P.1.008 - Differential role of phosphodiesterase inhibitors on AMPA receptor trafficking and downstream cyclic nucleotide signaling

Abstract

Previous studies have shown that the cGMP-selective PDE5 inhibitor, vardenafil, and the cAMP-selective PDE4 inhibitor, rolipram, could improve synaptic plasticity when they are applied in specific time windows [1,2]. Subsequently it is proposed that the distinction of long-term potentiation (LTP) into early and late phase relies on distinguish molecular cascades activating the cGMP/protein kinase G (PKG) and cAMP/protein kinase A (PKA), respectively. In the present study we aim to shed light into the mechanism that underlies the role of the above cascades in synaptic enhancement. A major player of mediating the effects of nucleotide signaling on synaptic plasticity is the glutamatergic AMPA receptor (AMPAR). Additionally, it was shown that phosphorylation of AMPARs at the S845 site by PKA and PKG promotes their trafficking into the membrane. Therefore, we hypothesize that the differential effect of the above inhibitors could be mediated by an increase in AMPAR synthesis and trafficking into the membrane. In order to examine the above hypothesis we incubate mouse hippocampal slices either with vardenafil or rolipram in 3 different times points during a chemical LTP (cLTP) induction protocol; 10 min before, 10 min after and 60 min after cLTP for targeting the acquisition-like, the early phase and the late phase of synaptic plasticity, respectively. Subsequently, membrane and total fractions of the GluR1 subunit of the AMPAR were assessed via separation of surface biotin-labeled proteins from the cytosolic unlabeled proteins using streptavidin magnetic beads. Our findings demonstrate that incubation of both PDE inhibitors 10 min before cLTP upregulates the number of AMPAR in the membrane without affecting the total number of the receptors. Considering that this upregulation is accompanied with increased AMPAR phosphorylation, a pre-requisite of its insertion in the membrane, we show that perfusion with both inhibitors before cLTP promotes the trafficking of an already existing pool of AMPAR without affecting the total number of the receptors. Interestingly, incubation with each of the inhibitors after induction of cLTP has a distinguishing effect. Specifically, vardenafil increases AMPAR synthesis and trafficking only when it is given 10 min, but not 60 min after induction of cLTP. On the contrary, application of rolipram has an opposite outcome, since it increases AMPAR synthesis and trafficking only when it is given at the late, but not at the early phase of cLTP. In both cases, the observed upregulation in AMPAR phosphorylation is due to increased total levels, indicating that the elevated AMPAR in the membrane fraction is mainly due to production of new receptors that subsequently travel to the membrane. Concluding, our study is the first to show that the distinctive role of the above PDE inhibitors and their downstream nucleotide signaling in improving plasticity is due to underlying cascades that promote AMPAR synthesis and subsequently insertion into the membrane.