Dystroglycan is involved in the activation of ERK pathway inducing the change of AQP4 expression in scratch-injured astrocytes.

We previously reported that aquaporin 4 (AQP4) played a critical role in formation of brain edema and the altered expression of dystroglycan (DG) could relate with AQP4 expression after traumatic brain injury (TBI). However the mechanisms of this process remain unclear. DG was showed could act as a scaffold involved in adhesion-mediated signaling in ERK/MAPK pathway. We hypothesize that after scratch, extracellular α-DG and transmembrane ß-DG may act as the scaffold in scratch mechanical force activating ERK pathway which may regulate the expression of AQP4. Use ERK inhibitor and activator to confirm whether the expression of AQP4 is regulated by the activation of ERK pathway in scratched astrocytes. Use DG siRNA to confirm whether DG takes part in the process that the extracellular signal transduces into cell and activates the ERK pathway. The significant increase of AQP4 and DG expression induced by scratch could be abolished by blocking ERK signaling and enhanced by activating ERK signaling. Blockade of DG by siRNA led to no obvious effect of scratched-injury on the ERK signaling pathway. It demonstrated that DG may act as the scaffold in scratch mechanical force activating ERK pathway which can regulate the expression of AQP4 in astrocytes after scratch.

Retinal ganglion cell axon sorting at the optic chiasm requires dystroglycan.

In the developing visual system, retinal ganglion cell (RGC) axons project from the retina to several distal retinorecipient regions in the brain. Several molecules have been implicated in guiding RGC axons in vivo, but the role of extracellular matrix molecules in this process remains poorly understood. Dystroglycan is a laminin-binding transmembrane protein important for formation and maintenance of the extracellular matrix and basement membranes and has previously been implicated in axon guidance in the developing spinal cord. Using two genetic models of functional dystroglycan loss, we show that dystroglycan is necessary for correct sorting of contralateral and ipsilateral RGC axons at the optic chiasm. Mis-sorted axons still target retinorecipient brain regions and persist in adult mice, even after axon pruning is complete. Our results highlight the importance of the extracellular matrix for axon sorting at an intermediate choice point in the developing visual circuit.

Neuronal Dystroglycan Is Necessary for Formation and Maintenance of Functional CCK-Positive Basket Cell Terminals on Pyramidal Cells.

Distinct types of GABAergic interneurons target different subcellular domains of pyramidal cells, thereby shaping pyramidal cell activity patterns. Whether the presynaptic heterogeneity of GABAergic innervation is mirrored by specific postsynaptic factors is largely unexplored. Here we show that dystroglycan, a protein responsible for the majority of congenital muscular dystrophies when dysfunctional, has a function at postsynaptic sites restricted to a subset of GABAergic interneurons. Conditional deletion of Dag1, encoding dystroglycan, in pyramidal cells caused loss of CCK-positive basket cell terminals in hippocampus and neocortex. PV-positive basket cell terminals were unaffected in mutant mice, demonstrating interneuron subtype-specific function of dystroglycan. Loss of dystroglycan in pyramidal cells had little influence on clustering of other GABAergic postsynaptic proteins and of glutamatergic synaptic proteins. CCK-positive terminals were not established at P21 in the absence of dystroglycan and were markedly reduced when dystroglycan was ablated in adult mice, suggesting a role for dystroglycan in both formation and maintenance of CCK-positive terminals. The necessity of neuronal dystroglycan for functional innervation by CCK-positive basket cell axon terminals was confirmed by reduced frequency of inhibitory events in pyramidal cells of dystroglycan-deficient mice and further corroborated by the inefficiency of carbachol to increase IPSC frequency in these cells. Finally, neurexin binding seems dispensable for dystroglycan function because knock-in mice expressing binding-deficient T190M dystroglycan displayed normal CCK-positive terminals. Together, we describe a novel function of dystroglycan in interneuron subtype-specific trans-synaptic signaling, revealing correlation of presynaptic and postsynaptic molecular diversity. SIGNIFICANCE STATEMENT: Dystroglycan, an extracellular and transmembrane protein of the dystrophin-glycoprotein complex, is at the center of molecular studies of muscular dystrophies. Although its synaptic distribution in cortical brain regions is long established, function of dystroglycan in the synapse remained obscure. Using mice that selectively lack neuronal dystroglycan, we provide evidence that a subset of GABAergic interneurons requires dystroglycan for formation and maintenance of axonal terminals on pyramidal cells. As such, dystroglycan is the first postsynaptic GABAergic protein for which an interneuron terminal-specific function could be shown. Our findings also offer a new perspective on the mechanisms that lead to intellectual disability in muscular dystrophies without associated brain malformations.

