Wnt-Ror-Dvl signalling and the dystrophin complex organize planar-polarized membrane compartments in C. elegans muscles.

Cell polarity mechanisms allow the formation of specialized membrane domains with unique protein compositions, signalling properties, and functional characteristics. By analyzing the localization of potassium channels and proteins belonging to the dystrophin-associated protein complex, we reveal the existence of distinct planar-polarized membrane compartments at the surface of C. elegans muscle cells. We find that muscle polarity is controlled by a non-canonical Wnt signalling cascade involving the ligand EGL-20/Wnt, the receptor CAM-1/Ror, and the intracellular effector DSH-1/Dishevelled. Interestingly, classical planar cell polarity proteins are not required for this process. Using time-resolved protein degradation, we demonstrate that -while it is essentially in place by the end of embryogenesis- muscle polarity is a dynamic state, requiring continued presence of DSH-1 throughout post-embryonic life. Our results reveal the unsuspected complexity of the C. elegans muscle membrane and establish a genetically tractable model system to study cellular polarity and membrane compartmentalization in vivo.

Use of Glucose-Fructose to Enhance the Exon Skipping Efficacy.

Exon-skipping antisense oligonucleotides (AOs) are promising treatments for muscle-related genetic ailments including Duchenne muscular dystrophy (DMD), but clinical translation is unfortunately hampered by insufficient systemic delivery. Here we describe that how one can employ a glucose-fructose injection mixture to improve muscle uptake and functional outcomes of DMD AOs in energy-deficient peripheral muscles of mdx mice. The potentiating effect of glucose-fructose on AOs in energy-deficient muscles offers a simple and economical method for enhancing AO potency, reducing screening costs for researchers and accelerating the translation of nucleic acid-based therapeutics in DMD and other muscular dystrophies.

ABC of multifaceted dystrophin glycoprotein complex (DGC).

Dystrophin protein in association with several other cellular proteins and glycoproteins leads to the formation of a large multifaceted protein complex at the cell membrane referred to as dystrophin glycoprotein complex (DGC), that serves distinct functions in cell signaling and maintaining the membrane stability as well as integrity. In accordance with this, several findings suggest exquisite role of DGC in signaling pathways associated with cell development and/or maintenance of homeostasis. In the present review, we summarize the established facts about the various components of this complex with emphasis on recent insights into specific contribution of the DGC in cell signaling at the membrane. We have also discussed the recent advances made in exploring the molecular associations of DGC components within the cells and the functional implications of these interactions. Our review would help to comprehend the composition, role, and functioning of DGC and may lead to a deeper understanding of its role in several human diseases.

Circulating miRNAs are generic and versatile therapeutic monitoring biomarkers in muscular dystrophies.

The development of medical approaches requires preclinical and clinical trials for assessment of therapeutic efficacy. Such evaluation entails the use of biomarkers, which provide information on the response to the therapeutic intervention. One newly-proposed class of biomarkers is the microRNA (miRNA) molecules. In muscular dystrophies (MD), the dysregulation of miRNAs was initially observed in muscle biopsy and later extended to plasma samples, suggesting that they may be of interest as biomarkers. First, we demonstrated that dystromiRs dysregulation occurs in MD with either preserved or disrupted expression of the dystrophin-associated glycoprotein complex, supporting the utilization of dystromiRs as generic biomarkers in MD. Then, we aimed at evaluation of the capacity of miRNAs as monitoring biomarkers for experimental therapeutic approach in MD. To this end, we took advantage of our previously characterized gene therapy approach in a mouse model for α-sarcoglycanopathy. We identified a dose-response correlation between the expression of miRNAs on both muscle tissue and blood serum and the therapeutic benefit as evaluated by a set of new and classically-used evaluation methods. This study supports the utility of profiling circulating miRNAs for the evaluation of therapeutic outcome in medical approaches for MD.

Novel Nuclear Protein Complexes of Dystrophin 71 Isoforms in Rat Cultured Hippocampal GABAergic and Glutamatergic Neurons.

