Abstract
Background
Amyloid plaques and neurofibrillary tangles (NFTs) are the defining pathological hallmarks of Alzheimer’s disease (AD). Increasing the quantity of the O-linked N-acetylglucosamine (O-GlcNAc) post-translational modification of nuclear and cytoplasmic proteins slows neurodegeneration and blocks the formation of NFTs in a tauopathy mouse model. It remains unknown, however, if O-GlcNAc can influence the formation of amyloid plaques in the presence of tau pathology.
Results
We treated double transgenic TAPP mice, which express both mutant human tau and amyloid precursor protein (APP), with a highly selective orally bioavailable inhibitor of the enzyme responsible for removing O-GlcNAc (OGA) to increase O-GlcNAc in the brain. We find that increased O-GlcNAc levels block cognitive decline in the TAPP mice and this effect parallels decreased β-amyloid peptide levels and decreased levels of amyloid plaques.
Conclusions
This study indicates that increased O-GlcNAc can influence β-amyloid pathology in the presence of tau pathology. The findings provide good support for OGA as a promising therapeutic target to alter disease progression in Alzheimer disease.
Similar content being viewed by others
Background
The formation of oligomers and aggregates of β-amyloid peptides derived from amyloid precursor protein (APP) is a causative early factor giving rise to Alzheimer Disease (AD) [1–3]. Neuritic plaques are the pathological feature associated with deposition of large aggregates of β-amyloid peptides. Mutations in the human APP gene can give rise to autosomal dominant early-onset forms of AD, which resemble late-onset AD (hereafter simply AD) both clinically and at the histopathological level [4–6]. Furthermore, some mutations in APP also confer protection against AD [7]. The deleterious APP mutations driving early-onset AD appear sufficient to promote, within humans, the formation of the other classical pathological hallmark of AD known as the neurofibrillary tangles (NFTs). NFTs are formed as a result of the self assembly of hyperphosphorylated microtubule-associated protein tau into paired helical filaments (PHFs) which then aggregate into larger structures known as NFTs [8–11]. Human mutations in the tau gene (MAPT), on their own, can also give rise to a group of neurodegenerative diseases referred to as frontotemporal dementia linked to chromosome-17 (FTDP-17), one group of a number of different types of tauopathy [12, 13]. However, human MAPT mutations do not cause the formation of neuritic plaques, thereby lending significant support for β-amyloid peptide formation as a factor upstream of tau in AD.
Studying the relationship between NFTs and neuritic plaques in AD is complicated by the fact that rodent models that carry human APP mutations do not generally display detectable NFT pathology [14, 15], even in the presence of neuritic plaques and cognitive impairment. Conversely, mouse models that express human MAPT mutations, such as the P301L mutant expressing JNPL3 mice, do not produce neuritic plaques [16]. For this reason several groups have developed mouse models of AD that recapitulate both pathological features of AD. One model, generated by Lewis et al., referred to as the TAPP mouse, resulted from crosses of JNPL3 tau mice with mice expressing the most common APP mutant (Swedish mutation K670N/M671L, APPSwe; Tg2576) [17]. Differing from the parent single transgenic models, TAPP mice brains contain both plaques and NFTs. Notably, these TAPP mice display enhanced tau pathology as compared to JNPL3 mice, suggesting that β-amyloid peptides accelerate the formation of NFTs [17], a proposal supported by other studies involving intracerebral injection of β-amyloid Aβ42 fibrils into P301L tau transgenic mice [18]. The TAPP mouse model is therefore well suited to study therapeutic strategies that might impact neuritic plaques or NFT formation in a setting that captures these two synergistic pathologies.
Previously, we reported a potential disease modifying approach aimed at reducing toxicity associated with tau aggregation and NFT formation. This approach involved increasing global levels of a little-studied post-translational modification known as the O-GlcNAc modification [19]. Chronic increases in the levels of O-GlcNAc modification in JNPL3 mouse brains and spinal cords slowed neuronal loss and reduced the number of NFTs formed over a treatment period of several months [20]. O-GlcNAc modification of proteins involves the attachment of single N-acetyl-D-glucosamine residues to the hydroxyl side chains of serine and threonine residues of proteins [19]. The O-GlcNAc modification differs from classical forms of glycosylation found on the outside of the cell and within the secretory pathway because O-GlcNAc is found in the nucleocytoplasm and it does not have additional sugar residues attached to it to form more complex structures [19]. The fact that O-GlcNAc can be added or removed from a particular protein multiple times during the lifespan of the protein makes it a dynamic modification [21] somewhat akin to protein phosphorylation, which is likewise reversible. Installation of O-GlcNAc on serine or threonine is carried out by a single glycosyltransferase referred to as O-GlcNAc transferase (OGT), which uses the high-energy donor sugar uridine 5’-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) as its substrate [22, 23]. A single glycoside hydrolase, O-GlcNAcase (OGA), is tasked with the hydrolytic cleavage of O-GlcNAc from modified proteins [24, 25]. To increase global O-GlcNAc levels in JNPL3 mice we made use of a potent (Ki =21 nM) and selective (37000-fold for human OGA over functionally related human β-hexosamindases) inhibitor of OGA referred to as Thiamet-G, which blocks removal of O-GlcNAc from modified proteins [26]. Thus, even as OGA is inhibited, OGT can continue adding O-GlcNAc onto to modified proteins resulting in elevated O-GlcNAc within cells [26, 27].
