biAb Mediated Restoration of the Linkage between Dystroglycan and Laminin-211 as a Therapeutic Approach for α-Dystroglycanopathies.

Patients with α-dystroglycanopathies, a subgroup of rare congenital muscular dystrophies, present with a spectrum of clinical manifestations that includes muscular dystrophy as well as CNS and ocular abnormalities. Although patients with α-dystroglycanopathies are genetically heterogeneous, they share a common defect of aberrant post-translational glycosylation modification of the dystroglycan alpha-subunit, which renders it defective in binding to several extracellular ligands such as laminin-211 in skeletal muscles, agrin in neuromuscular junctions, neurexin in the CNS, and pikachurin in the eye, leading to various symptoms. The genetic heterogeneity associated with the development of α-dystroglycanopathies poses significant challenges to developing a generalized treatment to address the spectrum of genetic defects. Here, we propose the development of a bispecific antibody (biAb) that functions as a surrogate molecular linker to reconnect laminin-211 and the dystroglycan beta-subunit to ameliorate sarcolemmal fragility, a primary pathology in patients with α-dystroglycan-related muscular dystrophies. We show that the treatment of LARGEmyd-3J mice, an α-dystroglycanopathy model, with the biAb improved muscle function and protected muscles from exercise-induced damage. These results demonstrate the viability of a biAb that binds to laminin-211 and dystroglycan simultaneously as a potential treatment for patients with α-dystroglycanopathy.

Multifaceted antibodies development against synthetic α-dystroglycan mucin glycopeptide as promising tools for dystroglycanopathies diagnostic.

Dystroglycanopathies are diseases characterized by progressive muscular degeneration and impairment of patient's quality of life. They are associated with altered glycosylation of the dystrophin-glycoprotein (DGC) complex components, such as α-dystroglycan (α-DG), fundamental in the structural and functional stability of the muscle fiber. The diagnosis of dystroglycanopathies is currently based on the observation of clinical manifestations, muscle biopsies and enzymatic measures, and the available monoclonal antibodies are not specific for the dystrophic hypoglycosylated muscle condition. Thus, modified α-DG mucins have been considered potential targets for the development of new diagnostic strategies toward these diseases. In this context, this work describes the synthesis of the hypoglycosylated α-DG mimetic glycopeptide NHAc-Gly-Pro-Thr-Val-Thr[αMan]-Ile-Arg-Gly-BSA (1) as a potential tool for the development of novel antibodies applicable to dystroglycanopathies diagnosis. Glycopeptide 1 was used for the development of polyclonal antibodies and recombinant monoclonal antibodies by Phage Display technology. Accordingly, polyclonal antibodies were reactive to glycopeptide 1, which enables the application of anti-glycopeptide 1 antibodies in immune reactive assays targeting hypoglycosylated α-DG. Regarding monoclonal antibodies, for the first time variable heavy (VH) and variable light (VL) immunoglobulin domains were selected by Phage Display, identified by NGS and described by in silico analysis. The best-characterized VH and VL domains were cloned, expressed in E. coli Shuffle T7 cells, and used to construct a single chain fragment variable that recognized the Glycopeptide 1 (GpαDG1 scFv). Molecular modelling of glycopeptide 1 and GpαDG1 scFv suggested that their interaction occurs through hydrogen bonds and hydrophobic contacts involving amino acids from scFv (I51, Y33, S229, Y235, and P233) and R8 and α-mannose from Glycopeptide 1.

A unique variant of lymphocytic choriomeningitis virus that induces pheromone binding protein MUP: Critical role for CTL.

Lymphocytic choriomeningitis virus (LCMV) WE variant 2.2 (v2.2) generated a high level of the major mouse urinary protein: MUP. Mice infected with LCMV WE v54, which differed from v2.2 by a single amino acid in the viral glycoprotein, failed to generate MUP above baseline levels found in uninfected controls. Variant 54 bound at 2.5 logs higher affinity to the LCMV receptor α-dystroglycan (α-DG) than v2.2 and entered α-DG-expressing but not α-DG-null cells. Variant 2.2 infected both α-DG-null or -expressing cells. Variant 54 infected more dendritic cells, generated a negligible CD8 T cell response, and caused a persistent infection, while v2.2 generated cytotoxic T lymphocytes (CTLs) and cleared virus within 10 days. By 20 days postinfection and through the 80-day observation period, significantly higher amounts of MUP were found in v2.2-infected mice. Production of MUP was dependent on virus-specific CTL as deletion of such cells aborted MUP production. Furthermore, MUP production was not elevated in v2.2 persistently infected mice unless virus was cleared following transfer of virus-specific CTL.

