Phosphorylation of microtubule-associated protein tau by Ca2+/calmodulin-dependent protein kinase II in its tubulin binding sites

The paired helical filaments (PHF) found in Alzheimer's disease (AD) brain are composed mainly of the hyperphosphorylated form of microtubule-associated protein tau (PHF-tau). It is well known that tau is a good in vitro substrate for Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II). To establish the phosphorylation sites, the longest human tau (hTau40) was bacterially expressed and phosphorylated by CaM kinase II, followed by digestion with lysyl endoprotease. The digests were subjected to liquid chromatography/mass spectrometry. We found that 5 of 22 identified peptides were phosphorylated. From the tandem mass spectrometry, two phosphorylation sites (serines 262 and 356) were identified in the tubulin binding sites. When tau was phosphorylated by CaM kinase II, the binding of tau to taxol-stabilized microtubules was remarkably impaired. As both serines 262 and 356 are reportedly phosphorylated in PHF-tau, CaM kinase II may be involved in hyperphosphorylation of tau in AD brain.     

Prostate-derived Sterile 20-like Kinases (PSKs/TAOKs) Phosphorylate Tau Protein and Are Activated in Tangle-bearing Neurons in Alzheimer Disease

In Alzheimer disease (AD), the microtubule-associated protein tau is highly phosphorylated and aggregates into characteristic neurofibrillary tangles. Prostate-derived sterile 20-like kinases (PSKs/TAOKs) 1 and 2, members of the sterile 20 family of kinases, have been shown to regulate microtubule stability and organization. Here we show that tau is a good substrate for PSK1 and PSK2 phosphorylation with mass spectrometric analysis of phosphorylated tau revealing more than 40 tau residues as targets of these kinases. Notably, phosphorylated residues include motifs located within the microtubule-binding repeat domain on tau (Ser-262, Ser-324, and Ser-356), sites that are known to regulate tau-microtubule interactions. PSK catalytic activity is enhanced in the entorhinal cortex and hippocampus, areas of the brain that are most susceptible to Alzheimer pathology, in comparison with the cerebellum, which is relatively spared. Activated PSK is associated with neurofibrillary tangles, dystrophic neurites surrounding neuritic plaques, neuropil threads, and granulovacuolar degeneration bodies in AD brain. By contrast, activated PSKs and phosphorylated tau are rarely detectible in immunostained control human brain. Our results demonstrate that tau is a substrate for PSK and suggest that this family of kinases could contribute to the development of AD pathology and dementia.     

Characterization of the phosphoproteome in androgen-repressed human prostate cancer cells by Fourier transform ion cyclotron resonance mass spectrometry

Androgen-repressed human prostate cancer, ARCaP, grows and is highly metastatic to bone and soft tissues in castrated mice. The molecular mechanisms underlying the aberrant responses to androgen are not fully understood. Here, we apply state-of-the-art mass spectrometry methods to investigate the phosphoproteome profiles in ARCaP cells. Because protein biological phosphorylation is always substoichiometric and the ionization efficiency of phosphopeptides is low, selective enrichment of phosphorylated proteins/peptides is required for mass spectrometric analysis of phosphorylation from complex biological samples. Therefore, we compare the sensitivity, efficiency, and specificity for three established enrichment strategies: calcium phosphate precipitation (CPP), immobilized metal ion affinity chromatography (IMAC), and TiO(2)-modified metal oxide chromatography. Calcium phosphate precipitation coupled with the TiO(2) approach offers the best strategy to characterize phosphorylation in ARCaP cells. We analyzed phosphopeptides from ARCaP cells by LC-MS/MS with a hybrid LTQ/FT-ICR mass spectrometer. After database search and stringent filtering, we identified 385 phosphoproteins with an average peptide mass error of 0.32 ± 0.6 ppm. Key identified oncogenic pathways include the mammalian target of rapamycin (mTOR) pathway and the E2F signaling pathway. Androgen-induced proliferation inhibitor (APRIN) was detected in its phosphorylated form, implicating a molecular mechanism underlying the ARCaP phenotype.     

