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PICK1-mediated glutamate receptor subunit 2 (GluR2) trafficking contributes to cell death in oxygen/glucose-deprived hippocampal neurons.

Dixon RM, Mellor JR, Hanley JG - J. Biol. Chem. (2009)

Bottom Line: Using biochemical and electrophysiological approaches, we show that peptides that interfere with PICK1 PDZ domain interactions block the OGD-induced switch in subunit composition, implicating PICK1 in restricting GluR2 from synapses during OGD.Furthermore, we show that GluR2-lacking AMPARs that arise at synapses during OGD as a result of PICK1 PDZ interactions are involved in OGD-induced delayed cell death.This work demonstrates that PICK1 plays a crucial role in the response to OGD that results in altered synaptic transmission and neuronal death and has implications for our understanding of the molecular mechanisms that underlie cell death during stroke.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council (MRC) Centre for Synaptic Plasticity, Department of Anatomy, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom.

ABSTRACT
Oxygen and glucose deprivation (OGD) induces delayed cell death in hippocampal CA1 neurons via Ca(2+)/Zn(2+)-permeable, GluR2-lacking AMPA receptors (AMPARs). Following OGD, synaptic AMPAR currents in hippocampal neurons show marked inward rectification and increased sensitivity to channel blockers selective for GluR2-lacking AMPARs. This occurs via two mechanisms: a delayed down-regulation of GluR2 mRNA expression and a rapid internalization of GluR2-containing AMPARs during the OGD insult, which are replaced by GluR2-lacking receptors. The mechanisms that underlie this rapid change in subunit composition are unknown. Here, we demonstrate that this trafficking event shares features in common with events that mediate long term depression and long term potentiation and is initiated by the activation of N-methyl-d-aspartic acid receptors. Using biochemical and electrophysiological approaches, we show that peptides that interfere with PICK1 PDZ domain interactions block the OGD-induced switch in subunit composition, implicating PICK1 in restricting GluR2 from synapses during OGD. Furthermore, we show that GluR2-lacking AMPARs that arise at synapses during OGD as a result of PICK1 PDZ interactions are involved in OGD-induced delayed cell death. This work demonstrates that PICK1 plays a crucial role in the response to OGD that results in altered synaptic transmission and neuronal death and has implications for our understanding of the molecular mechanisms that underlie cell death during stroke.

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OGD induces a rapid, PICK1-dependent removal of GluR2 from the surface of hippocampal neurons. A, GluR1 surface levels are unaffected by OGD. Dissociated hippocampal neurons were exposed to OGD for the times indicated followed by biotinylation of surface proteins. The top panel shows a representative Western blot of total GluR1 present in lysates and surface (biotinylated) GluR1 after OGD. The graph shows pooled data presented as ratios of surface over total GluR1. n = 5. B, GluR2 is rapidly removed from the surface in response to OGD. Methods are same as for A, above, except that the Western blot was probed for GluR2. n = 5, *, p < 0.01, **, p < 0.001. C, OGD-induced internalization of GluR2 is blocked by peptides that disrupt PICK1 PDZ domain interactions. Dissociated hippocampal neurons were transduced with Sindbis virus to express pep2-SVKE, pep2-SVKI, or pep2-EVKI. Cultures were exposed to OGD and analyzed as in B, above. n = 5, *, p < 0.005.
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fig3: OGD induces a rapid, PICK1-dependent removal of GluR2 from the surface of hippocampal neurons. A, GluR1 surface levels are unaffected by OGD. Dissociated hippocampal neurons were exposed to OGD for the times indicated followed by biotinylation of surface proteins. The top panel shows a representative Western blot of total GluR1 present in lysates and surface (biotinylated) GluR1 after OGD. The graph shows pooled data presented as ratios of surface over total GluR1. n = 5. B, GluR2 is rapidly removed from the surface in response to OGD. Methods are same as for A, above, except that the Western blot was probed for GluR2. n = 5, *, p < 0.01, **, p < 0.001. C, OGD-induced internalization of GluR2 is blocked by peptides that disrupt PICK1 PDZ domain interactions. Dissociated hippocampal neurons were transduced with Sindbis virus to express pep2-SVKE, pep2-SVKI, or pep2-EVKI. Cultures were exposed to OGD and analyzed as in B, above. n = 5, *, p < 0.005.

Mentions: The OGD-induced Reduction in Surface-expressed GluR2 Is PICK1-dependent—To directly analyze the surface AMPAR subunit switching induced by OGD, we carried out surface biotinylations in dissociated hippocampal cultures. Neurons were subjected to OGD followed immediately by biotinylation of surface proteins. We observed a time-dependent reduction in GluR2 surface expression by OGD (Fig. 3B), which is consistent with a previous study (17). 30 min of OGD was sufficient to induce this trafficking event, with the maximal effect seen at 120 min. GluR1 surface expression was unaffected by OGD at all time points tested (Fig. 3A), as demonstrated previously (17). To investigate the role of PICK1 PDZ interactions, we used the Sindbis virus to express GluR2 C-terminal peptides in cultured neurons. The viruses are bicistronic to express enhanced green fluorescent protein in addition to the peptides. By visualizing green fluorescent protein fluorescence, we consistently observed that 80–90% neurons were infected. We then assayed surface GluR2 following a 30-min period of OGD. The control peptide pep2-SVKE had no effect on OGD-induced GluR2 trafficking. However, pep2-SVKI and pep2-EVKI, both of which disrupt PICK1 PDZ interactions, completely blocked the reduction in surface-expressed GluR2 in response to OGD (Fig. 3C). These data strongly suggest that PICK1 plays a crucial role in the OGD-induced switch in surface AMPAR subunit composition.


