Pougnet JT et al. (2016), P2X-mediated AMPA receptor internalization and ...

XB-ART-52450

Sci Rep 2016 Sep 14;6:31836. doi: 10.1038/srep31836.

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P2X-mediated AMPA receptor internalization and synaptic depression is controlled by two CaMKII phosphorylation sites on GluA1 in hippocampal neurons.

Pougnet JT , Compans B , Martinez A , Choquet D , Hosy E , Boué-Grabot E .

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Plasticity at excitatory synapses can be induced either by synaptic release of glutamate or the release of gliotransmitters such as ATP. Recently, we showed that postsynaptic P2X2 receptors activated by ATP released from astrocytes downregulate synaptic AMPAR, providing a novel mechanism by which glial cells modulate synaptic activity. ATP- and lNMDA-induced depression in the CA1 region of the hippocampus are additive, suggesting distinct molecular pathways. AMPARs are homo-or hetero-tetramers composed of GluA1-A4. Here, we first show that P2X2-mediated AMPAR inhibition is dependent on the subunit composition of AMPAR. GluA3 homomers are insensitive and their presence in heteromers alters P2X-mediated inhibition. Using a mutational approach, we demonstrate that the two CaMKII phosphorylation sites S567 and S831 located in the cytoplasmic Loop1 and C-terminal tail of GluA1 subunits, respectively, are critical for P2X2-mediated AMPAR inhibition recorded from co-expressing Xenopus oocytes and removal of surface AMPAR at synapses of hippocampal neurons imaged by the super-resolution dSTORM technique. Finally, using phosphorylation site-specific antibodies, we show that P2X-induced depression in hippocampal slices produces a dephosphorylation of the GluA1 subunit at S567, contrary to NMDAR-mediated LTD. These findings indicate that GluA1 phosphorylation of S567 and S831 is critical for P2X2-mediated AMPAR internalization and ATP-driven synaptic depression.

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Species referenced: Xenopus
Genes referenced: camk2g p2rx2

