title: Prolonged adenosine A1 receptor activation in hypoxia and pial vessel disruption focal cortical ischemia facilitates clathrin-mediated AMPA receptor endocytosis and long-lasting synaptic inhibition in rat hippocampal CA3-CA1 synapses: differential regulation of GluA2 and GluA1 subunits by p38 MAPK and JNK.
abstract: Activation of presynaptic adenosine A1 receptors (A1Rs) causes substantial synaptic depression during hypoxia/cerebral ischemia, but postsynaptic actions of A1Rs are less clear. We found that A1Rs and GluA2-containing AMPA receptors (AMPARs) form stable protein complexes from hippocampal brain homogenates and cultured hippocampal neurons from Sprague Dawley rats. In contrast, adenosine A2A receptors (A2ARs) did not coprecipitate or colocalize with GluA2-containing AMPARs. Prolonged stimulation of A1Rs with the agonist N(6)-cyclopentyladenosine (CPA) caused adenosine-induced persistent synaptic depression (APSD) in hippocampal brain slices, and APSD levels were blunted by inhibiting clathrin-mediated endocytosis of GluA2 subunits with the Tat-GluA2-3Y peptide. Using biotinylation and membrane fractionation assays, prolonged CPA incubation showed significant depletion of GluA2/GluA1 surface expression from hippocampal brain slices and cultured neurons. Tat-GluA2-3Y peptide or dynamin inhibitor Dynasore prevented CPA-induced GluA2/GluA1 internalization. Confocal imaging analysis confirmed that functional A1Rs, but not A2ARs, are required for clathrin-mediated AMPAR endocytosis in hippocampal neurons. Pharmacological inhibitors or shRNA knockdown of p38 MAPK and JNK prevented A1R-mediated internalization of GluA2 but not GluA1 subunits. Tat-GluA2-3Y peptide or A1R antagonist 8-cyclopentyl-1,3-dipropylxanthine also prevented hypoxia-mediated GluA2/GluA1 internalization. Finally, in a pial vessel disruption cortical stroke model, a unilateral cortical lesion compared with sham surgery reduced hippocampal GluA2, GluA1, and A1R surface expression and also caused synaptic depression in hippocampal slices that was consistent with AMPAR downregulation and decreased probability of transmitter release. Together, these results indicate a previously unknown mechanism for A1R-induced persistent synaptic depression involving clathrin-mediated GluA2 and GluA1 internalization that leads to hippocampal neurodegeneration after hypoxia/cerebral ischemia.
authors.affiliation: Departments of Physiology and.
authors.forename: Zhicheng
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Departments of Physiology and.
authors.forename: Cherry
authors.identifier: http://orcid.org/0000-0003-0490-3461
authors.initials: C
authors.lastname: Xiong
authors.affiliation: Departments of Physiology and.
authors.forename: Cassandra
authors.initials: C
authors.lastname: Pancyr
authors.affiliation: Departments of Physiology and.
authors.forename: Jocelyn
authors.identifier: http://orcid.org/0000-0003-4197-340X
authors.initials: J
authors.lastname: Stockwell
authors.affiliation: Psychiatry, Neuroscience Research Group, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Walz
authors.affiliation: Departments of Physiology and frank.cayabyab@usask.ca.
authors.forename: Francisco S
authors.identifier: http://orcid.org/0000-0002-5058-7087
authors.initials: FS
authors.lastname: Cayabyab
mesh_terms: D058916:Adenosine A1 Receptor Antagonists / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D002966:Clathrin / Q000378:metabolism*; D004195:Disease Models, Animal; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology*; D020925:Hypoxia-Ischemia, Brain / Q000473:pathology / Q000503:physiopathology*; D066298:In Vitro Techniques; D036881:Long-Term Synaptic Depression / Q000502:physiology; D048670:MAP Kinase Kinase 4 / Q000378:metabolism*; D058906:Purinergic P1 Receptor Agonists / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D043682:Receptor, Adenosine A1 / Q000378:metabolism*; D018091:Receptors, AMPA / Q000378:metabolism*; D013569:Synapses / Q000187:drug effects / Q000502:physiology*; D013997:Time Factors; D014970:Xanthines / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D058916:Adenosine A1 Receptor Antagonists; D002966:Clathrin; D004791:Enzyme Inhibitors; D058906:Purinergic P1 Receptor Agonists; D043682:Receptor, Adenosine A1; D018091:Receptors, AMPA; D014970:Xanthines; C051360:1,3-dipropyl-8-cyclopentylxanthine; D048051:p38 Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4
keywords: AMPA receptor trafficking; adenosine A1 receptors; clathrin-mediated endocytosis; persistent synaptic depression; pial vessel disruption; stroke