Short-term treatment of golden retriever muscular dystrophy (GRMD) dogs with rAAVrh74.MHCK7.GALGT2 induces muscle glycosylation and utrophin expression but has no significant effect on muscle strength.

We have examined the effects of intravenous (IV) delivery of rAAVrh74.MHCK7.GALGT2 in the golden retriever muscular dystrophy (GRMD) model of Duchenne Muscular Dystrophy (DMD). After baseline testing, GRMD dogs were treated at 3 months of age and reassessed at 6 months. This 3-6 month age range is a period of rapid disease progression, thus offering a relatively short window to establish treatment efficacy. Measures analyzed included muscle AAV transduction, GALGT2 transgene expression, GALGT2-induced glycosylation, muscle pathology, and muscle function. A total of five dogs were treated, 4 at 2x1014vg/kg and one at 6x1014vgkg. The 2x1014vg/kg dose led to transduction of regions of the heart with 1-3 vector genomes (vg) per nucleus, while most skeletal muscles were transduced with 0.25-0.5vg/nucleus. GALGT2-induced glycosylation paralleled levels of myofiber vg transduction, with about 90% of cardiomyocytes having increased glycosylation versus 20-35% of all myofibers across the skeletal muscles tested. Conclusions from phenotypic testing were limited by the small number of dogs. Treated dogs had less pronounced fibrosis and overall lesion severity when compared to control groups, but surprisingly no significant changes in limb muscle function measures. GALGT2-treated skeletal muscle and heart had elevated levels of utrophin protein expression and GALGT2-induced expression of glycosylated α dystroglycan, providing further evidence of a treatment effect. Serum chemistry, hematology, and cardiac function measures were largely unchanged by treatment. Cumulatively, these data show that short-term intravenous treatment of GRMD dogs with rAAVrh74.MHCK7.GALGT2 at high doses can induce muscle glycosylation and utrophin expression and may be safe over a short 3-month interval, but that such treatments had only modest effects on muscle pathology and did not significantly improve muscle strength.

Alpha-dystroglycan staining pattern and mortality in patients undergoing radical prostatectomy with lymph node positive prostate cancer.

INTRODUCTION: Dystroglycan (DG) is a cell surface receptor for extracellular matrix proteins involved in tissue mechanical stability and matrix organization. Initial work has demonstrated that alpha-DG expression is decreased in many types of adenocarcinoma, including prostate, and potentially associated with the development of metastatic disease. However, the consistency between prostate and lymph node alpha-DG staining has not been previously reported. In addition, identification of an immunohistochemical marker associated with prostate cancer grade, stage, need for adjuvant or salvage therapy and mortality would have potential clinical value. MATERIALS AND METHODS: Node positive, margin negative radical prostatectomy specimens at a single institution from 1982 to 2012 were reviewed and identified 35 prostate specimens, including 26 patients with available tissue from both the primary prostatectomy and lymph node specimens. The expression levels of the alpha-DG subunit were analyzed using immunohistochemistry and graded from 0 to 4. Survival was compared in different staining pattern groups. RESULTS: Strength of alpha-DG staining was found to be consistent between prostate and lymph node specimens (p < 0.004). The median overall survival was shorter in those without alpha-DG staining in the prostate compared to those with positive staining, but this difference was not statistically significant (13.2 years versus 19.4 years, p = 0.21). In addition, negative staining was associated with higher mean PSA, pathologic T stage, Gleason grade and the need for adjuvant or salvage therapy compared to positive group but none reached statistical significance (16.06 ng/mL versus 11.67 ng/mL, p = 0.79; 89% versus 68%, p = 0.38; 33.3% versus 23.1%, p = 0.66; 88.9% versus 76.9%, p = 0.44). CONCLUSIONS: DG expression by immunohistochemistry staining was consistent between prostate and metastatic lymph node specimens. In a small cohort of prostate cancer patients with margin negative but node positive disease, DG staining was not associated with Gleason grade or with overall mortality.

Ribitol-phosphate-a newly identified posttranslational glycosylation unit in mammals: structure, modification enzymes and relationship to human diseases.

