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Blockade of NR2A-containing NMDA receptors induces Tau phosphorylation in rat hippocampal slices.

Allyson J, Dontigny E, Auberson Y, Cyr M, Massicotte G - Neural Plast. (2010)

Bottom Line: In the present study, we used selective NMDA receptor antagonists to investigate the involvement of NR2A and NR2B subunits in the modulatory effect of basal NMDA receptor activity on the phosphorylation of Tau proteins.This effect seemed to be Ser199 specific as there was no increase in phosphorylation at Ser262 and Ser409 residues located in the microtubule-binding and C-terminal domains of Tau proteins, respectively.From a mechanistic perspective, our study revealed that blockade of NR2A-containing receptors influences Tau phosphorylation probably by increasing calcium influx into neurons, which seems to rely on accumulation of new NR1/NR2B receptors in neuronal membranes and could involve the cyclin-dependent kinase 5 pathway.

View Article: PubMed Central - PubMed

Affiliation: Département de chimie-biologie, Université du Québec à Trois-Rivières, Trois-Rivières, QC, Canada.

ABSTRACT
Physiological activation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors has been proposed to play a key role in both neuronal cell function and dysfunction. In the present study, we used selective NMDA receptor antagonists to investigate the involvement of NR2A and NR2B subunits in the modulatory effect of basal NMDA receptor activity on the phosphorylation of Tau proteins. We observed, in acute hippocampal slice preparations, that blockade of NR2A-containing NMDA receptors by the NR2A antagonist NVP-AAM077 provoked the hyperphosphorylation of a residue located in the proline-rich domain of Tau (i.e., Ser199). This effect seemed to be Ser199 specific as there was no increase in phosphorylation at Ser262 and Ser409 residues located in the microtubule-binding and C-terminal domains of Tau proteins, respectively. From a mechanistic perspective, our study revealed that blockade of NR2A-containing receptors influences Tau phosphorylation probably by increasing calcium influx into neurons, which seems to rely on accumulation of new NR1/NR2B receptors in neuronal membranes and could involve the cyclin-dependent kinase 5 pathway.

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Related in: MedlinePlus

Blockade of NR2A-containing NMDA receptors is associated with increased levels of NR1 and NR2B subunits in biotinylated membranes. Glutamate receptor subunit levels were estimated by Western blotting on biotinylated membranes obtained from acute hippocampal slices treated with 50 nM NVP for 2 hours. Biotinylated NR1, NR2B and GluR1 subunit levels were normalized with respective total protein levels estimated in homogenates of hippocampal slices incubated with or without NVP. The data are means ± SEM of 3 measurements obtained from 4 different rats. Since these experiments were independently performed, we determined statistical significance using unpaired T-Test. **P < .01, ***P < .001, NVP-treated versus control.
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fig5: Blockade of NR2A-containing NMDA receptors is associated with increased levels of NR1 and NR2B subunits in biotinylated membranes. Glutamate receptor subunit levels were estimated by Western blotting on biotinylated membranes obtained from acute hippocampal slices treated with 50 nM NVP for 2 hours. Biotinylated NR1, NR2B and GluR1 subunit levels were normalized with respective total protein levels estimated in homogenates of hippocampal slices incubated with or without NVP. The data are means ± SEM of 3 measurements obtained from 4 different rats. Since these experiments were independently performed, we determined statistical significance using unpaired T-Test. **P < .01, ***P < .001, NVP-treated versus control.

Mentions: Taken together, the above findings indicate that blockade of NR2A-containing NMDA receptors promotes Tau phosphorylation at Ser199 residue, implicating both calcium and the Cdk5 signalling pathway. The exact mechanisms by which NVP induces calcium mobilization, however, remain to be clarified. One possibility is that blockade of NR2A-containing NMDA receptors could have led to calcium entrance in neurons by favouring the dysregulation of other glutamate receptor subtypes. Thus, we initiated a series of experiments to determine the effects of glutamate receptor antagonisms on NVP-induced Tau phosphorylation in rat hippocampal slices. Here, we report the results obtained with an antagonist acting on the AMPA subtype of glutamate receptors (NBQX) and the antagonist acting on NR2B-containing NMDA receptors (i.e., RO). Figure 4 shows that preexposure of hippocampal slices to 10 μM NBQX did not significantly reduce Tau phosphorylation at Ser199 residue resulting from inhibition of NR2A-containing receptors by NVP. However, NVP-induced Tau phosphorylation was completely reversed by the preincubation of slices in the presence of RO, suggesting that the ability of NVP to enhance phosphorylation of the Ser199 epitope is possibly dependent on alterations of NR2B-containing NMDA receptors. Indeed, we decided to test this scenario by measuring the surface expression of both AMPA and NMDA receptor subunits on biotinylated membranes [27]. As shown in Figure 5(a), the surface level of GluR1 subunits of AMPA receptors was not significantly modified in slices treated with NVP. In contrast, a significant effect on NR1 subunits of NMDA receptors was observed in hippocampal slices 2 hours after NVP exposure. The NR2A antagonist was found to increase NR1 subunit levels by more than 60% in biotinylated membranes prepared from hippocampal slices (Figure 5(b)), while similar results were obtained with NR2B subunit levels (Figure 5(c)).