Hepatocyte Growth Factor and Satellite Cell Activation.

Satellite cells are the "currency" for the muscle growth that is critical to meat production in many species, as well as to phenotypic distinctions in development at the level of species or taxa, and for human muscle growth, function and regeneration. Careful research on the activation and behaviour of satellite cells, the stem cells in skeletal muscle, including cross-species comparisons, has potential to reveal the mechanisms underlying pathological conditions in animals and humans, and to anticipate implications of development, evolution and environmental change on muscle function and animal performance.

Agrin mediates chondrocyte homeostasis and requires both LRP4 and α-dystroglycan to enhance cartilage formation in vitro and in vivo.

OBJECTIVES: Osteoarthritis (OA) is a leading cause of disability for which there is no cure. The identification of molecules supporting cartilage homeostasis and regeneration is therefore a major pursuit in musculoskeletal medicine. Agrin is a heparan sulfate proteoglycan which, through binding to low-density lipoprotein receptor-related protein 4 (LRP4), is required for neuromuscular synapse formation. In other tissues, it connects the cytoskeleton to the basement membrane through binding to α-dystroglycan. Prompted by an unexpected expression pattern, we investigated the role and receptor usage of agrin in cartilage. METHODS: Agrin expression pattern was investigated in human osteoarthritic cartilage and following destabilisation of the medial meniscus in mice. Extracellular matrix (ECM) formation and chondrocyte differentiation was studied in gain and loss of function experiments in vitro in three-dimensional cultures and gain of function in vivo, using an ectopic cartilage formation assay in nude mice. Receptor usage was investigated by disrupting LRP4 and α-dystroglycan by siRNA and blocking antibodies respectively. RESULTS: Agrin was detected in normal cartilage but was progressively lost in OA. In vitro, agrin knockdown resulted in reduced glycosaminoglycan content, downregulation of the cartilage transcription factor SOX9 and other cartilage-specific ECM molecules. Conversely, exogenous agrin supported cartilage differentiation in vitro and ectopic cartilage formation in vivo. In the context of cartilage differentiation, agrin used an unusual receptor repertoire requiring both LRP4 and α-dystroglycan. CONCLUSIONS: We have discovered that agrin strongly promotes chondrocyte differentiation and cartilage formation in vivo. Our results identify agrin as a novel potent anabolic growth factor with strong therapeutic potential in cartilage regeneration.

The extracellular matrix protein laminin α2 regulates the maturation and function of the blood-brain barrier.

Laminins are major constituents of the gliovascular basal lamina of the blood-brain barrier (BBB); however, the role of laminins in BBB development remains unclear. Here we report that Lama2(-/-) mice, lacking expression of the laminin α2 subunit of the laminin-211 heterotrimer expressed by astrocytes and pericytes, have a defective BBB in which systemically circulated tracer leaks into the brain parenchyma. The Lama2(-/-) vascular endothelium had significant abnormalities, including altered integrity and composition of the endothelial basal lamina, inappropriate expression of embryonic vascular endothelial protein MECA32, substantially reduced pericyte coverage, and tight junction abnormalities. Additionally, astrocytic endfeet were hypertrophic and lacked appropriately polarized aquaporin4 channels. Laminin-211 appears to mediate these effects at least in part by dystroglycan receptor interactions, as preventing dystroglycan expression in neural cells led to a similar set of BBB abnormalities and gliovascular disturbances, which additionally included perturbed vascular endothelial glucose transporter-1 localization. These findings provide insight into the cell and molecular changes that occur in congenital muscular dystrophies caused by Lama2 mutations or inappropriate dystroglycan post-translational modifications, which have accompanying brain abnormalities, including seizures. Our results indicate a novel role for laminin-dystroglycan interactions in the cooperative integration of astrocytes, endothelial cells, and pericytes in regulating the BBB.