Development of rabbit monoclonal antibodies for detection of alpha-dystroglycan in normal and dystrophic tissue.

Alpha-dystroglycan requires a rare O-mannose glycan modification to form its binding epitope for extracellular matrix proteins such as laminin. This functional glycan is disrupted in a cohort of muscular dystrophies, the secondary dystroglycanopathies, and is abnormal in some metastatic cancers. The most commonly used reagent for detection of alpha-dystroglycan is mouse monoclonal antibody IIH6, but it requires the functional O-mannose structure for recognition. Therefore, the ability to detect alpha-dystroglycan protein in disease states where it lacks the full O-mannose glycan has been limited. To overcome this hurdle, rabbit monoclonal antibodies against the alpha-dystroglycan C-terminus were generated. The new antibodies, named 5-2, 29-5, and 45-3, detect alpha-dystroglycan from mouse, rat and pig skeletal muscle by Western blot and immunofluorescence. In a mouse model of fukutin-deficient dystroglycanopathy, all antibodies detected low molecular weight alpha-dystroglycan in disease samples demonstrating a loss of functional glycosylation. Alternately, in a porcine model of Becker muscular dystrophy, relative abundance of alpha-dystroglycan was decreased, consistent with a reduction in expression of the dystrophin-glycoprotein complex in affected muscle. Therefore, these new rabbit monoclonal antibodies are suitable reagents for alpha-dystroglycan core protein detection and will enhance dystroglycan-related studies.

GTDC2 modifies O-mannosylated α-dystroglycan in the endoplasmic reticulum to generate N-acetyl glucosamine epitopes reactive with CTD110.6 antibody.

Hypoglycosylation is a common characteristic of dystroglycanopathy, which is a group of congenital muscular dystrophies. More than ten genes have been implicated in α-dystroglycanopathies that are associated with the defect in the O-mannosylation pathway. One such gene is GTDC2, which was recently reported to encode O-mannose ß-1,4-N-acetylglucosaminyltransferase. Here we show that GTDC2 generates CTD110.6 antibody-reactive N-acetylglucosamine (GlcNAc) epitopes on the O-mannosylated α-dystroglycan (α-DG). Using the antibody, we show that mutations of GTDC2 identified in Walker-Warburg syndrome and alanine-substitution of conserved residues between GTDC2 and EGF domain O-GlcNAc transferase resulted in decreased glycosylation. Moreover, GTDC2-modified GlcNAc epitopes are localized in the endoplasmic reticulum (ER). These data suggested that GTDC2 is a novel glycosyltransferase catalyzing GlcNAcylation of O-mannosylated α-DG in the ER. CTD110.6 antibody may be useful to detect a specific form of GlcNAcylated O-mannose and to analyze defective O-glycosylation in α-dystroglycanopathies.

Disruption of dystroglycan-laminin interactions modulates water uptake by astrocytes.

Cerebral edema is a serious complication of ischemic brain injury. Cerebral edema includes accumulation of extracellular fluid due to leakage of the brain's microvessel permeability barrier, and swelling of astrocytes as they absorb water from the extracellular space. Expression of matrix adhesion receptors in brain microvessels decreases in ischemic stroke; this contributes to increased microvessel permeability and detachment of astrocytes from the extracellular matrix (ECM). Since loss of the astrocyte adhesion receptor dystroglycan has been associated with disrupted polarization of ion and water channels, we hypothesized that adhesion of astrocytes to the ECM contributes to regulation of water uptake, and that disruption of matrix adhesion impairs the ability of astrocytes to direct water transport. To test this hypothesis, the capacity of astrocytes to take up water was measured using a fluorescence self-quenching assay under both oxygen/glucose deprivation (OGD) and direct antibody-mediated blockade of α-dystroglycan. Both conditions decreased the rate of water uptake. Moreover, inhibiting proteolytic cleavage of dystroglycan that occurs in OGD abrogated the effect of OGD, but not direct blockade of α-dystroglycan, indicating that interfering with dystroglycan-matrix binding itself affects water uptake. Activation of extracellular signal-related kinase (ERK) by OGD was dependent on α-dystroglycan binding, and inhibition of ERK activity with U0126 abrogated the loss of water uptake following OGD. These studies demonstrate for the first time that water uptake in astrocytes is regulated by dystroglycan-dependent signaling associated with matrix adhesion. This presents a novel potential approach to the treatment of cerebral edema.

