Molecular mechanisms of anterograde transport

描述(由申请人提供)： 顺行运输对神经元突触的维持至关重要。神经元突起的长距离顺行运输是由肌动蛋白、微管轨迹和分子马达、动蛋白、动力蛋白和肌球蛋白共同调节的。如何将货物附着到正确的马达上，以将其运送到细胞内的特定位置，是神经生理学中的一个主要问题。大量研究表明，淀粉样前体蛋白(APP)具有作用，当蛋白质分解时，它会产生阿尔茨海默病的有毒AB片段。我们已经开发了人类单纯疱疹病毒1型，作为一种工具来发现货物-运输相互作用的分子机制。在其生命周期的不同时期，HSV在神经元突起中要么逆行，要么顺行。我们通过将GFP-VP16标记的HSV注射到轴突内，并用共聚焦显微镜成像，重建了HSV在鱿鱼巨大轴突中的逆行运输。在这个项目中，GFP和mRFP1标记的HSV的顺行运输将在巨大的轴突中重建。这项测试将被用来发现货物-发动机相互作用的分子基础。在初步结果中，我们发现GFP-VP16标记的HSV也在巨大轴突中沿顺行方向运输。运动性病毒与全长APP共同净化--APP的提取去除了运动性。通过将APP的多肽片段包裹在荧光珠上，然后将其注射到轴突中，我们发现了一个足以调节珠子运输的15个氨基酸序列。在这一应用中，我们将：(1)分析单纯疱疹病毒顺行运输的方向和速度，并确定病毒使用的运动受体(S)；(2)通过向巨轴突内注射多肽包裹的荧光微球来确定APP顺行运动所需的多肽序列；(3)确定单纯疱疹病毒如何在培养细胞合成运动受体的过程中获得运动受体；以及(4)发现单纯疱疹病毒是否影响APP蛋白分解为有毒片段。这些研究的结果将明确确定HSV的运输机制和APP的作用。因此，这些结果将给出关于轴突运输的普遍问题的答案，关于疱疹发病机制的具体事实，以及关于APP在阿尔茨海默病中的作用的基本新信息。

DESCRIPTION (provided by applicant): Anterograde transport is crucial for maintenance of the synapse in neurons. Anterograde transport over long distances in neuronal processes is mediated by a combination of actin and microtubule tracks and molecular motors, kinesin, dynein and myosin. How cargo attaches to the correct motor to be transported to specific locations within the cell is a major question in neuronal physiology. Numerous studies suggest a role for amyloid precursor protein (APP), which when proteolyzed produces the toxic AB fragment of Alzheimer's disease. We have developed human herpes simplex virus 1, HSV, as a tool to discover molecular mechanisms of cargo-transport interactions. At different times in its life cycle, HSV travels in either the retrograde or the anterograde direction in neuronal processes. We reconstituted retrograde transport of HSV in the giant axon of the squid by injecting GFP-VP16 labeled HSV into axon and imaged transport by confocal microscopy. In this project, anterograde transport of GFP- and mRFP1- labeled HSV will be reconstituted in the giant axon. This assay will be used to discover the molecular basis for cargo-motor interactions. In Preliminary Results, we show that GFP-VP16-labeled HSV is also transported in the anterograde direction in the giant axon. Motile virus co-purifies with full length APP -- and extraction of APP removes motility. By coating fluorescent beads with peptide fragments of APP and injecting then into the axon, we discovered a 15 amino acid sequence sufficient to mediate transport of the beads. In this application, we will: (1) analyze the direction and velocity of HSV anterograde transport and identify the motor receptor(s) that the virus uses, (2) determine the peptide sequence of APP sufficient for anterograde motility using peptide-coated fluorescent beads injected into the giant axon, (3) determine how HSV acquires the motor receptor during its synthesis in cultured cells, and (4) discover whether HSV affects APP proteolysis into toxic fragments. Results from these studies will definitively identify the mechanism of HSV transport and the role of APP. Thus, these results will yield answers to universal questions on axonal transport, specific facts about herpes pathogenesis, and fundamental new information about the role of APP in Alzheimer's disease.