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Premature skeletal muscle aging in VPS13A deficiency relates to impaired autophagy

Acta Neuropathologica Communications volume 13, Article number: 83 (2025) Cite this article

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.

Introduction

VPS13A disease or chorea-acanthocytosis is an ultra-rare autosomal recessive disorder affecting 1,000 patients worldwide with the onset of symptoms during young adulthood [52]. Chorea-acanthocytosis manifests as a progressive, usually choreic, movement disorder accompanied by behavioural and cognitive changes, epilepsy and peripheral neuropathy and/or myopathy [52]. Chorea-acanthocytosis is caused by mutations in the chromosome 9 gene VPS13A (vacuolar protein sorting 13 homolog A) encoding the protein VPS13A (or chorein), and thus was recently renamed “VPS13A disease” [70]. Although our understanding of VPS13A structure and function has progressed, much about its homeostatic roles in VPS13A disease target organs such as basal ganglia, the erythropoietic niche and skeletal muscle remain to be investigated. Studies of cellular and animal models of the disease suggest that absence of VPS13A results in altered vesicle trafficking, impaired autophagy and accumulation of autophagic markers [38, 62]. Of note, cellular models expressing VPS13A revealed that it mediates direct bulk lipid transfer between membranes (see [27] for review). We recently reported that Vps13a−/− mice recapitulate hematologic and neurologic features of patients with VPS13A disease [53]. Of note, Vps13a−/− mice show acanthocytes with retention of multivescicular body, dyserythropoiesis and signs of both hyper and hypokinetic movement disorders [53]. In isolated Vps13a−/− basal ganglia we observed an impairment of autophagy involving the beclin-1 pathway, which was associated with accumulation of neurotoxic proteins such as active Lyn (phospho-Lyn), γ-synuclein and phospho-tau, suggesting dysfunctional proteostasis. Findings of neuroinflammation shown by Vps13a−/− mice are shared with those of Parkinson´s disease (PD) and others among the more common neurodegenerative disorders [53].

The early onset increase of serum muscle phosphocreatine kinase (CK) in VPS13A disease has been linked to the heterogeneous neuromuscular manifestations of the disease, characterized mainly by peripheral neuropathy, but primary myopathic alterations can also be detected [52, 54, 67]. Clinically, neuromuscular involvement of VPS13A disease manifests very variably, ranging from asymptomatic hyper-CK-emia and areflexia to severe muscle atrophy and immobilizing paresis [54, 67]. In some cases, neuromuscular involvement may be one of the main factors affecting quality of life and independent living. Although VPS13A disease is often primarily referred to as a neurodegenerative disorder of the basal ganglia, it is clearly a multisystem disease also involving the neuromuscular system. However, the pathophysiology of muscular VPS13A deficiency and the heterogeneity of clinical involvement are still poorly understood. Currently, data on skeletal muscle involvement in patients derive mainly from case series reporting (i) accumulation of tissue transglutaminase products, (ii) increase in number of small fibers associated with increased acid phosphatase activity; (iii) fascicular atrophy and (iv) selective fat infiltration [37, 44, 64, 67]. Although these reports describe patients with VPS13A disease at different disease stages, together they suggest a possible link between the VPS13A absence and autophagy perturbation in skeletal muscles from patients with VPS13A disease. The findings also resemble the reported acceleration of muscle aging by inhibition of autophagy [6, 8], characterized by increased oxidative damage and by accumulation of Neural Cell Adhesion Molecule (of NCAM) and other neuromuscular junction-enriched post-synaptic membrane components that are generally undetectable in adult myofibers. Of note, cytosolic and myolemmal relocalization of the accumulated NCAM is generally enriched near the neuromuscular junction, reflecting loss of myofiber innervation [6, 8].

Here, we bridge this gap by leveraging our recently described mouse model of VPS13A disease (Vps13a−/− mice) [53]. Although the neurologic phenotype in mice manifests beyond the age of 12 months, we have focused our skeletal muscle analysis on early stage disease in 8 month-old Vps13a−/− mice to minimize possible confounding effects of age [53]. Vps13a−/− mice exhibited increased muscle damage and depleted energy stores, marked by impaired fatty acid oxidation, oxidative stress and disrupted autophagy pathways. Rapamycin treatment mitigated autophagy deficits and oxidative markers, highlighting the role of VPS13A deficiency in accelerating muscle aging, which is corroborated by observations in patient muscle biopsies.

Materials and methods

Mouse model and design of the study

Experiments were performed on age- matched WT (C57BL/6J) and Vps13a−/− mice [53]. Since a gender effect on autophagy has been reported in models of neuromuscular disease such as Amyotrophic Lateral Sclerosis [19], we chose to study only female mice in the present study. WT and Vps13a−/− mice underwent 24 h starvation as indicated. Both starved and fed mice were treated with either vehicle or colchicine (CLC) at the dosage of 0.4 mg/kg by intraperitoneal injection (i.p.) every 12 h for 3 times, as previously reported [63]. Two subgroups of Vps13a−/− mice were treated with either vehicle or rapamycin (0.5 mg/Kg/d by i.p for 5 days) [35]. Isoflurane-anesthetized mice were randomly assigned to experimental groups and blindly analyzed [14]. Plasma creatine kinase (CK) was performed using standard biochemical assays [26]. Muscle wasting was evaluated as previously described [21]. Muscle MDA was determined as previously reported [39, 42]. We also studied muscle biopsies from 3 patients with VPS13A disease compared to 3 control subjects without neuromuscular diseases (normal “myohistology”). Demographic data are shown in Table 1 S.

