Hakima Moukhles

Chimaeric mice deficient in dystroglycans develop muscular dystrophy and have disrupted myoneural synapses

Mutations in the dystrophin gene (DMD) and in genes encoding several dystrophin-associated proteins result in Duchenne and other forms of muscular dystrophy. α-Dystroglycan (Dg) binds to laminins in the basement membrane surrounding each myofibre and docks with β-Dg, a transmembrane protein, which in turn interacts with dystrophin or utrophin in the subplasmalemmal cytoskeleton. α- and β-Dgs are thought to form the functional core of a larger complex of proteins extending from the basement membrane to the intracellular cytoskeleton, which serves as a superstructure necessary for sarcolemmal integrity. Dgs have also been implicated in the formation of synaptic densities of acetylcholine receptors (AChRs) on skeletal muscle. Here we report that chimaeric mice generated with ES cells targeted for both Dg alleles have skeletal muscles essentially devoid of Dgs and develop a progressive muscle pathology with changes emblematic of muscular dystrophies in humans. In addition, many neuromuscular junctions are disrupted in these mice. The ultrastructure of basement membranes and the deposition of laminin within them, however, appears unaffected in Dg-deficient muscles. We conclude that Dgs are necessary for myofibre survival and synapse differentiation or stability, but not for the formation of the muscle basement membrane, and that Dgs may have more than a purely structural function in maintaining muscle integrity.

Laminin-induced aggregation of the inwardly rectifying potassium channel, Kir4.1, and the water-permeable channel, AQP4, via a dystroglycan-containing complex in astrocytes

Dystroglycan (DG) is part of a multiprotein complex that links the extracellular matrix to the actin cytoskeleton of muscle fibers and that is involved in aggregating acetylcholine receptors at the neuromuscular junction. This complex is also expressed in regions of the central nervous system where it is localized to both neuronal and glial cells. DG and the inwardly rectifying potassium channels, Kir4.1, are concentrated at the interface of astroglia and small blood vessels. These channels are involved in siphoning potassium released into the extracellular space after neuronal excitation. This raises the possibility that DG may be involved in targeting Kir4.1 channels to specific domains of astroglia. To address this question, we used mixed hippocampal cultures to investigate the distribution of DG, syntrophin, dystrobrevin, and Kir4.1 channels, as well as aquaporin‐permeable water channels, AQP4. These proteins exhibit a similar distribution pattern and form aggregates in astrocytes cultured on laminin. Both DG and syntrophin colocalize with Kir4.1 channel aggregates in astrocytes. Similarly, DG colocalizes with AQP4 channel aggregates. Quantitative studies show a significant increase of Kir4.1 and AQP4 channel aggregates in astrocytes cultured in the presence of laminin when compared with those in the absence of laminin. These findings show that laminin has a role in Kir4.1 and AQP4 channel aggregation and suggest that this may be mediated via a dystroglycan‐containing complex. This study reveals a novel functional role for DG in brain including K+ buffering and water homeostasis.

Dystroglycan Is Not Required for Localization of Dystrophin, Syntrophin, and Neuronal Nitric-oxide Synthase at the Sarcolemma but Regulates Integrin α7B Expression and Caveolin-3 Distribution

Dystroglycan is part of the dystrophin-associated protein complex, which joins laminin in the extracellular matrix to dystrophin within the subsarcolemmal cytoskeleton. We have investigated how mutations in the components of the laminin-dystroglycan-dystrophin axis affect the organization and expression of dystrophin-associated proteins by comparing mice mutant for merosin (α2-laminin, dy), dystrophin (mdx), and dystroglycan (Dag1) using immunohistochemistry and immunoblots. We report that syntrophin and neuronal nitric-oxide synthase are depleted in muscle fibers lacking both dystrophin and dystroglycan. Some fibers deficient in dystroglycan, however, localize dystrophin at the cell surface at levels similar to that in wild-type muscle. Nevertheless, these fibers have signs of degeneration/regeneration including increased cell surface permeability and central nuclei. In these fibers, syntrophin and nitric-oxide synthase are also localized to the plasma membrane, whereas the sarcoglycan complex is disrupted. These results suggest a mechanism of membrane attachment for dystrophin independent of dystroglycan and that the interaction of sarcoglycans with dystrophin requires dystroglycan. The distribution of caveolin-3, a muscle-specific component of caveolae recently found to bind dystroglycan, was affected in dystroglycan- and dystrophin-deficient mice. We also examined alternative mechanisms of cell-extracellular matrix attachment to elucidate how the muscle basement membrane may subsist in the absence of dystroglycan, and we found the α7B splice variant of the α7 integrin receptor subunit to be up-regulated. These results support the possibility that α7B integrin compensates in mediating cell-extracellular matrix attachment but cannot rescue the dystrophic phenotype.