We previously showed that increasing O-GlcNAc levels has beneficial effects in the JNPL3 mouse model of tauopathy [20]. Recently APP has been found to be O-GlcNAc modified and one study suggested that O-GlcNAc might alter β-amyloid production by regulating APP processing [28, 29]. To study the role of O-GlcNAc on APP and β-amyloid production in mice exhibiting both tau and β-amyloid pathologies, we undertook a long-term study using Thiamet-G to increase the global levels of O-GlcNAc in TAPP mice. Here we show that Thiamet-G can increase O-GlcNAc levels in the TAPP mouse brain, leading to reductions in levels of both neuritic plaques and amyloidogenic β-amyloid peptides. Consistent with these findings we also find that Thiamet-G treatment blocks the onset of cognitive impairment in these animals. We also show, using cell models of β-amyloid peptide formation, that Thiamet-G does not alter the release of β-amyloid peptides from cells, suggesting that increased O-GlcNAc levels in mice mediate protection against β-amyloid peptides through a mechanism that is likely independent of Aβ42 release.
Results
Thiamet-G treatment improves performance in the Morris water maze (MWM)
To assess whether increased O-GlcNAc can influence amyloid deposition or cognitive impairment in bigenic TAPP mice, we divided 60 double transgenic TAPP mice into three groups (n = 20) receiving either 0, 200, or 500 mkd of Thiamet-G in their drinking water. We have previously shown oral Thiamet-G treatment of mice over a period of months leads to sustained O-GlcNAc increases in the brains of mice [20]. Parental transgenic Tg2576 mice, which harbor only the APPSwe mutation, develop pronounced memory impairment starting at 6 months of age [15, 30] as judged by performance in the Morris water maze (MWM). Therefore to determine whether increased O-GlcNAc can influence disease progression in TAPP mice we started dosing with Thiamet-G at 10-13 weeks of age and continued until 44-47 weeks of age. Effects of OGA inhibition on cognitive impairment were assessed by performing MWM testing on each of the animals in the three groups starting between 28-32 weeks of age. All data acquisition and data entry was performed blinded with the coding being held by non-experimenters. We find that in the acquisition phase, during which animals engage in spatial learning, the latency to solve the maze was not significantly different between the groups as judged by RM-ANOVA (F5,78 = 0.668, p =0.649) (Figure 1A). Differences in the distance traveled within the maze during this learning phase were significantly different (F5,78 = 2.389, p =0.045), though a Tukey’s post-hoc analysis did not reveal any specific differences between groups (Figure 1B). To clarify whether behavioural changes that might be observed in these studies are due to cognition per se and not due to motor differences between groups, we examined the time spent in the outer ring as a measure of anxiety and swim speed during these acquisition trials. The swim speed was significantly different between groups (F5,78 = 2.817, p =0.022), with the Tukey’s post-hoc analysis indicating that the 500 mkd Thiamet-G treated group was faster than the untreated wild-type control group (p =0.042), perhaps because treatment protects against neurodegeneration of motor neurons as previously observed in JNPL3 mice [16]. There was no difference between the groups in the time spent in the outer ring (F5,78 = 0.758, p =0.582) indicating the animals showed no apparent anxiety effects.
During the probe trials, where memories formed during the acquisition phase are tested, the 500 mkd Thiamet-G group spent significantly more time over the original platform location (F5,57 = 2.835, p =0.024) than did the untreated TAPP mice (p =0.03, Tukey’s post-hoc analysis) and the same amount of time as compared to wild-type control mice (Figure 1C), indicating increased O-GlcNAc blocks cognitive impairment in TAPP mice. Because the goal of the probe trial is for the animals to locate and remain within a very small area within the pool (~25 cm2), the differences between the 500 mkd Thiamet-G and the untreated TAPP mice are unlikely to result from motor differences between these groups. Consistent with this view, we did not observe any difference in the distance travelled during the probe trial (Figure 1D). These data indicate treatment with Thiamet-G resulted in cognitive enhancement compared to untreated animals, to a level matching that of wild-type animals.
