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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.

The α2δ‐1 subunit of voltage-gated calcium channels binds to gabapentin and pregabalin, mediating the analgesic action of these drugs against neuropathic pain. Extracellular matrix proteins from the thrombospondin (TSP) family have been identified as ligands of α2δ‐1 in the CNS. This interaction was found to be crucial for excitatory synaptogenesis and neuronal sensitisation which in turn can be inhibited by gabapentin, suggesting a potential role in the pathogenesis of neuropathic pain. Here, we provide information on the biochemical properties of the direct TSP/α2δ-1 interaction using an ELISA-style ligand binding assay. Our data reveal that full-length pentameric TSP-4, but neither TSP-5/COMP of the pentamer-forming subgroup B nor TSP-2 of the trimer-forming subgroup A directly interact with a soluble variant of α2δ-1 (α2δ-1S). Interestingly, this interaction is not inhibited by gabapentin on a molecular level and is not detectable on the surface of HEK293-EBNA cells over-expressing α2δ‐1 protein. These results provide biochemical evidence that supports a specific role of TSP-4 among the TSPs in mediating the binding to neuronal α2δ‐1 and suggest that gabapentin does not directly target TSP/α2δ-1 interaction to alleviate neuropathic pain.

The advent of molecularly targeted therapeutic agents has opened a new era in cancer therapy. However, many tumors rely on nondruggable cancer-driving lesions. In addition, long-lasting clinical benefits from single-agent therapies rarely occur, as most of the tumors acquire resistance over time. The identification of targeted combination regimens interfering with signaling through oncogenically rewired pathways provides a promising approach to enhance efficacy of single-agent–targeted treatments. Moreover, combination drug therapies might overcome the emergence of drug resistance. Here, we performed a focused flow cytometry–based drug synergy screen and identified a novel synergistic interaction between GLUT1-mediated glucose transport and the cell-cycle checkpoint kinases ATR and CHK1. Combined inhibition of CHK1/GLUT1 or ATR/GLUT1 robustly induced apoptosis, particularly in RAS-mutant cancer cells. Mechanistically, combined inhibition of ATR/CHK1 and GLUT1 arrested sensitive cells in S-phase and led to the accumulation of genotoxic damage, particularly in S-phase. In vivo, simultaneous inhibition of ATR and GLUT1 significantly reduced tumor volume gain in an autochthonous mouse model of KrasG12D-driven soft tissue sarcoma. Taken together, these findings pave the way for combined inhibition of GLUT1 and ATR/CHK1 as a therapeutic approach for KRAS-driven cancers. Significance: Dual targeting of the DNA damage response and glucose transport synergistically induces apoptosis in KRAS-mutant cancer, suggesting this combination treatment for clinical validation in KRAS-stratified tumor patients.

Background Knowledge about the neuroinflammatory state during months after sudden cardiac arrest is scarce. Neuroinflammation is mediated by cells that express the 18 kDa translocator protein (TSPO). We determined the time course of TSPO-expressing cells in a rat model of sudden cardiac arrest using longitudinal in vivo positron emission tomography (PET) imaging with the TSPO-specific tracer [18F]DAA1106 over a period of 6 months. Methods Five male Sprague Dawley rats were resuscitated from 6 min sudden cardiac arrest due to ventricular fibrillation, three animals served as shams. PET measurements were performed on day 5, 8, 14, 90, and 180 after intervention. Magnetic resonance imaging was performed on day 140. Imaging was preceded by Barnes Maze spatial memory testing on day 3, 13, 90, and 180. Specificity of [18F]DAA1106 binding was confirmed by Iba-1 immunohistochemistry. Results [18F]DAA1106 accumulated bilaterally in the dorsal hippocampus of all sudden cardiac arrest animals on all measured time points. Immunohistochemistry confirmed Iba-1 expressing cells in the hippocampal CA1 region. The number of Iba-1-immunoreactive objects per mm2 was significantly correlated with [18F]DAA1106 uptake. Additionally, two of the five sudden cardiac arrest rats showed bilateral TSPO-expression in the striatum that persisted until day 180. In Barnes Maze, the relative time spent in the target quadrant negatively correlates with dorsal hippocampal [18F]DAA1106 uptake on day 14 and 180. Conclusions After sudden cardiac arrest, TSPO remains expressed over the long-term. Sustainable treatment options for neuroinflammation may be considered to improve cognitive functions after sudden cardiac arrest.

