Endothelin B receptor inhibition rescues aging-dependent neuronal regenerative decline
- Department of Neuroscience, Washington University School of Medicine, United States
- CS27 Bioinformatics, United States
- Department of Neuroscience & Experimental Therapeutics, Texas A&M Health Science Center, United States
- Center of Regenerative Medicine, Washington University School of Medicine, United States
- Hope Center for Neurological Disorders, Washington University School of Medicine, United States
Figures
Figure 1 with 2 supplements
Ednrb is highly expressed in satellite glial cells.
(A) Representative whole-mount-stained images of DRG from Lycopersicon Esculentum Lectin (LEL) injected Fabp7CreER::Ai14 mice, labeled for TUJ1 (green), tdTomato (magenta), and LEL (gray). 3D reconstruction of blood vessels via LEL labeling (scale bars, 200 μm). Note that TUJ1 antibody staining is limited by penetration of the antibody in the whole mount. (B) Representative images of sectioned DRG from Lycopersicon Esculentum Lectin (LEL) injected C57BL/6 mice, labeled for TUJ1 (green), Fabp7 (red), and LEL (cyan) (Scale bars, 100 μm). (C) UMAP analysis of adult DRG 10 X sequencing data identified 8 cell clusters based on known marker genes. (D) Dot plot analysis showing the average gene expression (color coded) and number of expressing cells (dot size) for the marker genes. (E–H) UMAP overlay for expression of Ednra (E), Ednrb (F), Edn1 (G), and Edn3 (H). (I) Representative RNAScope in situ hybridization images showing Ednrb (red), Fabp7 (cyan), and DAPI (blue) of L4 DRGs from 3-month-old mice (scale bars, 50 μm).
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Figure 1—source data 1
Marker genes for 10x scRNA-seq analysis.
- https://cdn.elifesciences.org/articles/100217/elife-100217-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Sample QC and DEG analysis for 10x scRNA-Seq data.
(A, B) Sample QC for 10 x scRNA-Seq. (C) UMAP plots of genes used for cell cluster annotations. (D) UMAP overlay for expression of Ednrb in SGCs in mouse and human. Images generated from https://painseq.shinyapps.io/harmonized_painseq_v2/, related to Bhuiyan et al., 2024.
Figure 1—video 1
Z-stack video of whole mount preparation of DRG from Lycopersicon Esculentum Lectin (LEL) injected Fabp7CreER::Ai14 mouse, which labels SGCs with tdTomato, immunostained for TUJ1 (neurons).
Figure 2
Endothelin B receptor inhibition increases axonal growth in vitro and ex vivo.
(A) Representative images showing TUJ1 (black) immunostaining of neurons in DRG cultures (scale bars, 100 μm). (B) Representative image of TUJ1 (green) and Fabp7 (magenta) immunostaining of neurons and SGCs in control DRG cultures (scale bars, 50 μm). (C, D) Quantification of axonal radial length (B) and TUJ1+ area (C) per neuron. Different colors indicate biological replicates. N=246 (Veh; 8 replicates), 318 (BQ788; 8 replicates), 320 (IRL1620; 8 replicates), and 244 (BQ123; 8 replicates). Data presented as mean ± SD. (E) Scheme of drug treatment and DRG explant model. (F) Representative images of DRG explants 7 days after drug treatment, immunostained for TUJ1 (black) (scale bars, 1000 μm). (G) Quantification of radial length of the 35 longest axons from DRG explants from indicated groups N=36 explants from 6 individual mice (BQ788; 18 replicates, Veh; 18 replicates). The data are presented as mean ± SD. (H) Representative images of DRG explants immunostained for TUJ1 (green), FABP7 (magenta), and merged (scale bars, 50 μm).
Figure 3 with 2 supplements
Bosentan treatment improves axon regeneration after peripheral nerve injury in adult mice.
