Can a Little Sugar Keep Tau From Souring Neurons?

Neurofibrillary tangles, one of the toxic protein aggregates found in Alzheimer's disease (AD), comprise accumulations of the microtubule protein tau. How might those proteins be kept apart? Tiny sugars might do just that, suggests a paper in the February 26 Nature Chemical Biology online edition. For the first time, David Vocadlo, from the Simon Fraser University, Burnaby, British Columbia, Canada, and colleagues reported that chronically promoting a form of glycosylation in tau transgenic mice reduces neurofibrillary tangles and neurodegeneration. Contrary to previous results, however, the sugar seems to work without interfering with tau hyperphosphorylation, which promotes tau aggregation. The findings suggest that sugar moieties on tau—and perhaps on other proteins—prevent them from clumping together. "It suggests that one potential contributing factor to the aggregation of tau is the level of sugar modification inside cells," Vocadlo told ARF. "Perhaps this is a mechanism that could be exploited to prevent the pathological formation of toxic tau species." Another potential approach might be to inhibit modification of tau by acetylation. In the March 3 issue of the journal Brain, researchers from John Trojanowski’s lab at the University of Pennsylvania, Philadelphia, report that a specific, acetylated tau accompanies hyperphosphorylated forms of the protein in intracellular inclusions in several tauopathies, including AD. These authors suggest that this acetylated variant helps drive tau polymerization, injecting yet another form of post-translational modification into the process going from native to toxic tau.

Addition of N-acetylglucosamine (O-GlcNAc) to protein hydroxyl groups is a post-translational modification made to thousands of proteins in mammals. In many cases, addition of the sugar occurs at phosphorylation sites, and the two processes are thought to compete with one another (for a review, see Hart et al., 2011). In the brains of people with AD, hyperphosphorylated tau is mirrored by a decline in O-GlcNAc-modified tau (see Liu et al., 2009). One reason for the reduced glycosylation, which depends on adequate nutrient levels, could be alterations in glucose metabolism (see Schubert, 2005). Vocadlo and colleagues decided to test whether increasing O-GlcNAc modification of proteins would protect neurons and guard against neurofibrillary tangle (NFT) formation in a mouse model of tau neurodegeneration.

Co-first authors Scott Yuzwa and Xiaoyang Shan laced the drinking water of nine- to 12-week-old hemizygous JNPL3 mice with a compound developed in Vocadlo's lab. Thiamet-G inhibits the glycoside hydrolase (O-GlcNAcase) that removes N-acetylglucosamine from proteins. The transgenic mice carry the gene for human tau with the P301L mutation and develop neurodegeneration in the brain. Twenty mice imbibed the spiked water for 36 weeks, while 20 control mice lapped up untreated water.

JNPL3 mice show dramatic motor neuron loss in their spinal cords—ending up with about half the number that wild-type mice have—so the researchers first checked there for neuron rescue. At the end of the 36 weeks, thiamet-G-treated mice retained 1.4-fold more motor neurons in their cervical spinal cords than did untreated mice. The treated mice also maintained more of their body weight, losing less skeletal muscle than is common in this model.

What led to the protection? The AT8 antibody, which recognizes phosphorylated tau, detected 23 to 62 percent less phospho-tau immunoreactivity around neurofibrillary tangle-like structures in mouse brainstem, spinal cord, hypothalamus, and cortices. At the same time, O-GlcNAc jumped four- to fivefold in the brainstems of treated mice compared to controls. It seemed the neurons benefited from fewer NFTs, presumably through reduced phosphorylation, as previous research suggests. However, again using AT8 antibody, the researchers found no change in the global tau phosphorylation levels (including soluble forms) in homogenized JNPL3 brain tissue compared to control samples. That result led the team to hypothesize that added sugars reduced tau's clumping into tangles without blocking its phosphorylation sites. "One of O-GlcNAc's major functions might be to serve as a bumper to keep proteins from bumping into each other and aggregating," said Gerald Hart, Johns Hopkins University School of Medicine, Baltimore, Maryland, told ARF. Hart was not involved in the study.

