Fig 2: Muscle-resident glial cell activation in a mouse model of ALS.(A) Ngfr+ cell cytofluorimetric quantification was shown in the graphs as a percentage of Itga7+Sca1–Ln– population, in 90- and 140-day-old SODG93A mice muscle (n = 4, values represent mean ± SD. **P < 0.01; by 1-way ANOVA Tukey’s multiple-comparison test). Dotted line highlights percentage in WT mice muscle. (B) Representative immunofluorescence analysis of TA muscle cryosection derived from 90- and 140-day-old SODG93A and WT mice stained for neurofilament-L (Nfl, Cyan), Ngfr (red), and Plp1 (green). Nuclei were counterstained with DAPI. Scale bars: 100 μm. (C) Quantification graph of Ngfr+/Plp1+ cells in 90- and 140-day-old WT and SODG93A mice muscle (n = 3, values represent mean ± SD; ***P < 0.001; by 1-way ANOVA Tukey’s multiple-comparison test). (D) qPCR analysis for Ngfr, Gdnf, and Tnc expression in freshly isolated Itga7+Sca1–Ln– cells derived from WT and SODG93A muscle at 90 and 140 days of postnatal life. GAPDH was used as housekeeping gene (n = 4, values represent mean ± SD; *P < 0.05, **P < 0.01; by 1-way ANOVA Tukey’s multiple-comparison test). (E) Representative immunofluorescence analysis of TA muscle cryosection derived from 90- and 140-day-old SODG93A and WT mice stained for Tnc (green), Btx (red), and Caveolin-3 (Cav3, cyan). Arrows highlight Tnc and asterisk highlights Btx. Nuclei were counterstained with DAPI. Scale bar: 20 μm. (F) Quantification of Btx and Tnc colocalization in 90- and 140-day-old SODG93A mice muscle (n = 3, values represent mean ± SD *P < 0.05; by 2-tailed Student’s t test).

Fig 3: Activation of a neurotrophic signaling pathway in muscle glial cells upon denervation.(A) Distribution in UMAP-derived clusters of single cells (scRNA-Seq) of ltga7+Sca1–Ln– isolated cells from control (CTR, left) and 3-days denervated muscle (DEN, right). (B) RNA expression heatmap for Plp1 cell populations isolated from control and denervated muscle (column) and genes (row), sorted by clusters. (C) Distribution of Ngfr, Gdnf, Tnc, and Runx2 transcripts in UMAP-derived clusters of single cells (scRNA-Seq) of ltga7+Sca1–Ln– isolated cells from control (left) and denervated (right) muscle. (D) Representative cytofluorimetric plot of Ngfr+— gated within the ltga7+Sca1–Ln– population — cells in control (left) and denervated (right) muscle. (E) Quantification of Ngfr+ cells is shown in the graphs as a percentage of ltga7+Sca1–Ln– population (n = 8 CTR, n = 10 DEN, values represent mean ± SD, ***P < 0.001; by Mann-Whitney test).

Fig 4: Chronic NF-κB activation in OLs triggers non-cell-autonomous expansion and activation of microglia cells. a Cell number of isolated Cd11b+ microglia cells from IKK2-CAPLP−CreERT2 mice is significantly increased compared to Cd11b+ microglia cells derived from control mice at 3 and 20 wpi (3 wpi: Control: 0.37 ± 0.03 × 106, IKK2-CAPLP−CreERT2: 1.3 ± 0.24 × 106 n = 20–23, 20 wpi: Control: 0.5 ± 0.05 × 106, IKK2-CAPLP−CreERT2: 0.85 ± 0.1 × 106, n = 10–23, two brains with identical genotype were pooled and set to n = 1). b Principal component analysis determines distinct differential gene expression between control and IKK2-CAPLP−CreERT2 Cd11b+ microglia at 3 and 20 wpi. c IKK2-CAPLP−CreERT2 Cd11b+ microglia are activated towards an M1 pro-inflammatory phenotype. RNA Sequencing analysis reveals upregulation of M1-like and downregulation of M2-like marker genes in IKK2-CAPLP−CreERT2 Cd11b+ microglia compared to control Cd11b+ microglia as depicted in the heatmap. All deregulated genes shown reached significance at 3 wpi, while only Lgals3, IL1β and Cd14 reached significance at 20 wpi. d and e Seahorse analysis of Cd11b+ microglia from IKK2-CAPLP−CreERT2 mice revealed decreased oxidative phosphorylation (D, OCR normalized to control; IKK2-CAPLP−Cre−ERT2: 55.2 ± 4.9%, n = 3–4) and increased glycolysis (E, ECAR normalized to control; IKK2-CAPLPCreERT2: 128.2 ± 3.1%, n = 3–4) 3 wpi. fEx-vivo analysis of primary Cd11b+ microglia revealed distinct morphological differences between control and IKK2-CAPLP−CreERT2 Cd11b+ microglia. IKK2-CAPLP−CreERT2 Cd11b+ microglia show a rather bushy, activated phenotype. Data are shown as mean ± SEM. Grey bars, control; red bars, IKK2-CA.PLP−CreERT2. Statistical analysis: Two-way ANOVA multiple comparison test followed by Bonferroni´s post hoc test, Two-tailed Mann–Whitney-U Test, (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 NS, non-significant (p > 0.05))

Fig 5: Gene expression analysis of markers associated different enteric nervous system (ENS) cell types.(A) Schematic diagram demonstrating genes associated with particular clusters of neuronal (ELAVL4+), glial (PLP1+, SOX10+), progenitor (SOX10+), and non-neural cell identities. (B) Violin plot of key genes reflecting neuronal/glial/progenitor cell fates. (C) Violin plot of key genes for non-neural cell identities (fibroblast, melanocyte, epithelial, vascular muscle, mesenchymal, endothelial, and myeloid progenitor).