Premature skeletal muscle aging in VPS13A deficiency relates to impaired autophagy

Abstract

VPS13A disease (chorea-acanthocytosis), is an ultra-rare autosomal recessive neurodegenerative disorder caused by mutations of the VPS13A gene encoding Vps13A. Increased serum levels of the muscle isoform of creatine kinase associated with often asymptomatic muscle pathology are among the poorly understood early clinical manifestations of VPS13A disease. Here, we carried out an integrated analysis of skeletal muscle from Vps13a^-/- mice and from VPS13A disease patient muscle biopsies. The absence of Vps13A impaired autophagy, resulting in pathologic metabolic remodeling characterized by cellular energy depletion, increased protein/lipid oxidation and a hyperactivated unfolded protein response. This was associated with defects in myofibril stability and the myofibrillar regulatory proteome, with accumulation of the myocyte senescence marker, NCAM1. In Vps13a^-/- mice, the impairment of autophagy was further supported by the lacking effect of starvation alone or in combination with colchicine on autophagy markers. As a proof of concept, we showed that rapamycin treatment rescued the accumulation of terminal phase autophagy markers LAMP1 and p62 as well as NCAM1, supporting a connection between impaired autophagy and accelerated aging in the absence of VPS13A. The premature senescence was also corroborated by local activation of pro-inflammatory NF-kB-related pathways in both Vps13a^-/- mice and patients with VPS13A disease. Our data link for the first time impaired autophagy and inflammaging with muscle dysfunction in the absence of VPS13A. The biological relevance of our mouse findings, supported by human muscle biopsy data, shed new light on the role of VPS13A in muscle homeostasis.

Keywords: Autophagy; Energy; Inflammaging; Metabolome; NF-kB.

Fig. 1

Perturbation of autophagy is detectable in muscle biopsies from patients with VPS13A disease and in muscles from Vps13a^−/− mice. (a) Western-blot (Wb) analysis with specific antibody against Vps13A in muscle biopsies from patients with VPS13A disease (Dis.; P) and healthy controls (HC). GAPDH served as loading control. Below, densitometric analysis, means ± SEM (n = 3); **p < 0.002, paired Student t-test. (b) OxyBlot analysis of soluble fractions prepared from muscle biopsies of patients with VPS13A disease (Dis.; P) and controls. GAPDH, loading control. Representative of 3 similar experiments. Right, densitometric analysis, means ± SEM (n = 3); **p < 0.002, Student t-test. (c) Western-blot (Wb) analysis with specific antibody against LC3-I/II in muscle biopsies from patients with VPS13A disease (Dis.; P) and controls. GAPDH, loading control. Right, densitometric analysis, means ± SEM (n = 3); **p < 0.002, Student t-test. (d) Western-blot (Wb) analysis with specific antibodies against LAMP1, LAMP2 and P62 in muscle biopsies from patients with VPS13A disease (Dis.; P) and controls. GAPDH, loading control. Below, densitometric analyses, means ± SEM (n = 3); **p < 0.002 by paired Student t-test. (e) Western-blot (Wb) analysis with specific antibody against Vps13A (VPS13A) in quadriceps lysate from wild type (WT) and Vps13a^-/- mice. GAPDH, loading control. Below, densitometric analysis, means ± SEM (n = 10); **p < 0.002 by unpaired Student’s t test. (f) Plasma creatine kinase (CK) levels (U/L) in wild type (WT) and Vps13a^-/- mice aged 4 and 8 months (mo) (n = 6–10); * p < 0.05 by non-parametric Mann Whitney test. (g) Western-blot (Wb) analysis with specific antibody against LC3-I/II in quadriceps lysate from WT and Vps13a^-/- female mice at ages 4, 8 and 12 mo. GAPDH, loading control. Right, densitometric analysis, means ± SEM (n = 4); *p < 0.002 by Mann-Whitney analysis.

Fig. 2

Vps13a^−/− mouse skeletal muscle displays increased mitochondrial area with less but wider cristae, and signs of alteration autophagy-lysosome system associated with increased intracellular protein/ lipid oxidation. (a) EM analysis of WT (left) and Vps13a^-/- skeletal muscle (right). Upper panels: Vps13a^-/- muscle show the presence of altered myofiber structure and autophagic figures adjacent to mitochondria (asterisks), not detectable in WT muscles. Mitochondrial morphometry in WT and Vps13a^-/- 8-month-old quadriceps. Lower panels: higher magnification image highlighting mitochondrial ultrastructure. Scale bars: 2 μm for the upper panel, 200 nm for the lower panel. (b) Quantitative analysis of mitochondrial ultrastructure. Mitochondria show increased area and cristae number, but cristae appear wider. Unpaired Student’s t tests. (c) OxyBlot analysis of quadriceps from 8-month-old WT and Vps13a^-/- female mice. GAPDH, loading control. Representative of 4 similar experiments. (d) Malondialdehyde (MDA) content of WT and Vps13a^-/- quadriceps. Means ± SEM (n = 6), *p < 0.002 by unpaired Student’s t test. (e) Acid phosphatase staining of skeletal muscle samples from WT (top) and Vps13a^-/- mice. In Vps13a^-/- mice a few muscle fibers show multiple small foci of acid phosphatase reactivity (appearing red). Scale bar is 40 μm.