(A) Scheme of drug treatment and peripheral nerve injury model. (B) Quantification of the length of the 10 longest axons in indicated conditions. (C) Quantification of the regeneration index, calculated as the distance along the nerve where the SGC10 intensity is 50% of the SCG10 intensity at crush site. (D) Representative longitudinal sections of sciatic nerves 24 h after SNC, immunostained for SCG10, from mice with the indicated treatment. Dotted line indicates the crush site, determined as the maximal SGC10 intensity (scale bars, 200 μm). (E) Quantification of SCG10 intensity at the indicated distance normalized to the intensity at the crush site for each condition. N=5 mice/condition. (F) Scheme of long-term Bosentan treatment. (G) Representative images of hindpaw skin after long-term Bosentan treatment immunostained for PGP9.5 (white) and DAPI (blue) (scale bars, 50 μm). (H) Quantification of intraepidermal nerve fiber density (IENFD) 24 days after sciatic nerve crush from the indicated groups. N=5 mice/condition. (I) Scheme of adult DRG neuronal culture and treatments. (J, L) Representative images showing TUJ1 (black) immunostaining of neurons in DRG cultures (scale bars, 100 μm). (K, M) Quantification of axonal radial length (K) and total TUJ1+ area (L). Different colors represent different biological replicates. N (neuron number)=177 (vehicle; three biological replicates, naïve mice), 168 (Ambrisentan, three biological replicates, naïve mice), 183 (Bosentan; three biological replicates, naïve mice), 186 (vehicle; three biological replicates, injured mice), 204 (Ambrisentan, three biological replicates, injured mice), and 210 (Bosentan; three biological replicates, injured mice), respectively. The data are presented as mean ± SD.
Figure 3—figure supplement 1
Bosentan treatment improves axon regeneration 3 days after peripheral nerve injury.
(A) Scheme of drug treatment and peripheral nerve injury model. (B) Quantification of the 10 longest axons in indicated groups. (C) Quantification of 50% regenerative index, calculated as the distance along the nerve where the SCG10 intensity is 50% of the SCG10 intensity at crush site. (D) Representative longitudinal sections of sciatic nerves 3 d after SNC immunostained for SCG10 from mice with the indicated treatment. Dotted line indicates the crush site, determined as the maximal SCG10 intensity (scale bars, 200 μm). (E) Quantification of SCG10 intensity at the indicated distance normalized to the intensity at the crush site for each condition. N=5 mice/condition. The data are presented as mean ± SD. (F) RT-qPCR of Atf3, Aif1, Fabp7, Edn1, Ednra, and Ednrb gene in contralateral (CON) and ipsilateral DRGs at 3 days post injury (SNC). N (mouse number)=4/each group. (G) Western blot analysis and quantification of ETBR protein expression in DRGs from the mice with/without injury. (H) Quantification of ETBR expression normalized to GAPDH expression (folder of control mice). N (mouse number)=8/group. The data are presented as mean ± SD.
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Figure 3—figure supplement 1—source data 1
Original files for western blot analysis.
- https://cdn.elifesciences.org/articles/100217/elife-100217-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
PDF file containing original western blots, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/100217/elife-100217-fig3-figsupp1-data2-v1.zip
Figure 3—figure supplement 2
Bosentan treatment improves axon regeneration after dorsal root crush injury.
(A) Scheme of drug treatment and dorsal root crush injury model. (B) Representative longitudinal sections of sciatic nerves 3 d after DRC immunostained for SCG10 from mice with the indicated treatment. Dotted line indicates the crush site, determined as the maximal SCG10 intensity (scale bars, 200 μm). (C) Quantification of the 10 longest axons in indicated groups. (D) Quantification of 50% regenerative index, calculated as the distance along the nerve where the SCG10 intensity is 50% of the SCG10 intensity at crush site. (E) Quantification of SCG10 intensity at the indicated distance normalized to the intensity at the crush site for each condition. N=5 mice/condition. The data are presented as mean ± SD.
Figure 4 with 1 supplement
Bosentan treatment rescues aging-dependent neuronal regenerative decline.