Bacterial toxins are able to act on neurons to modulate signaling and function. Here, we find that nociceptive sensory neurons that mediate pain are enriched in the receptor for anthrax toxins, ANTXR2. Anthrax Edema Toxin (ET) induced cAMP and PKA signaling in Nav1.8+nociceptive neurons and modulated painin vivo. Peripherally administered ET mediated mechanical allodynia in naïve mice and duringB. anthracisinfection. Intrathecally administered ET produced analgesic effects, potently blocking pain-like behaviors in multiple mouse models of inflammatory and chronic neuropathic pain. Nociceptor-specific ablation of ANTXR2 attenuated ET-induced signaling and analgesia. Modified anthrax toxin successfully delivered exogenous protein cargo into nociceptive neurons, illustrating utility of the anthrax toxin system as a molecular platform to target pain. ET further induced signaling in human iPSC-derived sensory neurons. Our findings highlight novel interactions between a bacterial toxin and nociceptors that may be utilized for developing new pain therapeutics.SUMMARYANTXR2 expression on nociceptive neurons allows selective targeting and modulation of pain by native and engineered anthrax toxins.

Objective Osteoarthritis is a degenerative disease that affects synovial joints. Micro-injuries of articular structures initiate inflammatory processes, leading to persistent pain. Due to various risk factors, osteoarthritis is often diagnosed in multimorbid patients. This makes pain management one of the key challenges, with a consistent need for new therapeutic strategies. Hence, complementary and integrative methods such as hirudotherapy have become increasingly important, even though their mechanisms of action are not entirely understood. Methods We retrospectively analyzed the longitudinal effect of a single leech application on osteoarthritic joints in a heterogenic cohort of 24 cases with various chronic pain syndromes. We assessed articular pain intensity ratings and movability of the treated joint after one-time leeching for up to 12 months. We further investigated the effect of hirudotherapy on the systemic pain status and multimodal treatment strategies of the patients. Results There was a significant reduction in pain intensity ratings at the joint of leech application for up to 12 months after treatment. The improvements in pain intensities were independent of the form of osteoarthritis treated. In addition, we saw a considerable enhancement in local movability of the treated joint. Hirudotherapy did not seem to influence the systemic pain status as well as the previously established individualized multimodal treatment model of the patients. Conclusion Leeching as an adjuvant therapy has a great potential especially in terms of safety and long-term outcome.

Summary: Cdkn1a, which encodes p21, functions as a major route for p53-mediated cell-cycle arrest. However, the consequence of Cdkn1a gene dosage on tumor suppression has not been systematically investigated. Here, we employed BAC transgenesis to generate a Cdkn1aSUPER mouse, which harbors an additional Cdkn1a allele within its natural genomic context. We show that these mice display enhanced cell-cycle arrest and reduced apoptosis in response to genotoxic stress. Furthermore, using a chemically induced skin cancer model and an autochthonous Kras-driven lung adenocarcinoma model, we show that Cdkn1aSUPER mice display a cancer protection phenotype that is indistinguishable from that observed in Tp53SUPER animals. Moreover, we demonstrate that Tp53 and Cdkn1a cooperate in mediating cancer resistance, using a chemically induced fibrosarcoma model. Overall, our Cdkn1aSUPER allele enabled us to assess the contribution of Cdkn1a to Tp53-mediated tumor suppression. : Torgovnick et al. create a mouse model, carrying a third copy of Cdkn1a (p21), which shows enhanced cell-cycle arrest capacity and protection against DNA damage-induced apoptosis. The Cdkn1aSUPER animals display delayed epithelial regeneration and a robust cancer resistance phenotype, highlighting the importance of p21 in p53-dependent tumor suppression. Keywords: Cdkn1a, p21, p53, mouse model, cancer, tumor suppressor, cell cycle arrest, apoptosis, cancer protection

