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This project was supported by grants from the British Heart Foundation (PG/11/71/29091 and PG/16/68/31991) and the Orthopaedic Institute Ltd., (RJAH Orthopaedic Hospital, Oswestry, UK). Nesprins, nuclear envelope spectrin-repeat proteins encoded by the SYNE1 and SYNE2 genes, are involved in localization of nuclei. The short isoform, nesprin-1-alpha2, is required for relocation of the microtubule organizer function from centromeres to the nuclear rim during myogenesis. Using specific antibodies, we now show that both nesprin-1-alpha2 and nesprin-1-giant co-localize with kinesin at the junctions of concatenated nuclei and at the outer poles of nuclear chains in human skeletal myotubes. In adult muscle, nesprin-1-alpha2 was found, together with kinesin, only on nuclei associated with neuromuscular junctions, whereas all adult cardiomyocyte nuclei expressed nesprin-1-alpha2. In a proteomics study, kinesin heavy and light chains were the only significant proteins in myotube extracts pulled down by nesprin-1-alpha2, but not by a mutant lacking the highly-conserved STAR domain (18 amino-acids, including the LEWD motif). The results support a function for nesprin-1-alpha2 in the specific localization of skeletal muscle nuclei mediated by kinesins and suggest that its primary role is at the outer nuclear membrane. Publisher PDF

Representatives of academia, patient organisations, industry and the United States Food and Drug Administration attended a workshop on dystrophin quantification methodology. The aims of the workshop were to provide an overview of methods used to quantify dystrophin levels in human skeletal muscle and their applicability to clinical trial samples, outline the gaps with regards to validating the methods for robust clinical applications prior to regulatory agency review, and to align future efforts towards further optimizing these methods. The workshop facilitated a constructive but also critical discussion on the potential and limitations of techniques currently used in the field of translational research (western blot and immunofluorescence analysis) and emerging techniques (mass spectrometry and capillary western immunoassay). Notably, all participants reported variation in dystrophin levels between muscle biopsies from different healthy individuals and agreed on the need for a common reference sample.

BackgroundPopeye domain-containing proteins 1 and 2 (POPDC1 and POPDC2) are transmembrane proteins involved in cyclic AMP-mediated signalling processes and are required for normal cardiac pacemaking and conduction. In order to identify novel protein interaction partners, POPDC1 and 2 proteins were attached to beads and compared by proteomic analysis with control beads in the pull-down of proteins from cultured human skeletal myotubes.ResultsThere were highly-significant interactions of both POPDC1 and POPDC2 with XIRP1 (Xin actin binding repeat-containing protein 1), actin and, to a lesser degree, annexin A5. In adult human skeletal muscle, both XIRP1 and POPDC1/2 were present at the sarcolemma and in T-tubules. The interaction of POPDC1 with XIRP1 was confirmed in adult rat heart extracts. Using new monoclonal antibodies specific for POPDC1 and POPDC2, both proteins, together with XIRP1, were found mainly at intercalated discs but also at T-tubules in adult rat and human heart.ConclusionsMutations in humanPOPDC1,POPDC2and in humanXIRP1, all cause pathological cardiac arrhythmias, suggesting a possible role for POPDC1/2 and XIRP1 interaction in normal cardiac conduction.

Nebulin is a very large protein required for assembly of the contractile machinery in muscle. Mutations in the nebulin gene NEB are a common cause of nemaline myopathy. Nebulin mRNA is alternatively-spliced so that each mRNA contains either exon 143 or exon 144. We have produced monoclonal antibodies specific for the regions of nebulin encoded by these two exons, enabling analysis of expression of isoforms at the protein level for the first time. All antibodies recognized a protein of the expected size (600–900 kD) and stained cross-striations of sarcomeres in muscle sections. Expression of exon 143 is developmentally-regulated since newly-formed myotubes in cell culture expressed nebulin with exon 144 only; this was confirmed at the mRNA level by qPCR. In fetal muscle, nebulin with exon 143 was expressed in some myotubes by 12-weeks of gestation and strongly-expressed in most myotubes by 17-weeks. In mature human muscle, the exon 144 antibody stained all fibres, but the exon 143 antibody staining varied from very strong in some fibres to almost-undetectable in other fibres. The results show that nebulin containing exon 144 is the default isoform early in myogenesis, while regulated expression of nebulin containing exon 143 occurs at later stages of muscle development.

Emery-Dreifuss muscular dystrophy (EDMD) is a rare genetic disorder characterised by the early development of muscle contractures, progressive muscle weakness, and heart abnormalities. The latter may result in serious complications, or in severe cases, sudden death. Currently, there are very few effective treatment options available for EDMD and so there is a high clinical need for new therapies. Various genetic mutations have been identified in the development and causation of EDMD, each encoding proteins that are components of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, which spans the nuclear envelope and serves to connect the nuclear lamina to the cytoskeleton. Within this review, we examine how mutations in the genes encoding these proteins, including lamins A/C, emerin, nesprins 1/2, FHL1, and SUN1/2 lead to muscle cell differentiation and development pathway defects. Further work to identify conserved molecular pathways downstream of these defective proteins may reveal potential targets for therapy design.

