Emanuela Gussoni

Dystrophin expression in the mdx mouse restored by stem cell transplantation

The development of cell or gene therapies for diseases involving cells that are widely distributed throughout the body has been severely hampered by the inability to achieve the disseminated delivery of cells or genes to the affected tissues or organ. Here we report the results of bone marrow transplantation studies in the mdx mouse, an animal model of Duchennes muscular dystrophy, which indicate that the intravenous injection of either normal haematopoietic stem cells or a novel population of muscle-derived stem cells into irradiated animals results in the reconstitution of the haematopoietic compartment of the transplanted recipients, the incorporation of donor-derived nuclei into muscle, and the partial restoration of dystrophin expression in the affected muscle. These results suggest that the transplantation of different stem cell populations, using the procedures of bone marrow transplantation, might provide an unanticipated avenue for treating muscular dystrophy as well as other diseases where the systemic delivery of therapeutic cells to sites throughout the body is critical. Our studies also suggest that the inherent developmental potential of stem cells isolated from diverse tissues or organs may be more similar than previously anticipated.

Mutations in the Dystrophin-Associated Protein γ-Sarcoglycan in Chromosome 13 Muscular Dystrophy

Severe childhood autosomal recessive muscular dystrophy (SCARMD) is a progressive muscle-wasting disorder common in North Africa that segregates with microsatellite markers at chromosome 13q12. Here, it is shown that a mutation in the gene encoding the 35-kilodalton dystrophin-associated glycoprotein, γ-sarcoglycan, is likely to be the primary genetic defect in this disorder. The human γ-sarcoglycan gene was mapped to chromosome 13q12, and deletions that alter its reading frame were identified in three families and one of four sporadic cases of SCARMD. These mutations not only affect γ-sarcoglycan but also disrupt the integrity of the entire sarcoglycan complex.

β–sarcoglycan (A3b) mutations cause autosomal recessive muscular dystrophy with loss of the sarcoglycan complex

The dystrophin associated proteins (DAPs) are good candidates for harboring primary mutations in the genetically heterogeneous autosomal recessive muscular dystrophies (ARMD). The transmembrane components of the DAPs can be separated into the dystroglycan and the sarcoglycan complexes. Here we report the isolation of cDNAs encoding the 43 kD sarcoglycan protein β–sarcoglycan (A3b) and the localization of the human gene to chromosome 4q12. We describe a young girl with ARMD with truncating mutations on both alleles. Immunostaining of her muscle biopsy shows specific loss of the components of the sarcoglycan complex β–sarcoglycan, α–sarcoglycan (adhalin), and 35 kD sarcoglycan). Thus secondary destabilization of the sarcoglycan complex may be an important pathophysiological event in ARMD.

The fate of individual myoblasts after transplantation into muscles of DMD patients.

Muscle biopsies from six patients with Duchenne muscular dystrophy (DMD) participating in a myoblast transplantation clinical trial were reexamined using a fluorescence in situ hybridization (FISH)-based method. Donor nuclei were detected in all biopsies analyzed, including nine where no donor myoblasts were previously thought to be present. In three patients, more than 10% of the original number of donor cells were calculated as present 6 months after implantation. Half of the detected donor nuclei were fused into host myofibers, and of these, nearly 50% produced dystrophin. These findings demonstrate that although donor myoblasts have persisted after injection, their microenvironment influences whether they fuse and express dystrophin. Our methodology could be used for developing new approaches to improve myoblast transfer efficacy and for the analysis of future gene-and/or cell-based therapies of numerous genetic disorders.

Brown-fat paucity due to impaired BMP signalling induces compensatory browning of white fat

Maintenance of body temperature is essential for the survival of homeotherms. Brown adipose tissue (BAT) is a specialized fat tissue that is dedicated to thermoregulation. Owing to its remarkable capacity to dissipate stored energy and its demonstrated presence in adult humans, BAT holds great promise for the treatment of obesity and metabolic syndrome. Rodent data suggest the existence of two types of brown fat cells: constitutive BAT (cBAT), which is of embryonic origin and anatomically located in the interscapular region of mice; and recruitable BAT (rBAT), which resides within white adipose tissue (WAT) and skeletal muscle, and has alternatively been called beige, brite or inducible BAT. Bone morphogenetic proteins (BMPs) regulate the formation and thermogenic activity of BAT. Here we use mouse models to provide evidence for a systemically active regulatory mechanism that controls whole-body BAT activity for thermoregulation and energy homeostasis. Genetic ablation of the type 1A BMP receptor (Bmpr1a) in brown adipogenic progenitor cells leads to a severe paucity of cBAT. This in turn increases sympathetic input to WAT, thereby promoting the formation of rBAT within white fat depots. This previously unknown compensatory mechanism, aimed at restoring total brown-fat-mediated thermogenic capacity in the body, is sufficient to maintain normal temperature homeostasis and resistance to diet-induced obesity. These data suggest an important physiological cross-talk between constitutive and recruitable brown fat cells. This sophisticated regulatory mechanism of body temperature may participate in the control of energy balance and metabolic disease.

