Extracellular Collagen VI Has Prosurvival and Autophagy Instructive Properties in Mouse Fibroblasts

Collagen 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

Novel insights into the role of skeletal muscle autophagy in health and disease.

Structurally 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

Extracellular Collagen VI Has Prosurvival and Autophagy Instructive Properties in Mouse Fibroblasts

Collagen 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

Autophagy is Impaired in the Tibialis Anterior of Dystrophin Null Mice

Background 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

Multiple Mechanisms Converging on Transcription Factor EB Activation by the Natural Phenol Pterostilbene

: Pterostilbene (Pt) is a potentially beneficial plant phenol. In contrast to many other natural compounds (including the more celebrated resveratrol), Pt concentrations producing significant effects in vitro can also be reached with relative ease in vivo. Here we focus on some of the mechanisms underlying its activity, those involved in the activation of transcription factor EB (TFEB). A set of processes leading to this outcome starts with the generation of ROS, attributed to the interaction of Pt with complex I of the mitochondrial respiratory chain, and spreads to involve Ca2+ mobilization from the ER/mitochondria pool, activation of CREB and AMPK, and inhibition of mTORC1. TFEB migration to the nucleus results in the upregulation of autophagy and lysosomal and mitochondrial biogenesis. Cells exposed to several μM levels of Pt experience a mitochondrial crisis, an indication for using low doses in therapeutic or nutraceutical applications. Pt afforded significant functional improvements in a zebrafish embryo model of ColVI-related myopathy, a pathology which also involves defective autophagy. Furthermore, long-term supplementation with Pt reduced body weight gain and increased transcription levels of Ppargc1a and Tfeb in a mouse model of diet-induced obesity. These in vivo findings strengthen the in vitro observations and highlight the therapeutic potential of this natural compound

Reactivation of autophagy by spermidine ameliorates the myopathic defects of collagen VI-null mice

Autophagy 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

Role of adiponectin in the metabolism of skeletal muscles in collagen VI\u2013related myopathies

The role of adiponectin has been particularly deepened in diabetic muscles while the study of adiponectin in hereditary myop- athies has been marginally investigated. Here, we report the study about adiponectin effects in Col6a1 12/ 12 (collagen VI\u2013null) mice. Col6a1 12/ 12 mice show myophatic phenotype closer to that of patients with Bethlem myopathy, thus representing an excellent animal model for the study of this hereditary disease. Our findings demonstrate that Col6a1 12/ 12 mice have decreased plasma adiponectin content and diseased myoblasts have an impaired autocrine secretion of the hormone. Moreover, Col6a1 12/ 12 myo- blasts show decreased glucose uptake and mitochondria with depolarized membrane potential and impaired functionality, as supported by decreased oxygen consumption. Exogenous addition of globular adiponectin modifies the features of Col6a1 12/ 12 myoblasts, becoming closer to that of the healthy myoblasts. Indeed, globular adiponectin enhances glucose uptake in Col6a1 12/ 12 myoblasts, modifies mitochondrial membrane potential, and restores oxygen consumption, turning closer to those of wild-type myoblasts. Finally, increase of plasma adiponectin level in Col6a1 12/ 12 mice is induced by fasting, a condition that has been previously shown to lead to the amelioration of the dystrophic phenotype. Collectively, our results demonstrate that exogenous replenishment of adiponectin reverses metabolic abnormalities observed in Col6a1 12/ 12 myoblasts