The precise functional role of the dystrophin 71 in neurons is still elusive. Previously, we reported that dystrophin 71d and dystrophin 71f are present in nuclei from cultured neurons. In the present work, we performed a detailed analysis of the intranuclear distribution of dystrophin 71 isoforms (Dp71d and Dp71f), during the temporal course of 7-day postnatal rats hippocampal neurons culture for 1h, 2, 4, 10, 15 and 21 days in vitro (DIV). By immunofluorescence assays, we detected the highest level of nuclear expression of both dystrophin Dp71 isoforms at 10 DIV, during the temporal course of primary culture. Dp71d and Dp71f were detected mainly in bipolar GABAergic (≥60%) and multipolar Glutamatergic (≤40%) neurons, respectively. We also characterized the existence of two nuclear dystrophin-associated protein complexes (DAPC): dystrophin 71d or dystrophin 71f bound to ß-dystroglycan, α1-, ß-, α2-dystrobrevins, α-syntrophin, and syntrophin-associated protein nNOS (Dp71d-DAPC or Dp71f-DAPC, respectively), in the hippocampal neurons. Furthermore, both complexes were localized in interchromatin granule cluster structures (nuclear speckles) of neuronal nucleoskeleton preparations. The present study evinces that each Dp71's complexes differ slightly in dystrobrevins composition. The results demonstrated that Dp71d-DAPC was mainly localized in bipolar GABAergic and Dp71f-DAPC in multipolar Glutamatergic hippocampal neurons. Taken together, our results show that dystrophin 71d, dystrophin 71f and DAP integrate protein complexes, and both complexes were associated to nuclear speckles structures.

Finding the sweet spot: assembly and glycosylation of the dystrophin-associated glycoprotein complex.

The dystrophin-associated glycoprotein complex (DGC) is a collection of glycoproteins that are essential for the normal function of striated muscle and many other tissues. Recent genetic studies have implicated the components of this complex in over a dozen forms of muscular dystrophy. Furthermore, disruption of the DGC has been implicated in many forms of acquired disease. This review aims to summarize the current state of knowledge regarding the processing and assembly of dystrophin-associated proteins with a focus primarily on the dystroglycan heterodimer and the sarcoglycan complex. These proteins form the transmembrane portion of the DGC and undergo a complex multi-step processing with proteolytic cleavage, differential assembly, and both N- and O-glycosylation. The enzymes responsible for this processing and a model describing the sequence and subcellular localization of these events are discussed.

Ordered disorder of the astrocytic dystrophin-associated protein complex in the norm and pathology.

The abundance and potential functional roles of intrinsically disordered regions in aquaporin-4, Kir4.1, a dystrophin isoforms Dp71, α-1 syntrophin, and α-dystrobrevin; i.e., proteins constituting the functional core of the astrocytic dystrophin-associated protein complex (DAPC), are analyzed by a wealth of computational tools. The correlation between protein intrinsic disorder, single nucleotide polymorphisms (SNPs) and protein function is also studied together with the peculiarities of structural and functional conservation of these proteins. Our study revealed that the DAPC members are typical hybrid proteins that contain both ordered and intrinsically disordered regions. Both ordered and disordered regions are important for the stabilization of this complex. Many disordered binding regions of these five proteins are highly conserved among vertebrates. Conserved eukaryotic linear motifs and molecular recognition features found in the disordered regions of five protein constituting DAPC likely enhance protein-protein interactions that are required for the cellular functions of this complex. Curiously, the disorder-based binding regions are rarely affected by SNPs suggesting that these regions are crucial for the biological functions of their corresponding proteins.

Muscle ERRγ mitigates Duchenne muscular dystrophy via metabolic and angiogenic reprogramming.