To further test whether the added sugar reduced aggregation without hindering phosphorylation, the team did in-vitro tests on a truncated, fast-aggregating tau snippet. Glycosylating the snippet slowed that peptide's aggregation even in the absence of a phosphorylation effect. "We were surprised," Vocadlo told ARF. "It shows that the sugar modification on its own can influence the aggregation of tau, at least in vitro, in the absence of other factors, including phosphorylation." Vocadlo's team showed in a previous study that acutely treating wild-type rats with thiamet-G for a single day did reduce tau phosphorylation, while boosting the addition of O-GlcNAc to tau (see ARF related news story on Yuzwa et al., 2008). However, cells may adapt to treatment with chronic exposure, said Vocadlo, which could explain why continuous treatment of the JNPL3 mice is not accompanied by a reduction in tau phosphorylation. Since in this study, O-GlcNAc also hindered the heat-induced accumulation of an unrelated protein, the sTAB1 binding protein, the authors suggested that a general role of O-GlcNAc may be to prevent protein aggregation.

If glycosylation counteracts aggregation, what promotes it? Here, acetylation might come in, bespeaking the complexity of tau biology (see ARF related news story; also Min et al., 2010). In their new paper, the Trojanowski group extended earlier work showing that acetylation led to both a loss of one of tau’s major functions, i.e., promoting microtubule assembly, and a gain of toxic function, i.e., pathological tau aggregation. First author David Irwin and colleagues examined, postmortem, brains of people who had AD, corticobasal degeneration, or progressive supranuclear palsy, though no cognitively normal controls. They report that tau acetylated at lysine 280 appeared in similar structures as hyperphosphorylated tau in all cases. While acetylated tau appeared at all stages of AD, it was most prevalent in middle and late stages of the disease. Hence, it appears to occur mostly after tau phosphorylation, which may actually open the door for the acetylase to modify the protein, suggest the authors. They point out that most phosphorylation sites in tau flank the microtubule-binding repeat where lysine 280 sits. The UPenn authors suggest that acetylation may exacerbate loss of normal tau function and foster fibrillization, offering a new therapeutic target for AD and other tauopathies. If glycosylation modified those same phosphorylation sites, as has been proposed, it might also protect tau against subsequent acetylation. "We are interested in the potential interaction between different modifications on tau, but have not looked at the effects of acetylation on tau aggregation," Vocadlo told ARF in an e-mail.

Cheng-Xin Gong, New York State Institute for Basic Research, Staten Island, is not completely convinced of Vocadlo and colleagues’ finding that O-GlcNAc has no effect on tau phosphorylation. Since the researchers did not examine all of tau’s phosphorylation sites in their tests, other sites could have been modified, he pointed out. While further study is warranted on that point, "it is possible this drug may work through different pathways to benefit people with AD," he said, referencing a study suggesting that O-GlcNAc addition steers APP processing away from the amyloidogenic pathway (see Jacobsen and Iverfeldt, 2011). Recent research also suggests that O-GlcNAc plays a direct role in learning and memory, Hart pointed out (see Rexach et al,. 2012 and Kaleem et al., 2011). The Canadian team did not characterize cognition in these mice because they don't show the cognitive defects that other models do, Vocadlo said. As for motor function, treated mice fared no better than untreated. In the cage hang test of motor skills and coordination, untreated mice declined faster than treated mice, but the treated mice hung on for shorter times overall, possibly because they weigh more, so results are inconclusive, the authors note. In the rotarod test of strength and balance, both groups performed equally. The scientists plan to search for other inhibitors of O-GlcNAc addition, and test thiamet-G in other animal models to further probe its effects, Vocadlo said.

Proteins related to DNA transcription, protein translation, trafficking and degradation, cancer, and the cytoskeleton are all glycosylated and regulated by O-GlcNAc. The sugar's far-reaching influence prompts concerns about side effects when tilting the balance of glycosylation. For example, excess O-GlcNAc protein modification may worsen insulin resistance (see Vosseller et al., 2002). The team saw no negative effects on weight, food consumption, or motor neuron counts in wild-type mice treated with thiamet-G over 22 weeks. "This is hard to believe," given O-GlcNAc's range of effects, said Gong. He was unsure why that might be, but said that perhaps glycosylation less dramatically contorts protein conformation than phosphorylation, or that the body somehow buffers the compound's effects. "What is important here is they have shown that, in vivo, thiamet-G can reduce tau pathology," said Khalid Iqbal, New York Institute for Basic Research in Developmental Disabilities, Staten Island. Before the compound can be considered for human clinical trials, researchers will need to look for cognitive benefits in other animal models of tau pathology, especially ones that more closely mimic the disease mechanisms of sporadic AD, he said. The team will also need to do further work to elucidate the mechanism of thiamet-G, especially whether it has effects on phosphorylation, researchers agreed.—Gwyneth Dickey Zakaib

