No description available.

alpha- and beta-dystroglycan (DG) were first identified as members of an oligomeric, transmembrane complex expressed in muscle and linking laminin (LN) in the extracellular matrix (ECM) to dystrophin in the submembraneous cytoskeleton. This dystrophin-associated glycoprotein complex (DGC) has been proposed to perform a structural role in skeletal muscle, its loss leading to loss of membrane integrity, muscle fiber degeneration and muscular dystrophy. alpha- and beta-DG appear to form the core of the DGC since alpha-DG is a high affinity LN receptor while beta-DG is a transmembrane protein that anchors alpha-DG to the membrane and interacts with dystrophin intracellularly. In order to determine the involvement of DG in skeletal muscle homeostasis and in LN assembly, we generated mouse muscle cell lines deficient in DG expression. Extensive characterization of these cells revealed that DG is essential for LN assembly on the surface of mature myotubes but that it is not involved in the maintenance of membrane integrity in culture. However, DG-deficient cells show increased apoptotic cell death during and after the period of myoblast differentiation into myotubes, indicating that DG is important for muscle cell survival. / The ECM molecule agrin has been implicated in the induction of acetylcholine receptor (AChR) aggregation at the neuromuscular junction (NMJ). The C-terminus of agrin shares significant homology with the region of LN that interacts with alpha-DG; we therefore reasoned that alpha-DG could be an agrin receptor. Ligand overlay assays revealed that alpha-DG binds agrin with high affinity and antibody perturbation experiments indicated that alpha-DG is involved in agrin-induced aggregation of AChRs. The role of alpha-DG in AChR aggregation was further studied using the DG deficient muscle cell lines. We found that alpha-DG is involved in the later stages of agrin-induced AChR aggregation. / We further localized DG and two of its intracellular ligands, dystrophin and its autosomal homologue utrophin, to a synaptic layer in the retina suggesting a role for DG in central nervous system synapses. DG, utrophin and LN are also co-expressed around blood vessels indicating a possible function in blood-brain barrier homeostasis.

Oculopharyngeal Muscular Dystrophy (OPMD) is a late-onset dominant/recessive myopathy caused by the expansion of a polyalanine repeat in exon 1 of the PABPN1 gene. The expression of expanded PABPN1 (expPABPN1) triggers the formation of insoluble nuclear aggregates within muscle fiber nuclei of OPMD patients. These aggregates are enriched in poly(A)RNA and sequester molecular chaperones, ubiquitin and proteasome subunits. In addition to these cellular components, we first identified two novel PABPN1 interacting partners, hnRNPA1 and hnRNPA/B that also localized to the insoluble expPABPN1 aggregates. However, only hnRNPA1 was observed in inclusions of OPMD patients' muscle fiber nuclei. Following this finding, we next established the involvement of the ubiquitin-proteasome pathway in the clearance of misfolded expPABPN1 and provided more insights into the beneficial role of molecular chaperones in OPMD. The inhibition of proteasome correlated with an increase in the aggregation of expPABPN1, suggesting a possible proteasome impairment in OPMD. Conversely, the overexpression of Hsp70 and Hsp40 coincided with a decrease in nuclear aggregates concomitant with a reduced cellular toxicity, suggesting the therapeutic potential of manipulating molecular chaperones levels. Finally, we demonstrated that soluble forms of expPABPN1 are the primary toxic species in OPMD. In the presence of endogenous HSPs, a decrease in expPABPN1 aggregation correlated with an increased cellular toxicity. A defect in polyadenylation or ubiquitination significantly increased expPABPN1 solubility and cell death. Using live-cell imaging, we observed that nuclear aggregates prolonged the survival of expPABPN1-expressing cells, which led us to speculate that protein aggregates are subnuclear structures that preserve cellular homeostasis by depleting the expPABPN1 from the nuclear soluble pool. We propose that the polyalanine expansion in expPABPN1 could enable aberrant protein-protein interactions that would compromise the cellular function of nuclear factors and the expression of genes essential for muscle integrity and differentiation. For instance, expPABPN1 might compromise the function of hnRNP proteins and lead to altered mRNA processing and nucleocytoplasmic export, which can be detrimental to the cell.

No description available.

Duchenne and Becker muscular dystrophies (DMD, BMD) are allelic X-linked diseases which affect approximately one in 3500 male newborns. They are caused by mutations in a gene positioned on the short arm of the X chromosome at Xp21. The first indication of the location of this gene was the description of rare females expressing DMD and who were found to have constitutional X;autosome translocations with an X chromosome breakpoint at this site. There are now 24 such females known worldwide. They express DMD as a consequence of preferential inactivation of the normal X chromosome. In order to contribute to the understanding of the aetiology of mutations causing DMD and the aetiology of constitutional translocations, two types of study have been performed here. Firstly, the detailed mapping of the X chromosome breakpoints of DMD-associated X;autosome translocations has been investigated. The results of this study have been compared with data on the physical distribution of mutations causing DMD in male patients. Secondly, one translocation, an X;l translocation with an autosomal breakpoint at Ip34, has been selected for more detailed investigation and the DNA sequence has been determined at the site of the rearrangement. Translocation breakpoint mapping studies were performed by somatic cell hybrid analysis. Hybrids were karyotyped and this information was used to construct a hybrid panel for the purpose of determining the autosomal localisations of anonymous DNA probes. The mapping of seven probes using this panel is described. The work described in this thesis revealed that the distribution of translocation breakpoints within the DMD gene appears to be random and may differ from the distribution of mutations in male patients. The X;l translocation whose breakpoints are cloned and sequenced was found to involve two expressed loci, one coding for dystrophin on the X chromosome and one for the leukocyte antigen related protein on chromosome 1. Sequence data revealed that a deletion of four to seven nucleotides from the X chromosome and a duplication of two to five nucleotides are associated with the translocation. The possible involvement of trinucleotides adjacent to the breakpoints, and of a LINE, a SINE and a stretch of potential Z-DNA within 1 kb of the X chromosome or the chromosome 1 breakpoint, is discussed.