Skeletal Muscle-Specific Ablation of γcyto-Actin Does Not Exacerbate the mdx Phenotype

We previously documented a ten-fold increase in γcyto-actin expression in dystrophin-deficient skeletal muscle and hypothesized that increased γcyto-actin expression may participate in an adaptive cytoskeletal remodeling response. To explore whether increased γcyto-actin fortifies the cortical cytoskeleton in dystrophic skeletal muscle, we generated double knockout mice lacking both dystrophin and γcyto-actin specifically in skeletal muscle (ms-DKO). Surprisingly, dystrophin-deficient mdx and ms-DKO mice presented with comparable levels of myofiber necrosis, membrane instability, and deficits in muscle function. The lack of an exacerbated phenotype in ms-DKO mice suggests γcyto-actin and dystrophin function in a common pathway. Finally, because both mdx and ms-DKO skeletal muscle showed similar levels of utrophin expression and presented with identical dystrophies, we conclude utrophin can partially compensate for the loss of dystrophin independent of a γcyto-actin-utrophin interaction

Transgenic Rescue of the LARGEmyd Mouse: A LARGE Therapeutic Window?

LARGE is a glycosyltransferase involved in glycosylation of α-dystroglycan (α-DG). Absence of this protein in the LARGEmyd mouse results in α-DG hypoglycosylation, and is associated with central nervous system abnormalities and progressive muscular dystrophy. Up-regulation of LARGE has previously been proposed as a therapy for the secondary dystroglycanopathies: overexpression in cells compensates for defects in multiple dystroglycanopathy genes. Counterintuitively, LARGE overexpression in an FKRP-deficient mouse exacerbates pathology, suggesting that modulation of α-DG glycosylation requires further investigation. Here we demonstrate that transgenic expression of human LARGE (LARGE-LV5) in the LARGEmyd mouse restores α-DG glycosylation (with marked hyperglycosylation in muscle) and that this corrects both the muscle pathology and brain architecture. By quantitative analyses of LARGE transcripts we also here show that levels of transgenic and endogenous LARGE in the brains of transgenic animals are comparable, but that the transgene is markedly overexpressed in heart and particularly skeletal muscle (20–100 fold over endogenous). Our data suggest LARGE overexpression may only be deleterious under a forced regenerative context, such as that resulting from a reduction in FKRP: in the absence of such a defect we show that systemic expression of LARGE can indeed act therapeutically, and that even dramatic LARGE overexpression is well-tolerated in heart and skeletal muscle. Moreover, correction of LARGEmyd brain pathology with only moderate, near-physiological LARGE expression suggests a generous therapeutic window

Identification of Muscle-Specific MicroRNAs in Serum of Muscular Dystrophy Animal Models: Promising Novel Blood-Based Markers for Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder caused by mutations in the dystrophin gene, which encodes a cytoskeletal protein, dystrophin. Creatine kinase (CK) is generally used as a blood-based biomarker for muscular disease including DMD, but it is not always reliable since it is easily affected by stress to the body, such as exercise. Therefore, more reliable biomarkers of muscular dystrophy have long been desired. MicroRNAs (miRNAs) are small, ∼22 nucleotide, noncoding RNAs which play important roles in the regulation of gene expression at the post-transcriptional level. Recently, it has been reported that miRNAs exist in blood. In this study, we hypothesized that the expression levels of specific serum circulating miRNAs may be useful to monitor the pathological progression of muscular diseases, and therefore explored the possibility of these miRNAs as new biomarkers for muscular diseases. To confirm this hypothesis, we quantified the expression levels of miRNAs in serum of the dystrophin-deficient muscular dystrophy mouse model, mdx, and the canine X-linked muscular dystrophy in Japan dog model (CXMDJ), by real-time PCR. We found that the serum levels of several muscle-specific miRNAs (miR-1, miR-133a and miR-206) are increased in both mdx and CXMDJ. Interestingly, unlike CK levels, expression levels of these miRNAs in mdx serum are little influenced by exercise using treadmill. These results suggest that serum miRNAs are useful and reliable biomarkers for muscular dystrophy

Mediation and the Best Interests of the Child from the Child Law Perspective

What is the best interests of the child in family mediation and is mediation in the best interests of the child? In this article, I use child law and the United Nations Convention on the Rights of the Child combined with mediation theory to discuss these questions. Both mediation and the best interests of the child are open for multiple interpretations. Using facilitative and evaluative mediation theory and the legal concept ‘the best interests of the child’, I explore and compare the understandings of these concepts as they apply to family mediation. This includes a discussion of the advantages and disadvantages of facilitative as well as evaluative mediation orientations in terms of protecting the best interests of the child. Finnish court-connected family mediation is a combination of both mediation orientations, and the mediator is obliged to secure the best interests of the child. From a theoretical point of view, this seems to be a challenging combination.Peer reviewe

