Profiling sensory neuron microenvironment after peripheral and central axon injury reveals key pathways for neural repair

  1. Oshri Avraham
  2. Rui Feng
  3. Eric Edward Ewan
  4. Justin Rustenhoven
  5. Guoyan Zhao
  6. Valeria Cavalli

  • Curated by eLife

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    Evaluation Summary:

    Using single cell RNA seq, the authors characterize the sensory neuron microenvironment in dorsal root ganglia after sciatic nerve crush, dorsal root crush and dorsal column transection spinal cord injury 3 days after injury. The data revealed differentially expressed genes and pathways with sciatic nerve and dorsal root crushes co-clustering, whereas spinal cord injury largely co-clusters with uninjured. The results reveal influences of the tissue microenvironment and neuron extrinsic factors on axonal regeneration and also provide new insights into the role of PPARa signaling in regeneration after dorsal root crush. This is an impressive data collection effort across multiple cell types that will be of importance for generating new hypotheses in the field. The impact could be further broadened by increased attention to functional validation of the findings.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

Sensory neurons with cell bodies in dorsal root ganglia (DRG) represent a useful model to study axon regeneration. Whereas regeneration and functional recovery occurs after peripheral nerve injury, spinal cord injury or dorsal root injury is not followed by regenerative outcomes. Regeneration of sensory axons in peripheral nerves is not entirely cell autonomous. Whether the DRG microenvironment influences the different regenerative capacities after injury to peripheral or central axons remains largely unknown. To answer this question, we performed a single-cell transcriptional profiling of mouse DRG in response to peripheral (sciatic nerve crush) and central axon injuries (dorsal root crush and spinal cord injury). Each cell type responded differently to the three types of injuries. All injuries increased the proportion of a cell type that shares features of both immune cells and glial cells. A distinct subset of satellite glial cells (SGC) appeared specifically in response to peripheral nerve injury. Activation of the PPARα signaling pathway in SGC, which promotes axon regeneration after peripheral nerve injury, failed to occur after central axon injuries. Treatment with the FDA-approved PPARα agonist fenofibrate increased axon regeneration after dorsal root injury. This study provides a map of the distinct DRG microenvironment responses to peripheral and central injuries at the single-cell level and highlights that manipulating non-neuronal cells could lead to avenues to promote functional recovery after CNS injuries or disease.

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  1. Version published to 10.7554/elife.68457 on eLife
  2. Version published to 10.1101/2020.11.25.398537v2 on bioRxiv
  3. eLife

    Author Response:

    In this manuscript, Avraham et al. report their results of profiling different cell types in DRG from mice with different types of injury. In general, injuries occurred in PNS (sciatic nerves or dorsal roots) trigger more drastic effects on nearly all DRG cell types, comparing to those applied to CNS (spinal cord), albeit with some exceptions. Among these responding cell populations is a subset of macrophage expressing a satellite glial cell (SGC) marker, and an another population of SGCs, although their lineage and role remain unknown. Furthermore, fatty acid biosynthesis and PPARgamma signaling pathways are up-regulated after sciatic nerve injury, but down-regulated after dorsal root injury (again these observations are not verified and the underlying mechanisms are elusive). Application of a PPARgamma agonist is able to elevate axon regeneration after dorsal root injury. In general, this manuscript has a large amount of data from bioinformatics analysis but with limited functional verifications. Thus their biological meaning is less clear.

    Functional verifications were included in most figures. However, to address this point we have performed additional experiments and validations, as detailed below.

    Figure 3: In addition to the immunofluorescence for macrophage and proliferation markers in DRG sections from all injury conditions, we now validated with qPCR the downregulation of selected cytokines and genes involved in antigen processing and presentation and the upregulation of proliferation markers in macrophages following sciatic nerve crush injury (new Figure 3D).