Blockade of NR2A-containing NMDA receptors induces Tau phosphorylation in rat hippocampal slices.

Allyson J, Dontigny E, Auberson Y, Cyr M, Massicotte G - Neural Plast. (2010)

Blockade of NR2A-containing NMDA receptors is associated with increased levels of NR1 and NR2B subunits in biotinylated membranes. Glutamate receptor subunit levels were estimated by Western blotting on biotinylated membranes obtained from acute hippocampal slices treated with 50 nM NVP for 2 hours. Biotinylated NR1, NR2B and GluR1 subunit levels were normalized with respective total protein levels estimated in homogenates of hippocampal slices incubated with or without NVP. The data are means ± SEM of 3 measurements obtained from 4 different rats. Since these experiments were independently performed, we determined statistical significance using unpaired T-Test. **P < .01, ***P < .001, NVP-treated versus control.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2874924&req=5

fig5: Blockade of NR2A-containing NMDA receptors is associated with increased levels of NR1 and NR2B subunits in biotinylated membranes. Glutamate receptor subunit levels were estimated by Western blotting on biotinylated membranes obtained from acute hippocampal slices treated with 50 nM NVP for 2 hours. Biotinylated NR1, NR2B and GluR1 subunit levels were normalized with respective total protein levels estimated in homogenates of hippocampal slices incubated with or without NVP. The data are means ± SEM of 3 measurements obtained from 4 different rats. Since these experiments were independently performed, we determined statistical significance using unpaired T-Test. **P < .01, ***P < .001, NVP-treated versus control.
Mentions: Taken together, the above findings indicate that blockade of NR2A-containing NMDA receptors promotes Tau phosphorylation at Ser199 residue, implicating both calcium and the Cdk5 signalling pathway. The exact mechanisms by which NVP induces calcium mobilization, however, remain to be clarified. One possibility is that blockade of NR2A-containing NMDA receptors could have led to calcium entrance in neurons by favouring the dysregulation of other glutamate receptor subtypes. Thus, we initiated a series of experiments to determine the effects of glutamate receptor antagonisms on NVP-induced Tau phosphorylation in rat hippocampal slices. Here, we report the results obtained with an antagonist acting on the AMPA subtype of glutamate receptors (NBQX) and the antagonist acting on NR2B-containing NMDA receptors (i.e., RO). Figure 4 shows that preexposure of hippocampal slices to 10 μM NBQX did not significantly reduce Tau phosphorylation at Ser199 residue resulting from inhibition of NR2A-containing receptors by NVP. However, NVP-induced Tau phosphorylation was completely reversed by the preincubation of slices in the presence of RO, suggesting that the ability of NVP to enhance phosphorylation of the Ser199 epitope is possibly dependent on alterations of NR2B-containing NMDA receptors. Indeed, we decided to test this scenario by measuring the surface expression of both AMPA and NMDA receptor subunits on biotinylated membranes [27]. As shown in Figure 5(a), the surface level of GluR1 subunits of AMPA receptors was not significantly modified in slices treated with NVP. In contrast, a significant effect on NR1 subunits of NMDA receptors was observed in hippocampal slices 2 hours after NVP exposure. The NR2A antagonist was found to increase NR1 subunit levels by more than 60% in biotinylated membranes prepared from hippocampal slices (Figure 5(b)), while similar results were obtained with NR2B subunit levels (Figure 5(c)).

Bottom Line: In the present study, we used selective NMDA receptor antagonists to investigate the involvement of NR2A and NR2B subunits in the modulatory effect of basal NMDA receptor activity on the phosphorylation of Tau proteins.This effect seemed to be Ser199 specific as there was no increase in phosphorylation at Ser262 and Ser409 residues located in the microtubule-binding and C-terminal domains of Tau proteins, respectively.From a mechanistic perspective, our study revealed that blockade of NR2A-containing receptors influences Tau phosphorylation probably by increasing calcium influx into neurons, which seems to rely on accumulation of new NR1/NR2B receptors in neuronal membranes and could involve the cyclin-dependent kinase 5 pathway.

View Article: PubMed Central - PubMed

Affiliation: Département de chimie-biologie, Université du Québec à Trois-Rivières, Trois-Rivières, QC, Canada.

ABSTRACT
Physiological activation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors has been proposed to play a key role in both neuronal cell function and dysfunction. In the present study, we used selective NMDA receptor antagonists to investigate the involvement of NR2A and NR2B subunits in the modulatory effect of basal NMDA receptor activity on the phosphorylation of Tau proteins. We observed, in acute hippocampal slice preparations, that blockade of NR2A-containing NMDA receptors by the NR2A antagonist NVP-AAM077 provoked the hyperphosphorylation of a residue located in the proline-rich domain of Tau (i.e., Ser199). This effect seemed to be Ser199 specific as there was no increase in phosphorylation at Ser262 and Ser409 residues located in the microtubule-binding and C-terminal domains of Tau proteins, respectively. From a mechanistic perspective, our study revealed that blockade of NR2A-containing receptors influences Tau phosphorylation probably by increasing calcium influx into neurons, which seems to rely on accumulation of new NR1/NR2B receptors in neuronal membranes and could involve the cyclin-dependent kinase 5 pathway.

Show MeSH
Related in: MedlinePlus