Joseph J. Volpe - US grants

The objectives of this research are to utilize model systems in cell culture to provide insight into the following phenomena in differentiating astrocytes, oligodendroglia, and neurons: (1) the interrelations of cholestrerol and dolichol-dolichylphosphate (do1/do1-P) biosyntheses and the role of HMG-CoA reductase in modulating these interrelations, (2) the regulation of do1/do1-P metabolism and dolichol-linked oligosaccharide and glycoprotein (DLOG) biosyntheses, (3) the relation of do1/do1-P metabolism and DLOG biosyntheses to astrocytic, oligodendroglial, and neuronal differentiation , and (4) the role of thyroid hormone in the regulation of do1/do1-P metabolism and DLOG biosyntheses. In all of the experiments proposed, primary cultures of astrocytes, oligodendroglia or neurons, isolated as essentially homogenous preparations, will be utilized. Concerning the interrelations of cholesterol and do1/do-P biosyntheses, we will determine the extent to which these biosyntheses are regulated coordinately at a critical enzymatic step common to both pathways, i.e., HMG-CoA reductase. Concerning the regulation of do1/do-P metabolism and DLOG biosyntheses, we will define the enzymatic steps controlling the regulation of dolichyl phosphate levels and the utilization of dolicyl phosphate for oligosaccharide biosynthesis. Concerning the relation of do1/do1-P metabolism and DLOG biosyntheses to astroglial, oligodendroglial and nueuronal differentiation, two major series of experiments will be important. First, correlation of specific biochemical indicators of differentiation of the three cell types with specific aspects of dol/dol-P metaboslism and DLOG biosyntheses, described immediatlely above, will be accomplished. Second, we will determine whether these biochemical events in the dolichol-linked pathway are obligatory for the specific expressions of cellular differentiation. Thus, we will determine the effect on these expressions of differentiation of inhibition of either (a) dolichyl phophaste biosynthesis, i.e., by mevinolin, or (b) dolichol-linked oligosaccharide biosynthesis, i.e., by tunicamycin. Concerning the role of thyroid hormone importance of this work relates to previous studies which demonstrate the important role of thyroid hormone in brain development, particularly cellular differentiations, and to our preliminary data which indicate a regulatory role for thyroid hormone in the dolichol-linked pathway in at least the astrocytes and oligodendrogia.

DESCRIPTION (provided by applicant): This Center grant requests funding for seven core facilities to support a broadly-based research program in the study of mental retardation and developmental disabilities. The seven core facilities are: Administrative, Multimedia, Imaging, Cellular Neuroscience, Gene Manipulation, Molecular Genetics, and Cell Sorter. The Cores have undergone major development and support the research of 65 investigators. The research is in two programmatic areas, i.e., Genetics and Neuroscience. In Genetics, directed by Dr. Louis Kunkel, there are seven major research efforts, which include the molecular genetics of neuromuscular disease, somatic cell genetics, gene therapy and stem cell research. In Neuroscience, there are three major programs, i.e., Basic Neuroscience, Clinical Neuroscience/Behavior and Developmental Biology. The Basic Neuroscience Program, directed by Dr. Michael Greenberg, consists of 27 investigators whose research efforts span a broad spectrum of interdigitated research in molecular, cellular, and systems neurobiology, with a strong overall emphasis on development and developmental neurological disorders. A research project in Basic Neuroscience, proposed in this application as a "New Program", addresses "Signaling Mechanisms Mediating Axon Guidance Effects of Semaphorin 3A" (Zhigang He, Ph.D.). A second major program in Neuroscience, i.e., Clinical Neuroscience/Behavior, directed by the Center Director, includes 27 investigators whose research efforts include such areas as neonatal brain injury, behavioral development, learning disturbances, structural/functional brain imaging, and molecular genetics of human brain development. These areas of clinical research interdigitate with the basic research in the MRRC. A third exciting program in Neuroscience, i.e., Developmental Biology, directed by Dr. Merton Bernfield, addresses such areas as mechanisms of embryonic development, serpin biology and regulation of pregnancy duration. The multidisciplinary approaches to the research of this MRRC include the various basic science disciplines within Genetics and Neuroscience, and the clinical science fields of Neurology, Pediatrics, Neonatology, Infectious Disease, Endocrinology, Metabolism, Genetics, Cardiology, Neurosurgery, Neuropathology, Ophthalmology, Psychiatry, and Psychology. The basic science programs are housed in over 70,000 square feet of the Enders Pediatric Research Building. The clinical research programs are centered in the adjacent Children?s Hospital.

