Nintedanib treatment delays prostate dorsolateral lobe cancer progression in the TRAMP model: contribution to the epithelial-stromal interaction balance.

Prostate cancer (PCa) progression mechanism has been linked to epithelial proliferation, tumor invasion ability, and growth factors. Nintedanib (BIBF 1120) has been reported as being FGF and VEGF pathway inhibitors, exhibiting antitumor activity. Thus, the objective herein was to characterize the early Nintedanib treatment effects on the structure and molecules involved in the basal membrane, the extracellular matrix (ECM) maintenance, in addition to the angiogenesis and mitogenic processes at different grades of prostatic tumor development in TRAMP mice. Therefore, 45 male TRAMP mice were divided into control groups: 8-week-old mice (TC8), 12-week-old mice (TC12), and 16-week-old mice (TC16); and treated groups with 10 mg/kg/day Nintedanib dose for 4 weeks. The treated groups were euthanized at 12 (TN12) and 16 (TN16) weeks of age. Samples from the dorsolateral lobe were collected and processed for light microscopy, immunohistochemistry, Western blotting, and microvessel density analysis. The results showed that early Nintedanib treatment led to an increase of healthy epithelium frequency and a reduction of LGPIN and a maximum vascularization density in the TN12 group. Also, treatment led to a well-differentiated adenocarcinoma decrease and an α and ß dystroglycan and also laminin 1 increase in the TN16 group. IGFR1 decreased in the TN16 group. To conclude, early Nintedanib treatment led to a reduction in cancer severity, interfering in both ECM compounds and angiogenesis process to then contribute to a balance, not only in the prostatic epithelium and stroma, but also in the epithelial-stromal interaction during PCa progression.

Correlation Between Extravasation and Alterations of Cerebrovascular Laminin and ß-Dystroglycan Immunoreactivity Following Cryogenic Lesions in Rats.

The blood-brain barrier becomes "leaky" following lesions. Former studies revealed that following lesions the immunoreactivity of cerebrovascular laminin becomes detectable whereas that of ß-dystroglycan disappears. These alterations may be indicators of glio-vascular decoupling that may result in the impairment of the blood-brain-barrier. This study investigates correlation between the post-lesion extravasation and the above-mentioned immunohistochemical alterations. Following cryogenic lesions, the survival periods lasted 5, 10, 30 minutes, 1 or 12 hours, or 1 day. Some brains were fixed immediately post-lesion. Immunofluorescent reactions were performed in floating sections. The extravasation was detected with immunostaining for plasma fibronectin and rat immunoglobulins. When the survival period was 30 minutes or longer, the area of extravasation corresponded to the area of altered laminin and ß-dystroglycan immunoreactivities. Following immediate fixation some laminin immunoreactivity was already detected. The extravasation seemed to precede this early appearance of laminin immunoreactivity. The ß-dystroglycan immunoreactivity disappeared later. When the extravasation spread into the corpus callosum, vascular laminin immunoreactivity appeared but the ß-dystroglycan immunoreactivity persisted. It seems that extravasation separates the glial and vascular basal laminae, which results in the appearance of laminin immunoreactivity. The disappearance of ß-dystroglycan immunoreactivity is neither a condition nor an inevitable consequence of the 2 other phenomena.

A High-Throughput Assay for the Detection of α-Dystroglycan N-Terminus in Human Uterine Fluid to Determine Uterine Receptivity.

Embryo implantation requires a healthy embryo and a receptive uterus. In women, the uterus remains a hostile environment and must undergo functional changes to convert to a receptive state for embryo implantation. Determining uterine receptivity is vital in IVF treatment, as the timing of embryo transfer needs to be synchronized with uterine receptivity. However, to date, no reliable biochemical tests are available to determine uterine receptivity. We recently established that removal of α-dystroglycan N-terminus (α-DG-N) from the uterine surface plays an important role in the establishment of uterine receptivity. Importantly, the α-DG-N removed from the uterine tissue enters into the uterine fluid, and the levels correlate with the tissue status of receptivity. Detection of α-DG-N in uterine fluid may therefore provide a nonsurgical approach to assess uterine receptivity. In this study, we first validated three monoclonal antibodies raised against α-DG-N in our system, and then established a sandwich ELISA suitable for the detection of α-DG-N in human uterine fluid. This ELISA detected significantly higher concentrations of α-DG-N in uterine fluid of women in the receptive phase. We believe this newly established α-DG-N ELISA may provide an important tool in the development of noninvasive strategies to detect uterine receptivity in women.

