Nguyen thi Man

In vivo targeted repair of a point mutation in the canine dystrophin gene by a chimeric RNA/DNA oligonucleotide

In the canine model of Duchenne muscular dystrophy in golden retrievers (GRMD), a point mutation within the splice acceptor site of intron 6 leads to deletion of exon 7 from the dystrophin mRNA, and the consequent frameshift causes early termination of translation. We have designed a DNA and RNA chimeric oligonucleotide to induce host cell mismatch repair mechanisms and correct the chromosomal mutation to wild type. Direct skeletal muscle injection of the chimeric oligonucleotide into the cranial tibialis compartment of a six-week-old affected male dog, and subsequent analysis of biopsy and necropsy samples, demonstrated in vivo repair of the GRMD mutation that was sustained for 48 weeks. Reverse transcription–polymerase chain reaction (RT-PCR) analysis of exons 5–10 demonstrated increasing levels of exon 7 inclusion with time. An isolated exon 7-specific dystrophin antibody confirmed synthesis of normal-sized dystrophin product and positive localization to the sarcolemma. Chromosomal repair in muscle tissue was confirmed by restriction fragment length polymorphism (RFLP)–PCR and sequencing the PCR product. This work provides evidence for the long-term repair of a specific dystrophin point mutation in muscle of a live animal using a chimeric oligonucleotide.

Effect of pathogenic mis-sense mutations in lamin A on its interaction with emerin in vivo

Mutations in lamin A/C can cause Emery-Dreifuss muscular dystrophy (EDMD) or a related cardiomyopathy (CMD1A). Using transfection of lamin-A/C-deficient fibroblasts, we have studied the effects of nine pathogenic mutations on the ability of lamin A to assemble normally and to localize emerin normally at the nuclear rim. Five mutations in the rod domain (L85R, N195K, E358K, M371K and R386K) affected the assembly of the lamina. With the exception of mutant L85R, all rod domain mutants induced the formation of large nucleoplasmic foci in about 10% of all nuclei. The presence of emerin in these foci suggests that the interaction of lamin A with emerin is not directly affected by the rod domain mutations. Three mutations in the tail region, R453W, W520S and R527P, might directly affect emerin binding by disrupting the structure of the putative emerin-binding site, because mutant lamin A localized normally to the nuclear rim but its ability to trap emerin was impaired. Nucleoplasmic foci rarely formed in these three cases (<2%) but, when they did so, emerin was absent, consistent with a direct effect of the mutations on emerin binding. The lipodystrophy mutation R482Q, which causes a different phenotype and is believed to act through an emerin-independent mechanism, was indistinguishable from wild-type in its localization and its ability to trap emerin at the nuclear rim. The novel hypothesis suggested by the data is that EDMD/CMD1A mutations in the tail domain of lamin A/C work by direct impairment of emerin interaction, whereas mutations in the rod region cause defective lamina assembly that might or might not impair emerin capture at the nuclear rim. Subtle effects on the function of the lamina-emerin complex in EDMD/CMD1A patients might be responsible for the skeletal and/or cardiac muscle phenotype.

Diagnosis of X-linked Emery-Dreifuss muscular dystrophy by protein analysis of leucocytes and skin with monoclonal antibodies.

The X-linked form of Emery-Dreifuss muscular dystrophy (EDMD) was recently shown to be due to mutations in the STA gene on chromosome Xq28. We have demonstrated a simple test for the diagnosis of this condition, looking for altered expression of the protein, emerin, in leucocytes and skin with a monoclonal antibody. Full-length emerin is completely absent in affected boys from the EDMD families studied. The method has also enabled identification of a female carrier of the disease by reduced levels of the protein on the leucocyte Western blot and a mosaic pattern of expression by immunofluorescence microscopy of the skin biopsy.