Characterization of the in vitro phosphorylation of human tau by tau protein kinase II (cdk5/p20) using mass spectrometry

Hyperphosphorylated tau is an integral part of the neurofibrillary tangles that form within neuronal cell bodies, and tau protein kinase II is reported to play a role in the pathogenesis of Alzheimer's disease. Recently, we reported that tau protein kinase II (cdk5/p20)-phosphorylated human tau inhibits microtubule assembly, and tau protein kinase II (cdk5/p20) phosphorylation of microtubule-associated tau results in dissociation of phosphorylated tau from the microtubules and tubulin depolymerization. In the studies reported here, a combination of mass spectrometric techniques was used to study the phosphorylation of human recombinant tau by recombinant tau protein kinase II (cdk5/p20) in vitro. The extent of phosphorylation was determined by measuring the molecular mass of phosphorylated tau using mass spectrometry. Reaction of human recombinant tau with tau protein kinase II (cdk5/p20) resulted in the formation of two major species containing either five or six phosphate groups. The specific amino acid residues phosphorylated were determined by analyzing tryptic peptides by tandem mass spectrometry via either MALDI/TOF post-source decay or by electrospray tandem mass spectrometry. Based on these experiments, we conclude that tau protein kinase II (cdk5/p20) can phosphorylate human tau at Thr(181), Thr(205), Thr(212), Thr(217), Ser(396) and Ser(404).     

Role of glycosylation in hyperphosphorylation of tau in Alzheimer's disease

In Alzheimer's disease (AD) brain, microtubule-associated protein tau is abnormally modified by hyperphosphorylation and glycosylation, and is aggregated as neurofibrillary tangles of paired helical filaments. To investigate the role of tau glycosylation in neurofibrillary pathology, we isolated various pools of tau protein from AD brain which represent different stages of tau pathology. We found that the non-hyperphosphorylated tau from AD brain but not normal brain tau was glycosylated. Monosaccharide composition analyses and specific lectin blots suggested that the tau in AD brain was glycosylated mainly through N-linkage. In vitro phosphorylation indicated that the glycosylated tau was a better substrate for cAMP-dependent protein kinase than the deglycosylated tau. These results suggest that the glycosylation of tau is an early abnormality that can facilitate the subsequent abnormal hyperphosphorylation of tau in AD brain.     

Quantitative proteomics analysis of phosphorylated proteins in the hippocampus of Alzheimer's disease subjects

Phosphorylation on tyrosine, threonine and serine residues represents one of the most important post-translational modifications and is a key regulator of cellular signaling of multiple biological processes that require a strict control by protein kinases and protein phosphatases. Abnormal protein phosphorylation has been associated with several human diseases including Alzheimer's disease (AD). One of the characteristic hallmarks of AD is the presence of neurofibrillary tangles, composed of microtubule-associated, abnormally hyperphosphorylated tau protein. However, several others proteins showed altered phosphorylation levels in AD suggesting that deregulated phosphorylation may contribute to AD pathogenesis. Phosphoproteomics has recently gained attention as a valuable approach to analyze protein phosphorylation, both in a quantitative and a qualitative way. We used the fluorescent phosphospecific Pro-Q Diamond dye to identify proteins that showed alterations in their overall phosphorylation in the hippocampus of AD vs. control (CTR) subjects. Significant changes were found for 17 proteins involved in crucial neuronal process such as energy metabolism or signal transduction. These phosphoproteome data may provide new clues to better understand molecular pathways that are deregulated in the pathogenesis and progression of AD.     

Epitope mapping of mAbs AT8 and Tau5 directed against hyperphosphorylated regions of the human tau protein

Post-mortem diagnosis of Alzheimer's disease relies on high numbers of senile plaques and neurofibrillary tangles (NFTs) stained in distinct brain areas. NFTs mostly consist of hyperphosphorylated versions of the microtubule attached tau protein (PHF-tau) with more than 30 serine and threonine phosphorylation sites identified so far. Characterization of hyperphosphorylated tau regions and the hope to develop robust assays for early AD diagnosis relies mostly on phosphorylation-dependent monoclonal antibodies (mAbs) recognizing only disease-specific phosphorylation patterns. Here, we report that anti-PHF-tau mAb AT8 recognizes an epitope doubly phosphorylated at serine 202 and threonine 205, which was not influenced by a third phosphate group at serine 199. But mAb AT8 was cross-reactive to two doubly phosphorylated motifs containing either serines 199 and 202 or serines 205 and 208 of the human tau sequence. The epitope of anti-tau mAb Tau5 was mapped to the human tau sequence 218-225, which is not phosphorylated in vivo.     