Urologic and gastrointestinal symptoms in the dystroglycanopathies.

OBJECTIVE: To determine the frequency of urologic and gastrointestinal (GI) symptoms in a cohort of individuals with dystroglycanopathy compared with healthy household controls. METHODS: Participants in a North American dystroglycanopathy natural history study (NCT00313677) and other members of their households completed a questionnaire modified from validated instruments and clinical criteria. Urologic and GI symptom frequency, effect on patient life, and medications taken for these symptoms were assessed. Those younger than 4 years or not toilet trained were excluded. Healthy human bladder, esophagus, and duodenum from surgical specimens were immunostained for glycosylated α-dystroglycan. RESULTS: Thirty of 58 potential participants with dystroglycanopathy (51.7%) and 16 household controls participated. Subjects were aged 6 to 51 years (mean 26.7); 60.0% were female. Controls were aged 7 to 55 years (mean 34.6); 56.3% were female. The dystroglycanopathy cohort had higher frequency of urinary voiding symptoms (p = 0.02), higher urologic symptom scores (p = 0.05), and higher dysphagia symptom scores (p = 0.04). A correlation existed between urologic symptom score and effect on life (r = 0.71; 95% confidence interval 0.46, 0.85; p < 0.0001) and between dysphagia symptom score and effect on life (r = 0.72; 95% confidence interval 0.48, 0.86; p < 0.0001). Glycosylated α-dystroglycan was present in visceral smooth muscle of all normal tissues analyzed. CONCLUSIONS: Urologic symptoms and dysphagia are reported more frequently by individuals with dystroglycanopathies than by household controls. These symptoms can cause a perceived negative effect on patient life. Our results suggest urologic and GI dysfunction may be part of the dystroglycanopathy phenotype, and that questions about these symptoms should be incorporated into routine care because they may influence medical management.

A new monoclonal antibody DAG-6F4 against human alpha-dystroglycan reveals reduced core protein in some, but not all, dystroglycanopathy patients.

We generated a novel monoclonal antibody, DAG-6F4, against alpha-dystroglycan which immunolabels the sarcolemma in human muscle biopsies. Its seven amino-acid epitope, PNQRPEL, was identified using phage-displayed peptides and is located immediately after the highly-glycosylated mucin domain of alpha-dystroglycan. On Western blots of recombinant alpha-dystroglycan, epitope accessibility was reduced, but not entirely prevented, by glycosylation. DAG-6F4 immunolabelling was markedly reduced in muscle biopsies from Duchenne muscular dystrophy patients consistent with disruption of the dystroglycan complex. In a range of dystroglycanopathy patients with reduced/altered glycosylation, staining by DAG-6F4 was often less reduced than staining by IIH6 (antibody against the glycan epitope added by LARGE and commonly used to identify glycosylated alpha-dystroglycan). Whereas IIH6 was reduced in all patients, DAG-6F4 was hardly changed in a LARGE patient, less reduced than IIH6 in limb-girdle muscular dystrophy type 2I, but as reduced as IIH6 in some congenital muscular dystrophy patients. Although absence of the LARGE-dependent laminin-binding site appears not to affect alpha-dystroglycan stability at the sarcolemma, the results suggest that further reduction in aDG glycosylation may reduce its stability. These studies suggest that DAG-6F4 may be a useful addition to the antibody repertoire for evaluating the dystroglycan complex in neuromuscular disorders.

Correlation of deregulated like-acetylglucosaminyl transferase and aberrant α-dystroglycan expression with human tongue cancer metastasis.