Thiamet-G increases O-GlcNAc but does not impact tau phosphorylation
To better understand how Thiamet-G prevents impairment of cognitive performance, we first verified that O-GlcNAc levels were increased in treated animals (Figure 2). Immunoblotting with O-GlcNAc antibodies CTD110.6 and RL2 revealed dramatically increased O-GlcNAc levels in both the 200 and 500 mkd treated TAPP mice compared to the 0 mkd control TAPP mice (Figure 2A). We performed densitometric quantification of the O-GlcNAc immunoreactivity of the CTD110.6 antibody based on either all of the immunoreactive bands or only the low molecular weight (<50 kDa) immunoreactive bands (Figure 2B). This analysis revealed a trend toward lower O-GlcNAc levels in the 200 mkd TAPP mice group compared to the 500 mkd TAPP mice group when considering all of the immunoreactive bands. When only the low molecular weight bands are considered, O-GlcNAc levels were roughly 20% lower in the 200 mkd TAPP mice (p <0.05). These analyses reveal that there is a dose dependent effect of Thiamet-G on the levels of O-GlcNAc. We also performed immunohistochemistry (IHC) using the O-GlcNAc antibodies (CTD110.6 and RL2) on animals from the 0 and 500 mkd Thiamet-G treatment groups. The hippocampus and the cerebelleum have been previously shown to have high levels of expression of both OGA and OGT [31, 32] and O-GlcNAc levels in these structures, along with the pons and the amygdala, were particularly increased in the 500 mkd Thiamet-G treatment group (Figure 2C). Previously, we have shown that Thiamet-G does not block tau hyperphosphorylation in the transgenic parental JNPL3 mouse model [20]. We confirmed that this was also the case in the double transgenic TAPP mice by immunoblot (Figure 3A) and IHC (Figure 3B) analyses using various phosphorylation state-specific tau antibodies. Even though dramatically increased O-GlcNAc levels were observed (Figure 2) immunoblot analyses revealed that Thiamet-G treatment actually slightly increased the total amount of tau (92e) and the extent to which tau was phosphorylated but this did not reach statistical significance. In our previous study of JNPL3 mice [20], we found that Thiamet-G treatment reduced the amount of sarkosyl insoluble tau, which is known to correlate with the amount of fibrillar tau in 9 month old mice JNPL3 mice [33]. In a blinded experiment we therefore probed whether Thiamet-G can also reduce the amount of sarkosyl insoluble tau in the double transgenic TAPP mice. We observed a strong trend of less sarkosyl insoluble tau in the 500 mkd Thiamet-G treatment group (32% reduction), which was consistent in magnitude with our earlier JNPL3 study, although this difference did not reach statistical significance in the TAPP mice (Figure 4).
Thiamet-G treatment reduces the number of amyloid plaques and reduces Aβ levels
Previous work has shown that at 7-8 months of age the single transgenic JNPL3 mice generally perform no worse than age matched wild-type control animals in the MWM [34]. For this reason, and the absence of significant effects on tau phosphorylation, we felt that the cognitive effects of OGA inhibition in this TAPP model reflected in the MWM results may stem from effects of Thiamet-G treatment on accumulation of β-amyloid peptides and be reflected in the extent of amyloid plaque formation. To address this possibility we performed a number of experiments in which the experimenter was blinded using coded samples. The first study established whether Thiamet-G had any impact on the quantities of the amyloidogenic forms of β-amyloid 1-40 and 1-42 (Aβ40, Aβ42). Using widely used commercially available ELISA assays for Aβ40 and Aβ42, we determined that administration of 500 mkd Thiamet-G significantly reduced the quantity of Aβ42 while the 200 mkd treatment had no significant effect on this measure (Figure 5). We also observed a trend toward less Aβ40 in the 500 mkd treatment group that was not present in the 200 mkd group (Figure 5). As described above, O-GlcNAc levels in the 200 mkd group are slightly lower than the 500 mkd group and thus may indicate that a sustained increase in O-GlcNAc levels above a certain threshold must be reached in order to influence the levels of Aβ peptides within brain.
Finally, we evaluated whether the reduced Aβ42 levels correlated with fewer amyloid plaques within these animals. By quantitative analysis, we observed fewer amyloid plaques in both cortical and hippocampal regions of the brain in the 500 mkd Thiamet-G treated group (Figure 6). Interestingly however, in the cortex we found that 200 mkd was sufficient to reduce the number of amyloid plaques to the same level as seen in mice treated with 500 mkd Thiamet-G despite this dose being unable to significantly reduce levels of Aβ42 as assessed by ELISA (Figure 6B). It is interesting to note that O-GlcNAc levels are generally higher in the hippocampus (Figure 2C) than in the cortex, yet we observe larger reductions in the number of plaques in cortex in both dose groups than observed in the hippocampus. Perhaps different brain regions have differing capacities to increase O-GlcNAc levels and this may be coupled to tissue dependent differences in amyloid deposition that depend on O-GlcNAc levels. Further, perhaps differences in the levels of Aβ42 within certain brain regions are also present yet were not detected here because the ELISAs are conducted on homogenates obtained from complete brain hemispheres. Using whole brain homogenates might mask small differences in Aβ42 levels present in certain brain regions. These observations suggest that increased O-GlcNAc could influence both β-amyloid peptide production or clearance as well as assembly/clearance of amyloid plaques arising from Aβ42.
Thiamet-G treatment does not influence release of Aβ42 from cells
In an effort to address the question of how O-GlcNAc might affect β-amyloid peptide formation we turned to using a well-established cell culture model used to evaluate molecular pathways influencing APP processing [35–57] and cultured in primary neuron growth media (PNGM; Lonza). The astrocyte feeder layer for the neuronal co-culture was generated using neural progenitor cells as described [58]. A 50% volume media replacement was performed before neurons were treated with vehicle or 100 μM Thiamet-G for 24 hrs.
Immunohistochemistry
Free-floating brain sections were rinsed with PBS (pH 7.4) three times for 45 min first, then permeabilized with PBS containing 0.3% Triton X-100 (PBST) for 30 min. After blocking with 10% normal goat serum (NGS) and 2.5% BSA in PBST for 60 min, sections were incubated with mouse IgG monoclonal anti-β-amyloid antibody (6E10) at 4°C overnight, which reacts to human Aβ peptide (1-16 aa) and was generated in-house at the NYIBR and used at a dilution of 1:1000. After washing three times with PBS for 45 min, sections were incubated with Alexa488 goat anti mouse IgG (Invitrogen) secondary antibody at 1:1000 for 90 min. After 45 min washing, the sections were mounted on pre-coated slides (Adhesion superfrost plus, Brain Research Laboratories, Newton, MA), and cover-slipped with Vectashield MountingMedium (H-1000, Vector Laboratories). Sections examined in parallel but without being exposed to primary antibody served as experimental controls. The stained sections were examined using a Nikon C1 confocal system equipped with Nikon 90i fluorescent microscope. The images were acquired by the Nikon digital camera and Nikon C1 imaging system.
