Protein Components of the Synaptic Adhesive Scaffold

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Description (Provided by applicant): In the most contemporary view, the central nervous System synapse may be thought of as comprising 2 discrete subdomains which overlap structurally and functionally. The first domain is the synaptic "scaffold" which is observed by electron microscopy, consisting of apposed, rigorously parallel presynaptic and postsynaptic plasma membrane thickenings bound together by "crossbridges" that span the synaptic cleft. The scaffold is retained even after vigorous fractionation and detergent extraction of synaptosomes, and it seems clear that it is held together by adhesion molecules, whose identities remain unknown at present. The second subdomain is the neurotransmissional machinery through which the synapse mediates its primary physiological functions. This subdomain is superimposed upon the scaffold and interacts with it via molecular forces we don't understand as yet. Much effort has been focused on analyzing the physiological components of the synapse; however, the major constituents of the scaffold and how its intercellular adhesive components interact have remained elusive. This is because of inadequate fractionation techniques for the purification of intact CNS synaptic junctions, and the bewildering array of candidate proteins that may operate at different synapses. It is clear that efforts to recognize and evaluate changes in synaptic proteins which occur during degenerative processes must rely first on a complete catalog of the structural proteins involved in synaptic development and maintenance and how they interact with each other; and second, on an understanding of the intracellular binding partners for these scaffolding molecules which function in synaptic signaling phenomena, and in attachment to the underlying subsynaptic components. Long range, we want to understand exactly how molecular adhesive forces organize and stabilize the pre-to post-synaptic scaffold of the synaptic junctional complex in the CNS. We propose to: I) use a novel cell fractionation procedure we devised to purify synaptic junctional complexes in high yield, and then use newly developed methods in mass spectrometry to identify the component adhesion and adhesion associated molecules, and II) begin studies on the interactions between these molecules which lead to the assembly of the synaptic junctional complex in the CNS.