PICK1-mediated Glutamate Receptor Subunit 2 (GluR2) Trafficking Contributes to Cell Death in Oxygen/Glucose-deprived Hippocampal Neurons

Oxygen and glucose deprivation (OGD) induces delayed cell death in hippocampal CA1 neurons via Ca²⁺/Zn²⁺-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.Oxygen and glucose deprivation (OGD)³ associated with transient global ischemia induces delayed cell death, particularly in hippocampal CA1 pyramidal cells (1–3), a phenomenon that involves Ca²⁺/Zn²⁺-permeable, GluR2-lacking AMPARs (4). AMPARs are heteromeric complexes of subunits GluR1–4 (5), and most AMPARs in the hippocampus contain GluR2, which renders them calcium-impermeable and results in a marked inward rectification in their current-voltage relationship (6–8). Ischemia induces a delayed down-regulation of GluR2 mRNA and protein expression (4, 9–11), resulting in enhanced AMPAR-mediated Ca²⁺ and Zn²⁺ influx into CA1 neurons (10, 12). In these neurons, AMPAR-mediated postsynaptic currents (EPSCs) show marked inward rectification 1–2 days following ischemia and increased sensitivity to 1-naphthyl acetyl spermine (NASPM), a channel blocker selective for GluR2-lacking AMPARs (13–16). Blockade of these channels at 9–40 h following ischemia is neuroprotective, indicating a crucial role for Ca²⁺-permeable AMPARs in ischemic cell death (16).In addition to delayed changes in AMPAR subunit composition as a result of altered mRNA expression, it was recently reported that Ca²⁺-permable, GluR2-lacking AMPARs are targeted to synaptic sites via membrane trafficking at much earlier times during OGD (17). This subunit rearrangement involves endocytosis of AMPARs containing GluR2 complexed with GluR1/3, followed by exocytosis of GluR2-lacking receptors containing GluR1/3 (17). However, the molecular mechanisms behind this trafficking event are unknown, and furthermore, it is not known whether these trafficking-mediated changes in AMPAR subunit composition contribute to delayed cell death.AMPAR trafficking is a well studied phenomenon because of its crucial involvement in long term depression (LTD) and long term potentiation (LTP), activity-dependent forms of synaptic plasticity thought to underlie learning and memory. AMPAR endocytosis, exocytosis, and more recently subunit-switching events (brought about by trafficking that involves endo/exocytosis) are central to the necessary changes in synaptic receptor complement (7, 18–20). It is possible that similar mechanisms regulate AMPAR trafficking during OGD.PICK1 is a PDZ and BAR (Bin-amphiphysin-Rus) domain-containing protein that binds, via the PDZ domain, to a number of membrane proteins including AMPAR subunits GluR2/3. This interaction is required for AMPAR internalization from the synaptic plasma membrane in response to Ca²⁺ influx via NMDAR activation in hippocampal neurons (21–23). This process is the major mechanism that underlies the reduction in synaptic strength in LTD. Furthermore, PICK1-mediated trafficking has recently emerged as a mechanism that regulates the GluR2 content of synaptic receptors, which in turn determines their Ca²⁺ permeability (7, 20). This is likely to be of profound importance in both plasticity and pathological mechanisms. Importantly, PICK1 overexpression has been shown to induce a shift in synaptic AMPAR subunit composition in hippocampal CA1 neurons, resulting in inwardly rectifying AMPAR EPSCs via reduced surface GluR2 and no change in GluR1 (24). This suggests that PICK1 may mediate the rapid switch in subunit composition occurring during OGD (17). Here, we demonstrate that the OGD-induced switch in AMPAR subunit composition is dependent on PICK1 PDZ interactions, and importantly, that this early trafficking event that occurs during OGD contributes to the signaling that results in delayed neuronal death.