Steven A. Moore, MD, Indiana University 1982, PhD, Indiana University 1980 - US grants

Our objective is to develop an in vitro model of the blood brain barrier through which major hypotheses of vasogenic cerebral edema may be tested. Cerebral edema accompanies many different pathological insults to the CNS and is a prime determining factor of the ultimate morbidity or mortality associated with many of these insults. Several hypotheses have been proposed to explain the disruption of the blood brain barrier which precedes the formation of vasogenic cerebral edema. Arachidonic acid metabolism is a central issue in most of these hypotheses, but precise characterization of the metabolites involved, their sites of origin and/or action, and the mechanisms of their actions remains largely undetermined. Uniquely, mouse cerebral endothelium and vascular smooth muscle and rat C6 glioma cells are available in the laboratory for in vitro exploration of these hypotheses. Preliminary studies suggest that some cerebral endothelial properties are maintained in culture (i.e, impermeability to certain macromolecules), while others require co-culture of cerebral endothelium with C6 glioma cells for their expression (i.e., Gamma-GTP activity and polarity to amino acid transport). Further studies to characterize this in vitro blood brain barrier model will include: 1) permeability studies with small versus large M.W. and low versus high octanol/water partition co-efficient solutes, 2) ultrastructural probes for analysis of vesicular transport, 3) measurement of transendothelial electrical resistance, and 4) more extensive co-culture studies to assess intercellular interactions required for blood brain barrier differentiation and maintenance. Cell type specific studies of arachidonic acid metabolism utilizing primarily thin layer chromatography, high performance liquid chromatography, and radioimmunoassay will characterize sites of specific eicosanoid metabolism. Preliminary studies reveal differences between endothelium and smooth muscle. Knowledge of cerebrovascular eicosanoid metabolism will then be utilized to assess its effects on the in vitro model of the blood brain barrier developed earlier.

A growing number of oxygenated derivatives of long chain, polyunsaturated fatty acids are being described in the brain. Included among these compounds are prostaglandins, hydroxyeicosatetraenoic acids (HETEs), and leukotrienes. Although many of these eicosanoids mediate well- characterized physiological or pathophysiological events in other tissues, their role in the biological processes of the brain, and in particular, the cerebral blood vessels, is relatively unknown. A number functions have been proposed, ranging from the control of cerebrovascular tone, to action as transmitter substances or intracellular second messengers, to mediation of pathophysiologic events in stroke, brain trauma, and seizure. We and others have previously identified several metabolites or arachidonic acid and eicosapentaenoic acid produced by isolated brain microvessel preparations and by purified cultures of cerebromicrovascular endothelium and smooth muscle cells. Yet, the production of eicosanoids by cerebromicrovascular cells, the regulation of their production, and the function effects these arachidonic acid derivatives have on the cerebral circulation and adjacent brain parenchyma remain incompletely characterized. Even less is known about the metabolism and function of omega-3 fatty acids in cells of the cerebral circulation. Thus, the following aims are proposed in order to more completely understand the biology of long chain, polyunsaturated fatty acids in cerebral microvessels. First, the metabolism of leukotrienes and HETEs will be explored in cultured cerebral endothelium and isolated brain microvessels. These compounds are produced in many types of brain injury and are potential pathophysiologic mediators. Second, studies will be undertaken to identify the cerebrovascular metabolites of the omega-3 fatty acids, eicosapentaenic acid and docosahexaenoic acid, fatty acids that may prevent or ameliorate the pathology of cerebrovascular disease. Third, the regulation of eicosanoid biosynthesis by interactions among cellular components of the microvascular wall, astrocytes, smooth muscle cells, and endothelium, will be examined. Many aspects of cerebral endothelial differentiation are induced or modulated by astrocytes and similar relationships may exist between endothelium and smooth muscle. In addition, cells that comprise the cerebrovascular wall may share eicosanoid precursors or intermediates. Finally, the functional responses of cerebral microvessels to specific eicosanoids will be studied utilizing a combination of in vivo and in vitro pial vessel preparations and an in vitro model of the blood-brain barrier. Eicosanoids have great potential to act as autocrine or paracrine compounds within the cerebrovascular wall and may thus effect vascular tone and blood-brain barrier properties. This combination of in vitro and in vivo methods is unique and should prove to be a powerful approach to understanding eicosanoid biology in the cerebral microcirculation.

