Autophagy regulates neuronal excitability by controlling cAMP/protein kinase A signaling at the synapse.

Autophagy provides nutrients during starvation and eliminates detrimental cellular components. However, accumulating evidence indicates that autophagy is not merely a housekeeping process. Here, by combining mouse models of neuron-specific ATG5 deficiency in either excitatory or inhibitory neurons with quantitative proteomics, high-content microscopy, and live-imaging approaches, we show that autophagy protein ATG5 functions in neurons to regulate cAMP-dependent protein kinase A (PKA)-mediated phosphorylation of a synapse-confined proteome. This function of ATG5 is independent of bulk turnover of synaptic proteins and requires the targeting of PKA inhibitory R1 subunits to autophagosomes. Neuronal loss of ATG5 causes synaptic accumulation of PKA-R1, which sequesters the PKA catalytic subunit and diminishes cAMP/PKA-dependent phosphorylation of postsynaptic cytoskeletal proteins that mediate AMPAR trafficking. Furthermore, ATG5 deletion in glutamatergic neurons augments AMPAR-dependent excitatory neurotransmission and causes the appearance of spontaneous recurrent seizures in mice. Our findings identify a novel role of autophagy in regulating PKA signaling at glutamatergic synapses and suggest the PKA as a target for restoration of synaptic function in neurodegenerative conditions with autophagy dysfunction.

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

Hyperalgesic priming is characterized by enhanced nociceptor sensitization by pronociceptive mediators, prototypically PGE2 . Priming has gained interest as a mechanism underlying the transition to chronic pain. Which stimuli induce priming and what cellular mechanisms are employed remains incompletely understood. In adult male rats, we present the cytokine Oncostatin M (OSM), a member of the IL-6 family, as an inducer of priming by a novel mechanism. We used a high content microscopy based approach to quantify the activation of endogenous PKA-II and ERK of thousands sensory neurons in culture. Incubation with OSM increased and prolonged ERK activation by agents that increase cAMP production such as PGE2 , forskolin, and cAMP analogs. These changes were specific to IB4/CaMKIIα positive neurons, required protein translation, and increased cAMP-to-ERK signaling. In both, control and OSM-treated neurons, cAMP/ERK signaling involved RapGEF2 and PKA but not Epac. Similar enhancement of cAMP-to-ERK signaling could be induced by GDNF, which acts mostly on IB4/CaMKIIα-positive neurons, but not by NGF, which acts mostly on IB4/CaMKIIα-negative neurons. In vitro, OSM pretreatment rendered baseline TTX-R currents ERK-dependent and switched forskolin-increased currents from partial to full ERK-dependence in small/medium sized neurons. In summary, priming induced by OSM uses a novel mechanism to enhance and prolong coupling of cAMP/PKA to ERK1/2 signaling without changing the overall pathway structure.

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

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)-RIIß (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-RIIß, allowed subgroup segregation earlier than could be achieved by assessing the classical markers TRPV1 and Nav1.8 (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.

Pain modulators regulate the dynamics of PKA-RII phosphorylation in subgroups of sensory neurons.

Knowledge about the molecular structure of protein kinase A (PKA) isoforms is substantial. In contrast, the dynamics of PKA isoform activity in living primary cells has not been investigated in detail. Using a high content screening microscopy approach, we identified the RIIß subunit of PKA-II to be predominantly expressed in a subgroup of sensory neurons. The RIIß-positive subgroup included most neurons expressing nociceptive markers (TRPV1, NaV1.8, CGRP, IB4) and responded to pain-eliciting capsaicin with calcium influx. Isoform-specific PKA reporters showed in sensory-neuron-derived F11 cells that the inflammatory mediator PGE2 specifically activated PKA-II but not PKA-I. Accordingly, pain-sensitizing inflammatory mediators and activators of PKA increased the phosphorylation of RII subunits (pRII) in subgroups of primary sensory neurons. Detailed analyses revealed basal pRII to be regulated by the phosphatase PP2A. Increase of pRII was followed by phosphorylation of CREB in a PKA-dependent manner. Thus, we propose RII phosphorylation to represent an isoform-specific readout for endogenous PKA-II activity in vivo, suggest RIIß as a novel nociceptive subgroup marker, and extend the current model of PKA-II activation by introducing a PP2A-dependent basal state.