Publications (8)71.11 Total impact
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Article: Impaired autophagy contributes to muscle atrophy in glycogen storage disease type II patients.
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ABSTRACT: The autophagy-lysosome system is essential for muscle cell homeostasis and its dysfunction has been linked to muscle disorders that are typically distinguished by massive autophagic buildup. Among them, glycogen storage disease type II (GSDII) is characterized by the presence of large glycogen-filled lysosomes in the skeletal muscle, due to a defect in the lysosomal enzyme acid α-glucosidase (GAA). The accumulation of autophagosomes is believed to be detrimental for myofiber function. However, the role of autophagy in the pathogenesis of GSDII is still unclear. To address this issue we monitored autophagy in muscle biopsies and myotubes of early and late-onset GSDII patients at different time points of disease progression. Moreover we also analyzed muscles from patients treated with enzyme replacement therapy (ERT). Our data suggest that autophagy is a protective mechanism that is required for myofiber survival in late-onset forms of GSDII. Importantly, our findings suggest that a normal autophagy flux is important for a correct maturation of GAA and for the uptake of recombinant human GAA. In conclusion, autophagy failure plays an important role in GSDII disease progression, and the development of new drugs to restore the autophagic flux should be considered to improve ERT efficacy.Autophagy 08/2012; 8(11). · 7.45 Impact Factor -
Article: JunB transcription factor maintains skeletal muscle mass and promotes hypertrophy.
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ABSTRACT: The size of skeletal muscle cells is precisely regulated by intracellular signaling networks that determine the balance between overall rates of protein synthesis and degradation. Myofiber growth and protein synthesis are stimulated by the IGF-1/Akt/mammalian target of rapamycin (mTOR) pathway. In this study, we show that the transcription factor JunB is also a major determinant of whether adult muscles grow or atrophy. We found that in atrophying myotubes, JunB is excluded from the nucleus and that decreasing JunB expression by RNA interference in adult muscles causes atrophy. Furthermore, JunB overexpression induces hypertrophy without affecting satellite cell proliferation and stimulated protein synthesis independently of the Akt/mTOR pathway. When JunB is transfected into denervated muscles, fiber atrophy is prevented. JunB blocks FoxO3 binding to atrogin-1 and MuRF-1 promoters and thus reduces protein breakdown. Therefore, JunB is important not only in dividing populations but also in adult muscle, where it is required for the maintenance of muscle size and can induce rapid hypertrophy and block atrophy.The Journal of Cell Biology 10/2010; 191(1):101-13. · 10.26 Impact Factor -
Article: Mitochondrial fission and remodelling contributes to muscle atrophy.
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ABSTRACT: Mitochondria are crucial organelles in the production of energy and in the control of signalling cascades. A machinery of pro-fusion and fission proteins regulates their morphology and subcellular localization. In muscle this results in an orderly pattern of intermyofibrillar and subsarcolemmal mitochondria. Muscular atrophy is a genetically controlled process involving the activation of the autophagy-lysosome and the ubiquitin-proteasome systems. Whether and how the mitochondria are involved in muscular atrophy is unknown. Here, we show that the mitochondria are removed through autophagy system and that changes in mitochondrial network occur in atrophying muscles. Expression of the fission machinery is per se sufficient to cause muscle wasting in adult animals, by triggering organelle dysfunction and AMPK activation. Conversely, inhibition of the mitochondrial fission inhibits muscle loss during fasting and after FoxO3 overexpression. Mitochondrial-dependent muscle atrophy requires AMPK activation as inhibition of AMPK restores muscle size in myofibres with altered mitochondria. Thus, disruption of the mitochondrial network is an essential amplificatory loop of the muscular atrophy programme.The EMBO Journal 05/2010; 29(10):1774-85. · 9.20 Impact Factor -
Article: Autophagy inhibition induces atrophy and myopathy in adult skeletal muscles.