Dystroglycan depletion inhibits the functions of differentiated HL-60 cells.

Dystroglycan has recently been characterized in blood tissue cells, as part of the dystrophin glycoprotein complex but to date nothing is known of its role in the differentiation process of neutrophils. We have investigated the role of dystroglycan in the human promyelocytic leukemic cell line HL-60 differentiated to neutrophils. Depletion of dystroglycan by RNAi resulted in altered morphology and reduced properties of differentiated HL-60 cells, including chemotaxis, respiratory burst, phagocytic activities and expression of markers of differentiation. These findings strongly implicate dystroglycan as a key membrane adhesion protein involved in the differentiation process in HL-60 cells.

Mechanical and non-mechanical functions of Dystrophin can prevent cardiac abnormalities in Drosophila.

Dystrophin-deficiency causes cardiomyopathies and shortens the life expectancy of Duchenne and Becker muscular dystrophy patients. Restoring Dystrophin expression in the heart by gene transfer is a promising avenue to explore as a therapy. Truncated Dystrophin gene constructs have been engineered and shown to alleviate dystrophic skeletal muscle disease, but their potential in preventing the development of cardiomyopathy is not fully understood. In the present study, we found that either the mechanical or the signaling functions of Dystrophin were able to reduce the dilated heart phenotype of Dystrophin mutants in a Drosophila model. Our data suggest that Dystrophin retains some function in fly cardiomyocytes in the absence of a predicted mechanical link to the cytoskeleton. Interestingly, cardiac-specific manipulation of nitric oxide synthase expression also modulates cardiac function, which can in part be reversed by loss of Dystrophin function, further implying a signaling role of Dystrophin in the heart. These findings suggest that the signaling functions of Dystrophin protein are able to ameliorate the dilated cardiomyopathy, and thus might help to improve heart muscle function in micro-Dystrophin-based gene therapy approaches.

Dystroglycan function is a novel determinant of tumor growth and behavior in prostate cancer.

BACKGROUND: Dystroglycan is a ubiquitously expressed cell adhesion molecule frequently found to be altered or reduced in adenocarcinomas, however the mechanisms or consequences of dystroglycan loss have not been studied extensively. METHODS: We examined the consequence of overexpression or RNAi depletion of dystroglycan on properties of in vitro growth migration and invasion of LNCaP, PC3, and DU145 prostate cancer cell lines. RESULTS: Using LNCaP cells we observed cell density-dependent changes in ß-dystroglycan with the appearance of several lower molecular weight species ranging in size from 43 to 26 kDa. The bands of 31 and 26 kDa were attributed to proteolysis, whereas bands between 43 and 38 kDa were a consequence of mis-glycosylation. The localization of ß-dystroglycan in LNCaP colonies in culture also varied, cells with a mesenchymal appearance at the periphery of the colony had more pronounced membrane localization of dystroglycan. Whereas some cells demonstrated nuclear dystroglycan. Increased dystroglycan levels were inhibitory to growth in soft agar but promoted Matrigel invasion, whereas reduced dystroglycan levels promoted growth in soft agar but inhibited invasion. Similar results were also obtained for PC3 and DU145 cells. CONCLUSIONS: This study suggests that changes in ß-dystroglycan distribution within the cell and/or the loss of dystroglycan during tumorigenesis, through a combination of proteolysis and altered glycosylation, leads to an increased ability to grow in an anchorage independent manner, however dystroglycan may need to be re-expressed for cell invasion and metastasis to occur.

Loss of LARGE2 disrupts functional glycosylation of α-dystroglycan in prostate cancer.

