Kevin P. Campbell, Ph.D, University of Rochester 1976 - US grants

The overall goal of my research is to understand the structure and function of the junctional sarcoplasmic reticulum membrane of skeletal and cardiac muscle. My approach to the study of this specialized region of the sarcoplasmic reticulum is through purification and characterization of the protein constituents of this membrane. In skeletal muscle, the sarcoplasmic reticulum consists of three major proteins: Ca++ + Mg++ ATPase, calsequestrin and Mr 53,000 glycoprotein. Indirect immunofluorescence has shown us that both calsequestrin and Mr 53,000 glycoprotein are localized in the region of the junctional sarcoplasmic reticulum membrane. The proposed research concerns the structure, function and morphology of the Mr 53,000 glycoprotein. Lectin affinity chromatography will be used to purify the Mr 53,000 glycoprotein from rabbit skeletal muscle sarcoplasmic reticulum. Limited proteolysis will be performed to fragment the glycoprotein into two or three fragments which will then be purified using SDS chromatrography. Amino acid, carbohydrate, N-terminal sequence and COOH-terminal sequence analysis will be performed on purified fragments and the proteolytic fragments will be aligned NH2-terminal to COOH-terminal. Location of the sites of carbohydrate attachment, the site(s) of 8-N3-ATP labeling and the cytoplasmic associated domains in the proteolytic fragments of Mr 53,000 glycoprotein will be defined. Characterization of 8-N3-ATP binding will be completed and the functional role of the ATP binding site on the Mr 53,000 glycoprotein will be investigated. Ca++ binding properties of the Mr 53,000 glycoprotein will also be determined. The Mr 160,000 glycoprotein of the sarcoplasmic reticulum will be purified and compared to the Mr 53,000 glycoprotein. Experiments will be performed to test the hypothesis that the Mr 160,000 glycoprotein is a product of an inter-polypeptide cross-link between the Mr 53,000 glycoprotein and a sarcoplasmic reticulum or transverse tubular protein. Indirect immunoferritin labeling of ultrathin frozen sections will determine the ultrastructural localization of the Mr 53,000 glycoprotein in adult rat skeleta and papillary muscle. Finally, a preliminary characterization of the Mr 53,000 glycoprotein from canine cardiac sarocplasmic reticulum will be performed.

The overall goal of this project is the structural and functional characterization of the molecular components of the dihydropyridine receptor of the cardiac Ca2+ channel. In order to achieve this objective, we are developing a library of monoclonal antibodies to the dihydropyridine receptor of the cardiac Ca2+ channel. Identification and structural characterization of the protein components of the dihydropyridine receptor of the cardiac Ca2+ channel will be performed by indirect immunoperoxidase staining of protein blots of sarcolemma and by immunoprecipitation of 125I-labeled sarcolemma proteins. Structural characterization of the dihydropyridine receptor will involve proteolytic and glycosidase treatments of right-side-out and inside-out sarcolemma vesicles. The aim of these studies will be to determine the molecular weight and subunit composition of dihydropyridine receptor and the arrangement and orientation of the protein components of the dihydropyridine receptor of the cardiac Ca2+ channel. Ultrastructural localization of the dihydropyridine receptor of the cardiac Ca2+ channel using indirect immunogold labeling with monoclonal antibodies will be performed on canine ventricular muscle and isolated cardiac sarcolemma membranes. The aim of the ultrastructural studies will be to determine the distribution of the dihydropyridine receptor and to examine the arrangement and orientation of the molecular components of the dihydropyridine receptor in the sarcolemma membrane of canine cardiac muscle. Purification of the dihydropyridine receptor of the cardiac Ca2+ channel will be performed using monoclonal antibody affinity columns and/or nifedipine-Sepharose affinity columns. Subunits of the dihydropyridine receptor will be prepared by gel filtration in the presence of sodium dodecyl sulfate. The amino acid, carbohydrate and N-terminal amino acid sequence analysis will be performed on the purified receptor and/or the purified subunits. The isolation and characterization of cDNA clones for the dihydropyridine receptor will be performed using monoclonal antibodies and a library of cardiac cDNA cloned in Lambda gt11 expression vector. Functional characterization of the purified dihydropyridine receptor will involve determination of the binding properties of the various radioactive Ca2+ channel blockers to the purified dihydropyridine receptor, reconstitution of the purified dihydropyridine receptor in phospholipid vesicles and phosphorylation of the purified dihydropyridine receptor. The proposed studies will lead to an understanding of the structure and function of the cardiac Ca2+ channel which initiates and modulates the transmembrane influx of extracellular calcium during excitation-contraction coupling in cardiac muscle.

