Cinzia Bragato

Centronuclear myopathy related to dynamin 2 mutations: Clinical, morphological, muscle imaging and genetic features of an Italian cohort

Mutations in dynamin 2 (DNM2) gene cause autosomal dominant centronuclear myopathy and occur in around 50% of patients with centronuclear myopathy. We report clinical, morphological, muscle imaging and genetic data of 10 unrelated Italian patients with centronuclear myopathy related to DNM2 mutations. Our results confirm the clinical heterogeneity of this disease, underlining some peculiar clinical features, such as severe pulmonary impairment and jaw contracture that should be considered in the clinical follow-up of these patients. Muscle MRI showed a distinct pattern of involvement, with predominant involvement of soleus and tibialis anterior in the lower leg muscles, followed by hamstring muscles and adductor magnus at thigh level and gluteus maximus. The detection of three novel DNM2 mutations and the first case of somatic mosaicism further expand the genetic spectrum of the disease.

Zebrafish as a Model to Investigate Dynamin 2-Related Diseases

Mutations in the dynamin-2 gene (DNM2) cause autosomal dominant centronuclear myopathy (CNM) and dominant intermediate Charcot-Marie-Tooth (CMT) neuropathy type B (CMTDIB). As the relation between these DNM2-related diseases is poorly understood, we used zebrafish to investigate the effects of two different DNM2 mutations. First we identified a new alternatively spliced zebrafish dynamin-2a mRNA (dnm2a-v2) with greater similarity to human DNM2 than the deposited sequence. Then we knocked-down the zebrafish dnm2a, producing defects in muscle morphology. Finally, we expressed two mutated DNM2 mRNA by injecting zebrafish embryos with human mRNAs carrying the R522H mutation, causing CNM, or the G537C mutation, causing CMT. Defects arose especially in secondary motor neuron formation, with incorrect branching in embryos injected with CNM-mutated mRNA, and total absence of branching in those injected with CMT-mutated mRNA. Muscle morphology in embryos injected with CMT-mutated mRNA appeared less regularly organized than in those injected with CNM-mutated mRNA. Our results showing, a continuum between CNM and CMTDIB phenotypes in zebrafish, similarly to the human conditions, confirm this animal model to be a powerful tool to investigate mutations of DNM2 in vivo.

Anti-fibrotic effect of pirfenidone in muscle derived-fibroblasts from Duchenne muscular dystrophy patients.

AIMS Tissue fibrosis, characterized by excessive deposition of extracellular matrix proteins, is the end point of diseases affecting the kidney, bladder, liver, lung, gut, skin, heart and muscle. In Duchenne muscular dystrophy (DMD), connective fibrotic tissue progressively substitutes muscle fibers. So far no specific pharmacological treatment is available for muscle fibrosis. Among promising anti-fibrotic molecules, pirfenidone has shown anti-fibrotic and anti-inflammatory activity in animal and cell models, and has already been employed in clinical trials. Therefore we tested pirfenidone anti-fibrotic properties in an in vitro model of muscle fibrosis. MAIN METHODS We evaluated effect of pirfenidone on fibroblasts isolated from DMD muscle biopsies. These cells have been previously characterized as having a pro-fibrotic phenotype. We tested cell proliferation and migration, secretion of soluble collagens, intracellular levels of collagen type I and fibronectin, and diameter of 3D fibrotic nodules. KEY FINDINGS We found that pirfenidone significantly reduced proliferation and cell migration of control and DMD muscle-derived fibroblasts, decreased extracellular secretion of soluble collagens by control and DMD fibroblasts, as well as levels of collagen type I and fibronectin, and, in DMD fibroblasts only, reduced synthesis and deposition of intracellular collagen. Furthermore, pirfenidone was able to reduce the diameter of fibrotic-nodules in our 3D model of in vitro fibrosis. SIGNIFICANCE These pre-clinical results indicate that pirfenidone has potential anti-fibrotic effects also in skeletal muscle fibrosis, urging further studies in in vivo animal models of muscular dystrophy in order to translate the drug into the treatment of muscle fibrosis in DMD patients.

