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Novel insights into the role of skeletal muscle autophagy in health and disease.
No full textStructurally or functionally altered and unnecessary cellular components are physiologically removed from the cell through a self-degradative process named autophagy. An impairment of the autophagic machinery was shown to have a central role in the pathogenesis of several neurodegenerative, cardiac and age-related diseases. In the laboratory of Prof. Paolo Bonaldo, it was previously shown that an impairment of the autophagy pathway plays a key role in the pathogenesis of Bethlem Myopathy (BM) and Ullrich Congenital Muscular Dystrophy (UD), inherited human pathologies associated to collagen VI (COL6) deficiency and primarily affecting skeletal muscle. Moreover, autophagy reactivation by genetic, pharmacological and nutritional approaches was shown to rescue the dystrophic phenotype of Col6a1−/− mice, a model for human COL6-related myopathies.
Although a large number of stimuli and molecules induce autophagy, their efficiency in counteracting muscle disease progression need to be carefully evaluated at the preclinical and clinical level. During my PhD I took part to the evaluation of the outcomes of a normocaloric low protein diet (LPD)-based clinical trial for BM and UD patients, whose results are very promising. Due to the low level of daily protein intake, LPD is however not safe for pediatric BM and UD patients and alternative autophagy-inducing strategies need to be identified. Toward this aim, I devoted a large part of my PhD work to investigate the effects of spermidine, a naturally occurring non-toxic autophagy-inducing molecule, on Col6a1−/− mice. I demostrated that spermidine administration reactivates autophagy in this pathological context, and this is paralleled by structural and functional ameliorations in COL6-deficient myopathic muscles. This work paves the way for the formulation of novel diet-based therapies for the treatment of BM and UD.
Another major aim of my PhD project was investigating the mechanisms underlying autophagy regulation in skeletal muscle, focusing on AMBRA1 (activating molecule in BECN1-regulated autophagy). Studies in mice with a randomly mutated Ambra1 locus (Ambra1gt/gt) showed that this gene is essential for the development of the central nervous system, and may also play a key role for muscle development in zebrafish and mouse. To better clarify the role of AMBRA1 in skeletal muscles, we generated mice with a floxed Ambra1 allele (Ambra1f/f). I verified in vivo that the conditional allele for Ambra1 works properly, by breeding Ambra1f/f mice with a CAG-Cre transgenic line, which express Cre recombinase in the oocytes, to obtain the null allele for Ambra1. Mice bearing this allele either in homozygosity (Ambra1−/−) or in heterozygosity (Ambra1+/−) were preliminarily characterized. Moreover, I produced skeletal muscle-specific AMBRA1 null (Ambra1f/f::Mlc1f-Cre) mice and investigated muscle defects caused by AMBRA1 deficiency. Interestingly, data in adult Ambra1f/f::Mlc1f-Cre mice indicate that a high proportion of mature AMBRA1-deficient myofibers contain inclusion bodies. Ongoing experiments are aimed at identifying the composition of these inclusions and evaluating the activation status of autophagy and ubiquitin-proteasome system. The data I obtained until now exclude the presence of amyloid material in the inclusions, and highlight an impairment of both these degradation processes.
As side projects, during my PhD I also participated in the study of autophagy status in other hereditary muscle pathologies. First, I contributed to the identification of an autophagy impairment in the tibialis anterior of mdx mice, a model for Duchenne Muscular Dystrophy. Then I was involved in the characterization of an aggresome-autophagy process occurring in muscle biopsies from a patient carrying a p.C150R mutation in the FHL1 gene, and affected by severe sarcopenia and rigid spine syndrome (RSS). It emerges from these studies that autophagy is a commonly dysregulated process in different muscle pathologies, and that autophagy modulation may be a promising therapeutic strategy in different pathological contexts.