The overall goal of my research is to understand the mechanism of Ca2+ release from the junctional sarcoplasmic reticulum of cardiac muscle. The specific objective of this proposal is the structural and functional characterization of the ryanodine receptor of the junctional sarcoplasmic reticulum Ca2+ release channel of cardiac muscle. In order to accomplish our objectives, we are developing a library of monoclonal antibodies to the ryanodine receptor of the Ca2+ release channel from canine cardiac sarcoplasmic reticulum. Identification of the ryanodine receptor of the Ca2+ release channel will be performed by indirect immunoperoxidase staining of protein blots of cardiac sarcoplasmic reticulum proteins. Structural characterization of the ryanodine receptor will involve immunoblotting of cardiac sarcoplasmic reticulum following proteolytic and glycosidase treatments. Ultrastructural localization of the ryanodine receptor of the Ca2+ release channel using indirect immunogold labeling with monoclonal antibodies will be performed on canine ventricular muscle. Purification of the ryanodine receptor of the Ca2+ release channel from digitonin solubilized sarcoplasmic reticulum will be performed using monoclonal antibody affinity columns. The amino acid, carbohydrate and N-terminal amino acid sequence analysis will be performed on the purified receptor. The isolation and characterization of cDNA clones for the ryanodine receptor of the Ca2+ release channel will be performed using monoclonal antibodies and a library of cardiac cDNA cloned in lambda gt11 expression vector. Our final objective will be the functional characterization of the purified receptor. The dissociation constant and maximal binding of (3H) ryanodine and 45Ca2+ to the purified ryanodine receptor will be determine. Reconstitution of the purified receptor into phospholipid vesicles and planar lipid bilayers will be performed in order to demonstrate the identical nature of the ryanodine receptor and the Ca2+ release channel of the junctional sarcoplasmic reticulum. The proposed studies will lead to an understanding of the structure and function of the ryanodine receptor of the Ca2+ channel which initiates and modulates Ca2+ release from the junctional sarcoplasmic reticulum during excitation-contraction coupling in cardiac muscle.

Autosomal recessive limb-girdle muscular dystrophy (AR-LGMD) refers to a number of genetically and clinically heterogenous neuromuscular disorders that affect mainly skeletal muscle. Over the last few years, it has become clear that a number of genes encoding protein components of the sarcoglycan complex are responsible for several forms of AR-LGMDs. The sarcoglycans are expressed at the sarcolemma of muscle fibers and, along with other proteins, constitute the dystrophin-glycoprotein complex (DGC). These proteins are believed to play a role in maintaining the normal architecture of the muscle cell membrane by constituting a link between the subsarcolemmal cytoskeleton and the extracellular matrix. In particular, we have shown that alpha-sarcoglycan, a 50 kDa component of the DGC, is a deficient in skeletal muscle from patients having limb- girdle muscular dystrophy type 2D, and that the expression of all the other sarcoglycan proteins is also strongly reduced in muscle from these patients. Although these findings constitute great progress in our understanding of the genetic basis for AR-LGMDs, there have been no improvements in the treatment of these invalidating diseases. The long-term goal of this research proposal is the development of a gene transfer strategy for AR-LGMDs. We recently generated an animal model for LGMD2D by disrupting the alpha-sarcoglycan gene in mice and preliminary analyses of homozygous mutant mice indicate that their skeletal muscle displays a dystrophic phenotype, as expected, thus providing a valuable animal mode for LGMD2D. The overall objective of this pilot project is to develop a virally-mediated gene transfer of alpha-sarcoglycan and to investigate its therapeutic potential in alpha-sarcoglycan deficient mice. Our first aim will be the construction of recombinant adenovirus and adeno-associated virus vectors containing the human alpha-sarcoglycan deficient mice. Our first aim will be the construction of recombinant adenovirus and adeno-associated virus vectors containing the human alpha- sarcoglycan cDNA. These vectors will first be tested for their ability to induce expression of alpha-sarcoglycan, both in cultured myoblasts and myotubes. We will then proceed to in vivo experiments designed to test the following hypotheses: i) direct intra-muscular injections of adenoviral- based vectors containing the alpha-sarcoglycan cDNA will efficiently allow expression of the protein and restoration of the DGC in of skeletal muscle of mutant mice (Aim 2) and ii) gene transfer of alpha-sarcoglycan will support functional restoration of muscle fibers in these mice (Aim 3). Overall, the experiments outlined in our proposal will yield new information about alpha-sarcoglycan and the potential for virally-mediated alpha-sarcoglycan gene transfer in mutant mice. In addition, our findings should constitute a foundation for future investigations directed towards developing gene therapy for LGMD2D patients.

