Role of N-cadherin in synapse formation and maintenance

描述(申请人提供)：这项建议的目的是研究黏附分子N-钙粘附素在哺乳动物大脑中发育中的突触中的定位和作用。突触前和突触后特化的形成以及信号转导所需蛋白质的分子编排是在轴突与靶点接触后形成的，现在已知在没有神经传递的情况下发生。目前尚不清楚这一过程需要什么蛋白质和相互作用，但人们怀疑黏附分子将发挥关键作用。钙粘附素在细胞形态发生、轴突生长和轴突引导过程中调节重要的黏附相互作用，但钙粘附素在神经突触中的重要性仍不清楚。N-钙粘蛋白的分布和时间表达模式表明，这种黏附分子参与突触的形成。因此，在这项研究中，我们希望更准确地确定N-钙粘素聚集在突触部位的定位和时间，并确定它是否是突触形成所必需的。这些实验将在活的大鼠海马片上进行，并将包括各种技术，包括免疫细胞化学、GFP和DsRed融合蛋白的生物转染、使用显性负蛋白结构和延时共聚焦显微镜。本文提出的研究将进一步加深我们对细胞黏附分子作用的理解，也将阐明哺乳动物大脑发育中突触形成和维持的机制。

DESCRIPTION (provided by applicant): The aim of this proposal is to investigate the localization and role of the adhesion molecule N-cadherin at the developing synapse in the mammalian brain. Formation of pre- and postsynaptic specializations and the molecular orchestration of essential proteins for signal transduction follows contact of an axon with a target and is now known to occur in the absence of neurotransmission. It is not yet known what proteins and interactions are required for this process, but it is suspected that adhesion molecules will play a crucial role. Cadherins mediate important adhesive interactions during cellular morphogenesis, neurite outgrowth, and axonal guidance, but the importance of cadherins at the neural synapse remains unknown. The distribution and temporal expression patterns of N-cadherin implicate this adhesion molecule in synapse formation. Therefore, in this study we hope to determine more precisely the localization and timing of N-cadherin aggregation at synaptic sites and also determine if it is necessary for synapse formation. These experiments will be conducted in live rat hippocampal slices and will include a variety of techniques including immunocytochemistry, biolistic transfection of GFP and DsRed-fusion proteins, use dominant negative protein constructs, and time-lapse confocal microscopy. The studies proposed here will further our understanding of the roles of cell adhesion molecules and also elucidate mechanisms of synapse formation and maintenance in the developing mammalian brain.