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, 24 (22), 6492-504

Mitochondrial ATP Synthase Activity Is Impaired by Suppressed O-GlcNAcylation in Alzheimer's Disease

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Mitochondrial ATP Synthase Activity Is Impaired by Suppressed O-GlcNAcylation in Alzheimer's Disease

Moon-Yong Cha et al. Hum Mol Genet.

Abstract

Glycosylation with O-linked β-N-acetylglucosamine (O-GlcNAc) is one of the protein glycosylations affecting various intracellular events. However, the role of O-GlcNAcylation in neurodegenerative diseases such as Alzheimer's disease (AD) is poorly understood. Mitochondrial adenosine 5'-triphosphate (ATP) synthase is a multiprotein complex that synthesizes ATP from ADP and Pi. Here, we found that ATP synthase subunit α (ATP5A) was O-GlcNAcylated at Thr432 and ATP5A O-GlcNAcylation was decreased in the brains of AD patients and transgenic mouse model, as well as Aβ-treated cells. Indeed, Aβ bound to ATP synthase directly and reduced the O-GlcNAcylation of ATP5A by inhibition of direct interaction between ATP5A and mitochondrial O-GlcNAc transferase, resulting in decreased ATP production and ATPase activity. Furthermore, treatment of O-GlcNAcase inhibitor rescued the Aβ-induced impairment in ATP production and ATPase activity. These results indicate that Aβ-mediated reduction of ATP synthase activity in AD pathology results from direct binding between Aβ and ATP synthase and inhibition of O-GlcNAcylation of Thr432 residue on ATP5A.