(Adapted from the Applicant's Abstract) The ultimate aim of this research is to determine the potential role of dystroglycan dysfunction in congenital heart disease. The overall objective of this project is to investigate the function of dystroglycan in cardiac embryogenesis. Extracellular matrix components and receptors are critical for the morphogenesis of the heart and are excellent candidates for involvement in congenital heart disease including atrioventricular canal defects and perimembranous interventricular septal defects. Dystroglycan is a broadly expressed cell surface extracellular matrix receptor that is linked to the cytoskeleton. Recent studies have indicated that dystroglycan plays a critical role in myogenesis and organogenesis. To investigate the role of dystroglycan in developmental processes, the investigators disrupted the dystroglycan gene in the mouse. The null mutation results in early embryonic lethality, prior to the onset of gastrulation. This phenotype stems from the failed development of Reichert's membrane, an extraembryonic basement membrane structure in which dystroglycan is also expressed. From these studies, dystroglycan seems to be required for either the anchorage of cells to the extracellular matrix or the assembly of networks of extracellular matrix proteins. Dystroglycan's involvement with extracellular matrix and its expression in various cell types suggest that it could be involved in many aspects of heart morphogenesis. To begin to address the role of dystroglycan in cardiac embryogenesis, the investigators will first define the developmental expression of dystroglycan in the heart (Specific Aim 1). To directly examine dystroglycan's function in heart embryogenesis, the investigators have proposed experiments to circumvent the early lethality of the dystroglycan null mutation so that the investigators may analyze dystroglycan's role later during heart development (Specific Aims 2 to 4). The first specific aim will utilize normal mouse embryos to establish a map of the spatial and temporal pattern of expression of dystroglycan during heart development. The second aim will analyze the cellular role of dystroglycan in the development of cardiomyocytes in embryoid bodies. The third aim will use tetraploid complementation in order to rescue the Reichert's membrane defect and thus allow them to examine dystroglycan-null embryos that develop to later stages after the onset of heart development. The fourth aim will be the myocardium-specific disruption of the dystroglycan gene. The goal of the last two specific aims is to examine heart development in embryos that lack dystroglycan in all cells (Specific Aim 3) or just cardiomyocytes (Specific Aim 4). The complementary approaches outlined in these specific aims will yield a new understanding of the role of dystroglycan in heart development and will constitute a foundation for future investigations directed toward the identification of congenital heart disease patients with abnormalities in dystroglycan function.

DESCRIPTION (provided by applicant): The long-term goal of this project is to design a therapy for autosomal recessive limb-girdle muscular dystrophy type 2D (LGMD-2D), which is due to aberrations in the (-sarcoglycan gene (Sgca), and affects mainly limb and girdle muscles to lead to progressive muscle fiber necrosis and weakness, Alpha-Sarcoglycan interacts with beta-, gamma-, and delta-sarcoglycan to form a subcomplex that increases the overall stability of the dystrophin-glycoprotein complex (DGC). Epsilon-Sarcoglycan is a ubiquitously expressed homologue of the muscle-specific alpha- sarcoglycan, sharing 43% identity and a similar protein structure. Epsilon-Sarcoglycan expression is detected as early as day E8.5 in mouse embryos before the detection of alpha-sarcoglycan at E15, and preliminary data shows increased detection during muscle regeneration suggesting that epsilon-sarcoglycan is embryonic or developmental form of alpha-sarcoglycan. This project focuses on exploring the therapeutic potential of upregulating epsilon-sarcoglycan levels for the treatment of LGMD-2D. The First Aim hypothesizes that increased expression of epsilon-sarcoglycan will replace alpha-sarcoglycan within the DGC. Characterization of transgenic mice with targeted overexpression of epsilon-sarcoglycan to muscle will test this hypothesis. The Second Aim hypothesizes that increased expression of epsilon-sarcoglycan will compensate for an aberrant alpha-sarcoglycan to prevent the onset of muscular dystrophy. Analysis of Sgca-null mice overexpressing epsilon-sarcoglycan will test this hypothesis. The Final Aim hypothesizes that increased and sustained epsilon-sarcoglycan expression in alpha-sarcoglycan deficiency will prevent further muscle pathology and repair the primary membrane defect to allow for normal muscle function. Adult Sgca-null mice will be intramuscularly injected with rAAVl epsilon-sarcoglycan and analyzed. This project will investigate the functional and physiological consequences of increased levels of epsilon-sarcoglycan to prevent and treat muscular dystrophy in alpha-sarcoglycan deficiency. The overall results of these experiments will develop the possibility of increased epsilon-sarcoglycan expression to compensate for (-sarcoglycan in LGMD-2D patients. Focusing on an endogenous protein like epsilon-sarcoglycan will bypass any acquired therapeutic immune response, and provides a platform for new targets for therapy and drug treatments aimed at up-regulating epsilon-sarcoglycan