Comments

  1. Understanding the molecular mechanisms underlying Alzheimer’s disease (AD) and finding new therapeutic targets are of utmost interest to those trying to block or at least slow down the neurodegeneration process. Both of these issues are discussed in this article that focuses on the role of a post-translational modification, the O-linked β-N-acetylglucosamine (O-GlcNAc), as a molecular mechanism preventing tau aggregation—in contrast to phosphorylation, which promotes it. During the last 10 years, O-GlcNAc has emerged as a competitor of phosphorylation for several proteins, including the microtubule-associated tau (Arnold et al., 1996). In this paper, a small-molecule inhibitor of the O-GlcNAcase (OGA), the enzyme that removes the O-GlcNAc moiety added by its opposite functional counterpart, the O-GlcNAc transferase (OGT), serves as a valuable tool to evaluate the link between phosphorylation and O-GlcNAcylation in vivo. The authors use a transgenic mouse model that overexpresses human P301L tau mutant and exhibits AD-like neurodegeneration characterized by tau hyperphosphorylation and aggregation into neurofibrillary tangles (NFTs). Treatment with OGA inhibitor leads to a significant reduction of NFTs without altering tau phosphorylation at AD-relevant sites (AT8 and pS422). In a standard in-vitro aggregation assay using anionic species as oligomerization inducers and thioflavin S fluorescence as a monitor of aggregate formation, the recombinant O-GlcNAcylated amino-terminally truncated tau (tau244-441) exhibits a slower aggregation profile than the unmodified counterpart with serine 400 (S400) playing a key role. These data indicate that O-GlcNAc modifications significantly inhibit tau aggregation in vitro, potentially by preventing the formation of soluble cytotoxic species.

    Interestingly, in the P301L transgenic mouse model, reduction of NFTs concomitant with increasing level of O-GlcNAc due to OGA inhibition treatment is not accompanied by detectable changes in tau phosphorylation, at least in the proline-rich and carboxy-terminal regions, in contrast to what has been observed in a number of previous studies. In particular, S400 O-GlcNAcylation was found to both negatively regulate priming on S404 by CDK2/cyclinA3 and suppress GSK3β-mediated sequential phosphorylation of the carboxy-terminal epitope (S396/S400/S404) in vitro. Whereas implications resulting from such a disruption in the physiopathological phosphorylation process are of outmost importance in the context of AD, since GSK3β is a crucial kinase for tau phosphorylation at AD-relevant pro-directed sites, the finding that O-GlcNAc might function independently to phosphorylation suggests a new role of O-GlcNAc in AD pathogenesis. Hence, O-GlcNAc not only interferes with (hyper)phosphorylation, but also directly reduces the fibril formation on its own and, given the positive effect of increasing O-GlcNAc amounts, this study confirms the potential utility of new potent OGA inhibitors in the treatment of AD, as previously described by the authors (Yuzwa et al., 2008).