Treatment of Duchenne muscular dystrophy (DMD) by replacing mutant dystrophin or restoring dystrophin-associated glycoprotein complex (DAG) has been clinically challenging. Instead, identifying and targeting muscle pathways deregulated in DMD will provide new therapeutic avenues. We report that the expression of nuclear receptor estrogen-related receptor-γ (ERRγ), and its metabolic and angiogenic targets are down-regulated (50-85%) in skeletal muscles of mdx mice (DMD model) vs. wild-type mice. Corelatively, oxidative myofibers, muscle vasculature, and exercise tolerance (33%) are decreased in mdx vs. wild-type mice. Overexpressing ERRγ selectively in the dystrophic muscles of the mdx mice restored metabolic and angiogenic gene expression compared with control mdx mice. Further, ERRγ enhanced muscle oxidative myofibers, vasculature, and blood flow (by 33-66%) and improved exercise tolerance (by 75%) in the dystrophic mice. Restoring muscle ERRγ pathway ameliorated muscle damage and also prevented DMD hallmarks of postexercise muscle damage, hypoxia, and fatigue in mdx mice. Notably, ERRγ did not restore sarcolemmal DAG complex, which is thus dispensable for antidystrophic effects of ERRγ. In summary, ERRγ-dependent metabolic and angiogenic gene program is defective in DMD, and we demonstrate that its restoration is a potential strategy for treating muscular dystrophy.

Histone deacetylase inhibitor BML-210 induces growth inhibition and apoptosis and regulates HDAC and DAPC complex expression levels in cervical cancer cells.

Histone deacetylase inhibitors (HDACIs) represent a new class of targeted anti-cancer agents and different other diseases, like muscular disorders. A number of studies have shown that extracellular signal-activated kinases can target chromatin-modifying complexes directly and regulate their function. The molecular connection between the dystrophin-associated protein complex (DAPC) and chromatin has been described, by showing that NO signaling regulates histone deacetylase (HDAC) activity and influences gene expression in different cell types. In present study, we investigated HDACs changes in HeLa cells undergoing growth inhibition and apoptosis, caused by HDACI BML-210 and retinoic acid (ATRA). Cell cycle analysis indicated that HeLa cell treatment with 20 and 30 µM concentration of BML-210 increased the proportion of cells in G0/G1 phase, and caused accumulation in subG1, indicating that the cells are undergoing apoptosis. We determined down-regulation of HDAC 1-5 and 7 after treatment with BML-210. Also, we demonstrated expression of different isoforms of alpha-dystrobrevin (α-DB) and other components of DAPC such as syntrophin, dystrophin, beta-dystrobrevin (ß-DB) and NOS in HeLa cells after treatments. We determined changes in protein expression level of dystrophin, NOS1, α- and ß-DB and in subcellular localization of α-DB after treatments with BML-210 and ATRA. In conclusion, these results suggest that HDACI BML-210 can inhibit cell growth and induce apoptosis in cervical cancer cells, what correlates with down-regulation of HDAC class I and II and changes in the DAPC expression levels. This can be important for identifying target proteins in DAPC signaling to HDACs, as a target of pharmacological intervention for treatment of muscular dystrophies and other diseases.

The dystrophin-glycoprotein complex in brain development and disease.

In addition to muscle disease, defects in processing and assembly of the dystrophin-glycoprotein complex (DGC) are associated with a spectrum of brain abnormalities ranging from mild cognitive impairment (MCI) to neuronal migration disorders. In brain, the DGC is involved in the organisation of GABA(A) receptors (GABA(A)Rs) and aquaporin-4 (AQP4)-containing protein complexes in neurons and glia, respectively. During development, defects in the glycosylation of α-dystroglycan that impair its ability to interact with the extracellular matrix (ECM) are frequently associated with cobblestone lissencephaly and mental retardation. Furthermore, mutations in the gene encoding ɛ-sarcoglycan (SGCE) cause the neurogenic movement disorder myoclonus dystonia syndrome. In this review, we describe recent progress in defining distinct roles for the DGC in neurons and glia.

The atypical calpains: evolutionary analyses and roles in Caenorhabditis elegans cellular degeneration.

