Role of P70 S6 Kinase in the Formation of Tau Pathologies in Alzheimer’s Disease

One of the important neuropathological features of Alzheimer’s disease (AD) is the tau pathology seen as accumulation and hyperphosphorylation of this protein. Evidences showed that tau could be phosphorylated mostly at serine/threonine (S/T) residues by many kinases, including protein kinase B, glycogen synthase kinase (GSK)-3β, extracellular signal-regulated kinase (ERK1/2), ERK1/2 kinase (MEK1/2), c-Jun N-terminal kinase (JNK), and p38. However, there is a lack of evidence regarding tau accumulation caused by deregulation of tau protein translation. Since 70-kDa ribosomal protein S6 kinase (p70SK) is a S/T kinase that also plays a crucial role in the regulation of protein translation, it is of great interest to investigate whether or not this kinase is involved in the formation of tau-associated pathologies in AD brain, such as tau hyperphosphorylation, accumulation and assembly. Paper I investigated whether p70S6K activation is associated with PHF-tau accumulation in AD. By immunohistochemistry, we found that levels of phosphorylated (p) p70S6K at T389 or at T421/S424 were increased in accordance with the progressive sequence of neurofibrillary changes according to Braak's criteria. Confocal microscopy showed that in AD brain, p-p70S6K appeared especially in neurons that are known to later develop NFTs. By indirect enzyme-linked immunosorbent assay, the levels of pp70S6K (T389 or T421 / S424), total tau, and PHF-tau were found to significantly increased in AD as compared to control cases. Levels of p-p70S6K (T421/S424) showed significant correlations with both total tau and PHF-tau. Regression analyses revealed a significant dependence of total tau or PHF-tau on p-p70S6K at T421/S424 sites rather than at the T389 site. Levels of ribosomal protein S6 as well as levels of markers for the proteolytic systems were also significantly increased in AD as compared to control brains. In both SH-SY5Y neuroblastoma cells and primary cultured neurons, we found that zinc sulfate could induce p70S6K phosphorylation and activation, which results 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 p70S6K), attenuated zinc-induced effects. Paper II investigated whether a decrease in protein phosphatase (PP)-2A activity could induce activation of the mitogen-activated protein kinase (MAPK) pathway togther with tau phosphorylation. Rat brain slices kept under metabolically active conditions in oxygenated artificial cerebral spinal fluid were treated with 1.0 μM okadaic acid for 1h at 33C. Under this condition, PP-2A activity was decreased to ~35% of the vehicle-treated control slices, and activities of PP-1 and PP-2B were not affected. In the OA-treated slices, a dramatic increase of p-ERK1/2, p-MEK1/2 and p-p70S6K was observed both immunohistochemically and by Western blots. Treatment of 6-μm sections of the OA-treated slices with purified PP-2A reversed the phosphorylation / activation of these kinases. Tau hyperphosphorylation at several sites seen in AD brain was also affected. Paper III investigated the influences of various components of phosphatidylinositol 3-kinase (PI3K) and MAPK pathways on p70S6K and GSK-3β phosphorylation. We found that 100 μM zinc could induce an increase of p-p70S6K, p-PKB, p-GSK-3β, p-ERK1/2, p-JNK, and p-p38, especially in long-term treatment (4 to 8h) in serum-deprived SH-SY5Y cells. Treatment with different inhibitors including rapamycin, wortmannin, LY294002, and U0126, and their combinations indicated that p-p70S6K and pGSK-3β are regulated by rapamycin-dependent, PI3K and MAPK pathways. Furthermore, phosphorylation of p70S6K and GSK-3β affected levels of tau at Tau-1 and PHF-1 sites, and p70S6K phosphorylation affected total tau level. Paper IV investigated whether or not p70S6K mediates tau synthesis and phosphorylation. We found that: 1) p70S6K can directly phosphorylate tau at S262, S214, and T212 sites, and inhibit recombinant tau assembly in vitro; 2) the epitope T421/S424 of p-p70S6K is associated with tau phosphorylated at the S262 or S396/S404 sites; 3) over-expression of tau requires p70S6K phosphorylation / activation; 4) elevated levels of p-p70S6K (T421/S424) are significantly correlated with tau phosphorylated at S262, S214 and T212 sites in AD brains. These data suggest that p70S6K not only acts as a new tau kinase that directly phosphorylates tau at S262, S214, and T212 sites, but also mediates tau accumulation by up-regulation of tau translation. Activated p70S6K in NFT-bearing neurons might be caused by the aberrant regulation of PI3K and MAPK pathways, as well as the reduced activity of PP-2A in AD brain. LIST OF PUBLICATIONS This thesis is based on the following papers, which are referred to by their Roman numerals. I. An WL, Cowburn RF, Li L, Braak H, Alafuzoff I, Iqbal K, Grundke-Iqbal I, Winblad B, Pei J-J. Up-regulation of phosphorylated/activated p70 S6 kinase and its relationship to neurofibrillary pathology in Alzheimer's disease. Am J Pathol, 2003, 163,591-607 II. Pei J-J, Gong CX, An WL, Winblad B, Cowburn RF, Grundke-Iqbal I, Iqbal K.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. Am J Pathol, 2003, 163,845-858 III. An WL, Bjorkdahl C, Liu R, Cowburn RF, Winblad B, Pei J-J. Mechanism of zinc-induced phosphorylation of p70 S6 kinase and glycogen synthase kinase 3beta in SH-SY5Y neuroblastoma cells. J Neurochem, 2005, 92,1104-15 IV. An WL, Zhou X-W, Nishimura T, Norberg J, Benedikz E, Winblad B, Götz J, Pei J-J. P70 S6 kinase mediates tau phosphorylation and synthesis. (Submitted).