Molecular Biology of the Synapse

描述(申请人提供)：基本的突触传递是突触小泡周期，它涉及突触小泡与质膜融合以释放神经递质，随后通过网状蛋白介导的内吞作用恢复囊泡蛋白。笼蛋白介导的内吞作用还参与了突触传递和突触强度调节的其他重要过程，包括神经递质受体和转运体运输。在从酵母到人类的所有隔室细胞的运输中，笼蛋白包裹的囊泡也起着主要作用。因此，了解网状蛋白介导的囊泡运输机制引起了神经学家和细胞生物学家的广泛兴趣。分子筛包被囊泡循环包括从胞浆中招募分子，它们在膜上聚合形成包被分子囊，经过裂解反应后被内化为包被分子囊，随后被热休克蛋白70分子伴侣解聚以释放分子囊，从而使循环得以继续。我们下一个项目期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。随着我们更深入地研究突触小泡周期，我们也将扩大这项工作的影响，因为我们所了解的关于笼蛋白包裹的小泡和伴侣周期的影响将比我们最初对突触机制的探讨影响更广泛的研究领域。事实上，Hsp70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性疾病的理解。我们在下一个项目期间的目标是(1)表征网状蛋白与其固有的非结构化结合伙伴之间的相互作用。在这一目标中，我们将使用核磁共振波谱来表征网状蛋白重链的40kD N末端结构域与三种关键的内吞蛋白AP180、AP-2和两栖动物蛋白之间的相互作用。(2)明确Hsc70/Axlin脱膜包衣囊泡的物理机制。我们的工作处于有利地位，可以从根本上理解Hsc70如何促进脱涂层。(3)确定核苷酸交换因子在调节Hsc70中的作用：网织蛋白相互作用和突触小泡运输。我们最近发现，核苷酸交换因子促进了长寿命的Hsc70-clathrin复合体的解离。因此，在这个目标中，我们将确定哪个核苷酸交换因子参与突触小泡在神经末梢的运输，以及核苷酸交换因子：Hsc70结合和释放的循环是如何被控制的。这项工作有望促进我们对突触传递机制的基本理解，因此将是我们抗击神经障碍努力的关键部分。与公共健康相关：这项工作的重点是了解突触传递的基本机制，突触传递是神经元相互交流的过程。许多神经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还涉及许多疾病，这些疾病是由于受损的聚集蛋白(阿尔茨海默氏症、肌萎缩侧索硬化症、帕金森氏症、亨廷顿氏症等)积累的结果，因此这项工作也将与抗击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.