A growing number of oxygenated derivatives of long chain, polyunsaturated fatty acids are being described in the brain. Included among these compounds are prostaglandins, hydroxyeicosatetraenoic acids (HETEs), and leukotrienes. Although many of these eicosanoids mediate well- characterized physiological or pathophysiological events in other tissues, their role in the biological processes of the brain, and in particular, the cerebral blood vessels, is relatively unknown. A number functions have been proposed, ranging from the control of cerebrovascular tone, to action as transmitter substances or intracellular second messengers, to mediation of pathophysiologic events in stroke, brain trauma, and seizure. We and others have previously identified several metabolites or arachidonic acid and eicosapentaenoic acid produced by isolated brain microvessel preparations and by purified cultures of cerebromicrovascular endothelium and smooth muscle cells. Yet, the production of eicosanoids by cerebromicrovascular cells, the regulation of their production, and the function effects these arachidonic acid derivatives have on the cerebral circulation and adjacent brain parenchyma remain incompletely characterized. Even less is known about the metabolism and function of omega-3 fatty acids in cells of the cerebral circulation. Thus, the following aims are proposed in order to more completely understand the biology of long chain, polyunsaturated fatty acids in cerebral microvessels. First, the metabolism of leukotrienes and HETEs will be explored in cultured cerebral endothelium and isolated brain microvessels. These compounds are produced in many types of brain injury and are potential pathophysiologic mediators. Second, studies will be undertaken to identify the cerebrovascular metabolites of the omega-3 fatty acids, eicosapentaenic acid and docosahexaenoic acid, fatty acids that may prevent or ameliorate the pathology of cerebrovascular disease. Third, the regulation of eicosanoid biosynthesis by interactions among cellular components of the microvascular wall, astrocytes, smooth muscle cells, and endothelium, will be examined. Many aspects of cerebral endothelial differentiation are induced or modulated by astrocytes and similar relationships may exist between endothelium and smooth muscle. In addition, cells that comprise the cerebrovascular wall may share eicosanoid precursors or intermediates. Finally, the functional responses of cerebral microvessels to specific eicosanoids will be studied utilizing a combination of in vivo and in vitro pial vessel preparations and an in vitro model of the blood-brain barrier. Eicosanoids have great potential to act as autocrine or paracrine compounds within the cerebrovascular wall and may thus effect vascular tone and blood-brain barrier properties. This combination of in vitro and in vivo methods is unique and should prove to be a powerful approach to understanding eicosanoid biology in the cerebral microcirculation.

DESCRIPTION (Applicant's abstract): The dystrophin-glycoprotein complex (DGC) is a well characterized array of cytoplasmic, membrane spanning, and extracellular matrix proteins that form a critical linkage between the cytoskeleton and the basal lamina of striated muscle. Within the central nervous system (CNS), similar dystroglycan linkages to basal laminae are present at two interfaces formed by astrocytes: (1) foot processes abutting on cerebral blood vessels and (2) foot processes that form the glia limitans at the pial surface of the brain. The former interface is critical for formation and maintenance of the blood-brain barrier, while the latter is likely to play important roles in anchoring radial glia during neuronal migration. Basal lamina abnormalities at the glia limitans have been identified in some forms of congenital muscular dystrophy in humans (e.g. Fukuyama muscular dystrophy) and basal lamina disruption at the glia limitans leads to abnormal CNS development in animal models. In this proposal, we will focus attention on the central protein in the astrocyte-basal lamina linkage, dystroglycan. Our Specific Aims propose to identify protein elements of the astrocyte-dystroglycan complex, elucidate protein interactions within the complex, and demonstrate the importance of the astrocyte dystroglycan complex during CNS development. Through the use of Cre-lox methodology, we plan to create a novel murine model of CNS developmental disorders. This project is a cross-discipline collaboration among investigators with expertise in clinical neuropathology and in basic neuroscience, molecular biology, cell biology, and membrane physiology who are uniquely situated to carry out the proposed studies. Aim 1: To define the composition of the astrocyte-dystroglycan complex(es), we will test the hypothesis that one-or-more dystroglycan complexes are present in astrocytes using a combination of biochemical and immunohistochemical methods. These studies will utilize tissue sections and cultured astrocytes from wild type mice and mice with naturally occurring or genetically engineered mutations of one or more of the DGC components known to be expressed in astrocytes. Aim 2: To create a new model of CNS developmental abnormalities by selectively disrupting the astrocyte-dystroglycan complex. Dystroglycan +/-, dystroglycan lox/lox, and GFAP-Cre mice will be bred to produce GFAP-Cre/dystroglycan lox/- and GFAP-Cre/dystroglycan lox/lox mice. This strategy should disrupt the astrocyte DGC beginning in the latter half of embryonic development. We believe this strategy will produce mice with neuronal migration and cerebrovascular defects.

