The peripheral Atf3+ neuronal population is responsible for nerve regeneration at the early stage of nerve injury revealed by single-cell RNA sequencing

Abstract

Peripheral nerve injury (PNI) can transform primary somatosensory neurons to a regenerative state. However, the details of the transcriptomic changes associated with the nerve regeneration of somatosensory neurons remain unclear. In this study, single-cell RNA sequencing (scRNA-seq) is conducted on mouse dorsal root ganglion (DRG) cells after the early stage of nerve injury on day 3 after chronic constriction injury (CCI). We observe that a novel CCI-induced neuronal population (CIP) emerge and express high levels of activating transcription factor (Atf3), a neuronal injury marker. CIP neurons highly express regeneration-associated genes (RAGs) and are enriched in regeneration-related gene ontology (GO) terms, suggesting that these neurons can constitute a pro-regenerative population. Moreover, intercellular communication networks show that CIP neurons closely communicate with satellite glial cells (SGCs) and specifically transmit strong Fgf3-Fgfr1 signaling to SGCs, which could initiate regeneration-associated transcriptional changes in SGCs. We also confirm that regenerative progress occurs at the early stage of nerve injury because immunohistochemistry shows that the expression of ATF3 is significantly increased beginning at 3 days post-CCI and decreased at 1 month post-CCI. Our bioinformatics analysis at single-cell resolution advances the knowledge of regenerative dynamic transcriptional changes in DRG cells after injury and the underlying molecular mechanisms involved.

1. Introduction

Peripheral nerve injury (PNI) can result in motor and sensory disorders such as neuropathic pain, which affect 7%–10% of the general population and seriously reduce people’s quality of life [1,2]. Neuronal functional recovery requires axonal regrowth [3]. In contrast to the central nervous system (CNS), the peripheral nervous system (PNS) has intrinsic regeneration and repair abilities [3]. Currently, the preferred treatments are meticulous microsurgical repair by the use of tensionless epineurial sutures and autologous nerve grafting, which requires more extensive procedures and sacrifices the supply of healthy nerves and donor nerves [1,4]. However, although the understanding of neuropathophysiology has greatly improved, the principles of clinical treatment for nerve injury have not changed, and accordingly, clinical outcomes remain poor [5]. Therefore, a deeper understanding of the molecular and cellular mechanisms of nerve regeneration is urgently needed to explore new possibilities for treating nerve injury.

Many molecules and signaling pathways involved in regeneration have been investigated. For example, a previous report indicated that collapsin response mediator protein 4 (CRMP4) plays dual roles in the proximal and distal axon segments of severed DRG neurons to promote axon regeneration [6]. Another study suggested that the Klf2-Vav1-Rac1 axis induced by retrograde Ca2+ signaling from injured axons facilitates axon regeneration through activating Rac1 GTPase in adult DRG neurons [7]. Nevertheless, comprehensive insights into nerve regeneration remain unclear. Single-cell RNA sequencing (scRNA-seq) is a powerful technology for characterizing the transcriptome of individual cells and clarifying biological mechanisms at the cellular level. Recently, scRNA-seq has been used to study transcriptomic perturbations in DRG neurons following nerve injury [8,9]. However, there are still many important questions that have not been adequately addressed, especially the identification of nerve regeneration-associated neuronal subtypes and their mechanisms. CCI is a well-established model that includes compression, ischemia, inflammation, and axonal demyelination [10] and mimics the etiology of clinical conditions and causes symptoms similar to those of posttraumatic neuropathic pain in humans [11].