PURPOSE: The present study examined the correlation of α-dystroglycan (α-DG) expression and like-acetylglucosaminyl transferase (LARGE) with metastasis of human tongue cancer. MATERIALS AND METHODS: Fifty human tongue cancer tissues and 2 tongue squamous cell carcinoma cell lines (CAL27 and SCC4) were involved. Immunohistochemistry was used to detect the expression of α-DG and LARGE. Methylation-specific polymerase chain reaction was performed to assess the methylation status of the LARGE gene promoter. CAL27 and SCC4 cells were transfected with exogenous LARGE and treated with 5-aza-2'-deoxycytidine (Aza-dC), respectively. Glycol sites of α-DG were detected by western blotting. In addition, the laminin overlay assay, cell adhesion assay, and invasion assay were performed. RESULTS: Immunohistochemical results showed that decreased expression of VIA4-1 and IIH6 (antibodies that recognize the glycol sites of α-DG) were correlated with the lymph node metastasis of tongue cancer (n = 50; P = .016 and .025, respectively). Decreased LARGE expression and hypermethylation of the LARGE gene promoter were correlated with lymph node metastasis and α-DG glycosylation in human tongue cancer (n = 50; P = .043 and .015 respectively). In addition, LARGE overexpression and Aza-dC treatment actively led to restoration of functional α-DG expression, elevation of laminin binding, and decrease of migratory ability in cancer cells. CONCLUSION: The results suggested that absent α-DG expression and LARGE deregulation were closely associated with nodal metastasis of tongue cancer. Aberrant α-DG expression and glycosylation were attributed at least in part to the abnormal epigenetic modification of LARGE, especially the hypermethylation of its promoter.

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.

Identification of new dystroglycan complexes in skeletal muscle.

The dystroglycan complex contains the transmembrane protein ß-dystroglycan and its interacting extracellular mucin-like protein α-dystroglycan. In skeletal muscle fibers, the dystroglycan complex plays an important structural role by linking the cytoskeletal protein dystrophin to laminin in the extracellular matrix. Mutations that affect any of the proteins involved in this structural axis lead to myofiber degeneration and are associated with muscular dystrophies and congenital myopathies. Because loss of dystrophin in Duchenne muscular dystrophy (DMD) leads to an almost complete loss of dystroglycan complexes at the myofiber membrane, it is generally assumed that the vast majority of dystroglycan complexes within skeletal muscle fibers interact with dystrophin. The residual dystroglycan present in dystrophin-deficient muscle is thought to be preserved by utrophin, a structural homolog of dystrophin that is up-regulated in dystrophic muscles. However, we found that dystroglycan complexes are still present at the myofiber membrane in the absence of both dystrophin and utrophin. Our data show that only a minority of dystroglycan complexes associate with dystrophin in wild type muscle. Furthermore, we provide evidence for at least three separate pools of dystroglycan complexes within myofibers that differ in composition and are differentially affected by loss of dystrophin. Our findings indicate a more complex role of dystroglycan in muscle than currently recognized and may help explain differences in disease pathology and severity among myopathies linked to mutations in DAPC members.

Parietal epithelial cell activation marker in early recurrence of FSGS in the transplant.

BACKGROUND AND OBJECTIVES: Podocyte loss is key in glomerulosclerosis. Activated parietal epithelial cells are proposed to contribute to pathogenesis of glomerulosclerosis and may serve as stem cells that can transition to podocytes. CD44 is a marker for activated parietal epithelial cells. This study investigated whether activated parietal epithelial cells are increased in early recurrent FSGS in transplant compared with minimal change disease. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: CD44 staining in renal allograft biopsies from 12 patients with recurrent FSGS was performed and compared with native kidneys with minimal change disease or FSGS and normal control native and transplant kidneys without FSGS. CD44+ epithelial cells along Bowman's capsule in the parietal epithelial cell location and over the glomerular tuft in the visceral epithelial cell location were assessed. RESULTS: Cases with early recurrent FSGS manifesting only foot process effacement showed significantly increased CD44+ visceral epithelial cells involving 29.0% versus 2.6% of glomeruli in minimal change disease and 0% in non-FSGS transplants. Parietal location CD44 positivity also was numerically increased in recurrent FSGS. In later transplant biopsies, glomeruli with segmental lesions had more CD44+ visceral epithelial cells than glomeruli without lesions. CONCLUSIONS: Parietal epithelial cell activation marker is significantly increased in evolving FSGS versus minimal change disease, and this increase may distinguish early FSGS from minimal change disease. Whether parietal epithelial cell activation contributes to pathogenesis of sclerosis in idiopathic FSGS or is a regenerative/repair response to replace injured podocytes awaits additional study.

