Single-cell RNA-seq reveals distinct injury responses in different types of DRG sensory neurons

Abstract

Peripheral nerve injury leads to various injury-induced responses in sensory neurons including physiological pain, neuronal cell death, and nerve regeneration. In this study, we performed single-cell RNA-sequencing (scRNA-seq) analysis of mouse nonpeptidergic nociceptors (NP), peptidergic nociceptors (PEP), and large myelinated sensory neurons (LM) under both control and injury conditions at 3 days after sciatic nerve transection (SNT). After performing principle component and weighted gene co-expression network analysis, we categorized dorsal root ganglion (DRG) neurons into different subtypes and discovered co-regulated injury-response genes including novel regeneration associated genes (RAGs) in association with neuronal development, protein translation and cytoplasm transportation. In addition, we found significant up-regulation of the genes associated with cell death such as Pdcd2 in a subset of NP neurons after axotomy, implicating their actions in neuronal cell death upon nerve injury. Our study revealed the distinctive and sustained heterogeneity of transcriptomic responses to injury at single neuron level, implicating the involvement of different gene regulatory networks in nerve regeneration, neuronal cell death and neuropathy in different population of DRG neurons.

Figure 1. Single-cell RNA-seq analysis of sensory neurons with differential injury response after sciatic nerve transection.

(a) Unbiased PCA analysis of mRNA transcriptome in individual sensory neurons via single-cell RNA-seq analysis. Individual DRG neurons were annotated according to their different expression patterns of marker genes in Fig. 1f,i and Supplementary Fig. 4b–e. NP, nonpeptidergic nociceptors; PEP, peptidergic nociceptors; and LM, large myelinated sensory neurons. (b) Genes characterized in each cluster and DRG subtype based on their correlation with PC1 and PC2 (Each gray point refers to a gene. Known regeneration/inhibitory associated genes in cluster 1 and 2 are highlighted in blue and red circle respectively. Known neuronal subtype markers are highlighted in color point.). (c) Immunofluorescence confirming intact cells of SNT-side L5 DRG with positive PTEN expression. (d) Immunofluorescence showed normal nuclei (arrow head) in intact c-Jun⁻ neurons compared to peripherally migrated nuclei (arrow) in injured c-Jun⁺ neurons in SNT-side L5 DRG. (e) Representative gene expression in control, SNT-injury and SNT-intact DRG neurons. **P < 0.01, one-way ANOVA. (f–i) Relative expression level of DRG neuron type-specific marker was mapped back to each sample. Color key represents normalized gene expression with the highest expression marked red and the lowest marked gray.

Figure 2. Heterogeneous responses of DRG subtypes after nerve injury.

(a) Hierarchical clustering of genes with the highest loadings in the first and second principal components. Bottom: Scaled expression level of Nefh and P2rx3 in each single-cell sample. (b) Venn diagram showing numbers of overlapped differentially regulated genes in three types of DRG neurons after injury. (c) Boxplot showing expression patterns of genes in class I–IV of cluster 1 and cluster 2 neurons for each subtype.

Figure 3. Gene correlation networks reveal differential regulation patterns of injury induced genes.

(a,b) Heatmap showing relative expression of genes in seven gene modules identified by WGCNA of transcriptomes in control and injury-responsive neurons. (c,d) Boxplots showing expression patterns (scaled log₂TPM) of injury induced genes in each WGCNA module. Representative GO terms of each significantly-regulated module are listed.

Figure 4. Correlation of transcriptional gene regulation with axotomy-induced cell death and neuropathic pain.

(a-d) Boxplots showing the distribution of neuronal death/survival related gene expression of different DRG subtypes. *P < 0.05, **P < 0.01, unpaired t-test. (e,f) Boxplots showing the distribution of the gene expression of potassium channels in turquoise module among different DRG subtypes. (g) Immunofluorescence confirmed upregulation of apoptosis related PDCD2 in NEFH⁻ injured DRG neurons. PDCD2^high/NEFH⁻ and PDCD2^low/NEFH⁺ neurons (white arrow and white inset). (h–i) Immunofluorescence confirmed differential regulation of TFs in control and injured DRG neurons. NEFH⁺/c-Jun^high neuron (green arrow and green inset), NEFH⁻/c-Jun^high neuron (white arrow and white inset), NEFH⁻/STAT5b^high neuron (white arrow and white inset). (j) Correlation heatmap showing expression patterns of TFs in single DRG neurons.

Figure 5. Conserved injury responses in LM-subtype neurons at different time points of SNT.

(a) PCA analysis of mRNA transcriptome in LM neuron subtypes at 7 days after SNT respectively. Triangles represent DRG neurons collected on the SNT side, points represent DRG neurons on the control side. Individual DRG neurons were annotated according to their different expression patterns of marker genes as shown in Supplementary Fig. S6. (b) Hierarchical clustering of transcriptome profiles revealed closer cell-cell distance between day-3 and day-7 after SNT in LM neurons compared to other neuronal subtypes. (c) Heatmap showing relative expression of differentially expressed genes which are divided into 3 representative modules by WGCNA. Gene ontology (BP) analysis of each individual module is shown on the right. (d) Violin plot showing dynamic gene expression of gray, magenta and orange modules at different time points after SNT.