Behavioral test

A group of 26 (12 WT and 22 Vps13−/−) female mice were tested on the Rotarod apparatus followed by CatWalk XT test the following day. Behavioral tests were performed on a consistent time window (09.00 a.m. − 01.00 p.m.) during the dark phase of the inverted 12/12-h light-dark cycle; mice were acclimated to the testing room for at least 30 min.

Rotarod

Mice were tested on the Rotarod apparatus (Ugo Basile, Varese, Italy). Before the test phase, animals underwent a training session consisting of three trials at a constant speed of 4 rpm for 3 min. Each session was separated by 30-minute inter-trial intervals. ​At the end of the training session, mice were returned to their home cage for 2 h.​During the test phase, each animal underwent three trials (separated by 30-minute inter-trial intervals) at an accelerating speed from 4 to 40 rpm over 5 min. The mean latency to fall off the rotarod (average of the three trials) was used for statistical analysis. Pearson correlation analysis was used to assess the linear association between two continuous variables. The Pearson correlation coefficient (r) was employed as a quantitative measure of the strength and direction of the linear relationship between the variables.

CatWalk

Mice were tested on the CatWalk XT 10.7 (Noldus Information Technology BV, Wageningen, The Netherlands). The test consisted of three days of training to familiarize the mice to cross the walkway without hesitations, and a fourth day in which gait parameters were recorded (test day) [24]. On the test day gait parameters including run duration, run speed, swing speed, and stride length were evaluated in three runs without hesitation. All data analyses were performed with a pixel threshold value ≥ 25 arbitrary units.

Mouse histopathological analysis and electron microscopy

Histochemical and immunohistochemical analysis were carried out in skeletal muscles (tibialis and quadriceps) frozen in isopentane pre-cooled in liquid nitrogen and then sectioned at 8 μm in a cryostat. H&E, COX (cytochrome c oxidase), SDH (succinate dehydrogenase), acid phosphatase stains and p62 immunomicroscopy were performed by following standard protocols. Details are reported in Supplemental Materials.

Metabolomic analysis

Metabolomics analyses were performed as previously described [48]. Metabolites were extracted from snap-frozen muscle biopsies (10 mg) in 1 ml of ice-cold methanol/acetonitrile/water (5:3:2 v/v/v) [10], separated on a Kinetex XB-C18 column (Phenomenex), and analyzed using a UHPLC system (Vanquish, Thermo, San Jose, CA) coupled to a Q Exactive quadrupole-Orbitrap mass spectrometer (Thermo, San Jose, CA), as detailed by Nemkov et al. [48].

Immunoblot analysis

Immunoblot analysis was carried out on homogenized quadriceps as previously described [43, 53]. Details are reported in Supplemental materials.

RNA isolation, cDNA synthesis, and quantitative qRT-PCR

Quantitative RT-PCR (qRT-PCR) was performed by the SYBR-green method, following standard protocols with an Applied Biosystems ABI PRISM 7900HT Sequence Detection system. Details are reported in Supplemental materials.

Statistics

Two-tailed unpaired Student t-test or two-way analysis of variance with Tukey’s multiple comparisons were used for data analyses. Whenever indicated we used unpaired Student t-test or one-way ANOVA algorithm for repeated measures using Graph-pad 10.2. Normality was assessed with Shapiro-Wilk test. Data show values from individual mice and are presented as means ± SEM. Differences with p < 0.05 were considered significant.

Results

Muscle biopsies from patients with VPS13A disease suggest a perturbation of autophagy also detected in skeletal muscle from Vps13a −/− mice

