The aim of these continuing investigations is to correlate the structure, localization, biosynthesis and metabolism, and functional effects of nervous tissue proteoglycans as a means by which to better understand their roles in neurobiological processes such as cell interactions, the control of cell growth and differentiation, and nervous tissue histogenesis. Because we have found that monoclonal antibodies are currently the only reliable tool for the identification, isolation, and eventual biochemical characterization of the highly heterogeneous and complex population of chondroitin sulfate proteoglycans in brain, we now plan to prepare a new panel of monoclonal antibodies using as immunogen the chondroitin sulfate proteoglycans of 7-day postnatal rat brain from which three proteoglycans also present in adult brain have been immunodepleted. This work should provide unique reagents allowing the identification and biochemical characterization of chondroitin sulfate proteoglycans which may disappear during early postnatal brain development. In a second project, we plan to characterize the proteoglycans synthesized by a continuous line of human cerebral cortical neurons (HCN-1A) in their undifferentiated and differentiated states, and to examine the effects of transforming growth factor-beta on neuronal proteoglycans. The results from these studies should be of particular importance because HCN-1A cells are probably both considerably closer in their properties to """"""""normal"""""""" neurons than the neurotumor cell lines which are now widely employed as model systems in neurobiological investigations, and have the additional important property that they are of central nervous tissue origin. A third specific aim concerns the cloning and further characterization of one of the developmentally-regulated chondroitin sulfate proteoglycans of brain which we have identified with out monoclonal antibodies and biochemically characterized. These studies will give more detailed information concerning protein sequence homologies of potential functional importance (e.g., growth factor-like and lectin-like domains indicated by our preliminary data), on tissue- and species-distribution of message, as well as on the question of whether alternative splicing may account for the developmental changes in core protein size. In situ hybridization studies will be performed to identify the cellular origin of the extracellular form of the proteoglycan in early postnatal brain (and possibly also of alternatively spliced species), as compared to the intracellular (cytoplasmic) proteoglycans found in adult brain. These experiments provide a starting point for a molecular and functional analysis of nervous tissue proteoglycans at the nucleic acid level, and by inserting cloned antisense RNA into appropriate vectors for the extinction of proteoglycan gene expression, it should eventually be possible to determine the effect of targeted elimination (or expression) of specific proteoglycans on such processes as cell growth, cell interactions, and neuronal differentiation.
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