Protective role of Angiogenin in muscle regeneration in amyotrophic lateral sclerosis: Diagnostic and therapeutic implications

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease with no effective treatments, in part caused by variations in progression and the absence of biomarkers. Mice carrying the SOD1G93A transgene with different genetic backgrounds show variable disease rates, reflecting the diversity of patients. While extensive research has been done on the involvement of the central nervous system, the role of skeletal muscle remains underexplored. We examined the impact of angiogenin, including its RNase activity, in skeletal muscles of ALS mouse models and in biopsies from ALS patients. Elevated levels of angiogenin were found in slowly progressing mice but not in rapidly progressing mice, correlating with increased muscle regeneration and vascularisation. In patients, higher levels of angiogenin in skeletal muscles correlated with milder disease. Mechanistically, angiogenin promotes muscle regeneration and vascularisation through satellite cell-endothelial interactions during myogenesis and angiogenesis. Furthermore, specific angiogenin-derived tiRNAs were upregulated in slowly progressing mice, suggesting their role in mediating the effects of angiogenin. These findings highlight angiogenin and its tiRNAs as potential prognostic markers and therapeutic targets for ALS, offering avenues for patient stratification and interventions to mitigate disease progression by promoting muscle regeneration.

Keywords: Angiogenin; amyotrophic lateral sclerosis; biomarkers; skeletal muscle; tiRNAs.

FIGURE 1

The dynamic of skeletal muscle denervation atrophy discriminates the disease progression in ALS mice. (A) Schematic representation of the disease progression in the two models of ALS. Created with Biorender.com (B) Muscle mass was calculated by measure of tibialis anterior (TA) muscles weight of 129SvSOD1G93A mice; C57SOD1G93A mice and NTg littermates at different time points of the disease. Percent muscle wasting was calculated relative to NTg mice at PS, OS and SY stage. Data are reported as mean ± SEM. Significance was calculated with 2‐way ANOVA with uncorrected Fisher's LSD post‐analysis (*p ≤ 0.05, **p ≤ 0.01; ***p ≤ 0.001). (C) Real‐time qPCR analysis of AChRγ mRNA transcripts in the TA of C57SOD1G93A and 129SvSOD1G93A mice compared with NTg littermates. Data are reported as mean ± SEM and expressed as fold change (%) of 129Sv NTg mice. Significance was calculated with Two‐way ANOVA with uncorrected Fisher's LSD post‐analysis (*p ≤ 0.05, ****p ≤ 0.0001).

FIGURE 2

Disease progression in ALS mice is associated with differential ANG regulation. (A, B) Representative immunoblot images and relative densitometric analysis of Angiogenin protein expression in TA muscles of C57SOD1G93A and 129SvSOD1G93A mice compared with NTg littermates. Protein levels were normalised on the total amount of protein loaded. Data are reported as mean ± SEM. Significance was calculated with Two‐way ANOVA with uncorrected Fisher's LSD post‐analysis (*p ≤ 0.05, **p ≤ 0.01).

FIGURE 3

TsRNAs identified in RNA sequencing data from muscle tissue samples. (A) Normalised tRNA read counts identified in the NTg and SOD1G93A in C57 and 129Sv genetic backgrounds. (B) Summary of differentially expressed tsRNAs from the SOD1G93A versus NTg comparison in the C57 and 129Sv genetic background. The shape represents the regulation; upward facing arrow for upregulated, downward facing arrow for downregulated and a circle for not significant and the colour indicates the log2FC. (C) Summary of top differentially expressed tsRNAs with mean TPM greater than 100 from the SOD1G93A versus NTg comparison in the C57 and 129Sv genetic background. (D) Summary of top differentially expressed tiRNAs from the SOD1G93A versus NTg comparison in the C57 and 129Sv genetic background. (E) Coverage plots of 5' ValCAC, 5' GlyCCC, 5' LysCTT tiRNAs identified as top differentially expressed tiRNAs. The x‐axis represents the nucleotide position and the y‐axis represents the coverage in TPM. The two vertical dotted lines enclose the main tRNA segment of 70 bp, with an additional 50 bp included both upstream and downstream. The dotted line illustrates the mean coverage for NTg samples, while the solid line represents the mean coverage for the SOD1G93A condition, with the shaded areas indicating the standard deviation for both conditions.

