The Frizzled Nuclear Import Pathway in Synapse Development

描述(由申请者提供)：本项目的长期目标是了解突触发育的信号机制。在果蝇幼虫神经肌肉接头(NMJ)上使用强大的策略，我们发现了一个非规范的Wnt信号通路，Frizzled核输入(FNI)通路，这是正常突触发育所必需的。在上一个资金周期中，我们发现FNI途径与含有突触特异性mRNAs的大核糖核蛋白颗粒(MegaRNPs)的形成和核外流有关。在这一途径中，内源性转录物被包装在细胞核内的megaRNP颗粒中，然后通过核膜萌发将其输出到细胞质，这一过程类似于疱疹病毒的核外泄。这一应用中的方法构成了一种深刻的策略，以阐明这种新的核出口模式背后的分子机制。在揭示核孔复合体的分子成分导致理解转录调控、RNA加工和翻译方面取得重大进展的同时，我们预计解开核膜出芽的成分将为这些过程提供重要和新颖的视角。我们的研究发现Torsin是一种AAA-ATPase，在人类中与早发性肌张力障碍有关，是发芽所需核膜重塑的关键因素。我们还发现，细胞极性复合体的成分(非典型蛋白激酶C、Par3/Bazooka和Dap160/Intersectin)是核巨型RNP出口过程中核层局部重塑的重要决定因素。在这个项目中，我们提出了一个全面的策略来发现萌芽机械的其他组件，并确定它们对突触发育和功能的影响。本项目的具体目标是(1)通过核膜萌发来确定核megaRNP输出的分子连接点，(2)确定Torsin的功能作用并识别下游效应因子，以及(3)检验Dap160、Baz和Par6调节依赖于PKC的核膜局部重塑从而使megaRNPs能够发生核膜萌发的假设。通过为核膜相关营养不良(例如，椎板病和包膜病)、单纯疱疹病毒感染和肌张力障碍等疾病的机制基础提供新的见解，我们希望我们的研究将为治疗或治愈这些疾病的治疗设计打开新的窗口。

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand the signaling mechanisms underlying synapse development. Using powerful strategies at the Drosophila larval neuromuscular junction (NMJ) we have uncovered a non-canonical Wnt signaling pathway, the Frizzled Nuclear Import (FNI) pathway, which is required for proper synapse development. During the last funding cycle, we found that the FNI pathway is associated with the formation and nuclear egress of large ribonucleoprotein granules (megaRNPs) containing synapse-specific mRNAs. In this pathway, endogenous transcripts are packaged in megaRNP granules within the nucleus and are then exported to the cytoplasm by budding through the nuclear membrane in a process akin to the nuclear egress of Herpes-type viruses. The approach in this application constitutes an incisive strategy to elucidate the molecular machinery underlying this novel mode of nuclear export. In the same manner by which uncovering molecular components of the nuclear pore complex has led to major progress in understanding regulation of transcription, RNA processing and translation, we expect that unraveling constituents of nuclear envelope budding will shed important and novel perspectives on these processes. Our studies identify Torsin, a AAA-ATPase, which in humans is linked to early onset dystonia as a key element in the remodeling of nuclear membranes required for budding. We have also identified components of the cell polarity complex (atypical Protein Kinase C, Par3/Bazooka and Dap160/Intersectin) as important determinants of local remodeling of the nuclear lamina during nuclear megaRNP egress. In this project we propose a comprehensive strategy to uncover other components of the budding machinery and to determine their impact on synapse development and function. The specific aims of this project are (1) to identify the molecular linchpins of nuclear megaRNP export by nuclear envelope budding, (2) to determine the functional role of Torsin and identify downstream effectors and (3) test the hypothesis that Dap160, Baz and Par6 regulate aPKC- dependent local remodeling of the nuclear lamina to enable nuclear envelope budding of megaRNPs. By providing novel insight into the mechanistic basis of such diseases as nuclear envelope-associated dystrophies (e.g., laminopathies and envelopathies), HSV- type infections and dystonia, we expect our studies to open new windows for the design of therapies to treat or cure these conditions.