hDRG Spatial Transcriptomics
Paper Abstract
Nociceptors are specialized sensory neurons that detect damaging or potentially damaging stimuli and are found in the dorsal root ganglia (DRG) and trigeminal ganglia. These neurons are critical for the generation of neuronal signals that ultimately create the perception of pain. Nociceptors are also primary targets for treating acute and chronic pain. Single-cell transcriptomics on mouse nociceptors has transformed our understanding of pain mechanisms. We sought to generate equivalent information for human nociceptors with the goal of identifying transcriptomic signatures of nociceptors, identifying species differences and potential drug targets. We used spatial transcriptomics to molecularly characterize transcriptomes of single DRG neurons from eight organ donors. We identified 12 clusters of human sensory neurons, 5 of which are C nociceptors, as well as 1 C low-threshold mechanoreceptors (LTMRs), 1 Aβ nociceptor, 2 Aδ, 2 Aβ, and 1 proprioceptor subtypes. By focusing on expression profiles for ion channels, G protein-coupled receptors (GPCRs), and other pharmacological targets, we provided a rich map of potential drug targets in the human DRG with direct comparison to mouse sensory neuron transcriptomes. We also compared human DRG neuronal subtypes to nonhuman primates showing conserved patterns of gene expression among many cell types but divergence among specific nociceptor subsets. Last, we identified sex differences in human DRG subpopulation transcriptomes, including a marked increase in calcitonin-related polypeptide alpha (CALCA) expression in female pruritogen receptor-enriched nociceptors. This comprehensive spatial characterization of human nociceptors might open the door to development of better treatments for acute and chronic pain disorders.
Citation
Diana Tavares-Ferreira, Stephanie Shiers, Pradipta R Ray, Andi Wangzhou, Vivekanand Jeevakumar, Ishwarya Sankaranarayanan, Anna M Cervantes, Jeffrey C Reese, Alexander Chamessian, Bryan A Copits, Patrick M Dougherty, Robert W Gereau 4th, Michael D Burton, Gregory Dussor, Theodore J Price, Spatial transcriptomics of dorsal root ganglia identifies molecular signatures of human nociceptors. Sci. Transl. Med.14,eabj8186(2022). DOI:10.1126/scitranslmed.abj8186 (Article link)
Neuropathic Pain
Paper Abstract
Neuropathic pain is a leading cause of high-impact pain, is often disabling and is poorly managed by current therapeutics. Here we focused on a unique group of neuropathic pain patients undergoing thoracic vertebrectomy where the dorsal root ganglia is removed as part of the surgery allowing for molecular characterization and identification of mechanistic drivers of neuropathic pain independently of preclinical models. Our goal was to quantify whole transcriptome RNA abundances using RNA-seq in pain-associated human dorsal root ganglia from these patients, allowing comprehensive identification of molecular changes in these samples by contrasting them with non-pain-associated dorsal root ganglia.
We sequenced 70 human dorsal root ganglia, and among these 50 met inclusion criteria for sufficient neuronal mRNA signal for downstream analysis.
Our expression analysis revealed profound sex differences in differentially expressed genes including increase of IL1B, TNF, CXCL14 and OSM in male and CCL1, CCL21, PENK and TLR3 in female dorsal root ganglia associated with neuropathic pain. Coexpression modules revealed enrichment in members of JUN-FOS signalling in males and centromere protein coding genes in females. Neuro-immune signalling pathways revealed distinct cytokine signalling pathways associated with neuropathic pain in males (OSM, LIF, SOCS1) and females (CCL1, CCL19, CCL21). We validated cellular expression profiles of a subset of these findings using RNAscope in situ hybridization.
Our findings give direct support for sex differences in underlying mechanisms of neuropathic pain in patient populations.
Citation
Pradipta R Ray, Stephanie Shiers, James P Caruso, Diana Tavares-Ferreira, Ishwarya Sankaranarayanan, Megan L Uhelski, Yan Li, Robert Y North, Claudio Tatsui, Gregory Dussor, Michael D Burton, Patrick M Dougherty, Theodore J Price, RNA profiling of human dorsal root ganglia reveals sex differences in mechanisms promoting neuropathic pain, Brain, Volume 146, Issue 2, February 2023, Pages 749–766 (Journal article link)
hDRG Long Read Sequencing
Paper Abstract
Splicing is a post-transcriptional RNA processing mechanism that enhances genomic
complexity by creating multiple isoforms from the same gene. Diversity in splicing in the
mammalian nervous system is associated with neuronal development, synaptic function and
plasticity, and is also associated with diseases of the nervous system ranging from
neurodegeneration to chronic pain. We aimed to characterize the isoforms expressed in the
human peripheral nervous system, with the goal of creating a resource to identify novel
isoforms of functionally relevant genes associated with somatosensation and nociception. We
used long read sequencing (LRS) to document isoform expression in the human dorsal root
ganglia (hDRG) from 3 organ donors. Isoforms were validated in silico by confirming
expression in hDRG short read sequencing (SRS) data from 3 independent organ donors.
19,547 isoforms of protein-coding genes were detected using LRS and validated with SRS and
strict expression cutoffs. We identified 763 isoforms with at least one previously undescribed
splice-junction. Previously unannotated isoforms of multiple pain-associated genes, including
ASIC3, MRGPRX1 and HNRNPK were identified. In the novel isoforms of ASIC3, a region
comprising ~35% of the 5’UTR was excised. In contrast, a novel splice-junction was utilized in
isoforms of MRGPRX1 to include an additional exon upstream of the start-codon,
consequently adding a region to the 5’UTR. Novel isoforms of HNRNPK were identified which
utilized previously unannotated splice-sites to both excise exon 14 and include a sequence in
the 5’ end of exon 13. The insertion and deletion in the coding region was predicted to excise a
serine-phosphorylation site favored by cdc2, and replace it with a tyrosine-phosphorylation site
potentially phosphorylated by SRC. We also independently confirm a recently reported DRGspecific splicing event in WNK1 that gives insight into how painless peripheral neuropathy
occurs when this gene is mutated. Our findings give a clear overview of mRNA isoform
diversity in the hDRG obtained using LRS. Using this work as a foundation, an important next
step will be to use LRS on hDRG tissues recovered from people with a history of chronic pain.
This should enable identification of new drug targets and a better understanding of chronic
pain that may involve aberrant splicing events.
Citation
Arendt-Tranholm, A., Mwirigi, J. M., & Price, T. J. (2023). RNA isoform expression landscape of the human dorsal root ganglion (DRG) generated from long read sequencing. bioRxiv, 2023.2010.2028.564535. (Link)