Prenatal muscle development in a mouse model for the secondary dystroglycanopathies

The defective glycosylation of α-dystroglycan is associated with a group of muscular dystrophies that are collectively referred to as the secondary dystroglycanopathies. Mutations in the gene encoding fukutin-related protein (FKRP) are one of the most common causes of secondary dystroglycanopathy in the UK and are associated with a wide spectrum of disease. Whilst central nervous system involvement has a prenatal onset, no studies have addressed prenatal muscle development in any of the mouse models for this group of diseases. In view of the pivotal role of α-dystroglycan in early basement membrane formation, we sought to determine if the muscle formation was altered in a mouse model of FKRP-related dystrophy

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

Performance of Ultra-Deep Pyrosequencing in Analysis of HIV-1 pol Gene Variation

INTRODUCTION: Ultra-deep pyrosequencing (UDPS) has been used to detect minority variants within HIV-1 populations. Some aspects of the quality and reproducibility of UDPS have been previously evaluated, but comprehensive studies are still needed. PRINCIPAL FINDING: In this study the UDPS technology (FLX platform) was evaluated by analyzing a 120 base pair fragment of the HIV-1 pol gene from plasma samples from two patients and artificial mixtures of molecular clones. UDPS was performed using an optimized experimental protocol and an in-house data cleaning strategy. Nine samples and mixtures were analyzed and the average number of reads per sample was 19,404 (range 8,858-26,846). The two patient plasma samples were analyzed twice and quantification of viral variants was found to be highly repeatable for variants representing >0.27% of the virus population, whereas some variants representing 0.11-0.27% were detected in only one of the two UDPS runs. Bland-Altman analysis showed that a repeated measurement would have a 95% likelihood to lie approximately within ±0.5 log(10) of the initial estimate. A similar level of agreement was observed for variant frequency estimates in forward vs. reverse sequencing direction, but here the agreement was higher for common variants than for rare variants. UDPS following PCR amplification with alternative primers indicated that some variants may be incorrectly quantified due to primer-related selective amplification. Finally, the in vitro recombination rate during PCR was evaluated using artificial mixtures of clones and was found to be low. The most abundant in vitro recombinant represented 0.25% of all UDPS reads. CONCLUSION: This study demonstrates that this UDPS protocol results in low experimental noise and high repeatability, which is relevant for future research and clinical use of the UDPS technology. The low rate of in vitro recombination suggests that this UDPS system can be used to study genetic variants and mutational linkage

Transgenic Overexpression of LARGE Induces α-Dystroglycan Hyperglycosylation in Skeletal and Cardiac Muscle

BACKGROUND: LARGE is one of seven putative or demonstrated glycosyltransferase enzymes defective in a common group of muscular dystrophies with reduced glycosylation of α-dystroglycan. Overexpression of LARGE induces hyperglycosylation of α-dystroglycan in both wild type and in cells from dystroglycanopathy patients, irrespective of their primary gene defect, restoring functional glycosylation. Viral delivery of LARGE to skeletal muscle in animal models of dystroglycanopathy has identical effects in vivo, suggesting that the restoration of functional glycosylation could have therapeutic applications in these disorders. Pharmacological strategies to upregulate Large expression are also being explored. METHODOLOGY/PRINCIPAL FINDINGS: In order to asses the safety and efficacy of long term LARGE over-expression in vivo, we have generated four mouse lines expressing a human LARGE transgene. On observation, LARGE transgenic mice were indistinguishable from the wild type littermates. Tissue analysis from young mice of all four lines showed a variable pattern of transgene expression: highest in skeletal and cardiac muscles, and lower in brain, kidney and liver. Transgene expression in striated muscles correlated with α-dystroglycan hyperglycosylation, as determined by immunoreactivity to antibody IIH6 and increased laminin binding on an overlay assay. Other components of the dystroglycan complex and extracellular matrix ligands were normally expressed, and general muscle histology was indistinguishable from wild type controls. Further detailed muscle physiological analysis demonstrated a loss of force in response to eccentric exercise in the older, but not in the younger mice, suggesting this deficit developed over time. However this remained a subclinical feature as no pathology was observed in older mice in any muscles including the diaphragm, which is sensitive to mechanical load-induced damage. CONCLUSIONS/SIGNIFICANCE: This work shows that potential therapies in the dystroglycanopathies based on LARGE upregulation and α-dystroglycan hyperglycosylation in muscle should be safe

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

SIP metagenomics identifies uncultivated Methylophilaceae as dimethylsulphide degrading bacteria in soil and lake sediment.

