The Pain Neural Transcriptome

Clinical Center

Investigators

Linked publications, trials & patents

Abstract

Overview: The objectives of this project are to understand (a) the molecular biology of pain-sensing neurons in dorsal root ganglion (DRG), spinal cord, and damaged peripheral tissues at the transcriptome level, (b) the modulation of transcriptomic parameters in acute and chronic pain conditions and (c) to extend and verify animal model work using tissue from human patients, organ donors or post-mortem cases. Beyond these questions, the empirical framework we are developing forms the foundational knowledge basis for our translational projects (CL090033-07 Integrative and Molecular Studies of Pain and Pain Control and CL090034-07: Mechanisms of Pain and Immune Processes), and our clinical trials program involving the analgesic agent we developed, resiniferatoxin (RTX) (e.g., NCT0252261, periganglionic administration for cancer pain; NCT00804154, intrathecal administration for cancer pain; and NCT05695339 perineural administration for neuropathic pain). To meet these basic and clinical research objectives, we established protocols for patient recruitment and treatment, hardware and software infrastructure for analytical pipelines, collaborative arrangements for data analysis, various experimental models, and human investigative pilot protocols. We utilize cell biological and in vivo behavioral measurements in combination with deep RNA-Seq, multiplex fluorescent in situ hybridization, single nucleus sequencing, and spatial transcriptomics to probe nociceptive mechanisms, at present, mainly in the human. The resulting pain transcriptome/nociceptome encompasses primary afferent and spinal cord neurons, support and glial cells, and peripheral tissues such as skin and nerve. All have been reported in publications or are being intensively analyzed in ongoing investigations. In this cycle we have leveraged the spinal spatial transcriptomics of spinal projection neurons to extend our analysis to third order neurons in thalamus and will be discussed in the Xenium section. A main objective is to advance our understanding of human nociceptive neurons and circuits and translate this into pain therapies. In this cycle we performed an extensive set of fluorescent multiplex in situ and Xenium high density in situ hybridization analyses of human dorsal root ganglion and spinal cord for transcripts in nociceptive neurons. One insight that emerged was a new synthesis in which we were able to divide DRG nociceptors into those that contain the mu opioid receptor and those that do not. The data indicate that the Mu+ and TRPV1+ nociceptors transmit noxious sensations following tissue damage and that this type of pain can be controlled by opioid agonists. Pain sensations transmitted by the Mu-negative and TRPV1+ nociceptors are not responsive to opioids and we hypothesize that this population is active during neuropathic nerve injury pain. Pain in neuropathic disorders is notoriously insensitive to control by opioid agonists. This remarkable new level of insight into the molecular repertoire of nociceptors has been used in a drug development campaign that has now yielded both candidate receptors and through molecular docking several new potential non-opioid analgesics have been identified. Thus, the extensive foundational data acquired had facilitated and incisive program to generate new analgesics. Xenium high density spatial transcriptomics: This is a new technique that allows high spatial resolution for in situ hybridization of hundreds of gene transcripts at once on tissue sections. We use it to investigate cellular level expression in DRG and spinal cord neurons. Because each region contains specialized genes that are not found in brain or bodily organs, the Xenium investigation required generation of two custom probe panels, one for DRG, one for spinal cord (300 genes each). This method worked spectacularly well and complements and extends our whole tissue RNA sequencing. We also developed an analytical pipeline to cluster the transcripts within the individual neuronal cell bodies, satellite cells in DRG, or glial cells in cord. An example of how this method can be used for discovery research is seen in spinal cord. Here, using the spinal cord Xenium panel, we discovered that a specific population of spino-thalamic projection neurons expressed high levels of the gene, CARTPT, which codes for the neuropeptide cocaine and amphetamine regulated transcript. These large neurons are located in the marginal zone of the superficial dorsal horn and are known to be nociceptive specific. Because of the high-plex nature of the probe set we were able to determine that projection neurons express the Mu-opioid receptor