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Cloning and developmental expression analysis of ltd-1, the Caenorhabditis elegans homologue of the mouse kyphoscoliosis (ky) gene.
We have characterized the developmental expression pattern of the Caenorhabditis elegans homologue of the mouse ky gene. The Ky protein has a putative key function in muscle development and has homologues in invertebrates, fungi and a cyanobacterium. The C. elegans Ky homologue gene has been named ltd-1 for LIM and transglutaminase domains gene. The LTD-1::GFP construct is expressed in developing hypodermal cells from the twofold stage embryo through adulthood. These data define the ltd-1 gene as a novel marker for C. elegans epithelial cell development.
Testing of SHIRPA, a mouse phenotypic assessment protocol, on Dmd(mdx) and Dmd(mdx3cv) dystrophin-deficient mice.
The SHIRPA protocol was proposed as a rapid, comprehensive screening method for qualitatively abnormal phenotypes in the mouse (Rogers et al., Mamm Genome 8, 711, 1997). This screening technique is currently being used to identify mutants induced by N-ethylnitrosourea (ENU) mutagenesis (Brown and Nolan, Hum Mol Genet 7, 1627, 1998). SHIRPA can be used to identify mutants with neuromuscular abnormalities, but the sensitivity of the protocol is unknown. We tested two dystrophin-deficient mutants Dmd(mdx) and Dmd(mdx3cv), both of which are indistinguishable from wild-type by a simple visual assessment, at different ages, using the primary screen of the SHIRPA protocol. The most dramatic observation was that both Dmd(mdx) and Dmd(mdx3cv) mice showed extreme fatigue after testing, while mice from the same C57BL strains appeared unaffected. Each strain of dystrophin-deficient mice showed a different profile in locomotor activity and deficiencies in the wire maneuver, righting reflex, and negative geotaxis tests. Furthermore, the wire maneuver test indicated an earlier onset of muscular impairment in Dmd(mdx) than Dmd(mdx3cv) mice. These data suggest that the SHIRPA primary screen is effective not only in identifying subtle neuromuscular mutants, but also in distinguishing qualitative differences between mutants with neuromuscular abnormalities.
Assembly of multiple dystrobrevin-containing complexes in the kidney.
Dystrophin is the key component in the assembly and maintenance of the dystrophin-associated protein complex (DPC) in skeletal muscle. In kidney, dystroglycan, an integral component of the DPC, is involved in kidney epithelial morphogenesis, suggesting that the DPC is important in linking the extracellular matrix to the internal cytoskeleton of kidney epithelia. Here, we have investigated the molecular architecture of dystrophin-like protein complexes in kidneys from normal and dystrophin-deficient mice. Using isoform-specific antibodies, we show that the different cell types that make up the kidney maintain different dystrophin-like complexes. These complexes can be broadly grouped according to their dystrobrevin content: beta-dystrobrevin containing complexes are present at the basal region of renal epithelial cells, whilst alpha-dystrobrevin-1 containing complexes are found in endothelial and smooth muscle cells. Furthermore, these complexes are maintained even in the absence of all dystrophin isoforms. Thus our data suggest that the functions and assembly of the dystrophin-like complexes in kidney differ from those in skeletal muscle and implicate a protein other than dystrophin as the primary molecule in the assembly and maintenance of kidney complexes. Our findings also provide a possible explanation for the lack of kidney pathology in Duchenne muscular dystrophy patients and mice lacking all dystrophin isoforms.
Comparative genetic analysis: the utility of mouse genetic systems for studying human monogenic disease.
One of the long-term goals of mutagenesis programs in the mouse has been to generate mutant lines to facilitate the functional study of every mammalian gene. With a combination of complementary genetic approaches and advances in technology, this aim is slowly becoming a reality. One of the most important features of this strategy is the ability to identify and compare a number of mutations in the same gene, an allelic series. With the advent of gene-driven screening of mutant archives, the search for a specific series of interest is now a practical option. This review focuses on the analysis of multiple mutations from chemical mutagenesis projects in a wide variety of genes and the valuable functional information that has been obtained from these studies. Although gene knockouts and transgenics will continue to be an important resource to ascertain gene function, with a significant proportion of human diseases caused by point mutations, identifying an allelic series is becoming an equally efficient route to generating clinically relevant and functionally important mouse models.