Dystroglycan (DG) is a cell surface receptor for extracellular matrix proteins and is involved in cell polarity, matrix organization, and mechanical stability of tissues. Previous studies documented loss of DG protein expression and glycosylation in a variety of cancer types, but the underlying mechanisms and the functional consequences with respect to cancer progression remain unclear. Here, we show that the level of expression of the ßDG subunit as well as the glycosylation status of the αDG subunit inversely correlate with the Gleason scores of prostate cancers; furthermore, we show that the functional glycosylation of αDG is substantially reduced in prostate cancer metastases. Additionally, we demonstrate that LARGE2 (GYLTL1B), a gene not previously implicated in cancer, regulates functional αDG glycosylation in prostate cancer cell lines; knockdown of LARGE2 resulted in hypoglycosylation of αDG and loss of its ability to bind laminin-111 while overexpression restored ligand binding and diminished growth and migration of an aggressive prostate cancer cell line. Finally, our analysis of LARGE2 expression in human cancer specimens reveals that LARGE2 is significantly down-regulated in the context of prostate cancer, and that its reduction correlates with disease progression. Our results describe a novel molecular mechanism to account for the commonly observed hypoglycosylation of αDG in prostate cancer.

Dystroglycan organizes axon guidance cue localization and axonal pathfinding.

Precise patterning of axon guidance cue distribution is critical for nervous system development. Using a murine forward genetic screen for novel determinants of axon guidance, we identified B3gnt1 and ISPD as required for the glycosylation of dystroglycan in vivo. Analysis of B3gnt1, ISPD, and dystroglycan mutant mice revealed a critical role for glycosylated dystroglycan in the development of several longitudinal axon tracts. Remarkably, the axonal guidance defects observed in B3gnt1, ISPD, and dystroglycan mutants resemble several of the axon guidance defects found in mice lacking the axon guidance cue Slit and its receptor Robo. This similarity is explained by our observations that dystroglycan binds directly to Slit and is required for proper Slit localization within the basement membrane and floor plate in vivo. These findings establish a novel role for glycosylated dystroglycan as a key determinant of axon guidance cue distribution and function in the mammalian nervous system.

Drp2 and periaxin form Cajal bands with dystroglycan but have distinct roles in Schwann cell growth.

Cajal bands are cytoplasmic channels flanked by appositions where the abaxonal surface of Schwann cell myelin apposes and adheres to the overlying plasma membrane. These appositions contain a dystroglycan complex that includes periaxin and dystrophin-related protein 2 (Drp2). Loss of periaxin disrupts appositions and Cajal bands in Schwann cells and causes a severe demyelinating neuropathy in mouse and human. Here, we investigated the role of mouse Drp2 in apposition assembly and Cajal band function and compared it with periaxin. We show that periaxin and Drp2 are not only both required to form appositions, but they must also interact. Periaxin-Drp2 interaction is also required for Drp2 phosphorylation, but phosphorylation is not required for the assembly of appositions. Drp2 loss causes corresponding increases in Dystrophin family members, utrophin and dystrophin Dp116, although dystroglycan remains unchanged. We also show that all dystroglycan complexes in Schwann cells use the uncleaved form of ß-dystroglycan. Drp2-null Schwann cells have disrupted appositions and Cajal bands, and they undergo focal hypermyelination and concomitant demyelination. Nevertheless, they do not have the short internodal lengths and associated reduced nerve conduction velocity seen in the absence of periaxin, showing that periaxin regulates Schwann cell elongation independent of its role in the dystroglycan complex. We conclude that the primary role of the dystroglycan complex in appositions is to stabilize and limit the radial growth of myelin.

Cell-matrix interactions in muscle disease.

The extracellular matrix (ECM) provides a solid scaffold and signals to cells through ECM receptors. The cell-matrix interactions are crucial for normal biological processes and when disrupted they may lead to pathological processes. In particular, the biological importance of ECM-cell membrane-cytoskeleton interactions in skeletal muscle is accentuated by the number of inherited muscle diseases caused by mutations in proteins conferring these interactions. In this review we introduce laminins, collagens, dystroglycan, integrins, dystrophin and sarcoglycans. Mutations in corresponding genes cause various forms of muscular dystrophy. The muscle disorders are presented as well as advances toward the development of treatment.

Interstitial guidance of cancer invasion.

