Fig 2: (A) UMAP overlays of transcriptomes reported in Kimmel et al., Development 2020 (peach) and the datasets reported in this manuscript.(B) Marker-gene overlays showing canonical myogenic regulatory factors as well as S100b showing S100b is confined to the UMAP cluster containing primarily cells reported in this manuscript. (C) Pearson correlation coefficients across datasets reported herein as well as in Kimmel et al. shows high similarity overall, with three groups of highest correlation being the activated cells from Kimmel et al., our aged v3 datasets, and the young and aged quiescent v2 datasets. Some of this grouping is likely a result of differences in features and counts among the libraries. (D) Stacked bar graph showing origin of Pax7+S100b+ cells. Most are recovered in the aged v3 datasets we report, though several are also among the datasets reported by Kimmel et al., and in our young v3 dataset.

Fig 3: Single cell analysis of skeletal muscle stem cells (MuSCs) in age shows a subset express transcripts associated with the neuromuscular junction.(A) Experiment design schematic. MuSCs were fluorescent activated cell sorting (FACS) enriched from young and aged wild-type (WT) mice, then analyzed by IF, single cell mRNA sequencing (scRNA-Seq), or single-molecule fluorescence in situ hybridization (smFISH). (B) Left: Dimensional reduction and unsupervised clustering of young (2–3 months) and aged (22–24 months) WT FACS-isolated MuSCs and limb muscle cells from 24-month Tabula Muris Senis datasets colored by sample type showing overlap between FACS-enriched MuSCs and MuSC clusters among all mono-nucleated cells. Four and five independent replicates of young and age, respectively, were sequenced where each sample is composed of a pool from two mice. Cell types were identified according to markergene expression and labeled on the plot. NMJ – neuromuscular junction; TL – tenocyte; MSC – mesenchymal stromal cell; Immune – CD45+ immune cell; Endo – endothelial cell; SMC – smooth muscle cell. Right: Plots split by sample type showing contribution to uniform manifold approximation and projection (UMAP) and re-clustered UMAP of the NMJ-associated cluster. (C) Stacked bar plot representing the fraction of the sorted cell populations that clustered with each mono-nucleated cell type as well as the number of Pax7+S100b- (MuSC) and Pax7+S100b+ (NMJ) cells based on IF stains. A higher fraction of FACS purified cells from aged mice are associated with the NMJ cluster as compared to young. Quantification by FACS imaging of S100ß and Pax7 proteins shows that less than 5% of young MuSCs co-express both markers compared to 15% of aged MuSCs. **** denotes p < 0.0001 and ns denotes p > 0.05 by two-sided z-test for proportions with pairwise comparisons using Holm p-value adjustment. Threshold for significance was considered p < 0.05. Sample sizes (n) were 3568, 483, 6532, and 880 cells for scRNA-Seq young, FACS young, scRNA-Seq aged, and FACS aged, respectively. Representative IF images are shown in Figure 3—figure supplement 2. (D) Re-clustered UMAP diagrams of the NMJ-associated cluster and overlay of specific genes. (E) Representative pseudocolored images of MuSCs from young or aged mice, stained with DAPI (blue), probes against Myod1 mRNA (red, top panels), probes against Nrn1 mRNA (red, bottom panels), and probes against polyA RNA (yellow). Nuclear (cyan) and cell (yellow) boundaries are also represented. Scale bar, 2 µm. (F) Box plots of spots/cell, wherein the central line, box edges and bars represent the median, 25th and 75th quartiles, and data range respectively. p-Values are based on two-tailed t-test with Welch’s correction, ****p < 0.0001, **p<0.01, *p<0.1, NS – not significant, n=210 cells for young and n=210 cells for aged. (G) Volcano plot of differential gene expression between Pax7+ cells from the NMJ cluster and bulk RNA-Seq datasets from Schwann cells. Gene counts of the single cell datasets were merged across cells to generate ‘sudo-bulk’ expression matrices, where half of the cells from each sample were summed separately and considered a biological and technical replicate in order to achieve sufficient sample size. DESeq2 was used to calculate differential expression. More than 2500 genes were differentially expressed. n = 4 for Pax7+NMJ sequencing replicates and n = 5 for Schwann cell sequencing replicates.

Fig 4: AH induces release of circulating nucleic acids and expression of sterile inflammatory protein molecules.Level of SI markers in plasma was analysed after exposure to hypoxia for different time durations (0–24 hrs); (a) eDNA (µg/ml), (b) eRNA (µg/ml), (c) HMGB1 (ng/ml), (d) vWF (ng/ml), (e) HSP70 (ng/ml), (f) HSP90 (ng/ml), and (g) s100b (pg/ml). Data are shown as mean ± SEM (n = 5/group/each time point) from one experiment representative of three independent experiments, all performed in triplicate. One-way ANOVA revealed statistical significance in the results (*p< 0.05).

Fig 5: Neurodegeneration activates muscle stem cells (MuSCs) in a similar manner to aging and rescue of motor neurons partially reverses this activation state.(A) Schematic of neuromuscular junction (NMJ) in young (top) and aged or Sod1-/- (bottom) muscle, whereby NMJ becomes fragmented or partially denervated. (B) Representative images of Sod-/- and SynTgSod1-/- myofibers from uninjured and sciatic nerve transection (SNT) tibialis anterior muscles stained with DAPI (blue) and a-bungarotoxin (orange). (C) Quantification of the number of subsynaptic nuclei per synaptic area in uninjured control and SNT muscle fibers from both Sod1-/- and SynTgSod1-/- mice, normalized to the subsynaptic nuclei per area of Sod1-/- uninjured control (*p < 0.05, two-way ANOVA with Tukey post hoc test). (D) Uniform manifold approximation and projection (UMAP) dimensional reduction of fluorescent activated cell sorting (FACS)-isolated MuSCs (FSMs) from young and aged wild-type (WT) mice, 10-month Sod1-/- mice, 10-month Sod1-/- rescue (SynTgSod1-/-) mice, and public Tabula Muris Senis limb muscle datasets colored by sample type. One and two independent replicates of Sod1-/- and Sod1-/- rescue (SynTgSod1-/-) mice, respectively, were sequenced where each sample is composed of a pool from two mice. Cell types were identified according to markergene expression and labeled on the plot. TL – tenocyte; MSC – mesenchymal stromal cell; Immune – CD45+ immune cell; Endo – endothelial cell; SMC – smooth muscle cell. (E) Stacked bar plot representing the fraction of the sorted cell populations that clustered with each mono-nucleated cell type as well as the number of sorted cells that were Pax7+S100b- (MuSC) and Pax7+S100b+ (NMJ) cells based on IF staining. More FACS purified MuSCs from Sod1-/- mice cluster with NMJ-associated cells when compared to SynTgSod1-/- mice. **** denotes p < 0.0001 and * denotes p < 0.05 by two-sided z-test for proportions with pairwise comparisons using Holm p-value adjustment. Threshold for significance was considered p < 0.05. Sample sizes (n) were 6042, 1622, 10445, and 1614 cells for single cell mRNA sequencing (scRNA-Seq) Sod1-/-, FACS Sod1-/-, scRNA-Seq SynTgSod1-/-, and FACS SynTgSod1-/-, respectively. Representative IF images are shown in Figure 5—figure supplement 2. (F) Heatmap of the top 75 differentially expressed genes between MuSCs isolated from Sod1-/- and SynTgSod1-/- mice. All genes shown have a log fold change greater than 0.25 and a p_adjusted value less than 10-20.