The calpains are physiologically important Ca(2+)-activated regulatory proteases, which are divided into typical or atypical sub-families based on constituent domains. Both sub-families are present in mammals, but our understanding of calpain function is based primarily on typical sub-family members. Here, we take advantage of the model organism Caenorhabditis elegans, which expresses only atypical calpains, to extend our knowledge of the phylogenetic evolution and function of calpains. We provide evidence that a typical human calpain protein with a penta EF hand, detected using custom profile hidden Markov models, is conserved in ancient metazoans and a divergent clade. These analyses also provide evidence for the lineage-specific loss of typical calpain genes in C. elegans and Ciona, and they reveal that many calpain-like genes lack an intact catalytic triad. Given the association between the dysregulation of typical calpains and human degenerative pathologies, we explored the phenotypes, expression profiles, and consequences of inappropriate reduction or activation of C. elegans atypical calpains. These studies show that the atypical calpain gene, clp-1, contributes to muscle degeneration and reveal that clp-1 activity is sensitive to genetic manipulation of [Ca(2+)](i). We show that CLP-1 localizes to sarcomeric sub-structures, but is excluded from dense bodies (Z-disks). We find that the muscle degeneration observed in a C. elegans model of dystrophin-based muscular dystrophy can be suppressed by clp-1 inactivation and that nemadipine-A inhibition of the EGL-19 calcium channel reveals that Ca(2+) dysfunction underlies the C. elegans MyoD model of myopathy. Taken together, our analyses highlight the roles of calcium dysregulation and CLP-1 in muscle myopathies and suggest that the atypical calpains could retain conserved roles in myofilament turnover.

Progress in therapy for Duchenne muscular dystrophy.

Duchenne muscular dystrophy is a devastating muscular dystrophy of childhood. Mutations in the dystrophin gene destroy the link between the internal muscle filaments and the extracellular matrix, resulting in severe muscle weakness and progressive muscle wasting. There is currently no cure and, whilst palliative treatment has improved, affected boys are normally confined to a wheelchair by 12 years of age and die from respiratory or cardiac complications in their twenties or thirties. Therapies currently being developed include mutation-specific treatments, DNA- and cell-based therapies, and drugs which aim to modulate cellular pathways or gene expression. This review aims to provide an overview of the different therapeutic approaches aimed at reconstructing the dystrophin-associated protein complex, including restoration of dystrophin expression and upregulation of the functional homologue, utrophin.

The dystrophin-associated protein complex maintains muscle excitability by regulating Ca(2+)-dependent K(+) (BK) channel localization.

The dystrophin-associated protein complex (DAPC) consists of several transmembrane and intracellular scaffolding elements that have been implicated in maintaining the structure and morphology of the vertebrate neuromuscular junction (NMJ). Genetic linkage analysis has identified loss-of-function mutations in DAPC genes that give rise to degenerative muscular dystrophies. Although much is known about the involvement of the DAPC in maintaining muscle integrity, less is known about the precise contribution of the DAPC in cell signaling events. To better characterize the functional role of the DAPC at the NMJ, we used electrophysiology, immunohistochemistry, and fluorescent labeling to directly assess cholinergic synaptic transmission, ion channel localization, and muscle excitability in loss-of-function (lf) mutants of Caenorhabditis elegans DAPC homologues. We found that all DAPC mutants consistently display mislocalization of the Ca(2+)-gated K(+) channel, SLO-1, in muscle cells, while ionotropic acetylcholine receptor (AChR) expression and localization at the NMJ remained unaltered. Synaptic cholinergic signaling was also not significantly impacted across DAPC(lf) mutants. Consistent with these findings and the postsynaptic mislocalization of SLO-1, we observed an increase in muscle excitability downstream of cholinergic signaling. Based on our results, we conclude that the DAPC is not involved in regulating AChR architecture at the NMJ, but rather functions to control muscle excitability, in an activity-dependent manner, through the proper localization of SLO-1 channels.

Expression of dystrophins and the dystrophin-associated-protein complex by pituicytes in culture.

The dystrophin-associated-protein complex (DAPC) has been extensively characterized in the central nervous system where it is localized both in neuronal and glial cells. Few studies have characterized this complex in the neurohypophysis. To further study this complex in pituicytes, the resident astroglia of the neurophypophysis, we used adult pituicyte cultures and determined the expression and localization of dystrophins/utrophins and the DAPC by RT-PCR, western blotting and immunofluorescence. Our data show that the pituicytes express dystrophins, utrophins and several members of the DAPC including dystroglycans, δ-, γ-sarcoglycans, α-dystrobrevin-1 and α1-syntrophin. Double immunofluorescence analysis shows that laminin colocalizes with dystroglycan, suggesting that similarly to muscle and astrocytes, the DAPC interacts with the extracellular matrix in pituicytes. Collectively these findings show that dystrophins/utrophins and members of the DAPC are expressed in pituicytes where they may form multiprotein complexes and play a role in the retraction-reinsertion of pituicyte endfeet during specific physiological conditions.