Aβ40 and Aβ42 ELISAs
Commercially available enzyme linked immunosorbant assay (ELISA) kits for amyloidogenic β-amyloid 1-40 and 1-42 (Aβ40 and Aβ42) (Invitrogen) were used to determine the total human Aβ40 and Aβ42 levels in the brain homogenates. Aβ42 was detected in neuronal cell culture media exactly as described by the manufacturer’s instruction using an ELISA kit from Wako (Human/Rat β-Amyloid(42)). Protein concentrations of the brain homogenates were determined using the BCA method (Pierce) and the Aβ40 and Aβ42 concentrations were expressed as ng Aβ40 or Aβ42/g of protein in the homogenate.
Quantitative immunohistochemistry of amyloid plaques (APs)
One sagittal section per mouse was immuno-stained with 6E10 which recognizes amino acids 1-16 of β-amyloid. Immuno-positive APs were counted manually and expressed as the number of APs/section.
Immunohistochemical staining
Frozen mouse brain sections (50 μm) stored in DeOlomos buffer at -20°C were washed with TBS and treated with 1% H2O2 and 50% methanol for 30 min to inactivate endogenous peroxidase. After being blocked with 5% normal goat serum in TBS for 1 hour, sections were incubated with the appropriate primary antibodies (see Table 1), followed by biotin-labeled secondary antibodies (Vector Laboratories), HRP-labeled avidin-biotin complex (Vector Laboratories), and the substrate DAB (3,3'-Diaminobenzidine, Sigma). The specimens were observed under a microscope (Nikon) and images captured using a digital camera DS-L2 (Nikon).
Antibodies
All antibodies used in this study are described in the Table 1.
Quantitative image analysis
Phosphorylated tau was visualized in sagittal brain sections from 6-9 mice each group by IHC staining. Using the identical microscope and camera settings, at least four digital images per sample were taken to reflect the overall staining in the pons region of brain. For the cortex and hippocampus, images at 20X were used. Only one image for hippocampus and one for frontal cortex were taken. All images were analyzed using the commercially available software program Image-Pro Plus version 4.0 for Windows (Media Cybernetics, Silver Spring, MD). The total immuno-intensity of the selected immuno-positive area was divided by the area size, and the values relative to that of the 0 mkd TAPP group are presented in the graphs. The proposed method allowed numerical analysis of the immunostaining intensity. The data from each group was normalized by the 0 mkd TAPP group.
Statistics analysis
For the MWM testing repeated measures analysis of variance (RM-ANOVA) was used to examine the effects of Thiamet-G on the latency of mice to complete the maze, their distance traveled, their speed of movement, and their time in each of the quadrants, with Tukey’s post-hoc to determine group differences where appropriate. The probe trial was analyzed using a one-way ANOVA with Tukey’s post-hoc tests. For all other data two-tailed unpaired student’s t-tests were used except for the sarkosyl insoluble tau data where we had data from previous studies to inform the predicted direction of change thus allowing the use of a one-tailed test in this case.
Western blotting
Brain tissue was homogenized at 4°C in 9 volumes of buffer (Buffer H) containing (50 mM Tris-HCl (pH 7.4), 20 μM UDP, 0.5 μM PUGNAc, 2 mM sodium othovanadate, 0.1 M NaF, 1.0 mM EGTA, 0.5 mM AEBSF, 2.0 μg/ml aprotinin, 10 μg/ml Leupeptin, 2.0 μg/ml pepstatin A) and the protein concentrations of the homogenates were determined using the BCA method (Pierce). The levels of global O-GlcNAcylation (CTD110.6 and RL2) and tau phosphorylation at various sites were determined by Western blot analysis using the antibodies listed in Table 1. Primary neurons were lysed in RIPA buffer containing Complete Protease Inhibitor Cocktail (Roche). Samples (10 μg) were resolved on 16% SDS-polyacrylamide gels and transferred to nitrocellulose membranes for blotting of APP-CTF. 30 μg of protein lysates was loaded per lane of a 4-20% SDS-polyacrylamide gel for blotting of O-GlcNAcylation (CTD110.6). Membranes were incubated with the following primary antibodies overnight at 4°C: anti-APP-CTF (1:2500, Abcam), anti-O-GlcNAc antibody (CTD110.6, 1:3000, Covance) and anti-actin (1:1000; Li-Cor). Quantitative analysis of immunoreactivity was carried out using images obtained by Li-Cor fluorescence and quantified using ImageJ. Quantitation of the lysate concentrations was performed using the BioRad QuickStart Bradford assay for both neurons and the 20E2 cells and levels of proteins established as a ratio of protein to actin.