In summary, the data obtained with this PhD work underline the important pathogenetic role of autophagy dysregulation in congenital muscle pathologies, provide some basis for the development of novel therapeutic strategies based on autophagy modulation, and pave the way for unraveling the role of AMBRA1 in skeletal muscle.Componenti cellulari alterati o non più necessari vengono fisiologicamente rimossi dalla cellula attraverso un processo di auto-degradazione chiamato autofagia. E’ stato dimostrato che un difetto di autofagia può svolgere un ruolo centrale nella patogenesi di diverse malattie neurodegenerative, cardiache e legate all'invecchiamento. Nel nostro laboratorio è stato precedentemente dimostrato che una compromissione del flusso autofagico svolge un ruolo chiave nella patogenesi della miopatia di Bethlem (BM) e della distrofia muscolare congenita di Ullrich (UD), patologie umane congenite associate a carenza di collagene VI (COL6) che colpiscono principalmente il muscolo scheletrico. Inoltre è stato dimostrato che la riattivazione dell’autofagia attraverso strategie genetiche, farmacologiche o nutrizionali è in grado di migliorare il fenotipo miopatico dei topi Col6a1−/−, modello animale per le malattie dovute a deficit di COL6.
Nonostante siano noti molti stimoli e molecole che inducono autofagia, la loro efficienza nel contrastare la progressione delle patologie muscolari deve essere valutata attentamente caso per caso, sia a livello preclinico che in ambito clinico. Durante il mio dottorato ho partecipato alla valutazione degli effetti di un trial clinico effettuato somministrando una dieta normocalorica a basso contenuto proteico (Low Protein Diet, LPD) ad un gruppo di pazienti BM e UD. Nonostante i risultati siano molto promettenti, questa terapia non è adatta a pazienti pediatrici a causa del bassissimo apporto proteico giornaliero. Strategie alternative per contrastare la progressione di BM e UD devono essere identificate e testate, perciò ho dedicato buona parte del mio dottorato di ricerca allo studio degli effetti della spermidina. Questa molecola è naturalmente presente in molti alimenti, non è tossica ed è stato dimostrato che induce autofagia in molti organismi modello. La somministrazione di spermidina riattiva l'autofagia nel muscolo di topi Col6a1−/−, e questo è accompagnato da un miglioramento della struttura e della funzione dei muscoli miopatici privi di COL6. Questo lavoro apre la strada alla formulazione di nuove terapie basate sulla dieta per BM e UD.
Un altro obiettivo importante del mio progetto di dottorato è stato lo studio dei meccanismi alla base della regolazione dell’autofagia nel muscolo scheletrico. Mi sono concentrata in particolare sul chiarimento del ruolo di AMBRA1 (activating molecule in BECN1-regulated autophagy). Studi condotti su topi in cui il locus Ambra1 è stato mutato in modo casuale (Ambra1gt/gt) hanno dimostrato che questo gene è essenziale per lo sviluppo del sistema nervoso centrale, ed anche del muscolo scheletrico. Per chiarire meglio il ruolo di AMBRA1 nel muscolo scheletrico, abbiamo generato topi con un allele condizionale per Ambra1 (Ambra1f/f). Ho verificato in vivo il corretto funzionamento di questo allele, tramite incrocio con topi transgenici CAG-Cre, che esprimono la Cre ricombinasi negli ovociti, ottenendo l'allele null per Ambra1. Ho caratterizzato in modo preliminare topi omozigoti (Ambra1−/−) o eterozigoti (Ambra1+/−) per questo allele. Ho poi ottenuto topi knockout muscolo specifici per il gene Ambra1 (Ambra1f/f ::Mlc1f-Cre). I dati preliminari che ho sinora ottenuto sono molto interessanti: un'elevata percentuale di fibre muscolari prive di AMBRA1 contengono corpi di inclusione. Attualmente mi sto occupando di identificare la composizione di queste inclusioni e valutare lo stato di attivazione dell'autofagia e del sistema ubiquitina-proteasoma. I miei primi dati escludono la presenza di materiale amiloide nelle inclusioni, ed evidenziano un deficit in entrambi questi processi degradativi.
Durante il dottorato ho inoltre contribuito a due progetti nei quali la condizione dell’autofagia è stata studiata in altre patologie muscolari ereditarie, identificando un deficit autofagico nel tibiale anteriore di un modello murino della distrofia muscolare di Duchenne, il topo mdx. Ho inoltre contribuito allo studio di biopsie muscolari prelevate da una paziente con una mutazione p.C150R nel gene FHL1, affetta da grave sarcopenia e da sindrome della colonna vertebrale rigida (Rigid Spine Syndrome, RSS), nelle quali abbiamo evidenziato un’aumentata induzione di autofagia probabilmente volta alla degradazione degli aggregati presenti nelle miofibre. Questi studi dimostrano che una disregolazione dell’autofagia è un meccanismo patogenetico comune a svariate patologie muscolari su base genetica, e fornisce un’ulteriore conferma della validità della modulazione del flusso autofagico come strategia terapeutica.
