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Assembly of the microtubule-associated protein tau (tau) into paired helical filaments that ultimately give rise to neurofibrillary tangles (NFTs) makes up one half of the two hallmark pathologies of Alzheimer's disease (AD). Tau has been shown to be modified with O-linked N-acetylglucosamine residues (O-GlcNAc), which is the modification of serine and threonine residues of nucleocytoplasmic proteins with N-acetyl-D-glucosamine (GlcNAc) moieties. Increasing O-GlcNAc in mouse models of tauopathy has been shown to hinder the progression of symptoms in these mice and impair the aggregation of tau into NFTs. In order to study how O-GlcNAc on tau may contribute to the protective effects observed in tauopathy mouse models, it is beneficial to study O-GlcNAc modified tau in vitro. Here we describe a method for producing, purifying and enriching recombinant tau that is O-GlcNAc modified. These methods have enabled the identification of O-GlcNAc modification sites on tau including Ser400. We further describe the detection of Ser400 O-GlcNAc on tau from brain lysates.

The aggregation of the microtubule-associated protein tau into paired helical filaments to form neurofibrillary tangles constitutes one of the pathological hallmarks of Alzheimer's disease. Tau is post-translationally modified by the addition of N-acetyl-D-glucosamine O-linked to several serine and threonine residues (O-GlcNAc). Previously, increased O-GlcNAcylation of tau has been shown to block the accumulation of tau aggregates within a tauopathy mouse model. Here we show that O-GlcNAc modification of full-length human tau impairs the rate and extent of its heparin-induced aggregation without perturbing its activity toward microtubule polymerization. O-GlcNAcylation, however, does not impact the "global-fold" of tau as measured by a Förster resonance energy transfer assay. Similarly, nuclear magnetic resonance studies demonstrated that O-GlcNAcylation only minimally perturbs the local structural and dynamic features of a tau fragment (residues 353-408) spanning the last microtubule binding repeat to the major GlcNAc-acceptor Ser400. These data indicate that the inhibitory effects of O-GlcNAc on tau aggregation may result from enhanced monomer solubility or the destabilization of fibrils or soluble aggregates, rather than by altering the conformational properties of the monomeric protein. This work further underscores the potential of targeting the O-GlcNAc pathway for potential Alzheimer's disease therapeutics., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Oligomerization of tau is a key process contributing to the progressive death of neurons in Alzheimer's disease. Tau is modified by O-linked N-acetylglucosamine (O-GlcNAc), and O-GlcNAc can influence tau phosphorylation in certain cases. We therefore speculated that increasing tau O-GlcNAc could be a strategy to hinder pathological tau-induced neurodegeneration. Here we found that treatment of hemizygous JNPL3 tau transgenic mice with an O-GlcNAcase inhibitor increased tau O-GlcNAc, hindered formation of tau aggregates and decreased neuronal cell loss. Notably, increases in tau O-GlcNAc did not alter tau phosphorylation in vivo. Using in vitro biochemical aggregation studies, we found that O-GlcNAc modification, on its own, hinders tau oligomerization. O-GlcNAc also inhibits thermally induced aggregation of an unrelated protein, TAK-1 binding protein, suggesting that a basic biochemical function of O-GlcNAc may be to prevent protein aggregation. These results also suggest O-GlcNAcase as a potential therapeutic target that could hinder progression of Alzheimer's disease.

The microtubule-associated protein tau is known to be post-translationally modified by the addition of N-acetyl-D: -glucosamine monosaccharides to certain serine and threonine residues. These O-GlcNAc modification sites on tau have been challenging to identify due to the inherent complexity of tau from mammalian brains and the fact that the O-GlcNAc modification typically has substoichiometric occupancy. Here, we describe a method for the production of recombinant O-GlcNAc modified tau and, using this tau, we have mapped sites of O-GlcNAc on tau at Thr-123 and Ser-400 using mass spectrometry. We have also detected the presence of a third O-GlcNAc site on either Ser-409, Ser-412, or Ser-413. Using this information we have raised a rabbit polyclonal IgG antibody (3925) that detects tau O-GlcNAc modified at Ser-400. Further, using this antibody we have detected the Ser-400 tau O-GlcNAc modification in rat brain, which confirms the validity of this in vitro mapping approach. The identification of these O-GlcNAc sites on tau and this antibody will enable both in vivo and in vitro experiments designed to understand the possible functional roles of O-GlcNAc on tau.

The aggregation of the microtubule-associated protein tau into paired-helical filaments is the defining characteristic of the tauopathies. It has become apparent that the hyperphosphorylation of tau likely plays a role in the aggregation process and thus strategies to reduce tau phosphorylation are generating wide interest. The O-GlcNAc posttranslational modification of tau has been shown to be reciprocal to its phosphorylation; increasing O-GlcNAc leads to reductions in tau phosphorylation. In this mini-review, we highlight the use of chemical compounds as a means of understanding the reciprocal nature of tau phosphorylation and tau O-GlcNAcylation and highlight some recent progress in this area.

Pathological hyperphosphorylation of the microtubule-associated protein tau is characteristic of Alzheimer's disease (AD) and the associated tauopathies. The reciprocal relationship between phosphorylation and O-GlcNAc modification of tau and reductions in O-GlcNAc levels on tau in AD brain offers motivation for the generation of potent and selective inhibitors that can effectively enhance O-GlcNAc in vertebrate brain. We describe the rational design and synthesis of such an inhibitor (thiamet-G, K(i) = 21 nM; 1) of human O-GlcNAcase. Thiamet-G decreased phosphorylation of tau in PC-12 cells at pathologically relevant sites including Thr231 and Ser396. Thiamet-G also efficiently reduced phosphorylation of tau at Thr231, Ser396 and Ser422 in both rat cortex and hippocampus, which reveals the rapid and dynamic relationship between O-GlcNAc and phosphorylation of tau in vivo. We anticipate that thiamet-G will find wide use in probing the functional role of O-GlcNAc in vertebrate brain, and it may also offer a route to blocking pathological hyperphosphorylation of tau in AD.