Previous clinical case reports have suggested a possible link between the absence of VPS13A and perturbation of autophagy in the often mild or slowly progressive muscle symptoms of patients with VPS13A disease [29, 37, 44, 58, 64, 67]. We have therefore used our access to muscle biopsies from patients with VPS13A disease and healthy controls to further investigate this link. Patient clinical characteristics are reported in Table 1S. VPS13A was undetected in muscle biopsies from patients with VPS13A disease as compared with healthy controls (Fig. 1a). Patients with VPS13A disease exhibited increased muscle protein oxidation as determined by Oxyblot (Fig. 1b), associated with increased levels of the autophagy initiator LC3II (lipidated form of LC3) and with accumulation of the late phase autophagy markers LAMP1, LAMP2 and P62 (Fig. 1c, d). These data suggest the absence of VPS13A might sustain an impairment of autophagy, favoring a pro-oxidant environment in patients with VPS13A disease. To better understand the role of VPS13A in muscle homeostasis, we studied Vps13a−/− mice as a reliable model for VPS13A disease [53]. In Vps13a−/− skeletal muscle, Vps13a expression was undetectable as compared to wild-type (WT) muscle (Fig. 1e), consistent with previous reports on skeletal muscle biopsies from patients with VPS13A disease [37, 44, 64, 67]. We found significant increase of plasma CK (Fig. 1f) and decreased muscle mass (Fig. 1Sa) in 8-month-old Vps13a−/− mice compared to either 2-month-old Vps13a−/− mice or WT animals. 8-month-old Vps13a−/− mice exhibited a significantly worse performance on the Rotarod of Vps13a−/− mice when compared to either WT controls or 2-months old Vps13a−/− mice (Fig. 1Sb). Of note, worsening of performance of Vps13a−/− mice was strictly correlated to muscle weight (Fig. 1Sc). To overcome possible limitation related to mouse weight, we used CatWalk gait analysis, focusing on parameters independent to body weight. The gait analysis revealed significant alterations in locomotor parameters in Vps13a−/− mice when compared to WT controls. Specifically, affected mice exhibited a prolonged run duration (Fig. 1Sd), and a significant reduction in run speed, stride length and swing speed (Fig. 1Se, f, g). These findings align with previous studies showing locomotor deficits in models of VPS13A disease and mirror the motor symptoms observed in human patients [53, 66]. In Vps13a−/− mice (4–8 months of age), the muscle wasting was associated with significant increase of LC3-II when compared to matched WT animals, in agreement with human results (Fig. 1g). This difference was not found in 12-month-old mice, most likely related to age-dependent autophagy impairment as previously reported [6]. We therefore decided to focus further studies on 8 months old Vps13a−/− mice.

Fig. 1
figure 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.

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Morphologic analysis of skeletal muscle by H&E stain and enzyme histochemical assessment of COX and SDH activity revealed no obvious alterations in Vps13a−/− mice compared to WT animals (Fig. 2Sa). However, electron microscopic (EM) examination revealed the presence of several autophagic structures, which were not detected in WT muscles (Fig. 2a). In addition, EM analysis of mitochondria showed increased mitochondrial area, reduced number of mitochondrial cristae and increased cristae width in Vps13a−/− muscle compared to WT littermates, without change in length of cristae (Fig. 2b). These findings might reflect increased intracellular oxidation [30]. Accordingly, protein oxidation and lipid peroxidation in Vps13a−/− skeletal muscle was higher than in WT muscle (Fig. 2c, d). These data together indicate that absence of VPS13a is associated with alteration of autophagy-lysosome system promoting muscle dysfunction. This conclusion was corroborated by the presence of muscle fibers with multiple small foci of acid phosphatase reactivity (red) in Vps13a−/− muscle not-present in WT muscle (Fig. 2e).

Fig. 2
figure 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.

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Metabolomic analysis of skeletal muscles from Vps13a −/− mice revealed severe energy stress and impaired fatty acid metabolism

As autophagy perturbation might affect muscle cell homeostasis and promote metabolic reprogramming [9, 22], we compared the metabolomes of Vps13a−/− and WT skeletal muscle. Mass spectrometry-based metabolomics revealed severe reduction in high energy phosphate compounds in Vps13a−/− muscle (Fig. 3). Specifically, the observed depletion of ATP, ADP, GTP, GDP and UTP (lower half of the heat map in Fig. 3a and volcano plot in 3b) indicated severe impairment of energy metabolism, consistently reduced phosphocreatine pools. Reduced levels of NAD+ (Fig. 3a) and its nicotinate ribonucleotide precursor (Fig. 3a-b, and pathway analysis in 3c) were accompanied by accumulation of the NAD breakdown products ADP-ribose and nicotinamide (Fig. 3a and b), consistent with ADP-ribosyl cyclase-mediated catabolism of NAD [49]. Purine breakdown and oxidation products (allantoin) and salvage products (adenylosuccinate) accumulated in Vps13a−/− mice (Fig. 3a and pathway analyses in panels c-d). Urea cycle activation (ornithine, argininosuccinate) and elevation in free amino acids (L-lysine, L-methionine, L-threonine, L-tryptophan) were consistent with defective amino acid metabolism, perhaps a consequence of increased turnover or decreased de novo protein synthesis (one of the most ATP-intensive of intracellular processes) [4]. Impaired fatty acid oxidation was noted, with accumulation of several acyl-carnitines in Vps13a−/− muscle. Previous studies have shown that increased concentration of ACs is as marker of incomplete fatty acid oxidation to diagnose inborn fatty acid oxidation defects [40, 56]. More recently, incomplete fatty acid oxidation secondary to mitochondrial dysfunction has been recently shown in exercise intolerance in long COVID patients [25]. Of note, glycolytic precursors to ATP-synthesis steps, bisphoshoglycerate, phosphoglycerate and phosphoenolpyruvate were all depleted in Vps13a−/− muscle. Increased oxidative stress in Vps13a−/− muscle reflected not only increased purine deamination, but also depletion of the antioxidant ascorbate (vitamin C) and taurine. T The combination of cell energy depletion, impaired fatty acid metabolism and increased oxidation might promote cell membrane weakness [15], favoring the CK release from muscle into the circulation, which characterized both mice and humans with VPS13A disease.

