Collagen VI–NG2 axis in human tendon fibroblasts under conditions mimicking injury response

In response to injury, tendon fibroblasts are activated, migrate to the wound, and contribute to tissue repair by producing and organizing the extracellular matrix. Collagen VI is a microfibrillar collagen enriched in the pericellular matrix of tendon fibroblasts with a potential regulatory role in tendon repair mechanism. We investigated the molecular basis of the interaction between collagen VI and the cell membrane both in tissue sections and fibroblast cultures of human tendon, and analyzed the deposition of collagen VI during migration and myofibroblast trans-differentiation, two crucial events for tendon repair. Tendon fibroblast displayed a collagen VI microfibrillar network closely associated with the cell surface. Binding of collagen VI with the cell membrane was mediated by NG2 proteoglycan, as demonstrated by in vitro perturbation of collagen VI–NG2 interaction with a NG2-blocking antibody. Cultures subjected to wound healing scratch assay displayed collagen VI–NG2 complexes at the trailing edge of migrating cells, suggesting a potential role in cell migration. In fact, the addition of a NG2-blocking antibody led to an impairment of cell polarization and delay of wound closure. Similar results were obtained after in vitro perturbation of collagen VI extracellular assembly with the 3C4 anti-collagen VI antibody and in collagen VI-deficient tendon cultures of a Ullrich congenital muscular dystrophy patient carrying mutations in COL6A2 gene. Moreover, in vitro treatment with transforming growth factor β1 (TGFβ1) induced a dramatic reduction of NG2 expression, both at protein and mRNA transcript level, and the impairment of collagen VI association with the cell membrane. Instead, collagen VI was still detectable in the extracellular matrix in association with ED-A fibronectin and collagen I, which were strongly induced by TGFβ1 treatment. Our findings reveal a critical role of the NG2 proteoglycan for the binding of collagen VI to the surface of tendon fibroblasts. By interacting with NG2 proteoglycan and other extracellular matrix proteins, collagen VI regulates fibroblasts behavior and the assembly of tendon matrix, thereby playing a crucial role in tendon repair

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 defective in collagen VI muscular dystrophies and its reactivation rescues myofiber degeneration

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Unraveling the role of collagen VI in the central and peripheral nervous system