Genetic defects in muscular dystrophy.

The muscular dystrophies are a group of neuromuscular disorders associated with muscle weakness and wasting, which in many forms can lead to loss of ambulation and premature death. A number of muscular dystrophies are associated with loss of proteins required for the maintenance of muscle membrane integrity, in particular with proteins that comprise the dystrophin-associated glycoprotein (DAG) complex. Proper glycosylation of O-linked mannose chains on alpha-dystroglycan, a DAG member, is required for the binding of the extracellular matrix to dystroglycan and for proper DAG function. A number of congenital disorders of glycosylation have now been described where alpha-dystroglycan glycosylation is altered and where muscular dystrophy is a predominant phenotype. Glycosylation is also increasingly being appreciated as a genetic modifier of disease phenotypes in many forms of muscular dystrophy and as a target for the development of new therapies. Here we will review the mouse models available for the study of this group of diseases and outline the methodologies required to describe disease phenotypes.

Regulation of TRPC1 and TRPC4 cation channels requires an alpha1-syntrophin-dependent complex in skeletal mouse myotubes.

The dystrophin-associated protein complex (DAPC) is essential for skeletal muscle, and the lack of dystrophin in Duchenne muscular dystrophy results in a reduction of DAPC components such as syntrophins and in fiber necrosis. By anchoring various molecules, the syntrophins may confer a role in cell signaling to the DAPC. Calcium disorders and abnormally elevated cation influx in dystrophic muscle cells have suggested that the DAPC regulates some sarcolemmal cationic channels. We demonstrated previously that mini-dystrophin and alpha1-syntrophin restore normal cation entry in dystrophin-deficient myotubes and that sarcolemmal TRPC1 channels associate with dystrophin and the bound PDZ domain of alpha1-syntrophin. This study shows that small interfering RNA (siRNA) silencing of alpha1-syntrophin dysregulated cation influx in myotubes. Moreover, deletion of the PDZ-containing domain prevented restoration of normal cation entry by alpha1-syntrophin transfection in dystrophin-deficient myotubes. TRPC1 and TRPC4 channels are expressed at the sarcolemma of muscle cells; forced expression or siRNA silencing showed that cation influx regulated by alpha1-syntrophin is supported by TRPC1 and TRPC4. A molecular association was found between TRPC1 and TRPC4 channels and the alpha1-syntrophin-dystrophin complex. TRPC1 and TRPC4 channels may form sarcolemmal channels anchored to the DAPC, and alpha1-syntrophin is necessary to maintain the normal regulation of TRPC-supported cation entry in skeletal muscle. Cation channels with DAPC form a signaling complex that modulates cation entry and may be crucial for normal calcium homeostasis in skeletal muscles.

Sarcoglycan complex: implications for metabolic defects in muscular dystrophies.

The sarcoglycans are known as an integral subcomplex of the dystrophin glycoprotein complex, the function of which is best characterized in skeletal muscle in relation to muscular dystrophies. Here we demonstrate that the white adipocytes, which share a common precursor with the myocytes, express a cell-specific sarcoglycan complex containing beta-, delta-, and epsilon-sarcoglycan. In addition, the adipose sarcoglycan complex associates with sarcospan and laminin binding dystroglycan. Using multiple sarcoglycan null mouse models, we show that loss of alpha-sarcoglycan has no consequence on the expression of the adipocyte sarcoglycan complex. However, loss of beta- or delta-sarcoglycan leads to a concomitant loss of the sarcoglycan complex as well as sarcospan and a dramatic reduction in dystroglycan in adipocytes. We further demonstrate that beta-sarcoglycan null mice, which lack the sarcoglycan complex in adipose tissue and skeletal muscle, are glucose-intolerant and exhibit whole body insulin resistance specifically due to impaired insulin-stimulated glucose uptake in skeletal muscles. Thus, our data demonstrate a novel function of the sarcoglycan complex in whole body glucose homeostasis and skeletal muscle metabolism, suggesting that the impairment of the skeletal muscle metabolism influences the pathogenesis of muscular dystrophy.