DESCRIPTION (provided by applicant): The long-term goal of this project is to design a therapy for autosomal recessive limb-girdle muscular dystrophy type 2D (LGMD-2D), which is due to aberrations in the (-sarcoglycan gene (Sgca), and affects mainly limb and girdle muscles to lead to progressive muscle fiber necrosis and weakness, Alpha-Sarcoglycan interacts with beta-, gamma-, and delta-sarcoglycan to form a subcomplex that increases the overall stability of the dystrophin-glycoprotein complex (DGC). Epsilon-Sarcoglycan is a ubiquitously expressed homologue of the muscle-specific alpha- sarcoglycan, sharing 43% identity and a similar protein structure. Epsilon-Sarcoglycan expression is detected as early as day E8.5 in mouse embryos before the detection of alpha-sarcoglycan at E15, and preliminary data shows increased detection during muscle regeneration suggesting that epsilon-sarcoglycan is embryonic or developmental form of alpha-sarcoglycan. This project focuses on exploring the therapeutic potential of upregulating epsilon-sarcoglycan levels for the treatment of LGMD-2D. The First Aim hypothesizes that increased expression of epsilon-sarcoglycan will replace alpha-sarcoglycan within the DGC. Characterization of transgenic mice with targeted overexpression of epsilon-sarcoglycan to muscle will test this hypothesis. The Second Aim hypothesizes that increased expression of epsilon-sarcoglycan will compensate for an aberrant alpha-sarcoglycan to prevent the onset of muscular dystrophy. Analysis of Sgca-null mice overexpressing epsilon-sarcoglycan will test this hypothesis. The Final Aim hypothesizes that increased and sustained epsilon-sarcoglycan expression in alpha-sarcoglycan deficiency will prevent further muscle pathology and repair the primary membrane defect to allow for normal muscle function. Adult Sgca-null mice will be intramuscularly injected with rAAVl epsilon-sarcoglycan and analyzed. This project will investigate the functional and physiological consequences of increased levels of epsilon-sarcoglycan to prevent and treat muscular dystrophy in alpha-sarcoglycan deficiency. The overall results of these experiments will develop the possibility of increased epsilon-sarcoglycan expression to compensate for (-sarcoglycan in LGMD-2D patients. Focusing on an endogenous protein like epsilon-sarcoglycan will bypass any acquired therapeutic immune response, and provides a platform for new targets for therapy and drug treatments aimed at up-regulating epsilon-sarcoglycan

Description (provided by applicant): Muscular dystrophies are a diverse group of inherited disorders characterized by progressive muscle weakness and wasting. The overall goal of this Muscular Dystrophy Cooperative Research Center is to explore therapeutic strategies for the treatment of various muscular dystrophies. The Center will achieve this overall goal by enabling translational research on muscular dystrophies and providing advanced diagnostic services. The MDCRC is composed of three research projects, three cores and investigators with a proven track record of excellence and collaboration. The Director and Co-director, Kevin Campbell and Steven Moore, are investigators with established records in basic, translational and clinical research on muscular dystrophy. Project 1 (Campbell and Barresi) will use mouse models to explore the therapeutic potential of improving muscle membrane maintenance and repair for the treatment of Duchenne muscular dystrophy. Project 2 (Mathews, Campbell, Weiss and Romitti) will study muscular dystrophy patients with fukutin related protein mutations and develop mouse models in order to understand the pathogenesis of this disease and possible therapeutic strategies. Project 3 (Yang, Williamson and Barresi) will study the development of embryonic stem cells as therapeutic tools for stem cell treatments. Core A (Campbell and Moore) is an administrative core which will coordinate the activities within and outside the Center and will promote an interactive and collaborative research environment. Core B (Moore and Barresi) is a Muscle Tissue/Cell Culture/Diagnostics Core that will serve as a national tissue and cell culture resource for research as well as a laboratory for patient diagnostic and post-intervention biopsy evaluation for clinical trials. Finally, Core C (Williamson and Yang), the Human ES Cell Targeting Core, will use gene targeting strategies to produce ES cell lines with specific muscular dystrophy mutations, and thus serve as a national resource of targeted stem cells.