Nesprins-1 and -2 are highly expressed in skeletal and cardiac muscle and together with SUN (Sad1p/UNC84)-domain containing proteins and lamin A/C form the LInker of Nucleoskeleton-and-Cytoskeleton (LINC) bridging complex at the nuclear envelope (NE). Mutations in nesprin-1/2 have previously been found in patients with autosomal dominant Emery–Dreifuss muscular dystrophy (EDMD) as well as dilated cardiomyopathy (DCM). In this study, three novel rare variants (R8272Q, S8381C and N8406K) in the C-terminus of the SYNE1 gene (nesprin-1) were identified in seven DCM patients by mutation screening. Expression of these mutants caused nuclear morphology defects and reduced lamin A/C and SUN2 staining at the NE. GST pull-down indicated that nesprin-1/lamin/SUN interactions were disrupted. Nesprin-1 mutations were also associated with augmented activation of the ERK pathway in vitro and in hearts in vivo. During C2C12 muscle cell differentiation, nesprin-1 levels are increased concomitantly with kinesin light chain (KLC-1/2) and immunoprecipitation and GST pull-down showed that these proteins interacted via a recently identified LEWD domain in the C-terminus of nesprin-1. Expression of nesprin-1 mutants in C2C12 cells caused defects in myoblast differentiation and fusion associated with dysregulation of myogenic transcription factors and disruption of the nesprin-1 and KLC-1/2 interaction at the outer nuclear membrane. Expression of nesprin-1α2 WT and mutants in zebrafish embryos caused heart developmental defects that varied in severity. These findings support a role for nesprin-1 in myogenesis and muscle disease, and uncover a novel mechanism whereby disruption of the LINC complex may contribute to the pathogenesis of DCM.

Background Nesprin-1-giant (1008kD) is a protein of the outer nuclear membrane that links nuclei to the actin cytoskeleton via amino-terminal calponin homology domains. The short nesprin-1 isoform, nesprin-1-α2, is present only in skeletal and cardiac muscle and several pathogenic mutations occur within it, but the functions of this short isoform without calponin homology domains are unclear. The aim of this study was to determine mRNA levels and protein localization of nesprin-1-α2 at different stages of muscle development in order to shed light on its functions. Results mRNA levels of all known nesprin-1 isoforms with a KASH domain were determined by quantitative PCR. The mRNA for the 111 kD muscle-specific short isoform, nesprin-1-α2, was not detected in pre-differentiation human myoblasts but was present at significant levels in multinucleate myotubes. We developed a monoclonal antibody against the unique amino-terminal sequence of nesprin-1-α2, enabling specific immunolocalization for the first time. Nesprin-1-α2 protein was undetectable in pre-differentiation myoblasts but appeared at the nuclear rim in post-mitotic, multinucleate myotubes and reached its highest levels in fetal muscle. In muscle from a Duchenne muscular dystrophy biopsy, nesprin-1-α2 protein was detected mainly in regenerating fibres expressing neonatal myosin. Nesprin-1-giant was present at all developmental stages, but was also highest in fetal and regenerating fibres. In fetal muscle, both isoforms were present in the cytoplasm, as well as at the nuclear rim. A pathogenic early stop codon (E7854X) in nesprin-1 caused reduced mRNA levels and loss of protein levels of both nesprin-1-giant and (unexpectedly) nesprin-1-α2, but did not affect myogenesis in vitro. Conclusions Nesprin-1-α2 mRNA and protein expression is switched on during myogenesis, alongside other known markers of muscle differentiation. The results show that nesprin-1-α2 is dynamically controlled and may be involved in some process occurring during early myofibre formation, such as re-positioning of nuclei.

Mutations in the gene encoding fukutin protein cause Fukuyama muscular dystrophy, a severe congenital disorder that occurs mainly in Japan. A major consequence of the mutation is reduced glycosylation of alpha-dystroglycan, which is also a feature of other forms of congenital and limb-girdle muscular dystrophy. Immunodetection of endogenous fukutin in cells and tissues has been difficult and this has hampered progress in understanding fukutin function and disease pathogenesis. Using a new panel of monoclonal antibodies which bind to different defined sites on the fukutin molecule, we now show that fukutin has the predicted size for a protein without extensive glycosylation and is present at the Golgi apparatus at very low levels. These antibodies should enable more rapid future progress in understanding the molecular function of fukutin.

The 7th UK SMA researchers meeting at Lake Vyrnwy Hotel, Oswestry UK in October 2010, was focussed on two main aspects of current SMA research: (1) to understand the normal functions of SMN protein (Fig. 1) and the mechanism of what goes seriously wrong when SMN levels are reduced to less than 10–15% of normal; and (2) to generate novel treatment options for current SMA patients and preventative options for future sufferers. Possible targets for treatment are shown in Fig. 1. Spinal Muscular Atrophy (SMA) is an inherited neuromuscular disease displaying a wide range of severity and characterised by loss of function and degeneration of lower motor neurons in the anterior horn of the spinal cord. It is caused by reduced levels of the small ubiquitous survival of motor neuron (SMN) protein, which is encoded by two genes, SMN1 and SMN2 [1]. Most of the mRNA transcribed from the SMN2 gene is alternatively spliced to omit exon 7 and any protein translated from such “delta7” mRNA is unstable and rapidly degraded [1–3]. In SMA patients, the SMN1 gene is deleted and only a small amount of functional SMN is produced from the SMN2 gene, with the more severe phenotypes having the least SMN [4,5]. In theory, therefore, only small increases in functional SMN are needed to produce a less severe SMA phenotype. This means that a concentrated effort on therapies that might increase the SMN protein levels in patients could help to protect the motor neurons [6]. Additional approaches without increasing SMN include modifying factors or stabilisation of neuromuscular junctions [7]. Shortly after the SMN1 gene was identified as the affected gene in SMA, the SMN protein was shown to have an essential role in the assembly of U snRNPs in the cytoplasm and their transport to the nucleus, where they are further modified for participation in RNA splicing [8]. It