Sarkosyl extraction
Whole tissue homogenates described above were first centrifuged at 10,000 × g in an Eppendorf 5417C centrifuge for 20 min. Supernatants were adjusted to have a final concentration of 1% N-laurylsacrosinate (Sarkosyl, Sigma) and were incubated at 37°C for one hour with shaking. Samples were then centrifuged at 100,000 × g in a Beckman TLA-45 rotor in a Beckman TL-100 centrifuge at 4°C for 45 min. The supernatant was removed and the sarkosyl insoluble pellet was resuspended in SDS-PAGE loading buffer.
Abbreviations
- NFTs:
-
Neurofibrillary tangles
- AD:
-
Alzheimer Disease
- O-GlcNAc O:
-
-linked N-Acetylglucosamine
- APP:
-
Amyloid precursor protein
- OGA:
-
O-GlcNAcase
- FTDP-17:
-
Frontal temporal dementia linked to chromosome-17
- APPSwe:
-
Swedish APP mutation
- MWM:
-
Morris water maze
- ELISA:
-
Enzyme linked immunosorbant assay
- AP:
-
Amyloid plaque
- HEK:
-
Human embryonic kidney.
References
Glenner GG, Wong CW: Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun. 1984, 120: 885-890. 10.1016/S0006-291X(84)80190-4.
Hardy J, Selkoe DJ: The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science. 2002, 297: 353-356. 10.1126/science.1072994.
Masters CL, Simms G, Weinman NA, Multhaup G, McDonald BL, Beyreuther K: Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc Natl Acad Sci U S A. 1985, 82: 4245-4249. 10.1073/pnas.82.12.4245.
Goate A, Chartier-Harlin MC, Mullan M, Brown J, Crawford F, Fidani L, Giuffra L, Haynes A, Irving N, James L, Mant R, Newton P, Rooke K, Roques P, Talbot C, Pericak-Vance M, Roses A, Williamson R, Rossor M, Owen M, Hardy J: Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease. Nature. 1991, 349: 704-706. 10.1038/349704a0.
Hendriks L, van Duijn CM, Cras P, Cruts M, Van Hul W, van Harskamp F, Warren A, McInnis MG, Antonarakis SE, Martin JJ, Hofman A, Van Broeckhoven C: Presenile dementia and cerebral haemorrhage linked to a mutation at codon 692 of the beta-amyloid precursor protein gene. Nat Genet. 1992, 1: 218-221. 10.1038/ng0692-218.
Mullan M, Crawford F, Axelman K, Houlden H, Lilius L, Winblad B, Lannfelt L: A pathogenic mutation for probable Alzheimer's disease in the APP gene at the N-terminus of beta-amyloid. Nat Genet. 1992, 1: 345-347. 10.1038/ng0892-345.
Jonsson T, Atwal JK, Steinberg S, Snaedal J, Jonsson PV, Bjornsson S, Stefansson H, Sulem P, Gudbjartsson D, Maloney J, Hoyte K, Gustafson A, Liu Y, Lu Y, Bhangale T, Graham RR, Huttenlocher J, Bjornsdottir G, Andreassen OA, Jonsson EG, Palotie A, Behrens TW, Magnusson OT, Kong A, Thorsteinsdottir U, Watts RJ, Stefansson K: A mutation in APP protects against Alzheimer's disease and age-related cognitive decline. Nature. 2012, 488: 96-99. 10.1038/nature11283.
Alonso A, Zaidi T, Novak M, Grundke-Iqbal I, Iqbal K: Hyperphosphorylation induces self-assembly of tau into tangles of paired helical filaments/straight filaments. Proc Natl Acad Sci U S A. 2001, 98: 6923-6928. 10.1073/pnas.121119298.
Grundke-Iqbal I, Iqbal K, Quinlan M, Tung YC, Zaidi MS, Wisniewski HM: Microtubule-associated protein tau: a component of Alzheimer paired helical filaments. J Biol Chem. 1986, 261: 6084-6089.
Lindwall G, Cole RD: The purification of tau protein and the occurrence of two phosphorylation states of tau in brain. J Biol Chem. 1984, 259: 12241-12245.
Nukina N, Kosik KS, Selkoe DJ: Recognition of Alzheimer paired helical filaments by monoclonal neurofilament antibodies is due to crossreaction with tau protein. Proc Natl Acad Sci U S A. 1987, 84: 3415-3419. 10.1073/pnas.84.10.3415.
Hutton M, Lendon CL, Rizzu P, Baker M, Froelich S, Houlden H, Pickering-Brown S, Chakraverty S, Isaacs A, Grover A, Hackett J, Adamson J, Lincoln S, Dickson D, Davies P, Petersen RC, Stevens M, de Graaff E, Wauters E, van Baren J, Hillebrand M, Joosse M, Kwon JM, Nowotny P, Che LK, Norton J, Morris JC, Reed LA, Trojanowski J, Basun H, et al: Association of missense and 5'-splice-site mutations in tau with the inherited dementia FTDP-17. Nature. 1998, 393: 702-705. 10.1038/31508.
Lee VM, Goedert M, Trojanowski JQ: Neurodegenerative tauopathies. Annu Rev Neurosci. 2001, 24: 1121-1159. 10.1146/annurev.neuro.24.1.1121.