Dystroglycan promotes filopodial formation and process branching in differentiating oligodendroglia.

During central nervous system (CNS) development, individual oligodendrocytes myelinate multiple axons, thus requiring the outgrowth and extensive branching of oligodendroglial processes. Laminin (Lm)-deficient mice have a lower percentage of myelinated axons, which may indicate a defect in the ability to properly extend and branch processes. It remains unclear, however, to what extent extracellular matrix (ECM) receptors contribute to oligodendroglial process remodeling itself. In the current study, we report that the ECM receptor dystroglycan is necessary for Lm enhancement of filopodial formation, process outgrowth, and process branching in differentiating oligodendroglia. During early oligodendroglial differentiation, the disruption of dystroglycan-Lm interactions, via blocking antibodies or dystroglycan small interfering RNA (siRNA), resulted in decreased filopodial number and length, decreased process length, and decreased numbers of primary and secondary processes. Later in oligodendrocyte differentiation, dystroglycan-deficient cells developed fewer branches, thus producing less complex networks of processes as determined by Sholl analysis. In newly differentiating oligodendroglia, dystroglycan was localized in filopodial tips, whereas, in more mature oligodendrocytes, dystroglycan was enriched in focal adhesion kinase (FAK)-positive focal adhesion structures. These results suggest that dystroglycan-Lm interactions influence oligodendroglial process dynamics and therefore may regulate the myelination capacity of individual oligodendroglia.

Differential glycosylation of α-dystroglycan and proteins other than α-dystroglycan by like-glycosyltransferase.

Genetic defects in like-glycosyltransferase (LARGE) cause congenital muscular dystrophy with central nervous system manifestations. The underlying molecular pathomechanism is the hypoglycosylation of α-dystroglycan (α-DG), which is evidenced by diminished immunoreactivity to IIH6C4 and VIA4-1, antibodies that recognize carbohydrate epitopes. Previous studies indicate that LARGE participates in the formation of a phosphoryl glycan branch on O-linked mannose or it modifies complex N- and mucin O-glycans. In this study, we overexpressed LARGE in neural stem cells deficient in protein O-mannosyltransferase 2 (POMT2), an enzyme required for O-mannosyl glycosylation. The results showed that overexpressing LARGE did not lead to hyperglycosylation of α-DG in POMT2 knockout (KO) cells but did generate IIH6C4 and VIA4-1 immunoreactivity and laminin-binding activity. Additionally, overexpressing LARGE in cells deficient in both POMT2 and α-DG generated laminin-binding IIH6C4 immunoreactivity. These results indicate that LARGE expression resulted in the glycosylation of proteins other than α-DG in the absence of O-mannosyl glycosylation. The IIH6C4 immunoreactivity generated in double-KO cells was largely removed by treatment either with peptide N-glycosidase F or with cold aqueous hydrofluoric acid, suggesting that LARGE expression caused phosphoryl glycosylation of N-glycans. However, the glycosylation of α-DG by LARGE is dependent on POMT2, indicating that LARGE expression only modifies O-linked mannosyl glycans of α-DG. Thus, LARGE expression mediates the phosphoryl glycosylation of not only O-mannosyl glycans including those on α-DG but also N-glycans on proteins other than α-DG.

Hormonal therapy in the senescence: Prostatic microenvironment structure and adhesion molecules.