Mutations in the mismatch repair gene MSH2 underlie hereditary nonpolyposis colorectal cancer (Lynch syndrome). Whereas disruptive mutations are overtly pathogenic, the implications of missense mutations found in sporadic colorectal cancer patients or in suspected Lynch syndrome families are often unknown. Adequate genetic counseling of mutation carriers requires phenotypic characterization of the variant allele. We present a novel approach to functionally characterize MSH2 missense mutations. Our approach involves introduction of the mutation into the endogenous gene of murine embryonic stem cells (ESC) by oligonucleotide-directed gene modification, a technique we recently developed in our lab. Subsequently, the mismatch repair capacity of mutant ESC is determined using a set of validated functional assays. We have evaluated four clinically relevant MSH2 variants and found one to completely lack mismatch repair capacity while three behaved as wild-type MSH2 and can therefore be considered as polymorphisms. Our approach contributes to an adequate risk assessment of mismatch repair missense mutations. We have also shown that oligonucleotide-directed gene modification provides a straightforward approach to recreate allelic variants in the endogenous gene in murine ESC. This approach can be extended to other hereditary conditions.

Reduced levels of the survival of motor neurons protein (SMN), cause the inherited neuromuscular disorder, spinal muscular atrophy (SMA). The majority of therapeutic approaches to date have been focused on finding ways to increase expression of functional SMN protein, though stabilization of SMN protein may also be an important consideration. SMN interacts, directly or indirectly, stably or transiently, with a large number of other proteins, some of which contribute to SMN stability and may therefore be potential targets for SMA therapy. We recently characterized the nuclear SMN interactome using LC-MALDI-TOF/TOF analysis of anti-SMN pull-downs and identified myb-binding protein-1a (Mybbp1a) as a novel partner. In light of interest in cytoplasm-specific roles of the SMN complex, we have applied the same approach to characterise the cytoplasmic SMN interactome. We now show that SMN complexes from HeLa cytoplasmic extracts differ significantly from those found in nuclear extracts, with gemin5, importinbeta and annexin A2 easily detected only in the cytoplasmic extracts, whereas interaction of SMN with Mybbp1a appears to occur only in the nucleus. SMN is ubiquitinylated and we also found proteins of the ubiquitin-proteasome system associated with SMN in the cytoplasm.

In myotonic dystrophy, muscleblind-like protein 1 (MBNL1) protein binds specifically to expanded CUG or CCUG repeats, which accumulate as discrete nuclear foci, and this is thought to prevent its function in the regulation of alternative splicing of pre-mRNAs. There is strong evidence for the role of the MBNL1 gene in disease pathology, but the roles of two related genes, MBNL2 and MBNL3, are less clear. Using new monoclonal antibodies specific for each of the three gene products, we found that MBNL2 decreased during human fetal development and myoblast culture, while MBNL1 was unchanged. In Duchenne muscular dystrophy muscle, MBNL2 was elevated in immature, regenerating fibres compared with mature fibres, supporting some developmental role for MBNL2. MBNL3 was found only in C2C12 mouse myoblasts. Both MBNL1 and MBNL2 were partially sequestered by nuclear foci of expanded repeats in adult muscle and cultured cells from myotonic dystrophy patients. In adult muscle nucleoplasm, both proteins were reduced in myotonic dystrophy type 1 compared with an age-matched control. In normal human myoblast cultures, MBNL1 and MBNL2 always co-distributed but their distribution could change rapidly from nucleoplasmic to cytoplasmic. Functional differences between MBNL1 and MBNL2 have not yet been found and may prove quite subtle. The dominance of MBNL1 in mature, striated muscle would explain why ablation of the mouse mbnl1 gene alone is sufficient to cause a myotonic dystrophy.

Mutations in the gene encoding the inner nuclear membrane proteins lamins A and C produce cardiac and skeletal muscle dysfunction referred to as Emery Dreifuss muscular dystrophy. Lamins A and C participate in the LINC complex that, along with the nesprin and SUN proteins, LInk the Nucleoskeleton with the Cytoskeleton. Nesprins 1 and 2 are giant spectrin-repeat containing proteins that have large and small forms. The nesprins contain a transmembrane anchor that tethers to the nuclear membrane followed by a short domain that resides within the lumen between the inner and outer nuclear membrane. Nesprin's luminal domain binds directly to SUN proteins. We generated mice where the C-terminus of nesprin-1 was deleted. This strategy produced a protein lacking the transmembrane and luminal domains that together are referred to as the KASH domain. Mice homozygous for this mutation exhibit lethality with approximately half dying at or near birth from respiratory failure. Surviving mice display hindlimb weakness and an abnormal gait. With increasing age, kyphoscoliosis, muscle pathology and cardiac conduction defects develop. The protein components of the LINC complex, including mutant nesprin-1alpha, lamin A/C and SUN2, are localized at the nuclear membrane in this model. However, the LINC components do not normally associate since coimmunoprecipitation experiments with SUN2 and nesprin reveal that mutant nesprin-1 protein no longer interacts with SUN2. These findings demonstrate the role of the LINC complex, and nesprin-1, in neuromuscular and cardiac disease.

Nuclear speckles are storage sites for small nuclear RNPs (snRNPs) and other splicing factors. Current ideas about the role of speckles suggest that some pre-mRNAs are processed at the speckle periphery before being exported as mRNA. In myotonic dystrophy type 1 (DM1), the export of mutant DMPK mRNA is prevented by the presence of expanded CUG repeats that accumulate in nuclear foci. We now show that these foci accumulate at the periphery of nuclear speckles. In myotonic dystrophy type 2 (DM2), mRNA from the mutant ZNF9 gene is exported normally because the expanded CCUG repeats are removed during splicing. We now show that the nuclear foci formed by DM2 intronic repeats are widely dispersed in the nucleoplasm and not associated with either nuclear speckles or exosomes. We hypothesize that the expanded CUG repeats in DMPK mRNA are blocking a stage in its export pathway that would normally occur at the speckle periphery. Localization of the expanded repeats at the speckle periphery is not essential for their pathogenic effects because DM1 and DM2 are quite similar clinically.

