Mechanisms of adhesion and recognition at CNS synapses

中枢神经系统突触的粘附和识别机制

• 批准号: 6855109
• 负责人: GREG R PHILLIPS
• 金额: $ 19.6万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6855109
• 关键词: brain cell; cell adhesion molecules; cell cell interaction; cell membrane; central nervous system; chimeric proteins; electron microscopy; immunocytochemistry; in situ hybridization; laboratory rat; light microscopy; mass spectrometry; membrane proteins; molecular shape; neural plasticity; neural transmission; protein structure function; synapses; tissue /cell culture

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.

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悬而未决的重大问题之一。对这个问题的一些见解可以从突触连接的结构中收集到。每个兴奋性中枢神经系统突触都是一个不对称的细胞-细胞连接，在连接的两侧聚集着非常不同的分子。不同的分子如何靶向突触膜并保持彼此之间的直接相对位置尚不完全清楚，但跨突触相互作用在这一过程中一定起着重要作用，也可能涉及突触连通性的规范。我们假设突触间隙包含一些对跨突触相互作用重要的未表征分子。这些分子的鉴定将揭示突触前到突触后识别在突触结构维持和连通性产生中的作用。在这个R21提案中，我们将使用靶向生化方法来鉴定介导跨突触相互作用的分子，这是通过pi的初步研究实现的，在pi的初步研究中，从中枢神经系统中分离出完整的突触连接，保留跨突触分子及其相互作用。在Aim 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恢复突触裂蛋白。回收的候选突触断裂蛋白将进行电泳和质谱鉴定。在目标2中，我们将开发抗体来鉴定候选突触裂解蛋白，用于光和EM水平，以评估潜在裂解蛋白的突触定位。在完成这些目标后，我们期望手头上有一些与最突出的突触间隙分子相对应的试剂。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起始、成熟和可塑性以及其他关键分子向突触膜募集中的作用。