Fig. 3

Vps13a^−/− skeletal muscle metabolomics analysis highlights severe energy stress, impairment of fatty acid metabolism and depletion of phosphor-creatinine pools consistent with muscle wasting. (a) Hierarchical clustering analysis of the top 50 metabolites by unpaired t-test. (b) Volcano plot of muscle metabolites elevated (red) or reduced (blue) in Vps13a^-/- vs. WT mice. Dotted axes mark thresholds of statistical significance or log2(FC). (c) (d). Pathway analysis of the most down-regulated (c) or up-regulated pathways (d) in Vps13a^-/- vs. control muscle.

Fig. 4

Vps13a^−/− skeletal muscle metabolomics analyses suggest autophagy impairment leading to activation of pro-apoptotic pathways. (a) Hierarchical clustering analysis of the top 50 proteins by unpaired t-test. (b) Volcano plot of muscle proteins most downregulated (purple) or upregulated (red) in Vps13a^-/- vs. WT muscle. Dotted axes mark thresholds of statistical significance or log2(FC). (c) (d) (e) Pathway analysis of the most down-regulated pathways related to endoplasmic reticulum protein processing (c and d) and the most up-regulated pathways related to apoptotic processes (e) based on comparative proteomics of Vps13a^-/- and WT muscles.

Fig. 5

Peptide-level analysis of Vps13a^−/− vs. WT muscles reveals increased oxidative modification and ubiquitination of proteins. (a) Heat map of the top 500 (unpaired t-test) peptide-level changes in Vps13a^-/- mice reveals increased levels of modified peptides and depletion of unmodified peptides. (b) Volcano plot of peptide level changes (red, higher in Vps13a^-/- mice; blue, lower in Vps13a^-/- mice). Dotted axes mark thresholds of statistical significance or log2 (FC). (c) Cysteine modifications– including uncommon loss of ammonia– were increased in Vps13a^-/- mice. (d) Peptide level signatures of protein ubiquitination (K-GG) were increased in Vps13a^-/- mice, especially for myofibril light and heavy chain components.

Fig. 6

The absence of Vps13A promotes impaired autophagy, exacerbating muscle aging phenotype. (a) Western-blot (Wb) analysis with specific antibodies against LC3-I/II, ATG5, ATG7, RAB3, LAMP1, LAMP2 and P62 in quadriceps lysates from Vps13a^-/- and WT female mice aged 8 months. GAPDH, loading control. Right, densitometric analysis. Means ± SEM (n = 6); *p < 0.002, unpaired Student t-test. (b) Western-blot (Wb) analysis with specific antibodies against LC3-I/II and LAMP1 in quadriceps lysates from Vps13a^-/- and WT female mice aged 8 months, ad libitum fed or starved for 24 h. GAPDH, loading control. Below, densitometric analyses. Means ± SEM (n = 4); **p < 0.002, paired Student-t-test. (c) Western-blot (Wb) analysis with specific antibody against NCAM1 in quadriceps lysates of Vps13a^-/- and WT female mice aged 8 months. GAPDH, loading control. Below, densitometric analysis. Means ± SEM (n = 6); **p < 0.002, unpaired Student t-test. (d) Western-blot (Wb) analysis with specific antibodies against NCAM1, LAMP-1 and P62 in quadriceps lysates of 8-month-old Vps13a^-/- or WT female mice treated with rapamycin (2 mg/kg, intraperitoneally injected every 24 h for 5 days). GAPDH, loading control. Right, densitometric analyses. Means ± SEM (n = 6); **p < 0.002, unpaired t-test. (e) Western-blot (Wb) analysis with anti-NCAM1 antibody of lysates from muscle biopsies of patients with VPS13A disease (Dis.; P) and control. GAPDH, loading control. Right, densitometric analysis. Means ± SEM (n = 3); *p < 0.002, paired Student t-test.

Fig. 7

Skeletal muscles lacking Vps13A display overactivation of UPR and sustained local inflammatory response, supporting a contribution of inflammaging in muscle dysfunction of VPS13A disease. (a) Western-blot (Wb) analysis with specific antibodies against ATF4, GADD34 and CHOP in quadriceps lysates from WT and Vps13a^-/- female mice aged 8 months. GAPDH, loading control. Right, densitometric analyses. Means ± SEM (n = 6); * p < 0.05, **p < 0.002, one-way ANOVA. (b) Western-blot (Wb) analysis with specific antibodies against ATF4 and GADD34 in quadriceps lysates from 8-month-old wild type (WT) and Vps13a^-/- female ad libitum fed or starved for 24 h. GAPDH, loading control. Right, densitometric analyses. Means ± SEM (n = 6); *p < 0.05, paired Student t-test. (c) Western-blot (Wb) analysis with specific antibodies against phospho-NF-kB (Ser536), NF-kB, phospho-NRF2 (Ser40) and NRF2 in quadriceps lysates of WT and Vps13a^-/- female mice at 4, 8 or 12 months of age. GAPDH, loading control (see Fig. 4EV for densitometric analysis). (d) Quadriceps RNA levels of Il-6, Il-1b, Tnf-alfa, Nqo1 and Ho-1 normalized to Gapdh RNA in WT and Vps13a^-/- mice aged 8 months. Means ± SD (n = 3) (** p < 0.01, ANOVA test and post-hoc correction by Tukey’s multiple comparison tests). (e) Western-blot (Wb) analyses with specific antibodies against p-NF-kB (Ser536) and NF-kB in muscle biopsies from with VPS13A disease (Dis.; P) and controls. GAPDH, loading control. Below, densitometric analysis. Means ± SEM (n = 3); **p < 0.002, paired Student t-test.