Hormonal replacement has been utilized to minimize the harmful effects of hormonal imbalance in elderly men. The development and progression of prostatic diseases and their relation to hormone therapy is still unclear. Thus, the aim herewith was to characterize the structure and dystroglycan molecule (DGs) reactivities in the ventral prostatic lobe from elderly rats submitted to steroid hormone replacement. Male rats (Sprague-Dawley) were divided into one Young group and six senile groups. The Young group (YNG) (4 months old) received peanut oil (5mL/kg, s.c.). The senile rats (10 months old) were submitted to the following treatments: Senile group (SEN) (5mL/kg peanut oil, s.c.); Testosterone group (TEST) (5mg/kg testosterone cipionate, s.c.); Estrogen group (EST) (25µg/kg 17ß-estradiol, s.c.); Castrated group (CAS) (surgical castration); Castrated-Testosterone (CT) (surgical castration and treatment similar to TEST group); and Castrated-Estrogen (CE) (surgical castration and treatment similar to EST group). After 30 days treatment, blood samples were collected for hormonal analysis and ventral prostate samples were processed for light and transmission electron microscopies, morphometrical analysis, immunohistochemistry and Western Blotting. The results showed decreased serum testosterone levels in the senescence and increased testosterone and estrogen plasmatic levels after hormone administration in the TEST and EST groups, respectively, highlighting the therapy efficiency. Hypertrophied stroma and inflammatory cells were verified in the SEN group. After hormone replacement in the senescence or following castration, atrophic epithelium, epithelial cells with clear cytoplasmic halo around the nucleus, microacini and maintenance of hypertrophied stroma were seen. Decreased DG levels were verified in the senescence. After hormonal therapy, increased protein levels of these molecules were observed, especially in those groups which received estradiol. Thus, the occurrence of inflammatory cells, stromal hypertrophy and the presence of cells with clear halo around the nucleus after hormonal therapy probably indicated prostatic paracrine signaling imbalance, suggesting a stromal reactive microenvironment favorable to the development of glandular lesions. However, the increase of DG levels characterized positive effect of steroid hormone replacement on the prostate in the senescence. Thus, it could be concluded that despite having positive effects on important molecules involved in the maintenance of epithelial-stromal interaction and glandular cytoarchitecture, such as DGs, hormonal therapy enhanced structural changes associated with senescence, probably due to increased hormonal imbalance between androgens and estrogens in the prostatic tissue.

Quantitative organization of GABAergic synapses in the molecular layer of the mouse cerebellar cortex.

In the cerebellar cortex, interneurons of the molecular layer (stellate and basket cells) provide GABAergic input to Purkinje cells, as well as to each other and possibly to other interneurons. GABAergic inhibition in the molecular layer has mainly been investigated at the interneuron to Purkinje cell synapse. In this study, we used complementary subtractive strategies to quantitatively assess the ratio of GABAergic synapses on Purkinje cell dendrites versus those on interneurons. We generated a mouse model in which the GABAA receptor alpha1 subunit (GABAARalpha1) was selectively removed from Purkinje cells using the Cre/loxP system. Deletion of the alpha1 subunit resulted in a complete loss of GABAAR aggregates from Purkinje cells, allowing us to determine the density of GABAAR clusters in interneurons. In a complementary approach, we determined the density of GABA synapses impinging on Purkinje cells using alpha-dystroglycan as a specific marker of inhibitory postsynaptic sites. Combining these inverse approaches, we found that synapses received by interneurons represent approximately 40% of all GABAergic synapses in the molecular layer. Notably, this proportion was stable during postnatal development, indicating synchronized synaptogenesis. Based on the pure quantity of GABAergic synapses onto interneurons, we propose that mutual inhibition must play an important, yet largely neglected, computational role in the cerebellar cortex.

Post traumatic lesion absence of beta-dystroglycan-immunopositivity in brain vessels coincides with the glial reaction and the immunoreactivity of vascular laminin.

Following brain lesions, the gliovascular basal lamina undergoes destruction and the gliovascular connections 'decouple'. Laminin receptors, as dystroglycan, are essential in these processes. The present study compares the immunoreactivities of beta-dystroglycan, glial fibrillary acidic protein (GFAP), and laminin following stab wounds in adult rats. In intact brain the vessels were immunopositive to beta-dystroglycan, whereas the laminin of their basal lamina proved to be unavailable to immunoreactions. Following stab wound, however, the adjacent vessels lost their immunopositvity to beta-dystroglycan, whereas immunopositivity to laminin became detectable in them. In an advanced stage of glial reaction the territory of GFAP immunopositive reactive astrocytes coincided with the area where vessels lost their immunopositivity to beta-dystroglycan. When glial reaction regressed, the beta-dystroglycan immunopositivity re-appeared, and laminin immunopositivity became undetectable again. Post-lesional disappearance of vascular beta-dystroglycan immunostaining was described earlier, and was attributed to the cleavage of beta-dystroglycan by matrix metalloproteinases as a mechanism of the decoupling of the gliovascular connections. Our results, which were obtained in a different type of lesion support that the loss of vascular beta-dystroglycan immunopositivity is a general phenomenon following cerebral lesions, and an indirect marker of gliovascular decoupling. For the first time coincidences were presented between vascular immunonegativity to beta-dystroglycan, glial reaction and detectability of laminin. Manifestation of laminin immunoreactivity also indicates gliovascular decoupling. Coincidence between glial reaction and lack of vascular beta-dystroglycan suggests mutual enhancement between them. The observations may have clinico-pathologic importance since similar investigations may help to follow the progression and regression of post-lesion processes.