The recent identification of mutations in genes encoding demonstrated or putative glycosyltransferases has revealed a novel mechanism for congenital muscular dystrophy. Hypoglycosylated alpha-dystroglycan (alpha-DG) is commonly seen in Fukuyama-type congenital muscular dystrophy (FCMD), muscle-eye-brain disease (MEB), Walker-Warburg syndrome (WWS), and Large(myd) mice. POMGnT1 and POMTs, the gene products responsible for MEB and WWS, respectively, synthesize unique O-mannose sugar chains on alpha-DG. The function of fukutin, the gene product responsible for FCMD, remains undetermined. Here we show that fukutin co-localizes with POMGnT1 in the Golgi apparatus. Direct interaction between fukutin and POMGnT1 was confirmed by co-immunoprecipitation and two-hybrid analyses. The transmembrane region of fukutin mediates its localization to the Golgi and participates in the interaction with POMGnT1. Y371C, a missense mutation found in FCMD, retains fukutin in the ER and also redirects POMGnT1 to the ER. Finally, we demonstrate reduced POMGnT1 enzymatic activity in transgenic knock-in mice carrying the retrotransposal insertion in the fukutin gene, the prevalent mutation in FCMD. From these findings, we propose that fukutin forms a complex with POMGnT1 and may modulate its enzymatic activity.

A workshop on the nuclear envelope and Emery– Dreifuss muscular dystrophy (EDMD) was held at the Centre for Inherited Neuromuscular Disiease, Oswestry, UK, on 29th March 2006. Both structural defects and defects in signalling mechanisms that might link dysfunctional emerin and lamin A/C to muscle and heart disease were explored by researchers from eight different research groups within the UK. Quinlivan (Oswestry) welcomed visitors with an overview of the clinical services, pathology, locomotive assessment and molecular research undertaken by the Centre for Inherited Neuromuscular Disease (CIND) and described the new TORCH building that will house the Wolfson Neuromuscular Research Laboratory, the Keele University mass-spectrometry facility and a new gait laboratory from 2008. Emery (Oxford & Edinburgh) gave a historical account of ‘‘MD with contractures and heart block’’, as he modestly described the disease that bears his name, from the first family he studied in the USA in 1962 [1] to the identification of the genes responsible, emerin in 1994 [2] and lamin A/C in 1999 [3]. Morris (Oswestry) recalled that, when the emerin gene was first sequenced in 1994, people assumed it would encode a plasma membrane protein, like the

Emery-Dreifuss muscular dystrophy results from mutations in either emerin or lamin A/C and is caused by loss of some unknown function of emerin-lamin A/C complexes. This function must be of special importance in the skeletal and cardiac muscles that are affected by the disease. Some lamin A/C mutant proteins form 'nuclear foci' in the nucleoplasm when overexpressed by transient transfection and similar aggregates have been seen in cultured skin fibroblasts from patients with Emery-Dreifuss muscular dystrophy, suggesting that mis-assembly of the A-type lamina may be involved in the pathogenesis. Whereas an earlier study of cultured skin fibroblasts compared several different missense mutations in lamin A/C, we have chosen to study one particular Emery-Dreifuss mutation (R249Q) in greater detail. We found that the proportion of fibroblast nuclei containing abnormal lamin A/C aggregates can vary from 0.5 to 23.6% depending on the culture conditions. In particular, switching from a 'slow growth' medium to 'rapid growth' media increased both the number and size of nuclear aggregates. Similar results were obtained with fibroblasts from a second unrelated patient with the same mutation. In contrast to these aggregates of endogenous lamin A/C, 'nuclear foci' formed after transfection of mouse embryo fibroblasts by mutant lamin A/C were not affected by culture conditions. Faulty assembly of the nuclear lamina by mutated lamin A/C molecules could be partly responsible for the disease phenotype, though this has not been proven. The present study suggests that inappropriate lamin A/C assembly may be preventable by manipulation of cell growth conditions.

Shellfish are a common cause of food reactions in hypersensitive individuals and are among the eight foods that account for over 90% of food allergies. At present, the only way to prevent these serious consequences of food allergies is to avoid the foods that trigger the reactions. A sandwich-ELISA kit has been developed for the detection of crustacean meat in food, based on the major heat-stable shellfish allergen, tropomyosin. Tropomyosin was purified from whole prawn (Penaeus latisulcatus) and used to immunize rabbits after confirming its identity by MALDI-TOF MS. A sandwich-ELISA based on the rabbit antibodies takes less than 2 h to perform, including the food extraction, and has a detection limit of 1 ppm crustacean (prawn, lobster), without detectable cross-reactivity with fish or mammalian meat.

SIX5 is a member of the human SIX family of transcription factors, many of which are involved in eye development. However, SIX5 transcripts are known to be present at very low levels in cells and no study has yet convincingly demonstrated detection of endogenous SIX5 protein by Western blotting or immunolocalisation. We have produced a new panel of 18 monoclonal antibodies (mAbs) that recognise at least four different epitopes in order to identify authentic human SIX5 protein in cells and tissues. Phage-displayed peptide libraries were used to identify individual amino-acids important for antibody binding within each epitope. Endogenous SIX5 migrated in SDS-PAGE with an apparent M(r) of 100 kDa and was present at similar levels in all foetal tissues and cell lines tested. In HeLa cells, it was located in the nucleoplasm with a granular distribution. An mRNA for a shorter splicing isoform of SIX5, with an altered carboxy-terminus, has been described, but further mAbs specific for this isoform did not detect any endogenous protein. We conclude that the full-length isoform is the major functional protein in vivo while the putative shorter protein is undetectable and may not be expressed at all.