Steroid hormone receptors, matrix metalloproteinases, insulin-like growth factor, and dystroglycans interactions in prostatic diseases in the elderly men.

OBJECTIVES: The aim of this study was to evaluate the reactivity of steroid hormone receptors (SHRs), dystroglycans (DGs), matrix metalloproteinases (MMPs), insulin-like growth factor receptor (IGFR-1), and laminin (Lam) in both prostatic stromal and epithelial compartments showing different diseases in elderly men. METHODS: Sixty prostatic samples were obtained from 60- to 90-year-old patients (mean 63 years) with and without prostatic lesions from Hospital of the School of Medicine, State University of Campinas (UNICAMP). The Samples were divided into standard (no lesions); high grade prostatic intraepithelial neoplasia (HGPIN); prostatic cancer (PC); and benign prostatic hyperplasia (BPH) groups. The samples were submitted to immunohistochemistry and Western blotting analyses. Research Ethics Committee of the School of Medicine, University of Campinas/UNICAMP (number 0094.0.146.000-08). RESULTS: The results showed increased IGFR-1 and MMPs protein levels in the PC and HGPIN groups. Decreased αDG and ßDG protein levels were verified in the PC and HGPIN groups. Androgen receptor (AR) reactivity was similar among all groups. Estrogen receptor α (Erα) immunoreactivity was more intense in the epithelium in the PC and HGPIN groups. Estrogen receptor ß (ERß) immunoreactivity was weak in the epithelium of the HGPIN and PC groups. CONCLUSIONS: To conclude, there was an association among IGFR-1, MMPs, and SHRs, indicating IGFR-1 as a target molecule in prostate therapy, considering the IGF proliferative properties. Also, the distinct SHRs reactivities in the lesions in both prostatic compartments indicated different paracrine signals and pointed out the importance of estrogenic pathways in the activation of these disorders.

Differential expression of perlecan receptors, α-dystroglycan and integrin ß1, before and after invasion of oral squamous cell carcinoma.

OBJECTIVES: The deposition of perlecan, a heparan sulfate proteoglycan, is enhanced within oral carcinoma in situ (CIS) foci, while it dynamically switches from CIS foci to the stromal space in squamous cell carcinoma (SCC). Because α-dystroglycan and integrin ß1 have been identified as two of the perlecan receptors, we wanted to determine their differential distributions before and after invasion of oral SCC. METHODS: Eighty-two surgical tissue specimens of oral SCC containing different precancerous stages were examined by immunohistochemistry for perlecan, α-dystroglycan, integrin ß1, and Ki-67. In addition, α-dystroglycan mRNA signals were localized by in situ hybridization. RESULTS: In normal epithelia, α-dystroglycan and integrin ß1 were localized on the cell membrane of basal cells, while perlecan was faintly present in the intercellular spaces of parabasal cells. In epithelial dysplasia and CIS, α-dystroglycan and perlecan were well co-localized in the epithelial layer, especially in its lower half, and this co-localization was mostly overlapped with Ki-67-positive (+) cell zones. However, in SCC, α-dystroglycan was localized neither within carcinoma cell nests nor in the stroma, while perlecan disappeared from SCC foci but emerged in the stromal space, leaving integrin ß1+ and Ki-67+ cells only to the periphery of SCC foci. α-Dystroglycan mRNA signals were basically identical to the α-dystroglycan protein localizations. CONCLUSION: The findings suggest that α-dystroglycan and integrin ß1 act as perlecan receptors in oral precancerous lesions prior to invasion, and that the perlecan signals via the two different receptors function in cellular differentiation and proliferation of CIS cells, respectively.

Glial dystrophin-associated proteins, laminin and agrin, are downregulated in the brain of mdx mouse.