The overall objective of this proposal is to explore the therapeutic potential of improving muscle cell membrane maintenance and repair for the treatment of Duchenne muscular dystrophy. In particular, the proposal will test these hypotheses: 1) Overexpression of the glycosyltransferase, LARGE, will stabilize the sarcolemma in mdx muscle and thus prevent or delay muscle pathology; and 2) Overexpression of dysferlin will improve muscle membrane repair in mdx mice and thus prevent or delay muscle pathology. In Duchenne muscular dystrophy patients and mdx mice, the absence of dystrophin leads to a reduction of dystroglycan at the sarcolemma and disruption in the link between the sarcolemma and the extracellular matrix. We have previously demonstrated that Overexpression of LARGE will increase the glycosylation and laminin binding activity of alpha-dystroglycan in wild type muscle. We hypothesize that LARGE-dependant hyperglycosylation of alpha-dystroglycan in mdx muscle will maintain alpha-dystroglycan at the sarcolemma and thereby stabilize the muscle membrane and its link with extracelluar matrix in the absence of dystrophin. The first aim will test this hypothesis using viral-mediated Overexpression of LARGE in mdx muscle and LARGE transgenic mice crossed with mdx mice. In the second aim we propose that an increased and sustained expression of dysferlin in mdx muscle will prevent pathology by improving membrane repair. Recently, studies on dysferlin-null mice have revealed a new mechanism of muscle pathogenesis where membrane repair, and not structural integrity, of the sarcolemma is abnormal. Our preliminary data on dystrophin/dysferlin-null mice suggests that dysferlin may play a role in diminishing mdx muscle pathology because the dystrophic changes are more severe in the absence of both proteins. To test this hypothesis, mdx mice will be treated with dysferlin-expressing adenovirus or crossed with dysferlin transgenic mice. Therefore, this project will investigate the potentially beneficial functional and physiological consequences of increased expression of LARGE or dysferlin. Focusing on endogenous proteins like LARGE or dysferlin, whose activity could potentially be modulated, will provide a platform for new targets of therapy (possibly pharmacological treatments) aimed at upregulating LARGE or dysferlin expression and/or activity to treat Duchenne muscular dystrophy.

DESCRIPTION (provided by applicant): The long-term goal of this project is to design a therapy for autosomal recessive limb-girdle muscular dystrophy type 2D (LGMD-2D), which is due to aberrations in the (-sarcoglycan gene (Sgca), and affects mainly limb and girdle muscles to lead to progressive muscle fiber necrosis and weakness, Alpha-Sarcoglycan interacts with beta-, gamma-, and delta-sarcoglycan to form a subcomplex that increases the overall stability of the dystrophin-glycoprotein complex (DGC). Epsilon-Sarcoglycan is a ubiquitously expressed homologue of the muscle-specific alpha- sarcoglycan, sharing 43% identity and a similar protein structure. Epsilon-Sarcoglycan expression is detected as early as day E8.5 in mouse embryos before the detection of alpha-sarcoglycan at E15, and preliminary data shows increased detection during muscle regeneration suggesting that epsilon-sarcoglycan is embryonic or developmental form of alpha-sarcoglycan. This project focuses on exploring the therapeutic potential of upregulating epsilon-sarcoglycan levels for the treatment of LGMD-2D. The First Aim hypothesizes that increased expression of epsilon-sarcoglycan will replace alpha-sarcoglycan within the DGC. Characterization of transgenic mice with targeted overexpression of epsilon-sarcoglycan to muscle will test this hypothesis. The Second Aim hypothesizes that increased expression of epsilon-sarcoglycan will compensate for an aberrant alpha-sarcoglycan to prevent the onset of muscular dystrophy. Analysis of Sgca-null mice overexpressing epsilon-sarcoglycan will test this hypothesis. The Final Aim hypothesizes that increased and sustained epsilon-sarcoglycan expression in alpha-sarcoglycan deficiency will prevent further muscle pathology and repair the primary membrane defect to allow for normal muscle function. Adult Sgca-null mice will be intramuscularly injected with rAAVl epsilon-sarcoglycan and analyzed. This project will investigate the functional and physiological consequences of increased levels of epsilon-sarcoglycan to prevent and treat muscular dystrophy in alpha-sarcoglycan deficiency. The overall results of these experiments will develop the possibility of increased epsilon-sarcoglycan expression to compensate for (-sarcoglycan in LGMD-2D patients. Focusing on an endogenous protein like epsilon-sarcoglycan will bypass any acquired therapeutic immune response, and provides a platform for new targets for therapy and drug treatments aimed at up-regulating epsilon-sarcoglycan

The overall objective of this proposal is to explore the therapeutic potential of improving muscle cell membrane maintenance and repair for the treatment of Duchenne muscular dystrophy. In particular, the proposal will test these hypotheses: 1) Overexpression of the glycosyltransferase, LARGE, will stabilize the sarcolemma in mdx muscle and thus prevent or delay muscle pathology; and 2) Overexpression of dysferlin will improve muscle membrane repair in mdx mice and thus prevent or delay muscle pathology. In Duchenne muscular dystrophy patients and mdx mice, the absence of dystrophin leads to a reduction of dystroglycan at the sarcolemma and disruption in the link between the sarcolemma and the extracellular matrix. We have previously demonstrated that Overexpression of LARGE will increase the glycosylation and laminin binding activity of alpha-dystroglycan in wild type muscle. We hypothesize that LARGE-dependant hyperglycosylation of alpha-dystroglycan in mdx muscle will maintain alpha-dystroglycan at the sarcolemma and thereby stabilize the muscle membrane and its link with extracelluar matrix in the absence of dystrophin. The first aim will test this hypothesis using viral-mediated Overexpression of LARGE in mdx muscle and LARGE transgenic mice crossed with mdx mice. In the second aim we propose that an increased and sustained expression of dysferlin in mdx muscle will prevent pathology by improving membrane repair. Recently, studies on dysferlin-null mice have revealed a new mechanism of muscle pathogenesis where membrane repair, and not structural integrity, of the sarcolemma is abnormal. Our preliminary data on dystrophin/dysferlin-null mice suggests that dysferlin may play a role in diminishing mdx muscle pathology because the dystrophic changes are more severe in the absence of both proteins. To test this hypothesis, mdx mice will be treated with dysferlin-expressing adenovirus or crossed with dysferlin transgenic mice. Therefore, this project will investigate the potentially beneficial functional and physiological consequences of increased expression of LARGE or dysferlin. Focusing on endogenous proteins like LARGE or dysferlin, whose activity could potentially be modulated, will provide a platform for new targets of therapy (possibly pharmacological treatments) aimed at upregulating LARGE or dysferlin expression and/or activity to treat Duchenne muscular dystrophy.