Objectives Previously, we found in cultures of primary neurons and in animals that sensitized primary neurons can be desensitized by treatment with e.g. beta-adrenergic receptor agonists. We now tested whether also in human sensitization such as UVB-radiation induced sunburn-like hyperalgesia can be reduced by intradermal injection of the beta-adrenergic receptor agonist terbutaline. Methods In our prospective randomized study, 17 participants received an individual UVB dose to cause a defined local sunburn-like erythema at four locations, two on each forearm. Twenty-four hours later, the sensitized four areas were injected intradermally with terbutaline pH 4.3, terbutaline pH 7.0, saline pH 4.3 or saline pH 7.0, respectively. Pain thresholds were examined before and after induction of UVB-sensitization, and 15, 30 and 60 min after injection of the respective solution. Mechanical pain thresholds of the skin and of deeper tissues were determined by pinprick and pressure algometer measurements, respectively. Results UVB-irradiation decreased mechanical pain thresholds for pinprick and pressure algometer measurements demonstrating a successful sunburn-like sensitization. Intradermal injection of terbutaline pH 7.0 into the sensitized skin reduced the sensitization for all measured timepoints as determined by pinprick measurements. Pinprick measurements of sensitization were not reduced by injection of terbutaline pH 4.3, saline solution pH 7.0 or saline solution pH 4.3. Also, sensitization of deeper tissue nociceptors were not altered by any of the injections as measured with the pressure algometer. Conclusions Similar to our cellular observations, also in humans beta-adrenergic agonists such as terbutaline can reduce the sensitization of primary neurons in the skin. Significance We previously showed in model systems that beta-adrenergic stimulation can not only sensitize but also desensitize nociceptors. Our study shows that also in humans beta-adrenergic agonists desensitize if injected into UVB-sensitized skin. This indicates an analgesic activity of adrenergic agonists in addition to their vasoconstrictory function.

Background Contribution of the small intestine to systemic inflammation after cardiac arrest (CA) is poorly understood. The objective was to evaluate whether an in vivo rat model of 6 min CA is suitable to initiate intestinal ischaemia-reperfusion-injury and to evaluate histomorphological changes and inflammatory processes in the small intestinal mucosa resp. in sera. Methods Adult male Wistar rats were subjected to CA followed by cardio-pulmonary resuscitation. Proximal jejunum and serum was collected at 6 h, 24 h, 72 h and 7 d post return of spontaneous circulation (ROSC) and from a control group. The small intestine was evaluated histomorphologically. Cytokine concentrations were measured in jejunum lysates and sera. Results Histomorphological evaluation revealed a significant increase in mucosal damage in the jejunum at all timepoints compared to controls (p

Activity of endogenous protein kinase A (PKA) could never be analyzed directly in the cellular environment. Isensee et al. used antibodies to quantify conformational changes leading to an open conformation of endogenous PKA-II holoenzymes, which allowed them to analyze and model its activation cycle in primary sensory neurons., Type II isoforms of cyclic adenosine monophosphate (cAMP)–dependent protein kinase A (PKA-II) contain a phosphorylatable epitope within the inhibitory domain of RII subunits (pRII) with still unclear function. In vitro, RII phosphorylation occurs in the absence of cAMP, whereas staining of cells with pRII-specific antibodies revealed a cAMP-dependent pattern. In sensory neurons, we found that increased pRII immunoreactivity reflects increased accessibility of the already phosphorylated RII epitope during cAMP-induced opening of the tetrameric RII2:C2 holoenzyme. Accordingly, induction of pRII by cAMP was sensitive to novel inhibitors of dissociation, whereas blocking catalytic activity was ineffective. Also in vitro, cAMP increased the binding of pRII antibodies to RII2:C2 holoenzymes. Identification of an antibody specific for the glycine-rich loop of catalytic subunits facing the pRII-epitope confirmed activity-dependent binding with similar kinetics, proving that the reassociation is rapid and precisely controlled. Mechanistic modeling further supported that RII phosphorylation precedes cAMP binding and controls the inactivation by modulating the reassociation involving the coordinated action of phosphodiesterases and phosphatases.

A 74-year-old man with a history of prostate cancer with proven osseous metastatic disease underwent Ga-prostate-specific membrane antigen (PSMA) PET/CT under antiandrogen therapy. The scan revealed a long segment of increased PSMA tracer uptake within the right sciatic nerve, which appeared edematous and swollen, and the respective ganglia. Clinically, the patient suffered from pain and paresis in the right leg. As infiltration of a long segment of a single nerve seems unlikely, primarily neuronal disease such as neuritis (induced by metastases or radiotherapy) was considered. The observed uptake of PSMA-targeting PET tracers may then represent a peripheral nerve disorder.