    Figure 4: In addition to the immunofluorescence of DRG sections showing co-expression of the SGC marker FABP7 and the macrophage marker CD68, we added a flow cytometry experiment of genetically labeled SGC (BlbpCreER:Sun1GFP), labeled with 3 different macrophage specific markers to validate the scRNAseq analysis (new Figure 4E). These new results support the notion that a subset of macrophages express glial properties at the protein level.

    In Fig 3, the results imply different signaling involvement in DRG macrophages (cell cycle and DNA replication after SNC and steroid biosynthesis and glycolysis/gluconeogenesis pathways after DRC/SCI). Are these results due to differential resident/infiltrated macrophage in DRG after individual injury types?

    We thank the reviewer for highlighting this point. Previous studies have indicated that the number of macrophages increases in the DRG after peripheral nerve injury but not dorsal root injury (Kwon et al 2012). This increase in macrophages number after nerve injury results in part from proliferation of macrophages (Leonhard et al., 2002; Yu et al., 2020) and may also include myeloid cell proliferation (Yu et al., 2020) and infiltration of a small number of blood-borne myeloid cells (Kalinski et al 2020). We quantified all ki67 expressing cells in our scRNAseq, which demonstrates that the majority of cells proliferating following all injuries are macrophages, and that the number of proliferating macrophages is highest after sciatic nerve injury (Figure 3 K,L). Our results suggest that the different signaling responses result largely from proliferation of macrophages after nerve injury. Whether the proliferating macrophages originate from resident macrophages or from the infiltration of monocytes-derived macrophages remains to be determined and is beyond the scope of the current study.

    It is also intriguing to note that all injuries down-regulate genes related to antigen processing and presentation (Fig 3). This seems an interesting observation as macrophages often exhibit pro-inflammatory responses to injury. These results should be verified with independent methods.

    We agree and have performed a qPCR experiment to validate the downregulation of genes regulating antigen processing and presentation associated with class II major histocompatibility complex (MHC II) CD74, H2-Aa and Ctss (new Figure 3D. We also confirmed the downregulation of the cytokines Ccl2, Il1b and Tnf following sciatic nerve crush in qPCR experiments (new Figure 3D).

    In Fig. 4, the authors describe a subset of macrophages expressing glial markers whose numbers become increased after injury. Is it possible that these might be the macrophages with engulfed SGCs? To test this, perhaps the authors could compare the abundance of these type-specific RNAs or use other independent methods (with transgenic mice with GFP-labeled SGCs to see if any GFP signals are in these macrophages).

    We agree that it is important to exclude the possibility that immune glial cells simply result from phagocytosis of satellite glial cells by macrophages. We have performed additional experiments and additional analyses that strongly support the existence of subset of macrophages with glial properties. We renamed these cells Imoonglia, to reflect their immune properties and their crescent shape morphology typical of SGC surrounding sensory neurons. First, we performed a flow cytometry experiment to show that a subset of genetically labeled SGC (BLBP-creER: Sun1 GFP) express the specific macrophage markers Cd11b, F4/80 and cd45 (new figure 4E). Second, we included additional analyses showing that these immune glia cells express progenitor cell markers (Dhh, Sox2 and Foxd3), which are not expressed in macrophages (new figure 4C). Third, we performed a trajectory analysis demonstrating that Imoonglia express a transcriptome that position them between satellite glial cells and macrophages (new figure 4D). Fourth, we expanded the methods section to clarify that duplicate cells are filtered out from downstream analysis and also plotted total counts in all cell types (new figure 4- Figure supplement 1B), further excluding the possibility that these cells are satellite glial cells engulfed by macrophages. We believe that these additional experiments and analyses strongly support the characterization of this Imoonglia cell population.

    The results in Fig. 5 suggest that SGCs represent different cell populations. Again, their biological meaning remains unknown. An obvious possibility is these clusters might reflect their different activation states. It might be useful to apply single cell trajectory analysis to assess their relationship.