DESCRIPTION (provided by applicant): Dystroglycan is a widely expressed transmembrane glycoprotein that acts as a high- affinity receptor for both extracellular matrix proteins containing laminin-G domains and certain arenaviruses. Secondary dystroglycanopathies encompass a collection of muscular dystrophies characterized by impaired post-translational processing of dystroglycan. Profound muscle weakness and wasting as well as potential central nervous system impariment are typical pathologies associated with secondary dystroglycanopathies. Causative mutations for these disorders are found in known or putative glycosyltransferases that participate in the O-glycosylation of alpha- dystroglycan, a modificaiton required for functionality. Despite extensive efforts to understand the genetics and pathology of these diseases, the genetic causes of over half of these cases remain a mystery. Furthermore, there are no treatments available for patients. This proposal outlines approaches to elucidate remaining genetic causes of dystroglycanopathies, discover and validate small molecule and peptide effectors of dystroglycan glycosylation and provide the muscular dystrophy field with improved mouse models to sustain rapid future progress. These goals will be successfully met through collaboration with the Schultz laboratory at The Scripps Research Institute. The objective of Specific Aim 1 is to identify novel dystroglycanophy genetic loci using both high-throughput in vitro complementation and knockdown screens. Elucidation of new candidate genes will offer new opportunities for improved genetic diagnosis, new viable therapeutic targets and a better understanding of dystroglycan post-translational processing. Specific Aim 2 is designed to identify novel small molecule and secreted peptide effectors of dystroglycan glycosylation in a cell culture based, high-throughput manner. This unbiased approach will provide new directions for the development of therapeutic interventions for muscular dystrophy. Specific Aim 3 is targeted at both validation of previously and newly identified therapeutic strategies and the development of conditional and knockdown mouse models of dystroglycanopathy. The new mouse models will better capture both the genetic and phenotypic complexity of dystroglycanophies than the currently available cohort of mouse models. These models will serve the muscular dystrophy research community in efforts to explain the cellular mechanism and to develop viable treatment strategies for each genetic cause of dystroglycanopathy. These studies will provide the muscular dystrophy research field with improved tools and progress towards suitable means of improving dystroglycan function. This research meets the challenge of the National Institute of Neurological Disorders and Stroke mission statement to support "research on the causes, prevention, diagnosis, and treatment of neurological disorders and stroke, and supports basic research in related scientific areas". PUBLIC HEALTH RELEVANCE: Muscular dystrophies are a diverse set of inherited diseases characterized by progressive skeletal muscle weakness and wasting. Dystroglycan, a cell surface protein, requires extensive modification to serve as a link between the intracellular and extracellular cellular support network in muscle such that, when disrupted, it results in several forms of muscular dystrophy. This proposal is designed to identify new gene mutations that can cause these types of muscular dystrophy, discover small molecules that can improve dystroglycan function, develop needed mouse models of the disease and to validate both newly identified and currently known treatment strategies.