Fig. 3
figure 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.

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Proteomic analysis revealed defective autophagic responses and up-regulation of pro-apoptotic cascades

We next analyzed the proteomes of the same tissues to assess the abnormal proteostasis anticipated upon genetic ablation of Vps13a. Our results (Fig. 4) indicated down-regulation of metabolic enzymes (i.e. APRT for adenosine salvage/recycling– Fig. 4a). Volcano plot analysis of the proteomics results revealed downregulation of PFKP (phosphofructokinase, platelet isoform), the rate limiting enzyme of glycolysis and a low abundance isoform (~ 11% in circulating blood cells other than platelets, less abundant than the muscle isoform), recently linked to bioenergetics in the context of severe ATP depletion [49]. Elevated levels of enzymes involved in iron metabolism and ferroptosis, such as STEAP3 [13], were associated with compensatory upregulation of VPS25 and other VPS proteins, and upregulation of protein damage-repair enzyme PCMT1. Pathway analysis of the top-most down-regulated and up-regulated processes in Vps13a−/−mice identified impaired energetics, autophagy and elevated apoptotic cascades (Figs. 3c and 3Sb, c).

Fig. 4
figure 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.

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Increased protein oxidation in Vps13a −/− muscle reflects a defective clearance of ubiquitinylated components

The above findings prompted us to mine the proteomics results for post-translational modifications (PTMs). We first noted that proteins with PTM accumulated in Vps13a−/− muscles (Fig. 5a). Ubiquitinated proteins, advanced glycation end-products (carboxyethyl-lysine) and irreversible cysteine oxidation (to dehydroalanine and, rarely, ammonia loss) were increased in Vps13a−/− muscle (Fig. 5b-d), especially among mitochondrial electron transport chain complex components (NDUFS3, NDUV1, etc.), metabolic enzymes (ALDOA) and myofibril regulatory components (MYOM1, PYGM, RYR1, the last regulating calcium metabolism for myofibril contraction). The defective protein processing, down-regulated autophagic processes and up-regulated pro-apoptotic pathways in Vps13a−/− muscle prompted our hypothesis that protein accumulation of PTMs, especially irreversibly modified moieties, may reflect, at least in part, defects in redox-sensitive and ATP-dependent proteolysis, through either ubiqutinyl-transferase or the proteasome. Indeed, ATP pool depletion in Vps13a−/− mice was accompanied by elevations in ubiquitinylated proteins, including several myosin light (MYLPF), and heavy chains (MYH4, MYH6, MYH7b), regulatory elements (RYR1) or ATP metabolic enzymes (adenylate kinase - AK, ATP13A5), cytochrome C (CYC) and glycolytic enzymes (ENO1). The metabolomics and proteomics data of Vps13a-deficient muscle together depicted a state of impaired energy metabolism, protein synthesis, autophagic responses and mitochondrial metabolism, accompanied by defects in myofibril stability and the myofibrillar regulatory proteome.

Fig. 5
figure 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.

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Vps13a −/− muscle was characterized by impaired autophagy

The broadly abnormal metabolomic profile of Vps13a−/− muscle prompted further investigation of autophagy in Vps13a−/− mice. We observed not only an elevated LC3II fraction (Fig. 1g), but in addition found overactivation of the autophagy initiator ULK1, as increased ULK1 phospho-form at Ser555 [16, 71] (Fig. 4Sa). These findings were associated with Vps13a−/− muscle accumulation of VPS34 and ATG14, involved in the initial phase of autophagosome formation and of late phase autophagy-related proteins ATG5 and ATG7, RAB3, and lysosomal proteins LAMP1 and LAMP2 and P62 as compared to WT muscle (Figs. 6a, and 4Sb). Of note, in Vps13a−/− mice displayed few muscle fibers with multiple small foci of P62 immunoreactivity, supporting the accumulation of P62 in the absence of VPS13A in skeletal muscle (Fig. 4Sc). Active Lyn also accumulated in Vps13a−/− muscle to levels higher than in WT (Fig. 4Sd). These data taken together support a perturbation of autophagy-lysosome system in Vps13a−/− muscle as compared to WT muscle. As proof-of-concept, we evaluated the effect of the known autophagy trigger, starvation, on muscles of both mouse strains. As expected, starvation should increase the autophagosome formation and consequently the LC3II lipidation without altering the lysosomal content [41, 46]. As shown in Fig. 6b, we found increased LC3II fraction without changing LAMP1 in muscle of starved as compared to fed WT mice. In contrast, muscle LC3II levels were indistinguishable in fed and starved Vps13a−/− mice (Fig. 6b). This result was associated with LAMP1 accumulation in muscle from starved Vps13a−/− mice in the absence of significant change in Lamp1 gene expression when compared to starved WT animals (Fig. 5Sa). In order to test autophagy flux during the absence of nutrient, we blocked the autophagosome delivery to lysosome by CLC, causing the accumulation of autophagosome [32]. The autophagic flux results by the fold increase of LC3II band between treated and untreated mice. Figure 5Sb shows the combination of starvation and CLC increased LC3-II level in WT but not in Vps13a−/− muscle. The combined data consistently support the conclusion that autophagy is impaired in muscles lacking VPS13A.