    We thank the reviewer for this suggestion and have performed trajectory analysis to assess the activation state and the relationship of the different SGC subtypes. The results indicate a trajectory starting from cluster 3, to cluster 2, then cluster 1 and finally cluster 4 (new Figure 5E). This is very interesting in light of our comparison of SGC clusters to astrocytes and Schwann cells, showing that cluster 3 most resembles astrocytes while cluster 4 mostly resembles Schwann cells (Figure 5H,I). The trajectory analysis comparing different cell lineage genes suggests that all SGC subtypes present the same activation state (Figure 5E). The biological function of these different SGC clusters awaits further in-depth investigations that are beyond the scope of the current manuscript.

    Fig. 7, are the regeneration results after PPARa agonist comparable to those after sciatic nerve injury? Such information might provide insights as to its translational potential.

    It has been shown that dorsal root axonal growth occurs at half the rate of peripheral axons (Oblinger and Lasek 1984; Wujek and Lasek, 1983). In the experiment presented in Figure 7E-G, we observed that fenofibrate treatment almost doubled the length of dorsal root axons, suggesting that activating SGC with fenofibrate can increase axon growth. In our previous study, we showed that deleting the enzyme Fasn, which is upstream of PPARα activation, specifically in SGC, decreases axon growth in the sciatic nerve by about half (Avraham et al 2020). Altogether, these findings suggest that the lack of PPARα activation after dorsal root crush contributes to the low regeneration rates of axons in the dorsal root. We have edited the text in the discussion section (p.22) to provide insights into the translational potential of fenofibrate.

    Reviewer #2:

    Avraham et al. applied single cell RNA seq to characterize the sensory neuron microenvironment in dorsal root ganglia after sciatic nerve crush (SNC), dorsal root crush (DRC) and dorsal column transection spinal cord injury (SCI) 3 days after injury. The data revealed differentially expressed genes and pathways in endothelial cells, Schwann cells, macrophages and satellite glial cells (SGCs), etc. among the different injury models, with SNC and DRC co-clustering, and SCI and uninjured control co-clustering for the most part. While a number of cell types are implicated in the differential responses of the microenvironment to injury, the authors focused on the satellite glial cells (SGCs) in functional validation of PPARa signaling in regeneration after DRC using a PPARa agonist (fenofibrate).

    Strengths:

    1. Many strengths: contrasting injury models, scRNA seq, extensive bioinformatics analyses
    1. Many interesting pathways were found to be differentially expressed after different injuries (e.g. Arg1 in macrophages, the Hippo pathways in Schwann cells, etc).
    1. If immune glia cells prove to be a new subtype (of macrophages or SGCs?), it will be a very interesting finding indeed.
    1. It is interesting that PPARa signaling is upregulated in SNC, unchanged in DRC and reduced after SCI.
    1. The study illustrates the value of using single cell RNA seq to dissect the neuron microenvironment in response to injury and neuron extrinsic influences on axon regeneration.

    We thank the reviewer for highlighting the strengths. We agree that our approach highlights the importance of the microenvironment response and the potential extrinsic influence on axon regeneration. We have performed an additional analysis to highlight how the neuron microenvironment is affected by the different injuries. We examined the cell-cell interaction network based on ligand-receptor expression in the different cell types in injury conditions compared to naïve, which is now presented in new figure 1- Supplement Figure 1E. The molecular interactions between cells were identified based on CellPhoneDB repository (v 2.1.6). In this analysis, nodes (circles) in the figures represent cell clusters identified by Partek, and node size correlated with the relative cell counts in the cluster. Significant cell-cell interactions were predicted by CellPhoneDB and represented edges (arrows) in the network. The width and transparency of the edges correlated with the number of interactions defined by CellPhoneDB, and arrow indicates the directionality of ligand/receptor interactions. SNC changed significantly the cell-cell interaction network compared to naïve, and these changes are distinct from those elicited by DRC. SCI had limited influence on cell-cell interaction compared to naïve. This analysis, now presented in the new Figure1- figure supplement 1E further highlight the distinct neuron microenvironment responses to injuries. Additionally, we provide a detailed resource for the significant receptor-ligand interaction pairs for every cell population in all injury conditions in Figure 1- Source Data 2.

