HEPARAN SULFATE PROTEOGLYCANS IN NEURAL DEVELOPMENT

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The long-term goal of this project is to obtain an understanding of the role of heparan sulfate proteoglycans (HSPGs) in the regulation of cell interactions in the developing nervous system. Previous studies have documented a critical role for HSPG in the function of the neural cell adhesion molecule (NCAM). In view of this importance we have recently begun to characterize the HSPG of developing chick brain that interacts with NCAM. Recent studies have demonstrated that an abundant developmentally-regulated HSPG in chick brain, which contains a protein core of 250 kDa, is capable of binding to the heparin-binding domain (HBD) of NCAM. The HSPG has been purified by monoclonal antibody affinity chromatography and shown to promote cell adhesion when used as a substratum. Its role in cell adhesion requires heparan sulfate chains on the HSPG, since elimination of heparan sulfate from the molecule abolishes its ability to participate in cell adhesion. In order to begin to understand the molecular mechanisms by which this HSPG regulates NCAM function and cell adhesion, we have initiated molecular cloning of the core protein of the HSPG. These studies indicate that the core protein is identical to agrin, an extracellular matrix protein suggested to play a critical role in neuromuscular synaptogenesis. These findings are of interest since the function of agrin in brain is unclear, and our data suggest that brain agrin, as a HSPG, may regulate cell adhesion processes. To continue our characterization of this HSPG, now named PG-agrin (proteoglycan-agrin), we will address the following specific aims: l) Conduct a molecular and biochemical characterization of PG-agrin, with an emphasis on determining if alternatively spliced variants of agrin encode PG-agrin. These experiments will also examine the relationship of agrin to PG-agrin, and determine if other brain HSPGs are capable of interacting with NCAM. 2) Determine the distribution of PG-agrin protein and mRNA in developing chick nervous tissue. 3) Examine the molecular mechanisms by which PG-agrin modulates NCAM function, using non-neural and neural cell lines transfected with NCAM and/or PG-agrin cDNAs. 4) Characterize the role of PG-agrin in chick brain development in vivo. PG-agrin sense and antisense plasmid cDNA will be introduced into developing chick embryos using lipospermine, and the effect of these plasmids on neural development will be examined. The proposed studies are important since they will ultimately provide insight into the function of HSPGs in neural development, and will also provide crucial information about possible functions of agrin during CNS development.