Pharmacologically targeting the primary defect and downstream pathology in Duchenne muscular dystrophy.
DMD is a devastatingly progressive muscle wasting disorder of childhood that significantly shortens life expectancy. Despite efforts to develop an effective therapy that dates back over a century, clinical interventions are still restricted to management of symptoms rather than a cure. The rationale to develop effective therapies changed in 1986 with the discovery of the dystrophin gene. Since then extensive research into both the molecular basis and pathophysiology of DMD has paved the way not only for development of strategies which aim to correct the primary defect, but also towards the identification of countless therapeutic targets with the potential to alleviate the downstream pathology. In addition to gene and cell-based therapies, which aim to deliver the missing gene and/or protein, an exciting spectrum of pharmacological approaches aimed at modulating therapeutic targets within DMD muscle cells through the use of small drugs are also being developed. This review presents promising pharmacological approaches aimed at targeting the primary defect, including suppression of nonsense mutations and functional compensation by upregulation of the dystrophin homologue, utrophin. Downstream of the primary membrane fragility, inflammation and fibrosis are reduced by blocking NF-κB, TGF-α and TGF-β, and free radical damage has been targeted using antioxidants and dietary/nutritional supplements. There are new hopes that ACE and PDE5 inhibitors can protect against skeletal as well as cardiac pathology, and modulating Ca2+ influx, NO, BMP, protein degradation and the mitochondrial permeability pore hold further promise in tackling the complex pathogenesis of this multifaceted disorder.
Engineering multiple U7snRNA constructs to induce single and multiexon-skipping for Duchenne muscular dystrophy.
Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disorder caused by mutations in the dystrophin gene. Antisense-mediated exon skipping is one of the most promising approaches for the treatment of DMD but still faces personalized medicine challenges as different mutations found in DMD patients require skipping of different exons. However, 70% of DMD patients harbor dystrophin gene deletions in a mutation-rich area or "hot-spot" in the central genomic region. In this study, we have developed 11 different U7 small-nuclear RNA, to shuttle antisense sequences designed to mask key elements involved in the splicing of exons 45 to 55. We demonstrate that these constructs induce efficient exon skipping both in vitro in DMD patients' myoblasts and in vivo in human DMD (hDMD) mice and that they can be combined into a single vector to achieve a multi skipping of at least 3 exons. These very encouraging results provide proof of principle that efficient multiexon-skipping can be achieved using adeno-associated viral (AAV) vectors encoding multiple U7 small-nuclear RNAs (U7snRNAs), offering therefore very promising tools for clinical treatment of DMD.
MLP accumulation and remodelling in the infarcted rat heart.
Mutation of cytoskeletal protein genes results in abnormal protein function and causes cardiomyopathy. We hypothesised that cardiac levels of cytoskeletal proteins, such as dystrophin, desmin and muscle LIM protein (MLP), would be altered during remodelling caused by myocardial infarction (MI). We measured left-ventricular morphology, function and cytoskeletal protein levels 10 weeks after coronary artery ligation or sham operation in male Wistar rats. Two-dimensional echocardiography revealed significant impairment of systolic function and decreased ejection fraction in infarcted hearts compared with sham (47+/-5% versus 73+/-4%), commensurate with the development of heart failure. Western blotting was used to measure levels of beta-myosin heavy chain (beta-MyHC), a marker of hypertrophy, and levels of dystrophin, desmin, MLP, beta-tubulin, utrophin and syncoilin, using GAPDH for normalization. Relative to shams, beta-MyHC and MLP levels were increased 1.9-fold and 1.7-fold, respectively, in infarcted rat hearts, whereas the levels of other cytoskeletal proteins were unchanged. Both MLP and desmin protein levels correlated negatively with ejection fraction, with the strongest relation between MLP and ejection fraction (r=-0.95, n=13, p<0.0001). This work suggests that MLP may play an important compensatory role in cardiac remodelling following MI.
The artificial zinc finger coding gene 'Jazz' binds the utrophin promoter and activates transcription.