    Furthermore, O-GlcNAc is here presented as a general dynamic strategy employed by the cell to protect proteins from self-assembly by stabilizing their conformation. Molecular details are needed to determine the precise function of O-GlcNAc as exemplified by S400 O-GlcNAcylation. Could a single-site glycosylation change the conformation of tau even in the presence of vicinal phosphorylations so that oligomerization is hampered? Would more O-GlcNAc modifications elicit a more potent effect? In a peptide model, no conformational change was detected upon S400 glycosylation (Smet-Nocca et al., 2011). Site-specific introduction of O-GlcNAc at the S400 position using an expressed protein ligation strategy (Broncel et al., 2012) could answer this issue at both structural and functional levels. Moreover, the use of full-length tau protein will be informative of long-range structural effects upon S400 O-GlcNAcylation, i.e., whether it disrupts the paperclip-like conformation described by Mandelkow et al. (Mandelkow et al., 2007). This transient conformation of soluble tau explains the ability of so-called discontinuous antibodies (Alz50, MC1, TG3) that detect early stages of AD to map both amino- and carboxy-termini of tau in an intramolecular manner, and also reflects the conformation adopted in paired helical filaments of tau (Bibow et al., 2011). Hence, this more compact conformation could be the signal triggering the fibrillization processes through the production of soluble cytotoxic small oligomers. In this context, one can envision O-GlcNAcylation and O-phosphorylation as opposite mechanisms that could tune tau conformation in a way that either decreases or increases the propensity to adopt a pathological conformation, respectively. As already seen with a peptide fragment from the estrogen receptor β (ERβ), both post-translational modifications might modulate ERβ activity by shifting different local structures in the intrinsically disordered amino-terminal region of the transcription factor: The phosphorylated form has a stronger propensity to adopt an extended conformation, while the O-GlcNAcylated form promotes the formation of a turn around the modification site (Chen et al., 2006). Similarly, tau O-GlcNAcylation could promote a conformational change that hinders the oligomerization process, while phosphorylation would favor a pathological conformation (Bibow et al., 2011; Sibille et al., 2011). To understand the molecular mechanisms involved in tau aggregation, as well as in regulation of tau physiological function, models such as site-specific, homogeneously modified tau proteins are required. In this context, native chemical ligation techniques could offer access to large proteins with natural protein modifications (Hackenberger et al., 2008). During the past decade, O-GlcNAc modification of tau has offered remarkable perspectives in counteracting molecular events related to AD neurodegeneration and opened new routes for AD treatments that could rival kinase inhibitors or anti-aggregative compounds. Importantly, novel methodologies are being developed to improve detection and identification of O-GlcNAc sites, which are major steps in the characterization and understanding of cellular processes (Rexach et al., 2008).

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    Chen YX, Du JT, Zhou LX, Liu XH, Zhao YF, Nakanishi H, Li YM. Alternative O-GlcNAcylation/O-phosphorylation of Ser16 induce different conformational disturbances to the N terminus of murine estrogen receptor beta. Chem Biol. 2006 Sep;13(9):937-44. PubMed.

    Hackenberger CP, Schwarzer D. Chemoselective ligation and modification strategies for peptides and proteins. Angew Chem Int Ed Engl. 2008;47(52):10030-74. PubMed.

    Mandelkow E, von Bergen M, Biernat J, Mandelkow EM. Structural principles of tau and the paired helical filaments of Alzheimer's disease. Brain Pathol. 2007 Jan;17(1):83-90. PubMed.

    Rexach JE, Clark PM, Hsieh-Wilson LC. Chemical approaches to understanding O-GlcNAc glycosylation in the brain. Nat Chem Biol. 2008 Feb;4(2):97-106. PubMed.

    Sibille N, Huvent I, Fauquant C, Verdegem D, Amniai L, Leroy A, Wieruszeski JM, Lippens G, Landrieu I. Structural characterization by nuclear magnetic resonance of the impact of phosphorylation in the proline-rich region of the disordered Tau protein. Proteins. 2011 Sep 30; PubMed.

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    Yuzwa SA, Macauley MS, Heinonen JE, Shan X, Dennis RJ, He Y, Whitworth GE, Stubbs KA, McEachern EJ, Davies GJ, Vocadlo DJ. A potent mechanism-inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivo. Nat Chem Biol. 2008 Aug;4(8):483-90. PubMed.