Games D, Adams D, Alessandrini R, Barbour R, Borthelette P, Blackwell C, Carr T, Clemens J, Donaldson T, Gillespie F, Guido T, Hagopian S, Johnson-Wood K, Khan K, Lee M, Leibowitz P, Lieberburg I, Little S, Masliah E, McConlogue L, Montoya-Zavala M, Mucke L, Paganini L, Penniman E, Power M, Schenk D, Seubert P, Snyder B, Soriano F, Tan H, et al: Alzheimer-type neuropathology in transgenic mice overexpressing V717F beta-amyloid precursor protein. Nature. 1995, 373: 523-527. 10.1038/373523a0.
Hsiao K, Chapman P, Nilsen S, Eckman C, Harigaya Y, Younkin S, Yang F, Cole G: Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice. Science. 1996, 274: 99-102. 10.1126/science.274.5284.99.
Lewis J, McGowan E, Rockwood J, Melrose H, Nacharaju P, Van Slegtenhorst M, Gwinn-Hardy K, Paul Murphy M, Baker M, Yu X, Duff K, Hardy J, Corral A, Lin WL, Yen SH, Dickson DW, Davies P, Hutton M: Neurofibrillary tangles, amyotrophy and progressive motor disturbance in mice expressing mutant (P301L) tau protein. Nat Genet. 2000, 25: 402-405. 10.1038/78078.
Lewis J, Dickson DW, Lin WL, Chisholm L, Corral A, Jones G, Yen SH, Sahara N, Skipper L, Yager D, Eckman C, Hardy J, Hutton M, McGowan E: Enhanced neurofibrillary degeneration in transgenic mice expressing mutant tau and APP. Science. 2001, 293: 1487-1491. 10.1126/science.1058189.
Gotz J, Chen F, van Dorpe J, Nitsch RM: Formation of neurofibrillary tangles in P301l tau transgenic mice induced by Abeta 42 fibrils. Science. 2001, 293: 1491-1495. 10.1126/science.1062097.
Torres CR, Hart GW: Topography and polypeptide distribution of terminal N-acetylglucosamine residues on the surfaces of intact lymphocytes: evidence for O-linked GlcNAc. J Biol Chem. 1984, 259: 3308-3317.
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, 8: 393-399. 10.1038/nchembio.797.
Roquemore EP, Chevrier MR, Cotter RJ, Hart GW: Dynamic O-GlcNAcylation of the small heat shock protein alpha B-crystallin. Biochemistry. 1996, 35: 3578-3586. 10.1021/bi951918j.
Kreppel LK, Blomberg MA, Hart GW: Dynamic glycosylation of nuclear and cytosolic proteins. Cloning and characterization of a unique O-GlcNAc transferase with multiple tetratricopeptide repeats. J Biol Chem. 1997, 272: 9308-9315. 10.1074/jbc.272.14.9308.
Lubas WA, Frank DW, Krause M, Hanover JA: O-Linked GlcNAc transferase is a conserved nucleocytoplasmic protein containing tetratricopeptide repeats. J Biol Chem. 1997, 272: 9316-9324. 10.1074/jbc.272.14.9316.
Dong DL, Hart GW: Purification and characterization of an O-GlcNAc selective N-acetyl-beta-D-glucosaminidase from rat spleen cytosol. J Biol Chem. 1994, 269: 19321-19330.
Gao Y, Wells L, Comer FI, Parker GJ, Hart GW: Dynamic O-glycosylation of nuclear and cytosolic proteins: cloning and characterization of a neutral, cytosolic beta-N-acetylglucosaminidase from human brain. J Biol Chem. 2001, 276: 9838-9845. 10.1074/jbc.M010420200.
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, 4: 483-490. 10.1038/nchembio.96.
Haltiwanger RS, Grove K, Philipsberg GA: Modulation of O-linked N-acetylglucosamine levels on nuclear and cytoplasmic proteins in vivo using the peptide O-GlcNAc-beta-N-acetylglucosaminidase inhibitor O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate. J Biol Chem. 1998, 273: 3611-3617. 10.1074/jbc.273.6.3611.
Griffith LS, Mathes M, Schmitz B: Beta-amyloid precursor protein is modified with O-linked N-acetylglucosamine. J Neurosci Res. 1995, 41: 270-278. 10.1002/jnr.490410214.
Jacobsen KT, Iverfeldt K: O-GlcNAcylation increases non-amyloidogenic processing of the amyloid-beta precursor protein (APP). Biochem Biophys Res Commun. 2011, 404: 882-886. 10.1016/j.bbrc.2010.12.080.
Westerman MA, Cooper-Blacketer D, Mariash A, Kotilinek L, Kawarabayashi T, Younkin LH, Carlson GA, Younkin SG, Ashe KH: The relationship between Abeta and memory in the Tg2576 mouse model of Alzheimer's disease. J Neurosci. 2002, 22: 1858-1867.
Akimoto Y, Comer FI, Cole RN, Kudo A, Kawakami H, Hirano H, Hart GW: Localization of the O-GlcNAc transferase and O-GlcNAc-modified proteins in rat cerebellar cortex. Brain Res. 2003, 966: 194-205. 10.1016/S0006-8993(02)04158-6.
Liu K, Paterson AJ, Zhang F, McAndrew J, Fukuchi K, Wyss JM, Peng L, Hu Y, Kudlow JE: Accumulation of protein O-GlcNAc modification inhibits proteasomes in the brain and coincides with neuronal apoptosis in brain areas with high O-GlcNAc metabolism. J Neurochem. 2004, 89: 1044-1055. 10.1111/j.1471-4159.2004.02389.x.