Processing and secretion of the N-terminal domain of alpha-dystroglycan in cell culture media.

Alpha-dystroglycan (alpha-DG) plays a crucial role in maintaining the stability of muscle cell membrane. Although it has been shown that the N-terminal domain of alpha-DG (alpha-DG-N) is cleaved by a proprotein convertase, its physiological significance remains unclear. We show here that native alpha-DG-N is secreted by a wide variety of cultured cells into the culture media. The secreted alpha-DG-N was both N- and O-glycosylated. Finally, a small amount of alpha-DG-N was detectable in the normal human serum. These observations indicate that the cleavage of alpha-DG-N is a widespread event and suggest that the secreted alpha-DG-N might be transported via systemic circulation in vivo.

Sarcoglycan immunoreactivity is lacking in infantile hypertrophic pyloric stenosis. A confocal laser scanning microscopic study.

OBJECTIVES: The Dystrophin-Glycoprotein Complex (DGC) is a large multisubunit complex that plays a crucial role in maintaining the structural integrity and physiology of muscle fibers. Dystrophin has been reported to be absent in the pyloric muscle of infantile hypertrophic pyloric stenosis (IHPS) patients. The present study was designed to investigate the other two patterns of DGC (dystroglycan and sarcoglycan complexes) in normal pyloric muscle and their possible modifications in IHPS patients. METHODS: Ten pyloric muscle biopsies were obtained from babies operated for IHPS and five control pylorus biopsy taken at autopsy from cases without gastrointestinal disease. The DGC sub-complexes (beta-dystroglican and beta, delta- sarcoglycans) were localized immunohistochemically using specific monoclonal antibodies. The results were evaluated using a confocal laser scanning microscope. RESULTS: Positive immunolocalization of the two DGC sub complexes was demonstrated in the smooth muscle cells (SMCs) of the pyloric region of control patients. Similarly, a positive immune expression of beta-dystroglican was observed in the pyloric SMCs of IHPS patients. On the other hand a negative immunoreaction for sarcoglycans was recorded within the full thickness of the pyloric SMCs of these patients. CONCLUSIONS: The absence of sarcoglycans within the hypertrophied pyloric muscle may be a predisposing factor in the pathogenesis of IHPS since it could alter the normal physiology of SMCs through the modifications of structural integrity of sarcolemma and signaling between the extracellular and intracellular compartment.

A suspenseful game of 'hide and seek' between virus and host.

As part of the most important contributions to the understanding of viral immunity, Michael Oldstone recounts his pioneering work on lymphocytic choriomeningitis virus.

Binding of laminin alpha1-chain LG4-5 domain to alpha-dystroglycan causes tyrosine phosphorylation of syntrophin to initiate Rac1 signaling.