Long-term persistence of donor nuclei in a Duchenne muscular dystrophy patient receiving bone marrow transplantation

Duchenne muscular dystrophy (DMD) is a severe progressive muscle-wasting disorder caused by mutations in the dystrophin gene. Studies have shown that bone marrow cells transplanted into lethally irradiated mdx mice, the mouse model of DMD, can become part of skeletal muscle myofibers. Whether human marrow cells also have this ability is unknown. Here we report the analysis of muscle biopsies from a DMD patient (DMD-BMT1) who received bone marrow transplantation at age 1 year for X-linked severe combined immune deficiency and who was diagnosed with DMD at age 12 years. Analysis of muscle biopsies from DMD-BMT1 revealed the presence of donor nuclei within a small number of muscle myofibers (0.5-0.9%). The majority of the myofibers produce a truncated, in-frame isoform of dystrophin lacking exons 44 and 45 (not wild-type). The presence of bone marrow-derived donor nuclei in the muscle of this patient documents the ability of exogenous human bone marrow cells to fuse into skeletal muscle and persist up to 13 years after transplantation.

The Three Human Syntrophin Genes Are Expressed in Diverse Tissues, Have Distinct Chromosomal Locations, and Each Bind to Dystrophin and Its Relatives

The syntrophins are a biochemically heterogeneous group of 58-kDa intracellular membrane-associated dystrophin-binding proteins. We have cloned and characterized human acidic (α1-) syntrophin and a second isoform of human basic (β2-) syntrophin. Comparison of the deduced amino acid structure of the three human isoforms of syntrophin (together with the previously reported human β1-syntrophin) demonstrates their overall similarity. The deduced amino acid sequences of human α1- and β2-syntrophin are nearly identical to their homologues in mouse, suggesting a strong functional conservation among the individual isoforms. Much like β1-syntrophin, human β2-syntrophin has multiple transcript classes and is expressed widely, although in a distinct pattern of relative abundance. In contrast, human α1-syntrophin is most abundant in heart and skeletal muscle, and less so in other tissues. Somatic cell hybrids and fluorescent in situ hybridization were both used to determine their chromosomal locations: β2-syntrophin to chromosome 16q22-23 and α1-syntrophin to chromosome 20q11.2. Finally, we used in vitro translated proteins in an immunoprecipitation assay to show that, like β1-syntrophin, both β2- and α1-syntrophin interact with peptides encoding the syntrophin-binding region of dystrophin, utrophin/dystrophin related protein, and the Torpedo 87K protein.

Myoblast implantation in Duchenne muscular dystrophy: The San Francisco study

We evaluated myoblast implantation in 10 boys with Duchenne muscular dystrophy (DMD) and absent dystrophin (age 5–10 years) who were implanted with 100 million myoblasts in the anterior tibial muscle of one leg and placebo in the other. Cyclosporine (5 mg/kg/day) was administered for 7 months. Pre‐ and postimplantation (after 1 and 6 months) muscle biopsies were analyzed. Force generation (tetanic tension and maximum voluntary contraction) was measured monthly in a double‐blind design. There was increased force generation in both legs of all boys, probably due to cyclosporine. Using the polymerase chain reaction, evidence of myoblast survival and dystrophin mRNA expression was obtained in 3 patients after 1 month and in 1 patient after 6 months. These studies suggest a salutary effect of cyclosporine upon muscular force generation in Duchenne muscular dystrophy; however, myoblast implantation was not effective in replacing clinically significant amounts of dystrophin in DMD muscle.

A role for nephrin, a renal protein, in vertebrate skeletal muscle cell fusion

Skeletal muscle is formed via fusion of myoblasts, a well-studied process in Drosophila. In vertebrates however, this process is less well understood, and whether there is evolutionary conservation with the proteins studied in flies is under investigation. Sticks and stones (Sns), a cell surface protein found on Drosophila myoblasts, has structural homology to nephrin. Nephrin is a protein expressed in kidney that is part of the filtration barrier formed by podocytes. No previous study has established any role for nephrin in skeletal muscle. We show, using two models, zebrafish and mice, that the absence of nephrin results in poorly developed muscles and incompletely fused myotubes, respectively. Although nephrin-knockout (nephrinKO) myoblasts exhibit prolonged activation of MAPK/ERK pathway during myogenic differentiation, expression of myogenin does not seem to be altered. Nevertheless, MAPK pathway blockade does not rescue myoblast fusion. Co-cultures of unaffected human fetal myoblasts with nephrinKO myoblasts or myotubes restore the formation of mature myotubes; however, the contribution of nephrinKO myoblasts is minimal. These studies suggest that nephrin plays a role in secondary fusion of myoblasts into nascent myotubes, thus establishing a possible functional conservation with Drosophila Sns.

Stem cell therapy for muscular dystrophy

Muscular dystrophy is a heterogeneous group of neuromuscular disorders that manifests as progressive muscle weakness, muscle wasting and, in many cases, death. Although there has been enormous progress in the molecular understanding of muscular dystrophy, there is still no cure. There are, however, several different therapeutic options under investigation, including adult-derived stem cell transplantation. Encouraging and pioneering experiments in mouse models for Duchenne’s muscular dystrophy (DMD) demonstrated that myoblasts could be transplanted into dystrophic muscle; these myoblasts repaired a small proportion of damaged myofibres. Subsequent work has been devoted to optimisation of this technique. In doing so, a number of adult-derived stem cells have been isolated, characterised and used in promising animal transplantation experiments. Further research is ongoing, and is clearly necessary to make this therapy a viable treatment option for patients with muscular dystrophy.