Alzheimer disease-specific conformation of hyperphosphorylated paired helical filament-Tau is polyubiquitinated through Lys-48, Lys-11, and Lys-6 ubiquitin conjugation

One of the key pathological hallmarks of Alzheimer disease (AD) is the accumulation of paired helical filaments (PHFs) of hyperphosphorylated microtubule-associated protein Tau. Tandem mass spectrometry was employed to examine PHF-Tau post-translational modifications, in particular protein phosphorylation and ubiquitination, to shed light on their role in the early stages of Alzheimer disease. PHF-Tau from Alzheimer disease brain was affinity-purified by MC1 monoclonal antibody to isolate a soluble fraction of PHF-Tau in a conformation unique to human AD brain. A large number of phosphorylation sites were identified by employing a data-dependent neutral loss algorithm to trigger MS3 scans of phosphopeptides. It was found that soluble PHF-Tau is ubiquitinated at its microtubule-binding domain at residues Lys-254, Lys-311, and Lys-353, suggesting that ubiquitination of PHF-Tau may be an earlier pathological event than previously thought and that ubiquitination could play a regulatory role in modulating the integrity of microtubules during the course of AD. Tandem mass spectrometry data for ubiquitin itself indicate that PHF-Tau is modified by three polyubiquitin linkages, at Lys-6, Lys-11, and Lys-48. Relative quantitative analysis indicates that Lys-48-linked polyubiquitination is the primary form of polyubiquitination with a minor portion of ubiquitin linked at Lys-6 and Lys-11. Because modification by Lys-48-linked polyubiquitin chains is known to serve as the essential means of targeting proteins for degradation by the ubiquitin-proteasome system, and it has been reported that modification at Lys-6 inhibits ubiquitin-dependent protein degradation, a failure of the ubiquitin-proteasome system could play a role in initiating the formation of degradation-resistant PHF tangles.     

Passive Immunization with Phospho-Tau Antibodies Reduces Tau Pathology and Functional Deficits in Two Distinct Mouse Tauopathy Models

In Alzheimer’s disease (AD), an extensive accumulation of extracellular amyloid plaques and intraneuronal tau tangles, along with neuronal loss, is evident in distinct brain regions. Staging of tau pathology by postmortem analysis of AD subjects suggests a sequence of initiation and subsequent spread of neurofibrillary tau tangles along defined brain anatomical pathways. Further, the severity of cognitive deficits correlates with the degree and extent of tau pathology. In this study, we demonstrate that phospho-tau (p-tau) antibodies, PHF6 and PHF13, can prevent the induction of tau pathology in primary neuron cultures. The impact of passive immunotherapy on the formation and spread of tau pathology, as well as functional deficits, was subsequently evaluated with these antibodies in two distinct transgenic mouse tauopathy models. The rTg4510 transgenic mouse is characterized by inducible over-expression of P301L mutant tau, and exhibits robust age-dependent brain tau pathology. Systemic treatment with PHF6 and PHF13 from 3 to 6 months of age led to a significant decline in brain and CSF p-tau levels. In a second model, injection of preformed tau fibrils (PFFs) comprised of recombinant tau protein encompassing the microtubule-repeat domains into the cortex and hippocampus of young P301S mutant tau over-expressing mice (PS19) led to robust tau pathology on the ipsilateral side with evidence of spread to distant sites, including the contralateral hippocampus and bilateral entorhinal cortex 4 weeks post-injection. Systemic treatment with PHF13 led to a significant decline in the spread of tau pathology in this model. The reduction in tau species after p-tau antibody treatment was associated with an improvement in novel-object recognition memory test in both models. These studies provide evidence supporting the use of tau immunotherapy as a potential treatment option for AD and other tauopathies.     

Pseudophosphorylation of tau at serine 422 inhibits caspase cleavage: in vitro evidence and implications for tangle formation in vivo

The tangles of Alzheimer's disease (AD) are comprised of the tau protein displaying numerous alterations, including phosphorylation at serine 422 (S422) and truncation at aspartic acid 421 (D421). Truncation at the latter site appears to result from activation of caspases, a class of proteases that cleave specifically at aspartic acid residues. It has been proposed that phosphorylation at or near caspase cleavage sites could regulate the ability of the protease to cleave at those sites. Here, we use tau pseudophosphorylated at S422 (S422E) to examine the effects of tau phosphorylation on its cleavage by caspase 3. We find that S422E tau is more resistant to proteolysis by caspase 3 than non-pseudophosphorylated tau. Additionally, we use antibodies directed against the phosphorylation site and against the truncation epitope to assess the presence of these epitopes in neurofibrillary tangles in the aged human brain. We show that phosphorylation precedes truncation during tangle maturation. Moreover, the distribution of the two epitopes suggests that a significant length of time (perhaps as much as two decades) elapses between S422 phosphorylation and cleavage at D421. We further conclude that tau phosphorylation at S422 may be a protective mechanism that inhibits cleavage in vivo.     

