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ROLE OF AUTOPHAGY IN AGE-RELATED MUSCLE LOSS
Full text linkAutophagy is an ubiquitous degradation system, that is conserved through species. Cells activate autophagy to degrade long-lived proteins, damaged organelles or portions of cytoplasm, that are engulfed in double-membrane vesicles called autophagosomes, that ultimately fuse to lysosomes, where the cargo is degraded and breakdown products are recycled to sustain cellular energetic demands.
Skeletal muscle is the most abundant tissue in mammals and controls 80% of the blood glucose. We have recently shown that an efficient autophagy is required for muscle mass maintenance (Masiero et al., 2009).
During ageing, muscles inevitably undergo atrophy, a process named sarcopenia (Rossi et al. 2008). Moreover, it has been reported that autophagy declines with age (Tan et al., 2013). Since the mechanisms involved in age-related muscle loss remain obscure, we investigated whether autophagy impairment contributes to sarcopenia. In this work, the muscle-specific autophagy knockout (Atg7-/-MLC), that were recently generated in our laboratory, were characterized during ageing (Masiero et al., 2009). Aged Atg7-/- mice have reduced lifespan and exacerbated atrophic and myopathic phenotype. In vivo force measurements showed that they are weaker compared to age-matched control mice. Alteration of mitochondrial morphology is a typical feature of Atg7-/- muscles. Therefore, we studied mitochondrial function in adult mice. Mitochondria of Atg7-/- mice were dysfunctional, in fact they did not retain membrane potential upon inhibition of ATP synthase. This mitochondrial alteration induced an increase of oxidative stress. A proteomic approach on oxidized protein, in collaboration with Prof. Friguet at the University of Paris, revealed that contractile proteins, such as actin and myosin, were significantly more carbonylated when autophagy was blocked. Functional assays of force measurements on single isolated fibers and sliding properties of purified actin/myosin, performed in collaboration with Prof. Bottinelli at the University of Pavia, showed an impairment of these contractile proteins in Atg7-/- mice.
Atg7-/- mice also undergo spontaneous denervation, as confirmed by upregulation of denervation markers, such as Muscle Specific Kinase (MuSK), Acetylcholine Receptor gamma subunit (AchR-gamma) and Neural Cell Adhesion Molecule (NCAM). Moreover, in collaboration with Dr. Rudolf at Karlsruhe Institute of Technology (KIT), in Karlsrhue, we performed in vivo imaging of neuromuscular junction (NMJ), that revealed NMJ fragmentation and instability in autophagy-deficient mice. These findings suggest that inhibition of autophagy specifically in muscle generates a series of events that affect NMJ and causes a precocious denervation, contributing to sarcopenia. Since oxidative stress is an important feature of Atg7-/- mice and is believed to contribute to ageing, we treated adult mice with an antioxidant vitamin E analogue (Trolox), for 30 days, and we monitored the effects on the phenotype of Atg7-/- muscles. Trolox treatment reduced the level of protein carbonylation, restored the sliding properties of actin and myosin and brought back the force to normal level. Mitochondria function was also ameliorated but we did not find any benefit on atrophy and NMJ morphology. However, there was a small amelioration on NMJ stability.
These data showed that oxidative stress contributes only to some aspects of ageing features present in Atg7-/- mice. Therefore, other mechanisms are involved for the atrophy and the denervation aspects. We then hypothesized that muscles release neurotrophic factors that are critical for muscle-nerve interaction and stability. Initially, we tought for neurotrophic factors that were down-regulated in autophagy-deficient muscle both in adult and old mice. qRT-PCR identified FGF binding protein 1 (FGFBP1) to be the one that was always suppressed in Atg7-/- mice. FGFBP1 is protein involved in the bio-activation of FGF proteins, that are important pre-synaptic organizers. In order to investigate the role of FGFBP1 in NMJ instability we used loss and gain of function approaches. Down-regulation of FGFBP1 in control mice induced instability and fragmentation of NMJ. On the contrary FGFBP1 over-expression in Atg7-/- muscles reduced the number of denervated fibers and restored NMJ stability. Then we investigated the connection between autophagy impairment and FGFBP1 down-regulation, by analyzing MuSK activity, a kinase that is essential for NMJ maintenance. We observed an alterated MuSK clustering in NMJ of Atg7-/- mice. Moreover MuSK down-regulation in vivo leads to FGFBP1 suppression.
These results suggest that NMJ requires the secretion of FGFBP1 neurotrophic factor that is under MuSK regulation and that autophagy is critical for a normal MuSK localization and activity.