// Axel Lechner 1, 2, * , Hans Schloser 1, 3, * , Sacha I. Rothschild 1, 4, * , Martin Thelen 1 , Sabrina Reuter 1 , Peter Zentis 5 , Alexander Shimabukuro-Vornhagen 1, 6 , Sebastian Theurich 1, 6, 7 , Kerstin Wennhold 1 , Maria Garcia-Marquez 1 , Lars Tharun 8 , Alexander Quaas 8 , Astrid Schauss 5 , Jorg Isensee 9 , Tim Hucho 9 , Christian Huebbers 10 , Michael von Bergwelt-Baildon 1, 6, * and Dirk Beutner 2, * 1 Cologne Interventional Immunology, Department I of Internal Medicine, University Hospital of Cologne, Cologne, Germany 2 Department of Otorhinolaryngology, Head and Neck Surgery, University of Cologne, Cologne, Germany 3 Department of General, Visceral and Cancer Surgery, University of Cologne, Cologne, Germany 4 University Hospital Basel, Department of Internal Medicine, Medical Oncology, Basel, Switzerland 5 Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Cologne, Germany 6 Department I of Internal Medicine, Center for Integrated Oncology (CIO), University Hospital of Cologne, Cologne, Germany 7 Max-Planck-Institute for Metabolism Research, Cologne, Germany 8 Institute of Pathology, University of Cologne, Cologne, Germany 9 Department of Anesthesiology and Intensive Care Medicine, Experimental Anesthesiology and Pain Research, University Hospital of Cologne, University of Cologne, Germany 10 Jean-Uhrmacher Institute for Clinical ENT Research, University of Cologne, Cologne, Germany * These authors contributed equally to this work Correspondence to: Michael von Bergwelt-Baildon, email: michael.bergwelt@uk-koeln.de Keywords: squamous cell carcinoma, head and neck, microenvironment, T cells, immune checkpoint Received: January 10, 2017 Accepted: April 25, 2017 Published: May 16, 2017 ABSTRACT The composition of tumor-infiltrating lymphocytes (TIL) reflects biology and immunogenicity of cancer. Here, we characterize T-cell subsets and expression of immune checkpoint molecules in head and neck squamous cell carcinoma (HNSCC). We analyzed TIL subsets in primary tumors ( n = 34), blood (peripheral blood mononuclear cells (PBMC); n = 34) and non-cancerous mucosa ( n = 7) of 34 treatment-naive HNSCC patients and PBMC of 15 healthy controls. Flow cytometry analyses revealed a highly variable T-cell infiltration mainly of an effector memory phenotype (CD45RA − /CCR7 − ). Naive T cells (CD45RA + /CCR7 + ) were decreased in the microenvironment compared to PBMC of patients, while regulatory T cells (CD4 + /CD25 + /CD127 low and CD4 + /CD39 + ) were elevated. Furthermore, we performed digital image analyses of entire cross sections of HNSCC to define the ‘Immunoscore’ (CD3 + and CD8 + cell infiltration in tumor core and invasive margin) and quantified MHC class I expression on tumor cells by immunohistochemistry. Immune checkpoint molecules cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death 1 (PD-1) and programmed cell death 1 ligand 1 (PD-L1) were increased in TILs compared to peripheral T cells in flow-cytometric analysis. Human papillomavirus (HPV) positive tumors showed higher numbers of TILs, but a similar composition of T-cell subsets and checkpoint molecule expression compared to HPV negative tumors. Taken together, the tumor microenvironment of HNSCC is characterized by a strong infiltration of regulatory T cells and high checkpoint molecule expression on T-cell subsets. In view of increasingly used immunotherapies, a detailed knowledge of TILs and checkpoint molecule expression on TILs is of high translational relevance.

Pain sensitivity is strongly modulated by intracellular signalling. The analysis of pain signalling is hampered by small numbers of neurons, high cellular heterogeneity, and near absence of transfectability. HCS microscopy allowed us to proof that nociceptive neurons express specific signalling components, to analyze signalling activity of endogenous signalling components with single cell resolution, and thereby to suggest blockers of Nav1.7 as novel therapeutic approach to boost the efficacy of opioids.