Defects in the dystrophin gene cause the severe degenerative muscle disorder, Duchenne muscular dystrophy (DMD). Among the gene therapy approaches to DMD under investigation, a gene editing approach using oligonucleotide vectors has yielded encouraging results. Here, we extend our studies of gene editing with self-pairing, chimeric RNA/DNA oligonucleotides (RDOs) to the use of oligodeoxynucleotides (ODNs) to correct point mutations in the dystrophin gene. The ODN vectors offer many advantages over the RDO vectors, and we compare the targeting efficiencies in the mdx(5cv) mouse model of DMD. We found that ODNs targeted to either the transcribed or the non-transcribed strand of the dystrophin gene were capable of inducing gene repair, with efficiencies comparable to that seen with RDO vectors. Oligonucleotide-mediated repair was demonstrated at the genomic, mRNA and protein levels in muscle cells both in vitro and in vivo, and the correction was stable over time. Interestingly, there was a strand bias observed with the ODNs, with more efficient correction of the non-transcribed strand even though the dystrophin gene is not transcribed in proliferating myoblasts. This finding demonstrates that strand bias of ODN-mediated gene repair is likely to be due to the specific sequence of the target gene in addition to any effects of transcription. A better understanding of how the efficiency of gene editing relates to the target sequence will offer the opportunity for rational oligonucleotide design for further development of this elegant approach to gene therapy for DMD and other genetic diseases.

Nine patients with Duchenne or Becker muscular dystrophy were injected via the radialis muscle with a full-length human dystrophin plasmid, either once with 200 or 600 microg of DNA or twice, 2 weeks apart, with 600 microg of DNA. In the biopsies taken 3 weeks after the initial injection, the vector was detected at the injection site in all patients. Immunohistochemistry and nested reverse transcription-polymerase chain reaction indicated dystrophin expression in six of nine patients. The level of expression was low (up to 6% weak, but complete sarcolemmal dystrophin staining, and up to 26% partial sarcolemmal labeling). No side effects were observed, nor any cellular or humoral anti-dystrophin responses. These results suggest that exogenous dystrophin expression can be obtained in Duchenne/Becker patients after intramuscular transfer of plasmid, without adverse effects, hence paving the way for future developments in gene therapy of hereditary muscular diseases.

Most patients with the pediatric neurodegenerative disease spinal muscular atrophy have a homozygous deletion of the survival motor neuron 1 ( SMN1 ) gene, but retain one or more copies of the closely related SMN2 gene. The SMN2 gene encodes the same protein (SMN) but produces it at a low efficiency compared with the SMN1 gene. We performed a high-throughput screen of ∼47,000 compounds to identify those that increase production of an SMN2 -luciferase reporter protein, but not an SMN1 -luciferase reporter protein. Indoprofen, a nonsteroidal anti-inflammatory drug (NSAID) and cyclooxygenase (COX) inhibitor, selectively increased SMN2 -luciferase reporter protein and endogenous SMN protein and caused a 5-fold increase in the number of nuclear gems in fibroblasts from SMA patients. No other NSAIDs or COX inhibitors tested exhibited this activity.

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 (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.

As a target for gene therapy, Duchenne muscular dystrophy (DMD) presents many obstacles but also an unparalleled prospect for correction by alternative splicing. The majority of mutations in the dystrophin gene occur in the region encoding the spectrin-like central rod domain, which is largely dispensable. Thus, splicing around mutations can generate a shortened but in-frame transcript, permitting translation of a partially functional dystrophin protein. We have tested this idea in vivo in the mdx dystrophic mouse (carrying a mutation in exon 23 of the dystrophin gene) by combining a potent transfection protocol with a 2-O-methylated phosphorothioated antisense oligoribonucleotide (2OMeAO) designed to promote skipping of the mutated exon*. The treated mice show persistent production of dystrophin at normal levels in large numbers of muscle fibers and show functional improvement of the treated muscle. Repeated administration enhances dystrophin expression without eliciting immune responses. Our data establishes the realistic practicality of an approach that is applicable, in principle, to a majority of cases of severe dystrophinopathy.

Emery-Dreifuss muscular dystrophy is characterized by the clinical triad of early onset contractures of elbows, Achilles tendons and spine, wasting and weakness with a predominantly humero-peroneal distribution and life-threatening cardiac conduction defects and/or cardiomyopathy. Two main types of inheritance have been described: the X-linked form is caused by mutations in the STA gene on locus Xq28 and the gene for the autosomal dominant form (LMNA gene) has been localized on chromosome 1q11-q23. Recently, mutations in this LMNA gene have been also found to be responsible for the less frequent autosomal recessive form of the disease. Although all forms share a similar clinical presentation, some differences appear to exist between them as has been described recently in a large number of patients. We present the first documented Spanish family genetically confirmed to have autosomal dominant Emery-Dreifuss muscular dystrophy. Clinical, pathological and genetic data are described. We emphasize the difficulties in diagnosis, especially in sporadic cases or young patients in whom the clinical picture is not completely established.

Mutations in the SMN1 (survival motor neuron 1) gene cause spinal muscular atrophy (SMA). We now show that SMN protein, the SMN1 gene product, interacts directly with the tumor suppressor protein, p53. Pathogenic missense mutations in SMN reduce both self-association and p53 binding by SMN, and the extent of the reductions correlate with disease severity. The inactive, truncated form of SMN produced by the SMN2 gene in SMA patients fails to bind p53 efficiently. SMN and p53 co-localize in nuclear Cajal bodies, but p53 redistributes to the nucleolus in fibroblasts from SMA patients. These results suggest a functional interaction between SMN and p53, and the potential for apoptosis when this interaction is impaired may explain motor neuron death in SMA.