The Neuropathology Core for this Program Project Grant is a laboratory function dervied from neurohistochemistry and imunostaining capabilities that currently exist in the research laboratories of Drs. Davidson and Moore. Formalization of this laboratory function as the Neuropathology Core housed within Dr. Moore's research laboratory will centralize personnel, equipment, and reagents necessary to efficiently produce high quality brain sections and stains ready for microscopic evaluaton. The Neuropathology Core facility's overall objective is to support Drs. Davidson, Paulson, Kosik, Gonzalez-Alegre, and Dauer in the evaluation of mouse brains following transduction with viruses expressing specific shRNAs. This includes consultation with Program investigators concerning the processing of brains for microscopic evaluation, development of specific immunostaining or histochemical staining protocols, production of high quality brain sections, and staining sections for evaluation in individual investigator laboratories. The Neuropathology Core staff and investigators are in close contact through all phases of this process. Thus, the Core serves as both a research and development facility for histology-based methods and a service facility to provide brain sections ready for microscopic evaluation. In addition, Dr. Moore (a board-certified neuropathologist) will provide standard neuropathologic evaluation of brain sections as needed for each project. This latter responsibility is particularly important in the objective evaluation of transduced brains for untoward effects of RNAi. The main responsibilities of the Core will be: 1) Complete the processing of brains for histologic evaluation; 2) Produce microscopic sections specifically adapted to the needs of each project; 3) Produce histochemical or immunostained slides as needed by each project; 4) Store uncut tissue and unstained sections; 5) Evaluate brains for general neuropathological changes as needed by each project; 6) Develop new staining methods for use in individual projects.

The Muscle Tissue/Cell Culture/Diagnostics Core for this Wellstone Muscular Dystrophy Cooperative Research Center is a multifaceted laboratory that will be both a local and a national resource for muscular dystrophy research. There are two major goals of the Core. First, the Core will use approximately 4000 existing stored skeletal muscle biopsies to establish a muscle tissue repository. Among the existing muscle biopsies are more than 600 muscular dystrophy samples that include dystrophinopathies (Duchenne and Becker muscular dystrophy), limb-girdle muscular dystrophies, and congenital muscular dystrophies. We will accrue new specimens into the Core from a variety of sources: diagnostic muscle biopsies, therapeutic surgical procedures, endomyocardial biopsies, heart explants, skin biopsies, and autopsies. In addition to these tissues, we will establish fibroblast and myoblast cell lines from diagnostic muscle and skin biopsies. Patients with genetically defined muscular dystrophies will be recruited to undergo biopsies in order to maintain a widely representative spectrum of muscular dystrophy diagnoses. Well characterized tissues and cells from the Core will be made available to research investigators from other centers. The second goal of the Core is to establish a diagnostic resource. In this capacity it will provide diagnostic services that are not readily available through clinical laboratories, it will facilitate development of new diagnostic tests for transfer to the clinical laboratory, and will serve as a post-intervention biopsy evaluation resource for investigators conducting clinical trials. The Core will maintain patient confidentiality, and all testing will be quality controlled to meet CLIA licensing standards. Thus, the Muscle Tissue/Cell Culture/Diagnostics Core will establish a vital resource of muscular dystrophy tissues and cell cultures, provide a critical diagnostic link in expanding the number of patients with specific, molecular diagnoses, and serve as a national biopsy testing resource for clinical trials in muscular dystrophy patients.

[unreadable] DESCRIPTION (provided by applicant): This proposal seeks to obtain funding to support a Congenital Muscular Dystrophy Workshop that will be held at the University of Iowa, Iowa City, IA, USA in July 2008. Congenital muscular dystrophies (CMD) are a clinically and genetically heterogeneous group of muscle and developmental diseases with clinical features that include severe weakness, joint contractures, and devastating brain and eye malformations. Life expectancy ranges from a few months to a few years. The primary goal of this workshop is to bring together representatives of the clinical and basic science communities with an interest in CMD to assess the current state of knowledge and foster future collaborative efforts. Experts from the USA, several European countries, Australia, Canada and Japan have already agreed to attend. This workshop will serve as a forum for improving patient identification and genetic diagnosis, developing a patient registry, understanding disease pathology, and identifying pathways for the development of therapies for the full spectrum of CMD. A Workshop report will be published online through the Wellstone Muscular Dystrophy Cooperative Research Centers. Relevant to Public Health: This proposal seeks to obtain funding to support a Congenital Muscular Dystrophy Workshop that will serve as a forum for improving patient identification and genetic diagnosis, developing a patient registry, and brainstorming potential therapeutic approaches. The congenital muscular dystrophies are a heterogeneous group of inherited muscle and developmental diseases with clinical features that include severe weakness, joint contractures, and devastating brain and eye malformations. Life expectancy ranges from a few months to a few years. [unreadable] [unreadable] [unreadable] [unreadable]

