Background & Aims

Experiments using mouse Cre-driver lines have implicated specific populations of dorsal horn neurons in pain signaling and maladaptive pain states. In parallel, human genome-wide association studies (GWAS) have identified several loci confidently associated with the genetic predisposition to chronic pain. Previous studies have established some links between chronic pain-associated single nucleotide variants (SNVs) and their nearness to genes most expressed in specific cell types. However, most of the risk burden for chronic pain are not from genes but rather from several common, small effect-size variants residing in non-coding genomic regions, as in other complex polygenic diseases. Thus, to more fully account for the spinal cord’s potential genetic risk of chronic pain, and relate it to the cells studied in rodent models, the field requires a detailed single-cell, epigenetic resource to link human genetics to species-conserved cell types.

Methods

First, we generated a single-nucleus transcriptomic atlas of adult Rhesus macaque focused on the dorsal horn, and compared gene expression profiles to the publicly available mouse and human datasets. we introduced a new nomenclature for these cell types based on conserved marker genes that better reflects harmonization across datasets and species. We also compared the laminar patterns of these cell types in macaque, and mouse using two single-cell resolution assays, tradition in situ (RNAscope) and highly multiplexed in situ spatial transcriptomics (Xenium), respectively. Finally, we generated a single cell atlas of open chromatin in the mouse dorsal horn using the single-nucleus Assay for Transposase Accessible Chromatin (snATAC-seq), labeled cell types using RNA-ATAC integration, and mapped open chromatin profiles to human ortholog coordinates. We then partitioned heritability among single cell open chromatin profiles to identify cell types associated with human chronic pain markers.

Results

First, we find that our macaque snRNA-seq atlas identifies 18 dorsal horn neuron subtypes that are highly conserved in human and mouse datasets, and identify pairs of conserved marker genes for each cell type to provide names and potential targets for hybridization probes in labeling experiments. Next, we found in both RNAscope and Xenium, that the vast majority of conserved neuron subtypes had significant, consistent superficial versus deep location preferences in primate and mouse, with the rest having non-significant trends. Third, we found that conserved subtypes were identifiable in mouse snATAC-seq, and the majority of open chromatin peaks were mappable to human.

Finally, we found that open chromatin of nearly all conserved dorsal horn neuron subtypes were enriched for markers of several chronic pain traits from GWAS, which was not the case for any glial cell types or other comparator cells (human bulk liver cells, hippocampal or putamen neurons, or macrophages).

Conclusions

First, we find strong evidence that several groups of dorsal horn neurons are highly distinct subtypes based on molecular and open chromatin profiles as well as spatial distribution in primate and mouse.

Second, we found a novel association between chronic pain genetics and the distal regulatory regions of specific dorsal horn subtypes. Previously, to our knowledge, genetic variants associated with chronic pain have only been linked to regions in and around a small number of genes, but have not been linked to distal enhancers, which we have done comprehensively.

Finally, we provide a set of publicly available single-cell resources (macaque snRNA-seq, mouse Xenium, mouse snATAC-seq) for further of study of these conserved neuron subtypes.

References

1.Peirs, C., and Seal, R.P. (2016). Neural circuits for pain: Recent advances and current views. Science 354, 578–584. 10.1126/science.aaf8933.
2.Sathyamurthy, A., Johnson, K.R., Matson, K.J.E., Dobrott, C.I., Li, L., Ryba, A.R., Bergman, T.B., Kelly, M.C., Kelley, M.W., and Levine, A.J. (2018). Massively Parallel Single Nucleus Transcriptional Profiling Defines Spinal Cord Neurons and Their Activity during Behavior. Cell Reports 22, 2216–2225. 10.1016/j.celrep.2018.02.003.
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Presenting Author

Michael J. Leone

Poster Authors

Michael Leone

Carnegie Mellon University

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Cynthia Arokiaraj

Lead Author

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Michael Kleyman

Lead Author

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BaDoi Phan

Lead Author

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Vijay Cherupally

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Bettega Lopes

Lead Author

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Myung Chul Noh

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Kelly Corrigan

Lead Author

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Andreas Pfenning

Lead Author

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Rebecca Seal

Lead Author

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Topics

  • Mechanisms: Biological-Molecular and Cell Biology