lt of an inhibition of AKT by reducing its phosphorylation at Ser473 and Thr 308, which regulates its activity positively. AKT inhibition may also contribute to the thiamet-G�Cinduced decrease of tau phosphorylation at Thr212 and Ser214, because these two sites are also substrates of AKT. AKT phosphorylation was mainly catalyzed by the mTORC2 complex and PI3K�Cphosphoinositide-dependent protein kinase-1. Because we did not observe the corresponding decrease in PI3K, we speculate that the reduction/elimination of phosphorylation of AKT and GSK-3b after RWJ 64809 thiamet-G treatment may result from elevation of O-GlcNAcylation of AKT, PDK1 and/or mTOR. Alternatively, it could be off-target effect of the inhibitor when used at high doses. Phosphorylation of these kinases has been reported to be regulated negatively by O-GlcNAcylation. It is worth noting that different effects of OGA inhibition on phosphorylation of AKT and GSK-3 have been reported. Elevation of O-GlcNAcylation in skeletal muscles after OGA inhibition using another inhibitor, PUGNAc, does not significantly alter insulin-stimulated phosphorylation of AKT or GSK-3. In differentiated 3T3-L1 adipocytes, two different OGA inhibitors have been found to increase O-GlcNAc levels but not alter insulin-stimulated phosphorylation of AKT nor induce insulin resistance either. Therefore, it remains somewhat unclear as to the effects of OGA inhibition on alteration of GSK3b levels and AKT activity; the effects observed here could stem from high-dose inhibition of OGA, or alternatively from off-target effects of using the inhibitor at a high dose. Tau is abnormally hyperphosphorylated and aggregated in AD and other tauopathies. Previous studies from our and other groups have demonstrated differential roles of tau phosphorylation at various phosphorylation sites. A quantitative in vitro study demonstrated that phosphorylation of tau at Ser262, Thr231, and Ser235 inhibits its binding to microtubules by,35%,,25%, and,10%, respectively. In vitro kinetic studies of the binding between hyperphosphorylated tau and normal tau suggest that Ser199/Ser202/Thr205, Thr212, Thr231/Ser235, Ser262/ Ser356 and Ser422 are among the critical phosphorylation sites that convert tau to an inhibitory molecule that sequesters normal microtubule-associated proteins from microtubules. Further phosphorylation at Thr231, Ser396, and Ser422 promotes selfaggregation of tau into filaments. It is obvious that tau phosphorylation at various sites impacts tau activity and aggregation collectively. Our recent study has demonstrated that tau phosphorylation at the proline-rich region, which is located upstream of the microtubule-binding domains, inhibits its microtubule Niraparib assembly activity moderately and promotes its selfaggregation slightly. Tau phosphorylation at the C-terminal tail region increases its activity and promotes its self-aggregation markedly. Tau phosphorylation at both of these regions plus the microtubule-binding region nearly diminishes its activity and disrupts microtubules. Therefore, the overall impacts of thiamet-G on tau need to be further verified by its functional studies, and testing the effects of thiamet-G on cognitive function in mouse AD models, especially using different doses, becomes urgent before considering it to be a therapeutic agent for treating AD. In conclusion, thiamet-G is a specific OGA inhibitor and is very effective in elevating protein O-GlcNAcylation level in the mammalian brain. Because thiamet-G not only directly modulated tau phosphorylation inversely, but also stimulated GSK-3b activity likely via inhibition of AKT.