Figures

Figure 1.
Figure 1.
Decreased O-GlcNAcylation and ATP synthase activity in AD model brains. (A) O-GlcNAcylated ATP5A in the brains from AD patients (AD) and controls (Ctrl). Total proteins were extracted from the dissected entorhinal cortex of AD and control brains to determine the expression levels of the indicated proteins. In the top panel, total O-GlcNAcylated proteins were pulled down with sWGA-agarose beads from entorhinal cortical lysates and the level of O-GlcNAc-ATP5A was then assessed by western blotting with an anti-ATP5A mAb (n = 6 per group). (B) The intensities of total ATP5A and O-GlcNAc-ATP5A in (A) were analyzed using densitometry and the level of O-GlcNAc-ATP5A was normalized to ATP5A. (C) O-GlcNAcylated ATP5A in the Tg6799 (Tg) mouse brain. Total proteins were extracted from the dissected cortices of Tg6799 mice and LTs to determine the expression levels of the indicated proteins. In the top panel, total O-GlcNAcylated proteins were pulled down with sWGA-agarose beads from cortex lysates and the level of O-GlcNAc-ATP5A was then assessed by western blotting with an anti-ATP5A mAb (n = 5 per group). (D) The intensities of total ATP5A and O-GlcNAc-ATP5A in (C) were analyzed using densitometry and the level of O-GlcNAc-ATP5A was normalized to ATP5A. (E) ATP production was measured in brain extracts from Tg6799 mice (Tg) and LTs (n = 5 per group). The results were represented as mean ± standard error from three independent experiments. **P < 0.01; *P < 0.05; n.s.: non-significant.
Figure 2.
Figure 2.
O-GlcNAcylation of ATP5A in cultured cells. (A) Specificity of sWGA-agarose pull-down in a competition assay. All O-GlcNAcylated proteins were pulled down from total protein extracts (300 µg) of HeLa cells with agarose or sWGA-conjugated agarose in the presence or absence of free N-acetylglucosamine (GlcNAc) as a competitor. The level of ATP5A was measured by immunoblotting. (B) Specificity of sWGA-agarose pull-down by treatment with β-hexosaminidase (β-HEX). Total protein extracts (20 µg; left panel) or O-GlcNAc proteins pulled down with sWGA-agarose (right panel) were treated with β-HEX and analyzed for the total levels of O-GlcNAcylation (left panel) or O-GlcNAc-ATP5A in the indicated fractions (right panel) by western blotting. β-actin or total ATP5A were included as loading controls for each fraction. (C) Effect of changes of the O-GlcNAcylation machinery on O-GlcNAc-ATP5A in HeLa cells. HeLa cells were treated with an OGT inhibitor (ST045849; ST, 25 µM) or OGA inhibitor (Thiamet G; ThG, 1 µM) for 24 h. Samples or sWGA-agarose pull-down fractions were analyzed by western blotting with the indicated antibodies. Bar graphs show densitometric quantification of O-GlcNAcylated ATP5A in HeLa cells. (D) Effect of changes of the O-GlcNAcylation machinery on O-GlcNAc-ATP5A in primary cultured neurons. Primary cortical neurons were cultured from the brains of B6/SJL mice and treated as in (C). The level of O-GlcNAc-ATP5A was detected by western blotting. Bar graphs show densitometric quantification of O-GlcNAcylated ATP5A in neurons. (E, F) ATP production (E) and ATPase activity (F) from extracts of HeLa cell treated with OGA or OGT inhibitor. HeLa cells were incubated in the presence or absence of OGA or OGT inhibitor (1 µM ThG or 25 µM ST for 24 h). ATP production was measured as described in Materials and Methods (n = 3 per group). (G) Effect of OGT knockdown on O-GlcNAc-ATP5A in HeLa cells. HeLa cells were transfected with 20 nM siRNA against OGT. After 48 h, cells were fractionated and the mitochondrial and cytosolic fractions were analyzed by western blotting with the indicated antibodies. (H) Effect of mOGT overexpression on O-GlcNAc-ATP5A in HeLa cells. HeLa cells were transfected with a FLAG-mOGT construct. After 48 h, cells were fractionated and the mitochondrial fraction was analyzed by western blotting with the indicated antibodies. (I) Densitometric quantifications of O-GlcNAcylated ATP5A levels in OGT knockdown system (G) and mOGT overexpressing cells (H). (J, K) ATP production (J) and ATPase activity (K) from protein extracts of HeLa cells transfected with siRNA against OGT or FLAG-mOGT construct. ATP production and ATPase activity were measured as described in Materials and Methods (n = 3 per group). The results were represented as mean ± standard error from three independent experiments. ***P < 0.001; **P < 0.01; *P < 0.05; n.s.: non-significant.
Figure 3.
Figure 3.