Walker-Warburg syndrome

Walker-Warburg Syndrome (WWS) is a rare form of autosomal recessive congenital muscular dystrophy associated with brain and eye abnormalities. WWS has a worldwide distribution. The overall incidence is unknown but a survey in North-eastern Italy has reported an incidence rate of 1.2 per 100,000 live births. It is the most severe form of congenital muscular dystrophy with most children dying before the age of three years. WWS presents at birth with generalized hypotonia, muscle weakness, developmental delay with mental retardation and occasional seizures. It is associated with type II cobblestone lissencephaly, hydrocephalus, cerebellar malformations, eye abnormalities and congenital muscular dystrophy characterized by hypoglycosylation of α-dystroglycan. Several genes have been implicated in the etiology of WWS, and others are as yet unknown. Several mutations were found in the Protein O-Mannosyltransferase 1 and 2 (POMT1 and POMT2) genes, and one mutation was found in each of the fukutin and fukutin-related protein (FKRP) genes. Laboratory investigations usually show elevated creatine kinase, myopathic/dystrophic muscle pathology and altered α-dystroglycan. Antenatal diagnosis is possible in families with known mutations. Prenatal ultrasound may be helpful for diagnosis in families where the molecular defect is unknown. No specific treatment is available. Management is only supportive and preventive

Destabilization of the Dystrophin-Glycoprotein Complex without Functional Deficits in α-Dystrobrevin Null Muscle

α-Dystrobrevin is a component of the dystrophin-glycoprotein complex (DGC) and is thought to have both structural and signaling roles in skeletal muscle. Mice deficient for α-dystrobrevin (adbn−/−) exhibit extensive myofiber degeneration and neuromuscular junction abnormalities. However, the biochemical stability of the DGC and the functional performance of adbn−/− muscle have not been characterized. Here we show that the biochemical association between dystrophin and β-dystroglycan is compromised in adbn−/− skeletal muscle, suggesting that α-dystrobrevin plays a structural role in stabilizing the DGC. However, despite muscle cell death and DGC destabilization, costamere organization and physiological performance is normal in adbn−/− skeletal muscle. Our results demonstrate that myofiber degeneration alone does not cause functional deficits and suggests that more complex pathological factors contribute to the development of muscle weakness in muscular dystrophy

LARGE Expression Augments the Glycosylation of Glycoproteins in Addition to α-Dystroglycan Conferring Laminin Binding

Mutations in genes encoding glycosyltransferases (and presumed glycosyltransferases) that affect glycosylation and extracellular matrix binding activity of α-dystroglycan (α-DG) cause congenital muscular dystrophies (CMDs) with central nervous system manifestations. Among the identified genes, LARGE is of particular interest because its overexpression rescues glycosylation defects of α-DG in mutations of not only LARGE but also other CMD-causing genes and restores laminin binding activity of α-DG. It is not known whether LARGE protein glycosylates other proteins in addition to α-DG. In this study, we overexpressed LARGE in DG-deficient cells and analyzed glycosylated proteins by Western blot analysis. Surprisingly, overexpression of LARGE in α-DG-deficient cells led to glycosylation dependent IIH6C4 and VIA4-1 immunoreactivity, despite the prevailing view that these antibodies only recognize glycosylated α-DG. Furthermore, the hyperglycosylated proteins in LARGE-overexpressing cells demonstrated the functional capacity to bind the extracellular matrix molecule laminin and promote laminin assembly at the cell surface, an effect that was blocked by IIH6C4 antibodies. These results indicate that overexpression of LARGE catalyzes the glycosylation of at least one other glycoprotein in addition to α-DG, and that this glycosylation(s) promotes laminin binding activity