Fig. 6
figure 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.

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The muscle aging phenotype exacerbated by impaired autophagy can be rescued by Rapamycin

Previous studies have shown that the block of autophagy accelerates muscle senescence, contributing to neuromuscular junction (NMJ) instability. The resulting accumulation of neural cell adhesion molecule 1 (NCAM1) might be a marker of the skeletal muscle aging phenotype [6, 8]. Vps13a−/− muscle exhibited NCAM1 accumulation as compared to age-matched WT animals, supporting the hypothesis that impaired autophagy sustains protein oxidation to accelerate muscle aging (Fig. 6c). As a rescue experiment, we treated Vps13a−/− mice with the autophagy activator, rapamycin [33, 35]. As shown in Fig. 6d, rapamycin markedly reduced accumulation of NCAM1 as well as of terminal phase autophagy markers LAMP1 and p62 (Fig. 6d). These data together support the hypothesis that absence of VPS13A exacerbates the aging phenotype of skeletal muscles through impairment of autophagy-lysosome system. The importance of our mouse data is supported by our observation of NCAM1 accumulation in muscle biopsies from patients with VPS13A disease (Fig. 6e).

Overactivation of the unfolded protein response system (UPR) in Vps13a −/− mice further accelerates muscle aging

Activation of the UPR system in response to endoplasmic reticulum stress promoted by oxidation and accumulation of misfolded, damaged proteins is one of the hallmarks of skeletal muscle senescence [1, 36, 50]. The UPR is characterized by three branches driven by protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), inositol-requiring protein 1α (IRE1α), and activating transcription factor 6 (ATF6) [61]. Expression of ATF4, CHOP and GADD34 was higher in Vps13a−/− than in WT muscle, whereas muscle ATF6 expression was indistinguishable in the two mouse strains (Figs. 7a, and 6Sa). As expected, starvation increased expression of ATF4 and CHOP in WT but not in Vps13a−/− muscle. CHOP might be considered a pro-apoptotic factor common to the three branches of the UPR system, and prolonged CHOP expression might up-regulate expression of GADD34, another pro-apoptotic mechanism driving cells towards caspase activation [1, 36, 50]. Increased expression of both CHOP and GADD34 in Vps13a−/− muscle resulted in caspase 3 activation and increased caspase 8 expression as compared to WT muscle, associated with activation of the cell death pathway initiator p53 (Figs. 7a and 6Sb, c). Thus, Vps13a−/− skeletal muscle was characterized by a pro-apoptotic molecular signature sustained by impaired autophagy, UPR overactivation and pathologic metabolic reprogramming.

Fig. 7
figure 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.

Full size image

Inflammaging contributes to premature aging of Vps13a-deficient skeletal muscle

Previous studies have shown that muscle aging is associated with the local activation of pro-inflammatory and redox response transcriptional factors such as NF-kB and Nrf2 [2, 23, 57, 60, 68, 69]. As shown in Fig. 7c, we detected age-dependent activation of both NF-kB and Nrf2 in Vps13a−/− muscle as compared to WT muscle. NF-kB activation was higher in 12-month-old than in 2-month-old WT muscle (Figs. 7c and 7S), highlighting the potential role of NF-kB activation in inflammaging of muscle from Vps13a−/− mice. Indeed, Vps13a−/− muscle genes up-regulated at 8 months of age included the NF-kB-regulated pro-inflammatory cytokines Il-6, TNFα, Il-1b (Fig. 7d). Peak Nrf2 activation was found in Vps13a−/− muscle at 8 months of age when severe energy stress and protein oxidation were detected (Fig. 7c and 7S). Consistently, the expression of Nrf2-regulated Nqo1 and Ho-1 antioxidant systems were also induced (Fig. 7d). Of note, up-regulated muscle expression of IL-1β and other pro-inflammatory cytokines has been reported in a mouse model of Parkinson disease (PD), and is part of the senescence-associated phenotype contributing to PD sarcopenia [17, 51, 55]. In muscle biopsies from patients with VPS13A disease, we found activation of NF-kB as compared to healthy controls (Fig. 7e). Collectively, our data corroborates the working hypothesis that impaired autophagy in the absence of VPS13A is associated with a premature muscle aging phenotype.

