CSP alpha regulation of exo-endocytic cycle enhances synaptic stability

描述（由申请人提供）：CSP -调节外内吞周期增强突触稳定性。突触是一种复杂的结构，它会不断地发生结构变化。在健康的大脑中，突触是由活动依赖机制维持的。这些过程在阿尔茨海默病和帕金森病等神经退行性疾病中受损，导致疾病进展早期严重的突触丧失。本项目的目的是在缺乏共伴侣半胱氨酸弦蛋白（CSP）的小鼠中研究突触维持的突触前机制。这只老鼠的神经系统发育正常，但在老鼠成熟后突触迅速丢失，导致神经退化和早期死亡。我们对CSP¿客户进行了无偏筛选，这表明CSP¿与参与突触囊泡外吞和内吞作用的选定蛋白质相互作用。因此，我们假设CSP¿通过调节突触囊泡的外胞吞循环和与突触前细胞骨架相互作用来稳定突触。我将测试我们的假设，首先检查活动依赖突触囊泡循环使用刺激神经元培养，内吞标记和电镜分析。接下来，我将使用下拉实验建立CSP¿客户端蛋白的扩展列表，然后进行蛋白质组学分析。最后，使用客户端蛋白列表，我将测试是否有

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. PUBLIC HEALTH RELEVANCE: Neurodegenerative diseases are devastating for both patients and caretakers alike and will continue to affect an exponentially expanding portion of the population without novel therapeutics. A common denominator of neurodegenerative diseases is protein misfolding. By investigating an essential mechanism that assists synaptic protein folding, this project will characterize a novel pathway for prevention of neurodegenerative disease.