To investigate why tau phosphorylation at Ser199, Ser202, Ser396 and Ser422 was SCH772984 increased when O-GlcNAcylation was elevated upon thiametG treatment, we studied the major tau kinases. We first studied GSK-3b and its upstream regulating pathway, the PI3K-AKT signaling pathway. Which in turn is regulated by PI3K and other factors. We found that thiamet-G treatment did not significantly alter the level of GSK-3b except after treatment for 9�C24 h, but blocked its phosphorylation at Ser9 completely, suggesting that GSK-3b was markedly activated under these conditions. Phosphorylation of GSK-3b at Tyr216, which makes it more active, was also increased 24 h after thiamet-G treatment. Consistent with the almost complete dephosphorylation of GSK-3b at Ser9, Ser473 and Thr308 phosphorylation of AKT, which determines its kinase activity, was also blocked by thiamet-G treatment. However, we did not find any significant changes of either the level or the activation of PI3K, the major upstream kinase of AKT, in the mouse brain after thiamet-G treatment. These results suggest that thiamet-G induced over-activation of GSK-3b via inhibition of AKT phosphorylation. The over-activation of GSK-3b may explain the increased tau phosphorylation at Ser199, Ser202, Ser396 and Ser422, NVP-BKM120 because these sites are the phosphorylation sites catalyzed mainly by GSK-3b. CDK5 is the second most important tau kinase in the brainand is activated by p35/p25. We thus also studied the level of CDK5 and its activators. We found that thiamet-G did not alter either CDK5 or p35. P25, which is the truncated and a more active form of p35, was not detectable in the mouse brain. To elucidate the complex regulation of site-specific phosphorylation induced by thiamet-G, we employed cultured cells, because cell cultures are more easily manipulated. We first selected AHP cells because they are more close to brain neurons than tumor cell lines. As expected, treatment of AHP cells with 20 nM thiamet-G increased protein O-GlcNAcylation. While we observed decreased tau phosphorylation at several phosphorylation sites in the AHP cells after thiamet-G treatment, it did not induce any significant increase in tau phosphorylation at the phosphorylation sites studied except at a transient elevation of Ser396 at 30 min after the treatment. We then investigated the levels and the activation of GSK-3b and the upstream PI3K-AKT signaling transduction pathway, as well as CDK5/p35. We did not find any significant changes upon the treatments with thiamet-G in AHP cells. These results further support our conclusion above that the increased tau phosphorylation at some sites observed in the thiamet-G treated mouse brains was due to GSK3b activation. To learn whether the phenomena we observed in undifferentiated AHP cells were specific to these cells, we also performed similar experiments in differentiated AHP cells and differentiated PC12 cells. As seen in proliferating AHP cells, we did not observe any marked elevation of tau phosphorylation at any phosphorylation sites or changes of tau kinases upon thiamet-G treatments in these two types of cells. Thiamet-G is a highly specific OGA inhibitor that was synthesized based on rationale design. Initial studies indicated that this compound reduce tau phosphorylation at some phosphorylation sites that can be abnormally phosphorylated in AD, suggesting that OGA inhibition may offer a potential therapeutic approach for slowing tau-mediated neurodegeneration seen in AD and other tauopathies. Because tau phosphorylation at different sites has different impacts on tau’s function and pathology, investigating the role of thiamet-G on site-specific tau phosphorylation is needed.