Discussion

Here, we show for the first time that the absence of Vps13a in skeletal muscle impairs autophagy and secondarily promotes metabolic reprogramming similar to that of aging and other conditions [8]. The metabolome of Vps13a−/− skeletal muscle revealed severe depletion not only of phosphocreatine pools, but also of all high energy phosphate reservoirs. Altered adenylate pools could be explained in part by defective glycolysis, as evidenced by irreversible post-translational modifications to ALDOA and ENO1 and by severe depletion of PFKP, a PFK isoform upregulated when normal ATP levels fall > 80%. ATP depletion was likely exacerbated further by increased ubiquitination of AK, normally rate-limiting for adenylate pool preservation in the face of energetic challenge. Additional contributors to altered adenylate pools included dysregulated fatty acid metabolism as reflected in accumulation of acyl-carnitines. Purine breakdown and deamination was accompanied by depletion of NAD+ (and of its precursor nicotinate ribonucleotide) with elevation of its breakdown products ADP-ribose and nicotinamide, consistent with activation of ADP-ribosyl cyclases 1 and/or 2 (CD38/BST1) [49]. Of note, NAD pool reduction is a hallmark of aging and sarcopenia [11, 45] and oral NAD precursor supplementation has been proposed as a strategy to delay onset of age-related comorbidities [3].

Defects in autophagic cascades were incompletely compensated either by ATP-dependent proteasomal degradation of existing protein components or by de novo protein synthesis, arguably (along with ionic homeostasis) the most ATP-intensive of cellular processes. In this context, ER Ca2 + release channel and contractility regulator RYR1 of Vps13a-deficient muscle was irreversibly oxidized at cysteine residues and hyper-ubiquitinylated, as compared to WT muscle. Accumulation of ubiquitinylated myofibrillar light and heavy chain components, along with increased expression of protein damage repair enzymes such as the isoaspartate modifier L-isoaspartyl O-methyl transferase [12] was accompanied by increased levels of the ferrireductase STEAP3 [13] and other promoters of ferroptosis. The metabolic and proteomic profiles of Vps13a−/− muscle further support impairment of autophagy as a contributor to muscle dysfunction in (i) the lack of skeletal muscle autophagy activation in response to starvation and to starvation plus colchicine treatment; and (ii) the rescue of skeletal muscle autophagy in rapamycin-treated Vps13a−/− mice.

The impairment of autophagy in Vps13a−/− muscle resembled aspects of accelerated aging. Indeed, Vps13a−/− muscle exhibited accumulations of oxidized contractile proteins and NCAM1, both markers of early skeletal muscle senescence [6]. This observation was further corroborated by NCAM1 accumulation observed in muscle biopsies from patients with VPS13A disease, which suggests an instability of muscle nerve synapse. Overactivation of UPR (likely in response to sustained ER stress) served as additional evidence of premature muscle cell senescence in the absence of VPS13A. The insensitivity to starvation of ATF4 and GADD34 levels in Vps13a−/− skeletal muscle suggested UPR saturation, contributing to a pro-apoptotic profile including increased activities of caspase-3 and-8, and elevations in the ancillary senescence marker phospho-p53, in agreement with our proteomic data [7, 72]. Under physiologic condition, activation of UPR system by taking care of both protein folding and degradation within ER, contributes to skeletal muscle cell homeostasis. However, persistent overactivation of the UPR system results in accumulation of unfolded, oxidize proteins, contributing to cell muscle dysfunction. Whenever overactivation of UPR is combined with impaired autophagy, cells are re-directed towards pro-apoptotic pathways, ending in muscle waste and sarcopenia [18, 31]. In addition, sustained ER stress and overactivation of the UPR system can trigger a pro-inflammatory signaling through the activation of an NF-kB response [28, 34]. Indeed, Vps13a−/− mice display skeletal muscle activation of redox and inflammation-related transcriptional factors NFkB and NRF2, with up-regulation of their downstream gene targets such as Il-1b and Nqo1. Previous studies have shown that sterile inflammatory status activating NF-kB pro-inflammatory pathways generates a premature senescent environment (inflammaging theory) [20, 59]. This might contribute to age related muscle waste and sarcopenia [5, 65]. The activation of NRF2 pathway might try to cope chronic cell oxidation, to limit cell damage and ensure cell survival [47]. Collectively our data support a premature cell muscle aging in the absence of VPS13A. The clinical relevance of our novel findings in Vps13a−/− mice is supported by muscle biopsies from patients with VPS13A disease, which exhibit a premature aging phenotype, again characterized by (i) impaired autophagy with increased protein oxidation; (ii) accumulation of NCAM1; and (ii) chronic activation of NF-kB. Our data may contribute to clinical management of patients with VPS13A disease, suggesting a possible benefit of early exercise intervention to delay the muscle wasting that is expected to accelerate in response to subsequent/additional peripheral neuropathy.

Study limitations

Our study has two major limitations. First, the study focused on 8-month-old Vps13a−/− mice, younger in age than the 12–14-month-old mice with abnormal behavioral test results in our previous study [53]. As skeletal muscle activation of autophagy and NFkB was evident in 12-month-old WT mice, we chose 8 months of age as an optimal experimental time to avoid possible confounding effects of physiologic aging. However, histopathologic analysis of older mice might be considered for future study. Second, as VPS13A disease is an ultrarare disease, the number of muscle biopsies available for this study was limited.