Cancer cell invasion into healthy tissues develops preferentially along pre-existing tracks of least resistance, followed by secondary tissue remodelling and destruction. The tissue scaffolds supporting or preventing guidance of invasion vary in structure and molecular composition between organs. In the brain, the guidance is provided by myelinated axons, astrocyte processes, and blood vessels which are used as invasion routes by glioma cells. In the human breast, containing interstitial collagen-rich connective tissue, disseminating breast cancer cells preferentially invade along bundled collagen fibrils and the surface of adipocytes. In both invasion types, physical guidance prompted by interfaces and space is complemented by molecular guidance. Generic mechanisms shared by most, if not all, tissues include (i) guidance by integrins towards fibrillar interstitial collagen and/or laminins and type IV collagen in basement membranes decorating vessels and adipocytes, and, likely, CD44 engaging with hyaluronan; (ii) haptotactic guidance by chemokines and growth factors; and likely (iii) physical pushing mechanisms. Tissue-specific, resticted guidance cues include ECM proteins with restricted expression (tenascins, lecticans), cell-cell interfaces, and newly secreted matrix molecules decorating ECM fibres (laminin-332, thrombospondin-1, osteopontin, periostin). We here review physical and molecular guidance mechanisms in interstitial tissue and brain parenchyma and explore shared principles and organ-specific differences, and their implications for experimental model design and therapeutic targeting of tumour cell invasion.

[Recent Advances in α-dystroglycanopathy].

Fukuyama-type congenital muscular dystrophy (FCMD), muscle-eye-brain disease (MEB), and Walker-Warburg syndrome (WWS) are autosomal recessive disorders characterized by congenital muscular dystrophy with structural brain and eye abnormalities. Aberrant glycosylation of α-dystroglycan (α-DG) is a common pathomechanism of these disorders. In addition, genetic and glycobiological evidence has shown that abnormal glycosylation of α-DG is also seen in several forms of congenital and limb-girdle-type muscular dystrophies. These disorders are collectively called "α-dystroglycanopathy" and nowadays, this term is widely accepted because it is useful for illustrating a complicated genotype-phenotype correlation of these disorders. α-DG is a membrane-associated protein that interacts with extracellular matrix proteins such as laminin, and abnormal glycosylation of α-DG results in loss of its laminin-binding activity. A number of serine/threonine residues are present in the mucin-like domain of α-DG and are majorly composed of sugar chains. Among these glycans, an O-mannosyl tetrasaccharide (Neu5Ac-α2,3-Gal-ß1,4-GlcNAc-ß1,2-Man) is important for laminin-binding activity of α-DG. POMT1/2 and POMGnT1, protein products of causative genes of WWS and MEB, respectively, are enzymes that directly catalyze the biosynthesis of this glycan. Recent studies have suggested that a phosphodiester-linked structure on O-mannose is also important for the laminin-binding activity and that mutations in other causative genes of α-dystroglycanopathy, such as fukutin (originally identified as the gene responsible for FCMD) and LARGE, disrupt the post-phosphoryl structure. Here, we review the history of basic and clinical research on α-dystroglycanopathy and refine its clinical spectrum, which should be broadly extended. In addition, we reveal some progress in research on α-dystroglycanopathy including a novel disease mechanism and anti-sense oligonucleotide therapy for FCMD.

Age-related changes in dystrophin-glycoprotein complex and in utrophin are not correlated with intrinsic laryngeal muscles protection in mdx mice.

In this study we investigate whether dystrophic intrinsic laryngeal muscles (ILM) from aged mdx mice show alterations in dystrophin-glycoprotein complex (DGC) components.Immunofluorescence and immunoblotting analyses of beta-sarcoglycan, beta-dystroglycan, and utrophin showed that aged ILM had a similar pattern of changes in aged affected muscles (diaphragm and limb), suggesting that aging leads to changes in utrophin and DGC proteins in dystrophic ILM that cannot be correlated with their protection from dystrophic change.

New dystrophin/dystroglycan interactors control neuron behavior in Drosophila eye.