FGF2 and EGF Are Required for Self-Renewal and Organoid Formation of Canine Normal and Tumor Breast Stem Cells.

Recent studies suggest that human tumors are generated from cancer cells with stem cell (SC) properties. Spontaneously occurring cancers in dogs contain a diversity of cells that like for human tumors suggest that certain canine tumors are also generated from cancer stem cells (CSCs). CSCs, like normal SCs, have the capacity for self‐renewal as mammospheres in suspension cultures. To understand how cells with SC properties contribute to canine mammary gland tumor development and progression, comparative analysis between normal SCs and CSCs, obtained from the same individual, is essential. We have utilized the property of sphere formation to develop culture conditions for propagating stem/progenitor cells from canine normal and tumor tissue. We show that cells from dissociated mammospheres retain sphere reformation capacity for several serial passages and have the capacity to generate organoid structures ex situ. Utilizing various culture conditions for passaging SCs and CSCs, fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF) were found to positively or negatively regulate mammosphere regeneration, organoid formation, and multi‐lineage differentiation potential. The response of FGF2 and EGF on SCs and CSCs was different, with increased FGF2 and EGF self‐renewal promoted in SCs and repressed in CSCs. Our protocol for propagating SCs from normal and tumor canine breast tissue will provide new opportunities in comparative mammary gland stem cell analysis between species and anticancer treatment and therapies for dogs. J. Cell. Biochem. 118: 570–584, 2017.

FGF2 and EGF is required for self-renewal of canine normal and tumor breast stem/progenitor cells that have organoid formation potential

Recent studies suggest that human tumors are generated from cancer cells with stem cell (SC) properties. Spontaneously occurring cancers in dogs contain a diversity of cells that like for human tumors suggest that certain canine tumors are also generated from cancer stem cells (CSCs). CSCs, like normal SCs, have the capacity for self‐renewal as mammospheres in suspension cultures. To understand how cells with SC properties contribute to canine mammary gland tumor development and progression, comparative analysis between normal SCs and CSCs, obtained from the same individual, is essential. We have utilized the property of sphere formation to develop culture conditions for propagating stem/progenitor cells from canine normal and tumor tissue. We show that cells from dissociated mammospheres retain sphere reformation capacity for several serial passages and have the capacity to generate organoid structures ex situ. Utilizing various culture conditions for passaging SCs and CSCs, fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF) were found to positively or negatively regulate mammosphere regeneration, organoid formation, and multi‐lineage differentiation potential. The response of FGF2 and EGF on SCs and CSCs was different, with increased FGF2 and EGF self‐renewal promoted in SCs and repressed in CSCs. Our protocol for propagating SCs from normal and tumor canine breast tissue will provide new opportunities in comparative mammary gland stem cell analysis between species and anticancer treatment and therapies for dogs. J. Cell. Biochem. 118: 570–584, 2017.

Exosomes and exosomal miRNAs from muscle-derived fibroblasts promote skeletal muscle fibrosis

Exosomes, natural carriers of mRNAs, non-coding RNAs and proteins between donor and recipient cells, actively contribute to cell-cell communication. We investigated the potential pro-fibrotic role of exosomes released by muscle-derived fibroblasts of Duchenne muscular dystrophy (DMD) patients, and of miRNAs carried by exosomes. By fibrosis focused array analysis we found that exosomes from DMD fibroblasts, had significantly higher levels of miR-199a-5p, a miRNA up-regulated in fibrotic conditions, compared to control exosomes, while levels in myoblast-derived exosomes were not increased. In control fibroblasts, exposure to DMD fibroblast-derived exosomes induced a myofibroblastic phenotype with increase in α-smooth actin, collagen and fibronectin transcript and protein expression, soluble collagen production and deposition, cell proliferation, and activation of Akt and ERK signaling, while exposure to control exosomes did not. Transfecting control fibroblasts or loading control exosomes with miR-199a-5p mimic or inhibitor induced opposing effects on fibrosis-related mRNAs and proteins, on collagen production and Akt and ERK pathways. Finally, injection of DMD fibroblast-derived exosomes into mouse tibialis anterior muscle after cardiotoxin-induced necrosis, produced greater fibrosis than control exosomes. Our findings indicate that exosomes produced by local fibroblasts in the DMD muscle are able to induce phenotypic conversion of normal fibroblasts to myofibroblasts thereby increasing the fibrotic response. This conversion is related to transfer of high levels of miR-199a-5p and to reduction of its target caveolin-1; both, therefore, are potential therapeutic targets in muscle fibrosis.