FIGURE 4

Biochemical identification of stress responses in the muscle of fast and slow ALS mouse models. (A–C) Representative immunoblot images and relative densitometric analysis of (A, B) G3BP1 and (A, C) hSOD1 protein expression in TIF and SOL of the TA muscles of C57SOD1G93A and 129SvSOD1G93A mice compared with NTg littermates. Protein levels were normalised on GAPDH expression. Data are reported as mean ± SEM. Significance was calculated for each strain with the unpaired t‐test (*p ≤ 0.05).

FIGURE 5

Increased Angiogenin protein levels in the skeletal muscle of ALS mice correlates with enhanced muscle vascularisation. (A–C) Representative confocal micrographs showing the signal colocalisation of CD31 (capillaries) and ANG (A), CD31 (vessel) and ANG (B) and Pax7 (satellite cells) and ANG (C) in the skeletal muscle of C57SOD1G93A mice at the disease onset. Scale bars: A–C = 50 μm. (D, E) Representative confocal micrographs and percentage of CD31 coverage area in the TA of fast and slow transgenic mice and relative NTg littermates at the presymptomatic and onset disease stages. Scale bar: 50 μm Data are expressed as the mean ± SEM (n = 4). Significance was calculated with one‐way ANOVA with uncorrected Fisher's LSD post‐analysis (****p ≤ 0.0001).

FIGURE 6

Increased Angiogenin protein levels in the skeletal muscle of ALS mice correlates with enhanced myogenesis. (A–C) Representative immunoblot images and relative densitometric analysis of Pax7 (A, B) and MyoD (A, C) protein expression in TA muscles of C57SOD1G93A and 129SvSOD1G93A mice compared with NTg littermates. Protein levels were normalised on the total amount of protein loaded. Data are reported as mean ± SEM. Significance was calculated with Two‐way ANOVA with uncorrected Fisher's LSD post‐analysis (****p ≤ 0.0001). (D, E) Representative confocal images of embryonal muscle fibres (eMyHC) on TA coronal sections of C57SOD1G93A (D) and 129SvSOD1G93A (E) mice at onset disease stage, immunostained with laminin. (F) The percentage of embryonal muscle fibres was calculated relative to the total number of fibres in TA muscle and in relation to respective NTg mice. Data are expressed as the mean (±SEM). Significance was calculated with Two‐way ANOVA with uncorrected Fisher's LSD post‐analysis (**p ≤ 0.01).

FIGURE 7

In vitro evaluation of satellite cell proliferation and differentiation in the presence of Angiogenin. (A) Representative confocal micrograph showing the immunostaining for Ki67 (red) and DAPI (blue) on primary satellite cell (SC) cultures of C57SOD1G93A mice in growing medium for 72 h treated with Angiogenin or Vehicle for 24 h. Scale bar = 100 μm. (B) The proliferation index was calculated as No. of Ki67 and DAPI positive cells (n = 3). Data are reported by means ± SEM. **p < 0.01 by unpaired t‐test. (C) Representative confocal images showing the immunostaining for MF20‐MyHC (green) and DAPI (blue) on C57SOD1G93A SCs derived from muscles and cultured in DM for 48 h treated with Angiogenin or Vehicle for 24 h. Scale bar = 50 μm. (D) The fusion index was calculated as (No. nuclei present in MyHC⁺ cells with two or more nuclei/No. myotubes). Data are reported as the mean ± SEM from three independent experiments for each group. **p < 0.01 by unpaired t‐test. (E–H) Representative immunoblot images and relative densitometric analysis of pSMAD2/3 (E, F), Pax7 (E, G) and MyoG (E, H) protein expression in untreated C2C12 muscle cells, treated with TGFβ or ANG. Protein levels were normalised on GAPDH. Data are reported as mean ± SEM of three independent experiments per group. Significance was calculated with One‐way ANOVA with uncorrected Fisher's LSD post‐analysis (*p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001).

FIGURE 8

The Angiogenin expression correlates with the slow disease progression in sporadic ALS patients. (A) Bivariate analysis showing the strength of association between the muscular protein expression of Angiogenin assessed by immunoblot and the ΔFRS score of ALS patients. The higher is the ΔFRS, the faster is the disease progression. The data were analysed by nonparametric Spearman's rank correlation. (B, D) Representative confocal micrographs showing the signal colocalisation of CD31 (capillaries) and ANG in slow (B) and fast (D) progressing ALS patients. (C) Representative confocal micrograph showing CD31 (vessel) and ANG (C) in slow progressing ALS patient. Scale bars: B–C: 50 μm.