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取决于将大量蛋白质正确靶向特定的细胞作用位点，不正确的蛋白质靶向与各种疾病状态和异常有关，包括神经系统疾病。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。在阐明体树突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作用知之甚少。这项工作的目标是阐明进入极化神经元轴突的膜交通途径，并揭示其分子基础。具体来说，我们将分析轴突靶向的细胞粘附分子L1/NgCAM，介导轴突寻路和成束在大脑发育过程中。拟议的工作是基于我的实验室的结果表明，两个不同的后高尔基体运输路线的轴突存在L1/NgCAM。一种是通过体树突状细胞质膜和内体的间接胞吞途径，另一种是直接途径，没有体树突状细胞质膜上的中间站。我们的主要假设是L1/NgCAM是直接还是转胞吞到达轴突取决于其中包含的不同分选信号的阅读和执行。为了揭示控制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）详细绘制LI/NgCAM通过转胞吞到达轴突所需的靶向信号及其执行的具体顺序。(Aim 2）表征用于识别L1/NgCAM的靶向信号的分子机制。(Aim 3）剖析内吞途径的组织。我们的方法包括在培养的海马和其他神经元中表达腺病毒载体的突变分子（Aim 1），研究轴突运输动力学的新方法（Aim 1），荧光分子解剖通路的活细胞成像（Aim 3）和双杂交筛选和生物化学方法来识别分子组分（Aim 2）。我们工作的总体目标是详细说明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.