This exploratory neonatal brain disorders research grant is organized around the central unifying research theme, ischemic brain injury in the neonatal period. The overall program is composed of five interrelated projects, two in clinical neuroscience (Projects 1 and 2) and three in basic neuroscience (Projects 3-5). Project 1 addresses periventricular white matter injury in the premature infant. Considerable data suggest that this injury is caused by disturbances in cerebral blood flow (CBF) and the regulation thereof. However, a direct relationship between such disturbances and the occurrence of these lesions has not been established conclusively in human premature infants, primarily because of methodological deficiencies. We will utilize the new technique of near- infrared spectroscopy (NIRS) to determine whether abnormal reactivity of CBF to blood pressure and to PaCO2 is present in premature infants who develop these white matter lesions. Project 2 addresses the mechanisms of brain injury in mature infants undergoing cardiac surgery with deep hypothermia, cardiopulmonary bypass and circulatory arrest. Considerable data suggest that perioperative events are associated with the occurrence of both neuronal and white matter injury in these infants. We will utilize NIRS and related techniques to study cerebral hemodynamics and oxygen utilization during both the intraoperative and postoperative periods. Project 3 addresses mechanisms of oligodendroglial death studied in cell culture. Preliminary work has defined a maturation- dependent vulnerability of oligodendroglia to glutamate-induced cell death. Delineation of the development aspects of this vulnerability, the mechanisms of the toxicity and the means of prevention will be accomplished. Project 4 addresses the development of oligodendroglia in human brain and the delineation of the specific maturational state of the oligodendroglia particularly affected in periventricular white matter injury. The research will provide major insight into the cellular aspects of PVL. Project 5 addresses in the developing rat model of hypoxic-ischemic brain injury that is particularly relevant to the clinical research of project 2 concerning the infants undergoing cardiac surgery. Combinations of pharmacological agents, e.g., various excitatory amino acid antagonists, novel anti-oxidants, will be studied for their protective effects, with a particular emphasis on combinations that are both effective and likely to be clinically safe. At the end of this exploratory grant our accomplishments should provide major insight for the first time into the relationship of impaired regulation of CBF and the occurrence of periventricular white matter injury in the human premature infant, the mechanisms and timing of disturbances of cerebral hemodynamics and oxygen utilization leading to brain injury in mature infants undergoing cardiac surgery, the mechanisms of glutamate-induced oligodendroglial cell death and the prevention of thereof, the development of oligodendroglia in human brain and the cellular target in PVL, and the determination of an optimal combination of pharmacological agents for utilization in a clinical trial for prevention of brain injury in infants undergoing cardiac surgery. A strong multi-disciplinary core group of clinical and basic scientists will have been established and will form the nucleus for a major center grant on the study of neonatal brain disorders.