Collagen VI is an extracellular matrix protein expressed in different tissues, such as skin, peripheral nerves, cartilages and skeletal muscle. It consists of three alpha chains encoded by separate genes. Mutations of collagen VI genes in humans cause several muscle diseases, including Bethlem myopathy and Ullrich congenital muscular dystrophy. Collagen VI null (Col6a1–/–) mice display an early onset myopathic phenotype characterized by organelle defects, mitochondrial dysfunction and spontaneous apoptosis. During my first PhD year, I was involved in a large project aimed at elucidating collagen VI patho-molecular defects in muscle disorders. This led to demonstrate that the persistence of altered organelles and apoptosis in collagen VI deficient muscles is due to defective regulation of autophagy in myofibers. In particular, I performed experiments aimed at assessing the relationships between autophagy and apoptosis in wild-type and Col6a1–/– muscles, either in basal condition or after different genetic, nutritional and pharmacological treatments. During the second part of my PhD study, I decided to focus the study of collagen VI in another compartment, the nervous tissue. Indeed, alterations of the autophagic clearance machinery are know to cause detrimental effects in several organs, particularly in the central nervous system. Moreover, recent findings showed that collagen VI can protect neurons from the toxicity of Aβ peptides, a critical step in the pathogenesis of Alzheimer’s disease, and from UV-irradiation-induced apoptosis. To investigate the role of collagen VI in the central nervous system, I established cortical and hippocampal primary cultures from brains of neonatal wild-type and Col6a1–/– mice and analyzed some cellular processes and molecular pathways, which we previously found to be altered in the skeletal muscle in the absence of collagen VI. In wild-type cultures, collagen VI was found to be localized on the surface of both neurons and glial cells. Col6a1–/– neural cultures displayed a significant increase of spontaneous apoptosis, that could be rescued by plating them onto purified collagen VI. Biochemical analysis of autophagic markers and further experiments, carried out by directly monitoring LC3 lipidation and by measuring fluorescent autophagy puncta in neural cell cultures from transgenic GFP-LC3;Col6a1+/+ and GFP-LC3;Col6a1–/– under different conditions, allowed establishing that the autophagic flux of neural cultures is impaired in the absence of collagen VI. Furthermore, Col6a1–/– neural cell cultures displayed higher vulnerability to oxidative stress. These in vitro findings indicate that lack of collagen VI leads to increased apoptosis and to defective autophagic regulation in neural cells. Further in vivo studies carried out in brain sections from wild-type and Col6a1–/– mice of different ages suggested a protective role for collagen VI in the central nervous system during aging. Finally, during the last year, I carried out some studies also in the peripheral nervous system of wild-type and Col6a1–/– mice, considering previous work from our laboratory showing that Schwann cells express collagen VI during development. These studies showed that the protein is abundant in the endoneurium of sciatic nerves and that lack of collagen VI causes structural defects, including increased myelin thickness and abnormalities of Remak bundles, as well as motor and sensory functional defects. Altogether, these findings reveal an unforeseen new role for collagen VI in both central and peripheral nervous system compartments, where it seem to exert different structural and functional effects, thus opening the field for further investigations in patients.Il collagene Vi è una proteina della matrice extracellulare espressa in diversi tessuti come pelle, nervi periferici, cartilagine e muscolo scheletrico. La proteina è costituita dalle catene polipeptidiche, α1(VI), α2(VI) ed α3(VI), codificate da tre geni distinti. Mutazioni a carico di tali geni causano nell’uomo disordini muscolari come la miopatia di Bethlem e la distrofia muscolare congenita di Ullrich. I topi privi di collagene VI (Col6a1–/–) mostrano un fenotipo miopatico, caratterizzato dalla presenza, nei muscoli scheletrici, di difetti ultrastrutturali a carico di organelli cellulari, come mitocondri e reticolo sarcoplasmatico, di una disfunzione mitocondriale ed dall’insorgenza di apoptosi spontanea. Durante il primo anno del mio dottorato, ho partecipato ad un progetto volto alla comprensione dei meccanismi pato-molecolari alla base del difetto muscolare, in assenza del collagene VI. Questo lavoro ha dimostrato che nei topi Col6a1–/– la retenzione di organelli disfunzionali è dovuta ad una regolazione inefficiente del flusso autofagico nelle fibre muscolari. In particolare, in tale contesto, mi sono occupata degli esperimenti istologici e relativi all’analisi dell’apoptosi nei muscoli di topi wild-type e knockout in condizioni di controllo e sottoposti a diversi approcci di tipo genetico, nutrizionale e farmacologico. In seconda battuta, nel corso del periodo di dottorato ho focalizzato il mio interesse sullo studio del collagene VI in un tessuto diverso, quello nervoso. Infatti recentemente è stato dimostrato che questa proteina è in grado di proteggere specificamente i neuroni dalla tossicità degli Aβ-peptidi, agenti critici nell’insorgenza della malattia di Alzheimer, e dalla morte cellulare indotta da radiazioni UV. Inoltre, alterazioni a carico dell’apparato autofagico sono note essere particolarmente dannose nel sistema nervoso centrale (SNC). Al fine di comprendere il ruolo del collagene VI nel SNC ho allestito colture primarie da corteccia ed ippocampo di topi neonati wild-type e Col6a1–/– ed ho analizzato in vitro i segnali dimostratisi alterati nel muscolo scheletrico in assenza della proteina in esame. Nelle colture wild-type il collagene VI è localizzato sulla superficie delle cellule neuronali e gliali. Le cellule knockout mostrano un aumento significativo dell’apoptosi, effetto recuperato quando le colture sono piastrate su collagene VI purificato, come substrato. L’analisi biochimica di marcatori autofagici ed osservazioni condotte in colture primarie neurali derivate da topi transgenici GFP-LC3;Col6a1+/+ e GFP-LC3;Col6a1–/– hanno rivelato, inoltre, un’alterazione nel flusso autofagico in assenza di collagene VI. Infine, risultati preliminari indicano una maggiore vulnerabilità delle cellule Col6a1–/– allo stress ossidativo. Queste osservazioni in vitro suggeriscono che la mancanza di collagene VI induce morte cellulare ed un’alterata risposta autofagica in cellule primarie di derivazione nervosa. Di maggior interesse è quindi la dimostrazione in vivo di un ruolo protettivo del collagene VI nel SNC. Analisi condotte sul cervello di topi di diverse età confermano l’importanza della proteina nel SNC e suggeriscono un’implicazione durante il proceso di invecchiamento. Infine nell’ultimo anno, vista l’alta espressione del collagene VI nel sistema nervoso periferico (SNP), sono state condotte anche analisi a livello del nervo sciatico, volte alla comprensione del ruolo della proteina in questo tessuto. Precedenti studi, inoltre, dimostravano l’espressione della proteina durante il differenziamento delle cellule di Schwann. Le analisi finora condotte mostrano che la mancanza del collagene VI nel nervo sciatico inducono un ispessimento della mielina, alterazioni a livello ultrastrutturale nelle fibre nervose e deficit sia motori che sensoriali. Queste evidenze nell’insieme, dimostrano una crescente importanza del collagene VI sia nel sistema nervoso centrale che periferico, in cui la proteina svolge ruoli specifici, apparentemente differenti