    View all comments by Caroline Smet-Nocca

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References

News Citations

  1. Target Practice: A Trio of Papers to Ponder for Potential Therapies
  2. Tau Modification—Move Over Phosphate, Make Room for Acetylation

Paper Citations

  1. Hart GW, Slawson C, Ramirez-Correa G, Lagerlof O. Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease. Annu Rev Biochem. 2011;80:825-58. PubMed.
  2. Liu F, Shi J, Tanimukai H, Gu J, Grundke-Iqbal I, Iqbal K, Gong CX. Reduced O-GlcNAcylation links lower brain glucose metabolism and tau pathology in Alzheimer's disease. Brain. 2009 Jul;132(Pt 7):1820-32. PubMed.
  3. Schubert D. Glucose metabolism and Alzheimer's disease. Ageing Res Rev. 2005 May;4(2):240-57. PubMed.
  4. Yuzwa SA, Macauley MS, Heinonen JE, Shan X, Dennis RJ, He Y, Whitworth GE, Stubbs KA, McEachern EJ, Davies GJ, Vocadlo DJ. A potent mechanism-inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivo. Nat Chem Biol. 2008 Aug;4(8):483-90. PubMed.
  5. Min SW, Cho SH, Zhou Y, Schroeder S, Haroutunian V, Seeley WW, Huang EJ, Shen Y, Masliah E, Mukherjee C, Meyers D, Cole PA, Ott M, Gan L. Acetylation of tau inhibits its degradation and contributes to tauopathy. Neuron. 2010 Sep 23;67(6):953-66. PubMed.
  6. Jacobsen KT, Iverfeldt K. O-GlcNAcylation increases non-amyloidogenic processing of the amyloid-β precursor protein (APP). Biochem Biophys Res Commun. 2011 Jan 21;404(3):882-6. PubMed.
  7. Rexach JE, Clark PM, Mason DE, Neve RL, Peters EC, Hsieh-Wilson LC. Dynamic O-GlcNAc modification regulates CREB-mediated gene expression and memory formation. Nat Chem Biol. 2012;8(3):253-61. PubMed.
  8. Kaleem A, Hoessli DC, Haq IU, Walker-Nasir E, Butt A, Iqbal Z, Zamani Z, Shakoori AR, . CREB in long-term potentiation in hippocampus: role of post-translational modifications-studies In silico. J Cell Biochem. 2011 Jan;112(1):138-46. PubMed.
  9. Vosseller K, Wells L, Lane MD, Hart GW. Elevated nucleocytoplasmic glycosylation by O-GlcNAc results in insulin resistance associated with defects in Akt activation in 3T3-L1 adipocytes. Proc Natl Acad Sci U S A. 2002 Apr 16;99(8):5313-8. PubMed.

External Citations

  1. JNPL3 mice

Further Reading

Papers

  1. Liu F, Iqbal K, Grundke-Iqbal I, Hart GW, Gong CX. O-GlcNAcylation regulates phosphorylation of tau: a mechanism involved in Alzheimer's disease. Proc Natl Acad Sci U S A. 2004 Jul 20;101(29):10804-9. PubMed.
  2. Yang X, Ongusaha PP, Miles PD, Havstad JC, Zhang F, So WV, Kudlow JE, Michell RH, Olefsky JM, Field SJ, Evans RM. Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance. Nature. 2008 Feb 21;451(7181):964-9. PubMed.
  3. Vosseller K, Wells L, Lane MD, Hart GW. Elevated nucleocytoplasmic glycosylation by O-GlcNAc results in insulin resistance associated with defects in Akt activation in 3T3-L1 adipocytes. Proc Natl Acad Sci U S A. 2002 Apr 16;99(8):5313-8. PubMed.
  4. Rexach JE, Clark PM, Hsieh-Wilson LC. Chemical approaches to understanding O-GlcNAc glycosylation in the brain. Nat Chem Biol. 2008 Feb;4(2):97-106. PubMed.
  5. Yao PJ, Coleman PD. Reduction of O-linked N-acetylglucosamine-modified assembly protein-3 in Alzheimer's disease. J Neurosci. 1998 Apr 1;18(7):2399-411. PubMed.

Primary Papers

  1. Yuzwa SA, Shan X, Macauley MS, Clark T, Skorobogatko Y, Vosseller K, Vocadlo DJ. Increasing O-GlcNAc slows neurodegeneration and stabilizes tau against aggregation. Nat Chem Biol. 2012 Apr;8(4):393-9. PubMed.
  2. Irwin DJ, Cohen TJ, Grossman M, Arnold SE, Xie SX, Lee VM, Trojanowski JQ. Acetylated tau, a novel pathological signature in Alzheimer's disease and other tauopathies. Brain. 2012 Mar;135(Pt 3):807-18. PubMed.

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