The Injured Sciatic Nerve Atlas (iSNAT), insights into the cellular and molecular basis of neural tissue degeneration and regeneration
Abstract
Upon trauma, the adult murine PNS displays a remarkable degree of spontaneous anatomical and functional regeneration. To explore extrinsic mechanisms of neural repair, we carried out single cell analysis of naïve mouse sciatic nerve, peripheral blood mononuclear cells, and crushed sciatic nerves at 1-day, 3-days, and 7- days following injury. During the first week, monocytes and macrophages (Mo/Mac) rapidly accumulate in the injured nerve and undergo extensive metabolic reprogramming. Proinflammatory Mo/Mac in the injured nerve show high glycolytic flux compared to Mo/Mac in blood and dominate the early injury response. They subsequently give way to inflammation resolving Mac, programmed toward oxidative phosphorylation. Nerve crush injury causes partial leakiness of the blood-nerve-barrier, proliferation of endoneurial and perineurial stromal cells, and accumulation of select serum proteins. Micro-dissection of the nerve injury site and distal nerve, followed by single-cell RNA-sequencing, identified distinct immune compartments, triggered by mechanical nerve wounding and Wallerian degeneration, respectively. This finding was independently confirmed with Sarm1-/- mice, where Wallerian degeneration is greatly delayed. Experiments with chimeric mice showed that wildtype immune cells readily enter the injury site in Sarm1-/- mice, but are sparse in the distal nerve, except for Mo. We used CellChat to explore intercellular communications in the naïve and injured PNS and report on hundreds of ligand-receptor interactions. Our longitudinal analysis represents a new resource for nerve regeneration, reveals location specific immune microenvironments, and reports on large intercellular communication networks. To facilitate mining of scRNAseq datasets, we generated the injured sciatic nerve atlas (iSNAT): https://cdb-rshiny.med.umich.edu/Giger_iSNAT/
Data availability
All scRNA-seq datasets (fastq files and Seurat objects) are available online through the Gene Expression Omnibus (GEO) database https://www.ncbi.nlm.nih.gov/geo , accession number GSE198582.All code for iSNAT is available at https://github.com/GigerLab/iSNAT
-
Injured Sciatic Nerve Atlas (iSNAT)NCBI Gene Expression Omnibus, GSE198582.
Article and author information
Author details
Funding
Adelson Family Foundation (APNRR)
- Jeffery L Twiss
- Daniel H Geschwind
- Roman J Giger
NIH Blueprint for Neuroscience Research (MH119346)
- Roman J Giger
NIH Blueprint for Neuroscience Research (R01DC018500)
- Gabriel Corfas
NIH Blueprint for Neuroscience Research (T32 NS07222)
- Ashley L Kalinski
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Beth Stevens, Boston Children's Hospital, United States
Ethics
Animal experimentation: All procedures involving mice were approved by the Institutional Animal Care and Use Committees (IACUC) of the university of Michigan (PRO 00009851) and performed in accordance with guidelines developed by the National Institutes of Health
Version history
- Received: June 8, 2022
- Preprint posted: June 29, 2022 (view preprint)
- Accepted: November 30, 2022
- Accepted Manuscript published: December 14, 2022 (version 1)
- Version of Record published: January 9, 2023 (version 2)
- Version of Record updated: February 3, 2023 (version 3)
Copyright
© 2022, Zhao et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 4,694
- views
-
- 518
- downloads
-
- 21
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Cancer Biology
- Immunology and Inflammation
Pyrimidine nucleotide biosynthesis is a druggable metabolic dependency of cancer cells, and chemotherapy agents targeting pyrimidine metabolism are the backbone of treatment for many cancers. Dihydroorotate dehydrogenase (DHODH) is an essential enzyme in the de novo pyrimidine biosynthesis pathway that can be targeted by clinically approved inhibitors. However, despite robust preclinical anticancer efficacy, DHODH inhibitors have shown limited single-agent activity in phase 1 and 2 clinical trials. Therefore, novel combination therapy strategies are necessary to realize the potential of these drugs. To search for therapeutic vulnerabilities induced by DHODH inhibition, we examined gene expression changes in cancer cells treated with the potent and selective DHODH inhibitor brequinar (BQ). This revealed that BQ treatment causes upregulation of antigen presentation pathway genes and cell surface MHC class I expression. Mechanistic studies showed that this effect is (1) strictly dependent on pyrimidine nucleotide depletion, (2) independent of canonical antigen presentation pathway transcriptional regulators, and (3) mediated by RNA polymerase II elongation control by positive transcription elongation factor B (P-TEFb). Furthermore, BQ showed impressive single-agent efficacy in the immunocompetent B16F10 melanoma model, and combination treatment with BQ and dual immune checkpoint blockade (anti-CTLA-4 plus anti-PD-1) significantly prolonged mouse survival compared to either therapy alone. Our results have important implications for the clinical development of DHODH inhibitors and provide a rationale for combination therapy with BQ and immune checkpoint blockade.
-
- Immunology and Inflammation
We studied lysosomal Ca2+ in inflammasome. Lipopolysaccharide (LPS) + palmitic acid (PA) decreased lysosomal Ca2+ ([Ca2+]Lys) and increased [Ca2+]i through mitochondrial ROS, which was suppressed in Trpm2-KO macrophages. Inflammasome activation and metabolic inflammation in adipose tissue of high-fat diet (HFD)-fed mice were ameliorated by Trpm2 KO. ER→lysosome Ca2+ refilling occurred after lysosomal Ca2+ release whose blockade attenuated LPS + PA-induced inflammasome. Subsequently, store-operated Ca2+entry (SOCE) was activated whose inhibition suppressed inflammasome. SOCE was coupled with K+ efflux whose inhibition reduced ER Ca2+ content ([Ca2+]ER) and impaired [Ca2+]Lys recovery. LPS + PA activated KCa3.1 channel, a Ca2+-activated K+ channel. Inhibitors of KCa3.1 channel or Kcnn4 KO reduced [Ca2+]ER, attenuated increase of [Ca2+]i or inflammasome activation by LPS + PA, and ameliorated HFD-induced inflammasome or metabolic inflammation. Lysosomal Ca2+ release induced delayed JNK and ASC phosphorylation through CAMKII-ASK1. These results suggest a novel role of lysosomal Ca2+ release sustained by ER→lysosome Ca2+ refilling and K+ efflux through KCa3.1 channel in inflammasome activation and metabolic inflammation.