Mechanisms of adhesion and recognition at CNS synapses

中枢神经系统中突触连接的最终模式产生的机制是神经生物学中尚未回答的重大问题之一。从突触连接的结构中可以得到一些对这个问题的理解。每个兴奋性中枢神经系统突触是一个不对称的细胞-细胞连接，具有非常不同的分子集合聚集在连接的各自侧上。不同的分子如何靶向突触膜并保持彼此直接并置仍不完全清楚，但跨突触相互作用必须在这一过程中发挥重要作用，也可能参与突触连接的规范。我们假设，突触间隙中含有一些重要的跨突触相互作用的未知分子。这些分子的鉴定将揭示突触前到突触后识别在维持突触结构和产生连接中的作用。在这个R21提案中，我们将使用有针对性的生化方法来识别介导跨突触相互作用的分子，这是通过PI初步研究实现的，其中完整的突触连接从CNS中分离出来，保留跨突触分子及其相互作用。在目标1中，我们将利用细胞表面生物素化和固定化凝集素在分离的突触膜上回收突触间隙蛋白。筛选出的候选突触间隙蛋白将通过质谱法进行分析和鉴定。在目标2中，我们将开发用于在光和EM水平上使用的识别候选突触间隙蛋白的抗体，以评估潜在间隙蛋白的突触定位。在完成这些目标后，我们希望手头上有一些试剂对应于一些最突出的突触间隙分子。我们将使用这些试剂来研究细胞-细胞相互作用在突触连接的启动、成熟和可塑性以及在突触膜上募集其他关键分子中的作用。

The mechanisms by which the ultimate pattern of synaptic connections arises in the CNS is one of the great unanswered questions in neurobiology. Some insights into this question may be gleaned from the structure of synaptic junctions. Each excitatory CNS synapse is an asymmetric cell-cell junction, with very different sets of molecules clustered on the respective sides of the junction. How the different sets of molecules target to synaptic membranes and remain in direct apposition to each other is still not completely understood but trans-synaptic interactions must have an important role in this process and are also probably involved in the specification of synaptic connectivity. We hypothesize that the synaptic cleft contains a number of uncharacterized molecules important for trans-synaptic interactions. Identification of such molecules will shed new light on the role of pre-to-postsynaptic recognition in the maintenance of synaptic architecture and in the generation of connectivity. In this R21 proposal, we will use a targeted biochemical approach to identify the molecules that mediate trans-synaptic interactions, made possible by the PIs preliminary studies in which intact synaptic junctions were isolated from the CNS, preserving trans-synaptic molecules and their interactions. In Aim 1, we will recover synaptic cleft proteins on isolated synaptic membranes by utilizing cell surface biotinylation and immobilized lectins. Recovered candidate synaptic cleft proteins will be electrophoresed and identified by mass spectrometry. In Aim 2, we will develop antibodies to identified candidate synaptic cleft proteins for use at the light and EM levels to evaluate the synaptic localization of potential cleft proteins. Upon completion of these Aims, we expect to have in hand reagents corresponding to some of the most prominent synaptic cleft molecules. We will use these reagents to examine the role of cell-cell interactions in the initiation, maturation and plasticity of synaptic junctions and in the recruitment of other critical molecules to synaptic membranes.