Dosing regimen has a significant impact on the efficiency of morpholino oligomer-induced exon skipping in mdx mice.

Duchenne muscular dystrophy (DMD) is a myodegenerative disorder caused primarily by mutations that create premature termination of dystrophin translation. The antisense oligonucleotide approach for skipping dystrophin exons allows restoration of the correct reading frame in the dystrophin transcript, thus producing a shorter protein. A similar approach in humans would result in the conversion of DMD to the milder Becker muscular dystrophy. It has been demonstrated previously that repeated intravascular injection of phosphorodiamidate morpholino oligomers (PMOs) in the mdx mouse induces more dystrophin expression than a single injection, but this approach is costly, and data demonstrating the safety of high doses of systemically injected PMO are unavailable. Furthermore, several publications have demonstrated the efficacy of peptide-conjugated PMOs, but the clinical applicability of such compounds is unclear at this stage. Here, we report that multiple intravascular injections of low doses of naked PMO show significantly more dystrophin-positive fibers in a variety of muscle groups, 8 weeks after administration compared with a single dose of the same total amount. After administration of a total of 200 mg of PMO per kilogram, histological features, such as the cross-sectional area, centronucleation index, and expression of the dystrophin-associated protein complex, showed significant improvement in mice treated by repeated injection. Furthermore, four administrations of just 5 mg/kg induced a significant amount of dystrophin expression. These results clearly demonstrate the key role of the optimization of dosing regimen for the systemic administration of PMO in patients, and support the clinical feasibility of this approach with naked PMO.

Overexpression of Myostatin2 in zebrafish reduces the expression of dystrophin associated protein complex (DAPC) which leads to muscle dystrophy.

Myostatin, a member of the TGF-beta superfamily, is a potent negative regulator of skeletal muscle and growth. Previously, we reported Mstn1 from zebrafish and studied its influence on muscle development. In this study, we identified another form of Myostatin protein which is referred to as Mstn2. The size of Mstn2 cDNA is 1342 bp with 109 and 132 bp of 5' and 3'-untranslated regions (UTRs), respectively. The coding region is 1101 bp encoding 367 amino acids. The identity between zebrafish Mstn1 and 2 is 66%. The phylogenetic tree revealed that the Mstn2 is an ancestral form of Mstn1. To study the functional aspects, we overexpressed mstn2 and noticed that embryos became less active and the juveniles with bent and curved phenotypes when compared to the control. The RT-PCR and in situ hybridization showed concurrent reduction of dystrophin associated protein complex (DAPC). In cryosection and in situ hybridization, we observed the disintegration of somites, lack of transverse myoseptum and loss of muscle integrity due to the failure of muscle attachment in mstn2 overexpressed embryos. Immunohistochemistry and western blot showed that there was a reduction of dystrophin, dystroglycan and sarcoglycan at translational level in overexpressed embryos. Taken together, these results indicate the suitability of zebrafish as an excellent animal model and our data provide the first in vivo evidence of muscle attachment failure by the overexpression of mstn2 and it leads to muscle loss which results in muscle dystrophy that may contribute to Duchenne syndrome and other muscle related diseases.

A single gene network accurately predicts phenotypic effects of gene perturbation in Caenorhabditis elegans.

The fundamental aim of genetics is to understand how an organism's phenotype is determined by its genotype, and implicit in this is predicting how changes in DNA sequence alter phenotypes. A single network covering all the genes of an organism might guide such predictions down to the level of individual cells and tissues. To validate this approach, we computationally generated a network covering most C. elegans genes and tested its predictive capacity. Connectivity within this network predicts essentiality, identifying this relationship as an evolutionarily conserved biological principle. Critically, the network makes tissue-specific predictions-we accurately identify genes for most systematically assayed loss-of-function phenotypes, which span diverse cellular and developmental processes. Using the network, we identify 16 genes whose inactivation suppresses defects in the retinoblastoma tumor suppressor pathway, and we successfully predict that the dystrophin complex modulates EGF signaling. We conclude that an analogous network for human genes might be similarly predictive and thus facilitate identification of disease genes and rational therapeutic targets.