GLYCOSYLATION IS A CRUCIAL POSTTRANSLATIONAL MODIFICATION THAT IS INVOLVED IN NUMEROUS BIOLOGICAL EVENTS. THEREFORE, ABNORMAL GLYCOSYLATION CAN IMPAIR THE FUNCTIONS OF GLYCOPROTEINS OR GLYCOLIPIDS AND IS OCCASIONALLY ASSOCIATED WITH CELL DYSFUNCTION AND HUMAN DISEASES. FOR EXAMPLE, ABERRANT GLYCOSYLATION OF DYSTROGLYCAN (DG), A CELLULAR RECEPTOR FOR MATRIX AND SYNAPTIC PROTEINS, IS ASSOCIATED WITH MUSCULAR DYSTROPHY AND LISSENCEPHALY. DG SUGAR CHAINS ARE REQUIRED FOR HIGH-AFFINITY BINDING TO LIGAND PROTEINS, AND THUS DISRUPTION OF DG-LIGAND LINKAGES UNDERLIES DISEASE CONDITIONS. ALTHOUGH THEIR BIOLOGICAL SIGNIFICANCE IS WELL RECOGNIZED, THE SUGAR-CHAIN STRUCTURE OF DG AND ITS MODIFICATION ENZYMES HAVE LONG REMAINED INCOMPLETELY ELUCIDATED. HOWEVER, RECENT SEMINAL STUDIES HAVE FINALLY REVEALED A HIGHLY REGULATED MECHANISM FOR DG GLYCOSYLATION AND HAVE DISCOVERED A POSTTRANSLATIONAL UNIT, RIBITOL-PHOSPHATE, THAT WAS NOT PREVIOUSLY KNOWN TO BE USED IN MAMMALS. THIS REVIEW ARTICLE INTRODUCES THE STRUCTURE, MODIFICATION ENZYMES AND FUNCTIONS OF THE SUGAR CHAINS OF DG, AND THEN DISCUSSES THEIR RELATIONSHIP TO HUMAN DISEASES AND THERAPEUTIC STRATEGIES: .

Alterations in plasma membrane promote overexpression and increase of sodium influx through epithelial sodium channel in hypertensive platelets.

Platelets are small, anucleated cell fragments that activate in response to a wide variety of stimuli, triggering a complex series of intracellular pathways leading to a hemostatic thrombus formation at vascular injury sites. However, in essential hypertension, platelet activation contributes to causing myocardial infarction and ischemic stroke. Reported abnormalities in platelet functions, such as platelet hyperactivity and hyperaggregability to several agonists, contribute to the pathogenesis and complications of thrombotic events associated with hypertension. Platelet membrane lipid composition and fluidity are determining for protein site accessibility, structural arrangement of platelet surface, and response to appropriate stimuli. The present study aimed to demonstrate whether structural and biochemical abnormalities in lipid membrane composition and fluidity characteristic of platelets from hypertensive patients influence the expression of the Epithelial Sodium Channel (ENaC), fundamental for sodium influx during collagen activation. Wb, cytometry and quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) assays demonstrated ENaC overexpression in platelets from hypertensive subjects and in relation to control subjects. Additionally, our results strongly suggest a key role of ß-dystroglycan as a scaffold for the organization of ENaC and associated proteins. Understanding of the mechanisms of platelet alterations in hypertension should provide valuable information for the pathophysiology of hypertension.

Lost Polarization of Aquaporin4 and Dystroglycan in the Core Lesion after Traumatic Brain Injury Suggests Functional Divergence in Evolution.

OBJECTIVE: To understand how aquaporin4 (AQP4) and dystroglycan (DG) polarized distribution change and their roles in brain edema formation after traumatic brain injury (TBI). METHODS: Brain water content, Evans blue detection, real-time PCR, western blot, and immunofluorescence were used. RESULTS: At an early stage of TBI, AQP4 and DG maintained vessel-like pattern in perivascular endfeet; M1, M23, and M1/M23 were increased in the core lesion. At a later stage of TBI, DG expression was lost in perivascular area, accompanied with similar but delayed change of AQP4 expression; expression of M1, M23, and DG and the ratio of M1/M2 were increased. CONCLUSION: At an early stage, AQP4 and DG maintained the polarized distribution. Upregulated M1 and M23 could retard the cytotoxic edema formation. At a later stage AQP4 and DG polarized expression were lost from perivascular endfeet and induced the worst cytotoxic brain edema. The alteration of DG expression could regulate that of AQP4 expression after TBI.