    Weakness:

    1. The authors have previously shown that PPARa agonist rescues the reduced axon regeneration in fatty acid synthase (Fasn) conditional knockout mice after SNC, so the role of PPARa in SGCs to support regeneration is no longer novel.

    Yes, we agree that we previously unraveled the role of PPARα in SGC after peripheral nerve injury. However, here we show that PPARα is not activated in SGC after injury to the dorsal root, and that fenofibrate can increase axon growth in the dorsal root, which is novel and may have translational potential.

    1. This is not a weakness per se, but the two most interesting findings on immune glia cells and PPARa do not appear to be directly related.

    We agree, but as this is a resource paper that describes how the neuronal microenvironment respond to different injuries, we believe that even if unrelated, our findings are important for the community. We have revised the abstract to better highlight these two findings.

    1. Pharmacological test with fenofibrate does not address the cell type specific role of PPARa, so it cannot be firmly established that PPARa in SGCs is most important for regeneration.

    We agree that this is an important point. We previously addressed the specificity of fenofibrate by demonstrating that PPARα in the DRG is highly enriched in satellite glial cells (Avraham et al Nat Comm 2020). We showed in that prior study that PPARα and PPARα target genes are upregulated in SGC but not in neurons after injury. We also showed in an in vitro assay that fenofibrate does not promote growth in pure neuronal cultures, further supporting that PPARα is not expressed in neurons. Immunostaining for PPARα demonstrated that PPARα is expressed in SGC but not neurons, and that neither injury nor fenofibrate treatment led to PPARα expression in neurons. Furthermore, a transcriptional profiling study of sensory neurons at single cell resolution (Renthal W. et. al.,Neuron, 2020) confirms that PPARα is not expressed in neurons, neither in naïve conditions nor following sciatic nerve crush injury. In the current study, we performed additional analyses to examine PPARα expression in other cells. First, we plotted all the cells expressing PPARα by cell type and injury condition. This analysis reveals that the majority of PPARα expressing cells are SGC in any injury condition (Figure 7C). Although macrophages can express PPARa and PPARg, (Rigamonti et al, 2008), we did not detect expression of PPARα in DRG macrophages under naïve or injury conditions (Figure 7C). Second, we present violin plots of PPARα and selected PPAR specific target genes, which demonstrates higher expression of PPARα in SGC compared to all other cells in the DRG (figure 7, figure supplement 1C). Given that fenofibrate is a selective activator of PPARα and does not target other PPAR isoforms (Lee CH, Olson P, Evans RM. Minireview: lipid metabolism, metabolic diseases, and peroxisome proliferator-activated receptors. Endocrinology. 2003 Jun;144(6):2201-7), we believe that the pharmacological manipulations presented here sufficiently address the role of PPARα signaling in satellite glial cells.

  4. eLife

    Evaluation Summary:

    Using single cell RNA seq, the authors characterize the sensory neuron microenvironment in dorsal root ganglia after sciatic nerve crush, dorsal root crush and dorsal column transection spinal cord injury 3 days after injury. The data revealed differentially expressed genes and pathways with sciatic nerve and dorsal root crushes co-clustering, whereas spinal cord injury largely co-clusters with uninjured. The results reveal influences of the tissue microenvironment and neuron extrinsic factors on axonal regeneration and also provide new insights into the role of PPARa signaling in regeneration after dorsal root crush. This is an impressive data collection effort across multiple cell types that will be of importance for generating new hypotheses in the field. The impact could be further broadened by increased attention to functional validation of the findings.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  5. eLife

    Reviewer #1 (Public Review):

