scRNA-sequencing reveals subtype-specific transcriptomic perturbations in DRG neurons of PirtEGFPf mice in neuropathic pain condition

Abstract

Functionally distinct subtypes/clusters of dorsal root ganglion (DRG) neurons may play different roles in nerve regeneration and pain. However, details about their transcriptomic changes under neuropathic pain conditions remain unclear. Chronic constriction injury (CCI) of the sciatic nerve represents a well-established model of neuropathic pain, and we conducted single-cell RNA-sequencing (scRNA-seq) to characterize subtype-specific perturbations of transcriptomes in lumbar DRG neurons on day 7 post-CCI. By using PirtEGFPf mice that selectively express an enhanced green fluorescent protein in DRG neurons, we established a highly efficient purification process to enrich neurons for scRNA-seq. We observed the emergence of four prominent CCI-induced clusters and a loss of marker genes in injured neurons. Importantly, a portion of injured neurons from several clusters were spared from injury-induced identity loss, suggesting subtype-specific transcriptomic changes in injured neurons. Moreover, uninjured neurons, which are necessary for mediating the evoked pain, also demonstrated cell-type-specific transcriptomic perturbations in these clusters, but not in others. Notably, male and female mice showed differential transcriptomic changes in multiple neuronal clusters after CCI, suggesting transcriptomic sexual dimorphism in DRG neurons after nerve injury. Using Fgf3 as a proof-of-principle, RNAscope study provided further evidence of increased Fgf3 in injured neurons after CCI, supporting scRNA-seq analysis, and calcium imaging study unraveled a functional role of Fgf3 in neuronal excitability. These findings may contribute to the identification of new target genes and the development of DRG neuron cell-type-specific therapies for optimizing neuropathic pain treatment and nerve regeneration.

Spatial transcriptomics and single-nucleus RNA sequencing reveal a transcriptomic atlas of adult human spinal cord

“…The component heterogeneity of somatosensory neurons in the DRG is essential to relay different peripheral inputs to the central nervous system ( Abraira and Ginty, 2013 ; Gatto et al, 2019 ; Li et al, 2016 ; Todd, 2010 ). The heterogeneity in the molecular transcriptomes, neuronal types, and functional properties of DRG neurons in rodents has been well characterized ( Hu et al, 2016 ; Li et al, 2016 ; Renthal et al, 2020 ; Usoskin et al, 2015 ; Wang et al, 2021a ; Zhang et al, 2022 ; Zhou et al, 2022 ). Recently, emerging evidence has revealed the organization of human DRG somatosensory neurons ( Jung et al, 2023 ; Nguyen et al, 2021 ; Tavares-Ferreira et al, 2022 ; Yu et al, 2023 ).…”

Current understanding of the molecular mechanisms of chemotherapy-induced peripheral neuropathy

“…In contrast, recent advance in single-cell RNA sequencing (scRNA-seq) allows the characterization of transcriptomic changes across different functionally distinct cell types down to single-cell resolution in DRGs. Recently, scRNA-seq revealed heterogenous, subtype-specific transcriptomic changes in DRGs ( Hu et al, 2016 ; Zhang C. et al, 2022 ) and retinal ganglion cells ( Jacobi et al, 2022 ; Li et al, 2022 ) after axotomy. The distinct gene expression changes in different neuronal subtypes might account for the differences in the intrinsic growth capacity of different neuronal subtypes.…”

Single-cell transcriptomic profiling of dorsal root ganglion: an overview

“…(F) Neuronal subtypes of mouse DRG from the study of Zhou et al (2022) . (G) Neuronal subtypes of rat DRG by Zhang et al (2022) . NF, neurofilament; PEP, peptidergic; NP, non-peptidergic; TH, tyrosine hydroxylase; MHN, mechanoheat nociceptor; MI, mechanically insensitive; IS, itch-sensitive; MS, mechanically sensitive; MN, mechanical nociceptor; MR, mechanoreceptor; C-LTMR, C-fiber low-threshold MR; SST, somatostatin.…”

“…Although increasing number of studies have attempted to classify DRG neurons at single-cell resolution, the heterogeneity in sequencing methods, laboratory protocols, or sample treatments between studies led to some divergent results. In general, DRG neurons across species were classified into PEPs and NPs ( Usoskin et al, 2015 ; Hu et al, 2016 ; Renthal et al, 2020 ; Kupari et al, 2021 ; Wang et al, 2021 ; Zhang et al, 2022 ; Zhou et al, 2022 ; Jung et al, 2023 ; Yu et al, 2023 ), NFs ( Usoskin et al, 2015 ; Renthal et al, 2020 ; Zhang et al, 2022 ), and C-LTMRs ( Usoskin et al, 2015 ; Hu et al, 2016 ; Li et al, 2016 ; Renthal et al, 2020 ; Kupari et al, 2021 ; Tavares-Ferreira et al, 2022 ; Zhou et al, 2022 ; Jung et al, 2023 ; Yu et al, 2023 ) in most included studies of this review. Other common neuronal subtypes, such as TRPM8 ( Kupari et al, 2021 ; Zhou et al, 2022 ; Jung et al, 2023 ; Yu et al, 2023 ), SST ( Renthal et al, 2020 ; Zhou et al, 2022 ; Jung et al, 2023 ; Yu et al, 2023 ), proprioceptors ( Usoskin et al, 2015 ; Hu et al, 2016 ; Tavares-Ferreira et al, 2022 ; Jung et al, 2023 ; Yu et al, 2023 ), Aβ-LTMR ( Renthal et al, 2020 ; Tavares-Ferreira et al, 2022 ; Jung et al, 2023 ; Yu et al, 2023 ), Aδ-LTMR ( Tavares-Ferreira et al, 2022 ; Jung et al, 2023 ; Yu et al, 2023 ) were commonly identified in several studies.…”