In this study, we investigated the involvement of dystrophin-associated proteins (DAPs) and their relationship with the perivascular basement membrane in the brains of mdx mice and controls at the age of 2 months. We analyzed (1) the expression of glial DAPs α-ß-dystroglycan (DG), α-syntrophin, aquaporin-4 (AQP4) water channel, Kir 4.1 and dystrophin isoform (Dp71) by immunocytochemistry, laser confocal microscopy, immunogold electron microscopy, immunoblotting and RT-PCR; (2) the ultrastructure of the basement membrane and expression of laminin and agrin; and (3) the dual immunofluorescence colocalization of AQP4/α-ß-DG, and of Kir 4.1/agrin. The following results were observed in mdx brain as compared with controls: (1) a significant reduction in protein content and mRNA expression of DAPs; (2) ultrastructurally, a thickened and discontinuous appearance of the basement membrane and a significant reduction in laminin and agrin; and (3) a molecular rearrangment of α-ß-DG, coupled with a parallel loss of agrin and Kir 4.1 on basement membrane and glial endfeet. These data indicate that in mdx brain the deficiency in dystrophin and dystrophin isoform (Dp71) is coupled with a reduction of DAP components, coupled with an altered anchoring to the basement membrane.

Characterization of site-specific O-glycan structures within the mucin-like domain of alpha-dystroglycan from human skeletal muscle.

The glycosylation of the extracellular protein alpha-dystroglycan is important for its ligand-binding activity, and altered or blocked glycosylation is associated with several forms of congenital muscular dystrophies. By immunoprecipitation and sialic acid capture-and-release enrichment strategies, we isolated tryptic glycopeptides of alpha-dystroglycan from human skeletal muscle. Nano-liquid chromatography tandem mass spectrometry was used to identify both glycopeptides and peptides corresponding to the mucin-like and C-terminal domain of alpha-dystroglycan. The O-glycans found had either Hex-O-Thr or HexNAc-O-Ser/Thr anchored structures, which were often elongated and frequently, but not always, terminated with sialic acid. The HexNAc-O-Ser/Thr, but not Hex-O-Thr glycopeptides, displayed heterogeneity regarding glycan core structures and level of glycosylation site occupancy. We demonstrate for the first time glycan attachment sites of the NeuAcHexHexNAcHex-O structure corresponding to the anticipated Neu5Acalpha3Galbeta4GlcNAcbeta2Man-O-glycan (sLacNAc-Man), within the mucin-like domain of human alpha-dystroglycan from human skeletal muscle. Twenty-five glycopeptides were characterized from human alpha-dystroglycan, which provide insight to the complex in vivo O-glycosylation of alpha-dystroglycan.

Possible molecular mechanisms underlying age-related cardiomyocyte apoptosis in the F344XBN rat heart.

Despite advances in treatment, age-related cardiac dysfunction still remains a leading cause of cardiovascular death. Recent data have suggested that increases in cardiomyocyte apoptosis may be involved in the pathological remodeling of heart. Here, we examine the effects of aging on cardiomyocyte apoptosis in 6-, 30-, and 36-month-old Fischer344 x Brown Norway F1 hybrid rats (F344XBN). Compared with 6-month hearts, aged hearts exhibited increased TdT-mediated dUTP nick end labeling-positive nuclei, caspase-3 activation, caspase-dependent cleavage of alpha-fodrin and diminished phosphorylation of protein kinase B/Akt (Thr 308). These age-dependent increases in cardiomyocyte apoptosis were associated with alterations in the composition of the cardiac dystrophin glycoprotein complex and elevated cytoplasmic IgG and albumin immunoreactivity. Immunohistochemical analysis confirmed these data and demonstrated qualitative differences in localization of dystrophin-glycoprotein complex (DGC) molecules with aging. Taken together, these data suggest that aging-related increases in cardiac apoptotic activity model may be due, at least in part, to age-associated changes in DGC structure.

Congenital muscular dystrophies with defective glycosylation of dystroglycan: a population study.