Effect of Aβ on O-GlcNAcylation and the function of ATP synthase. HeLa cells were treated with vehicle (Vcl) or Aβ (3 µM, 24 h) and whole cell lysates were prepared. The expression levels of the indicated proteins (A), ATP production (B) and ATPase activity (C) were measured by western blotting or in vitro assays (n = 3 per group). Bar graph in (A) shows densitometric quantification of O-GlcNAcylated ATP5A in HeLa cells. The results were represented as mean ± standard error from three independent experiments. ***P < 0.001; **P < 0.01; n.s.: non-significant.
Figure 4.
Figure 4.
Mapping of the O-GlcNAc modification sites on ATP5A. (A) LC-MS/MS result for putative O-GlcNAcylation sites on ATP5A. (B) Validation of O-GlcNAcylation on ATP5A through site-directed mutagenesis. Cells were transfected with GFP, WT ATP5A or mutant ATP5A in which putative O-GlcNAcylation sites were replaced with Ala. After 48 h transfection, whole cell lysates were prepared and pulled down with sWGA-agarose beads. O-GlcNAcylated ATP5A was detected by western blotting. Bar graph shows densitometric quantification of O-GlcNAcylated ATP5A in cells. (C) O-GlcNAcylation of ATP5A on Thr432. (D, E) ATP production (D) and ATPase activity (E) from extracts of HeLa cell transfected with ATP5A WT or ATP5A T432 mutant construct (T432A). After 48 h, ATP production and ATPase activity were measured as described in Materials and Methods (n = 3 per group). The results were represented as mean ± standard error from three independent experiments. **P < 0.01; *P < 0.05; n.s.: non-significant.
Figure 5.
Figure 5.
Blocking of the interaction between ATP5A and mOGT by Aβ treatment. (A) Co-immunoprecipitation of FLAG-mOGT and ATP5A-GFP. Cells were co-transfected with FLAG-mOGT, ATP5A WT or ATP5A T432 mutant construct (T432A). After 48 h, cells were fractionated and the mitochondrial fraction was analyzed by western blotting with the indicated antibodies. Bar graph shows densitometric quantification of immunoprecipated band. (B) Cells overexpressing FLAG-mOGT and ATP5A-GFP were treated with 3 µM Aβ for 48 h. Prepared mitochondrial fraction was analyzed by western blotting with the indicated antibodies. Bar graph shows densitometric quantification of immunoprecipated band. The result was represented as mean ± standard error from three independent experiments. **P < 0.01; *P < 0.05. Stars indicate overexpressed protein.
Figure 6.
Figure 6.
Binding of Aβ to ATP synthase. (A) Overall view of glycosylated ATP synthase complex. O-GlcNAcylation of ATP5A (red) on Thr432 is shown as a stick model. The neighboring β subunit (ATP5B) is colored in green. (B) Enlarged view of the O-GlcNAcylation site. (C) Molecular docking simulation between ATP synthase and Aβ. ATP5A, red; ATP5B, green; Aβ, orange. (D) Validation of Aβ binding on ATP synthase. Cells were transfected with control GFP vector or GFP-tagged WT or deletion mutant ATP5A/B (DM) in combination with mitochondria-targeted Aβ (mito-Aβ). After 48 h transfection, whole cell lysates were prepared and immunoprecipitated with anti-GFP antibody. The co-immunoprecipitated Aβ was detected with anti-Aβ mAb (6E10). Star indicates overexpressed protein.
Figure 7.
Figure 7.
Restoration of impaired ATP5A O-GlcNAcylation by enhanced O-GlcNAcylation. HeLa cells, CHO cells and primary cultured cortical neurons were treated with Aβ (3 µM, 24 h) in the presence or absence of ThG and then whole cell lysates were prepared. The levels of O-GlcNAcylated and total ATP5A (A), ATP production (B) and ATPase activity (C) were measured by western blotting or in vitro assays, respectively (n = 3 per group). Bar graphs in (A) show densitometric quantification of O-GlcNAcylated ATP5A in each cell. The results were represented as mean ± standard error from three independent experiments. ***P < 0.001, **P < 0.01 and *P < 0.05 versus vehicle (Vcl)-treated; ##P < 0.01 and #P < 0.05 versus Aβ-treated; n.s.: not significant. (D) Total proteins were extracted from the dissected cortices of mice (Littermate + saline, LT; Tg6799 + saline, Tg; Tg6799 + ThG, Tg + ThG) to determine the indicated protein levels. Total O-GlcNAcylated proteins were pulled down with sWGA-agarose beads from cortex lysates and the level of O-GlcNAc-ATP5A was then assessed by western blotting with an anti-ATP5A mAb. Bar graph shows densitometric quantification of O-GlcNAcylated ATP5A (n = 4 per group). *P < 0.05 versus LT; ###P < 0.001 versus Tg; n.s.: not significant. (E) ATP production was measured in brain extracts from mice (Littermate + saline, LT; Tg6799 + saline, Tg; Tg6799 + ThG, Tg + ThG) (n = 4 per group). The results were represented as mean ± standard error from three independent experiments. ***P < 0.001 versus LT; #P < 0.05 versus Tg; n.s.: non-significant.
Figure 8.
Figure 8.
Schematic diagram.

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