The autosomal recessive neurodegenerative disease spinal muscular atrophy (SMA) results from low levels of survival motor neuron (SMN) protein; however, it is unclear how reduced SMN promotes SMA development. Here, we determined that ubiquitin-dependent pathways regulate neuromuscular pathology in SMA. Using mouse models of SMA, we observed widespread perturbations in ubiquitin homeostasis, including reduced levels of ubiquitin-like modifier activating enzyme 1 (UBA1). SMN physically interacted with UBA1 in neurons, and disruption of Uba1 mRNA splicing was observed in the spinal cords of SMA mice exhibiting disease symptoms. Pharmacological or genetic suppression of UBA1 was sufficient to recapitulate an SMA-like neuromuscular pathology in zebrafish, suggesting that UBA1 directly contributes to disease pathogenesis. Dysregulation of UBA1 and subsequent ubiquitination pathways led to β-catenin accumulation, and pharmacological inhibition of β-catenin robustly ameliorated neuromuscular pathology in zebrafish, Drosophila, and mouse models of SMA. UBA1-associated disruption of β-catenin was restricted to the neuromuscular system in SMA mice; therefore, pharmacological inhibition of β-catenin in these animals failed to prevent systemic pathology in peripheral tissues and organs, indicating fundamental molecular differences between neuromuscular and systemic SMA pathology. Our data indicate that SMA-associated reduction of UBA1 contributes to neuromuscular pathogenesis through disruption of ubiquitin homeostasis and subsequent β-catenin signaling, highlighting ubiquitin homeostasis and β-catenin as potential therapeutic targets for SMA.

Myotonic dystrophy (DM) is a multi-system neuromuscular disorder for which there is no treatment. We have developed a medium throughput phenotypic assay, based on the identification of nuclear foci in DM patient cell lines using in situ hybridization and high-content imaging to screen for potentially useful therapeutic compounds. A series of further assays based on molecular features of DM have also been employed. Two compounds that reduce and/or remove nuclear foci have been identified, Ro 31-8220 and chromomycin A3. Ro 31-8220 is a PKC inhibitor, previously shown to affect the hyperphosphorylation of CELF1 and ameliorate the cardiac phenotype in a DM1 mouse model. We show that the same compound eliminates nuclear foci, reduces MBNL1 protein in the nucleus, affects ATP2A1 alternative splicing and reduces steady-state levels of CELF1 protein. We demonstrate that this effect is independent of PKC activity and conclude that this compound may be acting on alternative kinase targets within DM pathophysiology. Understanding the activity profile for this compound is key for the development of targeted therapeutics in the treatment of DM.

We present our observations on the skeletal muscle pathology of nine cases from seven families of autosomal dominant Emery-Dreifuss muscular dystrophy (ADEDMD) with identified mutations in the lamin A/C gene, aged 2-35 years at the time of biopsy. The severity of pathological change was moderate and the most common features were variation in fibre size (hypertrophy and atrophy), an increase in internal nuclei and smaller diameter fibres with high oxidative enzyme activity. Only one case showed necrosis, which was present in two separate samples taken from the quadriceps and tibialis anterior, at different ages. Immunocytochemistry detected an age-related reduction of laminin beta1 on the muscle fibres in adolescent and adult cases. Antibodies to lamins A and A/C, and emerin did not reveal any detectable differences from controls. Electron microscopy of two out of three cases showed an abnormal distribution of heterochromatin in many fibre nuclei. Our results show that dystrophic changes in skeletal muscle are not a major feature of ADEDMD, and that nuclear abnormalities may be detected with electron microscopy. Immunodetection of reduced laminin beta1 may be a useful secondary marker in adults with this disorder, as immunocytochemistry of lamins is not yet of diagnostic use.

The giant isoforms of nesprins 1 and 2 are emerging as important players in cellular organization, particularly in the positioning of nuclei, and possibly other organelles, within the cytoplasm. The experimental evidence suggests that nesprins also occur at the inner nuclear membrane, where they interact with the nuclear lamina. In this paper, we consider whether this is consistent with current ideas about nesprin anchorage and about mechanisms for nuclear import of membrane proteins.

Most pathogenic mutations in Duchenne and Becker muscular dystrophies involve deletion of single or multiple exons from the dystrophin gene, so exon-specific monoclonal antibodies (mAbs) can be used to distinguish normal and mutant dystrophin proteins. In Duchenne therapy trials, mAbs can be used to identify or rule out dystrophin-positive "revertant" fibres, which have an internally-deleted dystrophin protein and which occur naturally in some Duchenne patients. Using phage-displayed peptide libraries, we now describe the new mapping of the binding sites of five dystrophin mAbs to a few amino-acids within single exons. The phage display method also confirmed previous mapping of MANEX1A (exon 1) and MANDRA1 (exon 77) by other methods. Of the 79 dystrophin exons, mAbs are now available against single exons 1, 6, 8, 12, 13, 14, 17, 21, 26, 28, 38, 41, 43, 44, 45, 46, 47, 50, 51, 58, 59, 62, 63, 75 and 77. Many have been used in clinical trials, as well as for diagnosis and studies of dystrophin isoforms.

