Avraham, Oshri

Cavalli, Valeria

Deng, Pan-Yue

Jones, Sara

Klyachko, Vitaly A.

Kuruvilla, Rejji

Semenkovich, Clay F.

English

Digital Commons@Becker

Supplementary Information  Satellite glial cells promote regenerative growth in sensory neurons  Oshri Avraham1, Pan-Yue Deng2, Sara Jones1, Rejji Kuruvilla3, Clay F. Semenkovich4,2, Vitaly A. Klyachko2 and Valeria Cavalli1,5,6,*      1 Department of Neuroscience, Washington University School of Medicine, St Louis, MO 63110, USA 2 Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, MO  63110, USA 3 Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA 4 Division of Endocrinology, Metabolism & Lipid Research, Washington University School of Medicine, St Louis, MO 63110, USA 5 Center of Regenerative Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA 6 Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO 63110, USA    Corresponding author: Valeria Cavalli, cavalli@wustl.edu   Supplementary Figures 1-6 SGC(605)SGC(605)Schwann(572)Astrocytes(500)Gene expressiona bcde fgControl Injury020406080100%NeuronsTrkATrkBTrkCSupplementary Figure 1: Cluster analysis from scRNAseq of mouse DRG, related to Figure 1 Pecam1/CD31 Aif1/Iba-1 CD68 CD3GIsl1 Des CD34 Col1aa) t-SNE overlay for expression of marker genes for different cell populations including Pecam/CD31 for endothelial cells, Aif1/Iba-1 and CD68 for Macrophages, CD3G for T-cells, Isl1 for neurons, Des for smooth muscle cells, CD34 for mesenchymal cell and Col1a1 for connective tissue. The relative levels of expression are presented as a blue color gradient on the left. b) Violin plots illustrate the gene counts (log) for Miki67 and Cdk1 genes of distinct cell populations after injury.  c) Fraction of neuronal type within control and injury condition by expression of Trk receptors. n=2 biologically independent experiments. Source data are provided as a Source Data file. d) Violin plots illustrate SGC marker genes signatures of distinct cell populations. e) Violin plots illustrate Schwann and SGC marker genes signatures in distinct cell populations. f) Top differentially expressed genes in the SGC cluster (605 genes) was compared to top differentially expressed genes in the Schwann cell cluster (572 genes). g) Top differentially expressed genes in the SGC cluster (605 genes) were compared to the top differentially expressed genes in astrocytes (500 genes).Kcnj10/Kir4.1 Cdh19 Glul/GSGene Counts (log)Mki67Fabp7Cdk1Gene Counts (log)Mpz Mbp Plp1Mag Prx NcmapSupplementary Figure 2: FABP7 is a specific marker for SGC in adult DRG, related to Figure 2 Plp1 FSchwann cellsGene expressionFabp7Plp1a bc df020406080100MeanFlourescenceIntensity nsnsControlFabp7KOTUJ1 FABP7yTUJ1 GS60708090100110MeanFlourescenceIntensity nsnsControlFabp7KOControlFabp7KOFABP7 TUJ1 DapiFABP7 TUJ1 TUJ1Merge MergeGSControlControlFabp7KOFabp7KO020406080100MeanFlourescenceIntensitns ControlFabp7KOp=0.0035Supplementary Figure 2: FABP7 is a specific marker for SGC in adult DRG, related to Figure 2 a) DRG sections from Fabp7KO and control mice were immunostained for FABP7 (green) and TUJ1 (red). Scale bar: 50 µm. Experiment was repeated independently 3 times. b) DRG sections from Fabp7KO and control mice were immunostained for Glutamine Synthase (GS) (green) and Tuj1 (red). c) Quantification of DRG mean fluorescence intensity of FABP7/GS and TUJ1. d) Longitudinal sections of sciatic nerves from Fabp7KO and control mice immunostained for FABP7 (green), TUJ1 (red) and Dapi. Scale bar: 50 µm. e) Quantification of nerves mean fluorescence intensity of FABP7 and TUJ1. f) t-SNE plot of injured sciatic nerve (9 days post injury) analyzed from the single cell data (Carr et al., 2018). t-SNE overlay for the expression of Plp1 gene, indicating the Schwann cell cluster, and Fabp7. Experiment was repeated independently 3 times in c and e. p-value by Two-way ANOVA in c and e. ns-non significant. Data are presented as mean values±SEM. Source data are provided as a Source Data file for c and e.FABP7TUJ10 2 4 6 8Protein digestion and absorptionPurine metabolismThyroid hormone signaling pathwayECM-receptor interactionp53 signaling pathwayUbiquitin mediated proteolysisCell cycleMacrophagesEnrichment Score0 2 4 6 8Wnt signaling pathwayPyruvate metabolismGap junctionECM-receptor interactionInsulin signaling pathwayPropanoate metabolismRelaxin signaling pathwayProlactin signaling pathwayFocal adhesionSchwannEnrichment Score0 5 10 15 20EndocytosisRNA transportNeurotrophin signaling pathwayHIF-1 signaling pathwayUbiquitin mediated proteolysisApoptosisProtein processing in endoplasmic reticulumOxidative phosphorylationNeuronsEnrichment Score0 5 10 15 20 25Regulation of actin cytoskeletonCircadian entrainmentcGMP-PKG signaling pathwayOxytocin signaling pathwayGlutathione metabolismProteasomeProtein processing