HDAC8 regulates canonical Wnt pathway to promote differentiation in skeletal muscles: FERRARI et al.

Histone deacetylase 8 (HDAC8) is a class 1 histone deacetylase and a member of the cohesin complex. HDAC8 is expressed in smooth muscles, but its expression in skeletal muscle has not been described. We have shown for the first time that HDAC8 is expressed in human and zebrafish skeletal muscles. Using RD/12 and RD/18 rhabdomyosarcoma cells with low and high differentiation potency, respectively, we highlighted a specific correlation with HDAC8 expression and an advanced stage of muscle differentiation. We inhibited HDAC8 activity through a specific PCI‐34051 inhibitor in murine C2C12 myoblasts and zebrafish embryos, and we observed skeletal muscles differentiation impairment. We also found a positive regulation of the canonical Wnt signaling by HDAC8 that might explain muscle differentiation defects. These findings suggest a novel mechanism through which HDAC8 expression, in a specific time window of skeletal muscle development, positively regulates canonical Wnt pathway that is necessary for muscle differentiation.

Modeling Cornelia de Lange Syndrome in vitro and in vivo reveals a role for cohesin complex in neuronal survival and differentiation

Abstract Cornelia de Lange syndrome (CdLS), which is reported to affect ˜1 in 10 000 to 30 000 newborns, is a multisystem organ developmental disorder with relatively mild to severe effects. Among others, intellectual disability represents an important feature of this condition. CdLS can result from mutations in at least five genes: nipped‐B‐like protein, structural maintenance of chromosomes 1A, structural maintenance of chromosomes 3, RAD21 cohesin complex component and histone deacetylase 8 (HDAC8). It is believed that mutations in these genes cause CdLS by impairing the function of the cohesin complex (to which all the aforementioned genes contribute to the structure or function), disrupting gene regulation during critical stages of early development. Since intellectual disorder might result from alterations in neural development, in this work, we studied the role of Hdac8 gene in mouse neural stem cells (NSCs) and in vertebrate (Danio rerio) brain development by knockdown and chemical inhibition experiments. Underlying features of Hdac8 deficiency is an increased cell death in the developing neural tissues, either in mouse NSCs or in zebrafish embryos.

FGF2 and EGF Are Required for Self-Renewal and Organoid Formation of Canine Normal and Tumor Breast Stem Cells: FGF2ANDEGF ESSENTIALFORCANINESC/CSC SELF-RENEWAL

Recent studies suggest that human tumors are generated from cancer cells with stem cell (SC) properties. Spontaneously occurring cancers in dogs contain a diversity of cells that like for human tumors suggest that certain canine tumors are also generated from cancer stem cells (CSCs). CSCs, like normal SCs, have the capacity for self‐renewal as mammospheres in suspension cultures. To understand how cells with SC properties contribute to canine mammary gland tumor development and progression, comparative analysis between normal SCs and CSCs, obtained from the same individual, is essential. We have utilized the property of sphere formation to develop culture conditions for propagating stem/progenitor cells from canine normal and tumor tissue. We show that cells from dissociated mammospheres retain sphere reformation capacity for several serial passages and have the capacity to generate organoid structures ex situ. Utilizing various culture conditions for passaging SCs and CSCs, fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF) were found to positively or negatively regulate mammosphere regeneration, organoid formation, and multi‐lineage differentiation potential. The response of FGF2 and EGF on SCs and CSCs was different, with increased FGF2 and EGF self‐renewal promoted in SCs and repressed in CSCs. Our protocol for propagating SCs from normal and tumor canine breast tissue will provide new opportunities in comparative mammary gland stem cell analysis between species and anticancer treatment and therapies for dogs. J. Cell. Biochem. 118: 570–584, 2017.