Crosstalk from cAMP to ERK1/2 emerges during postnatal maturation of nociceptive neurons and is maintained during aging

Abstract

Maturation of nociceptive neurons depends on changes in transcription factors, ion channels and neuropeptides. Mature nociceptors initiate pain in part by drastically reducing the activation threshold via intracellular sensitization signaling. Whether sensitization signaling also changes during development and aging remains so far unknown. Using a novel automated microscopy approach, we quantified changes in intracellular signaling protein expression and in their signaling dynamics, as well as changes in intracellular signaling cascade wiring, in sensory neurons from newborn to senescent (24 months of age) rats. We found that nociceptive subgroups defined by the signaling components protein kinase A (PKA)-RIIβ (also known as PRKAR2B) and CaMKIIα (also known as CAMK2A) developed at around postnatal day 10, the time of nociceptor maturation. The integrative nociceptor marker, PKA-RIIβ, allowed subgroup segregation earlier than could be achieved by assessing the classical markers TRPV1 and Na v 1.8 (also known as SCN10A). Signaling kinetics remained constant over lifetime despite in part strong changes in the expression levels. Strikingly, we found a mechanism important for neuronal memory -i.e. the crosstalk from cAMP and PKA to ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1, respectively) -to emerge postnatally. Thus, maturation of nociceptors is closely accompanied by altered expression, activation and connectivity of signaling pathways known to be central for pain sensitization and neuronal memory formation.

Identification of a Sacral, Visceral Sensory Transcriptome in Embryonic and Adult Mice

“…These included: Calb1, Ntn1, Snai2, and Sstr4 upregulated in sacral DRG; Htr2c, Trpm8, Bdnf, Nos1, and Chrm2 upregulated in lumbar DRG. Together, this maturational analysis further demonstrates the dynamic nature of sensory neuron phenotypes in the perinatal and postnatal periods (Rifkin et al, 2000;Hjerling-Leffler et al, 2007;Isensee et al, 2017).…”

PKA-RII subunit phosphorylation precedes activation by cAMP and regulates activity termination

“…The transient response to 5-HT, however, was substantially prolonged by IBMX, showing that the pRII decline depends on PDE activity after rapid deactivation of 5-HT 4 receptors (Fig. S1 B; Isensee et al, 2017b ).…”

“…1 I ). To confirm that H89 inhibited PKA, we analyzed the effect of H89 on PKA-dependent downstream signaling such as phosphorylation of CREB and cross talk to the MAPK cascade ( Isensee et al, 2014a , 2017b ). As expected, H89 substantially reduced CREB and ERK1/2 phosphorylation induced by Fsk or 5-HT ( Fig.…”

“…We also measured phosphorylation of ERK1/2 in the very same cells to analyze whether Rp-isomers also block PKA-dependent cross talk to the MAP cascade ( Isensee et al, 2017b ). Surprisingly, Rp-8-pCPT-cAMPS-pAB and Rp-8-Br-cAMPS-pAB with modifications at position eight of the adenine residue strongly induced ERK1/2 phosphorylation, whereas Rp-cAMPS-pAB had an inhibitory effect to concentrations up to 10 µM (Fig.…”

“…These neurons initiate the sensation of painful stimuli and make up ∼60% of sensory neurons within the dorsal root ganglion (DRG). Nociceptors specifically express high levels of PKA-RIIβ and show pRII responses to various G protein–coupled receptor (GPCR) agonists ( Isensee et al, 2014a , b , 2017a , b ). In contrast, the PKA-RIIα isoform was detected at lower levels in all cells of the DRG.…”