The Ubiquitin Proteasome Pathway & Synaptic Plasticity

描述（由申请人提供）：突触可塑性，即经验对突触强度和结构的调节，是许多生理和病理状态的核心，包括学习和记忆以及各种神经精神疾病。虽然许多研究集中在转录和翻译对突触可塑性的长期形式的贡献上，但通过泛素蛋白酶体途径（UPP）调节蛋白质降解提供了控制细胞蛋白质组成的另一种常见手段。由于UPP可以通过精确的空间和时间控制来调节蛋白质浓度，因此它可以很容易地在可变的时间域上以突触特异性的方式对可塑性方面做出贡献。这项R21探索性资助的目的是检验UPP在突触局部发挥作用以调节突触强度和生长的假设。我们已经发现，抑制UPP与几个浴应用蛋白酶体抑制剂在感觉运动培养物中产生突触强度的持久增加，增强阿托宁诱导的可塑性，并导致感觉和运动神经元之间的新的突触接触的生长。我们现在建议确定是否在突触的UPP功能局部调节突触的功效和结构。在具体的目标1中，我们将区分UPP在突触和在细胞体的作用，通过在突触或索马培养的感觉运动神经元的体细胞局部灌注蛋白酶体抑制剂，并测量对突触强度和对阿糖胞苷诱导的长期可塑性的影响。将使用通常被蛋白酶体降解的GFP报告基因构建体监测蛋白酶体抑制的空间限制。在具体目标2中，我们将抑制突触处的蛋白酶体，并通过延时显微镜确定对突触结构的影响。本提案中概述的实验将使我们能够阐明UPP在神经元可塑性过程中在突触处的作用。它们与R21机制的目的是一致的，因为它们也将使我们能够获得足够的数据来编写R01应用程序，我们预计其目的将涉及识别调节突触功效和生长的UPP底物，并分析这些底物是否以及如何被产生持久突触可塑性的刺激所调制。

DESCRIPTION (provided by applicant): Synaptic plasticity, the modulation of synaptic strength and structure with experience, is central to many physiological and pathological states, including learning and memory and a variety of neuropsychiatric diseases. While much research has focused on the contributions of transcription and translation to long-lasting forms of synaptic plasticity, regulated protein degradation through the ubiquitin proteasome pathway (UPP) provides another common means of controlling the protein composition of cells. Because the UPP can regulate protein concentration with exquisite spatial and temporal control, it could readily contribute to aspects of plasticity over variable time domains and in a synapse-specific manner. The aim of this R21 exploratory grant is to test the hypothesis that the UPP functions locally at the synapse to regulate synaptic strength and growth. We have found that inhibition of the UPP with several bath applied proteasome inhibitors in Aplysia sensory-motor cultures produces long-lasting increases in synaptic strength, enhances serotonin-induced plasticity, and leads to the growth of new synaptic contacts between sensory and motor neurons. We now propose to determine whether the UPP at the synapse functions locally to modulate synaptic efficacy and structure. In specific aim 1, we will differentiate between the role of the UPP at the synapse and at the cell body by locally perfusing proteasome inhibitors at the synapse or the soma of cultured Aplysia sensory-motor neurons and measuring the effect on synaptic strength and on serotonin-induced longterm plasticity. The spatial restriction of proteasome inhibition will be monitored using a GFP reporter construct that is normally degraded by the proteasome. In specific aim 2, we will inhibit the proteasome at the synapse and determine the effect on synaptic structure by time-lapse microscopy. The experiments outlined in this proposal will allow us to elucidate a role for the UPP at the synapse during neuronal plasticity. They are consistent with the purpose of the R21 mechanism in that they will also allow us to obtain a sufficient body of data to write an R01 application, whose aims we anticipate will involve identification of the substrates of the UPP that regulate synaptic efficacy and growth, and analysis of whether and how these substrates are modulated by stimuli that produce long-lasting synaptic plasticity.