Sahara N, Lewis J, DeTure M, McGowan E, Dickson DW, Hutton M, Yen SH: Assembly of tau in transgenic animals expressing P301L tau: alteration of phosphorylation and solubility. J Neurochem. 2002, 83: 1498-1508. 10.1046/j.1471-4159.2002.01241.x.
Arendash GW, Lewis J, Leighty RE, McGowan E, Cracchiolo JR, Hutton M, Garcia MF: Multi-metric behavioral comparison of APPsw and P301L models for Alzheimer's disease: linkage of poorer cognitive performance to tau pathology in forebrain. Brain Res. 2004, 1012: 29-41. 10.1016/j.brainres.2004.02.081.
Ly PT, Wu Y, Zou H, Wang R, Zhou W, Kinoshita A, Zhang M, Yang Y, Cai F, Woodgett J, Song W: Inhibition of GSK3beta-mediated BACE1 expression reduces Alzheimer-associated phenotypes. J Clin Invest. 2013, 123: 224-235. 10.1172/JCI64516.
Qing H, He G, Ly PT, Fox CJ, Staufenbiel M, Cai F, Zhang Z, Wei S, Sun X, Chen CH, Zhou W, Wang K, Song W: Valproic acid inhibits Abeta production, neuritic plaque formation, and behavioral deficits in Alzheimer's disease mouse models. J Exp Med. 2008, 205: 2781-2789. 10.1084/jem.20081588.
Qing H, Zhou W, Christensen MA, Sun X, Tong Y, Song W: Degradation of BACE by the ubiquitin-proteasome pathway. Faseb J. 2004, 18: 1571-1573.
Sun X, Wang Y, Qing H, Christensen MA, Liu Y, Zhou W, Tong Y, **ao C, Huang Y, Zhang S, Liu X, Song W: Distinct transcriptional regulation and function of the human BACE2 and BACE1 genes. Faseb J. 2005, 19: 739-749. 10.1096/fj.04-3426com.
Marshall S, Bacote V, Traxinger RR: Discovery of a metabolic pathway mediating glucose-induced desensitization of the glucose transport system. Role of hexosamine biosynthesis in the induction of insulin resistance. J Biol Chem. 1991, 266: 4706-4712.
Heiss WD, Szelies B, Kessler J, Herholz K: Abnormalities of energy metabolism in Alzheimer's disease studied with PET. Ann N Y Acad Sci. 1991, 640: 65-71.
Liu Y, Liu F, Iqbal K, Grundke-Iqbal I, Gong CX: Decreased glucose transporters correlate to abnormal hyperphosphorylation of tau in Alzheimer disease. FEBS Lett. 2008, 582: 359-364. 10.1016/j.febslet.2007.12.035.
Shulman JM, Chipendo P, Chibnik LB, Aubin C, Tran D, Keenan BT, Kramer PL, Schneider JA, Bennett DA, Feany MB, De Jager PL: Functional screening of Alzheimer pathology genome-wide association signals in drosophila. Am J Hum Genet. 2011, 88: 232-238. 10.1016/j.ajhg.2011.01.006.
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, 101: 10804-10809. 10.1073/pnas.0400348101.
Vignini A, Giulietti A, Nanetti L, Raffaelli F, Giusti L, Mazzanti L, Provinciali L: Alzheimer's disease and diabetes: new insights and unifying therapies. Curr Diabetes Rev. 2013, 9: 218-227. 10.2174/1573399811309030003.
Lefebvre T, Ferreira S, Dupont-Wallois L, Bussiere T, Dupire MJ, Delacourte A, Michalski JC, Caillet-Boudin ML: Evidence of a balance between phosphorylation and O-GlcNAc glycosylation of Tau proteins–a role in nuclear localization. Biochim Biophys Acta. 2003, 1619: 167-176. 10.1016/S0304-4165(02)00477-4.
Liu F, Shi J, Tanimukai H, Gu J, 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, 132: 1820-1832. 10.1093/brain/awp099.
Liu Y, Liu F, Grundke-Iqbal I, Iqbal K, Gong CX: Brain glucose transporters, O-GlcNAcylation and phosphorylation of tau in diabetes and Alzheimer's disease. J Neurochem. 2009, 111: 242-249. 10.1111/j.1471-4159.2009.06320.x.
Macauley MS, Stubbs KA, Vocadlo DJ: O-GlcNAcase catalyzes cleavage of thioglycosides without general acid catalysis. J Am Chem Soc. 2005, 127: 17202-17203. 10.1021/ja0567687.
Kim C, Nam DW, Park SY, Song H, Hong HS, Boo JH, Jung ES, Kim Y, Baek JY, Kim KS, Cho JW, Mook-Jung I: O-linked beta-N-acetylglucosaminidase inhibitor attenuates beta-amyloid plaque and rescues memory impairment. Neurobiol Aging. 2013, 34: 275-285. 10.1016/j.neurobiolaging.2012.03.001.
Oakley H, Cole SL, Logan S, Maus E, Shao P, Craft J, Guillozet-Bongaarts A, Ohno M, Disterhoft J, Van Eldik L, Berry R, Vassar R: Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer's disease mutations: potential factors in amyloid plaque formation. J Neurosci. 2006, 26: 10129-10140. 10.1523/JNEUROSCI.1202-06.2006.