Up-regulation of utrophin gene expression is recognized as a plausible therapeutic approach in the treatment of Duchenne muscular dystrophy (DMD). We have designed and engineered new zinc finger-based transcription factors capable of binding and activating transcription from the promoter of the dystrophin-related gene, utrophin. Using the recognition 'code' that proposes specific rules between zinc finger primary structure and potential DNA binding sites, we engineered a new gene named 'Jazz' that encodes for a three-zinc finger peptide. Jazz belongs to the Cys2-His2 zinc finger type and was engineered to target the nine base pair DNA sequence: 5'-GCT-GCT-GCG-3', present in the promoter region of both the human and mouse utrophin gene. The entire zinc finger alpha-helix region, containing the amino acid positions that are crucial for DNA binding, was specifically chosen on the basis of the contacts more frequently represented in the available list of the 'code'. Here we demonstrate that Jazz protein binds specifically to the double-stranded DNA target, with a dissociation constant of about 32 nM. Band shift and super-shift experiments confirmed the high affinity and specificity of Jazz protein for its DNA target. Moreover, we show that chimeric proteins, named Gal4-Jazz and Sp1-Jazz, are able to drive the transcription of a test gene from the human utrophin promoter.
Analysis of human neurological disorders using mutagenesis in the mouse.
The mouse continues to play a vital role in the deciphering of mammalian gene function and the modelling of human neurological disease. Advances in gene targeting technologies have facilitated the efficiency of generating new mouse mutants, although this valuable resource has rapidly expanded in recent years due to a number of major random mutagenesis programmes. The phenotype-driven mutagenesis screen at the MRC Mammalian Genetics Unit has generated a significant number of mice with potential neurological defects, and our aim has been to characterize selected mutants on a pathological and molecular level. Four lines are discussed, one displaying late-onset ataxia caused by Purkinje cell loss and an allelic series of three tremor mutants suffering from hypomyelination of the peripheral nerve. Molecular analysis of the causative mutation in each case has provided new insights into functional aspects of the mutated proteins, illustrating the power of mutagenesis screens to generate both novel and clinically relevant disease models.
Utrophin binds laterally along actin filaments and can couple costameric actin with sarcolemma when overexpressed in dystrophin-deficient muscle.
Dystrophin is widely thought to mechanically link the cortical cytoskeleton with the muscle sarcolemma. Although the dystrophin homolog utrophin can functionally compensate for dystrophin in mice, recent studies question whether utrophin can bind laterally along actin filaments and anchor filaments to the sarcolemma. Herein, we have expressed full-length recombinant utrophin and show that the purified protein is fully soluble with a native molecular weight and molecular dimensions indicative of monomers. We demonstrate that like dystrophin, utrophin can form an extensive lateral association with actin filaments and protect actin filaments from depolymerization in vitro. However, utrophin binds laterally along actin filaments through contribution of acidic spectrin-like repeats rather than the cluster of basic repeats used by dystrophin. We also show that the defective linkage between costameric actin filaments and the sarcolemma in dystrophin-deficient mdx muscle is rescued by overexpression of utrophin. Our results demonstrate that utrophin and dystrophin are functionally interchangeable actin binding proteins, but that the molecular epitopes important for filament binding differ between the two proteins. More generally, our results raise the possibility that spectrin-like repeats may enable some members of the plakin family of cytolinkers to laterally bind and stabilize actin filaments.
Function and genetics of dystrophin and dystrophin-related proteins in muscle.
The X-linked muscle-wasting disease Duchenne muscular dystrophy is caused by mutations in the gene encoding dystrophin. There is currently no effective treatment for the disease; however, the complex molecular pathology of this disorder is now being unravelled. Dystrophin is located at the muscle sarcolemma in a membrane-spanning protein complex that connects the cytoskeleton to the basal lamina. Mutations in many components of the dystrophin protein complex cause other forms of autosomally inherited muscular dystrophy, indicating the importance of this complex in normal muscle function. Although the precise function of dystrophin is unknown, the lack of protein causes membrane destabilization and the activation of multiple pathophysiological processes, many of which converge on alterations in intracellular calcium handling. Dystrophin is also the prototype of a family of dystrophin-related proteins, many of which are found in muscle. This family includes utrophin and alpha-dystrobrevin, which are involved in the maintenance of the neuromuscular junction architecture and in muscle homeostasis. New insights into the pathophysiology of dystrophic muscle, the identification of compensating proteins, and the discovery of new binding partners are paving the way for novel therapeutic strategies to treat this fatal muscle disease. This review discusses the role of the dystrophin complex and protein family in muscle and describes the physiological processes that are affected in Duchenne muscular dystrophy.
