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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.

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.

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.

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.

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.

Dystrophin is present in the outer plexiform layer of the retina and is required for normal retinal function as measured by electroretinography. We describe the identification of a novel isoform of dystrophin (Dp260) present in the mouse retina. The unique 5' terminus of the mRNA originates from a newly identified exon and is spliced in frame to exon 30 of the Duchenne muscular dystrophy (DMD) gene. The retinal isoform of dystrophin has 13 novel amino acids as its N-terminus followed by most of the dystrophin rod domain and the cysteine-rich C-terminal domains. Analysis of mouse tissues indicated this isoform of dystrophin is expressed in retina, brain and cardiac tissue. Comparison of retinal electrophysiology in mdx and mdx Cv3 mouse suggests that Dp260 is required for normal retinal function

Two promoters In the distal half of the Duchenne Muscular Dystrophy gene drive transcription of mRNAs which have novel first exons and encode the shortened forms of dystrophin, apo-dystrophln-1 (Dp71) and apodystrophln-2 (Dpi 16). Apo-dystrophin-1 has a G + C rich promoter and Is expressed in a wide range of cell types, whilst apo-dystrophln-2 is confined to peripheral nerve and brain. We have isolated and sequenced the unique 5' exon of rat apo-dystrophln-2 mRNA. Conceptual translation of this sequence Indicates that apo-dystrophin- 2 contains a unique 23 amino acid terminal peptide. Using specific probes derived from sequences at the 5' ends of apo-dystrophln-1 and apo-dystrophln-2 we have determined the expression of these two mRNAs during mouse embryonic development by RNA In situ hybridization. In contrast to full-length dystrophin, neither of these short dystrophin transcripts appear before organogenesis is well established. Apo-dystrophin-1 mRNA is detected in midllne cells of the ventral neural tube and later, in the ependymal cells lining the ventricles of the brain. These results suggest that apo-dystrophin-1 mRNA is associated with glial cells in the CNS. Apo-dystrophln-1 transcripts are also abundant in the teeth primordla throughout their development. In contrast apo-dystrophin-2 mRNA is largely undetectable during development, although transcripts are seen in the newborn brain. Western blots of late human fetal tissue extracts confirm that apo-dystrophin-2 is most abundant in brain and analysis of RNA and protein in cultured cell lines reveal expression of apo-dystrophln-1 and apo-dystrophin- 2 in glioma cells.

Apolipoprotein E (apoE) is a 34 kDa glycoprotein with three distinct isoforms in the human population (apoE2, apoE3 and apoE4) known to play a major role in differentially influencing risk to, as well as outcome from, disease and injury in the central nervous system. In general, the apoE4 allele is associated with poorer outcomes after disease or injury, whereas apoE3 is associated with better responses. The extent to which different apoE isoforms influence degenerative and regenerative events in the peripheral nervous system (PNS) is still to be established, and the mechanisms through which apoE exerts its isoform-specific effects remain unclear. Here, we have investigated isoform-specific effects of human apoE on the mouse PNS. Experiments in mice ubiquitously expressing human apoE3 or human apoE4 on a null mouse apoE background revealed that apoE4 expression significantly disrupted peripheral nerve regeneration and subsequent neuromuscular junction re-innervation following nerve injury compared with apoE3, with no observable effects on normal development, maturation or Wallerian degeneration. Proteomic isobaric tag for relative and absolute quantitation (iTRAQ) screens comparing healthy and regenerating peripheral nerves from mice expressing apoE3 or apoE4 revealed significant differences in networks of proteins regulating cellular outgrowth and regeneration (myosin/actin proteins), as well as differences in expression levels of proteins involved in regulating the blood‐nerve barrier (including orosomucoid 1). Taken together, these findings have identified isoform-specific roles for apoE in determining the protein composition of peripheral nerve as well as regulating nerve regeneration pathways in vivo.