Glycobiology of α-dystroglycan and muscular dystrophy.

Most proteins are modified by glycans, which can modulate the biological properties and functions of glycoproteins. The major glycans can be classified into N-glycans and O-glycans according to their glycan-peptide linkage. This review will provide an overview of the O-mannosyl glycans, one subtype of O-glycans. Originally, O-mannosyl glycan was only known to be present on a limited number of glycoproteins, especially α-dystroglycan (α-DG). However, once a clear relationship was established between O-mannosyl glycan and the pathological mechanisms of some congenital muscular dystrophies in humans, research on the biochemistry and pathology of O-mannosyl glycans has been expanding. Because α-DG glycosylation is defective in congenital muscular dystrophies, which also feature abnormal neuronal migration, these disorders are collectively called α-dystroglycanopathies. In this article, I will describe the structure, biosynthesis and pathology of O-mannosyl glycans.

Podocin and beta dystroglycan expression to study podocyte-podocyte and basement membrane matrix connections in adult protienuric states.

BACKGROUND: Podocytes can be the primary site of injury or secondarily involved in various protienuric states. Cross talk between adjacent foot processes and with basement membrane is important for slit diaphragm function. Does expression of podocyte associated proteins in kidney biopsies alter with site/type of primary injury? Genetic mutations of podocin result in steroid resistant FSGS. Can protein expression of podocin predict resistant cases to initiate further genetic evaluation? METHODS: Adult patients (n-88) with protienuria- minimal change disease(MCD)-22, focal segmental glomerulosclerosis(FSGS)-21,membranous glomerulonephritis(MGN)-25 and IgA nephropathy(IgAN)-20 were selected for immunohistochemistry with podocin and beta dystroglycan . Results were graded (0 - 3+scale )and compared with control biopsies and internal control. Treatment and follow up (6 months -2 ½ years) of FSGS and MCD cases were collected. RESULTS: There was intense to moderate staining of the podocytes with podocin and ß dystroglycan in the glomeruli in all cases (MCD, FSGS, IgAN and MGN) except for weak staining with ß dystroglycan in 3 cases of MCD. There was loss of immunostains in areas of segmental/global sclerosis. There was no significant difference in the staining pattern between the groups. In primary podocytopathies, staining pattern did not differ between steroid resistant, sensitive or dependent cases. CONCLUSIONS: Immunohistochemical expression of podocin and ß dystroglycan does not differ in nephropathies which have different site of injury depending on absence (MCD and FSGS) or presence of immune deposits and their localization (MGN and IgAN). Podocin and ß dystroglycan staining did not differentiate steroid sensitive and resistant cases, hence, does not give clue to initiate genetic studies. However, analysis of bigger cohort may be required. SUMMARY: Podocin and ß dystroglycan immunohistochemistry was done to analyze podocyte - podocyte and podocyte -basement membrane matrix connections in adult protienuric states. Primary podocytopathies i.e. MCD and FSGS and secondary podocytopathy due to immune complex deposition, i.e., MGN (subepithelial) and IgAN (mesangial) were analyzed. There was no difference in staining patterns between primary and secondary podocytopathies or between steroid sensitive, resistant and dependent cases of FSGS and MCD. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2258608781052786.

Suramin affects metalloproteinase-9 activity and increases ß-dystroglycan levels in the diaphragm of the dystrophin-deficient mdx mouse.

INTRODUCTION: In Duchenne muscular dystrophy and in the mdx mouse, muscle fiber degeneration and subsequent fibrosis lead to cardiorespiratory failure. Previously, we demonstrated that the anti-fibrotic agent suramin was effective in decreasing fibrosis in mdx muscles. In this study, we were interested to see whether suramin could affect metalloproteinases (MMP) and improve the functional activity of the mdx diaphragm muscle. METHODS: Zymography was performed to evaluate MMP-2 and MMP-9 activity. Western blotting was used to analyze the levels of beta-dystroglycan. Muscle function was assessed in hemidiaphragm in vitro preparations. RESULTS: We found that suramin affects metalloproteinase-9 activity and increases beta-dystroglycan. Furthermore, suramin also protects against diaphragm muscle fatigue over time. CONCLUSIONS: These results show the potential benefits of suramin in maintaining the structure of the dystrophin-glycoprotein complex.

