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Pharmacological inhibition of O-GlcNAcase (OGA) prevents cognitive decline and amyloid plaque formation in bigenic tau/APP mutant mice.

Yuzwa SA, Shan X, Jones BA, Zhao G, Woodward ML, Li X, Zhu Y, McEachern EJ, Silverman MA, Watson NV, Gong CX, Vocadlo DJ - Mol Neurodegener (2014)

Bottom Line: 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.The findings provide good support for OGA as a promising therapeutic target to alter disease progression in Alzheimer disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Dr, Burnaby, BC V5A 1S6, Canada. dvocadlo@sfu.ca.

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.

Show MeSH

Related in: MedlinePlus

Thiamet-G treatment of APPSwe-expressing 20E2 cells and primary hippocampal neurons does not alter APP processing. A. 20E2 cells were treated with Thiamet-G for 16 hours, and then the amount of Aβ42 in the cell culture media, collected at the indicated time points after the replacement of new culture media, was assessed using an ELISA assay and corrected for the amount of protein in the cell lysates from each condition. This analysis reveals that Thiamet-G does not alter the amount of Aβ42 that is produced. B. Little Aβ42 is detected in untransfected human embryonic kidney (HEK) cells indicating that validity of this culture model. C. O-GlcNAc levels in the 20E2 cells are increased at all time points analyzed (*indicates p <0.05, paired two-tailed t-test). D. Levels of APP-CTF were unchanged in 20E2 cells treated with Thiamet-G for 24 hours. E. Aβ42 production is not altered in primary hippocampal neurons treated with Thiamet-G for 24 hour. F. APP-CTF levels are unchanged in 24 hour Thiamet-G treated hippocampal neurons. G. O-GlcNAc levels in primary hippocampal neurons are increased after 24 hours of treatment with Thiamet-G. (***indicates p <0.001,, paired two-tailed t-test) Error bars represent standard error of the mean (± S.E.M). N.S. Not Significant. For all panels, N =3.
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Fig7: Thiamet-G treatment of APPSwe-expressing 20E2 cells and primary hippocampal neurons does not alter APP processing. A. 20E2 cells were treated with Thiamet-G for 16 hours, and then the amount of Aβ42 in the cell culture media, collected at the indicated time points after the replacement of new culture media, was assessed using an ELISA assay and corrected for the amount of protein in the cell lysates from each condition. This analysis reveals that Thiamet-G does not alter the amount of Aβ42 that is produced. B. Little Aβ42 is detected in untransfected human embryonic kidney (HEK) cells indicating that validity of this culture model. C. O-GlcNAc levels in the 20E2 cells are increased at all time points analyzed (*indicates p <0.05, paired two-tailed t-test). D. Levels of APP-CTF were unchanged in 20E2 cells treated with Thiamet-G for 24 hours. E. Aβ42 production is not altered in primary hippocampal neurons treated with Thiamet-G for 24 hour. F. APP-CTF levels are unchanged in 24 hour Thiamet-G treated hippocampal neurons. G. O-GlcNAc levels in primary hippocampal neurons are increased after 24 hours of treatment with Thiamet-G. (***indicates p <0.001,, paired two-tailed t-test) Error bars represent standard error of the mean (± S.E.M). N.S. Not Significant. For all panels, N =3.

Mentions: 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 [35–38]. Using a cellular model enables us to ascertain the effects of OGA inhibition on β-amyloid peptide release independent of other factors that could obscure such effects in animals, such as β-amyloid peptide clearance and sequestration into plaques. The 20E2 cells we used are an established HEK cell line stably expressing APPSwe. The mutant APPSwe is processed by endogenous α, β and γ-secretase enzymes leading to abundant and readily detectable release of Aβ42 (Figure 7A) as compared to untransfected HEK cells (Figure 7B). We treated this 20E2 cell line with 100 μM Thiamet-G overnight and in the morning provided fresh media while maintaining 100 μM Thiamet-G. This dose of Thiamet-G selected for use in culture studies was chosen because we have previously found this dose to be well beyond the saturation point of increased O-GlcNAc levels in PC-12 cells [26] suggesting that O-GlcNAc levels are highly elevated, which should therefore stimulate possible effects within cells during these experiments. The cells and culture media were collected at 4, 8 and 24 hours after changing the media. While we were able to detect significant accumulation of Aβ42 over time as compared to non-transfected HEK cells, Thiamet-G did not exert any influence on release of Aβ42. We further verified that O-GlcNAc levels were increased at all time points in this experiment (Figure 7C). These data suggest that there are no differences in the rate of production or clearance of Aβ42. Nevertheless, because Aβ42 can also be cleared or degraded through various processes we considered a scenario wherein Aβ42 levels may appear unchanged by ELISA assay even though rates of production of Aβ42 could be different, perhaps due to compensatory degradation mechanisms. To address this possibility, we noted that Aβ42 production results from the cleavage by γ-secretase and therefore we assessed the levels of the γ-secretase substrate, the APP C-terminal fragment (APP-CTF). Here we also observed no difference in the levels of APP-CTF in 20E2 cells treated for 24 hours (Figure 7D). If changes in O-GlcNAc influenced the rates of Aβ42 production in these cells but such changes were obscured in our assays by compensating Aβ42 degradation, then we would expect APP-CTF levels would differ. The absence of any difference in APP-CTF levels, or the α:β ratio of CTF fragments, upon increased O-GlcNAc therefore provides further support that Aβ42 release and APP processing are unaffected by increased O-GlcNAc within this cell line.Figure 7


Pharmacological inhibition of O-GlcNAcase (OGA) prevents cognitive decline and amyloid plaque formation in bigenic tau/APP mutant mice.