It has been consistently demonstrated that two lifestyle adaptations, namely caloric restriction and exercise, are able to extend lifespan and, in parallel, to mitigate age-related alterations in NMJ (Melov et al., 2007; Fontana et al., 2010; Sandri et al., 2013; Schiaffino et al., 2013; Coen et al., 2013; Toledo et al., 2013; Guarente, 2013). Moreover, both these conditions promote autophagy activation in skeletal muscles and in other tissues. It has also been reported that autophagy is required for exercise itself and for training-induced adaptations in glucose homeostasis (He et al., 2012). These findings remain controversial as skeletal muscle–specific autophagy-knockout mice show the opposite phenotype (Kim et al., 2013). In this scenario, it is still unknown whether it is whole body or muscle specific autophagy that is required to sustain contraction, maintain glucose homeostasis, and trigger exercise-induced benefits. For this reason, we used Tamoxifen-inducible, muscle-specific, Atg7 knockout mice (Atg7-/-HSA), that we have recently generated (Masiero et al., 2009), to investigate the role of autophagy in physical exercise. This inducible muscle-specific genetic model allows to minimize the chance of any adaptations and compensations that usually occur with constitutive deletion of genes. In order to investigate whether acute block of autophagy in muscle affects exercise performance, controls and autophagy-deficient mice were exercised on a treadmill. We used a concentric exercise protocol while monitoring the maximum distance ran to exhaustion. Surprisingly, we did not find any significant differences in running capacity between controls and inducible Atg7-/-. Thus, autophagy is not required to sustain muscle contraction during concentric physical activity. We hypothesized whether a damaging eccentric-type muscle contraction might unravel a novel role for autophagy during muscle repair after exercise. So we performed repeated bouts of eccentric exercise to exhaustion for three consecutive days to induce damaging eccentric contraction in controls and inducible Atg7-/- animals, and found out that in these conditions, autophagy-deficient mice ran significantly less than controls. Morphological analyses did not show any sign of inflammation or myofibre degeneration, thus suggesting that impaired performance of Atg7-/- muscles was not due to major structural alterations. We also looked for possible energetic imbalance upon exercise, by monitoring the activity of P-AMPK, one of the major sensor of energetic stress, and by checking glucose and lactate levels in the blood. However, no significant differences were observed, thus suggesting that autophagy is not required for metabolic regulation of skeletal muscle during exercise. Since autophagy is important for organelle quality control, we tested whether mitochondrial homeostasis was affected after exercise. Interestingly, isolated muscle fibers from inducible Atg7-/- animals contained dysfunctional mitochondria that well correlated with their impaired performance. Being mitochondria the main source of ROS in the cell, it was feasible to hypothesize that oxidative stress may play a role in this condition. To address that, we measured total protein carbonylation and ROS production in exercised muscles that indeed was higher in Atg7-/- muscles. All together these data showed that acute inhibition of autophagy led to accumulation of dysfunctional mitochondria, increased oxidative stress and reduced physical performance during eccentric contraction. Excessive oxidative stress impairs muscle function, thus potentially explaining the reduced physical performance of Atg7-/- mice. We therefore treated controls and inducible Atg7-/- mice with the anti-oxidant N-Acetyl Cysteine (NAC) for 6 weeks, and then exercised them eccentrically. Surprisingly, NAC treatment severely impaired performance of controls but did not elicit any benefit in inducible Atg7-/- animals. Moreover it impaired mitochondrial function of controls. This data were confirmed after treatment with another anti-oxidant (Mito-TEMPO), that was specific for mitochondria.
It has been reported that anti-oxidant treatment reduces activation of autophagy in control animals and that ROS are important for signalling pathways in the cell (Underwood et al., 2010; Owusu-Ansah et al., 2013). Our findings support these evidences, suggesting that physiological levels of ROS are important for the correct basal and stimulus-induced autophagy activation.
Our results highlight the role of autophagy in the maintenance of mitochondrial function but not in AMPK activation and exercise dependent glucose homeostasis, suggesting that autophagy is an adaptive response to exercise that ensures mitochondria-quality control during damaging contractions
Engineering an in vitro model of human muscle dystrophy for highthroughput screenings and development of therapeutic strategies
No full textAutophagy is not required to sustain exercise and PRKAA1/AMPK activity but is important to prevent mitochondrial damage during physical activity.
Full text linkPhysical activity has been recently documented to play a fundamental physiological role in the regulation of autophagy in several tissues. It has also been reported that autophagy is required for exercise itself and for training-induced adaptations in glucose homeostasis. These autophagy-mediated metabolic improvements are thought to be largely dependent on the activation of the metabolic sensor PRKAA1/AMPK. However, it is unknown whether these important benefits stem from systemic adaptations or are due solely to alterations in skeletal muscle metabolism. To address this we utilized inducible, muscle-specific, atg7 knockout mice that we have recently generated. Our findings indicate that acute inhibition of autophagy in skeletal muscle just prior to exercise does not have an impact on physical performance, PRKAA1 activation, or glucose homeostasis. However, we reveal that autophagy is critical for the preservation of mitochondrial function during damaging muscle contraction. This effect appears to be gender specific affecting primarily females. We also establish that basal oxidative stress plays a crucial role in mitochondrial maintenance during normal physical activity. Therefore, autophagy is an adaptive response to exercise that ensures effective mitochondrial quality control during damaging physical activity
Traffico umano ai fini dello sfruttamento sessuale in Italia. Il caso studio dell'associazione Lule: dall'emersione all'integrazione delle ragazze di strada
No full textLa tesi ha lo scopo di analizzare il fenomeno del traffico umano ai fini dello sfruttamento sessuale sul territorio italiano. Alcuni approfondimenti saranno dedicati a due fenomeni relazionati al tema della tratta: l'immigrazione e la prostituzione. L'ultima parte dell'elaborato prevede la descrizione delle attività svolte dall'associazione Lule che da anni opera contro lo sfruttamento sessuale in alcune aree del milanese e del pavese. Le attività svolte riguardano l'iter sociale delle ragazze di strada che vogliono uscire da una condizione di sfruttamento (il processo dell'emersione) e seguire dei percorsi di integrazione nella società ricevente
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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