    In this manuscript, Avraham et al. report their results of profiling different cell types in DRG from mice with different types of injury. In general, injuries occurred in PNS (sciatic nerves or dorsal roots) trigger more drastic effects on nearly all DRG cell types, comparing to those applied to CNS (spinal cord), albeit with some exceptions. Among these responding cell populations is a subset of macrophage expressing a satellite glial cell (SGC) marker, and an another population of SGCs, although their lineage and role remain unknown. Furthermore, fatty acid biosynthesis and PPARgamma signaling pathways are up-regulated after sciatic nerve injury, but down-regulated after dorsal root injury (again these observations are not verified and the underlying mechanisms are elusive). Application of a PPARgamma agonist is able to elevate axon regeneration after dorsal root injury. In general, this manuscript has a large amount of data from bioinformatics analysis but with limited functional verifications. Thus their biological meaning is less clear.
    In Fig 3, the results imply different signaling involvement in DRG macrophages (cell cycle and DNA replication after SNC and steroid biosynthesis and glycolysis/gluconeogenesis pathways after DRC/SCI). Are these results due to differential resident/infiltrated macrophage in DRG after individual injury types?

    It is also intriguing to note that all injuries down-regulate genes related to antigen processing and presentation (Fig 3). This seems an interesting observation as macrophages often exhibit pro-inflammatory responses to injury. These results should be verified with independent methods.

    In Fig. 4, the authors describe a subset of macrophages expressing glial markers whose numbers become increased after injury. Is it possible that these might be the macrophages with engulfed SGCs? To test this, perhaps the authors could compare the abundance of these type-specific RNAs or use other independent methods (with transgenic mice with GFP-labeled SGCs to see if any GFP signals are in these macrophages).

    The results in Fig. 5 suggest that SGCs represent different cell populations. Again, their biological meaning remains unknown. An obvious possibility is these clusters might reflect their different activation states. It might be useful to apply single cell trajectory analysis to assess their relationship.

    Fig. 7, are the regeneration results after PPARa agonist comparable to those after sciatic nerve injury? Such information might provide insights as to its translational potential.

  6. eLife

    Reviewer #2 (Public Review):

    Avraham et al. applied single cell RNA seq to characterize the sensory neuron microenvironment in dorsal root ganglia after sciatic nerve crush (SNC), dorsal root crush (DRC) and dorsal column transection spinal cord injury (SCI) 3 days after injury. The data revealed differentially expressed genes and pathways in endothelial cells, Schwann cells, macrophages and satellite glial cells (SGCs), etc. among the different injury models, with SNC and DRC co-clustering, and SCI and uninjured control co-clustering for the most part. While a number of cell types are implicated in the differential responses of the microenvironment to injury, the authors focused on the satellite glial cells (SGCs) in functional validation of PPARa signaling in regeneration after DRC using a PPARa agonist (fenofibrate).

    Strengths:

    1. Many strengths: contrasting injury models, scRNA seq, extensive bioinformatics analyses

    2. Many interesting pathways were found to be differentially expressed after different injuries (e.g. Arg1 in macrophages, the Hippo pathways in Schwann cells, etc).

    3. If immune glia cells prove to be a new subtype (of macrophages or SGCs?), it will be a very interesting finding indeed.

    4. It is interesting that PPARa signaling is upregulated in SNC, unchanged in DRC and reduced after SCI.

    5. The study illustrates the value of using single cell RNA seq to dissect the neuron microenvironment in response to injury and neuron extrinsic influences on axon regeneration.

    Weakness:

    1. The authors have previously shown that PPARa agonist rescues the reduced axon regeneration in fatty acid synthase (Fasn) conditional knockout mice after SNC, so the role of PPARa in SGCs to support regeneration is no longer novel.

    2. This is not a weakness per se, but the two most interesting findings on immune glia cells and PPARa do not appear to be directly related.

    3. Pharmacological test with fenofibrate does not address the cell type specific role of PPARa, so it cannot be firmly established that PPARa in SGCs is most important for regeneration.

  7. Version published to 10.1101/2020.11.25.398537v1 on bioRxiv