Ca2＋失调与阿尔茨海默病发生发展关系的研究进展

阿尔茨海默病（Alzheimer’s disease,AD）是全球老年人中最常见的神经退行性疾病。在对家族性AD的动物模型和个别AD患者死后脑组织的研究中发现,除了A1342水平升高外,神经元内Ca2＋信号失调,并且Ca2＋信号蛋白表达水平也发生了改变。淀粉样蛋白斑、神经元纤维缠结和神经元丢失是AD的后期标志,它们的共同点可能是破坏神经元钙离子信号。细胞内钙离子水平提高在功能上与淀粉样蛋白斑、早老素突变、tau缠结和突触功能障碍相关,基于这些研究,产生了AD的Ca2＋假说。主要介绍神经元内Ca2＋失调与AD的关系以及钙拮抗剂作为AD的潜在治疗方法。     

微管相关蛋白tau在动物死亡后被快速去磷酸化

tau蛋白是神经细胞中主要的微管相关蛋白, 它的异常过度磷酸化被认为是阿尔茨海默病 (AD) 致病过程中的关键因素. 由于法律、社会、家庭等诸多因素使得获取的人脑组织标本常常在死亡后2~3 h以上,因此了解死亡不同时间后tau蛋白磷酸化的改变,对研究tau蛋白的功能及在AD致病过程中作用显得十分重要. 用位点特异的、磷酸化依赖的抗tau蛋白抗体检测正常大鼠脑中tau蛋白磷酸化程度及死亡后其磷酸化的变化情况,再用非同位素的点印迹技术测定鼠脑中tau蛋白激酶、磷酸酶在不同温度下的活性. 结果发现,正常鼠脑中tau蛋白除了Ser262,Ser409,Ser422外,在Thr181,Ser199,Ser202,Thr205,Thr212,Ser214,Thr217,Ser396和Ser404存在不同程度的磷酸化,并且在死亡后3 h,出现tau的多位点的去磷酸化及tau蛋白迁移加快,6 h后更为明显,但tau蛋白水平即使在大鼠死亡后6 h,仍未见有明显的改变. 用点印迹测定蛋白激酶和磷酸酶活性结果显示,tau蛋白激酶、磷酸酶活性均有温度依赖性降低,在25℃时激酶活性降低远大于磷酸酶活性的降低,tau蛋白在死亡后的快速去磷酸化与相对高的磷酸酶作用有关.     

蛋白磷酸酯酶-1和-2A抑制剂对NG108-15细胞tau蛋白磷酸化的影响

Alzheimer痴呆(AD)的主要脑病理变化之一为由超磷酸化的tau蛋白组成的神经原纤维缠结(Neurofibrillary tangle,NFT)。AD的Tau蛋白异常磷酸化与蛋白磷酸酯酶(PP)-2A和-1缺陷有关。本文首先用免疫印迹法显示NG含两大组分的tau蛋白。MTT方法观察到PP-2A和PP-1抑制剂冈 田酸(Okadaic acid.OA)处理NG108-14细胞能导致细胞代谢明显下降,同时免疫印迹法显示OA能导致的NG细胞Tau蛋白磷酸化。该研究为建立AD样蛋白磷酸酯酶缺陷引起的tau蛋白磷酸化的细胞模型奠定了基础。     

The physiological link between metabolic rate depression and tau phosphorylation in mammalian hibernation

