Wen-Lin An

Up-Regulation of Phosphorylated/Activated p70 S6 Kinase and Its Relationship to Neurofibrillary Pathology in Alzheimer’s Disease

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 Alzheimers 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 Braaks 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.

Up-regulation of mitogen-activated protein kinases ERK1/2 and MEK1/2 is associated with the progression of neurofibrillary degeneration in Alzheimer’s disease

The abnormal hyperphosphorylation of tau in Alzheimers disease (AD) has been proposed to involve the extracellular-signal-regulated protein kinase (ERK) of the mitogen-activated protein (MAP) kinase family. ERK is phosphorylated and thereby activated by MAP kinase kinase (MEK). In the present study, we determined the intracellular and regional distribution of the active forms of both MEK1/2 and ERK1/2, i.e. p-MEK1/2 and p-ERK1/2 in the entorhinal, hippocampal, and temporal cortices of 49 brains staged for neurofibrillary changes according to Braak and Braaks protocol. We found that p-MEK1/2 and p-ERK1/2 were present in the initial stages of neurofibrillary degeneration in the projecting neurons in the transentorhinal region, and extended into other brain regions co-incident with the progressive sequence of neurofibrillary changes up to and including Braak stage VI. It appeared that the accumulation of p-MEK1/2 and p-ERK1/2 was initiated in the cytoplasm of pretangle neurons in varying size granules, which grew into large aggregates co-existing with the progressive development of neurofibrillary tangles. Accumulation of p-MEK1/2 and p-ERK1/2 was found in cases with stages I-III neurofibrillary degeneration, which were devoid of amyloid deposition. These data provide direct in situ evidence consistent with the possible involvement of MAP kinase pathway in the hyperphosphorylation of tau and the presence of this lesion before deposition of beta-amyloid in AD.

Role of protein kinase B in Alzheimer's neurofibrillary pathology

Protein kinase B (PKB) is an important intermediate in the phosphatidylinositol-3 kinase signaling cascade that acts to phosphorylate glycogen synthase kinase-3 (GSK-3) at its serine 9 residue, thereby inactivating it. Activated GSK-3 has been previously shown to be preferentially associated with neurofibrillary tangles (NFTs) in Alzheimers disease (AD) brain. In the present study, we performed immunohistochemistry with an antibody to the active form of PKB in brains with different stages of neurofibrillary degeneration. We found that the amount of activated PKB (p-Thr308) increased in correlation to the progressive sequence of AT8 immunoreactivity and neurofibrillary changes assessed according to Braaks criteria. By confocal microscopy, activated PKB (p-Thr308) was found to appear in particular in neurons that are known to later develop NFTs in AD. Western blotting showed that activated PKB was increased by more than 50% in the 16,000-g supernatants of AD brains as compared with normal aged and Huntingtons disease controls. This increase in PKB levels corresponded with a several-fold increase in the levels of total tau and abnormally hyperphosphorylated tau at the Tau-1 site. These studies suggest the involvement of PKB/GSK-3 signaling in Alzheimer neurofibrillary degeneration.

Okadaic-Acid-Induced Inhibition of Protein Phosphatase 2A Produces Activation of Mitogen-Activated Protein Kinases ERK1/2, MEK1/2, and p70 S6, Similar to That in Alzheimer’s Disease

In Alzheimers disease (AD) brain the activity of protein phosphatase (PP)-2A is compromised and that of the extracellular signal-regulated protein kinase (ERK1/2) of the mitogen-activated protein kinase (MAPK) family, which can phosphorylate tau, is up-regulated. We investigated whether a decrease in PP-2A activity could underlie the activation of these kinases and the abnormal hyperphosphorylation of tau. Rat brain slices, 400-microm-thick, kept under metabolically active conditions in oxygenated (95% O(2), 5% CO(2)) artificial CSF were treated with 1.0 micromol/L okadaic acid (OA) for 1 hour at 33 degrees C. Under this condition, PP-2A activity was decreased to approximately 35% of the vehicle-treated control slices, and activities of PP-1 and PP-2B were not affected. In the OA-treated slices, we observed a dramatic increase in the phosphorylation/activation of ERK1/2, MEK1/2, and p70 S6 kinase both immunohistochemically and by Western blots using phosphorylation-dependent antibodies against these kinases. Treatment of 6-microm sections of the OA-treated slices with purified PP-2A reversed the phosphorylation/activation of these kinases. Hyperphosphorylation of tau at several abnormal hyperphosphorylation sites was also observed, as seen in AD brain. These results suggest 1) that PP-2A down-regulates ERK1/2, MEK1/2, and p70 S6 kinase activities through dephosphorylation at the serine/threonine residues of these kinases, and 2) that in AD brain the decrease in PP-2A activity could have caused the activation of ERK1/2, MEK1/2, and p70 S6 kinase, and the abnormal hyperphosphorylation of tau both via an increase in its phosphorylation and a decrease in its dephosphorylation.