indicating participation endogenous opioid control by local circuit neurons (which were also identified) and in opioid-mediated analgesia resulting from exogenous administration of opioid analgesics like morphine. Furthermore, the CARTPT projection neurons express a second receptor that mediates analgesia, the adrenergic alpha2A receptor. Thus, these neurons participate in pain transmission and pain control our spatial transcriptomic data allow us to update the “gate control theory” of pain with precise molecular definition. And additional benefit of this approach is that, during this cycle, we were able to follow the projection to human thalamus. Staining for CART peptide in thalamus revealed a discrete focus of CART peptide staining The TRPV1 Transcriptome: One important focus is the subpopulation of DRG neurons expressing 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 ultra-potent capsaicin analog resiniferatoxin (RTX) can control cancer pain in canine and human patients by inactivating TRPV1 axons or nerve terminals, indicating a crucial role for TRPV1+ neurons in transmission of clinically relevant pain. In this cycle we were able to show that the TRPPv1 neurons were divisible into two populations: Those that expressed the mu-opioid receptor gene (OPRM1) and those that did not. We published these results and also have a commentary in press discussing the implications for analgesia. The strong efficacy of RTX implicates TRPV1+/OPRM1+ DRG neurons as the crucial population of nociceptive neurons for transmitting human clinical tissue damage pain. The TRPV1+/OPRM- population is likely involved in neuropathic pain. One ongoing objective is to identify new molecular routes within the two TRPV1 nociceptor populations to control various types of pain. The Xenium method is directly aimed at this question and we are augmenting this approach with a newer agnostic spatial sequencing technique called Visium HD. These data-informed routes provide rational, mechanism-based targeting for discovery of new effective pain control agents. Indeed, as mentioned, in this cycle three orphan GPCRs were identified and we are following up on agonist development for these receptors. The Spinal Pain Transcriptome: Xenium is a key method for expanding a data-driven approach to human spinal cord circuitry and was partially discussed above. Elaborating the circuitry in the different laminae is another result from the human Xenium studies. The high number of neurons in spinal lamina II that express proenkephalin and prodynorphin (in different neurons) is an example that reinforces the prominent role for the endogenous opioid system in modulation of pain in the human. Lamina V also contains spino-thalamic projection neurons which are molecularly distinct from those in the marginal zone. Our data suggest that they send substance P containing terminals to ventroposterolateral nucleus as distinct from mediodorsal nucleus. The implications of such a dual and distinct projection pattern are currently being explored. Peripheral Inflammatory and Surgical Incision Transcriptomes: The objectives are to understand peripheral tissue damage-induced processes at the molecular level to ascertain where and how pain starts. Early studies using transcriptomics disclosed multiple, temporally distinct waves of gene induction and recruitment of resident cells and infiltrating leukocytes and demonstrated the tissue damage secretome. We have now completed a human study to verify and extend the rodent results which is now in press. We worked with a team of surgeons from the NCI to obtain skin from the edge of the incision site at various intervals out to wound closure (between 6 and 8 hours) and have performed RNA-Seq, in situ hybridization and LC-MS-MS lipidomic profiling on the samples. The results provide a transcriptomic roadmap of objective biochemical readouts and tissue cell types that participate in the damage response and form a translational basis for targeted interventions. These studies are the initial observations that can inform a patient’s pain and wound healing status. Technical developments: Our emphasis on anatomical localization of genes in human PNS and CNS neurons made us confront the prominent problems of lipofuscin fluorescence and autofluorescence in human neurons. All researchers encounter this problem and it becomes worse with the age of the sample (e.g. Alzheimer brain tissue). The lipofuscin fluorescence is very bright and its emission occurs across most of the visible color spectrum. To eliminate this artifact, we developed a method and an apparatus for photobleaching tissue sections before staining. The techniques developed work extremely well with RNA being preserved. An extensive methods paper was published this cycle.