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	Figure 1. P2X2-mediated inhibition of AMPAR current is dependent upon AMPAR subunit composition.(A,B) Representative currents evoked by application of glutamate (Glu 1 mM for 5 s) in the presence of cyclothiazide (CTZ, 100 μM, 10 s of preincubation) before and 2 min after an ATP-induced current (100 μM) in oocytes co-expressing P2X2 and either GluA1 (A) or GluA3 subunits. (B) Summary of amplitude averages of AMPAR and P2X2 currents. (C) Superimposed AMPAR currents evoked in the same conditions as in A,B before (gray traces, unfilled areas) and 2 min after an ATP-induced current (black traces and shaded areas) for oocytes expressing P2X2R and indicated homomeric or heteromeric AMPARs. (D) Bar graphs summarizing the extent of inhibition (expressed as %) of homomeric or heteromeric AMPAR after activation of P2X2R. Statistical differences compared to GluA1 or GluA1A2 are indicated. P < 0.01; *P < 0.001; ns, P > 0.05; Error bars represent s.e.m.; Numbers of cells are indicated in parentheses. nf, non-functional.
	Figure 2. The carboxy tail of the GluA1 and Ser831 residue is necessary but not sufficient for P2X-mediated AMPAR depression.(A) Schematic of AMPAR subunit topology and sequence alignment of the intracellular carboxy-terminal tails (CT) of GluA1-A3 subunits. The three main phosphorylation sites on GluA1 known to contribute to synaptic plasticity are indicated by dots. (B,C) Chimeric GluA1 receptors with the intracellular CT of either GluA2 (B) or GluA3 (C) subunits were designed to determine the region involved in the inhibitory effect of P2X2 activation. Representative currents evoked by applications of glutamate (Glu 1 mM, 5 s) in the presence of cyclothiazide (CTZ, 100 μM, 10 s of preincubation) before and 2 min after an ATP-induced current (100 μM) in oocytes co-expressing P2X2 and chimeric GluA1CTA1 or GluA1CTA3 receptors. The mean amplitudes of currents are also indicated. (D) Superimposed glutamate-evoked currents before and after ATP-induced P2X2R current recorded in the same conditions as in (B) for point or double GluA1 mutants. Ser818, Ser831 and Ser845 were mutated into alanine (A) or phosphomimetic aspartate residues (D). Maximal amplitude after ATP-induce currents are indicated by black arrows. (E) Summary bar graph representing the percentage of P2X2-mediated AMPAR current inhibition for wild-type, chimeric and mutated GluA1 receptors. Statistical differences compared to GluA1 are indicated. p < 0.01; *p < 0.001 number of cells is indicated between brackets.
	Figure 3. S567 and S831 residues of GluA1 are involved in the P2X2-mediated AMPAR current inhibition.(A) Schematic representation of chimeric or mutant GluA1 receptors of the first intracellular loop and CT of GluA1. (B) Surimposed glutamate-evoked currents before and after ATP-induced P2X2R current recorded in the same conditions described in Fig. 2 for oocytes co-expressing P2X2R and the corresponding modified GluA1 subunits. Maximal amplitudes of AMPAR currents after ATP (shaded areas) are indicated by black arrows. (C) Bar graphs showing the percentage of P2X2-mediated AMPAR current inhibition for wild-type GluA1 and chimeric or mutated GluA1 receptors. Statistical differences compared to GluA1 are indicated. p < 0.05; **p < 0.001; Numbers of cells are indicated in parentheses.
	Figure 4. Chimeric GluA3 subunits with intracellular loop1 and CT of GluA1 confers P2X2-mediated inhibition on GluA3 receptors.(A) Schematic representation of chimeric GluA3 subunits bearing the first intracellular loop and/or the CT of GluA1. (B) Surimposed glutamate-evoked currents before and after ATP-induced P2X2R currents recorded from oocytes co-expressing P2X2R and the corresponding modified GluA3 subunits. Maximal amplitude of AMPAR currents after ATP (shaded areas) are indicated by black arrows. (C) Bar graph showing the percentage of P2X2-mediated AMPAR current inhibition for wild-type GluA3 and chimeric GluA3 receptors. Statistical differences compared to GluA3 are indicated. p < 0.05; **p < 0.001; Numbers of cells are indicated in parentheses.
	Figure 5. Decrease in number of dendritic and synaptic SEP-tagged GluA1 receptors triggered by activation of native P2XR in transfected hippocampal neurons is mediated by the S831 or S567 GluA1 residues.(A) Epifluorescence (upper panels) and super-resolution images (bottom panels) reconstructed from direct Stochastic Optical Reconstruction Microscopy (dSTORM) of wild-type (WT) SEP-tagged GluA1 in transfected hippocampal neurons labeled with surface anti-GFP antibodies before (control, left panel) and after ATP treatments (right panel). (B) Representative dSTORM images of spines from neurons expressing wild-type SEP-tagged GluA1 and mutant GluA1 S831A, S567L and double mutant in control conditions or 1a0 min after application of ATP (100 μM, 1 min) in presence of CGS15943 (3 μM) and TTX (0.5 μM). Scale bars, 1 μm. (C) Average density values of wild-type and mutant SEP-GluA1-containing AMPAR in synapses and dendrites in control condition (unshaded bars) and after P2XR activation (shaded bars). *P < 0.05; ns, P > 0.05; Error bars: s.e.m.
	Figure 6. Dephosphorylation of the S567 site of the AMPAR GluA1 subunit during P2XR-mediated synaptic depression in hippocampal slices.(A) Experimental design of ATP or NMDA- induced synaptic depression in hippocampal slices. (B) Crude membrane fractions from control hippocampal slices and ATP-induced synaptic depression or NMDAR-induced LTD slices taken at indicated times (5′ and/or 30′) after the application of ATP (300 μM for 10 min) or NMDA (20 μM for 3 min) respectively, separated under SDS-PAGE and blotted using antibodies against phospho-S831, phospho-S845 and actin. (C) Crude membrane fractions from hippocampal slices treated as described in A, immunoprecipated using anti-GluA1 before separation under SDS-PAGE and blotted using antibodies against anti-phospho S567 and anti-GluA1. Cropped blots are displayed (see Supplementary Fig. 1.) (D) Quantification of the relative amounts of phosphorylation of GluA1 on S831, S845 and S567 at indicated time points after ATP or NMDA treatments. Bars represent the ratio of the signals for the anti-phospho site specific antibodies and the actin or the anti-GluA1 normalized to control at each time point. The number of independent experiments is indicated in parentheses. *P < 0.05.