Activation of calcineurin and stress activated protein kinase/p38-mitogen activated protein kinase in hearts of utrophin-dystrophin knockout mice.
Dilated cardiomyopathy is a common complication of Duchenne and Becker muscular dystrophies, which are caused by mutations in the dystrophin gene. The mdx mouse is an animal model for Duchenne muscular dystrophy (DMD) and shows mildly dystrophic changes in the heart. By contrast, the utrophin-dystrophin knockout (dko) mouse shows severe dystrophic changes in cardiac muscle, that more closely resembles DMD cardiomyopathy than mdx mouse. However the pathogenesis of development has not been fully understood. Recently many reports have revealed that calcineurin and stress activated protein kinase (SAPK)/p38-mitogen activated protein kinase (MAPK) hypertrophic signalling pathways are associated with the development of some forms of hypertrophic and dilated cardiomyopathies. These signalling pathways may have some roles in the development of dystrophin-deficient cardiomyopathy. Here we report that calcineurin and SAPK/p38-MAPK signalling pathways were constantly activated in dko hearts, but the activation varied in mdx hearts. The pathogenesis of the development of dystrophin-deficient cardiomyopathy may be associated with the activation of these signalling pathways.
Plectin 1f scaffolding at the sarcolemma of dystrophic (mdx) muscle fibers through multiple interactions with beta-dystroglycan.
In skeletal muscle, the cytolinker plectin is prominently expressed at Z-disks and the sarcolemma. Alternative splicing of plectin transcripts gives rise to more than eight protein isoforms differing only in small N-terminal sequences (5-180 residues), four of which (plectins 1, 1b, 1d, and 1f) are found at substantial levels in muscle tissue. Using plectin isoform-specific antibodies and isoform expression constructs, we show the differential regulation of plectin isoforms during myotube differentiation and their localization to different compartments of muscle fibers, identifying plectins 1 and 1f as sarcolemma-associated isoforms, whereas plectin 1d localizes exclusively to Z-disks. Coimmunoprecipitation and in vitro binding assays using recombinant protein fragments revealed the direct binding of plectin to dystrophin (utrophin) and beta-dystroglycan, the key components of the dystrophin-glycoprotein complex. We propose a model in which plectin acts as a universal mediator of desmin intermediate filament anchorage at the sarcolemma and Z-disks. It also explains the plectin phenotype observed in dystrophic skeletal muscle of mdx mice and Duchenne muscular dystrophy patients.
A quantitative study of bioenergetics in skeletal muscle lacking utrophin and dystrophin.
Muscle energetics and function were investigated in the hindlimb of mice lacking dystrophin (mdx), utrophin and dystrophin (utr-dys) and controls (C57Bl/10) using 31P and 1H magnetic resonance techniques, electrical nerve stimulation and direct biochemical analysis. At rest, [adenosine triphosphate] and [total creatine] were lowest in utr-dys, while [inorganic phosphate] was elevated. Calculated [adenosine diphosphate] was 3-fold higher in mdx and 5-fold higher in utr-dys than in controls, consistent with an increased adenosine triphosphate requirement for ion pump activity. During stimulation, force production was low only in utr-dys, and this was reflected in the bioenergetic changes. Initial recovery rates of [phosphocreatine] and [adenosine diphosphate] after stimulation were rapid in all groups, indicative of normal mitochondrial adenosine triphosphate production in utr-dys and mdx. Recovery of pH was slow in utr-dys. The data indicate that the severe abnormalities which are present in the absence of utrophin and dystrophin leave basic muscle energetics intact and appear confined to processes involving the sarcolemma.
Isolation and culture of motor neurons from the newborn mouse spinal cord.
A protocol for the isolation and culture of motor neurons from postnatal day 1 mouse spinal cord is described. After 72 h in culture, phase contrast microscopy reveals healthy cells with motor neuronal morphology and extensive neuritic processes. These neurons express the 75-kDa low-affinity neurotrophin receptor (p75NTR) and choline acetyltransferase (ChAT), both proteins are specifically expressed by neonatal and embryonic motor neurons in vivo. This protocol can be adapted for various postnatal motor neuron assays.