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ABSTRACT: Autophagy is required for cellular survival and for the clearance of damaged proteins and altered organelles. Excessive autophagy activation contributes to muscle loss in different catabolic conditions. However, the function of basal autophagy for homeostasis of skeletal muscle was unknown. To clarify this issue we have generated conditional and inducible knockout mice for the critical gene Atg7, to block autophagy specifically in skeletal muscle. Atg7 null muscles reveal an unexpected phenotype which is characterized by muscle atrophy, weakness and features of myofiber degeneration. Morphological, biochemical and molecular analyses of our autophagy knockout mice show the presence of protein aggregates, abnormal mitochondria, accumulation of membrane bodies, sarcoplasmic reticulum distension, vacuolization, oxidative stress and apoptosis. Moreover, autophagy inhibition does not protect skeletal muscles from atrophy during denervation and fasting, but instead promotes greater muscle loss. In conclusion, autophagy plays a critical role for myofiber maintenance and its activation is crucial to avoid accumulation of toxic proteins and dysfunctional organelles that, in the end, would lead to atrophy and weakness.Autophagy 02/2010; 6(2):307-9. · 7.45 Impact Factor -
Article: Autophagy is required to maintain muscle mass.
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ABSTRACT: The ubiquitin-proteasome and autophagy-lysosome pathways are the two major routes for protein and organelle clearance. In skeletal muscle, both systems are under FoxO regulation and their excessive activation induces severe muscle loss. Although altered autophagy has been observed in various myopathies, the specific role of autophagy in skeletal muscle has not been determined by loss-of-function approaches. Here, we report that muscle-specific deletion of a crucial autophagy gene, Atg7, resulted in profound muscle atrophy and age-dependent decrease in force. Atg7 null muscles showed accumulation of abnormal mitochondria, sarcoplasmic reticulum distension, disorganization of sarcomere, and formation of aberrant concentric membranous structures. Autophagy inhibition exacerbated muscle loss during denervation and fasting. Thus, autophagy flux is important to preserve muscle mass and to maintain myofiber integrity. Our results suggest that inhibition/alteration of autophagy can contribute to myofiber degeneration and weakness in muscle disorders characterized by accumulation of abnormal mitochondria and inclusions.Cell metabolism 12/2009; 10(6):507-15. · 17.35 Impact Factor -
Article: Inducible activation of Akt increases skeletal muscle mass and force without satellite cell activation.
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ABSTRACT: A better understanding of the signaling pathways that control muscle growth is required to identify appropriate countermeasures to prevent or reverse the loss of muscle mass and force induced by aging, disuse, or neuromuscular diseases. However, two major issues in this field have not yet been fully addressed. The first concerns the pathways involved in leading to physiological changes in muscle size. Muscle hypertrophy based on perturbations of specific signaling pathways is either characterized by impaired force generation, e.g., myostatin knockout, or incompletely studied from the physiological point of view, e.g., IGF-1 overexpression. A second issue is whether satellite cell proliferation and incorporation into growing muscle fibers is required for a functional hypertrophy. To address these issues, we used an inducible transgenic model of muscle hypertrophy by short-term Akt activation in adult skeletal muscle. In this model, Akt activation for 3 wk was followed by marked hypertrophy ( approximately 50% of muscle mass) and by increased force generation, as determined in vivo by ankle plantar flexor stimulation, ex vivo in intact isolated diaphragm strips, and in single-skinned muscle fibers. No changes in fiber-type distribution and resistance to fatigue were detectable. Bromodeoxyuridine incorporation experiments showed that Akt-dependent muscle hypertrophy was accompanied by proliferation of interstitial cells but not by satellite cell activation and new myonuclei incorporation, pointing to an increase in myonuclear domain size. We can conclude that during a fast hypertrophic growth myonuclear domain can increase without compromising muscle performance.The FASEB Journal 09/2009; 23(11):3896-905. · 5.71 Impact Factor -
Article: FoxO3 controls autophagy in skeletal muscle in vivo.
