Molecular Biology of the Synapse

描述（由申请人提供）：潜在的突触传递是突触囊泡周期，其涉及突触囊泡与质膜融合以释放神经递质，随后通过网格蛋白介导的内吞作用回收囊泡蛋白。网格蛋白介导的内吞作用还涉及对突触传递和突触强度的调节重要的其他过程，包括神经递质受体和转运蛋白运输。网格蛋白包被的囊泡也在从酵母到人类的所有区室化细胞的运输中起主要作用。因此，了解网格蛋白介导的囊泡运输机制是神经科学家和细胞生物学家的广泛兴趣。网格蛋白包被的囊泡循环涉及从胞质溶胶中募集网格蛋白三聚体，它们在膜上聚合以形成网格蛋白包被的小凹，其在断裂反应后内化为网格蛋白包被的囊泡，随后通过Hsp 70伴侣解聚以释放三聚体，使得循环可以继续。我们下一个项目期间的目标将使我们在这个项目上的工作在深度和广度上达到一个新的水平。随着我们深入研究突触囊泡周期，我们也将扩大这项工作的影响，因为我们对网格蛋白包被的囊泡和伴侣蛋白周期的了解将影响比我们对突触机制的初步探索更广泛的研究领域。事实上，Hsp 70家族成员在解离与许多神经退行性疾病相关的蛋白质聚集体方面发挥着重要作用，因此这项工作也将影响我们对蛋白质聚集疾病的理解。我们在下一个项目期间的目标是（1）表征网格蛋白及其内在非结构化结合伙伴之间的相互作用。在这个目标中，我们将使用NMR光谱来表征网格蛋白重链的40 kD N-末端结构域和三个关键的内吞蛋白，AP 180，AP-2和amphiphysin之间的相互作用。(2)明确Hsc 70/auxilin对网格蛋白包被囊泡脱壳的物理机制。我们的工作是在一个点上，我们在一个强有力的位置，以了解如何Hsc 70促进脱壳在一个基本的水平。(3)定义核苷酸交换因子在调节Hsc 70中的作用：网格蛋白相互作用和突触囊泡运输。我们最近发现，核苷酸交换因子促进了一个长寿的Hsc 70-网格蛋白复合物的解离。因此，在这个目标中，我们将确定哪些核苷酸交换因子参与神经末梢中的突触囊泡运输，以及确定核苷酸交换因子Hsc 70结合和释放的周期是如何控制的。这项工作有望推进我们对突触传递机制的基本理解，因此将成为我们对抗神经系统疾病的关键部分。 公共卫生相关性：这项工作的重点是了解突触传递的基本机制，这是神经元相互交流的过程。许多神经系统疾病的一个共同特征是异常的突触传递，因此这项工作对于理解和开发能够进行临床干预的治疗策略将是重要的。此外，我们将研究的伴侣蛋白也涉及许多疾病，这些疾病是受损聚集蛋白积累的结果（阿尔茨海默氏症，ALS，帕金森氏症，亨廷顿氏症等），因此这项工作也将与对抗神经退行性疾病有关。

DESCRIPTION (provided by applicant): Underlying synaptic transmission is the synaptic vesicle cycle, which involves fusion of synaptic vesicles with the plasma membrane to release neurotransmitter and subsequent retrieval of vesicular proteins via clathrin mediated endocytosis. Clathrin mediated endocytosis is also involved in other processes important for synaptic transmission and the modulation of synaptic strength, including neurotransmitter receptor and transporter trafficking. Clathrin coated vesicles also play a major role in trafficking in all compartmentalized cells from yeast to humans. Understanding the mechanisms of clathrin mediated vesicular transport is therefore of broad interest to neuroscientists as well as cell biologists. The clathrin coated vesicle cycle involves recruitment of clathrin triskelia from the cytosol, their polymerization on a membrane to form clathrin coated pits that, following a scission reaction, are internalized as clathrin coated vesicles, and subsequently depolymerized by an Hsp70 chaperone to release triskelia, so that the cycle may continue. Our goals for the next project period will bring a new level of both depth and breadth to our work on this project. As we delve deeper into the synaptic vesicle cycle, we will also broaden the impact of the work, since what we learn about the clathrin coated vesicle and chaperone cycles will impact a wider range of research areas than our initial inquiry into synaptic mechanisms. Indeed, Hsp70 family members play important roles in dissociating the protein aggregates that are associated with many neurodegenerative diseases, so this work will also impact our understanding of protein aggregation disease. Our aims during this next project period are to (1) Characterize the interactions between clathrin and its intrinsically unstructured binding partners. In this aim, we will use NMR spectroscopy to characterize the interaction between the 40 kD N-terminal domain of the clathrin heavy chain and three key endocytic proteins, AP180, AP-2 and amphiphysin. (2) Define the physical mechanism of clathrin coated vesicle uncoating by Hsc70/auxilin. Our work is at the point where we are in a strong position to understand how Hsc70 promotes uncoating at a fundamental level. (3) Define the role of nucleotide exchange factors in regulating Hsc70:clathrin interactions and synaptic vesicle trafficking. We recently discovered that nucleotide exchange factors promote the dissociation of a long-lived Hsc70-clathrin complex. Therefore, in this aim we will determine which nucleotide exchange factor is involved in synaptic vesicle trafficking in nerve terminals, as well as determine how cycles of nucleotide exchange factor:Hsc70 binding and release are controlled. This work is expected to advance our fundamental understanding of the mechanisms that underlie synaptic transmission, and as such will be a critical part of our efforts to fight neurological disorders. PUBLIC HEALTH RELEVANCE: This work is focused on understanding the fundamental mechanisms of synaptic transmission, the process used by neurons to communicate with each other. A common feature of many neurological disorders is aberrant synaptic transmission, so this work will be important for understanding and developing therapeutic strategies that will enable clinical intervention. Moreover, the chaperone proteins that we will study are also involved in many diseases that are a consequence of the accumulation of damaged, aggregated proteins (Alzheimer's, ALS, Parkinson's, Huntington's, and others), so this work will also be relevant to the fight against neurodegenerative disorders.