Research Projects Role of Necdin in skeletal muscle regeneration and in the differentiation of mesoangioblast stem cells
Regeneration of muscle fibers, lost during pathological muscle degeneration or after injuries, is sustained by the production of new myofibers. An important cell type involved in muscle regeneration is the satellite cell. Necdin is a protein expressed in satellite cell-derived myogenic precursors during perinatal growth. However, its function in myogenesis is not known. We compared transgenic mice that over-express necdin in skeletal muscle with both wild type and necdin-null mice. Following muscle injury the necdin-null mice showed a significant defect in muscle healing while mice that over-express necdin showed a significant increase in myofiber regeneration. We also found that in muscle, necdin increases myogenin expression, accelerates differentiation, and counteracts myoblast apoptosis. Together these data clarify the function and mechanism of necdin in skeletal muscle and show the importance of necdin in muscle regeneration.
We are further investigating the mechanisms of action of this protein by searching for interacting molecules by yeast two hybrid screening and by identified target genes, by micro-array analysis.
In addition it appears that transient overexpression of Necdin increases the differentiation ability of mesoangioblasts in vitro. Mesoangioblasts are vessel-associated stem cells that have been shown to be ideal candidates for stem cell therapy of muscular dystrophies. However, despite of the identification of mesoangioblasts as potential source of skeletal muscle, detailed studies on the molecular pathways regulating their growth and differentiation are needed to improve their ability to differentiate and fuse to the fiber, to proliferate and resist to cell death and as such act as pool of resident stem cells. We are investigating the role of necdin in mesoangioblast biology, by isolating these cells from necdin gain and loss of function mice, and studying their properties in vitro and in vivo when transplanted in dystrophic mouse models.
Identifications of new molecular approaches to muscular dystrophies
The muscular dystrophies (MD) are a group of more than 30 genetic diseases characterized by progressive weakness and degeneration of the skeletal muscles that control movement. The current therapeutic approaches to muscular dystrophies involve steroids, such as prednisolone and deflazacort, and dantrolene, administered in various protocols. These treatments result in modest beneficial effects and are accompanied by severe side- Thus there is a need to identify therapeutic agents which slow the muscle fibre damage and delay the onset of disability in patients with muscular dystrophies more efficiently than the current therapies and causing a lesser degree of side effects. The beneficial effect of corticosteroids is believed to reside in the blocking of immune cell activation and infiltration which are precipitated by muscle fibre damage resulting from the disease.
Nitric oxide (NO) generated by a muscular NO synthase structurally and functionally linked to the dystrophin complex at the sarcolemma participates to physiological development and function of skeletal muscle. NSAIDs are known to reduce inflammation through inhibition of cyclooxygenases 1 and 2. We have found that the combination of nitric oxide-releasing compounds and NSAIDs is effective in retarding the development of, or reversing muscular dystrophies. Moreover these compounds, for which a novel use is proposed, are known to cause less side effects than current therapies for muscular dystrophy. We are currently further investigating several different coumpounds that combine NO and NSAIDS, on both the onset of muscle dystrophy and the transplantation of mesoangioblasts stem cells in dystrophic mouse model.
We are also investigating the molecular mechanism of the beneficial effect of these compounds, in particular on the maintenance of the satellite cells pool.
New pluripotency factors in embryonic stem cells
The maintenance of pluripotency in stem cells is dependent on both genetic and epigenetic factors, and the understandin of the molecules playing a role in this process is crucial for the manipulatiion of stem cell fate . In the early chick embryo and in chick embryonic stem cells, competitive interactions between three coiled-coil domain proteins (ERNI, BERT and Geminin) regulate Sox2 expression, resulting in neural induction and loss of pluripotency. It has been sugggested that these proteins modulate the recruitment of different HP1 transcriptional repressors to Brahma-containing chromatin remodelling complexes, which otherwise activate Sox2 expression.
Various bioinformatic attempt to identify a mouse homologue of ERNI have failed, therefore we have decided to look for a mouse functional homologue of the chicken protein ERNI by performing a two-hybrid screening assay. The “baits” are BERT and HP1gamma, both interacting with ERNI in chick ES cells and in the early chick embryo.
In the first screening using BERT as bait, we have identified and validated several positive interactors amongst which developmental pluripotency associated 5 (Dppa5), and nuclear receptor co-repressor 1 (Ncor1). We are now validating the interaction in mammalian cells by immunoprecipitation in vitro and in vivo using the bimolecular fluorescence system, and we will further investigate their function by assessing the effect of their expression on ES cells pluripotency and differention.
Bibliography
Brunelli, S., Tagliafico, E., De Angelis, F.G., Tonlorenzi, R., Ferrari, S., Bozzoni, I., Niinobe, M., Yoshikawa, K., Ferrari, S., Cossu, G. (2004) Msx2 and Necdin combined activities are required for smooth muscle differentiation in mesoangioblast stem cells. Circulation Research, .94: 1571-8.
Sciorati, C.*, Galvez, B.G.*, Brunelli, S.*, Tagliafico, E., Ferrari, S., Cossu, G. and Clementi, E. (2006) . An ex vivo treatment with nitric oxide that increases mesoangioblast therapeutic efficacy in muscular dystrophy. Journal of Cell Science. 119:5114-5123.
Brunelli, S. Rovere-Querini P., Sciorati , P., Manfredi, A., Clementi, C. (2007). Nitric oxide: emerging concepts about its use in cell-based therapies. Expert Opinion in Drug Development, 16:33-43.
Brunelli, S., Sciorati, C., D’Antona, G., Innocenzi, A., Covarello, D., Galvez, B.G., Monopoli, A., Sanvito, F., Bottinelli, R., Ongini, E., Cossu, G. and Clementi, E. (2007). Nitric Oxide release combined with non steroidal anti-inflammatory activity markedly ameliorates muscular dystrophy and potentiates the efficacy of stem cell therapy. PNAS, 104:264-9.
Deponti, D., Francois, S, Baesso, S., Sciorati, C., Innocenzi A., Broccoli, V., Muscatelli, F., Meneveri, R., Clementi, E., Cossu, G., Brunelli, S. (2007) Necdin mediate myoblasts survival and differentiation and accelerate skeletal muscle regeneration. Journal of Cell Biology. 179: 305–319.
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