinase (T421/S424) in neurons with different degrees of AT8-labeled PHF-tau involvement. In the normal-looking neurons, only active p70 S6 kinase was positive in granular form (A1, A2, and A3). In the pre-tangle neurons, some of the granular stainings of active p70 S6 kinase was partially overlapped with dotted AT8 labeled PHF-tau (B1, B2, and B3). In classic tangle bearing neurons, the granular staining of active p70 S6 kinase congregated, and some particle staining distributed along with the AT8-positive filamentous structures (C1, C2, and C3). At the advanced stage of tangle neurons, no dot-like active p70 S6 kinase staining could be seen, except for the faint filamentous structures partially overlapping with AT8 positive tangles (D1, D2, and D3). Bars, 10 μm.

Figure 6.

Levels of p70 S6 kinase and tau, and their correlations in human brain homogenates. Levels of active p70 S6 kinases (T389, T421/S424), total tau (labeled by R134 days), and PHF-tau (labeled by PHF-1 or AT8) were significantly increased in AD as compared with control (A and B). Levels of total p70 S6 kinase and normal tau (labeled by Tau-1) did not differ significantly between AD and controls (A and B). A and B: Mean ± SEM of 13 control and 22 AD cases. * indicates P < 0.05, and *** indicates P ≤ 0.001. The Pearson correlation analyses showed that levels of total p70 S6 kinase and active p70 S6 kinase (T421/S424) rather than active p70 S6 kinase (T389) showed significant correlations with total tau and PHF-tau (labeled by PHF-1 or AT8) in AD homogenates (C, D, and E; Table 4 ▶ ). Immunoreactivities of p70 S6 kinase and tau are presented as percentage of the OD values of control cases per 2 μg protein. ▴, AD cases and •, control individuals.

Figure 7.

Co-localization of p70 S6 kinase (T421/S424) and lysosomes (lamp-1). In Patterns 1 and 2 neurons (see Figure 5 ▶ ), there was no obvious overlap between p70 S6 kinase (T421/S424) and lysosomes (A1, A2, and A3 of panel A). In Pattern 3 neurons, partial co-localization with lamp-1 could be detected in p70 S6 kinase positive structures (B1, B2, and B3 of panel A). Twelve serial optical sections (1.5-μm interval) were obtained from the same neuron as B1, B2, and B3 of panel A by confocal microscope. Bars, 10 μm.

Figure 8.

Levels of immunoreactivity of ribosomal protein S6 (rpS6) and markers to proteolytic system in human brain homogenates. The immunoreactivities of both rpS6 (total) and phosphorylated rpS6 (S235/236), but not rpS6 (S240/244) were significantly increased in AD brains (A). Significant increase was also seen with antibodies to Rab5, Lamp-1 and ubiquitin in AD homogenates as compared with controls (B). The specificity of the antibodies was tested by Western blots, showing only one clear band corresponding to respective protein (B and D). Results represent the mean ± SEM of 13 control and 22 AD cases. * P < 0.05 and ** P < 0.01 compared with control group.

Figure 9.

The phosphorylation of p70 S6 kinase is associated with the accumulation and phosphorylation of tau protein. SH-SY5Y cells were cultured in DMEM/F12 (1:1) containing 5% fetal bovine serum (FBS) for 4 days, and then in media containing 0.5% FBS for 2 days. The time-dependent expression and phosphorylation of p70 S6 kinase (A) and tau (B) were detected by Western blots in SH-SY5Y cells treated with 100 μmol/L zinc sulfate. The phosphorylated p70 S6 kinase and tau protein showed instant increase after 5 minutes exposure with zinc sulfate, and the increase reached a significant high level at 30 minutes of treatment. Pretreatment of SH-SY5Y cells with 20 ng/ml rapamycin for 1 hour inhibited the effects of zinc sulfate on the phosphorylation of p70 S6 kinase (C) and tau phosphorylation and accumulation (D). Exposure of rat brain primary cultured neurons with 200 μmol/L zinc sulfate for 30 minutes showed significant increase of phosphorylated p70 S6 kinase at T421/S424, total tau (R134), normal tau (Tau-1), and phosphorylated tau (PHF-1). Pretreatment of the primary cultures with rapamycin for 1 hour resulted in decreased levels of phosphorylated p70 S6 kinase and tau induced by zinc sulfate treatment (G and H). The blots are representative from three or four experiments. Results in E, F, and H were expressed as percentage value relative to non-treated cells. E, F, and H: Means ± SEM of at least three independent experiments of C and D. Statistical analyses were made using analysis of variance with LSD post hoc test. * P < 0.05 and ** P < 0.01 compared with untreated cells; # P < 0.05 and ## P < 0.01 compared with zinc sulfate (100 μmol/L) 30-minute-treated group.

Figure 10.

Hypothetical scheme showing the effects of zinc-induced p70 S6 kinase activation by PI3K and MAPK pathways on the accumulation of tau protein. Zinc induces p70 S6 kinase activation, and then enhances the translation of TOP mRNA coding the translation apparatus, and results in the increase of translational capacity of tau protein. Even though the proteolytic system was also involved, it appears to be insufficient relative to the production for the degradation of tau