INTEGRIN LIGANDS IN SYNAPTIC PLASTICITY

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A. Specific Aims There were no significant changes in the Specific Aims of the funded proposal. B. Studies and Results Specific Aim 1 was to test for CA1 integrin ligands. To find integrin ligands we have continued to use hippocampal neurosynaptosomes as a resource for synaptic material. Our cross-linking experiments and cell blot experiments continued in the current budget year. We wanted to obtain results from more experiments in order to verify whether the data were reliably reproducible. The cell blot experiments have revealed four bands that support adhesion. The blot is conducted by resolving hippocampal, neurosynaptosomes by polyacrylamide gel electrophoresis, transferring the proteins to a membrane, and use of the membrane as a substratum. We tested several vehicles to incubate cells with the membranes, and found 2 useful methods. One, which we reported, was the use of syringes to incubate cells with the membrane. The other was a apparatus designed specifically for immunoblots, which we used for living cells. Both vehicles worked with similar efficiency. The bands identified migrate at positions corresponding to approximately 50, 75, 100 and 150 kDa. Mass spectrometry analysis of the 50 kDa region identified a number of proteins. Of the proteins in the 50 kDa region, one or more support cell attachment reliably. We are submitting more samples for mass spectrometry analysis in order to see whether there are cell adhesion is reproduced on molecules from the 50 kDa region isolated from neurosynaptosomes of different preparations, and test for ligand identity in the 75, 100, and 150 kDa regions of the gel. The experiments in Specific Aim 2 test for the location of integrin ligands. Using immuno-transmission electron microscopy we have located anti-integrin antibody in CA1. The data indicate integrins are at synapses. We are additionally testing for the location of a fluorophore-labeled GRGDSP integrin ligand peptide in LFS and HFS hippocampal slices. Our confocal images indicate that the ligand is located near CA1 apical dendrites. We are now testing for colocalizations with actin and synaptophysin. The experiments in Specific Aim 3 will test for effects on LTP when ligand in blocked. We have tested RNA1 oligonucleotides on cultured cells to see whether we can knock down integrins. Using standard transfection techniques we achieved a 60% reduction in integrin expression, as accessed by quantitative PCR and immunoblot analyses. This helps us set the stage to knockdown ligand identified by mass spectrometry. The knockdowns are a good approach to test for ligand roles in CA1 LTP. Anti-ligand antibodies, if available, will serve as a complementary approach. C. Significance Molecular processes underlying synaptic plasticity are complex and regulated by a number of different extracellular, cell-surface and intracellular molecules. There is compelling genetic and biochemical evidence that the cell-surface integrin receptors are important for CA! synaptic events. In hippocampal slices, anti-integrin antibodies and integrin ligand peptides antagonized the maintenance of LTP. Interestingly, in hippocampal slices, HFS triggers translocation of integrins that are in secretory vesicles to the cell surface. The translocation occurs within 1-2 minutes after LTP induction, so increased integrin expression is expected to foster important changes in the cytoskeleton, the shape of plasma membranes at and near synapses, and activate intracellular signaling pathways. Although integrin ligands are known in other systems, the identity and location of integrin ligand important for the maintenance of CA1 LTP is not clear. Our ultrastructural data indicates that integrins are at synapses, and mass spectrometry analysis found neurosynaptosome adhesion proteins. We are presently testing whether the findings are reproducible. If the finding is verified and ligand found, it will help us understand the when and where the signaling actions occur for the maintenance of LTP. The new information will fill in gaps in our present knowledge, and is expected to help us understand processes underlying neurodegenerative disorders.