Abnormal phosphorylation and aggregation of tau protein are hallmarks of a variety of neurological disorders, including Alzheimer's disease (AD). Increased tau phosphorylation is assumed to represent an early event in pathogenesis and a pivotal aspect for aggregation and formation of neurofibrillary tangles. However, the regulation of tau phosphorylation in vivo and the causes for its increased stage of phosphorylation in AD are still not well understood, a fact that is primarily based on the lack of adequate animal models. Recently we described the reversible formation of highly phosphorylated tau protein in hibernating European ground squirrels. Hence, mammalian hibernation represents a model system very well suited to study molecular mechanisms of both tau phosphorylation and dephosphorylation under in vivo physiological conditions. Here, we analysed the extent and kinetics of hibernation-state dependent tau phosphorylation in various brain regions of three species of hibernating mammals: arctic ground squirrels, Syrian hamsters and black bears. Overall, tau protein was highly phosphorylated in torpor states and phosphorylation levels decreased after arousal in all species. Differences between brain regions, hibernation-states and phosphosites were observed with respect to degree and kinetics of tau phosphorylation. Furthermore, we tested the phosphate net turnover of tau protein to analyse potential alterations in kinase and/or phosphatase activities during hibernation. Our results demonstrate that the hibernation-state dependent phosphorylation of tau protein is specifically regulated but involves, in addition, passive, temperature driven regulatory mechanisms. By determining the activity-state profile for key enzymes of tau phosphorylation we could identify kinases potentially involved in the differentially regulated, reversible tau phosphorylation that occurs during hibernation. We show that in black bears hibernation is associated with conformational changes of highly phosphorylated tau protein that are typically related to neuropathological alterations. The particular hibernation characteristics of black bears with a continuous torpor period and an only slightly decreased body temperature, therefore, potentially reflects the limitations of this adaptive reaction pattern and, thus, might indicate a transitional state of a physiological process.     

Characterization of tau in cerebrospinal fluid using mass spectrometry

The neurodegenerative disorder Alzheimer's disease (AD) is the most common cause of dementia in the elderly. The presence of neurofibrillary tangles, consisting of hyperphosphorylated tau protein, is one of the major neuropathologic characteristics of the disease, making this protein an attractive biomarker for AD and a possible target for therapy. Here, we describe an optimized immunoprecipitation mass spectrometry method that enables, for the first time, detailed characterization of tau in human cerebrospinal fluid. The identities of putative tau fragments were confirmed using nanoflow liquid chromatography and tandem mass spectrometry. Nineteen tryptic fragments of tau were detected, of which 16 are found in all tau isoforms while 3 represented unique tau isoforms. These results pave the way for clinical CSF studies on the tauopathies.     

A novel glycogen synthase kinase-3 inhibitor 2-methyl-5-(3-{4-[(S )-methylsulfinyl]phenyl}-1-benzofuran-5-yl)-1,3,4-oxadiazole decreases tau phosphorylation and ameliorates cognitive deficits in a transgenic model of Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative disorder leading to a progressive loss of cognitive function and is pathologically characterized by senile plaques and neurofibrillary tangles. Glycogen synthase kinase-3 (GSK-3) is involved in AD pathogenesis. GSK-3 is reported not only to phosphorylate tau, a major component of neurofibrillary tangles, but also to regulate the production of amyloid β, which is deposited in senile plaques. Therefore, pharmacological inhibition of GSK-3 is considered an attractive therapeutic approach. In this study, we report the pharmacological effects of a novel GSK-3 inhibitor, 2-methyl-5-(3-{4-[(S)-methylsulfinyl]phenyl}-1-benzofuran-5-yl)-1,3,4-oxadiazole (MMBO), which displays high selectivity for GSK-3 and brain penetration following oral administration. MMBO inhibited tau phosphorylation in primary neural cell culture and also in normal mouse brain. When administered to a transgenic mouse model of AD, MMBO significantly decreased hippocampal tau phosphorylation at GSK-3 sites. Additionally, chronic MMBO administration suppressed tau pathology as assessed by AT8-immunoreactivity without affecting amyloid β pathology. Finally, in behavioral assessments, MMBO significantly improved memory and cognitive deficits in the Y-maze and in novel object recognition tests in the transgenic AD mouse model. These results indicate that pharmacological GSK-3 inhibition ameliorates behavioral dysfunction with suppression of tau phosphorylation in an AD mouse model, and that MMBO might be beneficial for AD treatment.     