The 14kb dystrophin transcript from the Duchenne muscular dystrophy (DMD) locus, which encodes a 427kDa protein, is differentially spliced at the amino terminal end giving rise to alternative transcripts expressed in muscle and brain. Here we present evidence for a 4.8kb transcript from the DMD locus which is ubiquitously expressed but is particularly abundant in Schwannoma cells where dystrophin could not be detected. The hybridisation of Western blots with dystrophin antibodies also identifies a protein of approximately 80kDa of variable abundance in different human and mdx tissues. Immunocytochemistry studies confirm the expression of this protein in nerve cells, a tissue in which full length dystrophin is not detected. Sequencing of the 5' end of a clone isolated from a rat Schwannoma cDNA library, shows that the 4.8kb transcript shares exons with the carboxy terminal end of dystrophin but the 5' untranslated region is not contained within the dystrophin transcript. We propose that the 4.8kb gene product be called apodystrophin-1 as its expression is distinct from the dystrophin 14kb mRNA but it is transcribed from the same locus.

Spinal muscular atrophy (SMA) is caused by loss of the survival motor neuron gene (SMN1) and retention of the SMN2 gene. The copy number of SMN2 affects the amount of SMN protein produced and the severity of the SMA phenotype. While loss of mouse Smn is embryonic lethal, two copies of SMN2 prevents this embryonic lethality resulting in a mouse with severe SMA that dies 5 days after birth. Here we show that expression of full-length SMN under the prion promoter (PrP) rescues severe SMA mice. The PrP results in high levels of SMN in neurons at embryonic day 15. Mice homozygous for PrP-SMN with two copies of SMN2 and lacking mouse Smn survive for an average of 210 days and lumbar motor neuron root counts in these mice were normal. Expression of SMN solely in skeletal muscle using the human skeletal actin (HSA) promoter resulted in no improvement of the SMA phenotype or extension of survival. One HSA line displaying nerve expression of SMN did affect the SMA phenotype with mice living for an average of 160 days. Thus, we conclude that expression of full-length SMN in neurons can correct the severe SMA phenotype in mice. Furthermore, a small increase of SMN in neurons has a substantial impact on survival of SMA mice while high SMN levels in mature skeletal muscle alone has no impact.

Spinal muscular atrophy (SMA) is caused by mutations in the SMN (survival of motor neurons) gene and there is a correlation between disease severity and levels of functional SMN protein. Studies of structure-function relationships in SMN protein may lead to a better understanding of SMA pathogenesis. Self-association of the spinal muscular atrophy protein, SMN, is important for its function in RNA splicing. Biomolecular interaction analysis core analysis now shows that SMN self-association occurs via SMN regions encoded by exons 2b and 6, that exon 2b encodes a binding site for SMN-interacting protein-1 and that interaction occurs between exon 2- and 4-encoded regions within the SMN monomer. The presence of two separate self-association sites suggests a novel mechanism by which linear oligomers or closed rings might be formed from SMN monomers.

Emerin is a nuclear membrane protein which is missing or defective in Emery-Dreifuss muscular dystrophy (EDMD). It is one member of a family of lamina-associated proteins which includes LAP1, LAP2 and lamin B receptor (LBR). A panel of 16 monoclonal antibodies (mAbs) has been mapped to six specific sites throughout the emerin molecule using phage-displayed peptide libraries and has been used to localize emerin in human and rabbit heart. Several mAbs against different emerin epitopes did not recognize intercalated discs in the heart, though they recognized cardiomyocyte nuclei strongly, both at the rim and in intranuclear spots or channels. A polyclonal rabbit antiserum against emerin did recognize both nuclear membrane and intercalated discs but, after affinity purification against a pure-emerin band on a western blot, it stained only the nuclear membrane. These results would not be expected if immunostaining at intercalated discs were due to a product of the emerin gene and, therefore, cast some doubt upon the hypothesis that cardiac defects in EDMD are caused by absence of emerin from intercalated discs. Although emerin was abundant in the membranes of cardiomyocyte nuclei, it was absent from many non-myocyte cells in the heart. This distribution of emerin was similar to that of lamin A, a candidate gene for an autosomal form of EDMD. In contrast, lamin B1 was absent from cardiomyocyte nuclei, showing that lamin B1 is not essential for localization of emerin to the nuclear lamina. Lamin B1 is also almost completely absent from skeletal muscle nuclei. In EDMD, the additional absence of lamin B1 from heart and skeletal muscle nuclei which already lack emerin may offer an alternative explanation of why these tissues are particularly affected.