PICK1-mediated glutamate receptor subunit 2 (GluR2) trafficking contributes to cell death in oxygen/glucose-deprived hippocampal neurons.

Dixon RM, Mellor JR, Hanley JG - J. Biol. Chem. (2009)

OGD induces a rapid, PICK1-dependent removal of GluR2 from the surface of hippocampal neurons. A, GluR1 surface levels are unaffected by OGD. Dissociated hippocampal neurons were exposed to OGD for the times indicated followed by biotinylation of surface proteins. The top panel shows a representative Western blot of total GluR1 present in lysates and surface (biotinylated) GluR1 after OGD. The graph shows pooled data presented as ratios of surface over total GluR1. n = 5. B, GluR2 is rapidly removed from the surface in response to OGD. Methods are same as for A, above, except that the Western blot was probed for GluR2. n = 5, *, p < 0.01, **, p < 0.001. C, OGD-induced internalization of GluR2 is blocked by peptides that disrupt PICK1 PDZ domain interactions. Dissociated hippocampal neurons were transduced with Sindbis virus to express pep2-SVKE, pep2-SVKI, or pep2-EVKI. Cultures were exposed to OGD and analyzed as in B, above. n = 5, *, p < 0.005.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2682871&req=5

fig3: OGD induces a rapid, PICK1-dependent removal of GluR2 from the surface of hippocampal neurons. A, GluR1 surface levels are unaffected by OGD. Dissociated hippocampal neurons were exposed to OGD for the times indicated followed by biotinylation of surface proteins. The top panel shows a representative Western blot of total GluR1 present in lysates and surface (biotinylated) GluR1 after OGD. The graph shows pooled data presented as ratios of surface over total GluR1. n = 5. B, GluR2 is rapidly removed from the surface in response to OGD. Methods are same as for A, above, except that the Western blot was probed for GluR2. n = 5, *, p < 0.01, **, p < 0.001. C, OGD-induced internalization of GluR2 is blocked by peptides that disrupt PICK1 PDZ domain interactions. Dissociated hippocampal neurons were transduced with Sindbis virus to express pep2-SVKE, pep2-SVKI, or pep2-EVKI. Cultures were exposed to OGD and analyzed as in B, above. n = 5, *, p < 0.005.
Mentions: The OGD-induced Reduction in Surface-expressed GluR2 Is PICK1-dependent—To directly analyze the surface AMPAR subunit switching induced by OGD, we carried out surface biotinylations in dissociated hippocampal cultures. Neurons were subjected to OGD followed immediately by biotinylation of surface proteins. We observed a time-dependent reduction in GluR2 surface expression by OGD (Fig. 3B), which is consistent with a previous study (17). 30 min of OGD was sufficient to induce this trafficking event, with the maximal effect seen at 120 min. GluR1 surface expression was unaffected by OGD at all time points tested (Fig. 3A), as demonstrated previously (17). To investigate the role of PICK1 PDZ interactions, we used the Sindbis virus to express GluR2 C-terminal peptides in cultured neurons. The viruses are bicistronic to express enhanced green fluorescent protein in addition to the peptides. By visualizing green fluorescent protein fluorescence, we consistently observed that 80–90% neurons were infected. We then assayed surface GluR2 following a 30-min period of OGD. The control peptide pep2-SVKE had no effect on OGD-induced GluR2 trafficking. However, pep2-SVKI and pep2-EVKI, both of which disrupt PICK1 PDZ interactions, completely blocked the reduction in surface-expressed GluR2 in response to OGD (Fig. 3C). These data strongly suggest that PICK1 plays a crucial role in the OGD-induced switch in surface AMPAR subunit composition.

Bottom Line: Using biochemical and electrophysiological approaches, we show that peptides that interfere with PICK1 PDZ domain interactions block the OGD-induced switch in subunit composition, implicating PICK1 in restricting GluR2 from synapses during OGD.Furthermore, we show that GluR2-lacking AMPARs that arise at synapses during OGD as a result of PICK1 PDZ interactions are involved in OGD-induced delayed cell death.This work demonstrates that PICK1 plays a crucial role in the response to OGD that results in altered synaptic transmission and neuronal death and has implications for our understanding of the molecular mechanisms that underlie cell death during stroke.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council (MRC) Centre for Synaptic Plasticity, Department of Anatomy, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom.

ABSTRACT
Oxygen and glucose deprivation (OGD) induces delayed cell death in hippocampal CA1 neurons via Ca(2+)/Zn(2+)-permeable, GluR2-lacking AMPA receptors (AMPARs). Following OGD, synaptic AMPAR currents in hippocampal neurons show marked inward rectification and increased sensitivity to channel blockers selective for GluR2-lacking AMPARs. This occurs via two mechanisms: a delayed down-regulation of GluR2 mRNA expression and a rapid internalization of GluR2-containing AMPARs during the OGD insult, which are replaced by GluR2-lacking receptors. The mechanisms that underlie this rapid change in subunit composition are unknown. Here, we demonstrate that this trafficking event shares features in common with events that mediate long term depression and long term potentiation and is initiated by the activation of N-methyl-d-aspartic acid receptors. Using biochemical and electrophysiological approaches, we show that peptides that interfere with PICK1 PDZ domain interactions block the OGD-induced switch in subunit composition, implicating PICK1 in restricting GluR2 from synapses during OGD. Furthermore, we show that GluR2-lacking AMPARs that arise at synapses during OGD as a result of PICK1 PDZ interactions are involved in OGD-induced delayed cell death. This work demonstrates that PICK1 plays a crucial role in the response to OGD that results in altered synaptic transmission and neuronal death and has implications for our understanding of the molecular mechanisms that underlie cell death during stroke.

Show MeSH
Related in: MedlinePlus