Yuzwa SA, Shan X, Jones BA, Zhao G, Woodward ML, Li X, Zhu Y, McEachern EJ, Silverman MA, Watson NV, Gong CX, Vocadlo DJ - Mol Neurodegener (2014)

Thiamet-G treatment of APPSwe-expressing 20E2 cells and primary hippocampal neurons does not alter APP processing. A. 20E2 cells were treated with Thiamet-G for 16 hours, and then the amount of Aβ42 in the cell culture media, collected at the indicated time points after the replacement of new culture media, was assessed using an ELISA assay and corrected for the amount of protein in the cell lysates from each condition. This analysis reveals that Thiamet-G does not alter the amount of Aβ42 that is produced. B. Little Aβ42 is detected in untransfected human embryonic kidney (HEK) cells indicating that validity of this culture model. C. O-GlcNAc levels in the 20E2 cells are increased at all time points analyzed (*indicates p <0.05, paired two-tailed t-test). D. Levels of APP-CTF were unchanged in 20E2 cells treated with Thiamet-G for 24 hours. E. Aβ42 production is not altered in primary hippocampal neurons treated with Thiamet-G for 24 hour. F. APP-CTF levels are unchanged in 24 hour Thiamet-G treated hippocampal neurons. G. O-GlcNAc levels in primary hippocampal neurons are increased after 24 hours of treatment with Thiamet-G. (***indicates p <0.001,, paired two-tailed t-test) Error bars represent standard error of the mean (± S.E.M). N.S. Not Significant. For all panels, N =3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig7: Thiamet-G treatment of APPSwe-expressing 20E2 cells and primary hippocampal neurons does not alter APP processing. A. 20E2 cells were treated with Thiamet-G for 16 hours, and then the amount of Aβ42 in the cell culture media, collected at the indicated time points after the replacement of new culture media, was assessed using an ELISA assay and corrected for the amount of protein in the cell lysates from each condition. This analysis reveals that Thiamet-G does not alter the amount of Aβ42 that is produced. B. Little Aβ42 is detected in untransfected human embryonic kidney (HEK) cells indicating that validity of this culture model. C. O-GlcNAc levels in the 20E2 cells are increased at all time points analyzed (*indicates p <0.05, paired two-tailed t-test). D. Levels of APP-CTF were unchanged in 20E2 cells treated with Thiamet-G for 24 hours. E. Aβ42 production is not altered in primary hippocampal neurons treated with Thiamet-G for 24 hour. F. APP-CTF levels are unchanged in 24 hour Thiamet-G treated hippocampal neurons. G. O-GlcNAc levels in primary hippocampal neurons are increased after 24 hours of treatment with Thiamet-G. (***indicates p <0.001,, paired two-tailed t-test) Error bars represent standard error of the mean (± S.E.M). N.S. Not Significant. For all panels, N =3.
Mentions: 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 [35–38]. Using a cellular model enables us to ascertain the effects of OGA inhibition on β-amyloid peptide release independent of other factors that could obscure such effects in animals, such as β-amyloid peptide clearance and sequestration into plaques. The 20E2 cells we used are an established HEK cell line stably expressing APPSwe. The mutant APPSwe is processed by endogenous α, β and γ-secretase enzymes leading to abundant and readily detectable release of Aβ42 (Figure 7A) as compared to untransfected HEK cells (Figure 7B). We treated this 20E2 cell line with 100 μM Thiamet-G overnight and in the morning provided fresh media while maintaining 100 μM Thiamet-G. This dose of Thiamet-G selected for use in culture studies was chosen because we have previously found this dose to be well beyond the saturation point of increased O-GlcNAc levels in PC-12 cells [26] suggesting that O-GlcNAc levels are highly elevated, which should therefore stimulate possible effects within cells during these experiments. The cells and culture media were collected at 4, 8 and 24 hours after changing the media. While we were able to detect significant accumulation of Aβ42 over time as compared to non-transfected HEK cells, Thiamet-G did not exert any influence on release of Aβ42. We further verified that O-GlcNAc levels were increased at all time points in this experiment (Figure 7C). These data suggest that there are no differences in the rate of production or clearance of Aβ42. Nevertheless, because Aβ42 can also be cleared or degraded through various processes we considered a scenario wherein Aβ42 levels may appear unchanged by ELISA assay even though rates of production of Aβ42 could be different, perhaps due to compensatory degradation mechanisms. To address this possibility, we noted that Aβ42 production results from the cleavage by γ-secretase and therefore we assessed the levels of the γ-secretase substrate, the APP C-terminal fragment (APP-CTF). Here we also observed no difference in the levels of APP-CTF in 20E2 cells treated for 24 hours (Figure 7D). If changes in O-GlcNAc influenced the rates of Aβ42 production in these cells but such changes were obscured in our assays by compensating Aβ42 degradation, then we would expect APP-CTF levels would differ. The absence of any difference in APP-CTF levels, or the α:β ratio of CTF fragments, upon increased O-GlcNAc therefore provides further support that Aβ42 release and APP processing are unaffected by increased O-GlcNAc within this cell line.Figure 7

Bottom Line: 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.The findings provide good support for OGA as a promising therapeutic target to alter disease progression in Alzheimer disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Dr, Burnaby, BC V5A 1S6, Canada. dvocadlo@sfu.ca.

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.

Show MeSH
Related in: MedlinePlus