There is considerable confusion in the literature about the size of the myotonic dystrophy protein kinase (DMPK) and its localization within tissues. We have used a new panel of monoclonal antibodies (mAbs) to begin to resolve these issues, which are important for understanding the possible role of DMPK in myotonic dystrophy. Antisera raised against the catalytic and coil domains of DMPK recognized a major 55 kDa protein and a minor 72-80 kDa doublet on western blots of human skeletal muscle. Ten mAbs, five against the catalytic domain and five against the coil region, recognized only the 72-80 kDa doublet. The 72 kDa protein was present in all tissues tested, whereas the 80 kDa component was variably expressed, mainly in skeletal and cardiac muscles. The 72 kDa protein was absent in a DMPK knockout mouse and was greatly increased in a transgenic mouse overexpressing human DMPK, confirming its identity as authentic DMPK. Two mAbs against the catalytic domain recognized only the more abundant 55 kDa protein, which was found only in skeletal muscle. Nine out of 10 mAbs located DMPK to intercalated discs in human heart, an affected tissue in myotonic dystrophy. However, co-localization of DMPK with acetylcholine receptors at neuromuscular junctions was not observed with any of the mAbs. Subcellular fractionation and sedimentation analysis suggest that a major proportion of the DMPK in skeletal muscle and brain is cytosolic.

Seventeen families with Emery-Dreifuss muscular dystrophy (EDMD) have been studied both by DNA sequencing and by emerin protein expression. Fourteen had mutations in the X-linked emerin gene, while three showed evidence of autosomal inheritance. Twelve of the 14 emerin mutations caused early termination of translation. An in-frame deletion of six amino acids from the C-terminal transmembrane helix caused almost complete absence of emerin from muscle with no localization to the nuclear membrane, although mRNA levels were normal. This shows that mutant emerin proteins are unstable if they are unable to integrate into a membrane. A 22 bp deletion in the promoter region was expected to result in reduced emerin production, but normal amounts of emerin of normal size were found in leucocytes and lymphoblastoid cell lines. This shows that DNA analysis is necessary to exclude emerin mutations in suspected X-linked EDMD. Emerin levels in female carriers often deviated from the expected 50% and this was due, in at least two families, to skewed emerin mRNA expression from the normal and mutated alleles. In one family with a novel deletion of the last three exons of the emerin gene, a carrier had a cardiomyopathy and very low emerin levels (

A large fragment of emerin cDNA was prepared by PCR and expressed as a recombinant protein in Escherichia coli. Using this as immunogen, we prepared a panel of 12 monoclonal antibodies which recognise at least four different epitopes on emerin in order to ensure that emerin can be distinguished from non-specific cross-reacting proteins. All the mAbs recognised a 34 kDa protein in all tissues tested, though minor emerin-related bands were also detected in some tissues. Immunofluorescence microscopy showed that emerin is located at the nuclear rim in all tissues examined. A muscle biopsy from an Emery-Dreifuss muscular dystrophy (EMDM) patient showed complete absence of emerin by both Western blotting and immunohistochemistry, suggesting a simple diagnostic antibody test for EDMD families. Biochemical fractionation of brain and liver tissues showed that emerin was present in nuclei purified by centrifugation through 65% sucrose and was absent from soluble fractions (post-100,000 g). From these results, together with sequence and structural homologies between emerin, thymopoietins and the nuclear lamina-associated protein, LAP2, we suggest that emerin will prove to be one member of a family of inner nuclear membrane proteins.