In sintesi il mio lavoro evidenzia come una disregolazione dell’autofagia possa rivestire un ruolo chiave nello sviluppo di patologie muscolari, inoltre fornisce una base per lo sviluppo di nuove strategie terapeutiche basate sulla modulazione dell’autofagia nel muscolo scheletrico ed apre la strada per chiarire il ruolo di AMBRA1 in questo tessuto
A conditional mouse model and in vitro system to study Gba1 in myelinating glia: novel contribution for Gaucher Disease and Parkinson's Disease
No full textAutophagy is Impaired in the Tibialis Anterior of Dystrophin Null Mice
No full textBackground Duchenne muscular dystrophy is a lethal, progressive, muscle-wasting disease caused by mutations in the DMD gene. Structural remodelling processes are responsible for muscle atrophy and replacement of myofibers by fibrotic and adipose tissues. Molecular interventions modulating catabolic pathways, such as the ubiquitin-proteasome and the autophagy-lysosome systems, are under development for Duchenne and other muscular dystrophies. The Akt signaling cascade is one of the main pathways involved in protein synthesis and autophagy repression and is known to be up-regulated in dystrophin null mdx mice. Results We report that autophagy is triggered by fasting in the tibialis anterior muscle of control mice but not in mdx mice. Mdx mice show persistent Akt activation upon fasting and failure to increase the expression of FoxO3 regulated autophagy and atrophy genes, such as Bnip3 and Atrogin1. We also provide evidence that autophagy is differentially regulated in mdx tibialis anterior and diaphragm muscles. Conclusions Our data support the concept that autophagy is impaired in the tibialis anterior muscle of mdx mice and that the regulation of autophagy is muscle type dependent. Differences between muscle groups should be considered during the pre-clinical development of therapeutic strategies addressing muscle metabolism
Extracellular collagen VI has prosurvival and autophagy instructive properties in mouse fibroblasts
Full text linkCollagen VI (ColVI) is an abundant and distinctive extracellular matrix protein secreted by fibroblasts in different tissues. Human diseases linked to mutations on ColVI genes are primarily affecting skeletal muscle due to non-cell autonomous myofiber defects. To date, it is not known whether and how fibroblast homeostasis is affected by ColVI deficiency, a critical missing information as this may strengthen the use of patients' fibroblasts for preclinical purposes. Here, we established primary and immortalized fibroblast cultures from ColVI null (Col6a1-/-) mice, the animal model of ColVI-related diseases. We found that, under nutrient-stringent condition, lack of ColVI affects fibroblast survival, leading to increased apoptosis. Moreover, Col6a1-/- fibroblasts display defects in the autophagy/lysosome machinery, with impaired clearance of autophagosomes and failure of Parkin-dependent mitophagy. Col6a1-/- fibroblasts also show an increased activation of the Akt/mTOR pathway, compatible with the autophagy impairment, and adhesion onto purified ColVI elicits a major effect on the autophagic flux. Our findings reveal that ColVI ablation in fibroblasts impacts on autophagy regulation and cell survival, thus pointing at the new concept that this cell type may contribute to the pathological features of ColVI-related diseases
Reactivation of autophagy by spermidine ameliorates the myopathic defects of collagen VI-null mice
No full textAutophagy is a self-degradative process responsible for the clearance of damaged or unnecessary cellular components. We have previously found that persistence of dysfunctional organelles due to autophagy failure is a key event in the pathogenesis of COL6/collagen VI-related myopathies, and have demonstrated that reactivation of a proper autophagic flux rescues the muscle defects of Col6a1-null (col6a1(-/-)) mice. Here we show that treatment with spermidine, a naturally occurring nontoxic autophagy inducer, is beneficial for col6a1(-/-) mice. Systemic administration of spermidine in col6a1(-/-) mice reactivated autophagy in a dose-dependent manner, leading to a concurrent amelioration of the histological and ultrastructural muscle defects. The beneficial effects of spermidine, together with its being easy to administer and the lack of overt side effects, open the field for the design of novel nutraceutical strategies for the treatment of muscle diseases characterized by autophagy impairment
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
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