Lack of Collagen VI Promotes Wound-Induced Hair Growth

Collagen VI is an extracellular matrix molecule that is abundantly expressed in the skin. However, the role of collagen VI in hair follicle growth is unknown. Here, we show that collagen VI is strongly deposited in hair follicles, and is markedly upregulated by skin wounding. Lack of collagen VI in Col6a1−/− mice delays hair cycling and growth under physiological conditions, but promotes wound-induced hair regrowth without affecting skin regeneration. Conversely, addition of purified collagen VI rescues the abnormal wound-induced hair regrowth in Col6a1−/− mice. Mechanistic studies revealed that the increased wound-induced hair regrowth of Col6a1−/− mice is triggered by activation of the Wnt/β-catenin signaling pathway, and is abolished by inhibition of this pathway. These findings highlight the essential relationships between extracellular matrix (ECM) and hair follicle regeneration, and suggest that collagen VI could be a potential therapeutic target for hair loss and other skin-related diseases

Environmental Impact on Male (In)Fertility via Epigenetic Route

In the last 40 years, male reproductive health-which is very sensitive to both environmental exposure and metabolic status-has deteriorated and the poor sperm quality observed has been suggested to affect offspring development and its health in adult life. In this scenario, evidence now suggests that epigenetics shapes endocrine functions, linking genetics and environment. During fertilization, spermatozoa share with the oocyte their epigenome, along with their haploid genome, in order to orchestrate embryo development. The epigenetic signature of spermatozoa is the result of a dynamic modulation of the epigenetic marks occurring, firstly, in the testis-during germ cell progression-then, along the epididymis, where spermatozoa still receive molecules, conveyed by epididymosomes. Paternal lifestyle, including nutrition and exposure to hazardous substances, alters the phenotype of the next generations, through the remodeling of a sperm epigenetic blueprint that dynamically reacts to a wide range of environmental and lifestyle stressors. With that in mind, this review will summarize and discuss insights into germline epigenetic plasticity caused by environmental stimuli and diet and how spermatozoa may be carriers of induced epimutations across generations through a mechanism known as paternal transgenerational epigenetic inheritance