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ABSTRACT: Autophagy allows cell survival during starvation through the bulk degradation of proteins and organelles by lysosomal enzymes. However, the mechanisms responsible for the induction and regulation of the autophagy program are poorly understood. Here we show that the FoxO3 transcription factor, which plays a critical role in muscle atrophy, is necessary and sufficient for the induction of autophagy in skeletal muscle in vivo. Akt/PKB activation blocks FoxO3 activation and autophagy, and this effect is not prevented by rapamycin. FoxO3 controls the transcription of autophagy-related genes, including LC3 and Bnip3, and Bnip3 appears to mediate the effect of FoxO3 on autophagy. This effect is not prevented by proteasome inhibitors. Thus, FoxO3 controls the two major systems of protein breakdown in skeletal muscle, the ubiquitin-proteasomal and autophagic/lysosomal pathways, independently. These findings point to FoxO3 and Bnip3 as potential therapeutic targets in muscle wasting disorders and other degenerative and neoplastic diseases in which autophagy is involved.Cell Metabolism 01/2008; 6(6):458-71. · 13.67 Impact Factor -
Article: Role of Autophagy in the control of muscle mass
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ABSTRACT: Protein degradation in skeletal muscle cells is essentially mediated by the activity of two highly conserved pathways, the ubiquitin-proteasome and the autophagy-lysosome pathway. In the ubiquitin-proteasome pathway, target proteins are conjugated to multiple ubiquitin moieties and ubiquitin-tagged proteins are degraded within the proteasome complex (Lecker et al., 2006; Mammucari et al., 2007). The ubiquitin-proteasome system is constitutively active in normal skeletal muscle and is responsible for the turnover of most soluble and myofibrillar muscle proteins. In the autophagy-lysosome system, portions of cytoplasm and cell organelles are sequestered into vacuoles, called autophagosomes, that are delivered to the lysosomes for the degradation of their content by acidic hydrolases (Lum et al., 2005). Also the autophagy system is constitutively active in skeletal muscle. The ubiquitin-proteasome system is constitutively active in muscle but its activity increases significantly during muscle atrophy due to activation of two ubiquitin-ligases: Atrogin-1/Mafbx and Murf1 (Gomes et al., 2001). The activation of these two genes is regulated by the transcription factor FoxO3. This factor is normally phosphorylated and inactivated by AKT / PKB. Conversely when this pathway is suppressed (eg during muscle atrophy), FoxO3 translocates into the nucleus where it can transactivate its target genes (Sandri et al., 2004; Stitt et al., 2004). Alteration of autophagy has been observed in various myopathies caused by genetic defects of lysosomal components, e.g. Pompe’s and Danon’s disease, or by drugs that inhibit lysosomal function, such as chloroquine (Shintani and Klionsky, 2004). During muscle atrophy induced by various debilitating conditions (such as fasting and diabetes), there is activation of several genes, named "Atrophy-Related-Genes" or “Atrogenes”. Among the atrogenes, two most-induced are two ubiquitin-ligases, Atrogin-1 and Murf1. Several autophagy genes belong to the “Atrogenes”. These genes are: LC3, GABARAP and BNIP3. During the first part of my PhD we focused on the transcriptional regulation of the autophagy genes. Our hypothesis was that FoxO3 can coordinate the ubiquitin-proteasome and the autophagy-lysosome system. To characterize the mechanisms that control the autophagic/lysosomal pathway during muscle atrophy in vivo, we first determined whether the Akt/mTOR pathway is involved in the regulation of some of autophagy-related genes. During starvation and denervation, two different models of muscle wasting, the Autophagy-Related-Genes are induced. Moreover these autophagy-related genes are suppressed by Akt, and acute activation of Akt in transgenic mice inhibits autophagy in atrophying muscle. Importantly mTOR pathway did not appear to play a significant role in the activation of the autophagic/lysosomal pathway during muscle atrophy. Indeed the regulation of autophagy-related genes and the formation of autophagic vesicles are not induce either by rapamycin, an inhibitor of mTOR, or by knocking down of mTOR. These findings are in agreement with previous studies (Kochl et al., 2006; Mordier et al., 2000; Sarkar et al., 2007; Yamamoto et al., 2006). We used gain- and loss-of-function experiments to determine the role of FoxO3 in the autophagic/lysosomal pathway. These experiments found two novel FoxO3 targets that regulate autophagy. LC3 and Bnip3 promoters contain several potential FoxO binding sites and ChIP (Chromatin-ImmunoPrecipitation) experiments on atrophying muscles showed that FoxO3 binds chromatin of their promoters in specific sites. The regions of FoxO3 interaction were cloned upstream luciferase gene and functional studies confirmed that FoxO3 transactivates LC3 and BNIP3 genes. Moreover, loss-function experiments showed that BNIP3 upregulation is necessary for autophagy induction in adult muscle. Finally, we asked whether the induction of autophagy is secondary to the activation of the ubiquitin-proteasome system. Inhibition of ubiquitin-proteasome system by pharmacological or genetic approach, did not affect autophagy, suggesting that the two degradation pathways are independently controlled by FoxO3 (Mammucari et al., 2007). Thus, FoxO3 coordinates the two major proteolytic systems of the cell. In the second part of my PhD I focused my studies on the role of basal autophagy in skeletal muscle homeostasis. It is known that excessive activation of autophagy aggravates muscle wasting by removing portion of cytoplasm, proteins, and organelles (Dobrowolny et al., 2008; Mammucari et al., 2007; Wang et al., 2005; Zhao et al., 2007). Conversely, inhibition of lysosome-dependent degradation causes myopathies like Pompe and Danon diseases, and autophagy inhibition is thought to play a role in many myopathies with inclusions or with abnormal mitochondria (Levine and Kroemer, 2008; Temiz et al., 2009). To understand the exact role of autophagy in physiology of skeletal muscle we have generated conditional knockout for Atg7 gene to block autophagy specifically in skeletal muscle. The Atg7 protein is crucial for the formation of the autophagy vesicles by the activations of different Atg proteins and for the formation of the autophagosome. To understand the role of the autophagy in adult skeletal muscle, Atg7 floxed mice were crossed with mice that express the Cre-recombinase under the muscle-specific promoter Myosin light chain 1f. Muscle-specific deletion of Atg7, resulted in profound muscle atrophy, accumulation of protein aggregates that are positive for p62/SQSTM1 and age-dependent decrease in force. Moreover Atg7 null muscles showed accumulation of abnormal mitochondria, distension of sarcoplasmic reticulum, sarcomere disorganization, and formation of aberrant concentric membranous structures. Moreover, muscle loss is more exacerbated in autophagy knockout mice during denervation and fasting. These results suggest that the autophagy flux is important to preserve muscle mass and to maintain myofiber integrity. Moreover Atg7 null muscles showed activation of endoplasmic reticulum chaperones, such as BiP, as well as the phosphorylation of eIF2α, suggesting an ongoing unfolded protein response. The failure of protein-folding quality control in Atg7 null mice induces endoplasmic reticulum stress which can generate ROS, and suppression of protein synthesis which can contribute to muscle atrophy (Masiero et al., 2009). To further confirm our findings in adulthood, we generated a tamoxifen-inducible muscle-specific Atg7 knockout mice. In this case, the floxed Atg7 mice were crossed with mice expressing the Cre-recombinase fused with a modified estrogen receptor, under the control of a muscle-specific promoter (Human Skletal Muscle). When animals are treated with tamoxifen (an estrogen analogue that has a high affinity for the modified estrogen receptor), the Cre-recombinase is stabilized and can recombinate the loxP site. Identical results were obtained in inducible Atg7 null muscles. These mice showed p62/SQSTM1 accumulation, muscle atrophy and decrease in force generation. Morphological analyses revealed accumulation of abnormal mitochondria in small atrophic fibers and the number of centrally nucleated fibers were more abundant after acute Atg7 deletion than in non-inducible autophagy-deficient muscles (Masiero et al., 2009). Our results suggest that inhibition/alteration of autophagy can contribute to myofiber degeneration and weakness in muscle disorders characterized by accumulation of abnormal mitochondria and inclusions.
Top co-authors
Top Journals
- Autophagy (2)
- The EMBO Journal (1)
- The FASEB Journal (1)
- The Journal of Cell Biology (1)
- Cell Metabolism (1)
Institutions
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2010–2012
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University-Hospital of Padova
Padova, Veneto, Italy
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2009–2010
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Dulbecco Telethon Institute
Dublin, CA, USA
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2008–2010
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Venetian Institute of Molecular Medicine
Padova, Veneto, Italy
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