Overview: The objectives of this project are to understand the molecular biology of pain sensing neurons and peripheral tissues at the transcriptome level and modulation of transcriptomic parameters in acute and chronic pain models and in human patients or post-mortem samples. The laboratory has established research methodology and protocols, built an infrastructure of hardware and software, formed collaborative arrangements, trained a team of scientists and support personnel to utilize the methodology of RNA-Seq. We have performed hundreds of deep sequencing runs in various species and models and have obtained over 50 billion reads of transcriptome sequence information. We are intensively involved in the analysis of the resulting datasets from physiologically or genetically labeled pain-sensing neurons, neurons in dorsal spinal cord during peripheral inflammation, models of rheumatoid arthritis, inflamed peripheral tissue, axotomized DRG, dorsal and ventral spinal cords and peripheral nerve. We are also investigating transcriptional processes affected by general anesthesia in higher order brain regions. Multiple time points are sampled to follow the evolution and resolution of the intervention with enough samples at each point to permit statistical comparison. Because we isolated certain neuronal and non-neuronal cell populations we know which genes are in pain-sensing neurons and which are in mainly non-pain-sensing neurons such as proprioceptive primary afferents, supporting cells, or Schwann cells. The ability to form incisive hypotheses regarding pain physiology is greatly advanced by this type of tissue and neuron-specific information. We now have quantitative information on all the genes that mediate DRG and spinal cord sensory and motor functions and formation of the myelin sheath in the peripheral nervous system. TRPV1 Transcriptome: One important focus for our group is the subpopulation of DRG neurons that express the thermo-, chemo-, pH-, and lipid-responsive ion channel called TRPV1. This ion channel is also gated by capsaicin, the active ingredient in hot pepper. We have demonstrated that the potent capsaicin analog resiniferatoxin (RTX) can control cancer pain in dogs and humans indicating a crucial role for TRPV1+ neurons in transmission of clinical pain. Because of the efficacy of manipulations aimed at the TRPV1-expressing DRG neurons, we performed deep sequencing RNA-Seq on various animal and human ganglionic preparations targeting TRPV1 neurons. We published a report in mouse and rats using genetic techniques to FACS isolate TRPV1+ DRG neurons and to obtain the inverse population by killing the TRPV1+ neurons with diphtheria toxin or RTX treatment. and isolating TRPV1+ neurons by agonist-activated calcium fluorescence. Our initial publication outlines the transcriptome results from several of the above manipulations and provides a comprehensive transcriptomic profile of this clinically important population of nociceptive neurons. We followed this up in another publication in which we distinguished the contribution of Schwann cells versus neurons to the DRG transcriptome. A third publication has been submitted in which we isolated TRPV1+ neurons by agonist-activated calcium fluorescence and sequenced DRGs obtained at autopsy from one of our human cancer pain patients who had been treated with RTX and a cohort of canines with cancer pain who had been treated for pain with RTX. Interestingly these data demonstrated that the most sensitive neuronal component is the centrally projecting axons that contain RTX and the cell bodies in the ganglion are comparatively resistant to RTX. This important mechanistic insight was gained from transcriptomic analyses and is being used to fine-tune the administration protocol in our human clinical trial. Analgesia transcriptome: One of the most interesting aspects of the transcriptome analyses is quantitative insight provided by next-gen RNA-Seq. We now know the quantitative relationships between the exact genes that mediate the actions of known analgesic drugs such as morphine, clonidine, lidocaine, ibuprofen, and gabapentin and emerging targets such as nociceptor-neuron-specific sodium channels. Frequently it is not clear which molecular paralogs of ion channels or receptors are expressed by neurons in the pain pathway. Our data show that when expression for all the relevant genes are obtained quantitatively a more informative picture emerges. The transcriptome experiments also point to new targets for potential analgesic drug development. We identified an orphan GPCR that is well expressed in the nociceptive population, and are currently exploring its analgesic properties. We published a report that began with transcriptomic profiling of lipid generating genes to define a metabolic pathways for production of potentially neuroactive lipids. This novel approach predicted two new lipids which we identified using mass spectrometry. Correlative animal behavioral and human headache and psoriasis studies suggest roles in pain sensitization and itch. Transcriptomics of human pain sensitivity resulting from genetic variations: The RNA-Seq data provides a means for amplification of ongoing studies and informs all of our hypothesis-driven studies. We are in the process of characterizing human genetic copy number variations (CNVs) that produce pain insensitivity. The mechanisms can be probed, in part, using rat or mouse models of the CNVs. For example, we were able to evaluate the integrity of neurons in sensory ganglion and spinal cord in a rodent model of the hemideletion. In these studies, transcriptomic profiling allows for more precise hypothesis generation. Anesthesia Transcriptome: We are in the process of completing an assessment of the effects of inhalation general anesthesia on cortical and hippocampal transcriptomes and associated proteins identified from the gene analysis. This is the initial step to a larger study on general anesthesia and cognitive function in aged animals. In humans, general anesthesia can be deleterious to cognitive function and we hypothesize that mechanistic insight into the defect state can be obtained by understanding the molecular level changes induced by anesthesia and the capacity for recovery. Our results indicate that communication between synaptic input and nuclear transcriptional control is inhibited by general anesthesia and that the alterations are more pronounced in cortex than hippocampus. We detect widespread modulation of genes that mediate functional plasticity and memory formation. Corresponding decreases in several of the proteins are also observed. The data suggest that anesthesia can transiently uncouple synaptic activity from neuronal transcriptional control. Summary: The datasets acquired over the past several years provide unprecedented and extremely fine-grained detail on gene expression in pain-sensing circuits and anesthesia sensitive brain regions. The basic goal is to understand how we sense and control pain. We are determining exactly what molecules the different types of pain-sensing neurons make and how they work together to do their job. We have extended this approach to actions of anesthetic agents and observe a remarkable sensitivity of cerebral cortex to gaseous anesthetics compared to hippocampus. This suggests that executive function and working memory will be the most susceptible variables affected by general anesthesia. Taken together our data provide a transformative new resource for the pain research and anesthesia communities, and will allow more precise assessment and verification of experimental and clinical results.