(A) Scheme of drug treatment and DRG explant model. (B) Representative images of DRG explants 7 days after drug treatment (scale bars, 1000 μm). (C) Quantification of radial length of the 50 longest axons from DRG explants. N=36 explants from 6 individual mice (BQ788; 18 replicates, Veh; 18 replicates). (D) Representative longitudinal sections of sciatic nerves 3 d after SNC immunostained for SCG10 from mice with the indicated treatment. Dotted line indicates the crush site, determined as the maximal SCG10 intensity (scale bars, 200 μm). (E, F) Quantification of the 10 longest axons in indicated groups (E). Quantification of 50% regenerative index, calculated as the distance along the nerve where the SCG10 intensity is 50% of the SCG10 intensity at crush site (F). (G) Quantification of the SCG10 intensity at the indicated distance normalized to the intensity at the crush site for each condition. N (mouse number) = 4(adult, vehicle), 3 (aged, vehicle), and 3 (Bosentan +Age), respectively. The data are presented as mean ± SD.
Figure 4—figure supplement 1
ET-1 protein expression increases in DRGs of aged mice.
(A,B) Western blot analysis and quantification of ET1 (A) and ETBR (B) protein expression in DRG from adult and aged mice N=3 for each age group. (C) Scheme of drug treatment and explant culture model. (D) Quantification of radial length of the 10 longest axons from DRG explants. N=20 (Bosentan; 9 replicates, Veh; 11 replicates) (E) Representative images of DRG explants 4 days after culture (scale bars, 1000 μm). The data are presented as mean ± SD.
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Figure 4—figure supplement 1—source data 1
Original files for western blot analysis.
- https://cdn.elifesciences.org/articles/100217/elife-100217-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
PDF file containing original western blots, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/100217/elife-100217-fig4-figsupp1-data2-v1.zip
Figure 5 with 1 supplement
Aging alters SGC abundance and morphology.
(A) UMAP plot of adult and aged snRNA-seq identified 16 cell clusters based on known marker genes. (B) Dot plot analysis showing the average gene expression (color coded) and number of expressing cells (dot size) for the marker genes. (C, D) UMAP plot of DRG cells from adult (C) and aged (D) mice. (E). Bar plot of cell proportions in DRGs of adult and aged mice. (F) Representative TEM images of DRG sections from adult (2 M), middle-aged (12 M), and aged (21 M) mice showing neuronal cell bodies and the enveloping SGCs (SGCs are pseudo-colored in red; scale bars, 5 μm). (G) Quantification of the average width of SGC sheath per neuron soma. (H) Frequency of neuron soma in TEM images with 0, 1, 2, or 3 SGC nuclei in 2 M, 12 M, and 21 M old mice. The data are presented as mean ± SD.
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Figure 5—source data 1
Marker genes for Illumina snRNA-seq analysis.
- https://cdn.elifesciences.org/articles/100217/elife-100217-fig5-data1-v1.xls
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Figure 5—source data 2
DGE for SGCs in aged vs adult DRG.
- https://cdn.elifesciences.org/articles/100217/elife-100217-fig5-data2-v1.csv
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Figure 5—source data 3
GO pathway analysis for SGC in aged vs adult DRG.
- https://cdn.elifesciences.org/articles/100217/elife-100217-fig5-data3-v1.csv
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Figure 5—source data 4
KEGG pathway analysis for SGC ins aged vs adult DRG.
- https://cdn.elifesciences.org/articles/100217/elife-100217-fig5-data4-v1.csv
Figure 5—figure supplement 1
Quality control, DEG, and pathway analysis of aged vs. adult SGCs for Illumina snRNA-Seq.
(A, B) Sample QC for Illumina snRNA-Seq. (C) Volcano plot of upregulated and downregulated genes in SGCs of aged vs. adult mice. (D) Gene ontology analysis of upregulated genes in SGCs in aged mice compared to adults.
Figure 6 with 2 supplements
ETBR inhibition increases the expression of Cx43 in SGCs in adult and aged mice.
