Aggregation of presynaptic proteins during LTP

描述（由申请人提供）：海马突触的持久增强是突触可塑性的一种形式，被认为是哺乳动物学习和记忆的可能底物。为了研究增强作用的机制，我们最近在海马神经元的分离培养物中检测了增强作用期间突触前和突触后蛋白的免疫反应性（Antonova等人，2001年）。与最近的研究相一致，我们发现，在持久增强的开始时，突触后蛋白（GluR1，PSD 95）的簇（点）增加。然而，我们还发现，这些突触后的变化伴随着突触前蛋白（突触素，突触蛋白I，突触核蛋白）和前和突触后蛋白共定位，因此可能参与功能性突触的网站集群的快速增加。我们现在建议以两种方式扩展这些发现。首先，我们将通过测试新的突触前点是否立即参与新的功能性突触，或者它们是否是随着时间的推移成熟为功能性突触的结构的一部分，来研究新的突触前点的意义。由于共定位的突触前和突触后点的组装让人想起早期的突触发生，而增强的后期阶段伴随着新突触的生长，因此认为新的点可能代表该过程中的一个步骤是很有吸引力的。因此，我们建议调查是否持久的增强涉及协调组装的各种突触组件，发生在突触发育。 其次，我们将开始研究可能参与新斑点形成的逆行信使，包括自由扩散的分子如NO，细胞外信使如神经营养因子和粘附分子。我们还将研究这些信使可能的突触前效应物，重点关注已知参与调节肌动蛋白细胞骨架的效应物，肌动蛋白细胞骨架在突触前点的增加中起着关键作用。这些研究应该提供新的信息的功能意义和分子机制的一个新方面的突触可塑性，可能是重要的学习和记忆。

DESCRIPTION (provided by applicant): Long-lasting potentiation at hippocampal synapses is a form of synaptic plasticity that is thought to be a likely substrate of mammalian learning and memory. To investigate mechanisms of the potentiation, we recently examined immunoreactivity for pre- and postsynaptic proteins during potentiation in dissociated cultures of hippocampal neurons (Antonova et al., 2001). Consistent with recent studies, we found that there is an increase in clusters (puncta) of postsynaptic proteins (GluR1, PSD95) at the onset of long-lasting potentiation. However, we also found that these postsynaptic changes are accompanied by a rapid increase in clusters of presynaptic proteins (synaptophysin, synapsin I, synuclein) and sites where the pre- and postsynaptic proteins colocalize and therefore might participate in functional synapses. We now propose to extend these findings in two ways. First, we will examine the significance of the new presynaptic puncta by testing whether they participate in new functional synapses immediately, or whether they are part of structures that mature into functional synapses with the passage of time. Because the assembly of colocalized pre- and postsynaptic puncta is reminiscent of early synaptogenesis and the later stages of potentiation are accompanied by the growth of new synapses, it is attractive to think that the new puncta might represent a step in that process. We therefore propose to investigate whether long-lasting potentiation involves the coordinated assembly of a variety of synaptic components, as occurs during synaptic development. Second, we will begin to investigate retrograde messengers that may be involved in the formation of the new puncta, including freely diffusible molecules such as NO, extracellular messengers such as neurotrophins, and adhesion molecules. We will also investigate possible presynaptic effectors of these messengers, focusing on ones that are known to be involved in regulating the actin cytoskeleton, which plays a critical role in the increase in presynaptic puncta. These studies should provide new information about the functional significance and molecular mechanisms of a novel aspect of synaptic plasticity that may be important for learning and memory.