Dystroglycan contributes to the formation of multiple dystrophin-like complexes in brain

In muscle, dystrophin anchors a complex of proteins at the cell surface which includes α‐dystroglycan, β‐dystroglycan, syntrophins and dystrobrevins. Mutations in the dystrophin gene lead to muscular dystrophy and mental retardation. In contrast to muscle, little is known about the localization and the molecular interactions of dystrophin and dystrophin associated proteins (DAPs) in brain. In the present study, we show that α‐dystroglycan and dystrophin are localized to large neurones in cerebral cortex, hippocampus, cerebellum and spinal cord. Furthermore, we show that dystroglycan is a member of three distinct dystrophin‐containing complexes. Two of these complexes contain syntrophin and both dystrophin and syntrophin are enriched in post‐synaptic densities. These data suggest that dystrophin and DAPs may have a role in the organization of CNS synapses. Interestingly, the enrichment for syntrophin in post‐synaptic densities is not affected in mice mutant for all dystrophin isoforms. Thus in the brain, unlike in muscle, the association of syntrophin with dystrophin is not crucial for the DAP complex which suggests that it may be associated with other proteins.

Neural regulation of α-dystroglycan biosynthesis and glycosylation in skeletal muscle

Abstract:α‐Dystroglycan (α‐DG) is part of a complex of cell surface proteins linked to dystrophin or utrophin, which is distributed over the myofiber surface and is concentrated at neuromuscular junctions. In laminin overlays of muscle extracts from developing chick hindlimb muscle, α‐DG first appears at embryonic day (E) 10 with an apparent molecular mass of 120 kDa. By E15 it is replaced by smaller (∼100 kDa) and larger (∼150 kDa) isoforms. The larger form increases in amount and in molecular mass (>200 kDa) as the muscle is innervated and the postsynaptic membrane differentiates (E10‐E20), and then decreases dramatically in amount after hatching. In myoblasts differentiating in culture the molecular mass of α‐DG is not significantly increased by their replication, fusion, or differentiation into myotubes. Monoclonal antibody IIH6, which recognizes a carbohydrate epitope on α‐DG, preferentially binds to the larger forms, suggesting that the core protein is differentially glycosylated beginning at E16. Consistent with prior observations implicating the IIH6 epitope in laminin binding, the smaller forms of α‐DG bind more weakly to laminin affinity columns than the larger ones. In blots of adult rat skeletal muscle probed with radiolabeled laminin or monoclonal antibody IIH6, α‐DG appears as a >200‐kDa band that decreases in molecular mass but increases in intensity following denervation. Northern blots reveal a single mRNA transcript, indicating that the reduction in molecular mass of α‐DG after denervation is not obviously a result of alternative splicing but is likely due to posttranslational modification of newly synthesized molecules. The regulation of α‐DG by the nerve and its increased affinity for laminin suggest that glycosylation of this protein may be important in myofiber‐basement membrane interactions during development and after denervation.

Dystroglycan and Kir4.1 coclustering in retinal Müller glia is regulated by laminin-1 and requires the PDZ-ligand domain of Kir4.1.

Inwardly rectifying potassium (Kir) channels in Müller glia play a critical role in the spatial buffering of potassium ions that accumulate during retinal activity. To this end, Kir channels show a polarized subcellular distribution with the predominant channel subunit in Müller glia, Kir4.1, clustered in the endfeet of these cells at the inner limiting membrane. However, the molecular mechanisms underlying their distribution have yet to be identified. Here, we show that laminin, agrin and α‐dystroglycan (DG) codistribute with Kir4.1 at the inner limiting membrane in the retina and that laminin‐1 induces the clustering of α‐DG, syntrophin and Kir4.1 in Müller cell cultures. In addition, we found that α‐DG clusters were enriched for agrin and sought to investigate the role of agrin in their formation using recombinant C‐agrins. Both C‐agrin 4,8 and C‐agrin 0,0 failed to induce α‐DG clustering and neither of them potentiated the α‐DG clustering induced by laminin‐1. Finally, our data reveal that deletion of the PDZ‐ligand domain of Kir4.1 prevents their laminin‐induced clustering. These findings indicate that both laminin‐1 and α‐DG are involved in the distribution of Kir4.1 to specific Müller cell membrane domains and that this process occurs via a PDZ‐domain‐mediated interaction. Thus, in the basal lamina laminin is an essential regulator involved in clearing excess potassium released during neuronal activity, thereby contributing to the maintenance of normal synaptic transmission in the retina.

dystroglycan isoforms are differentially distributed in adult rat retina

α‐Dystroglycan (α ‐DG) is a laminin/agrin receptor expressed in skeletal muscle as well as in nervous system and other tissues. Glycosylation of the core protein of α‐DG is extensive, variable from tissue to tissue, and functionally relevant. To address differential glycosylation of α‐DG in the retina, we have investigated the distribution of this protein using two different antibodies: 1B7 directed against the core protein of α‐dystroglycan, and IIH6 directed against a carbohydrate moiety (Ervasti and Campbell [1993] J Cell Biol 122:809–823). Monoclonal antibody 1B7 recognizes a broader band than IIH6, which seems to recognize only a subset of α‐DG forms in retina. These data reflect the existence of differentially glycosylated isoforms of α‐DG. Monoclonal antibody 1B7 shows an extensive staining for α‐DG in the inner limiting membrane as well as in the ganglion cell and inner plexiform layers labeling Müller cell processes, whereas monoclonal antibody IIH6 staining is restricted to the inner limiting membrane and blood vessels. Our data indicate that there are distinct isoforms of α‐DG that are localized in apposition to basal lamina in the inner limiting membrane and blood vessels or within the parenchyma of the retina along Müller glia. Both isoforms are expressed in a Müller cell line in culture and coimmunoprecipitate with β‐dystroglycan. These data suggest that DGs may participate in organizing synapses and basement membrane assembly in the retina. J. Comp. Neurol. 420:182–194, 2000.