(A) UMAP overlay for expression of Gja1 in adult and aged mouse DRG. (B) Scheme of drug treatment and peripheral nerve injury model. (C) Quantification of the percentage of the Cx43/FABP7 expression area. (D) Quantification of the average number of Connexin 43 (Cx43) puncta per FABP7+ cell. The ratio of total Cx43 puncta to the number of FABP7+ cells surrounding a TUJ1+neuron was measured. N(cell number)=60(adult, uninjured), 62(aged, uninjured), 96(vehicle, adult, SNC), 117(bosentan, adult, SNC), 74(vehicle, aged, SNC), and 74 (bosentan, aged, SNC), respectively. The data are presented as mean ± SD. (E) Representative immunostaining images showing Connexin 43 (Cx43), FABP7, and TUJ1 in L4 DRGs from the indicated condition (scale bars, 50 μm). (F) Proposed model for the role of ETBR in age-dependent decline in axon regenerative capacity.
Figure 6—figure supplement 1
ETBR inhibition increases the expression of Cx43 in SGCs after DRC.
(A) Representative immunostaining images of connexin 43 (red), Fabp7 (gray), and TUJ1 (cyan) in L4 DRGs from mice of the indicated ages and treatments 3 d after dorsal root crush injury (scale bars, 50 μm). (B) Quantification of the percentage of the Cx43/FABP7 expression area in each condition. (C) Quantification of the average number of Connexin 43 (Cx43) puncta per FABP7+ cell. The ratio of total Cx43 puncta to the number of FABP7+ cells surrounding a TUJ1-positive neuron was measured. Different colors were used to indicate distinct biological replicates (mouse). N(cell number)=93 (uninjured), 97 (Vehicle, DRC), and 124 (Bosentan, DRC), respectively. The data are presented as mean ± SD.
Figure 6—video 1
Z-stack video of DRG section immunostained for FABP7 and CX43.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | C57BL/6 | Envigo; Jackson Laboratory | Envigo: 027; JAX: 000664 RRID:IMSR_CRL:027 RRID:IMSR_JAX:000664 | Female and male, used at various ages |
| Genetic reagent (M. musculus) | Ai14 | Jackson Laboratory | JAX: 007914 RRID:IMSR_JAX:007914 | B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J |
| Genetic reagent (M. musculus) | Fabp7CreER | Toshihiko Hosoya (gift) | Crossed with Ai14 to generate Fabp7CreER::Ai14 | |
| Biological sample (M. musculus) | DRG tissue | This paper | L3-L5, L4-L5 DRGs isolated from adult and aged mice | |
| Chemical compound, drug | Bosentan | Sigma-Aldrich | Sigma: PHR2708 | 10 mg/kg, oral gavage |
| Chemical compound, drug | Ambrisentan | Tocris | Tocris: 5828 | 10 mg/kg, oral gavage |
| Chemical compound, drug | BQ788 | Sigma-Aldrich | B157 | 1 µM for in vitro use |
| Chemical compound, drug | BQ123 | R&D Systems | 1188 | 1 mM for in vitro use |
| Chemical compound, drug | IRL620 | Sigma-Aldrich | SCP0135 | 100 nM for in vitro use |
| Antibody | Rabbit anti-ETBR (polyclonal) | Abcam | ab117529 RRID:AB_10902070 | WB (1:500) |
| Antibody | Mouse anti-ET-1 (monoclonal) | Invitrogen | MA3-005 RRID:AB_2096246 | WB (1:500) |
| Antibody | Rabbit anti-GAPDH (polyclonal) | Cell Signaling | 5174 s RRID:AB_10622025 | WB (1:5000) |
| Antibody | Rabbit anti-FABP7 (polyclonal) | Invitrogen | PA5-24949 RRID:AB_2542449 | IHC (1:1000) |
| Antibody | Rabbit anti-STMN2 (SCG10) (polyclonal) | Novus/Techne | NBP1-49461 RRID:AB_10011569 | IHC (1:1000) |
| Antibody | Rabbit anti-Cx43 (polyclonal) | Cell Signaling | 3512s RRID:AB_2294590 | IHC (1:200) |