Monoclonal antibodies against defined regions of the muscular dystrophy protein, dystrophin

Nineteen monoclonal antibodies which bind to native dystrophin in the plasma membrane of frozen muscle sections were obtained using a recombinant fusion protein as immunogen. On Western blots of normal mouse muscle extracts, the antibodies bind specifically to a 400 000 M r protein which is absent from dystrophic mouse (mdx) muscle. At least four distinct epitopes have been identified by cleavage mapping methods. Although the fusion protein contained 25% of the human dystrophin sequence (Cys816‐Asp1747; M r 108 000), most of the monoclonal antibodies (15 out of 19) recognize a single fragment of M r 27 500.

Utrophin-dystroglycan complex in membranes of adherent cultured cells.

In skeletal muscle, dystrophin binds to an oligomeric, transmembrane complex (DAGc; dystrophin-associated glycoprotein complex) which interacts with laminin in the extracellular matrix. We now present biochemical evidence for an association between utrophin (dystrophin-related protein, DRP) and a major DAGc component, beta-dystroglycan (43DAG) in cultured cell lines which contain little if any dystrophin. We have shown also that utrophin and beta-dystroglycan co-localise at or near the plasma membrane and that they co-sediment in large complexes on sucrose density gradients. On the lower plasma membrane, in contact with the substratum, part of the utrophin and beta-dystroglycan staining co-localised with alpha-actinin in a punctate distribution outside classical vinculin-rich focal adhesions. beta-dystroglycan, utrophin, syntrophin (59DAP), and alpha-actinin were found in all adhesion-competent cell lines studied, but levels of the last three proteins were greatly reduced in myeloma cells, which cannot readily attach to substrata. Possible roles for utrophin in cultured cells are considered in the light of recent evidence for involvement of utrophin-glycoprotein complexes in muscle in signal transduction and recruitment of acetylcholine receptors to neuromuscular junctions.

Epitopes in the interacting regions of β-dystroglycan (PPxY motif) and dystrophin (WW domain)

The dystroglycan gene produces two products from a single mRNA, the extracellular alpha-dystroglycan and the transmembrane beta-dystroglycan. The Duchenne muscular dystrophy protein, dystrophin, associates with the muscle membrane via beta-dystroglycan, the WW domain of dystrophin interacting with a PPxY motif in beta-dystroglycan. A panel of four monoclonal antibodies (MANDAG1-4) was produced using the last 16 amino acids of beta-dystroglycan as immunogen. The mAbs recognized a 43 kDa band on Western blots of all cells and tissues tested and stained the sarcolemma in immunohistochemistry of skeletal muscle over a wide range of animal species. A monoclonal antibody (mAb) against the WW domain of dystrophin, MANHINGE4A, produced using a 16-mer synthetic peptide, recognized dystrophin on Western blots and also stained the sarcolemma. We have identified the precise sequences recognized by the mAbs using a phage-displayed random 15-mer peptide library. A 7-amino-acid consensus sequence SPPPYVP involved in binding all four beta-dystroglycan mAbs was identified by sequencing 17 different peptides selected from the library. PPY were the most important residues for three mAbs, but PxxVP were essential residues for a fourth mAb, MANDAG2. By sequencing five different random peptides from the library, the epitope on dystrophin recognized by mAb MANHINGE4A was identified as PWxRA in the first beta-strand of the WW domain, with the W and R residues invariably present. A recent three-dimensional structure confirms that the two epitopes are adjacent in the dystrophin-dystroglycan complex, highlighting the question of how the two interacting motifs can also be accessible to antibodies during immunolocalization in situ.

Utrophin, the autosomal homologue of dystrophin, is widely-expressed and membrane-associated in cultured cell lines

Utrophin, the autosomal dystrophin‐related protein (DRP), is expressed in HeLa cells, smooth muscle‐like BC3HI cells from mouse brain, COS monkey kidney cells, the P38BD1 monocyte‐macrophage cell line and untransformed human skin fibroblasts, as well as in rat C6 glioma and Schwannoma cells. It was undetectable, however, in the Sp2/O mouse myeloma cell line and in hybridoma lines derived from it. Dystrophin was not detected in any of these cell lines. Although all utrophin‐containing cells were capable of forming monolayers in culture, no major effects of either attachment to substratum or length of time in culture (2–17 days) on utrophin levels were observed. After subcellular fractionation ofBC3HI or glioma cells, nearly all of the utrophin was found in the Triton‐soluble fraction, suggesting an association with cell membranes.