Conclusions

Our data shed new light on the mechanism of muscle dysfunction in VPS13A disease. We show for the first time that absence of VPS13A results in impaired autophagy in skeletal muscle, which markedly alters the metabolic profile. Cellular energy depletion combined with prolonged protein oxidation leads to inflammaging and muscle dysfunction in patients with VPS13A disease. Future studies in a larger cohort of patients with VPS13A disease may further confirm the link between impaired autophagy and the accelerated aging phenotype in the absence of VPS13A.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

VPS13A:

Vacuolar protein sorting 13 homolog A

NCAM1:

Neural Cell Adhesion Molecule

CLC:

Colchicine

CK:

Creatine Kinase

MDA:

Malondialdehyde

H&E:

Hematoxilin & Eosin

COX:

Cytochrome c oxidase

SDH:

Succinate dehydrogenase

LC3:

Microtubule-associated protein 1 A/1B-light chain 3

LAMP1:

Lysosomal associated membrane protein 1

LAMP2:

Lysosomal associated membrane protein 2

p62:

Sequestosome-1

ATP:

Adenosine triphosphate

ADP:

Adenosine diphosphate

GTP:

Guanosine triphosphate

GDP:

Guanosine diphosphate

UTP:

Uridine triphosphate

NAD:

Nicotinamide adenine dinucleotide

APRT:

Adenine phosphoribosyltransferase

PFKP:

Phosphofructokinase

VPS25:

Vacuolar protein sorting 25

PCMT1:

Protein-L-isoaspartate O-methyltransferase

PTM:

Post-translational modifications

NDUFS3:

NADH dehydrogenase [ubiquinone] iron-sulfur protein 3

NDUV1:

Ubiquinone oxidoreductase core subunit V1

ALDOA:

Aldolase A

MYOM1:

Myomesin-1

PYGM:

Myophosphorylase

RYR1:

Ryanodine receptor 1

MYLPF:

Myosin light chain, phosphorylatable, fast

MYH4:

Myosin-4

MYH6:

Myosin-6

MYH7b:

Myosin7b

CYC:

Cytochrome C

ENO1:

Enolase 1

ULK1:

Unc-51 Like Autophagy Activating Kinase 1

ATGs:

Autophagy related proteins

RAB3:

Ras related protein 3

NMJ:

Neuromuscular junction

UPR:

Unfolded protein response

PERK:

Protein kinase R (PKR)-like endoplasmic reticulum kinase

IRE1α:

Inositol-requiring protein 1α

ATF:

Activating transcription factor

CHOP:

C/EBP Homologous Protein

GADD34:

Arowth arrest and DNA damage-inducible protein

p53:

Tumor protein 53

NF-kB:

Nuclear factor kappa-light-chain-enhancer of activated B cells

Nrf2:

Nuclear erythroid trascription factor 2

Il-6:

Interleuchine 6

TNFα:

Tumor Necrosis Factorα

IL-1β:

Interleuchine 1β

Nqo1:

NAD(P)H dehydrogenase (quinone 1)

Ho-1:

Heme oxigenase-1

PFKP:

Phosphofructokinase

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Funding

This paper was supported by the Advocacy for Neuroacanthocytosis patients (LDF, RHW), FUR-UNIVR (LDF), Dept of Veterans Affairs (CX0002342, RHW). AH is supported by the Hermann und Lilly Schilling-Stiftung für medizinische Forschung im Stifterverband. KP was supported by Rostock Academy of Science (RAS).

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Authors and Affiliations

  1. Department of Engineering for Innovative Medicine- DIMI, University of Verona, Verona, Italy

    Veronica Riccardi, Enrica Federti, Angela Siciliano, Jacopo Ceolan, Valentina Salari, Paolo Fabene & Lucia De Franceschi

  2. Department of Bomedical Sciences, University of Padua, Padua, Italy

    Carlo Fiore Viscomi & Marco Sandri

  3. Venetian Institute of Molecular Medicine, Padova, Italy

    Marco Sandri

  4. Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO, USA

    Angelo D’Alessandro, Monika Dzieciatkowska & Daniel Stephenson

  5. Translational Neurodegeneration Section “Albrecht Kossel”, Department of Neurology, University Medical Center Rostock, University of Rostock, Rostock, Germany

