Abstract
Global cerebral ischemia results in oxygen and glucose deprivation (OGD) and consequent delayed cell death of vulnerable neurons, with hippocampal CA1 neurons more vulnerable than cortical neurons. Most AMPA receptors (AMPARs) are heteromeric complexes of subunits GluA1/GluA2 or GluA2/GluA3, and the presence of GluA2 renders AMPARs Ca2+-impermeable. In hippocampal CA1 neurons, OGD causes the synaptic expression of GluA2-lacking Ca2+-permeable AMPARs, contributing to toxic Ca2+ influx. The loss of synaptic GluA2 is caused by rapid trafficking of GluA2-containing AMPARs from the cell surface, followed by a delayed reduction in GluA2 mRNA expression. We show here that OGD causes endocytosis, lysosomal targeting and consequent degradation of GluA2- and GluA3-containing AMPARs, and that PICK1 is required for both OGD-induced GluA2 endocytosis and lysosomal sorting. Our results further suggest that GluA1-containing AMPARs resist OGD-induced endocytosis. OGD does not cause GluA2 endocytosis in cortical neurons, and we show that PICK1 binding to the endocytic adaptor AP2 is enhanced by OGD in hippocampal, but not cortical neurons. We propose that endocytosis of GluA2/3, caused by a hippocampal-specific increase in PICK1-AP2 interactions, followed by PICK1-dependent lysosomal targeting, are critical events in determining changes in AMPAR subunit composition in the response to ischaemia.
Highlights
AMPA receptors (AMPARs) mediate the majority of fast synaptic excitation in the brain, and the precise regulation of AMPA receptor (AMPAR) trafficking is crucial to excitatory neurotransmission, synaptic plasticity and the consequent formation and modification of appropriate neural circuits during learning and memory[1,2,3]
To investigate the fate of internalized GluA2-containing AMPARs after oxygen and glucose deprivation (OGD) in hippocampal neurons, we initially demonstrated a significant OGD-induced increase in the endocytosis of endogenous GluA2-containing AMPARs using an antibody-feeding assay (Fig. 1a,b) quantifying the proportion of endogenous GluA2 internalized from the cell surface
Our results indicate that OGD causes a selective PICK1-dependent endocytosis of endogenous AMPAR subunits GluA2 and GluA3 in hippocampal neurons, but not in cortical neurons, which can at least partly be explained by a cell-type specific OGD-induced increase in PICK1 binding to the endocytic adaptor complex AP2
Summary
AMPARs mediate the majority of fast synaptic excitation in the brain, and the precise regulation of AMPA receptor (AMPAR) trafficking is crucial to excitatory neurotransmission, synaptic plasticity and the consequent formation and modification of appropriate neural circuits during learning and memory[1,2,3]. The majority of synapses, especially on pyramidal neurons, do not express GluA2-lacking AMPARs under resting conditions, and precise regulation of their synaptic expression is important for Ca2+ signalling events, for example during Long-Term Potentiation (LTP) expression[8] Dysregulation of these processes can lead to a prolonged synaptic incorporation of CP-AMPARs, resulting in excessive Ca2+ influx, which causes synaptic dysfunction and cell death (excitotoxicity) in a number of diseases including brain ischemia, traumatic brain injury and chronic disorders such as Huntington’s disease[9]. Lysosomal sorting and degradation were blocked by acute application of a cell-permeant peptide that disrupts PICK1-GluA2 interactions We observed these trafficking events in hippocampal neurons, but not cortical neurons, which can be explained by an OGD-induced increase in PICK1-AP2 binding to drive AMPAR endocytosis in hippocampal neurons
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