Interdependence of Laminin-mediated Clustering of Lipid Rafts and the Dystrophin Complex in Astrocytes

Astrocyte endfeet surrounding blood vessels are active domains involved in water and potassium ion transport crucial to the maintenance of water and potassium ion homeostasis in brain. A growing body of evidence points to a role for dystroglycan and its interaction with perivascular laminin in the targeting of the dystrophin complex and the water-permeable channel, aquaporin 4 (AQP4), at astrocyte endfeet. However, the mechanisms underlying such compartmentalization remain poorly understood. In the present study we found that AQP4 resided in Triton X-100-insoluble fraction, whereas dystroglycan was recovered in the soluble fraction in astrocytes. Cholesterol depletion resulted in the translocation of a pool of AQP4 to the soluble fraction indicating that its distribution is indeed associated with cholesterol-rich membrane domains. Upon laminin treatment AQP4 and the dystrophin complex, including dystroglycan, reorganized into laminin-associated clusters enriched for the lipid raft markers GM1 and flotillin-1 but not caveolin-1. Reduced diffusion rates of GM1 in the laminin-induced clusters were indicative of the reorganization of raft components in these domains. In addition, both cholesterol depletion and dystroglycan silencing reduced the number and area of laminin-induced clusters of GM1, AQP4, and dystroglycan. These findings demonstrate the interdependence between laminin binding to dystroglycan and GM1-containing lipid raft reorganization and provide novel insight into the dystrophin complex regulation of AQP4 polarization in astrocytes.

Distribution of potassium ion and water permeable channels at perivascular glia in brain and retina of the Largemyd mouse

The dystroglycan protein complex provides a link between the cytoskeleton and the extracellular matrix (ECM). Defective O‐glycosylation of α‐dystroglycan (α‐DG) severs this link leading to muscular dystrophies named dystroglycanopathies. These are characterized not only by muscle degeneration, but also by brain and ocular defects. In brain and retina, α‐DG and ECM molecules are enriched around blood vessels where they may be involved in localizing the inwardly rectifying potassium channel, Kir4.1, and aquaporin channel, AQP4, to astrocytic endfeet. To investigate in vivo the role of ECM ligand‐binding to glycosylated sites on α‐DG in the polarized distribution of these channels, we used the Largemyd mouse, an animal model for dystroglycanopathies. We found that Kir4.1 and AQP4 are lost from astrocytic endfeet in brain whereas significant labeling for these channels is detected at similar cell domains in retina. Furthermore, while both α‐ and β1‐syntrophins are lost from perivascular astrocytes in brain, labeling for β1‐syntrophin is found in retina of the Largemyd mouse. These findings show that while ligand‐binding to the highly glycosylated isoform of α‐DG in concert with α‐ and β1‐syntrophins is crucial for the polarized distribution of Kir4.1 and AQP4 to functional domains in brain, distinct mechanisms may contribute to their localization in retina.

Localized Rho GTPase Activation Regulates RNA Dynamics and Compartmentalization in Tumor Cell Protrusions

mRNA trafficking and local protein translation are associated with protrusive cellular domains, such as neuronal growth cones, and deregulated control of protein translation is associated with tumor malignancy. We show here that activated RhoA, but not Rac1, is enriched in pseudopodia of MSV-MDCK-INV tumor cells and that Rho, Rho kinase (ROCK), and myosin II regulate the microtubule-independent targeting of RNA to these tumor cell domains. ROCK inhibition does not affect pseudopodial actin turnover but significantly reduces the dynamics of pseudopodial RNA turnover. Gene array analysis shows that 7.3% of the total genes analyzed exhibited a greater than 1.6-fold difference between the pseudopod and cell body fractions. Of these, only 13.2% (261 genes) are enriched in pseudopodia, suggesting that only a limited number of total cellular mRNAs are enriched in tumor cell protrusions. Comparison of the tumor pseudopod mRNA cohort and a cohort of mRNAs enriched in neuronal processes identified tumor pseudopod-specific signaling networks that were defined by expression of M-Ras and the Shp2 protein phosphatase. Pseudopod expression of M-Ras and Shp2 mRNA were diminished by ROCK inhibition linking pseudopodial Rho/ROCK activation to the localized expression of specific mRNAs. Pseudopodial enrichment for mRNAs involved in protein translation and signaling suggests that local mRNA translation regulates pseudopodial expression of less stable signaling molecules as well as the cellular machinery to translate these mRNAs. Pseudopodial Rho/ROCK activation may impact on tumor cell migration and metastasis by stimulating the pseudopodial translocation of mRNAs and thereby regulating the expression of local signaling cascades.