RNA-seq based transcriptome profiling of human and mouse dorsal root ganglion reveals a potential role for Protease Activated Receptor 3 (PAR3) in pain processing

The dorsal root ganglion (DRG) contains sensory neurons that innervate the surface of the body and many visceral organs. Included amongst these neurons are nociceptors, specialized neurons that detect damaging or potentially damaging stimuli, which are required for the detection of acute pain and play a key role in the development and maintenance of chronic pain states. RNA‐seq has recently been used to elucidate the transcriptome of this tissue in mouse and rat but the transcriptome of human DRG has not been explored. We obtained fresh, lumbar DRG tissue from female human donors and performed 75bp paired‐end polyA+ RNA‐sequencing on the Illumina platform. The sequenced fragments were mapped to the Gencode v14 reference transcriptome / hg19 reference genome to yield 80M mapped fragments, and relative transcript abundance in FPKM (Fragments Per Kilobase per Million mapped fragments) was quantified using the Tophat‐Cufflinks toolkit. We compared our RNA‐seq dataset to publicly available mouse DRG RNA‐seq data and performed integrative analysis with RNA‐seq data from several tissues associated with drug side effects (e.g. heart, small intestine, whole brain) to perform an unbiased search for conserved gene expression in DRG across both species. We find that there is broad conservation of known DRG and/or nociceptor enriched genes (e.g. P2XR3, SCN10A, SCN11A, NTRK1, MRGPRD) across mouse and human DRGs. Information theory approaches were used to identify tissue‐specific genes in human and mouse DRG compared to tissues 13 other tissues in human and mouse. We find strong correlation of expression across tissues between species for a few hundred DRG‐enriched transcripts, including known genes enriched in the DRG and previously unidentified ones. A conspicuous DRG enriched gene is F2RL2 which encodes PAR3. PAR3 expression in DRG is amongst the highest for all G protein coupled receptors (GPCRs) in human and mouse and mapping to existing cellular expression databases suggests neuronal expression in a population of nociceptors. To test the potential role of PAR3 in pain, we developed a novel ligand for this receptor using our synthetic tethered ligand discovery platform for PARs and show that it robustly activates calcium signaling in trigeminal ganglion neurons and causes mechanical hypersensitivity after hindpaw injection in mice but is devoid of activity at PAR2. Ongoing experiments are further evaluating the specificity of this compound. Our unbiased, human transcriptome approach to target discovery reveals PAR3 as a novel pain target. Support or Funding Information NIH grants NS073664 and NS065926