Targeting Pathways of Axonal Cell Adhesion Molecules

描述(由申请人提供)： 所有细胞类型的正常功能依赖于正确地将大量蛋白质靶向特定的细胞作用部位，错误的蛋白质靶向与各种疾病状态和异常有关，包括神经疾病。例如，在极化的脊椎动物神经元中，蛋白质需要正确地运输到轴突和树突。在阐明躯体树突状细胞蛋白的运输方面已经取得了一些进展，但对轴突蛋白的靶向了解很少。这项拟议工作的目标是阐明进入极化神经元轴突的膜运输通路，并揭示其分子基础。具体地说，我们将分析细胞黏附分子L1/NgCAM的轴突靶向，它在大脑发育过程中介导轴突的寻路和颤动。这项拟议的工作是基于我的实验室的结果，该结果证明L1/NgCAM存在两条不同的后高尔基体轴突运输路线。一种是通过体树突质膜和内小体的间接跨细胞途径，另一种是没有在体树突质膜上中间停留的直接途径。我们的主要假设是，L1/NgCAM是直接还是跨细胞地到达轴突，取决于其中包含的不同分类信号的读取和执行。为了揭示指导这一途径选择的分子原理，我们将追求三个具体目标：(目标1)详细映射LI/NgCAM通过细胞转运到达轴突所需的靶向信号及其执行的特定顺序。(目的2)描述识别L1/NgCAM靶向信号的分子机制。(目的3)剖析内吞途径的组织结构。我们的方法包括腺病毒载体在培养的海马神经元和其他神经元中表达突变分子(目标1)，研究轴突运输动力学的新方法(目标1)，荧光分子的活细胞成像以解剖通路(目标3)，以及双杂交筛选和生化方法来识别分子成分(目标2)。我们工作的总体目标是明确产生神经元极化结构的机制。

DESCRIPTION (provided by applicant): Proper functioning of all cell types depends on correctly targeting a large number of proteins to specific cellular sites of action, incorrect protein targeting has been linked to a variety of disease states and abnormalities, including neurological disorders. In polarized vertebrate neurons, for instance, proteins need to be trafficked correctly to axons and dendrites. Some progress has been made in elucidating the trafficking of somatodendritic proteins, but targeting of axonal proteins is poorly understood. The goal of the proposed work is to elucidate the membrane traffic pathways into the axon of polarized neurons and to uncover their molecular underpinnings. Specifically, we will analyze the axonal targeting of the cell adhesion molecule L1/NgCAM, which mediates axonal pathfinding and fasciculation during brain development. The proposed work is based on results from my laboratory that demonstrate that two distinct post-Golgi transport routes to the axon exist for L1/NgCAM. One is an indirect transcytotic route via the somatodendritic plasma membrane and endosomes, the other a direct route without intermediate stops on the somatodendritic plasma membrane. Our major hypothesis is that whether L1/NgCAM travels to the axon directly or transcytotically depends on the reading and execution of distinct sorting signals contained within it. In order to uncover the molecular principles that govern this pathway selection, we will pursue three specific aims: (Aim 1) Map in detail the targeting signals and the specific order of their execution required for LI/NgCAM to reach the axon via transcytosis. (Aim 2) Characterize the molecular machinery for recognizing L1/NgCAM's targeting signals. (Aim 3) Dissect the organization of endocytic pathways. Our approaches include expression of mutant molecules from adenovirus vectors in cultured hippocampal and other neurons (Aim 1), novel approaches to study the kinetics of axonal transport (Aim 1), live-cell imaging of fluorescent molecules to dissect pathways (Aim 3) and two-hybrid screens and biochemical approaches to identify molecular components (Aim 2). The overall goal of our work is to specify the mechanisms producing the polarized architecture of neurons.