Studying TRP channel expressing nociceptors requires the identification of the respective subpopulations as well as the quantification of dynamic cellular events. However, the heterogeneity of sensory neurons and associated nonneuronal cells demands the analysis of large numbers of cells to reflect the distribution of entire populations. Here we report a detailed workflow how to apply high-content screening (HCS) microscopy to signaling events in TRPV1-positive neurons as well as an approach to use the selective elimination of TRPV1 positive cells from dissociated rat sensory ganglia as base for transcriptomic analysis of TRPV1-positive cells and/or as control for TRPV1 antibody specificity.

Background The ion channel TRPV1 is mainly expressed in small diameter dorsal root ganglion (DRG) neurons, which are involved in the sensation of acute noxious thermal and chemical stimuli. Direct modifications of the channel by diverse signalling events have been intensively investigated, but little is known about the composition of modulating macromolecular TRPV1 signalling complexes. Here, we hypothesize that the novel adaptor protein ankyrin-rich membrane spanning protein/kinase D interacting substrate (ARMS) interacts with TRPV1 and modulates its function in rodent DRG neurons. Methods We used immunohistochemistry, electrophysiology, microfluorimetry and immunoprecipitation experiments to investigate TRPV1 and ARMS interactions in DRG neurons and transfected cells. Results We found that TRPV1 and ARMS are co-expressed in a subpopulation of DRG neurons. ARMS sensitizes TRPV1 towards capsaicin in transfected HEK 293 cells and in mouse DRG neurons in a PKA-dependent manner. Using a combination of functional imaging and immunocytochemistry, we show that the magnitude of the capsaicin response in DRG neurons depends not only on TRPV1 expression, but on the co-expression of ARMS alongside TRPV1. Conclusion These data indicate that ARMS is an important component of the signalling complex regulating the sensitivity of TRPV1. Significance The study identifies ARMS as an important component of the signalling complex regulating the sensitivity of excitatory ion channels (TRPV1) in peripheral sensory neurons (DRG neurons) and transfected cells.

Genetic loss of the voltage-gated sodium channel Na(v)1.7 (Nav1.7(-/-)) results in lifelong insensitivity to pain in mice and humans. One underlying cause is an increase in the production of endogenous opioids in sensory neurons. We analyzed whether Na(v)1.7 deficiency altered nociceptive heterotrimeric guanine nucleotide–binding protein–coupled receptor (GPCR) signaling, such as initiated by GPCRs that respond to serotonin (pronociceptive) or opioids (antinociceptive), in sensory neurons. We found that the nociceptive neurons of Na(v)1.7 knockout (Na(v)1.7(-/-)) mice, but not of Na(v)1.8 knockout (Na(v)1.8(-/-)) mice, exhibited decreased pro-nociceptive serotonergic signaling through the 5-HT(4) receptors, which are Gαs-coupled GPCRs that stimulate the production of cyclic adenosine monophosphate resulting in protein kinase A (PKA) activity, as well as reduced abundance of the RIIβ regulatory subunit of PKA. Simultaneously, the efficacy of anti-nociceptive opioid signaling mediated by the Gα(i)-coupled mu opioid receptor was increased. Consequently, opioids inhibited more efficiently tetrodotoxin-resistant sodium currents, which are important for pain-initiating neuronal activity in nociceptive neurons. Thus, Na(v)1.7 controls the efficacy and balance of GPCR mediated pro- and antinociceptive intracellular signaling, such that without Na(v)1.7, the balance is shifted toward antinociception, resulting in lifelong endogenous analgesia.

The cellular response to heat stress is an ancient and evolutionarily highly conserved defence mechanism characterised by the transcriptional up-regulation of cyto-protective genes and a partial inhibition of splicing. These features closely resemble the proteotoxic stress response during tumor development. The bromodomain protein BRD4 has been identified as an integral member of the oxidative stress as well as of the inflammatory response, mainly due to its role in the transcriptional regulation process. In addition, there are also several lines of evidence implicating BRD4 in the splicing process. Using RNA-sequencing we found a significant increase in splicing inhibition, in particular intron retentions (IR), following heat treatment in BRD4-depleted cells. This leads to a decrease of mRNA abundancy of the affected transcripts, most likely due to premature termination codons. Subsequent experiments revealed that BRD4 interacts with the heat shock factor 1 (HSF1) such that under heat stress BRD4 is recruited to nuclear stress bodies and non-coding SatIII RNA transcripts are up-regulated. These findings implicate BRD4 as an important regulator of splicing during heat stress. Our data which links BRD4 to the stress induced splicing process may provide novel mechanisms of BRD4 inhibitors in regard to anti-cancer therapies.