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.

Altered cytoskeletal structure of smooth muscle cells in ureteropelvic junction obstruction.

PURPOSE: Ureteropelvic junction obstruction is one of the most common causes of hydronephrosis in children. A malfunction of smooth muscle cells is believed to be the underlying mechanism causing obstruction. We investigated the expression of some integrins, talin and ß-dystroglycan, considered the main compound of smooth muscle cell cytoskeleton, and active caspase 3 at the level of the ureteropelvic junction obstruction. MATERIALS AND METHODS: Specimens were obtained at pyeloplasty in 12 children with ureteropelvic junction obstruction. Six control specimens were obtained during organ explantation. Specimens were divided into renal pelvis, ureteropelvic junction and ureter below the obstruction. Western blot analysis of active caspase 3, and immunofluorescence and polymerase chain reaction analysis were performed for α7A, ß1A, α7B and ß1D integrins, talin and ß-dystroglycan. RESULTS: Talin and ß-dystroglycan were slightly impaired in ureteropelvic junction obstruction, while α7B and ß1D integrins were severely reduced, and α7A, ß1A and active caspase 3 were significantly enhanced compared to controls. CONCLUSIONS: We demonstrated activation of apoptosis and a critical alteration of cytoskeleton that might explain the altered function and the increased apoptosis in smooth muscle cells in ureteropelvic junction obstruction. The delayed rearrangement of the cytoskeleton of smooth muscle cells in ureteropelvic junction obstruction might be linked to a postnatal splicing from α7A and ß1A to α7B and ß1D integrins, respectively. This relationship could explain the common clinical scenario of spontaneous improvement of hydronephrosis in children with suspected ureteropelvic junction obstruction.

Expression of alpha-dystroglycan correlates with tumour grade and predicts survival in oral squamous cell carcinoma.

AIMS: Dystroglycan (DG) is a non-integrin adhesion molecule connecting the extracellular matrix to the actin cytoskeleton. Decreased expression of DG has been reported in several human cancers and related to tumour aggressiveness. METHODS: Expression of the alpha-DG subunit was evaluated by immunostaining in a series of oral squamous cell carcinoma (OSCC) and its relation with traditional prognostic indicators and with the clinical outcome of the patients was evaluated. RESULTS: Alpha-DG expression was easily detected in normal epithelium with a mean percentage of positive cells >80% but was undetectable in a significant fraction (59%) of OSCC. Loss of alpha-DG staining correlated with higher tumour grade (p = 0.04) and stage (p = 0.01), with nodal involvement (p = 0.001) and with an increased risk of recurrence (p = 0.002) and death (p = 0.004) in a univariate analysis, but it was not confirmed as an independent predictor of clinical outcome in a multivariate analysis. CONCLUSIONS: Loss of alpha-DG expression, which corresponds to loss of a functional DG complex, is a frequent event in human OSCC. Further studies are warranted on the role of this molecule in the entire multistep process of oral squamous tumorigenesis.

Expression of sialidase and dystroglycan in human glomerular diseases.

BACKGROUND: alpha-Dystroglycan (alpha-DG) is a negatively charged glycoprotein that covers the surface of podocytes. A decreased glomerular expression of alpha-DG has been described in minimal change nephropathy (MCN), but not in focal segmental glomerulosclerosis (FSGS). This was suggested as a tool to distinguish these diseases. Sialic acid is a negatively charged carbohydrate extensively present on both alpha-DG and podocalyxin, which is also expressed on podocytes. Intrarenal perfusion with bacterial sialidase leads to foot process effacement and proteinuria. This is the first study on the expression of endogenous glomerular sialidase; furthermore, the expression of dystroglycan was re-evaluated. METHODS: The expression of alpha-DG and sialidase was investigated by immunofluorescence in kidney biopsies of patients with MCN (n = 5), FSGS (n = 15), proliferative lupus nephritis (LN, n = 9), membranous glomerulopathy (MG, n = 10) and normal human kidneys (NHK, n = 4). The urinary sialic acid concentration was measured using a newly developed LC-tandem mass spectrometry method. RESULTS: A 3-fold increased glomerular expression of sialidase was found in MG, accompanied with an increased urinary sialic acid concentration in two MG patients. However, we did not observe major changes in the expression of alpha-DG in patients with the above-mentioned glomerular diseases compared to NHK, also not between MCN and FSGS. CONCLUSIONS: Endogenous glomerular sialidase expression is increased in MG, which might represent a novel mechanism for the loss of negative charge in the glomerular capillary filter. The expression of dystroglycan cannot be used as a diagnostic tool to differentiate between glomerular diseases.

