Abstract
Chronic adenosine A1R stimulation in hypoxia leads to persistent hippocampal synaptic depression, while unopposed adenosine A2AR receptor stimulation during hypoxia/reperfusion triggers adenosine-induced post-hypoxia synaptic potentiation (APSP) and increased neuronal death. Still, the mechanisms responsible for this adenosine-mediated neuronal damage following hypoxia need to be fully elucidated. We tested the hypothesis that A1R and A2AR regulation by protein kinase casein kinase 2 (CK2) and clathrin-dependent endocytosis of AMPARs both contribute to APSPs and neuronal damage. The APSPs following a 20-min hypoxia recorded from CA1 layer of rat hippocampal slices were abolished by A1R and A2AR antagonists and by broad-spectrum AMPAR antagonists. The inhibitor of GluA2 clathrin-mediated endocytosis Tat-GluA2-3Y peptide and the dynamin-dependent endocytosis inhibitor dynasore both significantly inhibited APSPs. The CK2 antagonist DRB also inhibited APSPs and, like hypoxic treatment, caused opposite regulation of A1R and A2AR surface expression. APSPs were abolished when calcium-permeable AMPAR (CP-AMPAR) antagonist (IEM or philanthotoxin) or non-competitive AMPAR antagonist perampanel was applied 5 min after hypoxia. In contrast, perampanel, but not CP-AMPAR antagonists, abolished APSPs when applied during hypoxia/reperfusion. To test for neuronal viability after hypoxia, propidium iodide staining revealed significant neuroprotection of hippocampal CA1 pyramidal neurons when pretreated with Tat-GluA2-3Y peptide, CK2 inhibitors, dynamin inhibitor, CP-AMPAR antagonists (applied 5 min after hypoxia), and perampanel (either at 5 min hypoxia onset or during APSP). These results suggest that the A1R-CK2-A2AR signaling pathway in hypoxia/reperfusion injury model mediates increased hippocampal synaptic transmission and neuronal damage via calcium-permeable AMPARs that can be targeted by perampanel for neuroprotective stroke therapy.
Highlights
Ischemic stroke occurs when tissue perfusion is reduced to part of the CNS, which results in hypoxic conditions and can result in permanent damage to brain parenchyma [1]
Treatment of hippocampal slices with DPCPX significantly attenuated hypoxia-induced synaptic depression and Field EPSPs (fEPSPs) showed comparable levels to baseline before inducing hypoxia; synaptic transmission was ≈ 80% attenuated during hypoxia with slices treated with either control (DMSO) or the A2A receptor antagonist SCH442416
The biphasic response of hypoxia/reperfusion consists of two phases: it starts with A1 receptor (A1R)-dependent synaptic depression during hypoxia followed by A2A receptor (A2AR)-dependent potentiation of fEPSP during normoxic reperfusion, that we have termed adenosine-induced post-hypoxia synaptic potentiation (APSP)
Summary
Ischemic stroke occurs when tissue perfusion is reduced to part of the CNS, which results in hypoxic conditions and can result in permanent damage to brain parenchyma [1]. A significant reduction in synaptic transmission occurs [13,14,15], along with decreased cell surface expression of GluA1 and GluA2 AMPAR subunits after a 20-min hypoxic insult [12], which are both thought to be neuroprotective mechanisms during neuronal insult. If normoxic conditions were reintroduced following hypoxia, we found increased surface expression of GluA1-containing AMPARs, whereas GluA2containing AMPARs remained depressed [12], which is important because GluA2-lacking AMPARs are permeable to Ca2+ and can lead to excitotoxicity [17]. The cellular basis for this enhanced synaptic potentiation and neuronal damage remains to be fully elucidated
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