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 (grey). 3D reconstruction of blood vessels via LEL labeling (scale bars, 200 μm).

B. Representative z-stack 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 identified 7 cell clusters based on known marker genes.

D. Differential gene expression analysis revealed up- and down-regulated genes across cell clusters. Red or green indicates adjusted p < 0.01, while gray indicates p ≥ 0.01.

E-F. UMAP overlay for expression of Ednra (E) and Ednrb (F).

G. Dot plot analysis showing the average gene expression (color coded) and number of expressing cells (dot size) for the marker genes.

H. 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).

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-C. 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.

D. Scheme of drug treatment and DRG explant model.

E. Representative images of DRG explants 7 days after drug treatment, immunostained for TUJ1 (black) (scale bars, 1000 μm).

F. Quantification of radial length of the 35 longest axons from DRG explants from indicated groups N=36 (BQ788; 18 replicates, Veh; 18 replicates).

G. Representative images of DRG explants immunostained for TUJ1 (green), FABP7 (magenta), and merged (scale bars, 50 μm).

Bosentan treatment improves axonal 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 24h 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. Quantificaiton 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 adult DRG neuronal culture and treatments.

G. Representative images showing TUJ1 (black) immunostaining of neurons in DRG cultures (scale bars, 100 μm).

H-I. 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.

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 (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). Quantificaiton of 50% regenerative index, calculated as the disatnce 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.

ETBR inhibition increases the expression of Cx43 in SGCs in adult and aged mice.

A. UMAP overlay for expression of Gja1 in mouse DRG.

B. Box and whisker plot of Gja1 gene expression in 5 donors, each dot represents a single cell.

C. Plot showing the average gene expression of Gja1, Gjb2, Gjb6 and Gjc1 genes in SGCs in adult (2M), mid-aged (12M), and aged (21M) mice.

D. Scheme of drug treatment and peripheral nerve injury model.

E. Quantification of the percentage of the Cx43/FABP7 expression area.

F. 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.

G. Representative immunostaining images showing Connexin 43 (Cx43), FABP7 and TUJ1 in L4 DRGs from the indicated condition (scale bars, 50 μm).

Aging alters SGCs morphology and metabolism.

A. Representative TEM images of DRG sections from adult (2M), middle aged (12M) and aged (21M) mice showing neuronal cell bodies and the enveloping SGCs (SGCs are pseudo-colored in red) (scale bars, 5 μm).

B. Quantification of the average width of SGCs per neuron in μms.

C. Frequency of neurons with 0, 1, 2, or 3 SGC nuclei per neuron in TEM images for 2M, 12M, and 21M old mice.

D. UMAP analysis of 6430 SGCs from mice of different age.

E-F. Venn diagrams were constructed to compare the sets of differentially expressed genes (DEGs) that were upregulated (E) and downregulated (F) in the mid-aged vs. adult comparison and in aged vs. adult. (FDR ≤ 0.05, fold-change ≥ 2).

G-H. KEGG analysis was performed on the common upregulated DEGs (G) and common downregulated DEGs (H). The dots in the diagram represent the count of genes associated with each pathway.