The overall goal of Core C is to provide support for the research training and education mission of the MDCRC. In particular, Core C will support a Medical Student Fellowship, a Postdoctoral Fellowship and a two-day symposium to educate/engage patients and patient advocates. Both the medical student and postdoctoral fellow will have mentoring from the two core directors. This training effort is vital, so that there are basic scientists and qualified clinicians who can partner to test novel potential treatments. We are particularly interested in training the next generation of neurologists in muscular dystrophy translational research and thus have created the Medical Student Fellowship. This full-time fellowship will enable one medical student per year to be involved in all aspects of research in the MDCRC. The student will participate in the care of patients in the muscular dystrophy clinic and in the evaluation of patients participating in Project 2 under the supervision of Katherine Mathews. They will evaluate muscle biopsies with Steve Moore and study patient cells in Kevin Campbell's lab. Finally, they will present their findings from interesting cases at the Muscle Disease Neuropathology Conference once a month. To promote the development of basic scientists doing translational research in muscular dystrophy, the Postdoctoral Research Fellowship will provide an intensive translational research experience in the MDCRC for a PhD, MD or MD/PhD graduate in Kevin Campbell's laboratory. This research fellow will also observe patient care in the clinic and research settings to enhance their understanding of the clinical side of translational research. Finally, the lowa MDCRC will continue its tradition of involving patients and patient advocates in the center. Core C will host a two day symposium and Center open house with patients and their families. The goal of the symposium will be to educate the patients about the ongoing translational research in the MDCRC and provide a forum where their questions concerning translational research can be answered. The Medical Student and Postdoctoral Research fellows will participate in this symposium. Thus, this core will accelerate the education mission of the MDCRC and establish a training environment for clinician scientists.

The overall objectives of this proposal are to improve our basic understanding of how dystroglycan binds laminin, and to explore the effectiveness of expressing LARGE and applying drugs of several classes in treating dystroglycan-related muscular dystrophies. In particular, the role of a novel post-translational phosphate modification[unreadable]an O-linked mannose that is part of the laminin-binding glycan structure of alpha-dystroglycan[unreadable]in laminin binding will be investigated. Also, new antibodies that recognize only immature alpha-DG with a terminal phosphate modification will be developed and used to evaluate the post-translational modification status of alpha-DG in tissues or cells collected from individuals with various types of dystroglycanopathies (by immunofluorescence, immunoblotting, and radioisotope-labeling). These studies will improve our understanding of the modifications required for dystroglycan to serve as a laminin receptor, and advance patient diagnosis by improving biomarker correlations with clinical severity. In vitro experiments with patient cells have suggested that overexpression of the glycosyltransferase LARGE can bypass alpha-dystroglycan glycosylation defects in a broad range of dystroglycanopathies. This hypothesis will be corroborated In vivo by assessing the ability to prevent disease in different dystroglycanopathy mouse models through either systemic or muscle-specific expression of the Large transgene. This will provide a better understanding of the feasibility and likelihood of success of broadly applying a LARGE-based therapy to the genetically diverse group of dystroglycanopathies. Furthermore, existing clinical drugs will be tested for their potential to ameliorate disease in dystroglycanopathy mouse models. The group of drugs to be tested includes a premature stop-codon readthrough drug (PTC124), as well as steroids (Prednisone) and PDES inhibitors (Sildenafil);the latter two compounds have already shown promise in the treatment of non-dystroglycan related muscular dystrophies. The effectiveness of these treatments will be explored using a combination of cell culture and in vivo mouse studies and measuring dystroglycan function, muscle physiology and pathology. The proposed research will provide a platform for new therapeutic avenues, some of which it will be possible to implement directly in the care of dystroglycanopathy patients

Project 1 ? Project Summary The overall goal of our research is to develop therapeutic strategies for treating muscular dystrophy. Our current research focuses on the dystroglycanopathies ? muscular dystrophies in which aberrant post- translational modification of the ?-dystroglycan (?-DG) protein results in a reduction of its glycosylation, and thereby in loss of an essential link between it and its laminin-G domain-containing ligands in the extracellular matrix (ECM). At least eighteen genes encode post-translational enzymes that act on ?-DG, and mutations therein cause congenital/limb-girdle muscular dystrophies, conditions that can be accompanied by brain and eye abnormalities. Although great progress has been made on the identification and characterization of dystroglycanopathy genes, we still do not fully understand how mutations therein lead to the observed abnormalities in dystroglycan glycosylation and to muscular dystrophy. The overarching hypothesis of our research is that a thorough understanding of 1) the structure of an ECM ligand-binding glycan on native dystroglycan, 2) the abnormal glycan structure(s) in dystroglycanopathy patients, and 3) the pathological mechanisms underlying the abnormal glycosylation and receptor function in the dystroglycanopathies, are required to develop rational diagnostic and therapeutic strategies. We will approach our objectives using patient cells and state-of-the-art biological, cell biological and glycobiological analyses. Specific Aim 1 will establish the in vivo relevance of the LARGE repeats on ?-DG in skeletal muscle. These studies will reveal whether LARGE repeats are present on native ?-DG, and establish their contribution to ligand binding in vivo. Specific Aim 2 will define the ligand- and antibody-binding properties of LARGE repeats, establish the binding affinity and absolute stoichiometry of the LARGE-dependent glycan for its ECM ligands, and determine the specificity of antibodies that target the dystroglycan glycan. Specific Aim 3 will establish the post-translational modification status of ?-DG in various dystroglycanopathies. Identifying the molecular defects in ?-DG within different patient populations will improve our understanding of the modifications that will be required for therapeutic treatments. Finally, Specific Aim 4 will identify the mechanistic defects that result in abnormal ?-DG glycosylation. We will analyze the enzymatic activities of several dystroglycanopathy genes (LARGE, POMK, POMGNT2, B3GALNT2, and B4GAT1) and correlate these with functional glycosylation in patient cells and severity of the clinical phenotype. We will also prepare mutant constructs and test the enzymatic activity and sub-cellular localization of the translated proteins. Classification of the mutant enzymes as ?inactive? or ?defective for trafficking? will provide a framework for developing novel therapeutic strategies based on the functional defect. Collectively, these aims will provide a critical understanding of the pathological mechanisms underlying dystroglycanopathies and a rationale for future diagnostic and therapeutic strategies.

