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

Author:

Zhang Chi1ORCID,Hu Ming-Wen2,Wang Xue-Wei3ORCID,Cui Xiang1,Liu Jing1,Huang Qian1,Cao Xu3ORCID,Zhou Feng-Quan34,Qian Jiang2,He Shao-Qiu1ORCID,Guan Yun15ORCID

Affiliation:

1. Department of Anesthesiology and Critical Care Medicine, The Johns Hopkins University School of Medicine

2. Department of Ophthalmology, The Johns Hopkins University School of Medicine

3. Department of Orthopaedic Surgery, The Johns Hopkins University School of Medicine

4. The Solomon H. Snyder Department of Neuroscience, The Johns Hopkins University School of Medicine

5. Department of Neurological Surgery, The Johns Hopkins University School of Medicine

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.

Funder

National Institutes of Health

Johns Hopkins University

Publisher

eLife Sciences Publications, Ltd

Subject

General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Medicine,General Neuroscience

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