title: Subgroup-elimination transcriptomics identifies signaling proteins that define subclasses of TRPV1-positive neurons and a novel paracrine circuit.
abstract: Normal and painful stimuli are detected by specialized subgroups of peripheral sensory neurons. The understanding of the functional differences of each neuronal subgroup would be strongly enhanced by knowledge of the respective subgroup transcriptome. The separation of the subgroup of interest, however, has proven challenging as they can hardly be enriched. Instead of enriching, we now rapidly eliminated the subgroup of neurons expressing the heat-gated cation channel TRPV1 from dissociated rat sensory ganglia. Elimination was accomplished by brief treatment with TRPV1 agonists followed by the removal of compromised TRPV1(+) neurons using density centrifugation. By differential microarray and sequencing (RNA-Seq) based expression profiling we compared the transcriptome of all cells within sensory ganglia versus the same cells lacking TRPV1 expressing neurons, which revealed 240 differentially expressed genes (adj. p<0.05, fold-change>1.5). Corroborating the specificity of the approach, many of these genes have been reported to be involved in noxious heat or pain sensitization. Beyond the expected enrichment of ion channels, we found the TRPV1 transcriptome to be enriched for GPCRs and other signaling proteins involved in adenosine, calcium, and phosphatidylinositol signaling. Quantitative population analysis using a recent High Content Screening (HCS) microscopy approach identified substantial heterogeneity of expressed target proteins even within TRPV1-positive neurons. Signaling components defined distinct further subgroups within the population of TRPV1-positive neurons. Analysis of one such signaling system showed that the pain sensitizing prostaglandin PGD2 activates DP1 receptors expressed predominantly on TRPV1(+) neurons. In contrast, we found the PGD2 producing prostaglandin D synthase to be expressed exclusively in myelinated large-diameter neurons lacking TRPV1, which suggests a novel paracrine neuron-neuron communication. Thus, subgroup analysis based on the elimination rather than enrichment of the subgroup of interest revealed proteins that define subclasses of TRPV1-positive neurons and suggests a novel paracrine circuit.
authors.affiliation: Department of Anesthesiology and Intensive Care Medicine, Experimental Anesthesiology and Pain Research, University Hospital of Cologne, Cologne, Germany; Department for Human Molecular Genetics, Max Planck Institute for Molecular Genetics, Berlin, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Isensee
authors.affiliation: Department for Human Molecular Genetics, Max Planck Institute for Molecular Genetics, Berlin, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Wenzel
authors.affiliation: Department for Human Molecular Genetics, Max Planck Institute for Molecular Genetics, Berlin, Germany.
authors.forename: Rene
authors.initials: R
authors.lastname: Buschow
authors.affiliation: Department of Human Genetics, University Medicine Greifswald and Interfaculty Institute of Genetics and Functional Genomics, University of Greifswald, Greifswald, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Weissmann
authors.affiliation: Department of Human Genetics, University Medicine Greifswald and Interfaculty Institute of Genetics and Functional Genomics, University of Greifswald, Greifswald, Germany.
authors.forename: Andreas W
authors.initials: AW
authors.lastname: Kuss
authors.affiliation: Department of Anesthesiology and Intensive Care Medicine, Experimental Anesthesiology and Pain Research, University Hospital of Cologne, Cologne, Germany; Department for Human Molecular Genetics, Max Planck Institute for Molecular Genetics, Berlin, Germany.
authors.forename: Tim
authors.initials: T
authors.lastname: Hucho
mesh_terms: D000818:Animals; D001483:Base Sequence; D002211:Capsaicin / Q000494:pharmacology; D002478:Cells, Cultured; D005727:Ganglia, Spinal / Q000378:metabolism*; D020869:Gene Expression Profiling; D008297:Male; D009619:Nociceptors / Q000378:metabolism*; D010146:Pain / Q000235:genetics; D019899:Paracrine Communication / Q000235:genetics*; D010716:Phosphatidylinositols / Q000378:metabolism; D015221:Potassium Channels / Q000378:metabolism; D015230:Prostaglandin D2 / Q000096:biosynthesis / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D017422:Sequence Analysis, DNA; D050916:TRPV Cation Channels / Q000819:agonists / Q000235:genetics / Q000378:metabolism*
chemical_list: D010716:Phosphatidylinositols; D015221:Potassium Channels; D043562:Receptors, G-Protein-Coupled; D050916:TRPV Cation Channels; C493922:Trpv1 protein, rat; D015230:Prostaglandin D2; D002211:Capsaicin