BACKGROUND: Congenital muscular dystrophies (CMD) with reduced glycosylation of alpha-dystroglycan (alpha-DG) are a heterogeneous group of conditions associated with mutations in six genes encoding proven or putative glycosyltransferases. OBJECTIVES: The aim of the study was to establish the prevalence of mutations in the six genes in the Italian population and the spectrum of clinical and brain MRI findings. METHODS: As part of a multicentric study involving all the tertiary neuromuscular centers in Italy, FKRP, POMT1, POMT2, POMGnT1, fukutin, and LARGE were screened in 81 patients with CMD and alpha-DG reduction on muscle biopsy (n = 76) or with a phenotype suggestive of alpha-dystroglycanopathy but in whom a muscle biopsy was not available for alpha-DG immunostaining (n = 5). RESULTS: Homozygous and compound heterozygous mutations were detected in a total of 43/81 patients (53%), and included seven novel variants. Mutations in POMT1 were the most prevalent in our cohort (21%), followed by POMT2 (11%), POMGnT1 (10%), and FKRP (9%). One patient carried two heterozygous mutations in fukutin and one case harbored a new homozygous variant in LARGE. No clear-cut genotype-phenotype correlation could be observed with each gene, resulting in a wide spectrum of clinical phenotypes. The more severe phenotypes, however, appeared to be consistently associated with mutations predicted to result in a severe disruption of the respective genes. CONCLUSIONS: Our data broaden the clinical spectrum associated with mutations in glycosyltransferases and provide data on their prevalence in the Italian population.

Proteomic profiling of x-linked muscular dystrophy.

Progressive x-linked muscular dystrophy represents the most commonly inherited neuromuscular disorder in humans. Although the disintegration of the dystrophin-associated glycoprotein complex triggers the initial pathogenesis of Duchenne muscular dystrophy, secondary alterations in metabolic pathways, cellular signaling and the regulation of ion homeostasis are probably crucial factors that cause end-stage fibre degeneration. The application of mass spectrometry-based proteomics for the global cataloguing of muscle biomarkers has recently been applied to the analysis of the mdx animal model of muscular dystrophy and the biochemical evaluation of experimental exon skipping therapy. The fluorescence difference in-gel electrophoretic analysis of normal versus mdx diaphragm muscle revealed changed expression levels of proteins involved in nucleotide metabolism, Ca 2+-handling, the cellular stress response and key bioenergetic processes. The swift up-regulation of small heat shock proteins, such as cvHsp, seems to form an integral part of the repair mechanisms in dystrophic fibres and may be exploitable as a new option to treat inherited muscle degeneration. Importantly, the mass spectrometry-based profiling of mdx muscle following the specific removal of exon 23 in the mutated dystrophin gene transcript showed a partial reversal of important secondary changes. Experimental exon skipping restored the expression of the dystrophin isoform Dp427, its associated glycoprotein beta-dystroglycan, neuronal nitric oxide synthase, calsequestrin, adenylate kinase and the muscle-specific stress protein cvHsp. In the future, a well defined set of signature molecules could be used to improve diagnosis, monitor disease progression, identify new therapeutic pathways, and validate the effects of novel drugs or experimental treatments such as gene therapy.

Genes required for functional glycosylation of dystroglycan are conserved in zebrafish.

Mutations in human genes encoding proteins involved in alpha-dystroglycan glycosylation result in dystroglycanopathies: severe congenital muscular dystrophy phenotypes often accompanied by CNS abnormalities and ocular defects. We have identified the zebrafish orthologues of the seven known genes in this pathway and examined their expression during embryonic development. Zebrafish Large, POMT1, POMT2, POMGnT1, Fukutin, and FKRP show in situ hybridization patterns similar to those of dystroglycan, with broad expression throughout early development. By 30 h postfertilization (hpf), transcripts of all these genes are most prominent in the CNS, eye, and muscle, tissues that are predominantly affected in the dystroglycanopathies. In contrast, Large2 expression is more restricted and by 30 hpf is confined to the lens, cerebellum, and pronephric duct. We show that the monoclonal antibody IIH6, which recognizes a glycoform of dystroglycan, also detects the zebrafish protein. Injection of morpholino oligonucleotides against zebrafish Large2 resulted in loss of IIH6 immunostaining. These data indicate that the dystroglycan glycosylation pathway is conserved in zebrafish and suggest this organism is likely to be a useful model system for functional studies.

Developmental defects in a zebrafish model for muscular dystrophies associated with the loss of fukutin-related protein (FKRP).