    Andreas Hermann & Kevin Peikert

  6. Center for Transdisciplinary Neurosciences Rostock (CTNR), University Medical Center Rostock, Rostock, Germany

    Andreas Hermann & Kevin Peikert

  7. United Neuroscience Campus Lund-Rostock (UNC), Rostock site, Rostock, Germany

    Andreas Hermann & Kevin Peikert

  8. Clinical Genetics Unit, Department of Women’s and Children’s Health, University of Padova, Padova, Italy

    Leonardo Salviati

  9. Pediatric Research Institute (IRP) - Fondazione Città della Speranza, Padova, Italy

    Leonardo Salviati

  10. Azienda Ospedaliera Universitaria Integrata Verona, Verona, Italy

    Angela Siciliano, Jacopo Ceolan, Gaetano Vattemi & Lucia De Franceschi

  11. Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, Napoli, Italy

    Immacolata Andolfo & Achille Iolascon

  12. CEINGE Biotecnologie Avanzate Franco Salvatore, Napoli, Italy

    Immacolata Andolfo & Achille Iolascon

  13. Division of Nephrology, Beth Israel Deaconess Medical Center, Department of Medicine, Harvard Medical School, Boston, MA, USA

    Seth L. Alper

  14. Department of Neuroscience, Biomedicine and Movement Science, University of Verona, Verona, Italy

    Gaetano Vattemi

  15. Neurologische Klinik und Poliklinik, LMU Klinikum, LMU München, München, Germany

    Adrian Danek

  16. Department of Neurology, James J. Peters Veterans Affairs Medical Center, Bronx, NY, USA

    Ruth H. Walker

  17. Department of Neurology, Mount Sinai School of Medicine, New York City, NY, USA

    Ruth H. Walker

  18. Department of Neurology, Martin-Luther-University of Halle-Wittenberg, Halle (Saale), Germany

    Alexander Mensch & Markus Otto

  19. Department of Neurology, Klinikum Rechts der Isar, School of Medicine and Health, Technical University of Munich, München, Germany

    Marcus Deschauer

  20. Goethe University, University Hospital Frankfurt, Neurological Institute (Edinger Institute), Frankfurt am Main, Germany

    Moritz Armbrust

  21. Goethe University, University Hospital Frankfurt, University Cancer Center (UCT) Frankfurt-Marburg, Frankfurt am Main, Germany

    Moritz Armbrust

  22. Goethe University, University Hospital Frankfurt, Frankfurt Cancer Institute (FCI), Frankfurt am Main, Germany

    Moritz Armbrust

  23. German Cancer Consortium (DKTK), Partner site Frankfurt / Mainz and German Cancer Research Center (DKFZ), Heidelberg, Germany

    Moritz Armbrust

  24. Mitochondria and Metabolism Imaging Core, Department of Endocrinology, University of California, Los Angeles, USA

    Cristiane Beninca’,

  25. Section of Anatomy and Histology, Department of Excellence in Neurosciences, Biomedicine and Movement Sciences, University of Verona, Verona, Italy

    Paolo Fabene

Authors
  1. Veronica Riccardi
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  2. Carlo Fiore Viscomi
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  3. Marco Sandri
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  4. Angelo D’Alessandro
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Contributions

VR: performed animal experiments, Western-blots, analyzed data, wrote the paper; CFV: performed muscle histologic analysis and EM, discussed data and the design of the study, critically revised the paper; MS: discussed data and experimental plan, critically revised the paper; ADA: analyzed omics data, wrote the paper; MD and DS: performed omics and analyzed data; EF: carried out Western-blots and Ips, analyzed data; AH: collected clinical lab and clinical data of patients with VPS13A disease of the study, discussed data and critically revised the manuscript; LS: functional analyses; AS: performed animal experiments; IA: RT-PCR analyses, analyzed data and wrote the paper; SLA: discussed data and experimental plan, critically revised the paper; JC: data analyses, paper editing; AI: analyzed data and critically revised the paper; GV: carried out histologic analysis, wrote the paper; AD: critically revised the paper; RHW: critically revised the paper; AM, MO, MD, MA collected clinical lab and clinical data of patients with VPS13A disease of the study, performed muscle biopsies in healthy subjects and in patients with VPS13A disease; CB: performed muscle histologic analysis, analyzed data; KP: collected clinical lab and clinical data of patients with VPS13A disease of the study, discussed data and critically revised the manuscript; VS performed rotarod, behavior tests and analyzed data; PF revised and critically discussed data on rotarod and behavior tests; LDF: designed the study, analyzed and discussed data, and wrote the manuscript.

Corresponding author

Correspondence to Lucia De Franceschi.

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Ethics approval and consent to participate

The Institutional Animal Experimental Committee of University of Verona (CIRSAL) and Italian Ministry of health approved the experimental protocol (56DC9.12) following European directive 2010/63/EU and the federation for laboratory animal science associations guidelines and recommendations. The muscle biopsies of patients with VPS13A disease were obtained for routine diagnostic reasons (P1 and P2) and postmortem (P3). Retrospective scientific use of remaining biopsy specimens/muscle samples was approved by the institutional review boards of the University Medical Center Rostock (A 2022-0058) and University Medicine Halle (2021 − 101).

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The authors declare no competing interests.

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Riccardi, V., Viscomi, C.F., Sandri, M. et al. Premature skeletal muscle aging in VPS13A deficiency relates to impaired autophagy. acta neuropathol commun 13, 83 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s40478-025-01997-y

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Acta Neuropathologica Communications

ISSN: 2051-5960

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