Single Cell Transcriptome Data Analysis Defines the Heterogeneity of Peripheral Nerve Cells in Homeostasis and Regeneration

Abstract

The advances in single-cell RNA sequencing technologies and the development of bioinformatics pipelines enable us to more accurately define the heterogeneity of cell types in a selected tissue. In this report, we re-analyzed recently published single-cell RNA sequencing data sets and provide a rationale to redefine the heterogeneity of cells in both intact and injured mouse peripheral nerves. Our analysis showed that, in both intact and injured peripheral nerves, cells could be functionally classified into four categories: Schwann cells, nerve fibroblasts, immune cells, and cells associated with blood vessels. Nerve fibroblasts could be sub-clustered into epineurial, perineurial, and endoneurial fibroblasts. Identified immune cell clusters include macrophages, mast cells, natural killer cells, T and B lymphocytes as well as an unreported cluster of neutrophils. Cells associated with blood vessels include endothelial cells, vascular smooth muscle cells, and pericytes. We show that endothelial cells in the intact mouse sciatic nerve have three sub-types: epineurial, endoneurial, and lymphatic endothelial cells. Analysis of cell type-specific gene changes revealed that Schwann cells and endoneurial fibroblasts are the two most important cell types promoting peripheral nerve regeneration. Analysis of communication between these cells identified potential signals for early blood vessel regeneration, neutrophil recruitment of macrophages, and macrophages activating Schwann cells. Through this analysis, we also report appropriate marker genes for future single cell transcriptome data analysis to identify cell types in intact and injured peripheral nerves. The findings from our analysis could facilitate a better understanding of cell biology of peripheral nerves in homeostasis, regeneration, and disease.

Up-Regulation of CD146 in Schwann Cells Following Peripheral Nerve Injury Modulates Schwann Cell Function in Regeneration

“…Next, we examined cell type specific expression of CD146 in both intact and injured mouse sciatic nerves with analyzed single cell RNA sequencing data GSE147285, which studied cell types and gene expression in intact mouse sciatic nerve and in the distal sciatic nerve at day 3 after sciatic nerve transection injury ( Toma et al, 2020 ; Chen et al, 2021 ), and GSE120678 which studied cell types and gene expression in the distal sciatic nerves at day 9 after sciatic nerve transection injury ( Carr et al, 2019 ; Chen et al, 2021 ). Single cell transcriptomics analysis demonstrated that CD146 is expressed in non-myelinating Schwann cells, vasculature-associated smooth muscle (VSM) cells and pericytes of intact mouse sciatic nerve ( Figure 2 and Table 1 ).…”

“…*p-value < 0.05. Toma et al, 2020;Chen et al, 2021). Previously, we studied gene expression changes in both DRG and the distal nerve stump in the rat by microarray analysis, and identified CD146 as one of DEGs in the distal nerve stump (Yi et al, 2015).…”

“…Peripheral nerve injury induces vigorous gene expression changes not only in injured neurons but also in cells of the distal nerve stump ( Jessen and Mirsky, 2016 ; Renthal et al, 2020 ; Toma et al, 2020 ; Chen et al, 2021 ). Previously, we studied gene expression changes in both DRG and the distal nerve stump in the rat by microarray analysis, and identified CD146 as one of DEGs in the distal nerve stump ( Yi et al, 2015 ).…”

“…Single cell sequencing data for mouse distal nerve at day 9 after sciatic nerve transection injury were downloaded from GSE120678 ( Carr et al, 2019 ). Data were analyzed using Seurat v.3.2.1 1 and sctransform v.0.3 R packages v.4.0.2 as previously described ( Chen et al, 2021 ). CD146 dotplot, tSNE and violinplot were created using Seurat specific function.…”

A Novel NAD Signaling Mechanism in Axon Degeneration and its Relationship to Innate Immunity

“…The latent phase lasts up to 36 h wherein the axons appear normal and for much of this time retain the ability to conduct an action potential, although the axon becomes committed to degenerate ( Tsao et al, 1999 ; Beirowski et al, 2004 ; Beirowski et al, 2005 ; Conforti et al, 2014 ). This is also the stage in which the immune system is first seen to be involved; tissue-resident macrophages are activated and bone-marrow derived macrophages, neutrophils and phagocytes infiltrate into the nerve ( Perkins and Tracey, 2000 ; Sta et al, 2014 ; Zigmond and Echevarria, 2019 ; Boissonnas et al, 2020 ; Ydens et al, 2020 ; Chen et al, 2021 ). There is also an injury-associated increase in the levels of cytokines ( Yi et al, 2017 ).…”