L1 Interactions in Retino-Collicular Targeting

从眼睛到大脑的神经元轴突的抽象引导和靶向是通过一系列刚刚开始阐明的分子机制通过一系列不同的线索来协调的。这一建议的重点是神经细胞黏附分子L1调节视网膜神经节细胞轴突到大脑靶点的视网膜定位图的机制。有待检验的具体假设是，L1通过ankyrin与细胞骨架的联系是一个必要的机械决定因素，通过该决定因素，神经节细胞轴突被映射到上丘特定的局部靶点，以指定视觉连接。L1与神经元细胞骨架的相互作用在突触靶向中的作用将在一个新的L1突变小鼠L1(Y1229H)中进行测试，该小鼠缺乏结合ankyrin的能力，ankyrin是一种将L1连接到肌动蛋白细胞骨架的适配器。该突变体是第一个点突变破坏特定细胞骨架连接的例子，该连接扰乱了地形性轴突映射。目的1通过对L1(Y1229H)敲入小鼠上丘视网膜神经节细胞轴突的定位追踪，明确L1(Y1229H)敲入小鼠视网膜前后轴和内侧轴的定位模式，以了解L1对细胞骨架的锚定在轴突生长和突触定位中的作用。在目标2中，细胞和生化分析将确定L1与细胞骨架的结合所支配的蛋白质相互作用，这是轴突靶向和突触接触形成所必需的。具体的实验被设计用来分析L1-Ankyrin结合对整合素依赖的细胞黏附的调节作用以及对肾上腺素和Eph受体(A和B类受体)的吸引或排斥轴突引导。L1神经黏附分子与Ankyrin和EPhin/Eph受体的相互作用是一个新的概念，将促进对调节神经元连接的机制的分子理解。这项研究对于了解神经元连接的基本机制具有广泛的意义，因为L1是在大脑中广泛表达的黏附受体家族的原型，在那里它们可能通过不同的锚蛋白亚型介导突触连接。教育福利将包括培养2名本科生、2名研究生和1名博士后科学家。这笔赠款将增加妇女参与研究，增强多样性和少数群体代表性，因为所有受训人员都是妇女。对整个社会的普遍好处将包括提供新的知识，这将增加对神经元连接的基本机制的理解。这一知识将对神经再生和神经移植的更大领域产生广泛影响，并可能为改善动物和人类的生活和健康提供新的方法。

Abstract Guidance and targeting of neuronal axons from the eye to the brain is orchestrated through a diverse set of cues by molecular mechanisms that are just beginning to be elucidated. This proposal focuses on the mechanism by which the neural cell adhesion molecule L1, regulates the retinotopic map of retinal ganglion cell axons to targets in the brain. The specific hypothesis to be tested is that L1 linkage to the cytoskeleton through ankyrin is an esssential mechanistic determinant by which ganglion cell axons are mapped to topographically specific targets in the superior colliculus to specify visual connectivity. The role of L1 interactions with the neuronal cytoskeleton in synaptic targeting will be tested in a novel L1 mutant mouse L1(Y1229H), which lacks the capability to bind ankyrin, an adapter that links L1 to the actin cytoskeleton. This mutant is the first example of a point mutation disrupting a specific cytoskeletal linkage that perturbs topographic axon mapping. In Aim 1 axon tracing of retinal ganglion cell axons to topographic targets in the superior colliculus of L1(Y1229H) knock-in mice will be carried out to define the pattern of retinotopic mapping along both the anteroposterior and mediolateral axes in order to obtain insight into the role of L1 anchorage to the cytoskeleton in axonal outgrowth and synaptic targeting. In Aim 2, cellular and biochemical assays will identify the protein interactions governed by L1 binding to the cytoskeleton necessary for axonal targeting and synaptic contact formation. Specific experiments are designed to analyze L1-ankyrin binding effects on modulation of integrin-dependent cell adhesion and attractive or repulsive axon guidance to ephrins and Eph receptors (both A and B classes). The interaction of L1 neural adhesion molecules with ankyrin and ephrin/Eph receptors is a novel concept that will advance a molecular understanding of mechanisms that regulate neuronal wiring. This study has broad significance for understanding fundamental mechanisms of neuronal wiring, because L1 is the prototype of a family of adhesion receptors that are widely expressed in the brain, where they may mediate synaptic connections through different ankyrin isoforms. Educational benefits will include training of 2 undergraduates, 2 graduate students, and a postdoctoral scientist. The grant will increase the participation of women in research, enhancing diversity and minority representation, as all of the trainees are women. General benefits to the society at large will include provision of new knowledge that will increase understanding of fundamental mechanisms of neuronal connectivity. This knowledge will impact broadly on larger areas of nerve regeneration and nerve grafting, and may suggest new means to improve the life and health of animals and human beings.