This project is designed to determine the function and subcellular localization characteristics of a novel developmentally regulated antigen during neuronal differentiation, human brain development, and in Alzheimers disease (AD). The FAC 1 gene was identified by immunoscreening a human fetal brain cDNA expression library with the monoclonal antibody AIz-50. N- terminal FAC 1 gene sequences were not obtained from the fetal brain library. RT-PCR technologies will be utilized to isolate additional N- terminal FAC I sequences from human fetal cortex and fetal rat brain total RNA. The FAC 1 protein is expressed at high levels in the developing brain and at low levels in normal adult brain. In postnatal and adult brain FAC 1 protein is localize the nucleus of cells whereas during development the protein is located both in the nucleus and in the cytoplasm. The selective removal of FAC 1 from the cytoplasm in adult brain after birth represents a novel mechanism for regulated accumulation of nuclear proteins. In AD cortex FAC 1 protein is contained in a subset of neuritic plaques and in the cytoplasm of pyramidal cells. FAC 1 labeled neurites in AD brain may be a regenerative response to neuronal degeneration. By double label confocal microscopy we will determine if the cytoplasmic localization of FAC 1 in AD occurs in degenerating, regenerating or cells more resistant to damage. To test for FAC 1 expression and function during neurite outgrowth, immortalized neuronal cell lines will be-induced to differentiate. The appearance of FAC 1 protein in neurites will be determined by - immunocytochemistry, and the re-expression of FAC1 during differentiation will be monitored by Northern blot. A primary- goal of this proposal is to identify proteins that interact with FAC I both in the cytoplasm and in the nucleus. To accomplish this goal, FAC1 protein will be immunoaffinity purified from cytoplasmic fractions of human fetal brain. The co-purification of any cytoskeletal elements will be determined by immunoblot with antibodies to known cytoskeletal proteins. Monoclonal antibodies will be generated to novel proteins that co-purify with FAC I. Confirmation of protein-protein interactions will be performed by Co-immunoprecipitation and confocal microscopy studies. The intranuclear localization of FAC 1 will be determined by isolation of nuclear matrix components and confocal microscopy. Chances in the intranuclear localization of FAC I in degenerating cells of AD brain will also be determined by confocal microscopy. These results may yield new information about nuclear damage that occurs during AD. This project will provide novel insight into mechanisms that regulate neurite outgrowth and the selective expression and localization of proteins during both development and degeneration of the nervous system.
