Mechanisms of slit-roundabout mediated axon repulsion

描述（由申请人提供）：发育中的神经系统中的轴突如何成功地导航到正确的目标是神经生物学中的一个基本问题。轴突受到吸引和排斥信号的引导，它们是进化保守蛋白家族的成员。我们建议研究在排斥性轴突引导过程中起作用的信号机制。果蝇胚胎中枢神经系统（CNS）的中线为解决这些问题提供了一个理想的系统。就像它的结构类似物，脊椎动物的底板一样，苍蝇的中线是许多种类的导航轴突的中间目标，它们必须决定是否交叉。在果蝇的中枢神经系统中，保守的引导线索Slit及其神经元受体roundroughs （Robo）在中线轴突连接模式中发挥多种作用，主要作为轴突排斥剂。本研究的主要目的是：(1)验证蛋白质水解过程直接导致Robo排斥和信号终止的假设；(2)研究已确定的Robo信号成分Abelson、Son of Sevenless和CrossGAP如何共同协调Robo下游的信号传导；(3)解剖三种Robo家族成员不同排斥功能的机制。我们已经建立了特定金属蛋白酶和机器人排斥调节之间的遗传和直接生化联系，并进行了必要的遗传、生化和细胞生物学分析来研究蛋白质水解过程影响排斥的机制。在我们的实验室中，类似的方法已经建立起来，这将允许我们继续研究Robo下游的信号分子如何协调调节轴突排斥。先前成功的嵌合受体方法，即Robo1， Robo2和Robo3受体的不同部分相互交换，然后在转基因果蝇中分析其功能，将用于了解不同的Robo受体如何导致不同的排斥事件。总之，这些研究有望丰富我们对正常发育过程中Slit-Robo信号的理解，并可能为各种人类健康问题提供新的治疗靶点，包括神经系统发育障碍、脊髓损伤和中风。公共卫生相关性：拟议的研究有可能对理解神经系统发育障碍、损伤后再生做出重要贡献，此外还可能为了解各种癌症的恶性肿瘤提供见解。Slit-Roundabout信号系统在调节神经系统中的布线中具有良好的作用，这表明了解正常发育过程中的Slit-Robo信号可能为治疗各种人类健康问题提供新的靶点，包括中风和脊髓损伤，神经系统发育障碍和神经退行性疾病。

DESCRIPTION (provided by applicant): How axons in the developing nervous system successfully navigate to their correct targets is a fundamental problem in neurobiology. Axons are guided by both attractive and repulsive cues, which are members of evolutionary conserved protein families. We propose to study the signaling mechanisms that function during repulsive axon guidance. The midline of the Drosophila embryonic central nervous system (CNS) provides an ideal system to address these questions. Like its structural analog, the vertebrate floor plate, the fly midline is an intermediate target for many classes of navigating axons, which must decide whether or not to cross. In the Drosophila CNS, the conserved guidance cue Slit and its neuronal receptors the Roundabouts (Robo), play multiple roles in patterning axonal connections at the midline, acting primarily as axonal repellants. The primary aims of this proposal are to 1) test the hypothesis that proteolytic processing directly contributes to Robo repulsion and to signal termination, 2) to investigate how the identified Robo signaling components Abelson, Son of Sevenless and CrossGAP function together to coordinate signaling downstream of Robo and 3) to dissect the mechanisms underlying the distinct repulsive functions of the three Drosophila Robo family members. We have established genetic and direct biochemical links between a specific metalloprotease and regulation of Robo repulsion and have the necessary genetic, biochemical and cell biological assays to investigate the mechanism by which proteolytic processing influences repulsion. Similar kinds of approaches well established in our laboratory will allow for our continued investigation of how signaling molecules that function downstream of Robo coordinately regulate axon repulsion. A previously successful chimeric receptor approach where different portions of the Robo1, Robo2 and Robo3 receptors are exchanged with each other and then assayed for function in transgenic flies will be used to understand how different Robo receptors lead to distinct repulsive events. Together these studies promise to enrich our understanding of Slit-Robo signaling during normal development and may provide new therapeutic targets for diverse human health problems, ranging from developmental disorders of the nervous system to spinal cord injury and stroke. PUBLIC HEALTH RELEVANCE: The proposed research has the potential to make important contributions to the understanding of developmental disorders of the nervous system, regeneration after injury and in addition may provide insight into malignancy in various kinds of cancer. The Slit-Roundabout signaling system has a well-established role in regulating wiring in the nervous system, suggesting that understanding Slit-Robo signaling during normal development may provide new targets for therapies for diverse human health problems, ranging from stroke and spinal cord injury to developmental disorders of the nervous system and neurodegenerative disease.