Genomic instability can be a hallmark of both human genetic disease and cancer. We identify a deleterious UBQLN4 mutation in families with an autosomal recessive syndrome reminiscent of genome instability disorders. UBQLN4 deficiency leads to increased sensitivity to genotoxic stress and delayed DNA double-strand break (DSB) repair. The proteasomal shuttle factor UBQLN4 is phosphorylated by ATM and interacts with ubiquitylated MRE11 to mediate early steps of homologous recombination-mediated DSB repair (HRR). Loss of UBQLN4 leads to chromatin retention of MRE11, promoting non-physiological HRR activity in vitro and in vivo. Conversely, UBQLN4 overexpression represses HRR and favors non-homologous end joining. Moreover, we find UBQLN4 overexpressed in aggressive tumors. In line with an HRR defect in these tumors, UBQLN4 overexpression is associated with PARP1 inhibitor sensitivity. UBQLN4 therefore curtails HRR activity through removal of MRE11 from damaged chromatin and thus offers a therapeutic window for PARP1 inhibitor treatment in UBQLN4-overexpressing tumors.Control of MRE11 association with chromatin by UBQLN4 during double-strand break repair influences repair pathway choice and can be dysregulated in tumorigenesis., This work was funded through the Dr. M. and S.G. Adelson Medical Research Foundation, The Israel Science Foundation joint ISF-NSFC Research Program and The Israel Cancer Research Fund (to Y.S.), the German-Israeli Foundation for Research and Development (I-65-412.20-2016 to Y.S. and H.C.R.), the Deutsche Forschungsgemeinschaft (KFO-286-RP2 to H.C.R., KFO-286-CP2 to M.P., JA2439/1-1 to R.D.J., DI1731/2-1 to F.D.), the Else Kröner-Fresenius Stiftung (2014-A06 to H.C.R., 2016_Kolleg.19 to R.D.J.), the Deutsche Krebshilfe (1117240 to H.C.R. and the Mildred-Scheel Professorship to M.P.), the German Ministry of Education and Research (BMBF 01GM1211B to D.W., BMBF e:Med 01ZX1303A and 01ZX1406 to M.P., BMBF e:Med 01ZX1303 and 01ZX1307 to M.F.), and R+D+I grants from the Spanish Ministry of Economy and Competitivity (SAF2013-43255-P and SAF2016-74855-P to P.H.). Y.S. is a Research Professor of the Israel Cancer Research Fund.

All biological systems exhibit cell-to-cell variability. Frameworks exist for understanding how stochastic fluctuations and transient differences in cell state contribute to experimentally observable variations in cellular responses. However, current methods do not allow identification of the sources of variability between and within stable subpopulations of cells. We present a data-driven modeling framework for the analysis of populations comprising heterogeneous subpopulations. Our approach combines mixture modeling with frameworks for distribution approximation, facilitating the integration of multiple single-cell datasets and the detection of causal differences between and within subpopulations. The computational efficiency of our framework allows hundreds of competing hypotheses to be compared. We initially validate our method using simulated data with an understood ground truth, then we analyze data collected using quantitative single-cell microscopy of cultured sensory neurons involved in pain initiation. This approach allows us to quantify the relative contribution of neuronal subpopulations, culture conditions, and expression levels of signaling proteins to the observed cell-to-cell variability in NGF/TrkA-initiated Erk1/2 signaling. Loos et al. introduce a data-driven modeling framework for the mechanistic analysis of heterogeneous cell populations consisting of subpopulations. Applying the framework to single-cell microscopy data of primary sensory neurons, they analyze the influence of extracellular scaffolds onto sensitization signaling.

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 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.

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 RIIβ subunit of PKA-II to be predominantly expressed in a subgroup of sensory neurons. The RIIβ-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 RIIβ as a novel nociceptive subgroup marker, and extend the current model of PKA-II activation by introducing a PP2A-dependent basal state. ispartof: Journal of Cell Science vol:127 issue:1 pages:216-229 ispartof: location:England status: published