There are several lines of laboratory-based evidence emerging to suggest that purified polyphenol compounds such as resveratrol, found naturally in red grapes, epigallocatechin galate from green tea and curcumin from turmeric, might be useful for the treatment of various inherited neuromuscular diseases, including spinal muscular atrophy, Duchenne muscular dystrophy and Charcot–Marie–Tooth disease. Here, we critically examine the scientific evidence related to the known molecular effects that these polyphenols have on different models of inherited neuromuscular disease, with particular attention to problems with the validity of in vitro evidence. We also present proteomic evidence that polyphenols have in vitro effects on cells related to metal ion chelation in cell-culture media. Although their precise mechanisms of action remain somewhat elusive, polyphenols could be an attractive approach to therapy for inherited neuromuscular disease, especially since they may be safer to use on young children, compared with some of the other drug candidates.

Myotonic dystrophy (DM) is a multisystemic disorder caused by an inherited CTG repeat expansion which affects three genes encoding the DM protein kinase (DMPK), a homeobox protein Six5 and a protein containing WD repeats. Using a panel of 16 monoclonal antibodies against several different DMPK epitopes we detected DMPK, as a single protein of approximately 80 kDa, only in skeletal muscle, cardiac muscle and, to a lesser extent, smooth muscle. Many earlier reports of DMPK with different sizes and tissue distributions appear to be due to antibody cross-reactions with more abundant proteins. One such antibody, MANDM1, was used to isolate two related protein kinases, MRCK alpha and beta, from a human brain cDNA library and the shared epitope was located at the catalytic site of DMPK using a phage-displayed random peptide library. The peptide library also identified an epitope shared between DMPK and a 55 kDa muscle-specific protein. The results suggest that effects of the repeat expansion on the DMPK gene may be responsible for muscle and heart features of DM, whereas clinical changes in other tissues may be due to effects on the other two genes.

The spinal muscular atrophy protein, SMN, is a cytoplasmic protein that is also found in distinct nuclear structures called "gems." Gems are closely associated with nuclear coiled bodies and both may have a direct role in snRNP maturation and pre-RNA splicing. There has been some controversy over whether gems and coiled bodies colocalize or form adjacent/independent structures in HeLa and other cultured cells. Using a new panel of antibodies against SMN and antibodies against coilin-p80, a systematic and quantitative study of adult differentiated tissues has shown that gems always colocalize with coiled bodies. In some tissues, a small proportion of coiled bodies (10%) had no SMN, but independent or adjacent gems were not found. The most striking observation, however, was that many cell types appear to have neither gems nor coiled bodies (e.g., cardiac and smooth muscle, blood vessels, stomach, and spleen) and this expression pattern is conserved across human, rabbit, and pig species. This shows that assembly of distinct nuclear bodies is not essential for RNA splicing and supports the view that they may be storage sites for reserves of essential proteins and snRNPs. Overexpression of SMN in COS-7 cells produced supernumerary nuclear bodies, most of which also contained coilin-p80, confirming the close relationship between gems and coiled bodies. However, when SMN is reduced to very low levels in type I SMA fibroblasts, coiled bodies are still formed. Overall, the data suggest that gem/coiled body formation is not determined by high cytoplasmic SMN concentrations or high metabolic activity alone and that a differentiation-specific factor may control their formation.

Myotonic dystrophy (DM; OMIM 160900, also known as dystrophia myotonica, myotonia atrophica and Steinert disease) is an autosomal dominant myotonic myopathy associated with abnormalities of other organs, including eyes, heart, endocrine system, central and peripheral nervous systems, gastrointestinal organs, bone, and skin.1 The mutation underlying DM is an expansion of an unstable cytosine-thymine-guanine (CTG) trinucleotide repeat in the 3' untranslated region of the myotonic dystrophy protein kinase (DMPK) gene in chromosome 19q13.3.2-4 In 1994, Thornton et al.5 described an autosomal dominant disorder similar to DM without CTG repeat expansion at the DM locus. Ricker et al.6 named this disease "proximal myotonic myopathy" (PROMM; OMIM 600109) because of predominantly proximal muscle weakness without atrophy as opposed to the distal muscle involvement seen in DM. Subsequently, Meola et al.7 described a variant of PROMM with unusual myotonic and myopathic features, which they named "proximal myotonic myopathy syndrome," and Udd et al.8 described a PROMM-like family with dystrophic features, which they named "proximal myotonic dystrophy" (PDM). Researchers at the University of Minnesota9,10 found another multisystemic myotonic disorder that closely resembles DM with distal muscle weakness but no CTG repeat expansion. Because of the close phenotypic resemblance to DM, they called this disease "myotonic dystrophy type 2" (DM2; OMIM 602668). In 1998, Ranum et al.9 assigned the DM2 locus to chromosome 3q in a large kindred. Shortly after that, Ricker et al.11 found that the majority of German PROMM families show linkage to the DM2 locus. PDM was also mapped to this region (Krahe and Udd, personal communication, 1999). Whether PROMM, PDM, and DM2 represent different phenotypic expressions of a disease caused by the same mutation or if they are allelic disorders remains to be determined. It is also possible that these disorders are caused by mutations in different genes that are closely linked in the chromosome 3q region.12 Furthermore, the disease loci in some typical PROMM families11 and other families with multisystemic myotonic disorders have been excluded from both DM and DM2 loci. Because of the genetic and phenotypic heterogeneity in this group of disorders, it became necessary to establish a new nomenclature foreseeing the future discovery of new disease loci and phenotypic variability.