in endoplasmic reticulumProtein exportRetrograde endocannabinoid signalingThermogenesisOxidative phosphorylationConnective tissueEnrichment Score0 2 4 6 8Hippo signaling pathwaycGMP-PKG signaling pathwayOxytocin signaling pathwayInsulin secretionAldosterone synthesis and secretionGap junctionCircadian entrainmentMesenchymalEnrichment Score0 2 4 6 8AutophagyPeroxisomemTOR signaling pathwayThyroid hormone signaling pathwayAMPK signaling pathwayABC transportersVEGF signaling pathwayLongevity regulating pathwayErbB signaling pathwayEndothelialEnrichment Scorea bdce fgSupplementary Figure 3: Pathway analysis of differentially upregulated genes in major cell types, related to Figure 3 (a-f) Pathway analysis of differentially upregulated genes in major cell types in the DRG following nerve injury (KEGG 2016). g) Violin plots illustrate SGC injury induced genes Gja1 and Gfap signatures in distinct cell populations in control and injury conditions. Gja1 Gja1 Gfap GfapGene Counts (log)CASPASE3 TUJ1 DAPIa bc dNaive InjuryFasncKOControlBiocytin NF200 IB4 MergeControl FasncKONaiveInjuredTUJ1TUJ1SCG10MergeSupplementary Figure 4: Fasn deletion in SGC does not lead to neuronal cell death or abnormal functional properties, related to Figure 5 a) Representative images of longitudinal nerve sections from naïve and injured control and FasncKO mice,immunostained for TUJ1(red) and SCG10(green). Scale bar: 500 µm. b) DRG sections from FasncKO andcontrol mice in naïve and injured (3 days post injury) were immuonostained for Cleaved Caspase3 (green),TUJ1 (red) and DAPI (Blue).  Scale bar: 50 µm. c) Whole-cell recordings in dissociated co-cultures of DRGneurons and glia. Medium diameter neurons (control 19.19 ± 0.42 µm, n = 16 cells; FasncKO 19.27 ± 0.34 µm,n = 30 cells, p = 0.88; that were associated with at least one SGC, were targeted for recordings. two tailed t-test ns- non significant. Data are presented as mean values ±SEM. Source data are provided as a SourceData file. d) A subset of recorded cells was filled with biocytin (red) via the patch pipette for post hocverification of neuronal identity; IB4- nociceptors (green) NF200- Proprioceptors/LTMRs (Cyan). Scale bar: 50µm. Experiments were repeated independently 5 times in a and d and 3 times in b.Supplementary Figure 5:  Activation of PPARα in pure neuronal cultures does not enhance axon regeneration, related to Figure 7 GW7647ClofibrateFenofibrateDMSOFig.S4DAPIFABP7TUJ1Co-cultureNeurons onlyabca) Embryonic DRG were dissociated and plated as a spot without 5-fluorodeoxyuridine (FDU) and stained forFABP7 (green), TUJ1 (red) and DAPI (blue) at DIV6. Scale Bar: 250 µm.  Experiment was repeatedindependently 5 times. b) Embryonic DRG were dissociated and plated as a spot with 5-fluorodeoxyuridine(FDU). Experiment was repeated independently 5 times.  c) Embryonic DRG spot co-culture,supplemented with FDU, were axotomized at DIV7 after a 24 h pre-treatment with the indicated PPARαagonists fenofibrate (10µM), Clofibrate (100nM)  and GW7647 (10µM). Cultures were fixed after 24h andstained with SCG10. Scale Bar: 250 µm. Experiment was repeated independently 3 times.DAPI MergeATF3 TUJ1abcPPARα TUJ1 DAPIInjuredFenofibrateMergedSupplementary Figure 6:  Neuronal pro-regeneration genes expression in response to fenofibrate, related to figure 7, 8  0246810JunRelativemRNA ExpressionInjury:        - + - + - +Control FasncKOFenofibrate:   -        -            -        -           +        +p=0.0003 p=0.0004p<0.000105101520Gap43RelativemRNA ExpressionnsInjury:        - + - + - +Control FasncKOFenofibrate:  -        -          -         -           +        +p<0.0001p<0.0001 p<0.0001a) Representative images of DRG sections immunostained for PPARα (green) and TUJ1 (red) from mice that received a prior nerve injury or received fenofibrate treatment., Scale bar: 100 µm. b) Representative images of injured DRG sections (3 days post injury) immunostained for ATF3 (green), TUJ1(red) and DAPI(Blue) Scale Bar: 100 µm. c) qPCR analysis of Gap43 expression in DRG from control and FasncKO mice in naïve and 3 days after sciatic nerve injury with and without fenofibrate. ns-non significant.  d) qPCR analysis of Jun expression in DRG from control and FasncKO mice in naïve and 3 days after sciatic nerve injury with and without fenofibrate. n=3 independent animals in a and b. Experiments in c and d was repeated independently 3 times. p-values by One way ANOVA followed by Sidak's multiple comparisons test. Data are presented as mean values ±SEM. Source data are provided as a Source Data file for c and d.

Satellite glial cells promote regenerative growth in sensory neurons

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Satellite glial cells promote regenerative growth in sensory neurons

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