Le Corre S, Klafki HW, Plesnila N, Hubinger G, Obermeier A, Sahagun H, Monse B, Seneci P, Lewis J, Eriksen J, Zehr C, Yue M, McGowan E, Dickson DW, Hutton M, Roder HM: An inhibitor of tau hyperphosphorylation prevents severe motor impairments in tau transgenic mice. Proc Natl Acad Sci U S A. 2006, 103: 9673-9678. 10.1073/pnas.0602913103.
Mehdy A, Morelle W, Rosnoblet C, Legrand D, Lefebvre T, Duvet S, Foulquier F: PUGNAc treatment leads to an unusual accumulation of free oligosaccharides in CHO cells. J Biochem. 2012, 151: 439-446. 10.1093/jb/mvs012.
Stubbs KA, Macauley MS, Vocadlo DJ: A selective inhibitor Gal-PUGNAc of human lysosomal beta-hexosaminidases modulates levels of the ganglioside GM2 in neuroblastoma cells. Angew Chem Int Ed Engl. 2009, 48: 1300-1303. 10.1002/anie.200804583.
Borghgraef P, Menuet C, Theunis C, Louis JV, Devijver H, Maurin H, Smet-Nocca C, Lippens G, Hilaire G, Gijsen H, Moechars D, Van Leuven F: Increasing brain protein O-GlcNAcylation mitigates breathing defects and mortality of Tau.P301L mice. PLoS One. 2013, 8: e84442-10.1371/journal.pone.0084442.
Graham DL, Gray AJ, Joyce JA, Yu D, O'Moore J, Carlson GA, Shearman MS, Dellovade TL, Hering H: Increased O-GlcNAcylation reduces pathological tau without affecting its normal phosphorylation in a mouse model of tauopathy. Neuropharmacology. 2014, 79: 307-413.
Perrot-Sinal TS, Kostenuik MA, Ossenkopp KP, Kavaliers M: Sex differences in performance in the Morris water maze and the effects of initial nonstationary hidden platform training. Behav Neurosci. 1996, 110: 1309-1320.
Kaech S, Banker G: Culturing hippocampal neurons. Nat Protoc. 2006, 1: 2406-2415. 10.1038/nprot.2006.356.
Miranda CJ, Braun L, Jiang Y, Hester ME, Zhang L, Riolo M, Wang H, Rao M, Altura RA, Kaspar BK: Aging brain microenvironment decreases hippocampal neurogenesis through Wnt-mediated survivin signaling. Aging Cell. 2012, 11: 542-552. 10.1111/j.1474-9726.2012.00816.x.
Acknowledgements
This work was supported by grants from the National Institute on Aging/National Institutes of Health (R21AG031969 and R01AG027429), the U.S. Alzheimer’s Association (IIRG-10-170405), and the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant 00194522. This research was also supported by grants from NSERC (327100-06) and the Canadian Institutes of Health Research (CIHR; 90396) to M.A.S. M.L.W. was supported by a Vice-President of Research, Undergraduate Student Research Award from SFU. D.J.V. is the Canada Research Chair in Chemical Glycobiology and thanks NSERC for support through an E.W.R Steacie Memorial Fellowship. We also thank the technical staff at the Simon Fraser University Animal Care Facility and are also grateful to Lesley Chen for her expert technical assistance.
Author information
Authors and Affiliations
Additional information
Competing interests
DJV and EJM are cofounders of and hold equity in the company Alectos Therapeutics. DJV serves as CSO and Chair of the Scientific Advisory Board (SAB) of Alectos Therapeutics. EJM is the CEO of Alectos Therapeutics. CXG is a member of the SAB of Alectos Therapeutics. SAY, XS, EJM, and DJV may receive royalties from SFU for commercialization of technology relating to OGA inhibitors.
Authors’ contributions
SAY, XS, EJM performed the animal studies. SAY, XS, GZ, and XL performed immunohistochemical and immunoblot analyses of tissues, BAJ performed the behavioural studies. EJM synthesized inhibitor for the studies. SAY, MLW, YZ, and MAS performed the cell culture studies. SAY, XS, BAJ, MAS, YZ, EJM, NVW, CXG, and DJV designed experiments. All authors contributed to analysis of the data. SAY and DJV wrote the manuscript with input and revisions from all the authors. All authors read and approved the final manuscript.
Scott A Yuzwa, **aoyang Shan contributed equally to this work.
Authors’ original submitted files for images
Below are the links to the authors’ original submitted files for images.
Rights and permissions
This article is published under an open access license. Please check the 'Copyright Information' section either on this page or in the PDF for details of this license and what re-use is permitted. If your intended use exceeds what is permitted by the license or if you are unable to locate the licence and re-use information, please contact the Rights and Permissions team.
About this article
Cite this article
Yuzwa, S.A., Shan, X., Jones, B.A. et al. Pharmacological inhibition of O-GlcNAcase (OGA) prevents cognitive decline and amyloid plaque formation in bigenic tau/APP mutant mice. Mol Neurodegeneration 9, 42 (2014). https://doi.org/10.1186/1750-1326-9-42
Received:
Accepted:
Published:
DOI: https://doi.org/10.1186/1750-1326-9-42