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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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PICK1-mediated switching of AMPAR subunit composition during OGD contributes to OGD-induced neuronal death. A, blockade of GluR2-lacking receptors during OGD significantly reduces neuronal death. Dissociated hippocampal neurons were exposed to 45 min of OGD in the absence or presence of the GluR2-lacking AMPAR blocker NASPM (30 μm). Coverslips were washed extensively before returning them to normal conditioned growth medium. 30 h later, neurons were stained with Hoechst and PI, and the proportion of nuclei stained with PI was counted. The left panels show representative images for vehicle and NASPM-treated cultures. The graph shows pooled data, normalized to the vehicle-treated control. n = 7, **, p < 0.01. B, blockade of PICK1 PDZ domain interactions significantly reduces neuronal death. Dissociated hippocampal neurons were transfected with plasmids encoding the peptides pep2-SVKE (control), pep2-SVKI (wild type), and pep2-EVKI (PICK1-specific). Cultures were exposed to 45 min of OGD and stained with Hoechst and propidium iodide 30 h later, and the proportion of nuclei stained with PI was counted. Data are normalized to the pep2-SVKE condition. n = 4, *, p < 0.05.
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fig4: PICK1-mediated switching of AMPAR subunit composition during OGD contributes to OGD-induced neuronal death. A, blockade of GluR2-lacking receptors during OGD significantly reduces neuronal death. Dissociated hippocampal neurons were exposed to 45 min of OGD in the absence or presence of the GluR2-lacking AMPAR blocker NASPM (30 μm). Coverslips were washed extensively before returning them to normal conditioned growth medium. 30 h later, neurons were stained with Hoechst and PI, and the proportion of nuclei stained with PI was counted. The left panels show representative images for vehicle and NASPM-treated cultures. The graph shows pooled data, normalized to the vehicle-treated control. n = 7, **, p < 0.01. B, blockade of PICK1 PDZ domain interactions significantly reduces neuronal death. Dissociated hippocampal neurons were transfected with plasmids encoding the peptides pep2-SVKE (control), pep2-SVKI (wild type), and pep2-EVKI (PICK1-specific). Cultures were exposed to 45 min of OGD and stained with Hoechst and propidium iodide 30 h later, and the proportion of nuclei stained with PI was counted. Data are normalized to the pep2-SVKE condition. n = 4, *, p < 0.05.

Mentions: Activity of GluR2-lacking AMPARs during OGD Insult Contribute to Delayed Neuronal Death—To investigate the functional relevance of PICK1-mediated subunit switching during the OGD insult, we used NASPM to specifically block GluR2-lacking AMPARs. We applied the drug before OGD and washed it out immediately following the insult so that GluR2-lacking AMPARs would be blocked specifically during OGD. Cell death was then analyzed by PI staining 30 h later. NASPM application during OGD resulted in a significant reduction in delayed cell death (Fig. 4A), indicating that channel activity of GluR2-lacking AMPARs during OGD contributes to OGD-induced cell death.


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)

PICK1-mediated switching of AMPAR subunit composition during OGD contributes to OGD-induced neuronal death. A, blockade of GluR2-lacking receptors during OGD significantly reduces neuronal death. Dissociated hippocampal neurons were exposed to 45 min of OGD in the absence or presence of the GluR2-lacking AMPAR blocker NASPM (30 μm). Coverslips were washed extensively before returning them to normal conditioned growth medium. 30 h later, neurons were stained with Hoechst and PI, and the proportion of nuclei stained with PI was counted. The left panels show representative images for vehicle and NASPM-treated cultures. The graph shows pooled data, normalized to the vehicle-treated control. n = 7, **, p < 0.01. B, blockade of PICK1 PDZ domain interactions significantly reduces neuronal death. Dissociated hippocampal neurons were transfected with plasmids encoding the peptides pep2-SVKE (control), pep2-SVKI (wild type), and pep2-EVKI (PICK1-specific). Cultures were exposed to 45 min of OGD and stained with Hoechst and propidium iodide 30 h later, and the proportion of nuclei stained with PI was counted. Data are normalized to the pep2-SVKE condition. n = 4, *, p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2682871&req=5

fig4: PICK1-mediated switching of AMPAR subunit composition during OGD contributes to OGD-induced neuronal death. A, blockade of GluR2-lacking receptors during OGD significantly reduces neuronal death. Dissociated hippocampal neurons were exposed to 45 min of OGD in the absence or presence of the GluR2-lacking AMPAR blocker NASPM (30 μm). Coverslips were washed extensively before returning them to normal conditioned growth medium. 30 h later, neurons were stained with Hoechst and PI, and the proportion of nuclei stained with PI was counted. The left panels show representative images for vehicle and NASPM-treated cultures. The graph shows pooled data, normalized to the vehicle-treated control. n = 7, **, p < 0.01. B, blockade of PICK1 PDZ domain interactions significantly reduces neuronal death. Dissociated hippocampal neurons were transfected with plasmids encoding the peptides pep2-SVKE (control), pep2-SVKI (wild type), and pep2-EVKI (PICK1-specific). Cultures were exposed to 45 min of OGD and stained with Hoechst and propidium iodide 30 h later, and the proportion of nuclei stained with PI was counted. Data are normalized to the pep2-SVKE condition. n = 4, *, p < 0.05.
Mentions: Activity of GluR2-lacking AMPARs during OGD Insult Contribute to Delayed Neuronal Death—To investigate the functional relevance of PICK1-mediated subunit switching during the OGD insult, we used NASPM to specifically block GluR2-lacking AMPARs. We applied the drug before OGD and washed it out immediately following the insult so that GluR2-lacking AMPARs would be blocked specifically during OGD. Cell death was then analyzed by PI staining 30 h later. NASPM application during OGD resulted in a significant reduction in delayed cell death (Fig. 4A), indicating that channel activity of GluR2-lacking AMPARs during OGD contributes to OGD-induced cell death.

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