Dimethylsulphide (DMS) has an important role in the global sulphur cycle and atmospheric chemistry. Microorganisms using DMS as sole carbon, sulphur or energy source, contribute to the cycling of DMS in a wide variety of ecosystems. The diversity of microbial populations degrading DMS in terrestrial environments is poorly understood. Based on cultivation studies, a wide range of bacteria isolated from terrestrial ecosystems were shown to be able to degrade DMS, yet it remains unknown whether any of these have important roles in situ. In this study, we identified bacteria using DMS as a carbon and energy source in terrestrial environments, an agricultural soil and a lake sediment, by DNA stable isotope probing (SIP). Microbial communities involved in DMS degradation were analysed by denaturing gradient gel electrophoresis, high-throughput sequencing of SIP gradient fractions and metagenomic sequencing of phi29-amplified community DNA. Labelling patterns of time course SIP experiments identified members of the Methylophilaceae family, not previously implicated in DMS degradation, as dominant DMS-degrading populations in soil and lake sediment. Thiobacillus spp. were also detected in (13)C-DNA from SIP incubations. Metagenomic sequencing also suggested involvement of Methylophilaceae in DMS degradation and further indicated shifts in the functional profile of the DMS-assimilating communities in line with methylotrophy and oxidation of inorganic sulphur compounds. Overall, these data suggest that unlike in the marine environment where gammaproteobacterial populations were identified by SIP as DMS degraders, betaproteobacterial Methylophilaceae may have a key role in DMS cycling in terrestrial environments.HS was supported by a UK Natural Environment Research Council Advanced Fellowship NE/E013333/1), ÖE by a postgraduate scholarship from the University of Warwick and an Early Career Fellowship from the Institute of Advanced Study, University of Warwick, UK, respectively. Lawrence Davies is acknowledged for help with QIIME

Muscle Dystroglycan Organizes the Postsynapse and Regulates Presynaptic Neurotransmitter Release at the Drosophila Neuromuscular Junction

International audienceBACKGROUND: The Dystrophin-glycoprotein complex (DGC) comprises dystrophin, dystroglycan, sarcoglycan, dystrobrevin and syntrophin subunits. In muscle fibers, it is thought to provide an essential mechanical link between the intracellular cytoskeleton and the extracellular matrix and to protect the sarcolemma during muscle contraction. Mutations affecting the DGC cause muscular dystrophies. Most members of the DGC are also concentrated at the neuromuscular junction (NMJ), where their deficiency is often associated with NMJ structural defects. Hence, synaptic dysfunction may also intervene in the pathology of dystrophic muscles. Dystroglycan is a central component of the DGC because it establishes a link between the extracellular matrix and Dystrophin. In this study, we focused on the synaptic role of Dystroglycan (Dg) in Drosophila. METHODOLOGY/PRINCIPAL FINDINGS: We show that Dg was concentrated postsynaptically at the glutamatergic NMJ, where, like in vertebrates, it controls the concentration of synaptic Laminin and Dystrophin homologues. We also found that synaptic Dg controlled the amount of postsynaptic 4.1 protein Coracle and alpha-Spectrin, as well as the relative subunit composition of glutamate receptors. In addition, both Dystrophin and Coracle were required for normal Dg concentration at the synapse. In electrophysiological recordings, loss of postsynaptic Dg did not affect postsynaptic response, but, surprisingly, led to a decrease in glutamate release from the presynaptic site. CONCLUSION/SIGNIFICANCE: Altogether, our study illustrates a conservation of DGC composition and interactions between Drosophila and vertebrates at the synapse, highlights new proteins associated with this complex and suggests an unsuspected trans-synaptic function of Dg