One of the hallmark features of the nervous system is the dramatic variety of cell types and connections formed between cells. The overall goal of this research is to identify molecular regulators of the processes of development and differentiation in this system. Mechanisms are sought whereby this cell-type specificity and connectivity is generated and functions to maintain the dramatic plasticity of the system throughout life. Two anti-mouse monoclonal antibodies which specifically recognize neuronal nuclear proteins, called NeuN and F41, have been generated. The nervous system of mice develops in a manner similar to humans but over a greatly condensed time span so that these animals are an excellent model of human nervous system development and function. Indeed, the monoclonals described herein cross react with human neuronal nuclear antigens. The pattern of expression and biochemical properties of these mouse neuronal nuclear antigens suggest that they may be intimately involved in the regulation of nervous system differentiation. For example, the antigens display different but overlapping neuronal cell-type specificities. The genes encoding these antigens have been identified by screening recombinant cDNA expression libraries with the monoclonal antibodies. Full nucleotide sequence of the mRNA encoding these antigens will be obtained and exhaustively compared with known genetic sequence databases to determine if related genes have been described and to provide insight into the function of these antigens. Purified protein will be subjected to limited amino acid sequence analysis to confirm the cDNA sequence of these genes. Antisense oligonucleotides or cDNA constructs will be introduced to cell lines or primary neuronal cultures in an attempt to eliminate or reduce these proteins to directly assay protein function. In addition, cDNA clones will be used to explore the function of these antigens by introducing them into heterologous cell types. Factors such as phenotypic differentiation and changes in transcriptional activity will be assessed in the transfected cells. The possibility that the antigens bind to DNA specifically and regulate the expression of other genes will also be explored using antigen protein binding to known or putative regulator sequences. Antigen proteins will be further characterized biochemically by analyzing electrophoretic mobilities and the state of phosphorylation and glycosylation of the proteins. The antigenic protein will be purified using immunoaffinity chromatography and HPLC. These novel, nervous system specific nuclear proteins will illuminate mechanisms of regulation of the neuronal differentiation and development.
