Organization and function of neuronal endosomes

描述（由申请人提供）：受体的内体运输在神经元功能和神经发育以及神经系统疾病状态中起着重要作用。目前对内核体组织和功能的了解主要是基于酵母和成纤维细胞的研究。然而，我们最近的工作表明，神经元使用神经元特异性的内吞和内体机制。因此，目前对内体功能如何适应神经元的特定生理需求的理解存在根本差距。由于内体的特定组织决定了内吞后受体的转运（即信号传导、加工和/或降解），从而决定了特定的功能结果，因此该领域对内体系统的神经元适应性的无知构成了基础和疾病导向领域进展的重大障碍。我们的长期目标是揭示核内体对神经发育以及健康和患病大脑的功能贡献。提出这一建议的基本原理是，脊椎动物神经元在其核内体中表达神经元特异性蛋白（特别是NEEP21/Nsg-1和P19/Nsg-2），这些蛋白控制关键神经元膜蛋白的核内体运输，如在发育中重要的受体（轴突粘附分子L1/NgCAM）、突触功能（GluA2）和疾病（bAPP）。nsg蛋白是一个跨膜蛋白基因家族的成员，特异存在于神经元高尔基体和特征不明显的体突核内体（nsg核内体）中。该应用的核心概念是关键的神经元功能依赖于nsg核内体。我们的初步数据提出了一个特定的假设，即nsg蛋白在一个独特的非降解隔室中维持一个特定的内吞受体亚群，从那里，货物可以循环到多个位置，以调节轴突生长和突触功能。我们将使用创新的方法，包括定量单泡实时成像、超分辨率荧光显微镜和电子显微镜，结合初级神经元和啮齿动物皮层的功能干扰方法，以解决三个具体目标：目标1)nsg核内体是如何形成的，以及它们与其他体突核内体的关系如何？NEEP21/Nsg-1与哪些蛋白相互作用，哪些蛋白调控nsg -内体组织？目标2)NEEP21/Nsg-1和P19/Nsg-2的缺失如何影响内体组织和货物运输？目的3)NEEP21/Nsg-1和P19/Nsg-2的缺失是否影响皮层轴突和树突的发育？本研究的意义在于发现内体在健康和疾病中对神经元功能的贡献。获得的新见解不仅将导致理解神经元膜交通调节的基本进展，而且还将提高未来定制转化策略的新目标的可能性。

DESCRIPTION (provided by applicant): Endosomal trafficking of receptors plays fundamental roles in neuronal function and in neurodevelopment, as well as in disease states of the nervous system. The current understanding of endosome organization and function is based primarily on work in yeast and fibroblasts. However, our recent work showed that neurons employ neuronal-specific endocytic and endosomal machinery. There is thus presently a fundamental gap in the understanding of how endosomal function is adapted to cater to the specific physiological needs of neurons. Since the particular organization of the endosome determines postendocytic trafficking of receptors (i.e. signaling, processing, and/or degradation) and thereby specific functional outcomes, the field's ignorance of the neuronal adaptations of the endosomal system constitutes a significant barrier to progress in both basic and disease-oriented fields. Our long-term goals are to uncover the functional contributions of endosomes to neurodevelopment, and to the healthy and diseased brain. The rationale motivating this proposal is that vertebrate neurons express neuronal-specific proteins in their endosomes (in particular NEEP21/Nsg-1 and P19/Nsg-2) that control the endosomal trafficking of crucial neuronal membrane proteins, such as receptors important in development (the axonal adhesion molecule L1/NgCAM), synaptic function (GluA2), and disease (bAPP). Nsg-proteins are members of a gene family of transmembrane proteins found specifically in the neuronal Golgi and in poorly characterized somatodendritic endosomes ("Nsg-endosomes"). The central concept of this application is that crucial neuronal functions depend on Nsg-endosomes. Our preliminary data suggest the specific hypothesis that Nsg-proteins maintain a specific subset of endocytosed receptors in a distinct non-degradative compartment from where the cargo can recycle to multiple locations to regulate axon growth and synaptic function. We will use innovative approaches including quantitative single vesicle live imaging, super-resolution fluorescence microscopy, and electron microscopy in combination with functional interference approaches in primary neurons and in rodent cortex to address three specific aims: Aim 1) How are Nsg-endosomes formed and how do they relate to other somatodendritic endosomes? With which other proteins does NEEP21/Nsg-1 interact and which rabs regulate Nsg-endosomal organization? Aim 2) How does loss of NEEP21/Nsg-1 and P19/Nsg-2 affect endosomal organization and cargo trafficking? Aim 3) Does loss of NEEP21/Nsg-1 and P19/Nsg-2 affect axon and dendrite development in the cortex? The proposed research is significant because it will discover the contribution of endosomes to neuronal function in health and disease. The new insights gained will not only lead to fundamental advances in understanding the regulation of neuronal membrane traffic, but also raise the possibility of new targets for tailoring translational strateges in the future.