PROJECT SUMMARY The Administrative Core of the Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center will coordinate the activities within and outside the Center, as well as promote an interactive and collaborative research environment. The administrative responsibilities of Core A include the following: organization of the flow of project information; distribution of research effort; allocation of budgetary and other resources; preparation of annual budgets and projections; scheduling and facilitation of monthly Neuromuscular Disease Conference meetings; promotion and coordination of community outreach projects (including the annual Patient and Family Conference); and consultation with the Dean regarding progress, scientific direction, administrative issues and concerns, and future plans. The scientific responsibilities of Core A include the following: integration, coordination, and direction of research projects as needed; consultation with advisors and consultants concerning the importance and progress of Center research relative to other developments in the area of muscular dystrophy; identification of seminar speakers and coordination of their participation and contributions to the Center; and hosting of an annual family conference for patients and their families. This core will also facilitate expansion of our collaboration with national and international leaders in the field of congenital/limb-girdle muscular dystrophy, particularly researchers and clinicians at other Wellstone Centers throughout the United States. The Core A Administrator will coordinate an annual open house for the Muscular Dystrophy Association branch in Cedar Rapids, Iowa, at which seminars are held for approximately 100 patients/families; these describe current and planned research, as well as advancements in understanding and treating muscular dystrophy. The Administrator is also responsible for coordinating the Wellstone Steering Committee Face-to-Face Meeting when this is hosted by the University of Iowa, coordinating both the patient and family tours and the meetings among Core Directors and Project Leaders that take place during this event.

Project 1 Project Summary The overall goal of our research is to elucidate the cellular and molecular mechanisms that underly muscular dystrophies in order to facilitate the rational design of novel therapeutic strategies and quantitative assays to assess target engagement and the effectiveness of therapeutic approaches. Our current research focuses on the dystroglycanopathies, a group of congenital/limb-girdle muscular dystrophies caused by defects in post-translational processing of the extracellular matrix (ECM) receptor ?-dystroglycan (?-DG). ECM proteins that contain laminin-G-like (LG) domains bind to ?-DG via a unique heteropolysaccharide [-GlcA-?1,3-Xyl-?1,3-]n called matriglycan. Genetic studies have shown that mutations in any one of at least eighteen genes encoding enzymes required for ?-DG post-translational processing lead to the absence of or a reduction in matriglycan and thus impair ?-DG receptor function. Although significant progress has been made in the identification and characterization of dystroglycanopathy genes, we still do not fully understand the biochemical and physiological function of matriglycan, or how its absence or reduction in length causes muscular dystrophy. The overall objective of the proposed research is to provide mechanistic insights into the role matriglycan plays in ?-DG receptor function and in the pathophysiology of the dystroglycanopathies. The overarching hypothesis of our research is that a thorough understanding of (a) the shortened matriglycan structure and resulting ?-DG receptor dysfunction in dystroglycanopathy patients and (b) the mechanisms underlying the pathophysiology of the dystroglycanopathies, will lead to more reliable diagnostics and novel therapeutic strategies. Specific Aim 1 will establish the relationship between ?-DG matriglycan length and laminin-binding properties of matriglycan on ?-DG in control and dystroglycanopathy patient fibroblasts and muscle biopsies. These studies will reveal abnormalities in the post-translational processing of ?-DG that result in shorter matriglycan with reduced affinity for laminin leading to muscular dystrophy. Specific Aim 2 will define the biochemical regulation of matriglycan synthesis and its role in the receptor function of ?-DG. These studies will define the requirements for matriglycan synthesis and how its synthesis is regulated by protein-protein and protein-sugar interactions. Specific Aim 3 will define novel mechanisms underlying the pathophysiology of the dystroglycanopathies and determine the structure and laminin binding properties required to improve muscle function. These studies will use myd mice with established dystrophic muscle pathology that are evaluated before and after LARGE gene transfer. Collectively, these aims will provide an understanding of the pathological mechanisms underlying dystroglycanopathies, which will be needed to develop a rationale for the design of novel diagnostic and therapeutic strategies, as well as approaches to monitoring therapeutic engagement and efficacy.