A number of muscular dystrophies are associated with the defective glycosylation of alpha-dystroglycan and many are now known to result from mutations in a number of genes encoding putative or known glycosyltransferases. These diseases include severe forms of congenital muscular dystrophy (CMD) such as Fukuyama type congenital muscular dystrophy (FCMD), Muscle-Eye-Brain disease (MEB) and Walker-Warburg syndrome (WWS), which are associated with brain and eye abnormalities. The defective glycosylation of alpha-dystroglycan in these disorders leads to a failure of alpha-dystroglycan to bind to extra-cellular matrix components and previous attempts to model these disorders have shown that the generation of fukutin- and Pomt1-deficient knockout mice results in early embryonic lethality due to basement membrane defects. We have used the zebrafish as an animal model to investigate the pathological consequences of downregulating the expression of the putative glycosyltransferase gene fukutin-related protein (FKRP) on embryonic development. We have found that downregulating FKRP in the zebrafish results in embryos which develop a range of abnormalities reminiscent of the developmental defects observed in human muscular dystrophies associated with mutations in FKRP. FKRP morphant embryos showed a spectrum of phenotypic severity involving alterations in somitic structure and muscle fibre organization as well as defects in developing neuronal structures and eye morphology. The pathological phenotype was found to correlate with a reduction in alpha-dystroglycan glycosylation and reduced laminin binding. Further characterization of the developmental processes affected in FKRP morphant embryos may lead to a better understanding of the pathological spectrum observed in muscular dystrophies associated with mutations in the human FKRP gene.

Targeting of dystroglycan to the cleavage furrow and midbody in cytokinesis.

Dystroglycan is a cell adhesion molecule that interacts with ezrin family proteins and also components of the extracellular signal-regulated kinase pathway. Ezrin and extracellular signal-regulated kinase are both involved in aspects of the cell division cycle. We therefore examined the role of dystroglycan during cytokinesis. Endogenous dystroglycan colocalised with ezrin at the cleavage furrow and midbody during cytokinesis in REF52 cells. Live cell imaging of green fluorescent protein-tagged dystroglycan in Swiss 3T3 and Hela cells revealed a similar localisation. Live cell imaging of a dystroglycan lacking its cytoplasmic domain revealed an even membrane localisation but no cleavage furrow or midbody localisation. Deletion of a previously identified ezrin-binding site in the dystroglycan cytoplasmic domain however only resulted in a slight reduction in cleavage furrow localisation but loss of midbody staining. There was no apparent cytokinetic defect in cells depleted for dystroglycan, however apoptosis levels were considerably higher in dystroglycan knockdown cells. Cell cycle analysis showed a delay in G2/M transition, possibly caused by a more than 50% reduction in extracellular signal-regulated kinase levels in the knockdown cells. Dystroglycan may therefore not only have a role in organising the contractile ring through direct or indirect associations with actin, but can also modulate the cell cycle by affecting extracellular signal-regulated kinase levels.

Expression of the dystrophin-glycoprotein complex is a marker for human airway smooth muscle phenotype maturation.

Airway smooth muscle (ASM) cells may contribute to asthma pathogenesis through their capacity to switch between a synthetic/proliferative and a contractile phenotype. The multimeric dystrophin-glycoprotein complex (DGC) spans the sarcolemma, linking the actin cytoskeleton and extracellular matrix. The DGC is expressed in smooth muscle tissue, but its functional role is not fully established. We tested whether contractile phenotype maturation of human ASM is associated with accumulation of DGC proteins. We compared subconfluent, serum-fed cultures and confluent cultures subjected to serum deprivation, which express a contractile phenotype. Western blotting confirmed that beta-dystroglycan, beta-, delta-, and epsilon-sarcoglycan, and dystrophin abundance increased six- to eightfold in association with smooth muscle myosin heavy chain (smMHC) and calponin accumulation during 4-day serum deprivation. Immunocytochemistry showed that the accumulation of DGC subunits was specifically localized to a subset of cells that exhibit robust staining for smMHC. Laminin competing peptide (YIGSR, 1 microM) and phosphatidylinositol 3-kinase (PI3K) inhibitors (20 microM LY-294002 or 100 nM wortmannin) abrogated the accumulation of smMHC, calponin, and DGC proteins. These studies demonstrate that the accumulation of DGC is an integral feature for phenotype maturation of human ASM cells. This provides a strong rationale for future studies investigating the role of the DGC in ASM smooth muscle physiology in health and disease.