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

Abstract

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.

Fig. 1. Somatosensory neuron clusters in the macaque DRG.

a A schematic view of the workflow. b UMAP plots showing (left) the contribution of individual animals (N = 3) to STRT-2i-seq clusters and (right) final cluster numbers (m = male, f = female). c Violin plots showing total counts of unique transcripts, detected genes, and transcript counts for neuronal and satellite-glia marker genes in the neuronal clusters. Y-axes show detected genes per cell for nFeature_RNA; all others are raw UMI counts. Boxplot defines the median, interquartile range (IQR), and 1.5 × IQR (whiskers). d A hierarchically organized heatmap with the five most specific genes (by p-adj) for each cluster. e UMAPs showing mouse canonical marker gene expression in the STRT-2i-seq macaque clusters. f STRT-2i-seq macaque clusters named after most likely mouse counterparts. g Named Smart-seq2 clusters after label transfer from the STRT-2i-seq data. Image sources: freevectors.net (human silhouette), needpix.com (microwell plates), openclipart.org (Eppendorf tube).

Fig. 2. Consensus of mouse DRG neuron types across datasets and nomenclatures.

a–f UMAPs showing a Zeisel types with Zeisel nomenclature, b Zeisel data after label transfer from Sharma data, c Zeisel data with Usoskin nomenclature, d Sharma data with original nomenclature, e Sharma data after label transfer from Zeisel data, and f Sharma data with Usoskin nomenclature. g Probability scores of Sharma types against Zeisel trained module (performed with Usoskin nomenclature). h Probability scores of Sharma types against Zeisel trained module (performed with Zeisel nomenclature). i Probability scores of Zeisel types against Sharma trained module (performed with original nomenclature from Sharma). Boxplots in g–i define the median, interquartile range (IQR), and 1.5 × IQR (whiskers).

Fig. 3. Identification of primate correlates of mouse neuron types.

a, b Violin plots showing prediction percent probability scores for each macaque cluster against established mouse DRG cell types trained with Usoskin nomenclature. a STRT-2i-seq (n = 3), b Smart-seq2 (n = 5). c Violin plots of markers expressed in macaque clusters. Y-axis, raw UMI counts for STRT-2i-seq (STRT) and counts for Smart-seq2 (SS2). d Violin plots showing genes differentially expressed between macaque PEP2 and PEP3 clusters. Boxplots in a–d define the median, interquartile range (IQR), and 1.5 × IQR (whiskers). e–g LargeViz plot showing cross-species clustering of macaque STRT-2i-seq and mouse Zeisel et al. datasets on a shared plot. e Macaque,  f mouse, g merged plot (legend shows origin of samples, WG = macaque, Zeisel = mouse). h Probability plot of label propagation from mouse neuron types to macaque neuron types. Horizontal blue bars represent mean position for each distribution. i Synopsis of the corresponding DRG neuron types between mouse and macaque datasets and nomenclatures.

Fig. 4. In vivo validation and gene family expression in the macaque DRG neuron types.

a Naming the macaque clusters after the closest corresponding mouse cell types (left) with RNAScope validations (right). Scale bar = 20 µm. b Diameter of neuronal types in lumbar DRGs, mean ± SEM. c Percent of each neuronal type among neurons of all types identified in this study in lumbar DRG. In b, c, blue dots represent values for individual animals. d Heatmaps of gene expression profiles for selected gene families in the macaque STRT-2i-seq DRG neuron clusters. Source data for b, c are provided as a Source Data file.

Fig. 5. Comparison of gene expression profiles across species.

a Dot plots showing ten most specific shared markers of corresponding/homologous cell types between the species. b Dot plots showing ten most macaque specific markers for each corresponding cell-type pair. c Dot plots showing ten most mouse-specific markers for each corresponding cell-type pair. Dot sizes in a–c correspond to the percentage of cells expressing the gene in the cluster and color scale indicates log2FC. d Heatmap showing binary regulon activity in each of the macaque DRG cell types. e Heatmap showing binary regulon activity in the mouse DRG cell types. In d, e the number of target genes is indicated in the parenthesis.

Fig. 6. Contribution of macaque DRG cell types to partitioned heritability of human chronic pain sites.

a UK Biobank chronic pain sites mapped to the human body. Number of chronic case participants shown in parentheses. b Heatmap of FDR-corrected p values for enrichment in partitioned heritability of each macaque DRG cell type (n = 3 macaques) to each human chronic pain site. c Forest plot of partitioned heritability estimates for each macaque DRG cell type contributing to chronic pain. Shown are heritability coefficient estimates (circles) and their 95% confidence intervals (bars) for each pain site. Fixed-effect, standard error weighted, meta-analyzed heritability estimates (triangles) also shown, colored red when significant at Bonferroni-corrected level (corrected for nine cell types). d, e Top genes in type-specific cells contributing to specific chronic pain sites. Top genes highlighted in a scatter plot (pink, left). Scatter plots show human GWAS enrichment of gene (y-axis) as a function of macaque cluster enrichment (x-axis). f Bar plot of top pathways for PEP1 and NP2 cell types in the meta-analysis of the eight chronic pain sites. g, h Schematic illustrations of pathway-related genes in g PEP1 and h NP2 neuron types. Image sources: wikipedia.org (human silhouette).