Signaling Mechanisms of Retinal Axon Guidance

描述（由申请人提供）：在哺乳动物视觉系统的形成过程中，新生神经元延伸轴突，与视网膜和大脑内的特定靶区细胞形成定型连接。Eph受体及其肝配蛋白配体在视觉系统发育过程中发挥关键作用，将视网膜轴突正确引导至大脑。然而，介导Eph依赖性轴突靶向事件的细胞内信号传导事件仍不清楚。本研究的主要目标是识别和理解关键的细胞内信号事件，将细胞外轴突引导线索转化为适当的轴突转向事件。我们的初步研究表明，Vav和Ephexin家族RhoGEFs（Rho家族GT-鸟嘌呤核苷酸交换因子）是体外和体内Eh依赖性轴突排斥的重要调节因子。我们试图探索Vav GEFs在视网膜轴突到大脑的适当靶向中的确切作用，并测试Vav GEFs调节从吸引信号到排斥信号的开关的假设。为了解决这些想法并开始了解Vav GEFs在视网膜发育和轴突靶向中的作用和调节，我们提出了以下具体目标：1）通过分析发育过程中视交叉处Vav缺陷、EphB 1阳性轴突的行为，确定Vav GEF在体内视网膜神经轴突靶向中的作用，2）确定Vav GEF在体外和体内调节吸引性与排斥性Eph信号传导中的作用，和3）通过研究Vav 2的Eph激酶依赖性降解来确定Vav GEF在视网膜轴突引导期间的调节。在本研究中，我们将采用活体轴突追踪技术、生物化学和分子生物学方法以及细胞培养技术来研究Vav GEFs在视网膜发育中的作用。此外，我们已经开发了一种化学调节的，基于细胞培养的方法，以更容易地测试Eph受体信号传导事件的作用。这些研究的结果将为控制正常视觉系统发育的细胞信号传导过程提供有价值的新见解。了解这些视网膜轴突引导机制可能为视网膜发育障碍、视网膜神经损伤后轴突再生和导致部分或完全失明的神经退行性疾病的治疗方法提供重要的新见解。

DESCRIPTION (provided by applicant): During formation of the mammalian visual system, newly born neurons extend axons to make stereotyped connections with specific target field cells within the retina and brain. Eph receptors and their ephrin ligands play critical roles during visual system development in the proper guidance of retinal axons to the brain. However, the intracellular signaling events that mediate Eph-dependent axon targeting events has remained unclear. The main goal of this research proposal is to identify and understand the critical intracellular signaling events that convert the extracellular axon guidance cue into an appropriate axon turning event. Our preliminary studies have revealed that Vav and Ephexin family RhoGEFs (Rho family GTPase guanine nucleotide exchange factors) are essential regulators of Eph-dependent axon repulsion in vitro and in vivo. We seek to explore the precise role of Vav GEFs in the proper targeting of retinal axons to the brain, and to test the hypothesis that Vav GEFs regulate a switch from attractive to repulsive signaling. To address these ideas and to begin to understand the role and regulation of Vav GEFs in retinal development and axoon targeting, we propose the following specific aims: 1) to determine the role of Vav GEFs in retinogeniculate axon targeting in vivo by analyzing the behavior of Vav-deficient, EphB1 -positive axons at the optic chiasm during development, 2) to determine the role of Vav GEFs in regulating attractive vs. repulsive Eph signaling in vitro and in vivo, and 3) to determine the regulation of Vav GEFs during retinal axon guidance by studying the Eph kinase-dependent degradation of Vav2. In this proposal, in vivo axon tracing techniques, biochemical and molecular biology approaches combined with cell culture techniques will be employed to explore the role of Vav GEFs in retinal development. Moreover we have developed a chemically-regulated, cell culture-based approach to more easily test the role of Eph receptor signaling events. The results of these studies will provide valuable new insights into the cell signaling processes that control proper visual system development. Understanding these retinal axon guidance mechanisms might provide important new insights into therapeutic approaches to retinal development disorders, axon regeneration following retinal nerve injury and neurodegenerative diseases that lead to partial or total blindness.