The Muscle Tissue/Cell Culture/Diagnostics Core for this Wellstone Muscular Dystrophy Cooperative Research Center is a multifaceted laboratory that serves as a local and a national resource for muscular dystrophy research. There are two major goals of the Core. First, the Core will maintain and continue to grow the Repository of skeletal muscle biopsies and cell cultures established during the first five years of funding. Among the existing muscle biopsies are more than 1000 muscular dystrophy samples that include dystrophinopathies (Duchenne and Becker muscular dystrophy), limb-girdle muscular dystrophies, and congenital muscular dystrophies. More than 125 patients (primarily muscular dystrophy patients) have undergone skin biopsies to establish fibroblast cultures in the Repository. We will accrue new specimens into the Repository from a variety of sources: diagnostic muscle biopsies, therapeutic surgical procedures, endomyocardial biopsies, heart explants, skin biopsies, and autopsies. Patients with genetically defined muscular dystrophies are recruited to undergo biopsies in order to maintain a widely representative spectrum of muscular dystrophy diagnoses. Well-characterized tissues and cells from the Core are made available to research investigators at lowa and other academic centers. The second goal of the Core is to provide specialty diagnostic resources unavailable in clinical laboratories. This includes western blots from skeletal muscle as well as on-cell westerns and nuclear morphology assays in cultured cells. The Core facilitates development of new diagnostic tests for transfer to the clinical laboratory, and serves as a post-intervention biopsy evaluation resource for investigators conducting clinical trials. Thus, the Muscle Tissue/Cell Culture/Diagnostics Core maintains a vital resource of muscular dystrophy tissues and cell cultures, provides a critical diagnostic link in expanding the number of patients with specific, molecular diagnoses, and serves as a national biopsy testing resource for clinical trials in muscular dystrophy patients.

CORE B SUMMARY The Muscle Tissue/Cell Culture/Diagnostics Core of the Iowa Wellstone Muscular Dystrophy Cooperative Research Center is a multifaceted laboratory that serves as a local, national and international resource for research on muscular dystrophy and other neuromuscular diseases. This Core has two major goals. Firstly, it has established and will expand a tissue and cell culture Repository to support basic and translational research efforts. Currently, this Repository contains muscle biopsies from more than 3500 patients with neuromuscular disorders, as well as cryopreserved fibroblasts from more than 300 patients with myopathic disease, predominantly muscular dystrophies. Well-characterized tissues and cells from the Core are made available to research investigators at Iowa as well as other academic centers across the USA and around the world. Secondly, the Core provides specialty diagnostic services that are not readily available through clinical laboratories, and facilitates research on and the development of new diagnostic tests for eventual transfer into clinical laboratories. These diagnostic services are very closely integrated with clinical laboratory testing carried out in the Department of Pathology at the University of Iowa. Together, Core B and the Department of Pathology services provide state-of-the-art diagnostic testing for patients seen at the University of Iowa Hospitals and Clinics, and for patients referred to Iowa from throughout the USA and from several other countries. This infrastructure provides key support for clinical trials of neuromuscular disease.

CORE B SUMMARY The Muscle Tissue/Cell Culture/Diagnostics Core of the Iowa Wellstone Muscular Dystrophy Cooperative Research Center is a multifaceted laboratory that serves as a local, national and international resource for research on muscular dystrophy and other neuromuscular diseases. This Core has two major goals. Firstly, it has established and will expand a tissue and cell culture Repository to support basic and translational research efforts. Currently, this Repository contains muscle biopsies from more than 8500 patients with neuromuscular disorders, as well as cryopreserved fibroblasts from more than 425 patients with myopathic disease, predominantly muscular dystrophies. Well-characterized tissues and cells from the Core are made available to research investigators at Iowa as well as other academic centers across the USA and around the world. Secondly, the Core provides specialty diagnostic services that are not readily available through clinical laboratories, and facilitates research on and the development of new diagnostic tests for eventual transfer into clinical laboratories. These diagnostic services are very closely integrated with clinical laboratory testing carried out in the Department of Pathology at the University of Iowa. Together, Core B and the Department of Pathology services provide state-of-the-art diagnostic testing for patients seen at the University of Iowa Hospitals and Clinics, and for patients referred to Iowa from throughout the USA and from several other countries. This infrastructure provides key support for clinical trials of neuromuscular disease.