Valproate and bone loss: iTRAQ proteomics show that valproate reduces collagens and osteonectin in SMA cells.

Valproate is commonly used as an anticonvulsant and mood stabilizer, but its long-term side-effects can include bone loss. As a histone deacetylase (HDAC) inhibitor, valproate has also been considered for treatment of spinal muscular atrophy (SMA). Using iTRAQ labeling technology, followed by two-dimensional liquid chromatography and mass spectrometry analysis, a quantitative comparison of the proteome of an SMA cell line, with and without valproate treatment, was performed. The most striking change was a reduction in collagens I and VI, while over 1000 other proteins remained unchanged. The collagen I alpha-chain precursor was also reduced by more than 50% suggesting that valproate affects collagen I synthesis. The collagen-binding glycoprotein, osteonectin (SPARC, BM-40) was one of the few other proteins that were significantly reduced by valproate treatment. Collagen I is the main protein component of bone matrix and osteonectin has a major role in bone development, so the results suggest a possible molecular mechanism for bone loss following long-term exposure to valproate. SMA patients may already suffer bone weakness as a result of SMN1 gene deletion, so further bone loss would be undesirable.

A two-site ELISA can quantify upregulation of SMN protein by drugs for spinal muscular atrophy

Objectives: Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by loss of lower motor neurons during early or postnatal development. Severity is variable and is inversely related to the levels of survival of motor neurons (SMN) protein. The aim of this study was to produce a two-site ELISA capable of measuring both the low, basal levels of SMN protein in cell cultures from patients with severe SMA and small increases in these levels after treatment of cells with drugs. Methods: A monoclonal antibody against recombinant SMN, MANSMA1, was selected for capture of SMN onto microtiter plates. A selected rabbit antiserum against refolded recombinant SMN was used for detection of the captured SMN. Results: The ratio of SMN levels in control fibroblasts to levels in SMA fibroblasts was greater than 3.0, consistent with Western blot data. The limit of detection was 0.13 ng/mL and SMN could be measured in human NT-2 neuronal precursor cells grown in 96-well culture plates (3 × 104 cells per well). Increases in SMN levels of 50% were demonstrable by ELISA after 24 hours treatment of 105 SMA fibroblasts with valproate or phenylbutyrate. Conclusion: A rapid and specific two-site, 96-well ELISA assay, available in kit format, can now quantify the effects of drugs on survival of motor neurons protein levels in cell cultures. GLOSSARY: HDAC = histone deacetylase; SMA = spinal muscular atrophy; SMN = survival of motor neurons.

Full-length and short forms of utrophin, the dystrophin-related protein

All previous studies of the localization of utrophin (the dystrophin‐related protein) in muscle and other tissues have been performed only with antibodies against the C‐terminal region of the protein. Since several short forms of dystrophin, the apodystrophins, are produced from the 3′ end of the dystrophin gene, there is a possibility that similar short forms of utrophin exist and that these could be responsible for some of the many different localizations of ‘utrophin’ in muscle. We have produced a new panel of 15 mAbs against the N‐terminal region of utrophin and we have used it together with mAbs against the C‐terminal region to show that full‐length utrophin is present at neuromuscular junctions, in nerves, blood vessels and capillaries in normal muscle and in the sarcolemma of patients with muscular dystrophy and dermatomyositis. However, two of the 15 mAbs also recognised rat/mouse utrophin and both of these detected an additional 62 kDa protein on Western blots of rat C6 glioma cells. This potential 62 kDa ‘apo‐utrophin’ was not detected in human cerebral cortex, in rat Schwannoma cells nor in any of the non‐nerve cells and tissues tested.