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.

Oncostatin M induces hyperalgesic priming and amplifies signaling of cAMP to ERK by RapGEF2 and PKA

“…We established a High Content Screening (HCS) microscopy approach as a way to combine single cell analysis with cellular population analysis. Classical immunocytochemical fluorescence (ICF) labeling with phospho‐specific antibodies allows detection of changes of signaling events such as phosphorylation of endogenous ERK (pERK) and PKA‐RII (PKA‐pRII), on a single cell basis, for every cell in culture (Andres, Meyer, Dina, Levine, & Hucho, 2010; Isensee, Diskar, et al, 2014; Isensee & Hucho, 2017; Isensee et al, 2018; Isensee, Schild, Schwede, & Hucho, 2017; Isensee, Wenzel, et al, 2014; Loos, Moeller, Frohlich, Hucho, & Hasenauer, 2018). HCS microscopy allows investigation of large numbers of stimuli and analysis of more than 1 million neurons per study.…”

Scorpion toxin MeuNaTxα‐1 sensitizes primary nociceptors by selective modulation of voltage‐gated sodium channels

“…Therefore, overnight cultures of dissociated DRG were exposed to reconstituted venoms. We chose stimulations lasting for 5 or 30 min, which are time points matching well with maxima of signaling kinetics of PKA and ERK1/2, respectively, in response to common sensitizing compounds like serotonin and forskolin [25]. The cells were fixed and immunofluorescently labeled for the pan‐neuronal marker ubiquitin C‐terminal hydrolase L1 (UCHL1) for identification of neurons, phosphorylated type II regulatory subunit of PKA (pRII), phosphorylated ERK1/2 (pERK1/2), and with the nuclear stain 4′,6‐diamidino‐2‐phenylindole (DAPI; Fig.…”

“…1C). The adenylyl cyclase activator forskolin (10 µ m ) was used as a positive control as it activates PKA‐II with an EC 50 value of 1.7 µ m in the pRII assay resulting in subsequent ERK‐1/2 activity in DRG neurons [23,25]. We applied all venoms at a concentration of 100 µg·mL −1 .…”

“…Over the last years, an assay based on a high content screening (HCS) microscopy approach has been developed allowing the analysis of primary sensory neurons from dissociated dorsal root ganglia (DRG) [23–27]. Fluorescence intensities of labeled antibodies binding to different targets are quantified in tens of thousands of cells on a single‐cell level.…”

“…In addition, the two most commonly used pain therapeutic approaches, NSAIDs and opioids, share their inhibitory function on cAMP production [33,34]. Therefore, monitoring the activity of both pathway components allows detection of a very broad range of upstream activities potentially modulated by venoms or toxins [23,25,26]. Accordingly, we set out to analyze a collection of venoms for their potential to activate signal transduction in primary sensory neurons of rats using HCS microscopy.…”