Single cell transcriptomics of primate sensory neurons identifies cell types associated with chronic pain

ARTICLE https://doi.org/10.1038/s41467-021-21725-z OPEN Single cell transcriptomics of primate sensory neurons identifies cell types associated with chronic pain 1234567890():,; Jussi Kupari 1,7, Dmitry Usoskin1,7, Marc Parisien 2, Daohua Lou1, Yizhou Hu 1, Michael Fatt1, Peter Lönnerberg1, Mats Spångberg3, Bengt Eriksson3, Nikolaos Barkas 4, Peter V. Kharchenko4, Karin Loré 5,6, Samar Khoury2, Luda Diatchenko 2 ✉ & Patrik Ernfors 1 ✉ Distinct types of dorsal root ganglion sensory neurons may have unique contributions to chronic pain. Identification of primate sensory neuron types is critical for understanding the cellular origin and heritability of chronic pain. However, molecular insights into the primate sensory neurons are missing. Here we classify non-human primate dorsal root ganglion sensory neurons based on their transcriptome and map human pain heritability to neuronal types. First, we identified cell correlates between two major datasets for mouse sensory neuron types. Machine learning exposes an overall cross-species conservation of somatosensory neurons between primate and mouse, although with differences at individual gene level, highlighting the importance of primate data for clinical translation. We map genomic loci associated with chronic pain in human onto primate sensory neuron types to identify the cellular origin of chronic pain. Genome-wide associations for chronic pain converge on two different neuronal types distributed between pain disorders that display different genetic susceptibilities, suggesting both unique and shared mechanisms between different pain conditions. 1 Division of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden. 2 Alan Edwards Centre for Research on Pain, Department of Anesthesia, School of Medicine, School of Dentistry, McGill University, Montreá l, QC, Canada. 3 Astrid Fagraeus Laboratory, Comparative Medicine, Karolinska Institutet, Stockholm, Sweden. 4 Department of Biomedical Informatics, Harvard Medical School, Boston, MA, USA. 5 Division of Immunology and Allergy, Karolinska Institutet, Stockholm, Sweden. 6 Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden. 7These authors contributed equally: Jussi Kupari, Dmitry Usoskin. ✉email: ; NATURE COMMUNICATIONS | (2021)12:1510 | https://doi.org/10.1038/s41467-021-21725-z | www.nature.com/naturecommunications 1 ARTICLE T NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21725-z he dorsal root ganglion (DRG) consists of a variety of neuron types, each tuned to detect and transduce different physical stimuli. These neuron types can broadly be divided into low-threshold mechanosensitive neurons responsible for sensing touch and high-threshold nociceptors, which are involved in pain, temperature, and itch1–4. However, a comprehensive classification of DRG neurons is critical for understanding exactly how somatosensation works and for providing insights into the cellular basis for acute and chronic pain. Rodents represent the main species for studies on the cellular and molecular basis of nociception and the greatest insights with respect to molecular classification of neuronal types have been obtained from mouse, where single-cell RNA-sequencing (scRNA-seq) has led to a molecular taxonomy of existing types of sensory neurons5–9. This has enabled the identification of molecular types representing richly myelinated A-fiber low-threshold mechanoreceptors (LTMRs) and limb proprioceptors. The remaining neuronal types in the scRNA-seq are assigned as weakly myelinated or unmyelinated neurons. One of these is a C-fiber LTMR (C-LTMR) neuron type that expresses Vglut3 (Slc17a8) and tyrosine hydroxylase (Th) that likely is not involved in pain sensation1–5. Nociception is largely conferred through unmyelinated peptidergic C-fiber neuron types and a few lightly myelinated Aδ-nociceptors, a Trpm8 expressing cluster of neurons, as well as cell types marked by expression of Mrgprd, Mrgpra3, or Sst (named NP1, NP2, and NP3 types of neurons, respectively8). This molecular classification agrees remarkably well with previous studies based on myelination and conduction velocity, neurochemical features and termination patterns peripherally in the skin and centrally in the spinal cord and is also consistent with the known ontogeny of DRG neuron types5. As a result, there have been significant advances in understanding the cellular and molecular characteristics of sensory neurons found in mouse DRG. Much less is known about characteristics of human DRG. Apart from information on size of the ganglia along the rostrocaudal axis, micro-anatomy including neuron size10–12 and electrophysiological characteristics13–16, the molecular characterization of human DRG is still limited to bulk RNAsequencing17–19 and neurochemical analyses of gene products in a handful of studies20. Hence, the concordance of markers used in different studies and their relation to actual neuron types remain largely unknown. Nevertheless, by examining individual gene products, these studies suggest important species differences between human and mouse where, for example, Nav1.8, Nav1.9, P2X3 receptor, and TRPV1 are present in both small and large neurons in humans, but only small neurons in mouse, suggesting fundamental differences in molecular characteristics and principles of initiation and transduction of somatosensory stimuli between humans and rodent20. In humans, rare and drastic mutations that explain different types of congenital insensitivity to pain and erythromelalgia have been identified, such as, for example, SCN9A (Nav1.7), NTRK1 (TRKA), and SCN11A (Nav1.9)21–25. In addition to these rare causing mutations, it is known that the genetic risk for chronic pain is due to common variations with small effect size26. Close to half of the risk of developing chronic pain are attributable to genetic factors27–29, including musculoskeletal pain conditions28. For musculoskeletal pain there is statistical evidence for a diverse set of genes involved, with a marked overrepresentation of genes expressed in neurons and functionally associated with neurotransmission, indicating a strong heritable component caused by altered functions of neurons26. Pleiotropy of single-nucleotide polymorphisms (SNPs) among painful and non-painful conditions has also been shown30, even in human DRG31. It has recently become possible to connect genomic results to transcriptomics at the cellular level which allows for insights into the 2 cell types which are fundamental for disorders. Thus, taking advantage of scRNA-seq for mapping susceptibility genes to cell types, new insights have been made into the cell types involved, for example, in schizophrenia32,33, neuroticism34, intelligence35,36, and Alzheimer’s disease37, but such analyses have not been attempted for chronic pain conditions. Knowledge on the molecular and cellular characteristics of primate DRG a (...truncated)