Dynamic changes of the phosphoproteome in postmortem mouse brains

Protein phosphorylation is deeply involved in the pathological mechanism of various neurodegenerative disorders. However, in human pathological samples, phosphorylation can be modified during preservation by postmortem factors such as time and temperature. Postmortem changes may also differ among proteins. Unfortunately, there is no comprehensive database that could support the analysis of protein phosphorylation in human brain samples from the standpoint of postmortem changes. As a first step toward addressing the issue, we performed phosphoproteome analysis with brain tissue dissected from mouse bodies preserved under different conditions. Quantitative whole proteome mass analysis showed surprisingly diverse postmortem changes in phosphoproteins that were dependent on temperature, time and protein species. Twelve hrs postmortem was a critical time point for preservation at room temperature. At 4°C, after the body was cooled down, most phosphoproteins were stable for 72 hrs. At either temperature, increase greater than 2-fold was exceptional during this interval. We found several standard proteins by which we can calculate the postmortem time at room temperature. The information obtained in this study will be indispensable for evaluating experimental data with human as well as mouse brain samples.     

Developmental regulation of tau phosphorylation, tau kinases, and tau phosphatases

Tau is a neuronal microtubule-associated protein. Its hyperphosphorylation plays a critical role in Alzheimer disease (AD). Expression and phosphorylation of tau are regulated developmentally, but its dynamic regulation and the responsible kinases or phosphatases remain elusive. Here, we studied the developmental regulation of tau in rats during development from embryonic day 15 through the age of 24 months. We found that tau expression increased sharply during the embryonic stage and then became relatively stable, whereas tau phosphorylation was much higher in developing brain than in mature brain. However, the extent of tau phosphorylation at seven of the 14 sites studied was much less in developing brain than in AD brain. Tau phosphorylation during development matched the period of active neurite outgrowth in general. Tau phosphorylation at various sites had different topographic distributions. Several tau kinases appeared to regulate tau phosphorylation collectively at overlapping sites, and the decrease of overall tau phosphorylation in adult brain might be due to the higher levels of tau phosphatases in mature brain. These studies provide new insight into the developmental regulation of site-specific tau phosphorylation and identify the likely sites required for the abnormal hyperphosphorylation of tau in AD.     

Inhibition of protein phosphatase 2A overrides tau protein kinase I/glycogen synthase kinase 3 beta and cyclin-dependent kinase 5 inhibition and results in tau hyperphosphorylation in the hippocampus of starved mouse.

Hyperphosphorylated tau is the major component of paired helical filaments in neurofibrillary tangles found in Alzheimer's disease (AD) brain. Starvation of adult mice induces tau hyperphosphorylation at many paired helical filaments sites and with a similar regional selectivity as those in AD, suggesting that a common mechanism may be mobilized. Here we investigated the mechanism of starvation-induced tau hyperphosphorylation in terms of tau kinases and Ser/Thr protein phosphatases (PP), and the results were compared with those reported in AD brain. During starvation, tau hyperphosphorylation at specific epitopes was accompanied by decreases in tau protein kinase I/glycogen synthase kinase 3 beta (TPKI/GSK3 beta), cyclin-dependent kinase 5 (cdk5), and PP2A activities toward tau. These results demonstrate that the activation of TPKI/GSK3 beta and cdk5 is not necessary to obtain hyperphosphorylated tau in vivo, and indicate that inhibition of PP2A is likely the dominant factor in inducing tau hyperphosphorylation in the starved mouse, overriding the inhibition of key tau kinases such as TPKI/GSK3 beta and cdk5. Furthermore, these data give strong support to the hypothesis that PP2A is important for the regulation of tau phosphorylation in the adult brain, and provide in vivo evidence in support of a central role of PP2A in tau hyperphosphorylation in AD.     

Brain glucose transporters, O-GlcNAcylation and phosphorylation of tau in diabetes and Alzheimer's disease

Type 2 diabetes mellitus (T2DM) increases the risk for Alzheimer's disease (AD), but the underlying mechanism is unknown. In this study, we determined the levels of major brain glucose transporters, O -GlcNAcylation and phosphorylation of tau in the postmortem brain tissue from frontal cortices of 7 controls, 11 T2DM subjects, 10 AD subjects and 8 additional subjects who had both T2DM and AD. We found that the neuronal glucose transporter 3 was decreased to a bigger extent in T2DM brain than in AD brain. The O -GlcNAcylation levels of global proteins and of tau were also decreased in T2DM brain as seen in AD brain. Phosphorylation of tau at some of the AD abnormal hyperphosphorylation sites was increased in T2DM brain. These results suggest that T2DM may contribute to the increased risk for AD by impairing brain glucose uptake/metabolism and, consequently, down-regulation of O -GlcNAcylation, which facilitates abnormal hyperphosphorylation of tau.