L1 Interactions in Retino-Collicular Targeting

从眼睛到大脑的神经元轴突的引导和靶向是通过刚刚开始阐明的分子机制的各种线索来编排的。该建议的重点是神经细胞粘附分子L1调节视网膜神经节细胞轴突到大脑中靶点的视网膜定位图的机制。要测试的具体假设是，L1连接到细胞骨架通过锚蛋白是一个esssential机械决定因素，神经节细胞轴突被映射到地形特异性目标的上级丘，以指定视觉连接。 L1与神经元细胞骨架在突触靶向中的相互作用的作用将在一种新的L1突变小鼠L1（Y1229 H）中进行测试，该小鼠缺乏结合锚蛋白的能力，锚蛋白是一种将L1连接到肌动蛋白细胞骨架的适配器。这个突变体是第一个破坏特定细胞骨架连接的点突变的例子，该点突变扰乱了拓扑轴突映射。 在目的1中，将进行视网膜神经节细胞轴突追踪到L1（Y1229 H）敲入小鼠上级丘中的拓扑靶点的轴突追踪，以定义沿着前后轴和中间外侧轴的视网膜定位映射的模式，从而深入了解L1锚定到细胞骨架在轴突生长和突触靶向中的作用。在目标2中，细胞和生物化学测定将确定由L1结合到轴突靶向和突触接触形成所必需的细胞骨架所支配的蛋白质相互作用。设计特定实验以分析L1-锚蛋白结合对整联蛋白依赖性细胞粘附的调节以及对肝配蛋白和Eph受体（A和B类）的吸引或排斥轴突引导的作用。 L1神经粘附分子与锚蛋白和ephrin/Eph受体的相互作用是一个新的概念，将促进对调节神经元布线机制的分子理解。 这项研究对于理解神经元布线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粘附受体家族的原型，在那里它们可以通过不同的锚蛋白亚型介导突触连接。教育福利将包括2名本科生，2名研究生和一名博士后科学家的培训。由于所有受训人员都是妇女，这笔赠款将增加妇女参与研究，提高多样性和少数群体的代表性。对整个社会的一般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。这些知识将广泛影响神经再生和神经移植的更大领域，并可能提出改善动物和人类生活和健康的新方法。

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.