Immunohistochemical distribution of basement membrane proteins in the human inner ear from older subjects.

The immunolocalization of several basement membrane (BM) proteins was investigated in vestibular endorgans microdissected from temporal bones obtained from subjects with a documented normal auditory and vestibular function (n=5, average age=88 years old). Fluorescent immunostaining using antibodies directed at collagen IV alpha 2, nidogen-1, laminin-beta2, alpha-dystroglycan, and tenascin-C was applied to cryosections from human cochlea, cristae ampullares, utricular and saccular maculae. Collagen IV alpha 2, nidogen-1, and laminin-beta2 localized to all subepithelial cochlear BMs, Reissner's membrane, strial and spiral ligamental perineural and perivascular BMs, and the spiral limbus. Tenascin-C localized to the basilar membrane and the osseous spiral lamina. alpha-Dystroglycan localized to most cochlear BMs except those in the spiral ligament, basilar membrane and spiral limbus. Collagen IV, nidogen-1, and laminin-beta2 localized to the subepithelial BMs of the maculae and cristae ampullares, and the perineural and perivascular BMs within the underlying stroma. The BM underlying the transitional and dark cell region of the cristae ampullares also expressed collagen IV, nidogen-1, and laminin-beta2. Tenascin-C localized to the subepithelial BMs of the utricular maculae and cristae ampullares, and to calyx-like profiles throughout the vestibular epithelium, but not to the perineural and perivascular BMs. alpha-Dystroglycan colocalized with aquaporin-4 in the basal vestibular supporting cell, and was also expressed in the subepithelial BMs, as well as perivascular and perineural BMs. This study provides the first comprehensive immunolocalization of these ECM proteins in the human inner ear. The validity of the rodent models for inner ear disorders secondary to BM pathologies was confirmed as there is a high degree of conservation of expression of these proteins in the human inner ear. This information is critical to begin to unravel the role that BMs may play in human inner ear physiology and audiovestibular pathologies.

Reduced expression of sarcospan in muscles of Fukuyama congenital muscular dystrophy.

Expression profiles of sarcospan in muscles with muscular dystrophies are scarcely reported. To examine this, we studied five Fukuyama congenital muscular dystrophy (FCMD) muscles, five Duchenne muscular dystrophy (DMD) muscles, five disease control and five normal control muscles. Immunoblot showed reactions of sarcospan markedly decreased in FCMD and DMD muscle extracts. Immunohistochemistry of FCMD muscles showed that most large diameter myofibers expressed sarcospan discontinuously at their surface membranes. Immature small diameter FCMD myofibers usually did not express sarcospan. Immunoreactivity of sarcospan in DMD muscles was similarly reduced. With regard to dystroglycans and sarcoglycans, immunohistochemistry of FCMD muscles showed selective deficiency of glycosylated alpha-dystroglycan, together with reduced expression of beta-dystroglycan and alpha-, beta-, gamma-, delta-sarcoglycans. Although the expression of glycosylated alpha-dystroglycan was lost, scattered FCMD myofibers showed positive immunoreaction with an antibody against the core protein of alpha-dystroglycan. The group mean ratios of sarcospan mRNA copy number versus GAPDH mRNA copy number by real-time RT-PCR showed that the ratios between FCMD and normal control groups were not significantly different (P>0.1 by the two-tailed t test). This study implied either O-linked glycosylation defects of alpha-dystroglycan in the Golgi apparatus of FCMD muscles may lead to decreased expression of sarcoglycan and sarcospan molecules, or selective deficiency of glycosylated alpha-dystroglycan due to impaired glycosylation in FCMD muscles may affect the molecular integrity of the basal lamina of myofibers. This, in turn, leads to decreased expression of sarcoglycans, and finally of sarcospan at the FCMD myofiber surfaces.