A reporter mouse for optical imaging of inflammation in mdx muscles

BACKGROUND: Duchenne muscular dystrophy (DMD) is due to mutations in the gene coding for human DMD; DMD is characterized by progressive muscle degeneration, inflammation, fat accumulation, and fibrosis. The mdx mouse model of DMD lacks dystrophin protein and undergoes a predictable disease course. While this model has been a valuable resource for pre-clinical studies aiming to test therapeutic compounds, its utility is compromised by a lack of reliable biochemical tools to quantifiably assay muscle disease. Additionally, there are few non-invasive assays available to researchers for measuring early indicators of disease progression in mdx mice. METHODS: Mdx mice were crossed to knock-in mice expressing luciferase from the Cox2 promoter. These reporter mice (Cox2(FLuc/+)DMD(−/−)) were created to serve as a tool for researchers to evaluate muscle inflammation. Luciferase expression was assayed by immunohistochemistry to insure that it correlated with muscle lesions. The luciferase signal was quantified by optical imaging and luciferase assays to verify that the signal correlated with muscle damage. As proof of principle, Cox2(FLuc/+)DMD(−/−) mice were also treated with prednisolone to validate that a reduction in luciferase signal correlated with prednisone treatment. RESULTS: In this investigation, a novel reporter mouse (Cox2(FLuc/+)DMD(−/−) mice) was created and validated for non-invasive quantification of muscle inflammation in vivo. In this dystrophic mouse, luciferase is expressed from cyclooxygenase 2 (Cox2) expressing cells and bioluminescence is detected by optical imaging. Bioluminescence is significantly enhanced in damaged muscle of exercised Cox2(FLuc/+)DMD(−/−) mice compared to non-exercised Cox2(FLuc/+)DMD(+/+) mice. Moreover, the Cox2 bioluminescent signal is reduced in Cox2(FLuc/+)DMD(−/−) mice in response to a course of steroid treatment. Reduction in bioluminescence is detectable prior to measurable therapy-elicited improvements in muscle strength, as assessed by traditional means. Biochemical assay of luciferase provides a second means to quantify muscle inflammation. CONCLUSIONS: The Cox2(FLuc/+)DMD(−/−) mouse is a novel tool to evaluate the therapeutic benefits of drugs intended to target inflammatory aspects of dystrophic pathology. This mouse model will be a useful adjunct to traditional outcome measures in assessing potential therapeutic compounds

Retrograde trafficking of β-dystroglycan from the plasma membrane to the nucleus

β-Dystroglycan (β-DG) is a transmembrane protein with critical roles in cell adhesion, cytoskeleton remodeling and nuclear architecture. This functional diversity is attributed to the ability of β-DG to target to, and conform specific protein assemblies at the plasma membrane (PM) and nuclear envelope (NE). Although a classical NLS and importin α/β mediated nuclear import pathway has already been described for β-DG, the intracellular trafficking route by which β-DG reaches the nucleus is unknown. In this study, we demonstrated that β-DG undergoes retrograde intracellular trafficking from the PM to the nucleus via the endosome-ER network. Furthermore, we provided evidence indicating that the translocon complex Sec61 mediates the release of β-DG from the ER membrane, making it accessible for importins and nuclear import. Finally, we show that phosphorylation of β-DG at Tyr890 is a key stimulus for β-DG nuclear translocation. Collectively our data describe the retrograde intracellular trafficking route that β-DG follows from PM to the nucleus. This dual role for a cell adhesion receptor permits the cell to functionally connect the PM with the nucleus and represents to our knowledge the first example of a cell adhesion receptor exhibiting retrograde nuclear trafficking and having dual roles in PM and NE

Human first-trimester chorionic villi have a myogenic potential

First-trimester chorionic-villi-derived cells (FTCVs) are the earliest fetal material that can be obtained for prenatal diagnosis of fetal disorders such as Duchenne muscular dystrophy (DMD). DMD is a devastating X-linked disorder characterized by the absence of dystrophin at the sarcolemma of muscle fibers. Currently, a limited number of treatment options are available for DMD, although cell therapy is a promising treatment strategy for muscle degeneration in DMD patients. A novel candidate source of cells for this approach is FTCVs taken between the 9th and 11th weeks of gestation. FTCVs might have a higher undifferentiated potential than any other tissue-derived cells because they are the earliest fetal material. We examined the expression of mesenchymal stem cell and pluripotent stem cell markers in FTCVs, in addition to their myogenic potential. FTCVs expressed mesenchymal stem cell markers and Nanog and Sox2 transcription factors as pluripotent stem cell markers. These cells efficiently differentiated into myotubes after myogenic induction, at which point Nanog and Sox2 were down-regulated, whereas MyoD, myogenin, desmin and dystrophin were up-regulated. To our knowledge, this is the first demonstration that FTCVs can be efficiently directed to differentiate in vitro into skeletal muscle cells that express dystrophin as the last stage marker of myogenic differentiation. The myogenic potential of FTCVs reveals their promise for use in cell therapy for DMD, for which no effective treatment presently exists