Extracellular collagen type VI has prosurvival and autophagy instructive properties in mouse embryonic fibroblasts

Collagen VI is a major protein of the extracellular matrix with a broad distribution in many tissues, including skeletal muscle and connective tissues. It is composed of three distinct alpha chains, α1, α2 and α3(VI), ecoded by separate genes. Mutations of collagen VI genes in humans cause several muscle diseases, such as Bethlem myopathy and Ullrich congenital muscular dystrophy. Collagen VI null (Col6a1–/–) mice display a myopathic phenotype characterized by mitochondrial dysfunction, spontaneous apoptosis and autophagic impairments in myofibers. These findings indicate that collagen VI has a key role for skeletal muscle homeostasis. Before starting my PhD, I participated to a project aimed at investigating the effects of physical exercise on wild type and Col6a1–/– skeletal muscle. This work demonstrated for the first time that exercise is able to activate the autophagic response in muscle. Moreover, this study revealed that physical exercise it is detrimental for Col6a1–/– muscles. I decided to focus my main PhD work on investigating the role of collagen VI in fibroblasts, which are the major cell type responsible for the secretion and extracellular deposition of this protein, and elucidating the consequences on fibroblasts due to ablation of collagen VI. In patients affected by Bethlem myopathy and Ullrich congenital muscular dystrophy, the mutated forms of collagen VI are produced and retained by fibroblasts, suggesting a potential contribution for this cell type in the onset and progression of muscle defects. To assess how lack of collagen VI impacts on fibroblast functions, I generated stable mouse embryonic fibroblast (MEF) lines from wild type and Col6a1–/– mice and showed that collagen VI is necessary for autophagy regulation and has prosurvival properties in fibroblasts. Col6a1–/– MEFs displayed accumulation of LC3 both basally and following autophagy induction. To dissect the autophagic response of these cells, I studied the autophagy flux and the activity of the nutrient sensor kinase mTOR. I found that in Col6a1–/– MEFs the mTORC1 downstream targets, such as 4E-BP1 and S6, are persistently activated under nutrient depletion stimuli, leading to autophagy inhibition in starving cells. Furthermore, Col6a1–/– MEFs presented a general energy imbalance, leading to over-activation of the AMP-activated protein kinase. These signalling defects lead to massive accumulation of autophagosomes inside Col6a1–/– fibroblasts, due to a compromised autophagosome-lysosome fusion in association with the presence of enlarged lysosomes and LAMP-2 protein depletion. These lysosomal defects are also associated with aberrant localization and activity of TFEB, a master transcription factor for lysosome biogenesis and autophagy regulation. In addition, Col6a1–/– MEFs showed increased susceptibility to cell death, especially under nutrient stress, that ended with activation of the intrinsic pathway of apoptosis. This phenotype was specifically rescued by culturing cells onto purified collagen VI provided as an adhesive substrate . Lack of collagen VI also influenced the organization of the mitochondrial network, which has a key role in cell survival. Mitochondria of Col6a1–/– MEFs exhibited increased fragmented morphology, associated with Parkin translocation and defective mitophagy. These findings show that fibroblasts play a relevant role in the development of the pathophysiological defects of collagen VI null mice, a finding that provide a thus far undisclosed and valuable information for the diagnosis and therapy of inherited diseases associated with collagen VI gene mutations. Moreover, they reveal for the first time a direct effect of collagen VI on the regulation of autophagy and associated mechanisms in this cell type.