The role of proteasome-dependent protein degradation in fear memory reconsolidati

通过建议的研究和培训，我将提高我的研究技能，并继续调查记忆的复杂现象背后的神经基板。具体来说，本项目的长期目标是了解泛素-蛋白酶体介导的蛋白质降解对提取后杏仁核长期记忆稳定性的贡献。这一分子过程的贡献将使用两种方法进行检查。第一种方法是使用高度选择性GST-蛋白融合系统（这是一种体外蛋白纯化技术）和高度特异性组织分级分离程序（提供突触体膜部分）来量化记忆检索后的蛋白质降解速率。第二种方法是通过注入药物来操纵杏仁核中的特定分子过程，这些药物通过泛素-蛋白酶体系统阻止蛋白质降解，通过阻断翻译来阻止蛋白质合成，通过阻断NMDA受体来阻止多巴胺能活性，以及通过阻断几种蛋白激酶来阻止细胞内信号传导。将讨论四个具体目标。第一个目的是确定在提取背景和听觉恐惧记忆后，杏仁核中的蛋白质降解是否增加。为了实现这一目标，恐惧条件反射将被用来创建令人厌恶的上下文和基于记忆的记忆和蛋白质降解率将被量化的杏仁核检索后使用GST-蛋白质融合系统。第二个目标，即泛素-蛋白酶体系统的特定突触靶点在检索后是什么，将使用突触体膜制备和GST-蛋白融合系统实现。第三个目标，即杏仁核中的蛋白质降解是否对背景和听觉恐惧记忆的重新巩固至关重要，将通过靶向注射阻断蛋白质降解和蛋白质合成的药物来实现。最后的目的是确定什么分子机制的信号，这些增加蛋白质降解杏仁核后检索。为了实现这一目标，在回收前输注阻断NMDA受体活性和几种细胞内信号级联的药物，并在回收后使用GST-蛋白融合系统定量蛋白降解速率。 总的来说，这组实验将回答以下问题：蛋白酶体依赖性蛋白质降解是否1）参与突触后密度重排，2）是否严重参与杏仁核依赖性恐惧记忆的再巩固，3）是在再巩固过程中对蛋白质合成的需要的基础，4）是由调节蛋白质合成和“去稳定化”的相同机制触发的。记忆恢复后的记忆。

Through the proposed research and training I will improve my research skills and continue to investigate neural substrates underlying the complex phenomenon of memory. Specifically, the long-term goal of this project is to understand the contribution of ubiquitin-proteasome mediated protein degradation to the stability of long-term memory in the amygdala following retrieval. The contribution of this molecular process will be examined using two approaches. The first approach is to quantify the rate of protein degradation following memory retrieval using a highly selective GST-protein fusion system, which is an in vitro protein purification technique, and highly specific tissue fractionation procedure, which provides a synaptosomal membrane fraction. The second approach is to manipulate specific molecular processes in the amygdala with infusions of drugs that prevent protein degradation through the ubiquitin-proteasome system, protein synthesis by blocking translation, glutaminergic activity by blocking NMDA receptors, and intracellular signaling by blocking several protein kinases. Four specific aims will be addressed. The first aim is to determine whether protein degradation is increased in the amygdala following the retrieval of context and auditory fear memories. To achieve this aim, fear conditioning will be used to create aversive context- and auditory-based memories and the rate of protein degradation will be quantified in the amygdala following retrieval using the GST-protein fusion system. The second aim, which addresses what the specific synaptic targets of the ubiquitin- proteasome system are following retrieval, will be achieved using the synaptosomal membrane preparation and GST-protein fusion system. The third aim, which addresses whether protein degradation in the amygdala is critical for the reconsolidation of context and auditory fear memories, will be achieved using targeted infusions of drugs that block protein degradation and protein synthesis. The final aim is to determine what molecular mechanisms signal these increases in protein degradation within the amygdala following retrieval. To achieve this aim, infusions of drugs which block NMDA receptor activity and several intracellular signaling cascades will be given prior to retrieval and the rate of protein degradation will be quantified following retrieval using the GST-protein fusion system. Collectively, this set of experiments will answer questions about whether proteasome-dependent protein degradation 1) is involved in postsynaptic density rearrangement following retrieval, 2) is critically involved in amygdala-dependent fear memory reconsolidation, 3) is underlying the requirement for protein synthesis in the reconsolidation process and 4) is being triggered by the same mechanisms which regulate protein synthesis and the "destabilization" of stored memory following retrieval.