BACKGROUND: The Dystrophin Glycoprotein Complex (DGC) is a large multi-component complex that is well known for its function in muscle tissue. When the main components of the DGC, Dystrophin (Dys) and Dystroglycan (Dg) are affected cognitive impairment and mental retardation in addition to muscle degeneration can occur. Previously we performed an array of genetic screens using a Drosophila model for muscular dystrophy in order to find novel DGC interactors aiming to elucidate the signaling role(s) in which the complex is involved. Since the function of the DGC in the brain and nervous system has not been fully defined, we have here continued to analyze the DGC modifiers' function in the developing Drosophila brain and eye. RESULTS: Given that disruption of Dys and Dg leads to improper photoreceptor axon projections into the lamina and eye neuron elongation defects during development, we have determined the function of previously screened components and their genetic interaction with the DGC in this tissue. Our study first found that mutations in chif, CG34400, Nrk, Lis1, capt and Cam cause improper axon path-finding and loss of SP2353, Grh, Nrk, capt, CG34400, vimar, Lis1 and Cam cause shortened rhabdomere lengths. We determined that Nrk, mbl, capt and Cam genetically interact with Dys and/or Dg in these processes. It is notable that most of the neuronal DGC interacting components encountered are involved in regulation of actin dynamics. CONCLUSIONS: Our data indicate possible DGC involvement in the process of cytoskeletal remodeling in neurons. The identification of new components that interact with the DGC not only helps to dissect the mechanism of axon guidance and eye neuron differentiation but also provides a great opportunity for understanding the signaling mechanisms by which the cell surface receptor Dg communicates via Dys with the actin cytoskeleton.

Non-redundant function of dystroglycan and ß1 integrins in radial sorting of axons.

Radial sorting allows the segregation of axons by a single Schwann cell (SC) and is a prerequisite for myelination during peripheral nerve development. Radial sorting is impaired in models of human diseases, congenital muscular dystrophy (MDC) 1A, MDC1D and Fukuyama, owing to loss-of-function mutations in the genes coding for laminin α2, Large or fukutin glycosyltransferases, respectively. It is not clear which receptor(s) are activated by laminin 211, or glycosylated by Large and fukutin during sorting. Candidates are αß1 integrins, because their absence phenocopies laminin and glycosyltransferase deficiency, but the topography of the phenotypes is different and ß1 integrins are not substrates for Large and fukutin. By contrast, deletion of the Large and fukutin substrate dystroglycan does not result in radial sorting defects. Here, we show that absence of dystroglycan in a specific genetic background causes sorting defects with topography identical to that of laminin 211 mutants, and recapitulating the MDC1A, MDC1D and Fukuyama phenotypes. By epistasis studies in mice lacking one or both receptors in SCs, we show that only absence of ß1 integrins impairs proliferation and survival, and arrests radial sorting at early stages, that ß1 integrins and dystroglycan activate different pathways, and that the absence of both molecules is synergistic. Thus, the function of dystroglycan and ß1 integrins is not redundant, but is sequential. These data identify dystroglycan as a functional laminin 211 receptor during axonal sorting and the key substrate relevant to the pathogenesis of glycosyltransferase congenital muscular dystrophies.

The role of dystroglycan in PDGF-BB-dependent migration of activated hepatic stellate cells/myofibroblasts.

Hepatic stellate cells are embedded in the loose connective tissue matrix within the space of Disse. This extracellular matrix contains several basement membrane components including laminin, but its composition changes during liver injury because of the production of extracellular matrix components found in scar tissue. These changes in extracellular matrix composition and in cell-extracellular matrix interactions may play a key role in hepatic stellate cell transdifferentiation. In this communication we used early passages of mouse hepatic stellate cells (activated HSC/myofibroblasts) to study the platelet-derived growth factor BB (PDGF-BB)-dependent expression and regulation of ß-dystroglycan and its role in activated HSC/myofibroblast migration. We used Northern and Western analysis to study dystroglycan expression and confocal microscopy to investigate changes in subcellular distribution of the protein. Activated HSC migration was investigated using an in vitro wound-healing assay. PDGF-BB induced significant changes in dystroglycan regulation and subcellular distribution of the protein. Whereas steady-state levels of dystroglycan mRNA remained constant, PDGF-BB increased dystroglycan transcription but shortened the t(1/2) by 50%. Moreover, PDGF-BB changed dystroglycan and α5-integrin cellular distribution. Cell migration experiments revealed that PDGF-BB-dependent migration of activated HSC/myofibroblasts was completely blocked by neutralizing antibodies to fibronectin, α5-integrin, laminin, and ß-dystroglycan. Overall, these findings suggest that both laminin and fibronectin and their receptors play a key role in PDGF-BB-induced activated HSC migration.