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

描述（由申请人提供）：通过拟议的研究和培训，我将提高我的研究技能，并继续研究复杂记忆现象背后的神经基质。具体来说，该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杏仁核长期记忆恢复后稳定性的贡献。这个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选择性gst蛋白融合系统（这是一种体外蛋白质纯化技术）和高度特异性的组织分离程序（提供突触体膜部分）来量化记忆检索后蛋白质降解的速率。第二种方法是通过输注药物来控制杏仁核中的特定分子过程，这些药物通过泛素-蛋白酶体系统来阻止蛋白质降解，通过阻断翻译来阻止蛋白质合成，通过阻断NMDA受体来阻止谷氨酰胺能活性，以及通过阻断几种蛋白激酶来阻止细胞内信号传导。将处理四个具体目标。第一个目的是确定在检索情境和听觉恐惧记忆后，杏仁核中的蛋白质降解是否会增加。为了实现这一目标，恐惧条件反射将用于创建基于情境和听觉的厌恶记忆，并且在使用gst -蛋白质融合系统检索后，将量化杏仁核中蛋白质降解的速度。第二个目标是解决泛素-蛋白酶体系统在检索后的特定突触目标，将使用突触体膜制备和gst蛋白融合系统来实现。第三个目标是研究杏仁核中的蛋白质降解是否对情境和听觉恐惧记忆的重新巩固至关重要，将通过阻断蛋白质降解和蛋白质合成的靶向药物输注来实现。最终的目的是确定哪些分子机制表明在检索后杏仁核内蛋白质降解的增加。为了实现这一目标，将在检索之前给予阻断NMDA受体活性和几个细胞内信号级联的药物，并在检索后使用gst -蛋白质融合系统量化蛋白质降解率。总的来说，这组实验将回答以下问题：1)依赖蛋白酶体的蛋白质降解是否参与检索后的突触后密度重排，2)关键参与杏仁核依赖的恐惧记忆再巩固，3)在再巩固过程中对蛋白质合成的需求是潜在的，4)是否由调节蛋白质合成和检索后存储记忆的“不稳定”的相同机制触发。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Using the Pavlovian fear conditioning paradigm, the proposed experiments outlined in this document will expand the current literature on the physiological underpinnings of memory formation. Enhanced understanding of the biological mechanisms underlying memory is important not only for increasing our basic knowledge of this process but also for the potential translation of research findings to both normal and disordered memory in humans.