Transcripts encoding the 70-75 kDa C-terminal protein product of the dystrophin gene (Dp71) are alternatively spliced to generate multiple protein products in a number of adult human tissues. In this report, reverse transcriptase-polymerase chain reaction was used to clone and characterize a subpopulation of truncated Dp71 transcripts in adult human brain tissue which did not contain exons 71-74, resulting in an in-frame deletion of 330 bp encoding the syntrophin-binding domain. These truncated Dp71 transcripts are also alternatively spliced for exon 78. Immunoblot analysis, using dystrophin-specific C-terminal antibodies directed against epitopes in either exon 77 (MANDRA1), or 78 (1461), identified full-length dystrophin, Dp140 and Dp71, in total protein lysates from adult human brain tissue. In addition, a minor immunoreactive protein of approximately 58 kDa was also identified (designated Dp71 big up tri, open(110)). The observation that a monoclonal antibody directed against epitopes within exons 73-74 (MANEX7374A) failed to detect this 58 kDa protein provides definitive evidence that Dp71 big up tri, open(110) is derived from Dp71 transcripts deleted for the syntrophin-binding domain. These results, as well as previous findings, demonstrate that alternative splicing of Dp71 in the human brain generates a variety of mRNA transcripts encoding distinct protein variants of Dp71, and further supports the use of exon-specific antibodies in characterizing these variants. The presence of these Dp71 protein variants in brain tissue points to their interaction with various cellular proteins and their involvement in different cellular functions.

A novel method for cloning DNase I fragments into bacteriophage display vector fUSE2 was used to create libraries expressing hepatitis C virus (HCV) protein fragments on the phage surface. Selection by panning with a mixture of sera from five HCV-seropositive individuals enabled identification of antigenic determinants in NS3 (amino acids 1,383-1,415), NS4 (amino acids 1, 930-1,938), and NS5 (amino acids 2,088-2,104). The NS3 result is the most accurate location to date of a major conformational determinant that cannot be mimicked by short peptides. Any expressed sequence from the phage library can be excised with Bgl II and cloned directly into the Bgl II site of an appropriate plasmid for bacterial expression. This enables production of chimeric proteins containing multiple antigenic determinants, illustrated by co-expression of the NS4P (amino acids 1,930-1,938) epitope with an NS4N fragment (amino acids 1,644-1,812) containing at least three linear HCV epitopes. When used to screen 35 individual HCV-positive sera by enzyme-linked immunosorbent assay (ELISA), the chimeric antigen detected eight more positives than NS4N alone and gave increased immunoreactivity with others. This approach of identifying antigenic regions by phage display and then co-expressing them as chimeric proteins may be generally applicable to the production of improved diagnostic antigens and recombinant vaccines.

The homodimeric muscle isoform of creatine kinase (MM-CK) unfolds on exposure to low levels of guanidinium chloride (GdmCl) to yield a partly folded monomeric intermediate. Those regions of MM-CK that experience local unfolding were previously identified through an extensive study of antibody accessibility and protease sensitivity. Since these studies were completed, the coordinates of the rabbit isoform (MM-CK) were released. In light of this, we have determined the minimum changes to this structure required to explain our data on protease and epitope accessibility in the intermediate. We propose that the observed changes occur through (a) disruption of the monomer-monomer interface during dissociation, (b) separation and/ or unfolding of domains or subdomains, and (c) the partial unfolding of solvent-exposed helices. The proposed structure for the intermediate is consistent both with current models of unfolding intermediates and the results of independent studies pertaining to the unfolding of creatine kinase. Proteins 2001;42:269–278. © 2000 Wiley-Liss, Inc.

Emerin is the protein of the inner nuclear membrane that is affected by mutation in X-linked Emery-Dreifuss muscular dystrophy. The autosomal dominant form of the disease is caused by mutations in the lamin A/C gene. Several lines of circumstantial evidence have suggested an interaction of emerin with lamins in the nuclear lamina but direct interaction between the two proteins has not yet been demonstrated. We now demonstrate direct interaction between recombinant emerin and lamin A molecules using biomolecular interaction analysis (BIA) and monoclonal antibodies. An emerin–lamin A interaction system may be related in function to the LAP2-lamin B system at the inner nuclear rim.

This paper describes the Fiscal Environmental Integration Model (FEIM) and its use to examine the merits of introducing a set of air pollutant emission taxes and stringent abatement requirements based on best commonly available control technology. These environmental protection strategies are examined both independently and in combination. In addition, Hungary has very high VAT, employment, and income tax rates and therefore might receive more than the usual advantage from using environmental tax revenues to reduce other taxes. We therefore also examine the economic and environmental implications of different uses of the revenues generated by the air pollutant emission taxes. FEIM is a CGE model of the Hungarian economy that includes sectoral air pollution control costs functions and execution options that allow examination of the key policy choices involved. We developed and ran a baseline and seven scenarios with FEIM. The scenarios centered on introduction of environmental load fees (ELF) on emissions of SO2, NOx, and particulates and emission abatement requirements (EAR) for these pollutants.

Emery-Dreifuss muscular dystrophy has some remarkably specific features, with only cardiac and skeletal tissues being affected. Equally remarkably, the disease is caused by mutations in widely expressed genes for the nuclear membrane/lamina proteins, emerin and lamin A/C. How do mutations in proteins at the heart of the cell lead to stiff joints and sudden heart failure? This and related questions are the subject of this review.

We have determined the molecular basis for skeletal myopathy and dilated cardiomyopathy in two male German short-haired pointer (GSHP) littermates. Analysis of skeletal muscle demonstrated a complete absence of dystrophin on Western blot analysis. PCR analysis of genomic DNA revealed a deletion encompassing the entire dystrophin gene. Molecular cytogenetic analysis of lymphocytes from the dam and both dystrophic pups confirmed a visible deletion in the p21 region of the affected canine X chromosome. Utrophin is up-regulated in the skeletal muscle, but does not appear to ameliorate the dystrophic canine phenotype. This new canine model should further our understanding of the physiological and biochemical processes in Duchenne muscular dystrophy.