okadaic-acid and Reperfusion-Injury

Adenosine signaling via A1 receptor (A1R) and A2A receptor (A2AR) has shown promise in revealing potential targets for neuroprotection in cerebral ischemia. We recently showed a novel mechanism by which A1R activation with N(6)-cyclopentyl adenosine (CPA) induced GluA1 and GluA2 AMPA receptor (AMPAR) endocytosis and adenosine-induced persistent synaptic depression (APSD) in rat hippocampus. This study further investigates the mechanism of A1R-mediated AMPAR internalization and hippocampal slice neuronal damage through activation of protein phosphatase 1 (PP1), 2A (PP2A), and 2B (PP2B) using electrophysiological, biochemical and imaging techniques. Following prolonged A1R activation, GluA2 internalization was selectively blocked by PP2A inhibitors (okadaic acid and fostriecin), whereas inhibitors of PP2A, PP1 (tautomycetin), and PP2B (FK506) all prevented GluA1 internalization. Additionally, GluA1 phosphorylation at Ser831 and Ser845 was reduced after prolonged A1R activation in hippocampal slices. PP2A inhibitors nullified A1R-mediated downregulation of pSer845-GluA1, while PP1 and PP2B inhibitors prevented pSer831-GluA1 downregulation. Each protein phosphatase inhibitor also blunted CPA-induced synaptic depression and APSD. We then tested whether A1R-mediated changes in AMPAR trafficking and APSD contribute to hypoxia-induced neuronal injury. Hypoxia (20 min) induced A1R-mediated internalization of both AMPAR subunits, and subsequent normoxic reperfusion (45 min) increased GluA1 but persistently reduced GluA2 surface expression. Neuronal damage after hypoxia-reperfusion injury was significantly blunted by pre-incubation with the above protein phosphatase inhibitors. Together, these data suggest that A1R-mediated protein phosphatase activation causes persistent synaptic depression by downregulating GluA2-containing AMPARs; this previously undefined role of A1R stimulation in hippocampal neuronal damage represents a novel therapeutic target in cerebral ischemic damage.

Topics: Animals; Excitatory Postsynaptic Potentials; Furans; Hippocampus; Lipids; Male; Neurons; Okadaic Acid; Phosphoprotein Phosphatases; Polyenes; Protein Transport; Pyrones; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A1; Receptors, AMPA; Reperfusion Injury; Synaptic Transmission; Tacrolimus

Ischemia/reperfusion injury (IRI) induces an innate immune response, leading to an inflammatory reaction and tissue damage that have been attributed to engagement of the Toll-like receptor (TLR) 2 and 4. However, the respective roles of TLR2 and/or TLR4 in mediating downstream activation of mitogen-activated protein kinase (MAPK) pathways during IRI have not been fully elucidated. Here we show that extracellular signal-regulated kinase (ERK)1/2 is activated in both intact kidneys and cultured renal tubule epithelial cells (RTECs) from wildtype and Tlr4 knockout mice, but not those from Tlr2 knockout mice subjected to transient ischemia. Geldanamycin (GA), an inhibitor of heat shock protein 90 and reticulum endoplasmic-resident gp96, and gp96 mRNA silencing (siRNA), did not affect ERK1/2 activation in either post-hypoxic wild-type or Tlr4-deficient RTECs, but did restore its activation in post-hypoxic Tlr2-deficient RTECs. Immunoprecipitation studies revealed that gp96 co-immunoprecipitates with the serine-threonine protein phosphatase 5 (PP5), identified as a negative modulator of the mitogen extracellular kinase (MEK)-ERK pathway, in unstressed wild-type and post-hypoxic Tlr2-deficient RTECs. In contrast, PP5 co-immunoprecipitation with gp96 was strikingly reduced in post-hypoxic wild-type RTECs, suggesting that the inactivation of PP5 resulting from the dissociation of PP5 from gp96 allows the activation of ERK1/2 to occur. Inhibition of PP5 by okadaic acid, and Pp5 siRNA also restored TLR2-mediated phosphorylation of ERK1/2, and apoptosis signal-regulating kinase 1 (ASK1)/c-Jun N-terminal kinase (JNK)-mediated apoptosis in post-hypoxic Tlr2-deficient RTECs. These findings indicate that gp96 interacts with PP5 and controls TLR2-mediated induction of ERK1/2 in post-hypoxic renal tubule cells.

Topics: Animals; Apoptosis; Benzoquinones; Endoplasmic Reticulum; Enzyme Inhibitors; Epithelial Cells; Kidney Diseases; Kidney Tubules; Lactams, Macrocyclic; MAP Kinase Kinase Kinase 5; MAP Kinase Signaling System; Membrane Glycoproteins; Mice; Mice, Knockout; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nuclear Proteins; Okadaic Acid; Phosphoprotein Phosphatases; Reperfusion Injury; Toll-Like Receptor 2; Toll-Like Receptor 4

Topics: Animals; Creatinine; Enzyme Inhibitors; Female; Kidney; Okadaic Acid; Phosphoprotein Phosphatases; Rats; Rats, Inbred Lew; Reperfusion Injury