Previously, a signaling pathway was described [Oak, Zhou, and Jarrett (2003) J. Biol. Chem. 278, 39287-39295] that links matrix laminin binding on the outside of the sarcolemma to Grb2 binding to syntrophin on the inside surface of the sarcolemma and by way of Grb2-Sos1-Rac1-PAK1-JNK ultimately results in the phosphorylation of c-jun on Ser(65). How this signaling is initiated was investigated. Grb2-binding to syntrophin is increased by the addition of either laminin-1 or the isolated laminin alpha1 globular domain modules LG4-5, a protein referred to as E3. This identifies the LG4-5 sequences as the region of laminin responsible for signaling. Since laminin alpha1 LG4 is known to bind alpha-dystroglycan, this directly implicates alpha-dystroglycan as the laminin-signaling receptor. E3 or laminin-1 increase Grb2-binding and Rac1 activation. In the presence of E3 or laminin-1, syntrophin is phosphorylated on a tyrosine residue, and this increases and alters Grb2 binding. The alpha-dystroglycan antibody, IIH6, which blocks binding of laminins to alpha-dystroglycan, blocks both the laminin-induced Sos1/2 recruitment and syntrophin phosphorylation, showing that it is alpha-dystroglycan binding the LG4-5 region of laminin that is responsible. The C-terminal SH3 domain of Grb2 (C-SH3) binds only to nonphosphorylated syntrophin, and phosphorylation causes the Grb2 SH2 domain to bind and prevents SH3 binding. Syntrophin, tyrosine phosphate, beta-dystroglycan, and Rac1 all co-localize to the sarcolemma of rat muscle sections. A model for how this phosphorylation may initiate downstream events in laminin signaling is presented.

IgG marker of optic-spinal multiple sclerosis binds to the aquaporin-4 water channel.

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease that selectively affects optic nerves and spinal cord. It is considered a severe variant of multiple sclerosis (MS), and frequently is misdiagnosed as MS, but prognosis and optimal treatments differ. A serum immunoglobulin G autoantibody (NMO-IgG) serves as a specific marker for NMO. Here we show that NMO-IgG binds selectively to the aquaporin-4 water channel, a component of the dystroglycan protein complex located in astrocytic foot processes at the blood-brain barrier. NMO may represent the first example of a novel class of autoimmune channelopathy.

Localization of alpha-dystroglycan on the podocyte: from top to toe.

alpha-Dystroglycan (DG) is a negatively charged membrane-associated glycoprotein that links the cytoskeleton to the extracellular matrix. Previously, we described that alpha-DG covers the whole podocyte cell membrane in the rat. However, our finding was challenged by the description of a strictly basolateral localization in human kidney biopsies, using a different antibody against alpha-DG. Therefore, we studied the exact localization of glomerular alpha-DG by using these two antibodies in both species. The studies were performed by using monoclonal antibodies (MoAbs) IIH6 and VIA4.1 in immunofluorescence, confocal microscopy, and immunoelectron microscopy on both rat and human kidney sections, as well as on cultured mouse podocytes. The apical localization of alpha-DG on podocytes was more dominant than the basolateral localization. The basolateral staining with MoAb VIA4.1 was more pronounced than that of MoAb IIH6. With both MoAbs, the staining in rat kidneys was more prominent, in comparison to human kidneys. We conclude that alpha-DG is expressed at both the basolateral and apical sides of the podocyte. This localization suggests that alpha-DG plays a dual role in the maintenance of the unique architecture of podocytes by its binding to the glomerular basement membrane, and in the maintenance of the integrity of the filtration slit, respectively.

Immunodetection of partially glycosylated isoforms of alpha-dystroglycan by a new monoclonal antibody against its beta-dystroglycan-binding epitope.

The alpha/beta dystroglycan (DG) complex links the extracellular matrix to the actin cytoskeleton. The extensive glycosylation of alpha-DG is believed to be crucial for the interaction with its extracellular matrix-binding partners. We characterized a monoclonal antibody, directed against the beta-DG-binding epitope ( approximately positions 550-565), which recognizes preferentially hypoglycosylated alpha-DG. In Western blot, the antibody was able to detect a number of partially glycosylated alpha-DG isoforms from rat brain and chicken skeletal muscle tissue samples. In addition, we demonstrated its inhibitory effect on the interaction between alpha- and beta-DG in vitro and preliminary immunostaining experiments suggest that such hypoglycosylated alpha-DG isoforms could play a role within cells.

Mycobacterium leprae-induced demyelination: a model for early nerve degeneration.

The molecular events that occur at the early phase of many demyelinating neurodegenerative diseases are unknown. A recent demonstration of rapid demyelination and axonal injury induced by Mycobacterium leprae provides a model for elucidating the molecular events of early nerve degeneration which might be common to neurodegenerative diseases of both infectious origin and unknown etiology. The identification of the M. leprae-targeted Schwann cell receptor, dystroglycan, and its associated molecules in myelination, demyelination and axonal functions suggests a role for these molecules in early nerve degeneration.