This proposal for an Institutional Neurological Sciences Academic Development Award (NSADA) is presented by the Boston Children's Hospital. The Program Director of this NSADA program, Dr. Joseph Volpe, is the Chairman of the Department of Neurology and leader of the child neurology residency program at Children's Hospital/Harvard Medical School. The entire NSADA program will be carried out in the Longwood Medical Area, a physically and intellectually unified area composed of the Children's Hospital, Harvard Medical School, Beth Israel Hospital, Brigham and Women's Hospital and the Harvard School of Pubic Health. The primary goal of this proposal is to provide research development programs in basic and clinical neuroscience for child neurology residents committed to an academic career with a dominant career focus in research. The major components of the research development plan for both basic and clinical neuroscience are: 1) advice and guidance; 2) formal course work; 3) the research experience with the research mentor; 4) other intellectual activities; and 5) integration of components and ongoing evaluation and guidance. In basic neuroscience, the formal course work is derived from the Harvard Program in Neuroscience directed by Dr. Gerald Fischbach, Chairman of the Department of Neurobiology and Associate Director of this NSADA proposal. Course work will be continued in the first two years of the NSADA program for basic neuroscience development. The research experience with the research mentor will also begin during this interval and, in fact, will constitute the majority of the resident's total research effort. A remarkable richness of research opportunities are available in molecular, cellular, and systems/ integrative neurobiology from mentors at the Children's Hospital (primarily in the Department of Neurology), the Harvard Medical School (primarily in the Department of Neurobiology), and in the Departments of Neurology at the Beth Israel/Brigham and Women's Hospitals. This breadth and depth are crucial for the research development of the relatively undifferentiated child neurology resident and represent the greatest strengths of this proposal. In clinical neuroscience, the five elements of the research development program and the commitment to highly rigorous research training are identical to those features of the basic neuroscience research development program. The formal course work, however, is composed of study at the Harvard School of Public Health, consisting of a core and an elective curriculum, leading to a Master of Science degree at the School. Fundamental grounding in epidemiology, study design and biostatistics is thereby established. The research experience with the research mentor is carried out in one of seven well- developed clinical research programs at the Children's Hospital, i.e., cognitive neuroscience, epidemiology, epilepsy, neonatal neurology, neurocardiology, neurogenetics and neuro-oncology. The strong track record of the Program Director in fostering development of child neurology residents in research training in preparation for academic careers, the integrated nature of the Longwood Medical Area, including the Harvard Medical School and the Harvard School of Public Health, the commitment and established collaborative relation between the Program Director and the Associate Director, Dr. Fischbach, in research development of child neurology residents ensure the success of this NSADA program.

DESCRIPTION (provided by applicant): The competing renewal of this revised Program Project grant is organized around a unifying research theme, the pathogenesis and prevention of periventricular leukomalacia (PVL), the major form of brain injury in the premature infant. The proposed research is based primarily on the Program's many scientific discoveries of the previous cycle and on exciting preliminary data. The overall Program is composed of four integrated projects, one in clinical neuroscience and three in basic neuroscience. The first Project addresses the cellular and molecular pathology of PVL in human brain. Work hi the first cycle defined the key cellular target in PVL to be the pre-myelinating oligodendrocyte, i.e., pre-oligo, the death of which was shown to be associated with marked microgliosis and reactive astrocytosis and evidence for attack by reactive oxygen and nitrogen species (ROS/RNS). In the current proposal the degree of pre-oligo loss will be quantitated; the cellular origins of RNS identified; the role of microglia and astrocytes in ROS/RNS toxicity determined; and the extent and nature of neuronal/axonal impairment delineated. The second Project will address the cellular and molecular pathways of oxidative injury to pre-oligos, with an emphasis on microglia and RNS attack. Areas of focus include delineation of the mechanisms of injury to pre-oligos triggered by microglia, the molecular pathways responsible for RNS toxicity, and the role for RNS toxicity in in vivo white matter (WM) injury caused by hypoxia-ischemia or infection/inflammation or both insults. The third Project addresses the role of excitotoxicity in the pathogenesis of PVL. Emphases of the project are the expression of GluRs in normal human WM and in PVL, the toxic mechanisms downstream from ionotropic glutamate receptor (GluR) activation that are intrinsic to the pre-oligo or that involve other glial cell types, the sensitizing effect of subthreshold hypoxia on the impact of a subsequent insult, and the role of intracellular Ca2+and mitochondrial dysfunction in pre-oligo excitotoxicity. The fourth Project addresses the roles of exogenous and endogenous activators of microglia and of innate immunity in the pathogenesis of PVL. Research emphases include delineation of heat-shock protein 60 (HSP60) as a key endogenous activator of microglia and innate immunity and secondarily, thereby, injury to pre-oligos, the involvement of HSP60 in hypoxic-ischemic WM injury in the developing rodent and in human PVL, and the expression of key Toll-like receptors in developing human WM and in PVL. The progress of the overall Program in the first cycle and the proposed work building on that progress should lead to insights that allow formulation of preventative interventions suitable for clinical trial.