CSP alpha regulation of exo-endocytic cycle enhances synaptic stability

描述(由申请人提供)：CSP？调节胞外-内吞周期增强突触稳定性。突触是一种复杂的结构，不断地进行结构修改。在健康的大脑中，突触是由依赖活动的机制维持的。在阿尔茨海默氏症和帕金森氏症等神经退行性疾病中，这些过程受到损害，导致疾病进展早期严重的突触丢失。本项目的目的是利用缺乏辅助伴侣半胱氨酸串蛋白(CSP？)的小鼠来研究突触维持的突触前机制。这只小鼠的神经系统发育正常，但在小鼠成熟后突触迅速丢失，导致严重的神经退化和早期死亡。我们已经对CSP客户进行了无偏筛选，这表明CSP正在与参与突触小泡外吞和内吞的特定蛋白质相互作用。因此，我们假设CSP通过调节突触囊泡的胞外循环和与突触前细胞骨架的相互作用来稳定突触。我将首先通过刺激神经元培养、细胞内标记和电子显微镜分析来检验依赖活性的突触囊泡循环。接下来，我将使用下拉实验和蛋白质组学分析来建立一个扩展的CSP客户蛋白质列表。最后，使用客户蛋白质列表，我将测试是否有 单一客户或客户组合能够使用过度表达和基因敲除技术改变CSP基因敲除神经元中观察到的突触丢失表型。这项研究将为了解突触在健康神经系统中是如何维持的，以及在神经退行性疾病中突触是如何丢失的提供了洞察力。

DESCRIPTION (provided by applicant): CSP¿ regulation of exo-endocytic cycle enhances synaptic stability. Synapses are intricate structures that undergo structural modifications constantly. In healthy brains, synapses are maintained by activity-dependent mechanisms. These processes are compromised in neurodegenerative diseases such as Alzheimer's and Parkinson's diseases, leading to profound synapse loss early in disease progression. The purpose of this project is to investigate presynaptic mechanisms of synapse maintenance using a mouse lacking the co-chaperone cysteine string protein ¿ (CSP¿). The nervous system of this mouse develops normally, however synapses are rapidly lost after maturation of the mouse, leading to gross neurodegeneration and early death. We have performed an unbiased screen for CSP¿ clients, which indicates that CSP¿ is interacting with select proteins involved in synaptic vesicle exo- and endocytosis. We therefore hypothesize that CSP¿ stabilizes synapses by regulating the exo-endocytic cycling of synaptic vesicles and interacting with the presynaptic cytoskeleton. I will test our hypothesis by first examining activity-dependent synaptic vesicle cycling using stimulated neuron cultures, endocytic labeling and electron microscopic analysis. Next, I will establish an expanded list of CSP¿ client proteins using pulldown experiments followed by proteomic analysis. Finally, using the list of client proteins, I will test whether any single client or a combination of clients are able to modify the synapse loss phenotype observed in CSP¿ knockout neurons using overexpression and knockdown techniques. This study will provide insight into how synapses are maintained in healthy nervous systems and how synapses are lost in neurodegenerative diseases.