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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 induces Tau phosphorylation at Ser199 site in rat hippocampal slices. Phosphorylation and protein levels were estimated by Western blotting on cell extracts (40 μg proteins) obtained from acute hippocampal slices treated with 2 NMDA receptor antagonists for periods ranging from 1 to 3 hours.  Phosphorylated Tau levels at Ser199, expressed relative to total Tau (Tau-5) levels, were measured in slices treated with 50 nM NVP and 1 μM RO. The data were expressed as percentage of control values and are means ± SEM of 3 measurements per cell extract obtained from 7 different rats. Statistical analysis using two-way ANOVA followed by the post hoc Bonferroni test revealed that there was a main effect between treatment (F(2,54) = 16.370, P < .0001), no effect between time (F(2,54) = 2.509, P = .091) and no significant interaction between treatment and time (F(4,54) = 1.337, P = .268). *P < .05, ***P < .001, drug-treated versus control.
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fig1: Blockade of NR2A-containing NMDA receptors induces Tau phosphorylation at Ser199 site in rat hippocampal slices. Phosphorylation and protein levels were estimated by Western blotting on cell extracts (40 μg proteins) obtained from acute hippocampal slices treated with 2 NMDA receptor antagonists for periods ranging from 1 to 3 hours. Phosphorylated Tau levels at Ser199, expressed relative to total Tau (Tau-5) levels, were measured in slices treated with 50 nM NVP and 1 μM RO. The data were expressed as percentage of control values and are means ± SEM of 3 measurements per cell extract obtained from 7 different rats. Statistical analysis using two-way ANOVA followed by the post hoc Bonferroni test revealed that there was a main effect between treatment (F(2,54) = 16.370, P < .0001), no effect between time (F(2,54) = 2.509, P = .091) and no significant interaction between treatment and time (F(4,54) = 1.337, P = .268). *P < .05, ***P < .001, drug-treated versus control.

Mentions: In this study, we investigated the influence of tonic NMDA receptor activity on Tau status by quantifying phosphorylation and protein levels in the hippocampus. Acute hippocampal slices from rats were treated for different time periods with NMDA receptor antagonists and then processed by Western blotting. We first examined Tau phosphorylation levels on Ser199 after preincubating hippocampal slices with NVP-AAM077 (NVP) and R025-6981 (RO), 2 compounds that preferentially block, respectively, NR2A- and NR2B-containing NMDA receptors. Our experiments were performed with 50 nM NVP and 1 μM RO, concentrations that are known to be highly selective for NR2A and NR2B, respectively [25, 26]. In initial experiments, we observed that hippocampal tissues were strongly and consistently stained with an antibody recognizing the phosphorylated Ser199 epitope of a Tau isoform estimated to 62 kDa (Figure 1, top panels). As presented in the Figure 1 histogram, we observed that this Tau isoform became progressively hyperphosphorylated at the Ser199 residue after blockade of NR2A-containing NMDA receptors with NVP (Figure 1, black bars). In fact, when normalized with Tau-5 (an antibody that recognized phosphate-independent epitopes of Tau), it became evident that overtime NVP elevated phosphorylated Tau levels at its Ser199 site, with a maximal increase observed in slices preincubated for a period of 2 hours (n = 5, P < .01). Time-course analysis showed, however, that Ser199 was not subjected to substantial change in phosphorylation after exposure to the NR2B antagonist RO (Figure 1, grey bars). It is noteworthy that treatments of rat hippocampal slices with both NVP and RO failed to produce significant changes in Tau-5 staining intensity (Figure 1, top panels), indicating that Ser199 hyperphosphorylation resulting from blockade of NR2A-containing receptors is not dependent on variations in Tau synthesis and/or degradation.


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 induces Tau phosphorylation at Ser199 site in rat hippocampal slices. Phosphorylation and protein levels were estimated by Western blotting on cell extracts (40 μg proteins) obtained from acute hippocampal slices treated with 2 NMDA receptor antagonists for periods ranging from 1 to 3 hours.  Phosphorylated Tau levels at Ser199, expressed relative to total Tau (Tau-5) levels, were measured in slices treated with 50 nM NVP and 1 μM RO. The data were expressed as percentage of control values and are means ± SEM of 3 measurements per cell extract obtained from 7 different rats. Statistical analysis using two-way ANOVA followed by the post hoc Bonferroni test revealed that there was a main effect between treatment (F(2,54) = 16.370, P < .0001), no effect between time (F(2,54) = 2.509, P = .091) and no significant interaction between treatment and time (F(4,54) = 1.337, P = .268). *P < .05, ***P < .001, drug-treated versus control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Blockade of NR2A-containing NMDA receptors induces Tau phosphorylation at Ser199 site in rat hippocampal slices. Phosphorylation and protein levels were estimated by Western blotting on cell extracts (40 μg proteins) obtained from acute hippocampal slices treated with 2 NMDA receptor antagonists for periods ranging from 1 to 3 hours. Phosphorylated Tau levels at Ser199, expressed relative to total Tau (Tau-5) levels, were measured in slices treated with 50 nM NVP and 1 μM RO. The data were expressed as percentage of control values and are means ± SEM of 3 measurements per cell extract obtained from 7 different rats. Statistical analysis using two-way ANOVA followed by the post hoc Bonferroni test revealed that there was a main effect between treatment (F(2,54) = 16.370, P < .0001), no effect between time (F(2,54) = 2.509, P = .091) and no significant interaction between treatment and time (F(4,54) = 1.337, P = .268). *P < .05, ***P < .001, drug-treated versus control.
Mentions: In this study, we investigated the influence of tonic NMDA receptor activity on Tau status by quantifying phosphorylation and protein levels in the hippocampus. Acute hippocampal slices from rats were treated for different time periods with NMDA receptor antagonists and then processed by Western blotting. We first examined Tau phosphorylation levels on Ser199 after preincubating hippocampal slices with NVP-AAM077 (NVP) and R025-6981 (RO), 2 compounds that preferentially block, respectively, NR2A- and NR2B-containing NMDA receptors. Our experiments were performed with 50 nM NVP and 1 μM RO, concentrations that are known to be highly selective for NR2A and NR2B, respectively [25, 26]. In initial experiments, we observed that hippocampal tissues were strongly and consistently stained with an antibody recognizing the phosphorylated Ser199 epitope of a Tau isoform estimated to 62 kDa (Figure 1, top panels). As presented in the Figure 1 histogram, we observed that this Tau isoform became progressively hyperphosphorylated at the Ser199 residue after blockade of NR2A-containing NMDA receptors with NVP (Figure 1, black bars). In fact, when normalized with Tau-5 (an antibody that recognized phosphate-independent epitopes of Tau), it became evident that overtime NVP elevated phosphorylated Tau levels at its Ser199 site, with a maximal increase observed in slices preincubated for a period of 2 hours (n = 5, P < .01). Time-course analysis showed, however, that Ser199 was not subjected to substantial change in phosphorylation after exposure to the NR2B antagonist RO (Figure 1, grey bars). It is noteworthy that treatments of rat hippocampal slices with both NVP and RO failed to produce significant changes in Tau-5 staining intensity (Figure 1, top panels), indicating that Ser199 hyperphosphorylation resulting from blockade of NR2A-containing receptors is not dependent on variations in Tau synthesis and/or degradation.

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