| Antibody | Mouse anti-TUJ1 (βIII tubulin) (monoclonal) | Biolegend | 801202 RRID:AB_2313773 | IHC (1:1000) |
| Antibody | Rabbit anti-PGP9.5 (polyclonal) | LS Bio | LS-B5981-50 | IHC (1:500) |
| Antibody | Secondary antibodies Alexa Fluor 488/594/647 | Invitrogen | Various | IHC (1:500) |
| Commercial assay, kit | RNAscope Fluorescent Multiplex Kit | ACD (Advanced Cell Diagnostics) | For RNA in situ hybridization | |
| Commercial assay, kit | RNeasy Mini Kit | QIAGEN | 74104 | RNA extraction |
| Commercial assay, kit | High-Capacity cDNA Reverse Transcription Kit | Thermo Fisher | 4368814 | cDNA synthesis |
| Commercial assay, kit | PowerUp SYBR Green Master Mix | Thermo Fisher | A25780 | qPCR |
| Commercial assay, kit | LIVE/DEAD Fixable Aqua Dead Cell Stain Kit | Thermo Fisher | L34965 | Cell viability stain |
| Chemical compound | DAPI | Sigma-Aldrich | D9542 | (300 nM) nuclear stain |
| Commercial assay, kit | ProLong Gold Antifade Mountant | Invitrogen | P36930 | For mounting fluorescent samples |
| Chemical compound | PFA (paraformaldehyde) | Various | 4% used for fixation | |
| Chemical compound | OCT compound | Tissue-Tek | For cryosectioning | |
| Peptide, recombinant protein | NGF (Nerve Growth Factor) | Alomone | N-240 | Used in DRG explants |
| Chemical compound | HBSS | Thermo Fisher, Gibco | 14175–079 | Dissection medium |
| Chemical compound | HEPES | Thermo Fisher, Gibco | 15630080 | Buffering agent |
| chemical compound | Papain | Worthington Biochemical | LS003126 | For tissue dissociation |
| Chemical compound | L-cysteine | Sigma | C7352 | Added to dissociation mix |
| Chemical compound | DNase I | Worthington Biochemical | LS002139 | For DNA degradation during dissociation |
| Chemical compound | Collagenase | Sigma | C6885 | Enzyme for tissue dissociation |
| Chemical compound | Neurobasal-A Medium | Thermo Fisher, Gibco | 12349015 | Culture medium |
| Commercial assay, kit | B-27 Plus Supplement | Thermo Fisher, Gibco | A3582801 | Culture supplement |
| Commercial assay, kit | GlutaMAX Supplement | Thermo Fisher, Gibco | 35050061 | Glutamine substitute |
| Chemical compound | Poly-D-lysine | Coating coverslips | ||
| Biological sample (M. musculus) | DRG explants | This paper | For explant culture | |
| Software, algorithm | Fiji | Schindelin et al., 2012 | RRID:SCR_002285 | Image analysis |
| Software, algorithm | QuantStudio 6 Flex System | Thermo Fisher | For qPCR analysis | |
| Software, algorithm | Seurat v5.1.0 | Satija Lab | RRID:SCR_007322 | For sc/snRNA-seq analysis |
| Software, algorithm | CellRanger v7.1.0 | 10 X Genomics | RRID:SCR_017344 | For scRNA-seq processing |
| Software, algorithm | Pipseeker v3.3.0 | Fluent BioSciences | For snRNA-seq processing | |
| Software, algorithm | Imaris v9.7 | Oxford Instruments | RRID:SCR_007370 | Vessel quantification |
Additional files
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Supplementary file 1
List of qPCR Primers.
- https://cdn.elifesciences.org/articles/100217/elife-100217-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/100217/elife-100217-mdarchecklist1-v1.docx
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Endothelin B receptor inhibition rescues aging-dependent neuronal regenerative decline
eLife 13:RP100217.
https://doi.org/10.7554/eLife.100217.3