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	Figure 1. P2X2-mediated inhibition of AMPAR current is dependent upon AMPAR subunit composition.(A,B) Representative currents evoked by application of glutamate (Glu 1 mM for 5 s) in the presence of cyclothiazide (CTZ, 100 μM, 10 s of preincubation) before and 2 min after an ATP-induced current (100 μM) in oocytes co-expressing P2X2 and either GluA1 (A) or GluA3 subunits. (B) Summary of amplitude averages of AMPAR and P2X2 currents. (C) Superimposed AMPAR currents evoked in the same conditions as in A,B before (gray traces, unfilled areas) and 2 min after an ATP-induced current (black traces and shaded areas) for oocytes expressing P2X2R and indicated homomeric or heteromeric AMPARs. (D) Bar graphs summarizing the extent of inhibition (expressed as %) of homomeric or heteromeric AMPAR after activation of P2X2R. Statistical differences compared to GluA1 or GluA1A2 are indicated. P < 0.01; *P < 0.001; ns, P > 0.05; Error bars represent s.e.m.; Numbers of cells are indicated in parentheses. nf, non-functional.
	Figure 2. The carboxy tail of the GluA1 and Ser831 residue is necessary but not sufficient for P2X-mediated AMPAR depression.(A) Schematic of AMPAR subunit topology and sequence alignment of the intracellular carboxy-terminal tails (CT) of GluA1-A3 subunits. The three main phosphorylation sites on GluA1 known to contribute to synaptic plasticity are indicated by dots. (B,C) Chimeric GluA1 receptors with the intracellular CT of either GluA2 (B) or GluA3 (C) subunits were designed to determine the region involved in the inhibitory effect of P2X2 activation. Representative currents evoked by applications of glutamate (Glu 1 mM, 5 s) in the presence of cyclothiazide (CTZ, 100 μM, 10 s of preincubation) before and 2 min after an ATP-induced current (100 μM) in oocytes co-expressing P2X2 and chimeric GluA1CTA1 or GluA1CTA3 receptors. The mean amplitudes of currents are also indicated. (D) Superimposed glutamate-evoked currents before and after ATP-induced P2X2R current recorded in the same conditions as in (B) for point or double GluA1 mutants. Ser818, Ser831 and Ser845 were mutated into alanine (A) or phosphomimetic aspartate residues (D). Maximal amplitude after ATP-induce currents are indicated by black arrows. (E) Summary bar graph representing the percentage of P2X2-mediated AMPAR current inhibition for wild-type, chimeric and mutated GluA1 receptors. Statistical differences compared to GluA1 are indicated. p < 0.01; *p < 0.001 number of cells is indicated between brackets.
	Figure 3. S567 and S831 residues of GluA1 are involved in the P2X2-mediated AMPAR current inhibition.(A) Schematic representation of chimeric or mutant GluA1 receptors of the first intracellular loop and CT of GluA1. (B) Surimposed glutamate-evoked currents before and after ATP-induced P2X2R current recorded in the same conditions described in Fig. 2 for oocytes co-expressing P2X2R and the corresponding modified GluA1 subunits. Maximal amplitudes of AMPAR currents after ATP (shaded areas) are indicated by black arrows. (C) Bar graphs showing the percentage of P2X2-mediated AMPAR current inhibition for wild-type GluA1 and chimeric or mutated GluA1 receptors. Statistical differences compared to GluA1 are indicated. p < 0.05; **p < 0.001; Numbers of cells are indicated in parentheses.
	Figure 4. Chimeric GluA3 subunits with intracellular loop1 and CT of GluA1 confers P2X2-mediated inhibition on GluA3 receptors.(A) Schematic representation of chimeric GluA3 subunits bearing the first intracellular loop and/or the CT of GluA1. (B) Surimposed glutamate-evoked currents before and after ATP-induced P2X2R currents recorded from oocytes co-expressing P2X2R and the corresponding modified GluA3 subunits. Maximal amplitude of AMPAR currents after ATP (shaded areas) are indicated by black arrows. (C) Bar graph showing the percentage of P2X2-mediated AMPAR current inhibition for wild-type GluA3 and chimeric GluA3 receptors. Statistical differences compared to GluA3 are indicated. p < 0.05; **p < 0.001; Numbers of cells are indicated in parentheses.
	Figure 5. Decrease in number of dendritic and synaptic SEP-tagged GluA1 receptors triggered by activation of native P2XR in transfected hippocampal neurons is mediated by the S831 or S567 GluA1 residues.(A) Epifluorescence (upper panels) and super-resolution images (bottom panels) reconstructed from direct Stochastic Optical Reconstruction Microscopy (dSTORM) of wild-type (WT) SEP-tagged GluA1 in transfected hippocampal neurons labeled with surface anti-GFP antibodies before (control, left panel) and after ATP treatments (right panel). (B) Representative dSTORM images of spines from neurons expressing wild-type SEP-tagged GluA1 and mutant GluA1 S831A, S567L and double mutant in control conditions or 1a0 min after application of ATP (100 μM, 1 min) in presence of CGS15943 (3 μM) and TTX (0.5 μM). Scale bars, 1 μm. (C) Average density values of wild-type and mutant SEP-GluA1-containing AMPAR in synapses and dendrites in control condition (unshaded bars) and after P2XR activation (shaded bars). *P < 0.05; ns, P > 0.05; Error bars: s.e.m.
	Figure 6. Dephosphorylation of the S567 site of the AMPAR GluA1 subunit during P2XR-mediated synaptic depression in hippocampal slices.(A) Experimental design of ATP or NMDA- induced synaptic depression in hippocampal slices. (B) Crude membrane fractions from control hippocampal slices and ATP-induced synaptic depression or NMDAR-induced LTD slices taken at indicated times (5′ and/or 30′) after the application of ATP (300 μM for 10 min) or NMDA (20 μM for 3 min) respectively, separated under SDS-PAGE and blotted using antibodies against phospho-S831, phospho-S845 and actin. (C) Crude membrane fractions from hippocampal slices treated as described in A, immunoprecipated using anti-GluA1 before separation under SDS-PAGE and blotted using antibodies against anti-phospho S567 and anti-GluA1. Cropped blots are displayed (see Supplementary Fig. 1.) (D) Quantification of the relative amounts of phosphorylation of GluA1 on S831, S845 and S567 at indicated time points after ATP or NMDA treatments. Bars represent the ratio of the signals for the anti-phospho site specific antibodies and the actin or the anti-GluA1 normalized to control at each time point. The number of independent experiments is indicated in parentheses. *P < 0.05.