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, 2010, 340168

Blockade of NR2A-containing NMDA Receptors Induces Tau Phosphorylation in Rat Hippocampal Slices

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Blockade of NR2A-containing NMDA Receptors Induces Tau Phosphorylation in Rat Hippocampal Slices

Julie Allyson et al. Neural Plast.

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.

Figures

Figure 1
Figure 1
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.
Figure 2
Figure 2
Blockade of NR2A-containing NMDA receptors is not associated with increased Tau phosphorylation levels at Ser409 and Ser262 sites. Phosphorylation and protein levels were estimated by Western blotting on cell extracts (40 μg proteins) obtained from acute hippocampal slices treated with 50 nM NVP for 2 hours. Phosphorylated Tau levels, expressed relative to total Tau (i.e., Tau-5) levels, were measured using antibodies raised against Tau phosphorylated at Ser199, Ser262, and Ser409. The data were expressed as percentage of control values and are means ± SEM of 3 measurements per cell extract obtained from 6 different rats. Since these experiments were independently performed, we determined statistical significance using unpaired T-Test. *P < .05, ***P < .0001, NVP-treated versus control.
Figure 3
Figure 3
NVP-induced Tau phosphorylation is mediated by calcium and the Cdk5 pathway. (a) Phosphorylated Tau levels at Ser199 were estimated by Western blotting on cell extracts obtained from acute hippocampal slices treated with 50 nM NVP for 2 hours alone or in combination with 10 μM BAPTA-AM or 10 μM BAPTA. The data are expressed relative to total Tau (i.e., Tau-5) levels. (b) As in A, except for the GSK-3β inhibitor SB216367 (10 μM), the Cdk5 inhibitor roscovitine (10 μM), or the calpain inhibitor calpeptine (10 μM) were employed. The data were expressed as percentage of control values and are means ± SEM of 3 measurements per cell extract obtained from 5 different rats. Statistical analysis was performed by one-way ANOVA followed by Neuman-Keuls' post hoc test. ***P < .001, drug-treated versus control.
Figure 4
Figure 4
NR2B-containing receptors play a role in Tau phosphorylation induced by NVP. Phosphorylated Tau levels at Ser199 were estimated by Western blotting on cell extracts obtained from acute hippocampal slices treated with 50 nM NVP for 2 hours alone or in combination with 10 μM NBQX and 10 μM RO25-6981. The data, expressed relative to total Tau (i.e., Tau-5) levels, are means ± SEM of 3 measurements per cell extract obtained from 4 different rats. Statistical analysis was performed by one-way ANOVA followed by Neuman-Keuls' post hoc test. *P < .05, ***P < .001, drug-treated versus control.
Figure 5
Figure 5
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.
Figure 6
Figure 6
Working model of NVP-induced Tau phosphorylation. Blockade of NR2A-containing receptors appears to initiate the insertion of new functional NMDA receptors containing a high proportion of NR2B subunits in neuronal plasma membranes, promoting calcium accumulation. Through an unknown mechanism, Cdk5 activity would selectively enhance the phosphorylation of Ser199 residue in the proline-rich domain of Tau. In parallel, blockade of NR2A-containing receptors may reduce specific phosphatase activity which could have an impact on Tau phosphorylation at Ser199.

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