Protein Components of the Synaptic Adhesive Scaffold

描述（由申请人提供）：在最现代的观点，中央 神经系统突触可以被认为包括2个离散的亚结构域 它们在结构和功能上重叠。第一个结构域是突触 “支架”，其通过电子显微镜观察，由并置的， 突触前和突触后质膜增厚严格平行 通过跨越突触间隙的“横桥”连接在一起。所述支架是 即使在剧烈分馏和洗涤剂萃取后， 突触体，似乎很明显，它是由粘附在一起 分子，其身份目前仍然未知。第二个子域是 神经传递机制，通过它突触介导其 主要生理功能。此子域叠加在 支架并通过我们还不了解的分子力与它相互作用。 许多努力都集中在分析的生理组成部分， 突触;然而，支架的主要成分及其 细胞间粘附组分相互作用仍然是难以捉摸。这是 由于用于纯化完整的 中枢神经系统突触连接，以及一系列令人困惑的候选蛋白质， 可以在不同的突触处工作。显然，承认和 评估退行性过程中发生的突触蛋白的变化 必须首先依赖于一个完整的结构蛋白目录， 突触的发育和维持以及它们如何相互作用;以及 第二，对这些细胞内结合伴侣的理解， 在突触信号传导现象中起作用的支架分子， 连接到潜在的突触下成分。 从长远来看，我们想确切地了解分子粘合力 组织和稳定突触的突触前到突触后支架， 中枢神经系统中的连接复合体。我们建议：I）使用一种新的细胞分级分离法 我们设计了一种高产量纯化突触连接复合物的方法， 然后使用新开发的质谱分析方法来鉴定成分 粘附和粘附相关分子，和II）开始研究 这些分子之间的相互作用导致突触的组装 中枢神经系统中的连接复合体。

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.