Transduction of full-length dystrophin to multiple skeletal muscles improves motor performance and life span in utrophin/dystrophin double knockout mice.

Duchenne muscular dystrophy (DMD) is a fatal, progressive, muscle-wasting disease caused by defects in the dystrophin. No viral vector except the helper-dependent adenovirus vector (HDAdv) can package 14-kilobase (kb) full-length dystrophin complementary DNA (cDNA), and HDAdv is considerably safer than old-generation adenovirus vectors because of the large-size deletion in its genome. We have generated HDAdv that carries myc-tagged murine full-length dystrophin cDNA (HDAdv-myc-mFLdys). We injected it into multiple proximal muscles of 7-day-old utrophin/dystrophin double knockout mice (dko mice) (which typically show symptoms quite similar to human DMD) because the proximal muscles are affected in DMD patients. Eight weeks after the injections, the transduced dystrophin was widely expressed, and we found a significant reduction in centrally nucleated myofibers and the restoration of the dystrophin-associated proteins, beta-dystroglycan (beta-DG) and alpha-sarcoglycan (alpha-SG), as well as neuronal nitric oxide synthase (nNOS). The injected dko mice also showed an increase in body weight, an improvement in motor performance, and a prolongation of life span. Using HDAdv, we could treat DMD model mice even by transferring the therapeutic gene into multiple skeletal muscles. Our results suggest that multiple intramuscular administrations of HDAdv carrying full-length dystrophin cDNA may reduce symptoms and compensate for lost functions in DMD patients.

Alpha-dystroglycan is involved in positive selection of thymocytes by participating in immunological synapse formation.

Alpha-dystroglycan has been proved to be involved in lymphocyte activation by participating in immunological synapse (IS) formation. Considering the existence of IS formation in thymic development, we questioned whether alpha-dystroglycan was expressed in thymus and influenced thymic development. In this study, we demonstrated that alpha-dystroglycan was expressed on fetal thymocytes, especially on double-positive (DP, CD4(+)CD8(+)) cells. Blocking alpha-dystroglycan by treatment of fetal thymus organ culture (FTOC) with anti-alpha-dystroglycan antibody IIH6C4 decreased the number of DP cells compared with nontreated or isotype antibody controls. Down-regulation of alpha-dystroglycan by retroviruses carrying antisense cDNA of dystroglycan in reaggregate thymus organ culture (RTOC) further confirmed these results. Enhanced apoptosis of DP cells was observed after blocking alpha-dystroglycan. Interestingly, we found that blocking alpha-dystroglycan reduced IS formation between DP cells and thymic epithelial cells. Furthermore, blocking alpha-dystroglycan up-regulated CD95/CD95L expression and reduced Bcl-2 expression on DP cells in the developing thymus. Finally, the increase in the apoptosis of DP cells was associated with a consequent decrease in the positive selection, as indicated by the reduction of both ERK phosphorylation in DP cells and single-positive (SP, CD4(+) or CD8(+)) cell outcome. Altogether, these results indicated that alpha-dystroglycan was involved in positive selection of thymocytes by participating in the IS formation.

The rapid decrease in astrocyte-associated dystroglycan expression by focal cerebral ischemia is protease-dependent.

During focal cerebral ischemia, the detachment of astrocytes from the microvascular basal lamina is not completely explained by known integrin receptor expression changes. Here, the impact of experimental ischemia (oxygen-glucose deprivation (OGD)) on dystroglycan expression by murine endothelial cells and astrocytes grown on vascular matrix laminin, perlecan, or collagen and the impact of middle cerebral artery occlusion on alphabeta-dystroglycan within cerebral microvessels of the nonhuman primate were examined. Dystroglycan was expressed on all cerebral microvessels in cortical gray and white matter, and the striatum. Astrocyte adhesion to basal lamina proteins was managed in part by alpha-dystroglycan, while ischemia significantly reduced expression of dystroglycan both in vivo and in vitro. Furthermore, dystroglycan and integrin alpha6beta4 expressions on astrocyte end-feet decreased in parallel both in vivo and in vitro. The rapid loss of astrocyte dystroglycan during OGD appears protease-dependent, involving an matrix metalloproteinase-like activity. This may explain the rapid detachment of astrocytes from the microvascular basal lamina during ischemic injury, which could contribute to significant changes in microvascular integrity.