Mechanism of zinc-induced phosphorylation of p70 S6 kinase and glycogen synthase kinase 3β in SH-SY5Y neuroblastoma cells

We have previously reported an aberrant accumulation of activated protein kinase B (PKB), glycogen synthase kinase (GSK)‐3β, extracellular signal‐regulated kinase (ERK1/2), c‐Jun N‐terminal kinase (JNK), p38 and p70 S6 kinase (p70S6K) in neurons bearing neurofibrillary tangles (NFTs) in Alzheimers disease (AD). However, the mechanism by which these tau candidate kinases are involved in the regulation of p70S6K and GSK‐3β phosphorylation is unknown. In the current study, 100 μm zinc sulfate was used, and influences of various components of phosphatidylinositol 3‐kinase (PI3K) and mitogen‐activated protein kinase (MAPK) pathways on p70S6K and GSK‐3β phosphorylation have been investigated in serum‐deprived SH‐SY5Y neuroblastoma cells. We found that zinc could induce an increase of phosphorylated (p) p70S6K, p‐PKB, p‐GSK‐3β, p‐ERK1/2, p‐JNK and p‐p38, especially in long‐term treatment (4–8 h). Treatment with different inhibitors including rapamycin, wortmannin, LY294002, and U0126, and their combinations, indicated that phosphorylation of p70S6K and GSK‐3β is regulated by rapamycin‐dependent, PI3K and MAPK pathways. Furthermore, phosphorylation of p70S6K and GSK‐3β affected levels of tau unphosphorylated at the Tau‐1 site and phosphorylated at the PHF‐1 site, and p70S6K phosphorylation affected the total tau level. Thus, 100 μm zinc might activate PKB, GSK‐3β, ERK1/2, JNK, p38 and p70S6K, that are consequently involved in tau changes in SH‐SY5Y cells.

P70 S6 kinase mediates tau phosphorylation and synthesis

Currently, we found that the 70‐kDa p70 S6 kinase (p70S6K) directly phosphorylates tau at S262, S214, and T212 sites in vitro. By immunoprecipitation, p‐p70S6K (T421/S424) showed a close association with p‐tau (S262 and S396/404). Zinc‐induced p70S6K activation could only upregulate translation of total S6 and tau but not global proteins in SH‐SY5Y cells. The requirement of p70S6K activation was confirmed in the SH‐SY5Y cells that overexpress wild‐type htau40. Level of p‐p70S6K (T421/S424) was only significantly correlated with p‐tau at S262, S214, and T212, but not T212/S214, in Alzheimers disease (AD) brains. These suggested that p70S6K might contribute to tau related pathologies in AD brains.

Phosphorylated eukaryotic translation factor 4E is elevated in Alzheimer brain.

Eukaryotic translation factor 4E (eIF4E) plays a key role in regulating protein translation. It was thought that in order to maintain neuronal functions, tau protein is continuously generated to compensate those being hyperphosphorylated and compromised in its ability to promote and maintain microtubule assembly in Alzheimers disease. If eIF4E is involved in tau mRNA translation, level of eIF4E phosphorylation should be changed. In the current study, we found a dramatic increase of phosphorylated eIF4E in Alzheimers disease, especially in those cases with late stages of neurofibrillary changes. Level of eIF4E phosphorylation is significantly correlated with total- and Alzheimer hyperphosphorylated taus. These data suggest that the increase of eIF4E phosphorylation is involved in formation of Alzheimer neurofibrillary changes.