Charles J. Epstein, M.D. - US grants

Studies will be continued on the mechanisms whereby chromosome imbalance results in abnormalities of development and function. The research goals for the coming year are: a) The isolation and analysis of trisomic and normal cell populations from normal reversibly yields trisomy 17 mouse chimeras for H-2 antigen synthesis and integration into membranes. b) The formation and analysis of normal reversibly yields monosomy 19 (1,12) chimeras to determine whether monosomy results in cell lethality. c) The analysis of protein synthesis in monosomic mouse embryos by 2-dimensional gel electrophoresis and ultrastructural examination of monosomy 19 embryos. d) The quantitation of interferon induced products in normal and trisomy 21 cells.

The international conference on Down Syndrome research will be held in the United States under the auspices of the National Down Syndrome Society. The meeting will review and document the current status of the entire field of Down Syndrome research and examine the directions that future research about this genetic disorder should take. The meeting will focus on five major topics: l) the structure of human chromosome 21, 2) the cause(s) and mechanisms of non.disjunction and prenatal detection, 3) the components of the Down Syndrome phenotype, 4) mental retardation, and 5) Alzheimer disease in Down Syndrome. There will also be two poster sessions for scientists to describe work on specific topics. Both the speakers and attendees will include Americans and representatives of countries throughout the world. The meeting will provide an opportunity for the participants to network with one another to share their knowledge, develop new collaborations, and facilitate the thrusting of Down Syndrome research forward in new directions. The conference will be convened under the direction of the National Down Syndrome Society Science Advisory Board.

animal care; biomedical facility; genetically modified animals; laboratory mouse;

The proposed studies are based on the hypothesis that cellular and organ aging are, to a significant degree, the consequences of oxygen free radical-mediated mitochondrial aging or impairment resulting from the accumulation of mitochondrial DNA mutations. Despite much indirect evidence, there is, as yet, no direct proof of the mitochondrial hypothesis of aging. However, the ability to modulate the activity of Mn superoxide dismutase (MnSOD) within mitochondria by genetic means makes it possible to assess the role of oxygen free radicals generated by the mitochondria in the aging process. To achieve this modulation of MnSOD activity, mutant mice completely lacking in MnSOD and mice with a low level of MnSOD expression from a transgene bred into the MnSOD deficient stock will be used. The finding that fetal fibroblasts lacking MnSOD are sensitive to atmospheric oxygen and have a shorter replicative life span than normal cells indicates that intramitochondrial superoxide levels can be affected by alteration of MnSOD activity. Mutant mice lacking MnSOD quickly develop a dilated cardiomyopathy and metabolic acidosis and die within 10 days after birth. However, in Specific Aim I, chimeras composed of cells completely lacking MnSOD and wild type cells will be formed, and the survival of the MnSOD-deficient cells exposed to a high level of superoxide will be followed in several organs and in the brain. In this manner it will be possible to assess the vulnerability of different types of cells and of different regions of the brain to free- radical induced mitochondrial damage. In Specific Aim II, the effects of chronically increased exposure to intra-mitochondrial superoxide will be studied in appropriately constructed transgenic animals with 10-30% of normal MnSOD activity. The longevity of these animals will determined, and the long term effects on mitochondrial DNA and the electron transport system will be assessed. In addition, behavioral studies will be carried out to determine whether any changes observed in the brain are correlated with alterations in behavior. These studies represent a new and innovative approach to the direct examination of the role of oxygen free radicals in producing mitochondrial mutations and dysfunction and, in turn, organ and organismal aging. Evidence that mitochondrial free radicals are indeed involved in the aging process would constitute a basis for searching for therapeutic approaches to the modulation of oxygen free radical levels within mitochondria.

The objective of this project is to determine the effects of alterations in the balance of oxygen free radical metabolizing enzymes on the aging process. There is considerable interest in the role which oxygen free radicals may play in the aging process, as well as in the genesis of many types of degenerative processes and reactions to trauma. A powerful approach to the investigation of these issues is to perturb the system for handling oxygen free radicals, and many ways of doing so have been used. Genetically modified animals have been used for this purpose and have been found to be a particularly powerful method for producing the types of perturbations desired, because of both the reproducibility of the changes produced and their stability over time. In this proposal are described a series of studies of the effects of altered CuZn- superoxide dismutase (CuZnSOD) and/or MnSOD activities on age-related and induced degenerative changes in the brain, heart, and other organs, on the accumulation of mitochondrial and nuclear DNA mutations and of other biomarkers of oxidative stress, and on various aspects of mitochondrial function. Particular attention will be paid to the effects of free radical imbalance on the central nervous system, and age-dependent changes in learning and memory and in the function of the basal forebrain cholinergic system will be assessed. The animals to be studied will include transgenic mice overexpressing either CuZnSOD or MnSOD, mutant mice completely lacking in CuZnSOD, and mice with absent or very low levels of MnSOD. To engineer the mice with low levels of MnSOD activity, the tetracycline-regulated transgenic transactivator system will be used and will be adapted to produce low expression of the gene for MnSOD. The strains of genetically altered mice which will be generated in this project, along with those which already exist, constitute a unique set of model animals which will make it possible to study the effects on age-related degenerative processes of intracellular SOD activities ranging from absent to greatly elevated and of the resulting alterations of superoxide levels.