Dystroglycan protein distribution coincides with basement membranes and muscle differentiation during mouse embryogenesis.

Using immunohistochemistry, we have examined beta-Dystroglycan protein distribution in the mouse embryo at embryonic stages E9.5 to E11.5. Our data show that Dystroglycan expression correlates with basement membranes in many tissues, such as the notochord, neural tube, promesonephros, and myotome. In the myotome, we describe the timing of Dystroglycan protein re-distribution at the surface of myogenic precursor cells as basement membrane assembles into a continuous sheet. We also report on non-basement-membrane-associated Dystroglycan expression in the floor plate and the myocardium. This distribution often corresponds to sites of expression previously reported in adults, suggesting that Dystroglycan is continuously produced during development.

Altered expression of natively glycosylated alpha dystroglycan in pediatric solid tumors.

Altered glycosylation and/or expression of dystroglycan have been reported in forms of congenital muscular dystrophy as well as in cancers of the breast, colon, and oral epithelium. To date, however, there has been no study of the expression of dystroglycan in pediatric solid tumors. Using a combination of immunostaining on tissue microarrays and immunoblotting of snap-frozen unfixed tissues, we demonstrate a significant reduction in native alpha dystroglycan expression in pediatric alveolar rhabdomyosarcoma (RMS), embryonal RMS, neuroblastoma (NBL), and medulloblastoma, whereas expression of beta dystroglycan, which is cotranslated with alpha dystroglycan, is largely unchanged. Loss of native alpha dystroglycan expression was significantly more pronounced in stage 4 NBL than in pooled samples of stage 1 and stage 2 NBL, suggesting that loss of native alpha dystroglycan expression increases with advancing tumor stage. Neuroblastoma and RMS samples with reduced expression of native alpha dystroglycan also showed reduced laminin binding in laminin overlay experiments. Expression of natively glycosylated alpha dystroglycan was not altered in several other pediatric tumor types when compared with appropriate normal tissue controls. These data provide the first evidence that alpha dystroglycan glycosylation and laminin binding to alpha dystroglycan are altered in certain pediatric solid tumors and suggest that aberrant dystroglycan glycosylation may contribute to tumor cell biology in patients with RMS, medulloblastoma, and NBL.

Aberrant expression of alpha-dystroglycan in cervical and vulvar cancer.

OBJECTIVES: Cervical and vulvar cancers develop through well-defined precursor lesions but their exact pathogenesis is still unknown. The dystroglycan complex is a transmembrane glycoprotein that forms a continuous link from the extracellular matrix to the actin cytoskeleton. Deregulated expression of dystroglycan has been reported in human malignancies and related to tumor differentiation and aggressiveness. In this study, expression of dystroglycan was evaluated in the multistep cervical and vulvar tumorigenesis. METHODS: Expression of the dystroglycan complex was evaluated by immunostaining in lesions representing different stages of vulvar and cervical tumorigenesis using a monoclonal antibody which recognizes carbohydratic epitopes on the alpha-dystroglycan subunit. RESULTS: alpha-dystroglycan was constantly detected in normal cervical epithelium with a mean percentage of positive cells higher than 80%. A progressive significant reduction in the mean percentage of positive cells was observed in low (67%) and high grade SIL (14%) and in invasive carcinomas (2.6%) of the cervix. In cancers, no differences were observed in terms of percentage of positive cells when cases were stratified according with either tumor grade or stage. A progressive significant reduction in the mean percentage of positive cells was also observed from normal vulvar epithelium (90%) to VIN1 (66%), VIN2 (28%) and invasive vulvar carcinomas (22%). No significant decrease in the alpha-dystroglycan staining was observed in squamous cell hyperplasia lesions (85%) while lichen sclerosus displayed a percentage of positive cells (47%) significantly lower than normal epithelium. CONCLUSIONS: Detection of alpha-dystroglycan is frequently lost in human cervical and vulvar tumorigenesis and further studies are warranted to verify whether evaluation of this molecule might serve as marker of risk progression of preneoplastic lesions and to better understand its significance in terms of cancer development.