Protein kinase C–dependent and independent signaling pathways regulate synaptic GluR1 and GluR4 AMPAR subunits during in vitro classical conditioning

Abstract

Protein kinase C (PKC) signal transduction pathways have been implicated in mechanisms of synaptic plasticity and learning, however, the roles of the different PKC family isoforms remain to be clarified. Previous studies showed that NMDAR-mediated trafficking of GluR4-containing AMPARs supports conditioning and that the mitogen-activated protein kinases (MAPKs) have a central role in the synaptic delivery of GluR4 subunits. Here, an in vitro model of classical conditioning was used to assess the role of PKC isoforms in mechanisms underlying this form of learning. We show that the PKC antagonists chelerythrine and bisindolylmaleimide I attenuated conditioned response (CR) acquisition and expression, as did the PKCζ pseudosubstrate peptide inhibitor ZIP. Analysis of protein expression revealed that PKCζ is activated in early stages of conditioning followed shortly afterward by increased levels of PKCα/β and activation of ERK MAPK. Data also suggest that PKCζ is upstream from and activates ERK. Finally, protein localization studies using confocal imaging indicate that inhibitors of ERK, but not PKC, suppress colocalization of GluR1 with synaptophysin while inhibitors of PKC and ERK attenuate colocalization of GluR4 with synaptophysin. Together, these data suggest that acquisition of conditioning proceeds by two stages of AMPAR trafficking. The first is PKC-independent and ERKdependent synaptic delivery of GluR1 subunits to activate silent synapses. This is followed by PKCdependent and ERK-dependent synthesis and delivery of GluR4 subunits that supports the acquisition of CRs. Therefore, there is a selective role for PKC and MAPK signaling pathways in multistep AMPAR trafficking that mediates acquisition of classical conditioning.

Molecular mechanisms controlling synaptic recruitment of GluA4 subunit-containing AMPA-receptors critical for functional maturation of CA1 glutamatergic synapses

“…The MPR has been previously implicated in GluA4 trafficking (Boehm et al, 2006) and incorporates the established interaction sites for the protein 4.1N (Coleman et al, 2003), PKCγ (Correia et al, 2003), α-actinin-1 and IQGAP1 (Nuriya et al, 2005). The MPR sequence has been reported to promote GluA4 surface expression (Carvalho et al, 1999;Coleman et al, 2003;Gomes et al, 2007;Zheng and Keifer, 2008;2014) as well as phosphorylation by PKCγ at Ser862 (Gomes et al, 2007). However, using a RKR/SSS mutant to selectively knock out 4.1N and PKCg interactions, we found that these proteins alone are not sufficient for PKA-dependent synaptic delivery of GluA4 at immature CA3-CA1 synapses.…”

Pain after Discontinuation of Morphine Treatment Is Associated with Synaptic Increase of GluA4-Containing AMPAR in the Dorsal Horn of the Spinal Cord

“…A recent study using PKA-selective small interference RNA confirms that PKA activity in the spinal cord is essential for the development of morphine-induced hypersensitivity (Tumati et al, 2011). GluA4 can also be phosphorylated at Ser842 by PKC-g (Gomes et al, 2007;Zheng and Keifer, 2008). Deletion of the gene encoding PKC-g abolishes opioid-induced hypersensitivity (Célérier et al, 2004), whereas repeated administration of m-opioid agonists increases PKC-g activity (Chakrabarti et al, 2005;Narita et al, 2001).…”

Oligomeric Amyloid-β Inhibits the Proteolytic Conversion of Brain-derived Neurotrophic Factor (BDNF), AMPA Receptor Trafficking, and Classical Conditioning

“…These data indicate that oligomeric A␤ treatment interferes with the expression of BDNF during in vitro conditioning. (24,25), we showed that PKA, CaMKII and CaMKIV, CREB, ERK, and the isoform of PKC were activated during conditioning. To determine whether any of these signaling pathways are affected by oligomeric A␤ treatment during conditioning, Western blot analysis was performed.…”